Oxidative stress-induced evolutionary engineering of Leuconostoc mesenteroides WiKim0121 improves freeze-drying and storage stability via polysaccharide accumulation

ABSTRACT Storage stability, including freeze-drying (FD) tolerance and post-storage viability, is crucial for the industrial use of lactic acid bacteria (LAB) starter cultures. This study applied adaptive laboratory evolution (ALE) to develop a Leuconostoc mesenteroides strain with enhanced viability after FD and subsequent storage. Leu. mesenteroides WiKim0121 was subjected to oxidative stress through H 2 O 2 exposure to induce beneficial genotypic and phenotypic traits. The evolved strain and its wild-type (WT) counterpart were freeze-dried using a pilot-scale system and stored for 8 weeks at 4°C or 25°C with or without cryoprotectants. The evolved strain consistently exhibited higher viability than the WT under all tested conditions. Notably, when stored at 25°C for 8 weeks with soy powder as a cryoprotectant, it showed only a 0.43 log reduction in viability. Comparative genomics revealed 187 mutations in the evolved strain, including nine genes potentially linked to oxidative stress resistance. Phenotypic analysis further showed a 51.4% increase in exopolysaccharide layer thickness and a 227.7% increase in intracellular polysaccharide content relative to the WT. These findings demonstrate that oxidative stress-driven ALE markedly enhances FD tolerance and storage stability, offering a practical strategy to improve the industrial applicability of LAB starter cultures.