Spontaneous fermentations contribute to the identity of ancestral mezcal, although duration and yield may vary. This study selected an indigenous yeast and evaluated it as a starter candidate. Four native isolates (Torulaspora delbrueckii A1GM and A6GM, Kluyveromyces marxianus A2GM, and Saccharomyces cerevisiae A5GM) and a spontaneous control were compared in Agave cupreata microfermentations. Selection considered distillate alcohol content and preliminary ethanol-tolerance assays. A5GM was subsequently evaluated in three production batches containing A. angustifolia + A. karwinskii, A. angustifolia, or A. cupreata + A. angustifolia, with three independent vats per treatment. Fermentation profiles, distillate composition, yield, and sensory acceptance were assessed. A5GM produced the highest microfermentation distillate alcohol content (43.91 ± 0.89% Alc. Vol.) and grew at 16% ethanol (v/v). In the first two production batches, inoculated vats showed higher volumes standardized to 45% Alc. Vol. and lower calculated agave requirements than the controls; these were batch-specific observations under the operating conditions studied. Sensory evaluation used pooled vat-derived distillate composites, and the A5GM composites received the highest mean hedonic scores. Because strain-level establishment and persistence of A5GM were not confirmed, the observed outcomes cannot be attributed exclusively to this strain. Sensory interpretation is limited by fixed presentation order. Further independent batches, standardized management, and strain-level monitoring are needed to assess reproducibility.

Spontaneous fermentation is central to ancestral mezcal production, yet the bacterial and fungal communities associated with Agave cupreata must remain insufficiently characterized. This exploratory study described community composition and predicted functional profiles at three stages of a traditional fermentation defined by sugar depletion: 10 °Brix (0 h), 5 °Brix (72 h), and 2 °Brix (144 h). Ambient temperature ranged from 21.7 to 23.7 °C, must temperature from 28.5 to 29.9 °C, pH from 4.6 to 5.2, and relative humidity from 51.3% to 58.7%. At each stage, material collected from two wooden vats and three vertical positions was pooled into one composite sample. Bacterial 16S rRNA gene and fungal ITS1 amplicons were sequenced and analyzed using QIIME 2. At 10 °Brix, five dominant bacterial genera accounted for more than 93% of relative abundance. Lactic and acetic acid bacteria represented more than 91% at 5 °Brix, while Pediococcus, Lactiplantibacillus, and Loigolactobacillus were prominent at 2 °Brix. Seven fungal genera represented more than 98% of the initial profile, whereas Saccharomyces, Debaryomyces, and Maudiozyma accounted for more than 98% at 2 °Brix. PICRUSt2 predicted pathways related to carbohydrate degradation and fermentation, whereas FUNGuild assignments were predominantly saprotrophic at the later stages. Because one pooled sample was analyzed per stage, these patterns are descriptive and require validation in replicated fermentations. Despite these limitations, this study provides baseline information on the microbial communities associated with ancestral A. cupreata fermentation and identifies taxa and predicted functions that merit future isolation and experimental validation.

The purpose of this research is to characterize the antibacterial and antioxidant activity of medicinal plant extracts and compare their biological activity with reported plant extracts.