Alterations in intracellular metabolites of Saccharomyces cerevisiae during freeze-drying and strategies for enhancing viability

The efficient preservation of industrial microbial strains is essential for maintaining their metabolic stability and functional performance. In particular, Saccharomyces cerevisiae , widely used in the food industry, may undergo subtle yet critical metabolic changes during drying processes. Therefore, monitoring both intracellular and extracellular metabolite profiles is important for evaluating the suitability of preservation techniques. In this study, we compared the effects of three drying methods—freeze-drying (FD), fluidized bed drying (FBD), and encapsulation (Enp)—on the metabolites profiles of S. cerevisiae . We applied high-resolution LC-MS/MS-based metabolomics to analyze intracellular and extracellular metabolites, and integrated the results with multivariate statistical analysis. The results revealed that while extracellular metabolite profiles remained relatively unchanged across all drying methods, freeze-drying significantly altered the intracellular metabolite profile. Notably, amino acids such as proline and glutamic acid were markedly depleted in freeze-dried samples. Supplementation of these metabolites prior to drying led to a 5.71-fold and 3.04-fold increase in yeast survival, respectively. These findings indicate that freeze-drying induces specific metabolic shifts in yeast and that targeted metabolite supplementation can improve post-drying cell viability without the need for additional cryoprotectants. This study highlights the importance of metabolite-level monitoring for optimizing preservation strategies in industrial yeast applications. • Dried yeast metabolites profiles were characterized by LC-MS/MS-based metabolomics • Yeast extracellular profile was not changed by freeze drying process • Freeze-drying induced alterations in yeast intracellular metabolism • Proline and glutamic acid alleviated yeast cell death induced by freeze-drying