Antimicrobial activity of 150 lactic acid bacteria from vacuum packaged processed meats was evaluated against 26 potential food spoilage microorganisms. Four bacteriocin producers were isolated and characterized. Leuconostoc carnosum (LA54a, TA26b and TAlla), and Leuconostoc mesenteroides TA33a each produced diffusible, proteinaceous bacteriocins with a narrow inhibitory spectrum. Crude bacteriocins were heat stable (100�C for 30 minutes) with activity unaffected from pH 2 to 7, bacteriolytic, and sensitive to proteolytic enzymes but not non-proteolytic enzymes or chloroform. Bacteriocin production was plasmid mediated and growth associated. Leuconostoc mesenteroides TA33a produced three bacteriocins (leucocins A-, B-, and C-TA33a) with different inhibitory activity spectra. Genetic determinants of leucocin A-TA33a were located on a 55.3 kb plasmid. DNA sequence analysis revealed ORFs 1 and 2, which encode proteins of 61 (bacteriocin structural gene) and 113 amino acids (immunity gene), respectively. ORF 3 encodes a protein of 149 amino acids, with homology to the thioredoxin superfamily of proteins. The partial sequences of ORFs 4 and 5 show sequence homology to proteins of the HlyB family of ATP-binding cassette transporters, and accessory proteins of other bacteriocins, respectively. Genetic organisation of these genes resembled other Leuconostoc bacteriocin operons. Furthermore, the deduced amino acid sequence of leucocin A-TA33a was identical to that of leucocin A-UAL 187 (KYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW). All three bacteriocins were purified to homogeneity by adsorption/desorption from producer cells and reversed-phase high-performance liquid chromatography. The amino acid sequence of leucocin B-TA33a was determined up to the 31st residue as KGKGFWSWASKATSWLTGPQQPGSPLLKKHR by automated Edman degradation (calculated molecular mass 3466 Da). The partial amino acid sequence of leucocin C-TA33a was determined up to the 36th residue (measured molecular mass 4598 Da) as KNYGNGVHCTKKGCSVDWGYAATNIANNSVMNGLTG (calculated molecular mass 3744 Da). Purified bacteriocins were heat stable (92�C for 30 minutes) with activity unaffected up to pH 7. Circular dichroism spectra of leucocins in aqueous solution and micellar sodium dodecyl sulphate indicated a structural transition when in a membrane-mimicking environment. Theoretical predictions from circular dichroism data suggested that leucocins A-, B- and C-TA33a adopted a β structure in membrane-mimicking environments.
|Degree Type||Doctoral degree|