Influence of driven fermentation of cacao in bioreactors on quality: decoding the effect of temperature, mixing, and pH on metabolomic, sensory, and volatile profiles

Controlled cacao fermentation is limited by traditional methods, hindering understanding of flavour formation dynamics. Therefore, this study investigated the influence of controlled fermentation variables—temperature profile, initial pH setting, and mixing frequency—on cacao flavour development, evaluated through sensory analysis, volatiles analysis, and untargeted metabolomics. Internal pH dynamics were also monitored. A fully controlled fermentation system using stirred-tank bioreactors enabled precise regulation of process conditions. Eight treatments were tested, varying temperature (constant 45 °C vs. stepped gradient 35–45 °C), initial pH (controlled vs. spontaneous), and mixing frequency. The results showed that temperature and initial pH were the primary drivers of internal acidification and flavour differentiation, while mixing frequency showed negligible effects. Constant-temperature (45 °C) fermentation with spontaneous pH produced chocolates with enhanced fruity, nutty, woody, and floral attributes. Conversely, fermentation under a stepped gradient with controlled pH intensified bitterness and astringency. Uncontrolled pH under the same gradient led to chocolates with pronounced acidity and lack of distinctive attributes. Extended fermentation (144–192 h) under spontaneous conditions resulted in off-flavours. Metabolomic and volatile profiles aligned with sensory data, revealing four treatment-specific clusters and identifying peptides and volatiles as candidate biomarkers of cacao quality. • Controlled bioreactor fermentation modulates chocolate flavour profiles. • Internal pH dynamics and temperature are key drivers of flavour development. • Bioreactor processing at 45 °C with uncontrolled initial pH enhances flavour quality. • Controlled pH at 45 °C leads to higher bitterness and astringency in chocolate. • Mixing frequency has limited influence on flavour-related transformations.