Resumo

The contribuition of lactic acid bacteria (LAB) to improvement of food safety and stability of fermented foods has long been known. Lactobacilli are classified among the LAB and have been studied extensively as biopreservative in fermented foods due to production of various antimicrobial compounds. In particular, Lactobacillus curvatus was shown to produce different bacteriocins, a ribosomally synthesized antimicrobial peptides. The objective of this study was to purify and characterize the bacteriocins produced by two L. curvatus strains isolated from salami in Brazil (L. curvatus MBSa2 and MBSa3). The RAPD-PCR using five primers showed that the two strains are distinct. The purification was carried out using a three chromatographic steps: cation-exchange, reversed-phase and high performance reversed-phase. Listeria ivanovii was used as indicator strain in tests of the antimicrobial activity. The molecular weight and partial amino acid sequence of the purified bacteriocins were determined by Q-TOF-MS and MS/MS, respectively. The proteinaceous nature of the purified bacteriocins was confirmed by treatment with proteinase K and trypsin (1 mg/mL), and the possible presence and importance for activity of disulfide bonds was tested using 100 mM dithiothreitol (DTT). Peptide sequence analyses showed that both MBSa2 and MBSa3 strains produce two active peptides each, identical in the two strains. One peptide of 4457.9 Da contains the partial sequence AAANWATGGNAG and a second peptide of 4360.1 Da contains partial sequence AVANLTTGGAAG, also present in sakacin P and sakacin X, respectively. Another non active peptide of MW of 2228.16 Da was also detected and the partial sequence MAGNSSNFIHKIKQIFTHR was identical to the sequence of the bacteriocin-type signal sequence domain protein. Treatment with proteinases caused complete inactivation of the peptides, but the antimicrobial activities were only moderately reduced in the presence of DTT, suggesting that the disulfide bond is important but not essential for antimicrobial activity.