Optimization of cryopreservation protocols for cold-adapted yeasts relevant to the brewing industry.

Most studies on yeast cryopreservation have focused on Saccharomyces cerevisiae; therefore, long-term viability and stability of wild strains from other yeast species remain uncertain. Standard protocols typically use glycerol as a cryoprotectant and a CoolCell® container to maintain slow cooling rates. However, limited evidence exists regarding the efficacy of glycerol and the advantages of using controlled-rate containers such as Mr. Frosty™ or CoolCell® compared to direct freezing from ambient temperature to -80 °C or liquid nitrogen (-196 °C) for other yeast species, and particularly for environmentally isolated strains (non-domesticated). In this study, the cryotolerant wild yeast S. eubayanus, relevant for the brewing industry for being the parental species of the Lager beer yeast, was employed to: (1) characterize growth kinetics and cell size dynamics, (2) evaluate the impact of glycerol exposure on survival, and (3) assess the effects of different cryopreservation protocols on survival, vitality, phenotypic traits, and genetic stability. S. eubayanus was found to have a cell volume at least 41% smaller than S. cerevisiae. Regarding glycerol exposure, a significant decrease in survival was observed with increasing glycerol concentration: at 10% w/w, survival declined from 81.28% to 60.90% after 15 min, whereas at 20% w/w, it dropped from 76.99% to 26.22%. Among cryopreservation protocols, the highest survival was achieved using a cooling rate of 6.4 °C min-1 with glycerol as cryoprotectant. No evidence of genetic mutations or changes in fermentation capacity was observed. In conclusion, protocols employing cooling rates exceeding those achieved by the CoolCell® device were equally or more effective in maintaining survival, vitality, and phenotypic and genetic stability. These findings support a more practical and cost-effective approach for the long-term cryopreservation of S. eubayanus.