Impact of Freeze-Drying on the Viability and Microbial Community Structure of Traditional Mexican Pulque

Pulque is a traditional Mexican fermented beverage produced by the spontaneous fermentation of the sap (aguamiel) produced by several Agave (maguey) species. Pulque fermentation starts with the addition of freshly collected aguamiel (harvested twice daily) into a traditional container known as the tinacal, which contains previously fermented pulque serving as a microbial inoculum; the native microbiota associated with both the aguamiel and the inoculum ferments the available sugars, driving the development of the beverage’s characteristic sensorial properties. However, the preservation of its complex microbiota for research, fermentation standardization, and long-term conservation has not been systematically evaluated. In this study, we assessed the impact of freeze-drying on the viability, taxonomic composition, and diversity of the bacterial and yeast communities of pulque across five independent batches. Viable counts revealed no systematic loss of cultivable populations across major guilds. High-throughput sequencing of the V3-V4 16S rDNA and ITS1 regions demonstrated that the global taxonomic structure of pulque is preserved mainly after freeze-drying, with dominant genera, including Lactobacillus, Acetobacter, Zymomonas, Lactococcus, Saccharomyces, and Kazachstania, remaining stable. A modest decrease in richness, without major shifts in community architecture, was observed among minor yeasts, indicating that freeze-drying effectively preserves the core microbiota of pulque. Moreover, preserving pulque biomass safeguards the microbial dimension of this ancestral biocultural resource while enabling future efforts to standardize fermentation and establish microbial biobanks.