Saccharomyces eubayanus is a cryotolerant wild yeast known as the cold-adapted parent of S. pastorianus, the hybrid responsible for lager beer production. Due to its industrial relevance, efficient cryopreservation methods are essential to improve culture viability during long-term storage. The standard protocol (CoolCell® devices) submits cryovials at a cooling rate of 1 °C/min. An alternative technique is the direct freezing from ambient temperature to −80 °C (DU), which avoids manipulation and is cost-effective.
Both methods involve different freezing stages governed by transient heat conduction that depend on overall heat transfer coefficients (U) which reflect conductive and convective heat exchange between the cryovial and its environment. The cryopreservation protocol can be represented by the characteristic freezing time (tc) which is inversely proportional to the freezing rate, and is defined as the time elapsed between the initial freezing temperature and a reference temperature (-40°C).
The objectives were to i)determine tc and heat transfer coefficients for DU and CoolCell® (CU) protocols; ii)correlate these parameters with the viability, vitality, phenotype, and genetic stability of S. eubayanus CRUB 1568T.
Cells were harvested at early stationary phase, suspended in 10% glycerol, and stored for one year at -80°C. Viability and vitality were evaluated, and experimental fermentations and PCR fingerprinting were performed to assess phenotypic and genetic stability. Thermal histories of cryovials were recorded using thermocouples to measure tc. Heat transfer coefficients were determined by computational modeling using the finite element method, incorporating temperature-dependent properties.
For the CU method, the obtained values were tc=26.85 min, U=4.72 W/m²K, and viability=51.2%. For DU, a higher cooling rate was observed (smaller tc=10.86 min) associated with higher values of U=18.75 W/m²K and viability (71.7%), with no observed genetic or fermentation alterations of the tested strain.
In conclusion, DU, with higher cooling rates, improved cryopreservation of S. eubayanus and was a simpler and cost-saving process.
