Cold-Active Postbiotics from Fermented Food-Derived Lactobacillus fuchuensis H.Y.35 Exhibit Multifunctional In Vitro Bioactivities.

Postbiotics are stable, non-living microbial products that provide health benefits similar to probiotics and have lower safety concerns. Despite the expanding recognition and use of postbiotics, little is known about the specific properties and applications of postbiotics derived from cold-active (psychrophilic or psychrotolerant) microbial strains. To identify the molecular mechanisms that underpin therapeutic applications, we isolated cold-active probiotic candidates at 4 °C from fermented foods (feta cheese and kefir) sourced from local markets. Cell-free supernatant (CFS) of the isolates was initially screened for antioxidant activity, then followed by assessment of antimicrobial effects against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis. Cold-active Lactobacillus fuchuensis H.Y.35 showed strong antioxidant and antimicrobial capacities compared to other isolates. Antifreeze and cryoprotective assays confirmed that H.Y.35 postbiotics inhibited ice recrystallization and reduced drip loss in frozen cells. Untargeted metabolomics of H.Y.35 CFS identified bioactive compounds, including phenolic acids, fatty acyl derivatives, and amino acid-based metabolites. These bioactive compounds are linked to radical scavenging, antimicrobial activity, and cryoprotection. Complementary proteomic analysis further revealed the enrichment of proteins involved in stress adaptation, antimicrobial defense, and cryoprotection. These detected compound include pseudouridine synthase, cyclic-di-AMP phosphodiesterase, RNA polymerase sigma factor SigA, and the Sakacin Q immunity protein. These findings provide molecular evidence supporting the multifunctionality of cold-active postbiotics. The Lactobacillus fuchuensis H.Y.35 postbiotics/CFS also demonstrated potent wound-healing activity in vitro at 1% and 10% (v/v) concentrations, achieving up to 100% closure within 24 h (p < 0.01). This multifunctionality highlights the biotechnological and therapeutic potential of cold-active postbiotics as natural agents for health and industrial applications.