Development of Stabilizing Solution for Long-Term Storage of Bacteriophages at Room Temperature and Application to Control Foodborne Pathogens

Abstract

Bacteriophages (phages) have gained considerable attention as effective antimicrobial agents that infect and kill pathogenic bacteria. Based on this feature, phages have been increasingly used to achieve food safety. They are stored in a medium or buffer to ensure stability; however, they cannot be directly applied to food under these conditions due to reasons such as regulatory considerations and concerns about marketability. This study developed a stabilizing solution that allowed the maintenance of phage activity for extended periods at room temperature while being directly applicable to food. The stability of phages stored in distilled water was relatively low. However, adding a stabilizer composed of sugars and salts improved the survival rates of phages significantly, resulting in stability for up to 48 weeks at room temperature. When Escherichia coli O157:H7-contaminated vegetables were washed with tap water containing phages, the phages reduced the pathogenic E. coli count by over 90% compared with washing with tap water alone. Additionally, when pathogenic E. coli-contaminated vegetables were placed in a phage-coated container and exposed to water, the coating of the container dissolved, releasing phages and lysing the pathogenic E. coli. This led to a significant 90% reduction in pathogenic E. coli contamination compared to that after water rinsing. These results suggest an effective and economical method for maintaining phage activity and establishing the potential for commercialization through application in the food industry.

Keywords: bacteriophage; biocontrol; long-term storage; sanitizer; stabilizer.

Figure 1

Phage stability in different solutions suitable for food consumption. Two types of phages were dispersed in sodium chloride–magnesium sulfate (SM) buffer (circle), saline (rectangular), distilled water (DW; triangle), or tap water (diamond) and preserved at either room temperature or in the refrigerator. Analyses of phage plaque counts were carried out periodically on representative samples. (A) E. coli phage LEC1, room temperature; (B) E. coli phage LEC1, refrigerator; (C) S. aureus phage LSA5, room temperature; and (D) S. aureus phage LSA5, refrigerator. Values represent the means with the standard deviations of three trials.

Figure 2

Synergistic effect of ions and sugar on phage stability during long-term storage. The numbers of phage LEC1 (A) and LSA5 (B) suspended in DW with or without 10% sorbitol or 0.1 M NaCl and 8 mM MgSO₄ were measured at 1, 21, or 63 days of storage. ND; not detected (limit of quantification; 10 PFUs/mL). The plaque numbers of phage LEC1 (C) and LSA5 (D) stored in DW with 10% sorbitol, 0.1 M NaCl, and 8 mM MgSO₄ were monitored for up to 48 weeks of storage at room temperature. Values represent the means with standard deviations of three trials. (ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

Figure 3

Efficacy of the phage solution in reducing E. coli O157:H7 levels in vegetables. Different concentrations of phage solution were directly dropped on E. coli-contaminated strawberries (A) and kimchi cabbage (B). After 30 min, the numbers of remnant E. coli on the vegetables were counted. MOI (multiplicity of infection) represents the ratio of phage to bacterial quantities. Values represent the means with standard deviations of three trials. The marks above the bars, * or **, represent significant differences at p < 0.05 or p < 0.01, respectively.

Figure 4

Efficacy of the phage solution as a washing material for the reduction of E. coli O157:H7 levels in vegetables. Brussels sprouts contaminated with pathogenic E. coli were washed with tap water containing the phage solution. The numbers of the remnant E. coli were counted immediately after washing and 24 h after refrigerated storage following the wash. The antibacterial activity of the phage solution is shown in comparison to tap water as a negative control. Values represent the means with standard deviations of three trials. * Significant at p < 0.05.

Figure 5

Stability of hydrated phages. Phages were coated with pullulan, carboxymethyl cellulose (CMC), or polyvinyl alcohol (PVA), and the number of plaques of the rehydrated phages were compared (A). The plaque number under the water-dried condition without the coating (negative control) was set to 100 for each phage, and the relative values were represented accordingly. For pullulan and CMC, the effect of stabilizer addition was also assessed (B). After applying a coating for 5 days, the number of plaques of the hydrated phages was counted. The relative values were obtained after the minimum value per phage was normalized to 100. Values represent the means with standard deviations of three trials.

Figure 6

Stability of coated phages during storage at room temperature. Phages LEC1 (A) and LSA5 (B) were mixed with pullulan with or without stabilizer and applied as a coating on 6-well microplates. After drying, the plaque numbers of rehydrated phages were measured as a function of the extended storage time. Values represent the means with standard deviations of three trials.

Figure 7

Efficacy of phage-coated rinsing containers in reducing E. coli O157:H7 levels in vegetables. Pathogenic E. coli-contaminated Brussels sprouts (A) and broccoli (B) were washed in an LEC1-coated container. After 10 min, the numbers of remnant E. coli on the vegetables were counted. Values represent the means with standard deviations of three trials. The marks above the bars, * or ***, represent significant differences at p < 0.05 or p < 0.001, respectively.

Figure 8

Schematic diagrams and example photos of the phage solution and coating application methods. Schematic diagram of the procedure for utilizing the phage solution (with stabilizer) in food washing (A) and example photo of the phage solution product forms (B). Schematic diagram of the washing method using phage-coated food containers (C) and example photos of a phage-coated container and the washing method using it (D). While the coating described in the manuscript is transparent, for experimental purposes, it was intentionally colored blue using edible dye to confirm the presence of the coating and its complete dissolution in water.