Isolation, identification and oenological characterization of non-Saccharomycesyeasts in a Mediterranean island

Abstract

Significance and Impact of the Study: In recent years, interest is growing for wine production by nonSaccharomyces yeasts, both in research and in the industry. This study describes the yeast population of the grapes in a small-secluded island in the Mediterranean Sea, useful site for the search of new strains. Evaluation of fundamental oenological characters identifies potential best yeasts to assay in experimental vinifications. We also describe, for the first time, 14 new colony morphologies on WL Nutrient Agar, culture medium used to monitor the yeast population dynamics. AbstractWe isolated, identified and characterized yeast strains from grapes, and their fermented musts, sampled in the small island of Linosa, where there are no wineries and therefore the possibility of territory contamination by industrial strains is minimal. By traditional culture-dependent methods, we isolated 3805 colonies, distinguished by molecular methods in 17 different species. Five hundred and fortyfour isolates were analysed for the main oenological characteristics such as fermentative vigour with and without sulphites, sugar consumption and production of alcohol, volatile acidity, hydrogen sulphide, glycerol and b-glucosidase. This analysis identified Kluyveromyces marxianus (seldomly used in winemaking) as the most interesting candidate yeast for the production of innovative wines.

Use of Kluyveromyces marxianus to Increase Free Monoterpenes and Aliphatic Esters in White Wines

“…Instead, we found greater differences when considering volatile compounds. In the Muscat of Alexandria wines (Table 4), we found significant and substantial differences in the amount of free terpenes: among the compounds present well above their odor threshold, linalool is more abundant in wines made with K. marxianus and, on the whole, free terpenes are 50% more abundant in these wines compared to those produced with only S. cerevisiae; again, this is consistent with the ability of the strain to produce β-glucosidase as previously reported [26]. In wines produced with the strain Km L2009, aliphatic esters as a whole are one and a half times more abundant than in wines made by spontaneous fermentation and twice as much if compared to wines made by the commercial S. cerevisiae strain.…”

“…With the limitations deriving from the differences in residual sugars in the various fermentations, statistically significant differences were observed between wines produced using only S. cerevisiae and those produced by K. marxianus. In particular, in fermentations by only K. marxianus or mixed by K. marxianus with S. cerevisiae, we found a higher amount of linalool and of total free terpenes, consistently with the reported production of β-glucosidase by strain Km L2009 [26]. This could affect the overall quality of the wines, even in the case of compounds that are present at concentrations below their perception threshold, because of their synergistic effect; in fact it is known that terpenes interact in such a way that a component can increase the aroma of another, and that a mixture is more aromatic than the single most aromatic component of this mixture [55].…”

“…Samples of fermenting must were serially diluted into sterile 0.1% peptone and plated on WL Nutrient Agar, Lysine Agar and WL Differential Agar (Oxoid, Basingstoke, Hampshire, UK). WL Nutrient Agar allows the growth of all yeasts but with different colony morphology as a function of the genus or species [26,[32][33][34][35]. Lysine Agar allows growth of all yeast except Saccharomyces [36].…”

“…Lysine Agar allows growth of all yeast except Saccharomyces [36]. WL Differential Agar is WL Nutrient Agar with 10 mg/L cycloheximide (Sigma-Aldrich, St. Louis, MO, USA): at this concentration S. cerevisiae yeasts do not grow, according to Di Maio et al [37], but K. marxianus Km L2009 still grows well, showing its typical colony morphology [26]: the concentration of all other non-Saccharomyces yeasts was calculated as the difference between the concentration of all non-Saccharomyces yeasts (detected on Lysine Agar) and the concentration of K. marxianus (detected on WL Nutrient Agar and/or on WL Differential Agar). To check the absence of microbial species able to alter wine characteristics, further microbiological analyses were performed on WL Nutrient Agar, Lysine Agar, MRS Agar and Tomato Juice Agar (Oxoid, Basingstoke, Hampshire, UK) before and after bottling (data not shown).…”

“…Previously we reported on the isolation of K. marxianus Km L2009 strain in the island of Linosa (Italy) and on its ability to produce β-glucosidase [26]; this enzyme increases the release of monoterpenols from their flavorless, non-volatile glycosidic precursors, thus helping to increase wine aromatic content [27].…”