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Despite the current appreciation of pulque as a probiotic fermented beverage, pulque has been also regarded as a poor-quality product, particularly due to the lack of sanitary control during its elaboration. To address this problem, a semi-continuous fermentation system was established, emulating the artisanal production process. Microfiltration-sterilized aguamiel was employed as the substrate, whereas a good-quality pulque was used as the fermentation inoculum. During the fermentation, the physicochemical, microbiological (lactic acid and Leuconostoc-type bacteria and yeasts) and sensory characteristics of the must were monitored. The isolated microorganisms were identified by molecular biology and MALDI-MS techniques. The sterilization of aguamiel by microfiltration did not negatively affect its physicochemical attributes. After 6–8 days of operation of the semi-continuous bioreactor, the fermentation reached a quasi-stationary state considering most of the parameters monitored during the experiment. The final fermentation product presented similar physicochemical, microbial and sensory properties to those of the pulque inoculum. The genera identified were Leuconostoc, Lentilactobacillus, Lactobacillus, Liquorilactobacillus, Fructilactobacillus and Saccharomyces. The strains Lentilactobacillus diolivorans and Liquorilactobacillus capillatus and uvarum have not been previously isolated from pulque. In conclusion, the fermentation system developed in this work was effective to standardize the quality of pulque while preserving the positive attributes of the artisanal process, thus harnessing the probiotic properties of pulque. [ABSTRACT FROM AUTHOR]