Fernando Astudillo-Melgar

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.

The traditional Mexican fermented beverage pulque has been considered a healthy product for treating gastrointestinal disorders. Lactic acid bacteria (LAB) have been identified as one of the most abundant microbial groups during pulque fermentation. As traditional pulque is consumed directly from the fermentation vessel, the naturally associated LABs are ingested, reaching the consumer’s small intestine alive, suggesting their potential probiotic capability. In this contribution, we assayed the probiotic potential of the strain of Lactiplantibacillus plantarum LB1_P46 isolated from pulque produced in Huitzilac, Morelos State, Mexico. The characterization included resistance to acid pH (3.5) and exposure to bile salts at 37 °C; the assay of the hemolytic activity and antibiotic resistance profiling; the functional traits of cholesterol reduction and β-galactosidase activity; and several cell surface properties, indicating that this LAB possesses probiotic properties comparable to other LAB. Additionally, this L. plantarum showed significance in in vitro antimicrobial activity against several Gram-negative and Gram-positive bacteria and in vivo preventive anti-infective capability against Salmonella in a BALB/c mouse model. Several functional traits and probiotic activities assayed were correlated with the corresponding enzymes encoded in the complete genome of the strain. The genome mining for bacteriocins led to the identification of several bacteriocins and a ribosomally synthesized and post-translationally modified peptide encoding for the plantaricin EF. Results indicated that L. plantarum LB1_P46 is a promising probiotic LAB for preparing functional non-dairy and dairy beverages.

Pulque is the most-studied traditional Mexican alcoholic beverage prepared by fermentation of the fresh sap (aguamiel, AM) extracted from different Agave species (maguey) cultivated for pulque production. This beverage has been produced mainly in the Central Mexico Plateau since pre-Columbian times. In this contribution, we report the analysis of the bacterial and fungal diversity through 16S rRNA gene V3–V4 fragment amplicon and ITSR1 sequencing associated with the tissue of the walls (metzal) of the cavity or cajete, where the sap accumulates in producing plants for its daily extraction, in AM, and during four fermentation stages for pulque production. The results led to determining which microorganisms detected in the plant tissue are present in AM and maintained during the fermentation process. The results showed that eight bacterial OTUs (Lactobacillus, Leuconostoc, Weisella, Lactococcus, Acetobacter, Gluconobacter, Zymomonas, and Obesumbacterium) and five fungal OTUs (Kazachstania, Kluyveromyces, Saccharomyces, Hanseniaspora, and the OTU O_Saccharomycetales) were present from metzal to AM and during all the stages of the fermentation analyzed. The detected diversity was considered the microbial core for pulque fermentation, comprising up to ~84% of the total bacterial diversity and up to ~99.6% of the fungal diversity detected in the pulque produced from three plants of A. salmiana from the locality of Huitzilac, Morelos, Mexico. This study provides relevant information on the potential microorganisms responsible for pulque fermentation, demonstrating that the core of microorganisms is preserved throughout the elaboration process and their association with the AM and fermented beverage physiochemical profile.

Palm wine is obtained by fermentation of palm tree sap. In the Pacific coast of Mexico, palm wine is called Tuba and it is consumed as a traditional fermented beverage. Tuba has empirical applications such as an auxiliary in gastrointestinal diseases and a good source of nutrients. In the present study, a next-generation sequencing of the V3-V4 regions of the 16S rRNA gene was employed to analyze bacterial diversity and population dynamics during the fermentation process of Tuba, both in laboratory controlled conditions and in commercial samples from local vendors. Taxonomic identification showed that Fructobacillus was the main genus in all the samples, following by Leuconostoc , Gluconacetobacter , Sphingomonas, and Vibrio . Alpha diversity analysis demonstrated variability between all the samples. Beta diversity clustered the bacterial population according to the collection origin of the sample. Metabolic functional profile inference showed that the members of the bacterial communities may present the vitamin, antibiotic and antioxidant biosynthesis genes. Additionally, we further investigated the correlation between the predominant genera and some composition parameters of this beverage. This study provides the basis of the bacterial community composition and functionality of the fermented beverage.