Romanian Biotechnological Letters Vol. 16, No.1, 2011, Supplement Copyright © 2011 University of Buchare Printed in Romania. All rights reserved ORIGINAL PAPER Yeast biodiversity evolution over decades in Dealu Mare-Valea Calugareasca vineyard Received for publication, November 6, 2010 Accepted, February 11, 2011 MATEI RADOI FLORENTINA 1, BRINDUSE ELENA2, NICOALE GETUTA1, TUDORACHE AURELIA2, TEODORESCU RAZVAN 3 1 University of Agronomical Sciences and Veterinary Medicine of Bucharest, Faculty of Biotechnology 59, Marasti Ave., Bucharest, 010464 Romania, e-mail: [email protected] 2ICDVV Valea Calugareasca, Prahova, Romania 3University of Agronomical Sciences and Veterinary Medicine of Bucharest, FIFIM Abstract Belonging to a wider national project of yeast biodiversity conservation from consecrated Romanian vineyards, this study has been performed in a red wine recognized area, Valea Calugareasca from Dealu Mare County. The main goal has been to isolate yeasts from the vineyard, from the grape and during different phases of alcoholic fermentation and to perform their identification. The yeast isolation has been done from vine plantation of Cabernet Sauvignon, Merlot, Feteasca Neagra and Pinot Noir grape wines. We have used classical methodology for isolation and purification on YEPD, finally obtaining 262 isolates. The identification has followed two approaches: one by classical morphological and cultural analysis, using Barnett J.L. (2000) guide and the second one, employed rapid physiological tests of API 20 C Biomerieux galleries. A comparison between the two identification methods has been performed. A secondary goal of our study, but very important in the context to microbiota evolution, has been to compare our results (2007-2009) to former results obtained by Dr. Kontek A. in the years 1970- 1974 in the same areal. Because the former work was performed by classical identification methods, for a realistic comparison we have used the same methods. We should have taken into account also the changes in the wine yeast taxonomy. During the last 4 decades, the yeast biodiversity have changed, by the species number increase. In the years 70s in the beginning of the fermentation non-Saccharomyces species were Kloeckera apiculata (40%) and Pichia norvegensis (14%), while in the last 3 years new species have been identified, as Candida famata (32,5%), Debaryomyces hansenii (22,5%). During the middle of the fermentation, Saccharomyces species were dominant, as it was expected. Keywords: wine yeast, biodiversity evolution, Dealu Mare vineyard Introduction In the field of wine yeast, main studies have been usually focused on Saccharomyces strains as being responsible for the alcoholic fermentation. But, former or more recent works, proves the involvement in this process of other non-Saccharomyces yeasts during the initial steps, while they have an important influence on the final organoleptic properties of the wines (Pretorius, 2000). The role of non-Saccharomyces species in wine industry may be an interesting debating subject for the researchers. Initially, it has been thought that this species die during the first phase of the alcoholic fermentation because of the high alcoholic content due the Saccharomyces cerevisiae activity. The last decade’s researches have proved that non- Saccharomyces species can survive during the alcoholic fermentation and can reach a population of 106-108 CFU/ml. These species can improve the final wine organoleptic properties, mainly on aromatic level, by producing specific enzymes and volatile products for different vineyard [Zohre, 2002). In the last years has been reported the isolation during the 113 MATEI RADOI FLORENTINA, BRINDUSE ELENA, NICOALE GETUTA, TUDORACHE AURELIA, TEODORESCU RAZVAN alcoholic fermentation of non-Saccharomyces species (Hanseniaspora, Candida, Kluyveromyces) of high ethanol tolerance, similar to Saccharomyces cerevisiae [Pina et al., 2004; Xufre et al., 2006; Nisiotou et al., 2007]. Belonging to a wider national project of yeast biodiversity conservation from consecrated Romanian vineyards, this study has been performed in a red wine recognized area, Valea Calugareasca from Dealu Mare County. The main goal has been to isolate yeasts from the vineyard, from the grape and during different phases of alcoholic fermentation and to perform their identification. Wine production in most countries is based on the use of commercial strains leading to the colonization of the wineries by these strains with the consequent reduction of autochthonous biodiversity. The vineyard could be an important source of native yeasts of oenological interest. The specific yeast microbiota biodiversity study in a DOC winemaking areal as Dealu Mare – Valea Calugareasca is an important goal for the fundamental research and moreover to define the wine characteristics (“terroir” aspects) in the area. In Romania, few works had reported the biodiversity of yeast in different vineyards during the years 50’s and 60’s (Lepadatu V, Septilici G, Teodorescu S), but a large study has been done in Dealu Mare region by Dr. Kontek in 70’s. Only in the 90’s Dr Burcea M. has started a new era in the wine yeast study in Romania, isolating valuable oenological strains from Panciu Region [Burcea, 1998; Burcea, 2005]. In this regard, another goal of our study, but very important in the context of microbiota evolution, has been to compare our results (2007-2009) to former results obtained by Dr. Kontek A. in the years 1970-1974 in the same areal. Because the former work was performed by classical identification methods, for a realistic comparison we have used the same methods. Materials and methods The origin of the yeasts has been Dealu Mare (Grand Hill in Romanian language) vineyard, Valea Calugareasca center (Fig.1). Dealu Mare vineyards make up the most compact Romanian vine culture and wine making area (spreads on approximately 400 square kilometers over sub-Carpathian Hills). Dealu Mare is the homeland of red wines, where the red grapes found those suitable conditions that would stimulate their qualities. A number of factors are responsible for this: the plantations stretch over hill-slopes facing the light and heat of the sun and looking south and south-east, the temperature is higher during the period of vegetation of the vines, the rain does not fall in excess, the soils are not very rich and contain a high level of iron oxides. Figure 1. Dealu Mare wine area in the Romanian vineyards assembly (squared area) As biologic material have been used grape varieties for red wines recommended in Valea Calugareasca centre, respectively Cabernet Sauvignon (ecological and non-ecological plantation), Merlot, Feteasca Neagra and Pinot Noir. Sampling and yeast isolation. The sampling has been performed during 3 harvest years, respectively 2007, 2008 and 2009. The grape berries have been harvested in September 114 Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011) Yeast biodiversity evolution over decades in Dealu Mare-Valea Calugareasca vineyard and October with pedicel. For each grape variety have been harvested samples from 3 points of the plot, from different levels of the rope: ground, middle and top. The samples have been prepared in two ways. For the first series the berries have been harvested in aseptic conditions and introduced in an Erlenmeyer with sterile distillated water in order to collect the yeast from the grape surfaces. After 24 hours of shaking, from the suspension, plates with YEPD have been inoculated and cultivated during 48–72 hours at 27oC. For the second series, the berries have been crushed in aseptic conditions and the natural fermentation has occurred. During the fermentation, three moments for sampling have been chosen: beginning, middle and final phase of the fermentation. After applying Domerq dilutions, the samples have been inoculated on YEPD. Every isolated colony has been passed in slant on GYP medium and pure cultures have been obtained which were the identification subjects. Yeast identification and characterization. Two approaches have been followed for the identification: classical morphological and cultural tests and rapid biochemical tests. In the case of classical identification there have been examined morphological and cultural aspects (shape and cell dimension of the cell) of each strain on liquid media after 3 days cultivation on Wickerham media at 25oC and on solid media YEPD; also, the pseudomycelia formation on potato-agar media after 12 cultivation days have been observed; another parameter was the sporulation on Gorodkova media after 30 cultivation days. The physiological characters have been studied by rapid test API 20 C AUX (Biomerieux), which allows testing the assimilation of 19 substrates (Fig.2). The obtained data have been analyzed by the APILAB PLUS software. Figure 2. Aspects of API 20 C galleries (yeast physiological identification) In recent years, to improve the conventional methods, rapid kits for yeast identification have been developed. However, they were initially designed for clinical diagnosis and their application is restricted to 40-60 yeast species of medical interest [Deak,1993]. The reproducibility of these techniques is questionable due to the fact that they depend on the physiological state of the yeast cells. When the API 20C tests have not been clear, classical fermentation and assimilation tests have been done for sugars and nitrates. The taxonomical identification has been performed following the Barnett J.A.