Evaluating the temporal stability of the core microbiota in traditional Mexican pulque fermentation

Pulque is a traditional Mexican alcoholic beverage produced by the spontaneous fermentation of aguamiel, the sap extracted from various Agave species. In a previous study, we identified a core microbiota across key stages of pulque production, but its temporal stability remained unclear. Here, we evaluated the persistence and functional relevance of this core across four seasonal samplings (2021–2022) in Huitzilac, Morelos, Mexico. Microbial diversity was characterized in metzal (the residual scraped maguey pineapple), aguamiel, overnight-fermented pulque, and laboratory-controlled fermentations. Microbial diversity was analyzed by 16S rRNA (V3–V4) and ITS1 amplicon sequencing. Fermentable sugars, ethanol, and organic acids were quantified by UPLC/HPLC. The core microbiota comprises the OTUs Zymomonas, Lactobacillus, Leuconostoc, Lactococcus, Acetobacter, Kazachstania gamospora, Kluyveromyces marxianus , Saccharomyces cerevisiae , and an unidentified Saccharomycetales OTU, which overlaps with the 2019 report and is consistently dominant across all samples and time points. Transient OTUs were season-specific and originated in metzal, highlighting their ecological role as a microbial reservoir. Spearman correlation analysis confirmed the functional contributions of dominant taxa, including strong positive associations between Zymomonas and ethanol, Lactobacillus and lactate, and negative correlations between fermentative yeasts and sugars. Despite seasonal shifts in diversity, the taxonomic and functional composition of the core remained stable. These findings establish a robust ecological framework for understanding pulque fermentation and support the selection of native consortia to develop starter cultures. The persistence and functionality of this core underscore its potential for applications in microbial ecology and traditional fermented food biotechnology.