Deletion of the DEAD-box helicase CshA in Lacticaseibacillus paracasei elicits heightened low-temperature sensitivity...

While cold chain logistics remains the primary strategy to mitigate post acidification in fermented dairy products, maintaining reliable refrigeration systems presents substantial challenges, especially in resource-limited settings. Biological approaches targeting strain low-temperature sensitivity modulation for post-acidification control are increasingly recognized as promising alternatives. This study utilized CRISPR/Cas9-mediated knockout of DEAD-box helicase genes in Lacticaseibacillus paracasei, successfully identifying a low-temperature-sensitive strain, ∆AF91_12,710. Under optimal conditions, ∆AF91_12,710 showed comparable growth patterns and pH profiles to the wild-type strain. However, under low-temperature conditions, ∆AF91_12,710 demonstrated significantly impaired growth capacity and distinct pH regulation patterns. Metabolomic analysis of post-acidification revealed 39 unique differential metabolites in ∆AF91_12,710 fermented milk over 21 days, featuring decreased ATP, γ-linolenic acid, and dGMP concentrations, alongside elevated glucose-6-phosphate, cAMP, and D-galactose levels. These metabolic alterations indicate impaired glycolysis, likely resulting from DEAD-box helicase gene deletion-induced RNA instability and reduced metabolic activity in ∆AF91_12,710 under cold stress. The AF91_12,710 deletion significantly enhanced low-temperature sensitivity while reducing post-acidification capability in fermented milk. This research provides valuable insights into post-acidification attenuation in low-temperature-sensitive strains and demonstrates the potential for developing robust starter cultures in dairy applications.