Trehalose and maltodextrin preserve microbial community structure in freeze-dried fecal samples for fecal microbiota...

Fecal microbiota transplantation (FMT) is a promising approach for restoring gut microbial balance in both humans and animals. However, the logistical limitations of transplanting fresh fecal samples have increased interest in freeze-dried (lyophilized) fecal material as a transplant inoculum. While lyophilization facilitates storage, it can compromise bacterial viability, which is essential for FMT effectiveness. Lyoprotectants are often used to protect bacterial cultures during freeze-drying, but their effect on complex microbial communities remains unclear, as they may preferentially preserve some taxa over others. This study investigated the impact of four lyoprotectants-mannitol, maltodextrin, trehalose, and a maltodextrin-trehalose mixture-on bacterial viability and community structure in pig fecal samples post-lyophilization. Propidium monoazide (PMA) treatment combined with 16S rRNA sequencing (PMAseq) was used to differentiate viable from non-viable bacteria. In the total community (without PMA), microbial profiles appeared similar across treatment groups. However, when focusing on the viable community (PMA-treated), lyoprotectant choice significantly influenced the post-lyophilization community composition. Gram-negative bacterial viability was especially sensitive to lyophilization. Trehalose and maltodextrin preserved bacterial viability and community structure more effectively than mannitol. Mannitol-treated samples had reduced viable bacterial cells and altered community composition, while trehalose and maltodextrin better maintained diversity and structure of the viable (PMA-treated) communities. Taken together, lyoprotectants have differential effects on microbial composition during lyophilization. Among those tested, trehalose and maltodextrin best preserved both viability and community structure, making them promising candidates for FMT applications. Future research should explore optimizing lyoprotectant formulations to enhance microbiome stability and functional outcomes.