The advantages of using phages as an alternative to antibiotics are evident in the response to the growing multidrug resistance of microorganisms. The research community has shown great interest in the use of green solvents and efficient extraction techniques. In this study, Klebsiella pneumoniae phage was efficiently isolated and purified from the crude phage lysate using the sugaring-out extraction technique. The sugaring-out extraction system, composed of triethyl citrate and glucose, was optimized based on phage recovery and impurity removal. The distribution behavior of the phage, phase separation, and the extraction kinetics were investigated. In this study, the recovery of the phage phiKpS2 in intermediate phase reached to 82.42%, and most proteins (91.78%), cells (96.23%), and endotoxins (81.09%) were effectively removed using a sugaring-out extraction system consisting of 28% (w/w) glucose and 30% (w/w) triethyl citrate. The concentration multiplicity of the phage was 46.67, and the phage separation factors were 149.72, 109.64, and 5.42 for proteins, cells, and endotoxins, respectively. Furthermore, the conductivity detection indicated that the sugaring-out extraction 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 minutes without the need for centrifugation. Finally, this sugaring-out extraction system is also a viable alternative for the isolation and purification of phages λ, phiSM29, and phiSM30. Understanding the mechanism of the sugaring-out extraction process is crucial for its application and scale-up in phage isolation.
