Separation and enrichment of phages at the interface between two phases in a green solvent-based sugaring-out extraction system.

The advantages of using phages as an alternative to antibiotics are evident in the response to the growing multidrug resistance of microorganisms. Green and efficient extraction methods for phages have attracted considerable attention for large-scale production. In this study, a novel sugaring-out extraction (SOE) system was developed to isolate and purify Klebsiella pneumoniae phage from the crude phage lysate. Phages were preferentially enriched in a middle phase formed between the triethyl citrate-rich top phase and the glucose-rich bottom phase. The SOE system composed of triethyl citrate and glucose was optimized based on phage recovery and impurity removal. The distribution behavior of the phages, phase separation, and extraction kinetics were investigated. The optimized system achieved a middle-phase recovery of 82.4 ± 1.6% for phage phiKpS2, and most proteins (91.8 ± 0.9%), cells (96.2 ± 1.5%), and endotoxins (81.1 ± 1.3%) were effectively removed using a SOE system consisting of 28% (w/w) glucose and 30% (w/w) triethyl citrate. The phage concentration factor reached 46.7-fold, and the separation factors of phage relative to proteins, cells, and endotoxins were 149.7, 109.6, and 5.4, respectively. Furthermore, the conductivity measurements indicated that the SOE gradually shifted phage phiKpS2 from the bottom phase to the middle phase as the phase volume ratio increased. The extraction kinetics showed that the partitioning behavior could be completed within 40 min without centrifugation. Finally, this SOE system also proved applicable for the isolation and purification of phages λ, phiSM29, and phiSM30, which were successfully enriched in the middle phase. Understanding the mechanism of the SOE process is important for its further application and industrial scale-up in phage purification.