Encapsulation of E. coli phage ZCEC5 in chitosan-alginate beads as a delivery system in phage therapy

Bacteriophages can be used successfully to treat pathogenic bacteria in the food chain including zoonotic pathogens that colonize the intestines of farm animals. However, harsh gastric conditions of low pH and digestive enzyme activities affect phage viability, and accordingly reduce their effectiveness. We report the development of a natural protective barrier suitable for oral administration to farm animals that confers acid stability before functional release of bead-encapsulated phages. Escherichia coli bacteriophage ZSEC5 is rendered inactive at pH 2.0 but encapsulation in chitosan-alginate bead with a honey and gelatin matrix limited titer reductions to 1 log₁₀ PFU mL^-1. The encapsulated phage titers were stable upon storage in water but achieved near complete release over 4-5 h in a simulated intestinal solution (0.1% bile salt, 0.4% pancreatin, 50 mM KH₂PO₄ pH 7.5) at 37 °C. Exposure of E. coli O157:H7 to the bead-encapsulated phage preparations produced a delayed response, reaching a maximal reductions of 4.2 to 4.8 log₁₀ CFU mL^-1 after 10 h at 37 °C under simulated intestinal conditions compared to a maximal reduction of 5.1 log₁₀ CFU mL^-1 at 3 h for free phage applied at MOI = 1. Bead-encapsulation is a promising reliable and cost-effective method for the functional delivery of bacteriophage targeting intestinal bacteria of farm animals.