Characterization and production of amylovorin L471, a bacteriocin purifiedfrom Lactobacillus amylovorus DCE 471 by a novel three-step method

The strongly hydrophobic bacteriocin amylovorin L471 from Lactobacillus amy lovorus DCE 471 was isolated and purified to homogeneity from complex cultu re broth by a novel, rapid and simple three-step protocol including (i) amm onium sulphate precipitation, (ii) chloroform/methanol extraction/precipita tion and (iii) reversed-phase HPLC, the only chromatographic step involved, The molecular mass of the peptide was determined to be 4876.9 Da by electr ospray mass spectrometric analysis, N-terminal amino acid sequencing identi fied 35 amino acid residues as being identical to the N-terminal sequence o f lactobin A, a bacteriocin from another L. amylovorus strain. These non-id entical strains produce bacteriocins that display small differences in mole cular mass and inhibitory spectrum. The amino acid sequence of amylovorin L 471 shared significant homology with lactacin X, one of the two bactericida l peptides produced by Lactobacillus johnsonii VPI11088, A purified amylovo rin L471 preparation permitted confirmation of the inhibitory spectrum prev iously established with a crude extract. It displayed a bactericidal mode o f action on lactobacilli after an extremely rapid adsorption to the target cells. Two Listeria spp. were only weakly sensitive, Amylovorin L471 appear s to be produced constitutively. Ethanol not only stimulated specific bacte riocin production but also prevented adsorption of the bacteriocin molecule s to the producer cells upon prolonged fermentation. The latter result supp orts the hypothesis that the apparent inactivation of bacteriocin observed during the stationary phase of batch fermentations is due to adsorption.