Profiling the Metschnikowia yeast populations in spontaneous fermentation of Amarone della Valpolicella

Abstract

The microbial diversity during spontaneous grape must fermentation has a determinant influence on the chemical composition and sensory properties of wine. Therefore, yeast diversity is an important target to better understand wine regionality. Hence, the aim of this study was to isolate, identify, and characterize the yeast core microbiota in grape must during the early stage of lab-scale spontaneous fermentation of withered grapes to produce Amarone della Valpolicella wine (Verona, Italy). At the end of the withering process, chosen grape bunches, mainly of Corvina and Corvinone varieties, were pressed and transferred to glass bottles in the laboratory for spontaneous fermentation (SF). To investigate the impact of grape washing on microbial removal, one batch of grapes was submitted to a washing step in aqueous solution (1% w/v citric acid) at the winery before pressing. Microbial composition was investigated during the first five days of fermentation, a stage known for greater variability of microorganisms, isolating yeast colonies from WL agar plates. Overall, 67 colonies were purified and the partial 26S rRNA gene sequencing allowed the identification of six different species, among which Metschnikowia spp. was prevalent. Indeed, 42 isolates of this genus were obtained, deriving from musts of washed grapes (22) and from non-washed grapes (20). Interestingly, the washing step did not impact on the presence of pulcherrimin-producing isolates. A more in-depth characterization was carried out on those 42 isolates, as Metschnikowia spp. are acknowledged to contribute to the diversity and complexity of wine taste. A strain level analysis was performed by means of fingerprinting profiles (primer (GTG)5) and phenotypic characterization (sulfite reductase, β-glucosidase, and esterase enzymatic activities). 11 genotypic profiles and 6 different phenotypic combinations were observed among the 42 isolates. Considering both approaches, it was possible to define the presence of 19 strains of Metschnikowia spp., most of them isolated only once, but some present in both washed and non-washed grapes, throughout the whole sampling period. Results obtained in this study shed light on the native Metschnikowia yeast community of washed and non-washed withered grapes, that is composed by diverse strains, and highlight that this biodiversity can be underestimated if only genotypic or phenotypic properties are investigated. This diversity represents a reservoir of strains with enological/pro technological significance that could be applied and combined to improve the sensory characteristics of wine and fermented beverages.