Antagonistic activity of Lactobacillus helveticus postbiotics against mastitis causing and dairy spoilage bacteria and their produced biofilms

INTRODUCTION. Lactobacillus helveticus is a lactic acid bacterium widely used in the production of fermented dairy products, and several studies have demonstrated that its live cultures, exopolysaccharides, and cell-free supernatants exhibit inhibitory effects against pathogenic bacteria. However, little is known about the antibiofilm potential of L. helveticus postbiotics in dairy-associated environments. This study evaluated postbiotics derived from commercially available L. helveticus against biofilms formed by pathogenic and milk-spoilage microorganisms.

METHODS. Freeze-dried L. helveticus (LMG26307; THT031102) was precultured in MRS broth at 37°C. After 18 h, cultures were centrifuged, and biomass was reinoculated (5% w/v). Following incubation, cells were collected, washed, thermally inactivated, and ethanol-treated, yielding postbiotics consisting of inactivated cell biomass. Cell-free supernatant was obtained by centrifugation and sterile filtration (0.22 μm). Indicator strains (Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, and Bacillus subtilis) were cultured and standardized to 0.5 McFarland. Preparations were diluted (1:10), mixed with bacterial suspensions (1:1 v/v), and incubated for 48 h at 37 °C in 96-well plates. Planktonic growth (OD620) and biofilm biomass were measured.

RESULTS. Both preparations affected bacterial growth and biofilm formation. The supernatant inhibited planktonic growth in all tested species and reduced biofilm formation in K. pneumoniae, P. mirabilis, and P. aeruginosa. Postbiotics increased planktonic OD values due to cell-derived turbidity but reduced biofilm formation in K. pneumoniae and P. aeruginosa, although less effectively than supernatant. S. aureus, B. subtilis, E. coli, and E. faecalis strains did not form biofilms under tested conditions.

CONCLUSION. L. helveticus supernatant exhibited strong antimicrobial and antibiofilm activity, while postbiotics showed a more limited but still notable effect.

ACKNOWLEDGEMENTS. 1.1.1.9 Research application No 1.1.1.9/LZP/1/24/162 of the Activity "Post-doctoral Research" "Development of postbiotics’ compositions including the evaluation of its bioactivity against pathogenic bacteria and their produced biofilms in the dairy industry"