Extracellular matrix affects mature biofilm and stress resistance of psychrotrophic spoilage Pseudomonas at cold temperature.

Psychrotrophic Pseudomonas as the dominant spoilage bacteria, have biofilm forming ability, increasing persistence and contamination in the chilled food. Biofilm formation of spoilage Pseudomonas at cold temperature was documented, however, role of extracellular matrix in mature biofilm and stress resistance of psychrotrophic Pseudomonas are much less abundant. The aim of this study was to investigate the biofilm forming characteristics of three spoilers P. fluorescens PF07, P. lundensis PL28, and P. psychrophile PP26 at 25 °C, 15 °C and 4 °C, and to explore their stress resistance to chemical and thermal treatments of mature biofilms. The results showed that biofilm biomass of three Pseudomonas at 4 °C was significantly higher than that at 15 °C and 25 °C. The secretion of extracellular polymeric substances (EPS) greatly increased in those Pseudomonas under low temperature, of which extracellular protein constituted about 71.03%-77.44%. Compared to 25 °C, the mature biofilms were observed to more aggregation and thicker spatial structure at 4 °C ranging from 42.7 to 54.6 μm, in contrast to 25.0-29.8 μm at 25 °C, especially strain PF07. These Pseudomonas biofilms switched into moderate hydrophobicity, and their swarming and swimming were significantly inhibited at low temperature. Furthermore, the resistance to NaClO and heating at 65 °C apparently enhanced for mature biofilm formed at 4 °C, indicating the difference in EPS matrix production influenced the stress resistance of biofilm. In addition, three strains contained alg and psl operons for exopolysaccharide biosynthesis, and biofilm related genes of algK, pslA, rpoS, and luxR were significantly up-regulated, while flgA gene was down-regulated at 4 °C compared to 25 °C, consistent with the above phenotype changes. Thus, the dramatic increase of mature biofilm and their stress resistance in psychrotrophic Pseudomonas were associated with large secretion and protection of extracellular matrix under low temperature, which provide a theoretical basis for subsequent biofilm control during cold chain.