Combined effects of cold and acid on dual-species biofilms of Pseudomonas fluorescens and Listeria monocytogenes under simulated chilled beef processing conditions.

Interactions across bacterial species boundaries are usually influenced by environmental stresses, yet little has been evaluated regarding multifactorial stresses on the fate of dual-species biofilm formation in food industry. In this study, the processing conditions of chilled beef were established as a combination of cold and acid stresses (4 °C and pH 5.4), with pH 7.0 or 25 °C serving as the controls, to investigate the interaction of dual-species biofilm between Pseudomonas fluorescens and Listeria monocytogenes. Dual-species biofilms significantly increased biofilm formation at 72 h under the condition of 25°C-pH7.0 and 25°C-pH5.4 (P < 0.05). Compared with mono-species biofilms, the cell numbers of L. monocytogenes in dual-species biofilms were lower at 25 °C (P < 0.05), however, the adherent cells of L. monocytogenes was higher in dual-species biofilms at 4 °C (P < 0.05). Furthermore, the amount of extracellular polysaccharides and proteins secreted by single P. fluorescens biofilms at 4 °C was more than three times than those at 25 °C. The surface-enhanced Raman spectroscopy further profiled the variability of extracellular polymeric substances (EPS) composition. Additionally, RT-qPCR results revealed an upregulation of biofilm-related and genes in co-culture species. It provides valuable insights into the strategies for removing mixed biofilms under diverse stressful conditions in practical food processing.