Sequential fermentation with immobilized Hanseniaspora opuntiae in fungal pellets and Saccharomyces cerevisiae for reduced-ethanol wine production

Increased grape sugar concentrations associated with climate change are contributing to higher ethanol levels in wines (up ∼2% v/v), potentially affecting sensory balance. This study investigates a controlled microbiological approach to modulate ethanol concentration and enhance aromatic complexity during alcoholic fermentation through sequential inoculation with Hanseniaspora opuntiae TR-5, a non-commercialnon-Saccharomyces yeast, immobilized in inactive Aspergillus oryzae pellets (Microbial Biocapsules), followed by inoculation with a commercial Saccharomyces cerevisiae strain (Opale). The performance of the immobilized system was compared with sequential fermentation using free (suspended) cells and with spontaneous fermentation. Sequential fermentation with immobilized H. opuntiae TR-5 resulted in a final ethanol concentration of 13.0 ± 0.02% (v/v), lower than that observed in spontaneous fermentation (13.5 ± 0.01% v/v) and in sequential fermentation with free cells (13.2 ± 0.02% v/v). The immobilization strategy enabled the physical removal of the non-Saccharomyces yeast at a defined stage, limiting its metabolic contribution, the interference with S. cerevisiae and allowing controlled ethanol modulation. Wines produced under immobilized conditions exhibited increased concentrations of certainvolatile compounds, including isoamyl alcohols and 2-phenylethanol, as well as higher glycerol levels, which exceeded its sensory perception threshold only in this treatment. Sensory evaluation indicated improved fruity and floral attributes and better gustatory balance in sequential fermentations, particularly in the immobilized system, compared to the spontaneous control. These results demonstrate that the immobilization of H. opuntiae TR-5 in inactive fungal pellets provides a precise and effective strategy to modulate ethanol concentration and enhance the wine's aroma profile, yielding an ethanol reduction of 0.5 ± 0.02% (v/v), which, although representing 25% of the initial target and thus not the optimal result, is nonetheless an encouraging outcome considering the simultaneous attainment of an improved sensory profile.