Large-Scale Purification of a Bacteriocin Produced by Leuconostoc Mesenteroides Subsp Cremoris Using Diatomite Calcium Silicate

Abstract

Leuconostoc mesenteroides subsp. cremoris strain W3, isolated from wine, was previously found to produce a bacteriocin with inhibitory activity towards malolactic bacteria and hence inhibit malolactic fermentation in model wine medium. The bacteriocin, which we termed mesentericin W3, was purified by multistep chromatography and found in a previous study to be similar to mesentericin Y105, albeit with a low recovery (3.2%). In this study, we aimed to achieve large-scale isolation of mesentericin W3 by adsorption of the bacteriocin from culture supernatant onto Micro-Cel (diatomite calcium silicate) and then by subsequent desorption. Water-soluble surfactants including Tween 80, Triton X-100, sodium deoxycholate, sodium dodecyl sulfate (SDS) in distilled water, organic solvents (methanol, ethanol, acetonitrile), and distilled water with a pH range of 2-10 were tested for the desorption of the bacteriocin from Micro-Cel. Mesentericin W3 was successfully desorbed from Micro-Cel with SDS, while other tested reagents were not as successful. The bacteriocin desorbed from Micro-Cel showed the same inhibitory activity as the culture supernatant. The desorbed bacteriocin was applied to an SP Sepharose Fast Flow column for final purification. MALDI-TOF mass spectrometry confirmed the identity of the bacteriocin.