Analysis of non-Saccharomycesyeast populations isolated from grape musts from Sicily (Italy)

Abstract

Aims: The aim of this study was to identify the non‐Saccharomyces yeast populations present in the grape must microflora from wineries from different areas around the island of Sicily. Methods and Results: Yeasts identification was conducted on 2575 colonies isolated from six musts, characterized using Wallerstein Laboratory (WL) nutrient agar, restriction analysis of the amplified 5·8S‐internal transcribed spacer region and restriction profiles of amplified 26S rDNA. In those colonies, we identified 11 different yeast species originating from wine musts from two different geographical areas of the island of Sicily. Conclusions: We isolated non‐Saccharomyces yeasts and described the microflora in grape musts from different areas of Sicily. Moreover, we discovered two new colony morphologies for yeasts on WL agar never previously described. Significance and Impact of the Study: This investigation is a first step in understanding the distribution of non‐Saccharomyces yeasts in grape musts from Sicily. The contribution is important as a tool for monitoring the microflora in grape musts and for establishing a new non‐Saccharomyces yeast collection; in the future, this collection will be used for understanding the significance of these yeasts in oenology.

A Method to Discriminate Between the Candida stellata and Saccharomyces cerevisiae in Mixed Fermentation on WLD and Lysine Agar Media

“…On WL all the other yeasts were also identifiable by different colony morphologies, at least while the S. cerevisiae colonies were not so abundant as to make it impossible to recognise any other species. Only genus Hanseniaspora was found on WLD together with C. stellata, confirming that this genus is widespread in the early stages of the spontaneous fermentation of Sicilian grapes (Romancino et al, 2008): their different morphologies allowed their easy and rapid distinction during the counting of the colonies. From the second day, wild Saccharomyces yeasts were found on WL and, as expected, they did not grow on WLD and lysine agar.…”

“…mtDNA-RFLP analyses demonstrated the massive presence (92 to 100%) of the Cs3 strain in the C. stellata population of the fermenting must (data not shown). CONCLUSIONS C. stellata clearly is able to grow at low cycloheximide concentrations, but with important strain-dependent differences: for the C. stellata IRVV strains, which showed a higher resistance compared to the available data (Cavazza & Poznansky, 1998), this phenotypical evidence is a probable sign of their common geographical origin (Romancino et al, 2008). Indeed, studies on S. cerevisiae cycloheximide resistance show that mutations in different genetic loci can induce this antimycotic resistance (Del Pozo et al, 1991;Mortimer et al, 1991).…”

“…The yeasts used in this study belong to various genera and species: 14 Saccharomyces strains, including some belonging to different collections and most from commercial origins, as shown in Table 1; 42 non-Saccharomyces yeast isolates from the IRVV collection (Romancino et al, 2008), including 14 Candida stellata (Cs1-Cs14), four Hanseniaspora guilliermondii (Hg1-Hg4), five H. uvarum (Hu1-Hu4 and Hu03), three Issatchenkia terricola (It1-It3), four K. thermotolerans (Kt1-Kt4), three Metschnikowia pulcherrima (Mp1-Mp3), three T. delbrueckii (Td1-Td3), three Zygosaccharomyces bailii (Zb1-Zb3) and three Zygoascus hellenicus (Zh1-Zh3); and finally the C. stellata 6714, H. guilliermondii 6796 and K. thermotolerans 6232 strains of the University of Perugia's Industrial Yeasts Collection DBVPG (www.agr.unipg.it/dbvpg). The commercial S. cerevisiae NDA21 yeast was also selected by the IRVV for the production of high quality wines (Di Maio et al, 2006;Oliva et al, 2006).…”

“…We demonstrated that it is possible to collect data about a single C. stellata strain in a fermentation with Saccharomyces and non-Saccharomyces yeasts using WL, WLD and lysine agar. Furthermore, this method has the potential to distinguish between C. stellata and K. thermotolerans, both generally found in grape musts, growing on WL with very similar morphologies (Romancino et al, 2008) but showing very different levels of resistance to cycloheximide. Finally, this method allows the distinction between inoculated and wild C. stellata, if coupled with molecular analyses of C. stellata strains (Pramateftaki et al, 2000).…”

“…Currently, different culture agar media are used, either selective or differential, which allows some discrimination between the different microorganism species involved in a wine fermentation. WL nutrient agar (Cavazza et al, 1992;Pallmann et al, 2001;Romancino et al, 2008), YM agar (Soden et al, 2000;Moreira et al, 2005) and YEPD (Ciani et al, 2006;Perez-Nevado et al, 2006) media are generally used to count total yeasts present in an oenological sample. The same media appropriately modified by the addition of an antimycotic or antiseptic (Moreira et al, 2005;Perez-Nevado et al, 2006) are selective and allow the counting of resistant yeasts.…”