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Biologie UNIVERSITATEA DIN CRAIOVA 9 Horticultură UNIVERSITY of CRAIOVA 9 Tehnologia Prelucrării Produselor Agricole 9 Ingineria Mediului Vol

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Biologie UNIVERSITATEA DIN CRAIOVA 9 Horticultură UNIVERSITY of CRAIOVA 9 Tehnologia Prelucrării Produselor Agricole 9 Ingineria Mediului Vol Seria: 9 Biologie UNIVERSITATEA DIN CRAIOVA 9 Horticultură UNIVERSITY OF CRAIOVA 9 Tehnologia prelucrării produselor agricole 9 Ingineria mediului Vol. XV ( XLXI ) - 2010 CALORIMETRIC DETERMINATION OF THE QUANTITATIVE INHIBITORY PARAMETERS OF ETHANOL FOR SOME YEAST STRAINS SELECTED IN VALEA CALUGAREASCA AND PANCIU REGIONS FOR WINE PRODUCTION Antoce Arina Oana 1,*, Nămoloşanu I.1, Rădoi-Matei Florentina2, Brânduşe Elena3 KEY WORDS: ethanol, inhibition parameters, winemaking, microbial calorimetry, yeast selection ABSTRACT Tolerance to ethanol is a key parameter for any yeast strain that is to be used for winemaking. In this work the quantitative inhibitory parameters regarding ethanol for 9 yeast strains selected in Valea Călugărească and Panciu vineyards and wines were determined by the use of an isothermal microbial calorimetry technique. A group of strains belonging to the Saccharomyces genus, with good tolerance to ethanol, was thus identified. These yeasts, after some further testing for the sensory qualities of the wines they produce, could, in principle, be recommended for the production of wines with a local character. A number of non-Saccharomyces yeast strains were also identified, which belong rather to the category of spoilage yeasts. Some of the strains of this last group can also be, in principle, recommended for wine production, but before that they should be tested further on wines for the evaluation of the chemical and sensory profile. INTRODUCTION Most quality wines are obtained nowadays by controlled fermentations in the presence of specially selected yeasts which confer good and stable oenological characteristics (Rosini, 1984; Goldfarb, 1994; Rainieri and Pretorius, 2000). As a result, many yeasts are at present selected (Heard, 1999) and commercialized by several companies from abroad, each yeast strain having certain traits worth to be taken into consideration for a certain style of wine. For many years the most important wine producing countries changed their traditional winemaking practices by introducing the inoculation of grape must with starter (Kunkee and Amerine, 1970), especially form S. cerevisiae species. However, most often these strains selected in another geographical location, from another grape variety or even from other sources (Rainieri et al., 1998; Castellari et al., 1994), can significantly change the sensory profile of a local wine, wiping away the sensory characteristics that account for their typicality. Finding an yeast strain 1 University of Agronomical Sciences and Veterinary Medicine of Bucharest, Department of Viticulture and Enology, Faculty of Horticulture, Center for the Wine and Vine Studies and Sensory Analysis, 59, Mărăşti Ave. Sector 1, 011464 Bucharest. 2 University of Agronomical Sciences and Veterinary Medicine of Bucharest, Department of Viticulture and Enology, Faculty of Biotechnology, 59, Mărăşti Ave. Sector 1, 011464 Bucharest. 3 ICDVV Valea Călugarească, Str. Valea Mantei nr. 4, Jud. Prahova. *Corresponding author: E-mail address: [email protected] 5 possessing an ideal combination of technological and qualitative traits is not an easy task. Therefore, there are still many winemakers who favour spontaneous fermentation and, in 1999 was estimated that in the world around 80% of the wines were made without the addition of a starter culture (Heard, 1999). A way to avoid losing regional typicality is the selection of a yeasts from local vineyards (Romano et al., 2002), able to ferment the musts in good conditions, without confering off-flavours or causing sluggish or stuck fermentations. One of the most important characteristics of a selected yeast is the tolerance to the end-product of fermentation, that is ethanol (Zambonelli, 2003). Therefore, after the isolation of the yeasts from local vineyards, in order to select the most suitable for wine fermentation, the inhibition parameters of ethanol should be determined (Jimenez and Benitez, 1986). In this work a calorimetric method is applied for the determination of growth inhibitory parameters in the presence of ethanol for nine yeast strains selected in Romanian vineyards. MATERIAL AND METHODS The yeasts evaluated in this work were isolated and selected in two Romanian vineyards, Valea Călugărească and Panciu, either in conventional monovarietal plantations, or in ecological plantations. The yeast strains isolated were identified as belonging to Saccharomyces genus, but also to some other genera. The isolation location and grape variety, codification and the species are included in Table 1. For the determination of the ethanol tolerance, the yeast growth in the presence of ethanol in various concentrations was determined by monitoring the heat evolved during culture growth in a microbial isothermal calorimeter that can record the signals of 24 cultures in a single experiment. Each yeast was inocculated in vials containing 5 ml liquid medium with 20 g/l of glucose. Aside of the 4 control vials, that contained no added ethanol, in the rest of the 20 vials there ethanol was added in increasing concentrations (v./v.): 1.75% (3 vials); 2.62% (3 vials); 3.49% (2 vials); 4.36% (2 vials); 5.24% (2 vials); 6.11%; 6.98% (2 vials); 7.85% (2 vials); 8.73% (2 vials). For each strain the quantitative inhibition parameters for ethanol were calculated taking into consideration both the reduction of growth activity and the retardation of growth (Antoce et al., 1996a). Both methods provide reliable quantitative parameters, the first method being the most used, while the second one is referred to only in the case of a bactericidal type of inhibition, when the growth rate remains constant and the method based on reduction of growth activity cannot be applied. The quantitative parameters that are derived by processing the signals recorded by the calorimeter's computer during yeast growth are the following: - the specific growth activity μi/μm where μi represents the growth rate constant of a culture in the presence of ethanol in concentration i and μm is the growth rate constant reached in the absence of ethanol (maximum growth rate constant); - the specific growth retardation tα(0)/tα(i) where tα(0) represents the time required for a culture to reach the growth level α in the absence of ethanol (minimum time) and tα(i) is the time required for a culture to reach the same α level of growth in the presence of ethanol in concentration i; - the concentration of inhibitor at which the growth activity of the yeast cells is inhibited by 50%, Kμ, the value derived form the μi/μm drug potency curve (variation of μi/μm with the inhibitor concentration), after fitting with the drug potency equation: mμ μμ mi += Ki μ )/1/(1/ 6 - the concentration of the drug which doubles the time required for the culture to reach a certain level of growth, Kθ, the value derived form the tα(0)/tα(i) drug potency curve (variation of tα(0)/tα(i) with the inhibitor concentration) after fitting with the drug potency equation: m /)0( += θ /1/(1)( Kiitt ) α α θ - the minimum inhibitory concentration MICμ, representing the concentration at which the growth activity of the microorganisms is completely inhibited, 1/m determined by fitting the μi/μm drug potency curve with the MIC equation: 1 MICμ = ()1/ k1 - the minimum inhibitory concentration MICθ, also representing the concentration of inhibitor at which the growth activity of the microorganisms is completely inhibited, but determined by fitting the tα(0)/tα(i) drug potency curve with the MIC 1/m equation: 2 MICθ = ()1 / k 2 - the cooperativity degree mμ and mθ, resulting also during the regression analysis applied for the deduction of Kμ and Kθ; they are derived from the same drug potency equations and are related to the steepness of the drug potency curves and represent a measure of the cooperativity in the drug-cell interaction. A complete description of these parameters and the theoretical background are given extensively elsewhere (Antoce, 1998). RESULTS AND DISCUSSION In this work the calorimetric technique was applied for a rapid determination of the quantitative inhibitory parameters of ethanol, in order to select from the isolated yeasts the most resistant and hence more suitable ones for the local wine production. In the same time calorimetry allowed for the evaluation of the growth rate, a parameter related to the adaptability of a yeast to a growth medium and its capability of growing rapidly and competing with other strains, as it is the case of the grape must. For each strain the ethanol inhibition was followed in triplicate and the calculated inhibitory parameters are reported in Table 2 as mean values and standard deviations. The drug potency curves that were fitted with the drug potency equations and MIC equations are presented for all the evaluated yeast strains, the first five in Fig. 1 and the other five in Fig. 2. In each figure, on the left side there are the drug potency curves derived from the specific growth activity μi/μm and in the right side the curves obtained on the basis of the specific growth retardation, tα(0)/tα(i). Each set of data points was fitted first with the drug potency equation (continuous line) in order to derive the 50% inhibitory concentration, Kμ and Kθ, respectively, then with the MIC equation (dotted line), in order to obtain the minimum inhibitory concentration, MICμ or MICθ, at the interception of the line with the ethanol concentration axis. As stated before (Antoce et al., 1997a; Antoce et al., 1997b) the most used parameters for the evaluation of the effect of an inhibitor are those derived from the specific growth activity, μi/μm, that is, the parameters Kμ and MICμ. These best describe the effect of an inhibitor that acts based on a bacteriostatic inhibition mechanism. Actually, theoretically speaking, in the case of a pure bacteriostatic action, the calculated parameters do not differ signifficantly from the complementary ones evaluate from the data points of specific growth retardation, that is Kθ and MICθ.
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