Title : Synergy between the phage endolysin LysH 5 and nisin to kill Staphylococcus 1 aureus in pasteurized milk

26 27 Phage-encoded endolysins are recently considered as new biocontrol tools to inhibit 28 pathogens in food. In this work, we have studied the ionic requirements for optimal lytic 29 activity of LysH5, the endolysin encoded by the staphylococcal bacteriophage phi30 SauS-IPLA88. LysH5 activity was inhibited by the presence of Mn and Zn and 31 enhanced by Ca, Mg and NaCl. When LysH5 was combined with nisin, a 32 bacteriocin currently used as a biopreservative in food, a strong synergistic effect was 33 observed. The Minimum Inhibitory Concentrations of nisin and LysH5 were reduced 34 64and 16-fold, respectively, as determined in checkerboard microtitre tests. In 35 addition, nisin enhanced 8-fold the lytic activity of LysH5 on cell suspensions. The 36 synergy observed in vitro was confirmed in challenge assays in pasteurized milk 37 contaminated with S. aureus Sa9. Clearance of the pathogen was only achieved by the 38 combined activity of both antimicrobials. As far as we know, this is the first study that 39 exploits the possibilities of hurdle technology combining a phage-encoded endolysin 40 and the bacteriocin nisin for efficient S. aureus inhibition in milk. 41 42