Analysis and direct quantification of Saccharomyces cerevisiae and Hanseniaspora guilliermondii populations during alcoholic fermentation by fluorescence in situ hybridization, flow cytometry and quantitative PCR Imma Andorra a, b , Margarida Monteiro a , Braulio Esteve-Zarzoso b , Helena Albergaria a, * , Albert Mas b a LNEG, Unidade Bioenergia, Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal b Universidad Rovira & Virgili, Dept Bioquim & Biotecnol, Fac Enologia, Tarragona 43007, Spain article info Article history: Received 30 November 2010 Received in revised form 29 July 2011 Accepted 7 August 2011 Available online 12 August 2011 Keywords: Wine Culture-independent methods RNA stability Yeast population dynamics abstract Traditionally, it was assumed that non-Saccharomyces (NS) yeasts could only survive in the early stages of alcoholic fermentations. However, recent studies applying culture-independent methods have shown that NS populations persist throughout the fermentation process. The aim of the present work was to analyze and quantify Saccharomyces cerevisiae (Sc) and Hanseniaspora guilliermondii (Hg) populations during alcoholic fermentations by plating and culture-independent methods, such as fluorescence in situ hybridization (FISH) and quantitative PCR (QPCR). Species-specific FISH probes labeled with fluorescein (FITC) were used to directly hybridize Sc and Hg cells from single and mixed cultures that were enumerated by epifluorescence microscopy and flow cytometry. Static and agitated fermentations were performed in synthetic grape juice and cell density as well as sugar consumption and ethanol production were determined throughout fermentations. Cell density values obtained by FISH and QPCR revealed the presence of high populations (10 7 e10 8 cells/ml) of Sc and Hg throughout fermentations. Plate counts of both species did not show significant differences with culture-independent results in pure cultures. However, during mixed fermentations Hg lost its culturability after 4e6 days, while Sc remained cul- turable (about 10 8 cells/ml) throughout the entire fermentation (up to 10 days). The rRNA content of cells during mixed fermentations was also analyzed by flow cytometry in combination with FISH probes. The fluorescence intensity conferred by the species-specific FISH probes was considerably lower for Hg than for Sc. Moreover, the rRNA content of Hg cells, conversely to Sc cells, remained almost unchanged after boiling, which showed that rRNA stability is species-dependent. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction The transformation of grape must into wine involves the coex- istence and succession of different yeast species. The microorgan- isms present on the berry surfaces are composed mainly of non-Saccharomyces (NS) yeasts which predominate during the early stages of the alcoholic fermentation. These are soon overtaken by the growth of Saccharomyces cerevisiae (Sc) which dominates the mid to final stages of the fermentation (Fleet and Heard, 1993; Fleet, 2003). This typical growth pattern has long been accepted and established mainly by plating methods. Indeed, more recent studies have questioned this pattern after using molecular methods that reveal the persistence of NS populations throughout the fermen- tation process (Fernández et al., 1999; Cocolin et al., 2000; Andorrà et al., 2010; Zott et al., 2010). The causes of the early displacement of NS wine yeasts are still controversial. Previously, it was thought that this was mainly due to the lower tolerance of NS species toward the increasingly adverse conditions (low pH values, high levels of ethanol and organic acids, nutrient depletion, etc.) established in the medium as the fermentation progresses (Fleet and Heard, 1993). More recently, the dominance of Sc has been attributed to other factors such as growth arrest mediated by cell-to-cell contact mechanisms (Nissen et al., 2003) and the secretion of toxic compounds (Pérez-Nevado et al., 2006; Albergaria et al., 2010). However, most of these studies have been carried out by using classic plating methods which are laborious, time-consuming, somewhat unreliable (Giraffa, 2004) and which only detect culturable populations. Molecular techniques have been developed and used to control microbial growth and to characterize the microflora of different processes and environments. These methods are generally faster, more specific, more sensitive, and more accurate and allow the precise study of microbial populations and their diversity (Justé * Corresponding author. Tel.: þ351 210924721; fax: þ351 217163636. E-mail address: helena.albergaria@ineti.pt (H. Albergaria). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm 0740-0020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.08.009 Food Microbiology 28 (2011) 1483e1491