Cocoa Pulp Juice as a Fermentation Substrate for Potential Probiotic Lacticaseibacillus paracasei WU2502: Genomic Characterization, Metabolite Profiling, and Antimicrobial Activity Against Foodborne Pathogens

Cocoa pulp juice is an underutilized by-product generated during cocoa bean fermentation that is rich in fermentable sugars and phytochemicals but remains largely unexplored as a substrate for microbial fermentation. This study aimed to evaluate cocoa pulp juice as a sustainable fermentation substrate for the potential probiotic Lacticaseibacillus paracasei WU2502 using whole-genome sequencing (WGS), carbohydrate-active enzyme (CAZyme) analysis, untargeted LC–QTOF-MS-based metabolite profiling, and antimicrobial evaluation. L. paracasei WU2502 showed substantial growth in cocoa pulp juice, reaching a maximum viable count of 10.70 log CFU/mL in 25% juice and >11 log CFU/mL in 50% and 100% juice, while the pH decreased from approximately 3.9–4.0 at Day 0 to 3.1–3.2 during fermentation. Fermented cocoa pulp juice showed antibacterial activity against Bacillus cereus, Staphylococcus aureus, Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7 with MIC and MBC values of 12.5–25% (v/v). In a preliminary qualitative assessment, neutralization reduced the observed inhibitory activity, whereas Proteinase K treatment produced little apparent change, suggesting a possible pH-dependent contribution to the antimicrobial activity. WGS resulted in a 3.02 Mb draft genome with a GC content of 46.27%, 3114 coding sequences and genes relevant to glucose metabolism, acid tolerance, oxidative stress response and antimicrobial activity. Genome mining identified putative bacteriocin-associated regions containing Carnocin CP52- and Thermophilin A-related sequences and a RiPP-like biosynthetic region. No acquired antimicrobial resistance genes or virulence-associated factors were detected. CAZyme analysis revealed glycoside hydrolases and glycosyltransferases that may be involved in carbohydrate metabolic pathways and adaptation to plant-derived substrates. LC–MS/MS analysis of unfermented cocoa pulp juice provided baseline chemical characterization of the starting substrate, with representative putative annotations including sucrose, maltotriose, myo-inositol, citric acid, pyroglutamic acid, and choline. These findings support the feasibility of cocoa pulp juice as a fermentation substrate for L. paracasei WU2502 and provide a basis for further evaluation of this strain and fermented matrix for potential food applications.