Untargeted Metabolomics of Yerba Mate Fermentation by Microbial Consortium: pilot study

Kombucha is an ancient fermented beverage made from an aqueous infusion of dry plant material enriched with sucrose and fermented by a symbiotic culture of bacteria and yeast (SCOBY). Yerba mate (Ilex paraguariensis) is a notable South American botanical matrix recently adopted as an alternative substrate for kombucha due to its high density of phenolic compounds, methylxanthines, and triterpenic saponins. Kombucha possesses a rich and dynamic biodiversity of non-conventional microorganisms during its fermentation, with a consensus on the presence of diverse yeasts and acetic acid bacteria, alongside the facultative presence of lactic acid bacteria. Among them, yeasts of the Brettanomyces and Pichia genera, which represent a promising biotechnological strategy for the production of molecules of interest in fermented beverages, were evaluated to verify their potential contribution to the functional and sensory aspects of the beverage. This study aimed to map the potential biotransformation pathways and track the metabolic signatures of microorganisms isolated and identified from a local SCOBY, they were evaluated regarding the chemical compounds found in the product in yerba mate kombucha after 20 days of fermentation. The chemical profile of the initial infusion (T0) and the fermented beverage (T20) was evaluated using tandem mass spectrometry (LC-MS/MS). For untargeted metabolomics data mining, the fragmentation spectra were submitted to the GNPS (Global Natural Products Social Molecular Networking) platform for the identification of the present compounds. The Classical Molecular Networking approach allowed the structural annotation of metabolites against public libraries and the semi-quantitative evaluation of consumption and biosynthesis pathways via spectral counting. Molecular mapping of key chemical compounds evidenced the expressive modulation of the matrix by the yeasts, highlighting the intense β-glucosidase activity in the depolymerization of glycosylated flavonols and complex saponins into highly bioavailable aglycones. Simultaneously, the tracking confirmed the consumption of native hydroxycinnamic acids from the plant, elucidating the cinnamate decarboxylase pathway, responsible for the synthesis of phenolic derivatives with high sensory and bioactive impact. The results demonstrate that the integration of metabolomics based on spectral similarity networks is a robust tool for bioprospecting, evidencing the potential of these three yeasts as agents in the sensory and functional biotransformation of alternative matrices for fermented beverages. This research was supported by CNPq/MCTI project 407279/2023-7 and Araucária Foundation. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.