Devitrification of lyoprotectants: A critical determinant for bacteriophages inactivation in freeze-drying and storage

Bacteriophages as promising natural antibacterial additives are widely used in food processing and storage. Although freeze-drying is an economical and efficient way to preserve phages, so far there is limited data for phage freeze-drying and key factors that inactivate phages during freeze-drying and storage remain unknown. Here we systemically compared different types of saccharides/polyols (dextran 5000, glucose, sucrose, trehalose, mannitol, and xylitol) as lyoprotectants and their potential ratios for phage freeze-drying. The pH and osmotic pressure tolerance of bacteriophages were determined and all lyoprotectant solutions were within the tolerance range of phages. Combined with thermodynamic data, it was found that only completely vitrified formulations (glucose, sucrose, and trehalose) could preserve phages during freeze-drying. Selected freeze-dried phages were further arranged for an accelerated stability study. Most formulations stored at higher temperatures (& GE;25 & DEG;C) presented devitrification, resulting in a significant drop in phage titer. 10% (w/v) of sucrose was recommended as the best formulation for freeze-dried phage storage with less devitrification and a better fitting coefficient (R2 = 0.9592) to the Arrhenius equation, predictively reaching shelf-time as 1093.3 days at 4 & DEG;C storage. These findings implied that the devitrification of lyoprotectants was the critical determinant for bacteriophage inac-tivation both in freeze-drying and storage.