Uncovering metabolic interactions between yeasts during wine alcoholic fermentation by non-targeted extracellular metabolomics

Abstract

During wine fermentation, different yeast species coexist and interact, shaping metabolomic profiles through species-specific metabolism, thereby influencing wine quality1. These effects are driven by metabolic exchanges that remodel their surroundings2,3. Characterizing these extracellular metabolite profiles is essential for elucidating the physicochemical mechanisms behind interspecies interactions. In this study, it was introduced an experimental framework that enables systematic characterization of the extracellular metabolome at the end of alcoholic fermentation, moving beyond traditional pairwise co-culture designs to investigate complex tri-species yeast interactions involving Saccharomyces cerevisiae, Torulaspora delbrueckii, and Hanseniaspora uvarum. Combined approach was employed (LC-MS and GC-MS) to obtain comprehensive metabolic fingerprints of extracellular cultures. These techniques enabled the analysis of both non-volatile and volatile compounds, facilitating the detection of condition-specific and interaction-driven changes in secreted metabolites4,5. This strategy provides deeper insight into the metabolic mechanisms of microbial interactions. Comparison of the exometabolomic footprints of monoculture and their corresponding two and three species co-cultures allowed the identification of 527 significant biomarkers out of 2,857 detected features. Of these, 336 were associated with monoculture, while 115 and 76 were enriched in two and three species co-culture, respectively. These differences reveal distinct exometabolomic profiles characterized by pronounced shifts in metabolite composition, resulting from the differential regulation of 36 common and 27 unique metabolic pathways across conditions. The most affected pathways were related to ABC transporters, byosinthesis of amino acids and carbon metabolism, demonstrating the impact of yeast interspecies interactions during alcoholic fermentation. Furthermore, this metabolomic remodeling was reflected in the volatilome profile. A total of 23 volatile compounds were significantly affected across culture conditions, with esters, alcohols and monoterpenes being the most impacted chemical families. These findings demonstrate that yeast interspecies interactions extensively remodel the extracellular metabolome and affect volatile compound production. The metabolic signatures observed in co-cultures highlight the key role of microbial interactions in modulating biochemical pathways.

References

1. Sun, Y., Lu, Y., Joseph, L., Ma, L., Bisson, F., & Liu, Y. (2024). Metabolomic analysis reveals the interactions between Chinese indigenous and commercial Saccharomyces cerevisiae strains during wine co-fermentations at low YAN concentration. Food Bioscience, 60, 104362. https://doi.org/10.1016/j.fbio.2024.104362

2. Roullier-Gall, C., David, V., Hemmler, D., Schmitt-Kopplin, P., & Alexandre, H. (2020). Exploring yeast interactions through metabolic profiling. Scientific reports, 10(1), 6073. https://doi.org/10.1038/s41598-020-63182-6

3. Del Fresno, M., Morata, A., Loira, I., Bañuelos, A., Escott, C., Benito, S., & Suárez-Lepe, J. (2017). Use of non-Saccharomyces in single-culture, mixed and sequential fermentation to improve red wine quality. European Food Research and Technology, 243(12), 2175-2185. https://doi.org/10.1007/s00217-017-2920-4

4. Puyo, M., Scalabrino, L., Romanet, R., Simonin, S., Klein, G., Alexandre, H., & Tourdot-Maréchal, R. (2024). Competition for Nitrogen Resources: An Explanation of the Effects of a Bioprotective Strain Metschnikowia pulcherrima on the Growth of Hanseniaspora Genus in Oenology. Foods, 13(5), 724. https://doi.org/10.3390/foods13050724

5. Bordet, F., Romanet, R., Bahut, F., Ballester, J., Eicher, C., Peña, C., & Alexandre, H. (2023). Expanding the diversity of Chardonnay aroma through the metabolic interactions of Saccharomyces cerevisiae cocultures. Frontiers in Microbiology, 13, 1032842. https://doi.org/10.3389/fmicb.2022.1032842