Improved identification of Saccharomyces species and hybrids using MALDI-TOF MS

Accurate identification of Saccharomyces species is critical for food fermentation and brewing, especially for closely related species and interspecific hybrids with distinct technological properties. In this study, a custom MALDI-TOF MS database was constructed to improve the identification of Saccharomyces yeasts and was systematically evaluated using laboratory collections. Compared with a commercial reference library, the customized database markedly enhanced species-level discrimination across the genus. While automated database matching showed limitations in distinguishing the hybrid Saccharomyces pastorianus from its parental species, detailed peak-level analysis revealed robust and reproducible hybrid-specific spectral patterns. Specifically, S. pastorianus consistently displayed the co-occurrence of parental diagnostic peaks (6210 m/z and 6185 m/z), providing a biologically interpretable signature that was conserved across reference strains and commercial lager yeasts. Intraspecific analysis of S. cerevisiae further revealed that mass spectral profiles are structured by phylogenetic origin, ecological niche, and fermentation context, suggesting that MALDI-TOF MS can capture signatures linked to ecological adaptation. This study demonstrates that MALDI-TOF MS, when combined with targeted database construction and informed spectral interpretation, is a powerful tool for identifying Saccharomyces species of relevance to food and fermentation systems.