Comparative analysis of different preservation techniques for the storage of Staphylococcus phages aimed for the industrial development of phage-based antimicrobial products

Bacteriophages have been proven as effective antimicrobial agents in the treatment of infectious diseases and in other biocontrol applications including food preservation and disinfection. The extensive use of bacteriophages requires improved methodologies for medium-and long-term storage as well as for easy shipping. To this aim, we have determined the stability of four Staphylococcus phages (philPLA88, philPLA35, philPLA-RODI and philPLA-C1C) with antimicrobial potential at different temperatures (20 degrees C/25 degrees C, 4 degrees C, -20 degrees C, -80 degrees C, -196 degrees C) and during lyophilization (freeze drying) using several stabilizing additives (disaccharides, glycerol, sorbitol and skim milk). Differences between phages were observed at different temperatures (20 degrees C/25 degrees C, 4 degrees C and -20 degrees C), where phages were less stable. At lower temperatures (-80 degrees C and -196 degrees C), all phages showed good viability after 24 months regardless of the stabilizer. Differences between phages were also observed after lyophilization although the addition of skim milk yielded a dry powder with a stable titer after 24 months. As an alternative to facilitate storage and transportation, phage encapsulation has been also explored. Phage philPLA-RODI encapsulated in alginate capsules retained high viability when stored at 4 degrees C for 6 months and at 20 degrees C for 1 month. Moreover, the spray-dryer technique allowed obtaining dry powders containing viable encapsulated phages (philPLA-RODI and philPLA88) in both skim milk and trehalose for 12 months at 4 degrees C. Storage of phages at 20 degrees C was less effective; in fact, philPLA88 was stable for at least 12 months in trehalose but not in skim milk, while philPLA-RODI was stable only for 6 months in either stabilizer. These results suggest that encapsulated phages might be a suitable way for shipping phages.