Separation and purification of Klebsiella phage by two-step salting-out extraction

The bacterial resistance caused by the irrational usage of antibiotic has become a global public health crisis. Phage therapy is expected to be an alternative to antibiotics for the treatment of drug-resistant infections. However, industrial separation and purification of phage are cumbersome, time-consuming, high cost, or low efficiency. In this study, two-step salting-out extraction of Klebsiella phage was evaluated. In the first step, under the optimal conditions of 10 wt% sodium citrate/30 wt% ethyl acetate, 99 ± 4.86% of phage was recovered in the bottom phase. Meanwhile, most of the cells and proteins could be removed. Subsequently, n-propanol was added into the bottom phase for the second salting-out extraction. Under the optimal conditions, 77 ± 1.3% of the phage can be recovered in the intermediate phase, and the concentration factor can be as high as 475 ± 17.6. Through the two-step salting-out extraction, the purification fold of phage to total proteins, cells and endotoxins was 19.31 ± 1.26, 18.04 ± 1.3 and 5.19 ± 0.49, respectively, as well as a total phage recovery of 77 ± 2.6%, a total concentration factor of 500 ± 24 and no significant reduction in infectivity of phage. This work provides a potential method for industrial phage purification and has a great significance for the future development of phage therapy.