Long-Term Antimicrobial Activity of Phage–Sugar Glasses is Closely Tied to the Processing Conditions

Bacteriophage-based antimicrobials have proven to be effective in the control of foodborne contaminants at various stages of the food production chain. Incorporating bacteriophages onto food-processing surfaces and food packaging can significantly impact food safety. One of the major roadblocks toward using phage as a natural antimicrobial/preservative in food packaging is phage sensitivity to desiccation. We recently developed a coating (film) composed of pullulan and trehalose that proved to be highly effective in preserving the antimicrobial activity of phage for more than 8 weeks. During this investigation, we realized that the conditions under which the films were formed and stored significantly affected the protective ability of these films toward bacteriophage antimicrobials, leading us to design a more in-depth investigation to identify and evaluate the major factors that affect the efficacy of these antimicrobial films. In this work, we report the outcome of a four-factor, two-level full-factorial randomized design of experiments, in which we examined the effects of four processing/storage factors on the short-term and long-term antimicrobial activity of phage-embedded pullulan/trehalose films. The drying method used to make the films and the storage humidity are the two factors that have the most significant impact on the long-term antimicrobial activity of pullulan/trehalose. The long-term viability of the bacteriophage (as measured by loss of titer) can be increased by over 1000-fold by using dry vacuum to form the films and then storing (for 28 days at room temperature) them in a closed container (tube) with low humidity in the headspace. The presence of divalent ions during the drying process and modification of the surface onto which the films were cast did not have a significant effect on long-term phage viability. Coatings, such as a pullulan/trehalose film, can help realize the promise of phage in the food industry. Insights in the control and stabilization of active ingredients in pullulan/trehalose films can also be applied to stabilization of viral vaccines and diagnostic bioassays.