Metabolomics-driven insights into the multi-target antibacterial mechanisms of 2-methoxycinnamaldehyde against Bacillus cereus and its application in pork preservation.

Bacillus cereus is a major foodborne pathogen characterized by robust biofilm formation and increasing antimicrobial resistance. This study identified 2-Methoxycinnamaldehyde (MCA) from Toona sinensis as the principal antibacterial compound and evaluated its inhibitory mechanisms and preservation potential. MCA exhibited potent activity against B. cereus ATCC 11778 (MIC = 150 μg/mL; MBC = 200 μg/mL) and resistant strains. In vitro assays demonstrated that MCA induced concentration-dependent membrane disruption, DNA damage, and intracellular protein leakage. Furthermore, treatment at 1 MIC and 2 MIC significantly impeded biofilm maturation; the secretion of extracellular proteins was reduced by 45.3% and 54.5%, polysaccharides by approximately 93%, and extracellular DNA (eDNA) by over 99%. Correspondingly, biofilm metabolic activity declined by 85.9% and 90.3%, and initial cellular adhesion was reduced by up to 88.1%. Untargeted metabolomic analysis revealed that these phenotypic defects stem from profound disturbances in amino acid biosynthesis, the TCA cycle, and nucleotide metabolism. Molecular docking revealed that MCA targets multiple essential bacterial enzymes, including ribonucleotide reductase, lysyl-tRNA synthetase, pyruvate kinase, betaine aldehyde dehydrogenase, and 5'-nucleotidase, through stable hydrogen bonding and π-interactions. In a refrigerated pork model, MCA completely eliminated B. cereus by day 7 while significantly delaying pH increases and color deterioration. These findings provide the first evidence for MCA as an antibacterial and antibiofilm agent against B. cereus, highlighting its potential in food preservation.