Characterization of the Clostridium perfringens phage endolysin cpp-lys and its application on lettuce

Clostridium perfringens is an important foodborne pathogen that can have severe consequences, including mortality and economic losses. In this study, the gene encoding cpp-lys, an endolysin from the C. perfringens phage cpp has been cloned and overexpressed. The encoded protein was characterized, and then its efficacy in controlling C. perfringens on lettuce was evaluated. The endolysin cpp-lys presented lytic activity against seven strains of C. perfringens that produce different types of toxins. It maintained stability across a wide range of temperatures (4 °C - 50 °C), and demonstrated tolerance to varying pH levels (4-9). Storage of endolysin cpp-lys under room-temperature conditions (16 °C-25 °C) and cold-temperature conditions (4 °C, -20 °C, and -80 °C) for 30 days did not affect its lytic activity. However, the lytic activity of cpp-lys decreased by 40 % and 18 % after storage for 30 d at 42 °C and 37 °C, respectively. The endolysin cpp-lys did not display cytotoxic activity against normal eukaryotic cells. The bacterial viability on lettuce was significantly lower in the group treated with endolysin cpp-lys than in the PBS group, and >4-log of C. perfringens J1 were removed within 15 min. Cpp-lys plus Zn²⁺ inhibited the activity of cpp-lys. The EDTA-treated cpp-lys significantly reduced the number of bacteria by up to 0.6-log CFU compared with the endolysin cpp-lys group. The findings of this study demonstrated that endolysin cpp-lys has potential applications in controlling C. perfringens in the food industry.