DFT-based evaluation of cryoprotectants and their role in lyophilization success

Cryopreservation and lyophilization are essential for preserving and transporting biological samples, including bacterial cultures. These processes face severe challenges due to the formation of ice crystals that can damage cellular structures. Cryoprotectants (CPAs) are vital in reducing this damage by inhibiting ice nucleation and growth. The present study examines the use of various CPAs, focusing on their ability to enhance the survival of Bacillus coagulans and Streptococcus thermophilus during cryo-freezing and lyophilization. Using density functional theory (DFT) calculations, we analyzed interactions between water molecules. We selected CPAs (sucrose, fructosofuranose, glucose, fructose, and glycerol) to predict their effectiveness in forming protective hydrate shells around bacterial cells. Our results indicate that sucrose, which has a low Gibbs free energy of solvation at low temperatures, provides the most effective cryoprotection. Experimental validation showed that a 12 % sucrose solution improves bacterial survival after lyophilization. We also investigated the effect of different freezing protocols on bacterial survival. Initial freezing at -18 °C and subsequent storage at -80 °C were the optimal methods, maximizing cell survival. The findings contribute to a deeper understanding of cryopreservation and lyophilization processes, with potential applications in biotechnology and biomedical fields.