Impact of temperature on endolysin LysRODI inactivation and killing dynamics of Staphylococcus aureus in milk

Artisanal cheese production requires the implementation of strict control measures to inhibit growth of pathogenic and spoilage bacteria, while preserving the viability of microorganisms involved in fermentation and ripening. In this context, phage lytic proteins can be a very interesting antimicrobial choice due to their specificity, while exhibiting a broader spectrum of action than phages themselves. Several studies have demonstrated the potential of lytic proteins against Staphylococcus aureus in milk. However, the impact of the existing environmental conditions during treatment must be considered to maximize its efficacy. Temperature is a particularly crucial parameter in dairy production, and it is also known to influence the enzymatic activity and stability of lysins. Here, we estimated different parameters related to the killing activity and stability of protein LysRODI at different temperatures (12 ºC, 25 ºC, 32 ºC and 37 ºC). The results obtained at 12 ºC revealed good protein stability but lack of enzymatic activity and very slow bacterial growth. At the other temperatures, bacterial growth and death due to endolysin killing were estimated based on previous data. Interestingly, the killing rates estimated at the three temperatures were similar, indicating a similar enzymatic activity within the analyzed range. However, the protein displayed much greater stability at 25 ºC compared to 32 ºC and especially to 37 ºC. The estimated parameter values will be subsequently used to develop a mathematical model to further study the endolysin killing dynamics under different production scenarios, considering temperature variation during milk storage and fermentation.