Purification and characterization of the Lactobacillus acidophilus bacteriocin lactacin B

Abstract

Parameters for production and purification of a bacteriocin produced by Lactobacillus acidophilus N2 are described. Production of lactatin B was pH dependent, with maximum activity detected in broth cultures maintained at pH 6. Lactacin B was purified by ion-exchange chromatography, ultrafiltration, and successive gel filtrations in the presence of 8 M urea and then 0.1% sodium dodecyl sulfate. The molecular weight of lactacin B was ca. 6,000 to 6,500, and the purified compound showed maximum absorbance at 211 nm. The activity of purified lactacin B was bactericidal to sensitive cells and restricted to members of the family Lactobacilliaceae, L. keichmannii, L. bulgaricus, L. helveticus, and L. lactis. Characteristics identified for lactacin B indicated that it was a peptide and confirmed its identity as a bacteriocin.Bacteriocins are antagonistic proteins or peptides that show bactericidal activity against closely related species (23). Although numerous bacteriocins from other bacterial genera have been isolated and characterized (10,18,20,27), few have been studied extensively in the lactobacilli. 4,5) and Upreti and Hinsdill (24,25) identified and characterized bacteriocins produced by Lactobacillusfermentum and L. helveticus, respectively. In crude form both compounds were nondialyzable, heat stable, insensitive to catalase, sensitive to proteolytic enzymes, and inhibited only members of the family Lactobacilliaceae. Upon subsequent purification, both bacteriocins were identified as large lipopolysaccharide protein complexes of ca. 200,000 in molecular weight. Because the L. fermenti bacteriocin could not be dissociated without loss of activity, DeKlerk and Smit (5) concluded that the complex molecule was essential for activity. Chromatography of the L. helveticus bacteriocin in the presence of sodium dodecyl sulfate (SDS) showed that the active moiety was a glycoprotein with a molecular weight of 12,400 (24).We reported previously that a bacteriocin-like antagonist was produced when L. acidophilus N2 was propagated anaerobically in agar cultures (1). Crude preparations of lactacin B were stable at 100°C for 60 min, insensitive to catalase, sensitive to protease, and bactericidal to sensitive species. In contrast to recent reports of bacteriocin-type compounds that are produced by lactobacilli and antagonize Clostridium (13), Neisseria (16) compound showed that lactacin B was a peptide with 'actericidal activity against the four indicator species. MATERIALS AND METHODSCultures and media. The bacteriocin producer L. acidophilus N2, the sensitive indicator L. leichmannii 4797, and all other cultures used in this study were described previously (1). Lactobacilli were maintained as frozen stocks at -76°C (9) and propagated twice (1% inoculum) through MRS broth (Difco Laboratories, Detroit, Mich.) for 14 to 16 h at 37°C before experimental use.Liquid media used were either MRS broth or a semidefined medium. The basal semi-defined medium included (per liter of solution) Casitone (Difco), 10 g; sodium acetate, 5 g;...

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