Controlling the microorganisms involved in alcoholic fermentation during wine production can be achieved by adding a small quantity of spontaneously fermenting must to freshly crushed grapes, a technique known as pied de cuve (PdC). This method not only serves as an inoculation starter but also enhances the microbial footprint unique to each wine region. Recent studies have confirmed that wines inoculated with PdC exhibit efficient fermentation kinetics comparable to those inoculated with commercial strains of Saccharomyces cerevisiae. However, further research is required to draw robust conclusions about the chemical and sensory impacts of PdC-inoculated wines. In this study, we examined the chemical and sensory effects of the PdC technique across three different harvests: Muscat of Alexandria (Spain, harvests 2022 and 2023) and Sauvignon Blanc (Chile, harvest 2023). Each PdC was prepared using various stressors (sulfur dioxide, ethanol, and temperature). Our findings revealed that wines produced with PdC exhibited similar fermentation kinetics and sensory profiles to those inoculated with commercial strains. Notably, PdC fermentations resulted in lower concentrations of acetic acid compared to both the commercial strain and spontaneous fermentations. The sensory analysis indicated that PdC wines significantly differed from those made with commercial strains, with PdC wines displaying more pronounced tropical notes. These results suggest that the PdC technique, particularly when using specific stressors, can maintain desirable fermentation characteristics while enhancing certain sensory attributes, offering a viable alternative to traditional inoculation methods.

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 winemaking stage 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.