Microbiol. Res. (1999) 154, 199-204 hup://www.urbanfischer.de/journals/microbiolres © Urban & Fischer Verlag Purification and amino acid sequence of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705 J. Palacios, G. Vignolo, M. E. Farias, A. P. de Ruiz Holgado, G. Oliver, F. Sesma Centro de Referencia para Lactobacilos (CERELA) - CONICET, San Miguel de Tucuman, Tucuman, Argentina. Accepted: June 17, 1999 Abstract Lactobacillus casei CRL 705, isolated from a dry fermented sausage, produces an antibacterial peptide which is active against Listeria monocytogenes. Previous studies have shown that this compound is potentially useful to control food-borne pathogens in ground meat. In view of the potential application of this antimicrobial substance in food fennentation, a detailed biochemical analysis of this peptide is required. In this work, the purification and amino acid sequence of this bacteriocin is presented. The adsorption-desorption pH-dependent property of lactocin 705 was exploited for purification. The active extract was further subjected to RP-HPLC and SDS-PAGE. The active antimicrobial band was electroeluted from an SDS- PAGE gel and its amino acid sequence determined. Lactocin 705 had an estimated molecular weight of 3357.80 and an iso- electric point of 10.03. The peptide contains a high ratio of glycine residues and does not show any modified amino acids, like lanthionine or The sequence was unique when compared to several databases. Key words: Lactobacillus casei - bacteriocin - pathogen control Introduction Besides their economic importance in the processing of fermented foods, lactic acid bacteria also playa role in preservation of food through the production of organic acids, pH decrease and excretion of several inhibitory compounds. Excluding lactic acid, bacteriocins are one of the most important class of compounds among the variety of inhibitors synthesized by lactic acid bacteria Corresponding author: F. Sesma, FaxlPhone: 54-81-311720, E-mail: fsesma@cerela.org.ar 0944-5013/99/154/02-199 $12.00/0 (LAB). They are one of the most promising natural bio- preservatives in foods, and have promoted a great inter- est in health care and pharmaceutical applications (De Vuyst and Vandamme 1994; DaescheI1993). Bacteriocins are antimicrobial molecules of pro- teinaceous nature with inhibitory activity targeted to- ward species related to the producer culture (Jack et al. 1995). However, several bacteriocins show a broader spectrum, displaying inhibitory activity against some food-borne pathogens, such as Listeria monocytogenes and Staphylococcus aureus (Klaenhammer 1993). We have been involved in the study of the properties of lactocin 705, a broad-spectrum bacteriocin produced by L. casei CRL 705, isolated from sausage (Vignolo etal. 1993). Recent work showed that this antimicrobial peptide is effective in inhibiting growth of L. monocyto genes in ground beef (Vignolo et al. 1996). Because this peptide may serve as food preservative, it is necessary to know in more details its structure, mode of action, and the biochemical and genetic aspects of its production. In this study we report the purification and sequencing of lactocin 705. Materials and methods Bacterial strains and media. The bacterial strain used as indicator microorganism for bacteriocin activity was L. plantarum CRL 691 (CRL = CERELA stock collec- tion), which was propagated at 30°C in MRS medium (de Man etat. 1960). The bacteriocin-producing strain, L. casei CRL 705 was grown in a modified MRS broth, without proteose peptone at 30°C. Overlay agar for testing antimicrobial activity was MRS broth with 0.7% agar, (hereafter referred to as soft MRS agar). Microbiol. Res. 154 (1999) 2 199