Purification, characterization and N-terminal amino acid sequencing of Sakacin 1, a bacteriocin produced by Lactobacillus sakei 1

Biopreservation using lactic acid bacteria (LAB) and/or their metabolites represents an alternative for improving food safety. LAB can synthesize and excrete antimicrobial peptides denominated bacteriocins, but only nisin has been largely commercially used as a food preservative. There are limitations in using this bacteriocin, in part due to its inactivation in meat products. Current research on LAB bacteriocins are conducted aiming to broad their application as natural food preservatives. Lactobacillus sakei 1, is a LAB previously isolated in our laboratory from pork sausage and it is capable to inhibit Listeria monocytogenes, by bacteriocin production. In this work, L. sakei 1 was grown in MRS broth and its bacteriocin was extracted by adsorption on its own cells and purified by cation-exchange, followed by hydrophobic interaction and high performance reverse-phase chromatography. An estimate of the molecular weight for the partially purified peptide (3,420 Da) was carried out using SDS-PAGE stained by silver stain and with a bioindicator (L. monocytogenes). The purified bacteriocin was also analyzed by mass espectrometry (MALDI-TOF-MS) and the molecular weight was determined as 4,438.83 Da. Approximately 31 N-terminal amino acid residues were sequenced by Edman degradation and the motif YGNGV (commonly found in Class IIa bacteriocins) was found in the N-terminal sequence. Several antimicrobial peptides produced by lactic acid bacteria have been purified and they present potential for food applications. However, the only peptide largely applied commercially is the bacteriocin nisin. To widen the use of bacteriocins in food biopreservation, it is important to study bacteriocin-producing strains that can be well adapted to foods where they intend to be used and to elucidate the structure of the inhibitory molecule, to allow production in large scale if the purified peptide is to be used. In the present work, the bacteriocin produced by the Brazilian meat isolate Lactobacillus sakei 1 has been characterized.