Grape ripeness shapes microbial community dynamics and fermentation outcomes during modified pied de cuve wine...

Pied de cuve (PdC) is a traditional inoculation strategy consisting of adding a small volume of actively fermenting must to initiate alcoholic fermentation (AF) in the main vat. In this study, we evaluated the effect of grape ripeness on microbial community composition, fermentation performance, and Saccharomyces cerevisiae strain diversity using a modified spontaneous PdC prepared with sulphur dioxide (SO₂) and ethanol and fermented at 18 °C. PdC inoculation was compared with inoculation using the commercial S. cerevisiae strain QA23 and with spontaneous fermentation. Experiments were performed at three ripening stages corresponding to potential alcohol content (PAC) of 10%, 12% and 13% (v/v). Amplicon sequencing revealed that both early winemaking sampling point and grape ripeness significantly shaped bacterial and fungal community composition, whereas ripeness had a limited effect on fungal alpha diversity. Despite differences in PdC development among maturity levels, PdC inoculation consistently improved fermentation kinetics compared with spontaneous fermentation, particularly at 13% PAC. Interdelta-PCR analysis showed that PdC fermentations preserved higher S. cerevisiae strain diversity than commercial inoculation, while an indigenous strain recurrently detected across fermentations displayed strong competitive ability against QA23. Differences in inoculation strategy were also reflected in wine chemistry and sensory properties, with PdC wines showing controlled acetic acid concentrations and favourable sensory profiles. Overall, these results demonstrate that grape ripeness acts as an ecological driver of fermentation and that modified PdC inoculation represents a practical strategy for balancing fermentation reliability with the preservation of indigenous fermentative diversity.