Short communication Yeast ecology in French cider and black olive natural fermentations Emmanuel Coton a, * , Monika Coton a , Delphine Levert a , Serge Casaregola b , Danie `le Sohier c a ADRIA NORMANDIE, boulevard du 13 juin 1944, 14310 Villers-Bocage, France b Laboratoire de Microbiologie et Ge ´ne ´tique Mole ´culaire, CNRS/INA-PG UMR2585, INRA UMR1238, BP01, F-78850 Thiverval-Grignon, France c ADRIA De ´veloppement, Z.A. de Cre ´ac’h Gwen, 29196 Quimper, France Received 28 June 2004; received in revised form 28 September 2005; accepted 12 October 2005 Abstract In this study, rDNA ITS restriction analysis was used to identify yeasts from two naturally fermented products: French ciders and black olives. In cider musts and bottled ciders, the PCR-RFLP method generated 15 different ITS/RFLP profiles for a total of 208 isolates. The predominant yeasts corresponded to Saccharomyces bayanus , Saccharomyces cerevisiae , Lachancea cidri and Dekkera anomala . Three identified species: Candida sake , Candida tropicalis and Kluyveromyces marxianus had never been described before in ciders. For the black olive fermentation, the method allowed for identification of 11 profiles for a total of 137 isolates. A sequential apparition of yeasts was observed with Pichia anomala , Candida boidinii and Debaryomyces etchellsii being the predominant species. Four isolates did not correspond to any known species based on the sequencing of the D1/D2 region of the 26S rRNA gene. By using the rDNA ITS method, valuable information on yeast population biodiversity and dynamics in the naturally fermented food products studied was obtained. D 2005 Elsevier B.V. All rights reserved. Keywords: Identification; PCR-RFLP; Fermentation; Food; Beverage; Yeast 1. Introduction For centuries, yeasts have been present in fermented foods and beverages such as bread, cheese, olives, wine, beer and cider (Boekhout and Robert, 2003). In these products, some yeasts are beneficial (‘‘technological flora’’) to the quality of the final product while others are detrimental (spoilage flora). Although some industries using yeast fermented products (bread, soft cheese, wine, beer) have selected technological strains as starters (Angelov et al., 1996; Lancashire and Gopal, 1998; Prior et al., 1999; Ferreira and Viljoen, 2003), other industries (cider and black olives industries) are still relying on indigenous flora to perform the fermentations (Fernandez Gonzalez et al., 1992; Laplace et al., 2001). Therefore, yeast identification is of great importance for the selection of technological yeasts as well as for the study of biodiversity and population dynamics of fermentative ecosystems. Concerning ciders, natural fermentations are still in use in France, Spain and Ireland. In these natural fermentations, a succession of yeast species is observed (Beech, 1993). The fermentation begins with apiculate yeasts (Hanseniaspora/ Kloeckera sp.) soon overgrown by Saccharomyces sp., especially Saccharomyces uvarum , more adapted to the anaerobic conditions and alcohol content. In the final stage, also called maturation, Brettanomyces /Dekkera species often dominate (Morrissey et al., 2004). Concerning natural black olives, yeasts are responsible for the fermentation process, contrary to green olives in which the fermentation is performed by lactic acid bacteria (Garrido- Fernandez et al., 1997). Black olive fermentations have been studied in different countries, mainly Spain, Portugal, Greece and Morocco and showed some yeast species biodiversity, yet a few species were dominant: Pichia membranifaciens , Saccha- romyces oleaginosus , Pichia anomala , Candida boidinii and Torulaspora delbrueckii (Marquina et al., 1992; Fernandez Gonzalez et al., 1992; Kotzekidou, 1997; Marquina et al., 1997). Yeast ecology in French black olives (Nyons area) has never been reported so far. The polymorphism of the rDNA internal transcribed spacer (ITS), covering the conserved 5.8S rRNA gene region and the two variable flanking regions ITS1 and ITS2, has been used in 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.10.016 * Corresponding author. Tel.: +33 231 25 43 01; fax: +33 231 77 49 43. E-mail address: ecoton@adrianie.org (E. Coton). International Journal of Food Microbiology 108 (2006) 130 – 135 www.elsevier.com/locate/ijfoodmicro