Deletion of the amidase domain of endolysin LysRODI enhances antistaphylococcal activity in milk and during fresh cheese production

Abstract

Milk contamination with Staphylococcus aureus can lead to food poisoning in consumers. One strategy to minimize this risk is the use of phage-derived lysins, which are innocuous for humans and do not readily select for resistant variants. However, it remains necessary to find new candidate lysins and define the conditions for their utilization. This study compares the potential of LysRODI and its derivative LysRODIΔAmi (lacking the amidase domain), which displays high activity and storage stability, to successfully decrease staphylococcal contamination in milk under different conditions. Our results show that the engineered protein is more efficacious than the parent endolysin in practically all cases. For instance, while LysRODI only decreased the number of cells by 1–2 log units in different types of commercial milk and contamination levels, the chimeric lysin eliminated them below detection. Also, LysRODIΔAmi was more active against four strains with varying degrees of susceptibility. Regarding incubation temperature, both proteins were faster at 32 °C and 37 °C. Significantly, the engineered lysin eliminated detectable contamination in just 15 min. Finally, LysRODIΔAmi proved very successful at reducing staphylococcal contamination below detection during lab-scale fresh cheese production by enzymatic coagulation. Our data show that LysRODIΔAmi is a promising candidate for biocontrol in milk.