Honey possesses unique properties, characterized by its high sugar concentration and the synergistic interaction among nectar, pollen, bees, and yeasts. These features render it an exceptional substrate for exploring microbial diversity for bioprospecting purposes. In this study, we characterized fermentative yeast populations from 19 honey samples collected in Northern Patagonia, Argentina. A total of 380 yeast isolates were obtained, identifying eight yeast species. Starmerella magnoliae emerged as the dominant species, found in 76% of samples and representing 63% of total isolates. Intraspecific diversity analysis, using mtDNA-RFLP and sequencing of nuclear genes (FSY1 and FFZ1), revealed the presence of two distinct phylogeographic populations. Phenotypic assays indicated that most S. magnoliae strains tolerate high sulfite and ethanol concentrations, alongside exhibiting broad temperature tolerance, with some strains thriving even at 37 °C. Despite the fact that none of the strains completed the fermentation, microfermentation trials confirmed the fructophilic nature of this species and highlighted intraspecific variability in glycerol and acetic acid production. These findings underscore S. magnoliae as a promising non-Saccharomyces yeast for the fermented beverage industry.

Torulaspora delbrueckii is a promising non-Saccharomyces yeast for beverage diversification, although its diversity and application in cider production remain poorly explored. This study characterized Patagonian T. delbrueckii isolates from wine- and cider-associated environments and evaluated their fermentative potential in apple must. Mitochondrial DNA-RFLP analysis showed that mitochondrial profiles varied according to the isolation source, while physiological characterization revealed differences in temperature, sulfite, and ethanol responses among isolates from different sources. Compared with wine isolates, cider isolates generally exhibited greater tolerance to high temperatures, lower sulfite tolerance, and shorter lag phases under ethanol stress. Eleven representative strains completed apple must microfermentations. Compared with a commercial Saccharomyces cerevisiae strain, T. delbrueckii produced higher glycerol and erythritol concentrations while achieving comparable or higher ethanol levels. Two contrasting strains were further evaluated in sequential mixed fermentations with S. cerevisiae. NPCC1340 enhanced glycerol and erythritol production, whereas NPCC1608 increased malic and succinic acids while maintaining low acetic acid levels. In mixed fermentations, ciders retained the characteristic metabolic profiles of each T. delbrueckii strain, whereas S. cerevisiae improved fermentation performance, producing intermediate fermentation kinetics. These findings reveal phenotypic diversity among Patagonian T. delbrueckii strains and suggest differences associated with their isolation sources, highlighting their potential as starter cultures for producing ciders with distinct chemical characteristics.