Formation of Biofilm by Tetragenococcus halophilus Benefited Stress Tolerance and Anti-biofilm Activity Against S. aureus and S. Typhimurium

<i>Tetragenococcus halophilus</i>, a halophilic lactic acid bacterium (LAB), plays an important role in the production of high-salt fermented foods. Generally, formation of biofilm benefits the fitness of cells when faced with competitive and increasingly hostile fermented environments. In this work, the biofilm-forming capacity of <i>T. halophilus</i> was investigated. The results showed that the optimal conditions for biofilm formation by <i>T. halophilus</i> were at 3-9% salt content, 0-6% ethanol content, pH 7.0, 30°C, and on the surface of stainless steel. Confocal laser scanning microscopy (CLSM) analysis presented a dense and flat biofilm with a thickness of about 24 μm, and higher amounts of live cells were located near the surface of biofilm and more dead cells located at the bottom. Proteins, polysaccharides, extracellular-DNA (eDNA), and humic-like substances were all proved to take part in biofilm formation. Higher basic surface charge, greater hydrophilicity, and lower intracellular lactate dehydrogenase (LDH) activities were detected in <i>T. halophilus</i> grown in biofilms. Atomic force microscopy (AFM) imaging revealed that biofilm cultures of <i>T. halophilus</i> had stronger surface adhesion forces than planktonic cells. Cells in biofilm exhibited higher cell viability under acid stress, ethanol stress, heat stress, and oxidative stress. In addition, <i>T. halophilus</i> biofilms exhibited aggregation activity and anti-biofilm activity against <i>Staphylococcus aureus</i> and <i>Salmonella Typhimurium</i>. Results presented in the study may contribute to enhancing stress tolerance of <i>T. halophilus</i> and utilize their antagonistic activities against foodborne pathogens during the production of fermented foods.