Comparison of several methods for preserving ETEC-S3 bacteriophage and its application to food

Food contamination caused by pathogenic bacteria is commonly controlled using chemical preservatives and physical preservation, which may negatively impact food quality. Bacteriophages are viruses that specifically infect bacteria, it can be used as an alternative antimicrobial agents. For further applications, phage need to be preserved. Phages show varying sensitivities to different preservation conditions, hence it is important to develop reliable long-term phage preservation methods. The ETEC-S3 phage, isolated from soil from previous study, showed lytic activity against Enterotoxigenic Escherichia coli (ETEC) as its original host. However, its preservation methods had not been investigated. This study investigates the effects of lyophilization and low-temperature storage, using trehalose, sucrose, or glycerol as stabilizing agents, on the titer stability of ETEC-S3, and evaluates their application in reducing ETEC populations in various food matrices. Low-temperature storage at −80 °C with sucrose or glycerol, and lyophilization with sucrose, effectively maintained ETEC-S3 stability, with titer reductions of 0.04, 0.08, and 0.26 log units, respectively, after 24 weeks. Lower temperatures performed more effective in preserving phage titers compare to higher temperatures. Stabilizers provided protection, which helped maintain phage viability throughout storage. Furthermore, lyophilized ETEC-S3 with the sucrose addition still performed high lytic activity after being applied to artificially contaminated various food samples. We found that food samples which high moisture content allowed easier phage diffusion, which resulted in higher bacterial reductions. The highest bacterial reductions were observed in tofu at 28 °C (99.86 %), followed by pasteurized milk at the same temperature (94.37 %). These findings indicate the potential preservation condition of ETEC-S3 as a natural food preservative.