Introduction: Salmonella enterica subsp. enterica is one of the most prevalent food-borne bacterial pathogens with the highest incidence reported in poultry. The ability of lytic phages to infect and lyse their bacterial target with high specificity makes them ideal candidates for pathogen biocontrol in food production. However, despite their promising features, some hurdles exist to exploiting phage technology for biocontrol purposes to enhance food safety such as the development of phage resistant mutants and having a stable, feasible and effective delivery techniques.

Purpose: The purpose of this study is to design and evaluate a multi-receptor phage cocktail encapsulated in electrospun polyethylene oxide (PEO) microfibers as a potential solution to combat the high variability and emergence of phage resistance in Salmonella. The study aims to assess the antimicrobial effectiveness of the phage cocktail in vitro and in chicken meat, specifically targeting Salmonella Enteritidis.

Methods: Five phages were selected for cocktail composition. This cocktail targets four different receptors: O-antigen, BtuB, OmpC, and rough Salmonella strains. Salmonella Enteritidis was used to study growth inhibition at different environmental conditions and to investigate the development of phage resistant mutants. Furthermore, the phage cocktail biocontrol efficiency was evaluated on chicken skin. Chicken skin pieces inoculated with 4 log₁₀ CFU/cm² were dipped into cocktail suspension containing either 5 or 7 log₁₀ PFU/mL and incubated at three temperatures (25, 15, and 4°C) for 48 hours. To study the antimicrobial effect of the encapsulated phage cocktail, polyethylene oxide (PEO) phage-loaded microfibers were produced using electrospinning. Phage cocktail was electrospun in 12% PEO at 30.3 kV a Elmarco:Nanospider™. Finally, the effect of the phage-loaded electrospun fibers was evaluated in chicken meat. Chicken breast pieces were inoculated to a final concentration of 3 log₁₀ CFU/cm² and wrapped in the phage loaded fibers. Wrapped meat was stored at three temperatures (25, 15, and 4°C) and the bacterial reduction was evaluated for up to 48 hours.

Results: Bacteria challenge experiments using S. Enteritidis treated with different phage concentrations (MOIs 10^-1-10³) showed complete growth inhibition at 25°C and 15°C for 48 and 96 hours, respectively. No cross-resistance to all phages in the cocktail was observed. Biocontrol experiments showed a 3.5 log₁₀ CFU/cm² reduction after 48 hours with treatments of 7 log₁₀ PFU/mL at 25 and 15°C, and 2.5 log₁₀ CFU/cm² at 4°C. An average of 1 log₁₀ PFU/mg reduction post-electrospinning was observed for all phages in the cocktail. Overall, fibers containing 6 log₁₀ PFU/mg were obtained with the electrospinning method. The PEO phage-loaded fibers showed an immediate release of viral particles upon contact with an aqueous solution. The fibers showed to be stable for up to four months at 4°C. The phage fibers showed an antimicrobial effect against S. Enteritidis in chicken meat with 2 log₁₀ CFU/cm² reduction observed at 25 and 15°C when compared to the control.

Significance: The bacteriophage cocktail developed in this study holds great promise as a biocontrol tool against Salmonella, making it a valuable addition to poultry processing practices for enhanced food safety. Furthermore, this study highlights the potential of phage-loaded microfibers as an innovative packaging material for controlling the growth of Salmonella in packed poultry products, presenting a novel approach to ensure product safety.