Metabolic reprogramming of whey into fatty acid-based nitric oxide donors for controlling of recalcitrant multi-species biofilms in meat systems

Whey fermentation by lactic acid bacteria is a sustainable strategy to upcycle dairy byproducts into functional biomaterials; however, the molecular mechanisms underlying its anti-biofilm potential remain elusive. This study demonstrated the inhibitory mechanism of whey fermented by Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus GG against multi-species biofilm formed by Escherichia coli O157:H7 and Listeria monocytogenes . Fermented whey induced the active dispersal of mature biofilms by upregulating motility. This dispersal was driven by an increase in intracellular nitric oxide (NO) levels, which stimulated phosphodiesterase activity and triggered cyclic-di-GMP depletion. LC-MS/MS-based metabolomics revealed that fermentation mediated the enzymatic hydrolysis of complex whey lipids, resulting in free fatty acid accumulation. KEGG pathway analysis indicated that the subsequent catabolism of these fatty acids increased intracellular NADH levels and consequently promoted the enzymatic reduction of nitrite to NO; thus, it acts as the metabolic trigger for biofilm dispersal. Furthermore, validation in meat models demonstrated that fermented whey significantly reduced viable biofilm cells on fresh pork and beef surfaces under both refrigeration (4 °C) and temperature abuse (37 °C) conditions. Collectively, these findings suggest fermented whey as a sustainable, fatty acid-based NO donor for controlling recalcitrant biofilms in food systems.