Novel isolation campaign to explore microbial biodiversity associated to a Pied de Cuve for Amarone wine

Pied de Cuve (PdC) is a winemaking technique where a small amount of fermenting must is used to start the main fermentation and spontaneous PdC fermentation, driven by resident microbiota, is considered to positively express the “terroir” concept. The study explores the microbial biodiversity of spontaneously fermented PdC for “Amarone della Valpolicella” wine through culture-dependent methods, to determine presence, dominance and persistence of winemaking-related microbiota. Nine samples were collected during PdC fermentation and after inoculum in total mass. Non-Saccharomyces yeasts were isolated from PdC after grape crushing and before inoculation. Saccharomyces and bacteria were sampled during the PdC fermentation at 12,8% alcohol (v/v) and after inoculation. Cell counts were performed on WL and MRS agar. The cell population remained around 5.5 log CFU/mL until the beginning of alcoholic fermentation in PdC, reaching a peak of 8 log CFU/mL at 1.89%. Colonies from countable plates were selected based on morphology and differentiated into Saccharomyces or non-Saccharomyces yeasts using lysine agar, resulting in 118 isolates: 26 putative non-Saccharomyces yeasts, 89 putative Saccharomyces yeasts, and 3 bacteria. Dominance and persistence of different Saccharomyces strains during fermentation were evaluated with Interdelta-targeted PCRs, while non-Saccharomyces isolates were dereplicated and identified through sequencing of the D1/D2 domains of 26S rRNA gene. This study highlighted, for the first time, the biodiversity associated with spontaneous PdC fermentation in Amarone wine and identified the dominant Saccharomyces strains, which will be further characterized for enological traits.