epsilon-Poly-L-lysine (epsilon-PL) has been widely used in the food industry as a biological preservative like Nissin and natamycin for many years. However, few studies have focused on the separation and purification of epsilon-PL from fermentation broth. In the present study, a successive process for epsilon-PL separation and purification from fermentation broth was proposed, and two key operation units of solid-liquid separation and ion exchange chromatography were systematically investigated. Solid-liquid separation was successfully achieved by filtration for fermentation broth flocculated by the addition of 800 mg/L sodium polyacrylate at pH 1.5. The adsorption and desorption of epsilon-PL on Amberlite IRC-50 resin were optimized, and the optimal conditions are as follows: initial epsilon-PL concentration of 15 g/L, pH 8.5, and a flow rate of 1.5 bed volume (BV)/h for adsorption; 0.25 M HCI and a flow rate of 7.0 BV/h for desorption. On the basis of the above results and those from our previous studies, a successive process including flocculation, filtration, ultrafiltration, ion-exchange chromatography, and decoloration was employed to extract epsilon-PL from fermentation broth; 90.2% purity was attained with 75% recovery. This is the first report on the separation and purification of epsilon-PL from fermentation broth containing a high concentration of epsilon-PL, which will provide guidance for its industrial manufacture. (C) 2015 Elsevier Ltd. All rights reserved.