Characterization of bacteriocins produced by lactic acid bacteria isolated from spoiled black olives

Bacteriocin-producing strains of Lactobacillus plantarum ST23LD and ST341LD, Enterococcus faecium ST311LD and Leuconostoc mesenteroides subsp. mesenteroides ST33LD were isolated from the brine of spoiled black olives. The bacteriocins produced by all four strains inhibited the growth of Gram-positive bacteria (E. faecalis, L. casei and Streptococcus pneumoniae), but also Escherichia coli and Pseudomonas aeruginosa. Strain ST23LD produced two bacteriocins (ST23LDa and ST23LDb of approximately 3.0 and 14.0 kDa, respectively), with a combined maximum level of activity of 25,600 AU/ml after 18 h of growth. The same level of activity was recorded for bacteriocin ST341LD (approximately 3.0 kDa), but after 16 h. Bacteriocins ST311LD (ca. 2.3 kDa) and ST33LD (ca. 2.7 kDa) were produced at much lower levels (6400 AU/ml), and only after 20 h of growth. Bacteriocin activity was destroyed after treatment with proteolytic enzymes and Triton X, but not when treated with alpha-amylase, SDS, Tween 20, Tween 80, urea and EDTA, or when heated for 20 min at 121 degrees C. Addition of bacteriocins ST23LD, ST341LD and ST311LD to cells of Lactobacillus casei LHS in logarithmic phase resulted in growth inhibition for one hour, followed by a slight increase in optical density over the next seven hours. Bacteriocin ST33LD also inhibited the growth of strain LHS, but to a lesser extent. Bacteriocins ST23LD, ST341LD and ST33LD remained at the same level of activity for 6 h at pH<4.0. However, the activity of bacteriocin ST311LD decreased by 50% within 2 h at pH 4.4. The possibility of the bacteriocin adsorbing to the producer cell and proteolytic degradation is unlikely.