Letters in Applied Microbiology 2000, 30, 42–46 Triton X-114 phase partitioning for the isolation of a pediocin- like bacteriocin from Carnobacterium divergens A. Me ´ tivier 1,2 , P. Boyaval 2 , F. Duffes 2 , X. Dousset 3 , J.-P. Compoint 1 and D. Marion 1 1 I.NRA, Laboratoire de Biochimie et Technologie des Prote ´ ines, BP71627, Nantes, 2 INRA, Laboratoire de Recherches de Technologie Laitie ` re, Rennes, and 3 ENITIAA, Laboratoire de Microbiologie, rue de la Ge ´ raudie ` re, Nantes, France 2274/99: received 5 August 1999 and accepted 17 September 1999 A. METIVIER, P. BOYAVAL, F. DUFFES, CX. DOUSSET, J.-P. COMPOINT AND D. MARION. 2000. A new procedure combining Triton X-114 phase partitioning and cation exchange chromatography was developed to purify a bacteriocin from a complex culture medium. This pediocin-like bacteriocin, secreted by Carnobacterium divergens and named divercin V41, was entirely recovered in the lower detergent-rich phase whereas all other substances (compounds from culture medium, bacterial metabolites) remained in the upper detergent- poor phase. Subsequent cation-exchange chromatography of the TX-114-rich phase allowed recovery of the pure active bacteriocin and also detergent removing. This new purification method is versatile, fast (only two steps) and can be carried out on whole broth. INTRODUCTION Increasing consumer interest for food preserved against pathogenic bacteria has led to a considerable increase in the number of scientific papers dealing with bacteriocins, natural antimicrobial peptides and proteins produced by bacteria. Most of these papers have focused on the in vitro inhibitory activities, molecular and genetic characterization of new pep- tides active against the pathogenic Listeria strains. Few reports are related to the development of efficient methods compatible with the large-scale production of these active molecules, an essential key for their use to preserve foodstuffs against microbial spoilage. All bacteriocins have been purified by standard methods with three, four or more steps including ammonium sulphate precipitation, cation exchange or hydrophobic interaction chromatography and C18 reversed-phase HPLC. At the end of purification, the bacteriocin is pure to nearly 100% but peptide recovery is generally low (about 100 mgl -1 of culture supernatant). With such small quantities of purified peptide, the characterization of peptide is limited and, in any case, such purification procedures cannot fulfil the quantities required to validate the bacteriocin efficiency in food products. Generally bacteriocins, as other antimicrobial peptides Correspondence to: Didier Marion, INRA, Laboratoire de Biochimie et Technologie des Prote ´ines, BP71627, Nantes cedex 03, France (marion@nantes.inra.fr). © 2000 The Society for Applied Microbiology induce drastic changes in the permeability of the cytoplasmic membrane of sensitive cells creating the depletion of proton motive force and the lost of small molecules such as minerals, ATP, sugars and amino acids (Van Belkum et al. 1991; Bruno and Montville 1993; Chikindas et al. 1993; Maftah et al. 1993; Chen and Montville 1995). It is assumed that such effects are due to the formation of pores in the membrane of sensitive strains and, in a sense, bacteriocins can anchor in lipid bilayers as transmembrane proteins (Bhugaloo-Vial et al. 1996). Transmembrane proteins are generally extracted from their lipid environment by non-ionic or zwitterionic detergent. Bordier (1981) has shown for the first time that trans- membrane proteins can be specifically and quantitatively recovered by Triton X-114 (TX-114) phase partitioning. Briefly, heating of aqueous solution of TX-114 above 22 °C, a critical temperature called cloud point, leads to the for- mation of large aggregates which deposit rapidly after low- speed centrifugation. An upper detergent-poor phase and a lower detergent-rich phase are formed. The latter phase contains membrane lipids and transmembrane proteins. With regard to its versatility and its performance, this technique is now used commonly in membranology (Pryde 1986; Sanchez- Ferrer et al. 1994). Interestingly, this phase partitioning has also been shown to be useful in isolating non-membrane plant proteins which are capable of interacting spontaneously with membrane lipids (Blochet et al. 1993; Dubreil et al. 1997). In this work we have applied this phase-partitioning tech-