Abrogating the adenine methylation ability of Lacticaseibacillus paracasei improves its freeze-drying and storage resistance.

Freeze-drying is a widely adopted method for the long-term storage of starter cultures in the food industry but can cause cell instability and a decline in post-storage viability. We used an unmethylated Lacticaseibacillus paracasei Zhang mutant lacking adenine-specific DNA-methyltransferase. This mutant was subjected to freeze-drying and stored at 30 °C for two distinct durations (30 and 60 days), Our analysis revealed the unmethylated mutant outperformed the wild-type in cell viability and survival following freeze-drying and post-freeze-drying storage. And significant metabolic pathway differences between the stored mutant and wild-type bacteria. These differences were evident in the phosphotransferase system, carbohydrate, and amino acid metabolism, and fatty acid biosynthesis, and were consistent across transcriptomic, proteomic, and metabolomic analyses. This is achieved by modulating key metabolic pathways within the bacteria. This study contributes to the limited literature on the role of bacterial adenine methylation in industrial strain application and starter culture storage.