Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococci bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method. To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium (LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible microorganisms. Recovery yields using filtration were found to range between 53 and 68%, a high recovery performance. The bacteriocin activity of crude extracts of all the three lactobacilli were between 95 and 105 IU ml(-1). When the substances were separated using ultrafiltration membrane (4 kDa MWCO) their activity was enhanced to 145-150 IU ml(-1), achieving a total potency yield of 44-53%. Further enhancement of yields up to 36% was attained employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml(-1). Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields, optimising their activity as well as presenting a realistic option towards the formulation of these as commercially available antibacterial agents. (C) 2013 Elsevier Ltd. All rights reserved.