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

Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Department of Applied Biological Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium¹
Laboratory of Microbial Gene Technology, Department of Biotechnological Sciences, Agricultural University of Norway, Post Box 5051, N-1432 s, Norway²
Laboratory of Protein Biochemistry and Protein Engineering, Faculty of Sciences, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium³

Author for correspondence: Luc De Vuyst. Tel: +32 2 629 32 45. Fax: +32 2 629 27 20. e-mail: ldvuyst{at}vub.ac.be

The strongly hydrophobic bacteriocin amylovorin L471 from Lactobacillus amylovorus DCE 471 was isolated and purified to homogeneity from complex culture broth by a novel, rapid and simple three-step protocol including (i) ammonium sulphate precipitation, (ii) chloroform/methanol extraction/precipitation and (iii) reversed-phase HPLC, the only chromatographic step involved. The molecular mass of the peptide was determined to be 4876·9 Da by electrospray mass spectrometric analysis. N-terminal amino acid sequencing identified 35 amino acid residues as being identical to the N-terminal sequence of lactobin A, a bacteriocin from another L. amylovorus strain. These non-identical strains produce bacteriocins that display small differences in molecular mass and inhibitory spectrum. The amino acid sequence of amylovorin L471 shared significant homology with lactacin X, one of the two bactericidal peptides produced by Lactobacillus johnsonii VPI11088. A purified amylovorin L471 preparation permitted confirmation of the inhibitory spectrum previously established with a crude extract. It displayed a bactericidal mode of action on lactobacilli after an extremely rapid adsorption to the target cells. Two Listeria spp. were only weakly sensitive. Amylovorin L471 appears to be produced constitutively. Ethanol not only stimulated specific bacteriocin production but also prevented adsorption of the bacteriocin molecules to the producer cells upon prolonged fermentation. The latter result supports the hypothesis that the apparent inactivation of bacteriocin observed during the stationary phase of batch fermentations is due to adsorption.

Keywords: lactic acid bacteria, Lactobacillus amylovorus, amylovorin L471, bacteriocin purification, bacteriocin production and adsorption

Abbreviations: RP, reversed-phase; TFA, trifluoroacetic acid

The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is P81927.

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