Integrated Genomic, Structural, and Metabolomic Profiling of Freeze-Dried Postbiotic Supernatants Reveals Multi-Target Anticancer Activity Against Caco-2 Cells.

Postbiotic metabolites derived from probiotics bacteria have gained increasing attention as multitargeted anticancer agents with improved safety and stability compared to live microorganisms. In this study, we evaluated the anticancer activity of freeze-dried cell-free supernatants (CFS) obtained from Bifidobacterium bifidum and Lacticaseibacillus rhamnosus against human colorectal adenocarcinoma (Caco-2) cells and integrated functional, molecular, and omics-based analyses. Cytotoxicity assessment using the MTT assay demonstrated concentration-dependent growth inhibition, with IC₅₀ values of 94.38 µg/mL and 395.19 µg/mL for B. bifidum and L. rhamnosus CFS, respectively. Flow cytometry and morphological analyses confirmed apoptosis induction and G₀/G₁ cell-cycle arrest. Immunocytochemical evaluation revealed increased E-cadherin and caspase-3 expression accompanied by vimentin downregulation, indicating suppression of epithelial-mesenchymal transition (EMT). Genomic screening identified apoptosis-related proteases and metalloproteases, supported by three-dimensional structural prediction, suggesting a functional role in postbiotic bioactivity. Metabolomic profiling using HPLC and GC-MS revealed strain-specific signatures dominated by organic acids, volatile compounds, and fatty acids associated with oxidative, inflammatory, and apoptotic modulation. Collectively, these findings demonstrate that freeze-dried CFS exert anticancer effects through coordinated transcriptomic-independent, proteomic, and metabolomic mechanisms, highlighting their potential as multifunctional postbiotic candidates for colorectal cancer intervention.