Pulque is a traditional Mexican fermented beverage associated with numerous health benefits. Over time, there has been considerable interest in studying the bacterial diversity of pulque, and microbial characterization has been carried out using traditional microbiological and molecular methods. Therefore, the objective of this research was to characterize the microbiota of artisanal pulque obtained from the Zacatlán region in Puebla, México, by the means of Illumina sequencing, and to compare it with the microbial diversity of aguamiel (sap before fermentation), commercial pulque (supplemented with additives to increase its yield), and its seed (batch of pulque previously fermented). An analysis of the Shannon index showed medium diversity for both aguamiel and pulque samples (score > 2), while the Chao 1 index exhibited a non-significant difference between them. On the other hand, a principal components analysis confirmed the role of the seed as an essential inoculum to define the microbial diversity of pulque, emphasizing the importance of its preservation as a quality standard during the elaboration process. In addition, results showed that the dominant phyla in artisanal and commercial pulque were Firmicutes and Bacteroidetes. As the fermentation process progressed, it was possible to observe an increase in the population of lactic acid bacteria (LAB) in both types of pulque compared to those detected in aguamiel. Of these, the species Lactobacillus, Leuconostoc, and Lactococcus represented almost 95% of the total LAB. Finally, even though the safety of pulque has been in question due to its non-aseptic manufacturing process, the present study confirmed that less than 1% of its microbiota corresponds to the genera with a pathogenic potential such as γ-proteobacteria (Enterobacter and Hafnia), which decreases as the fermentation process advances.
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