PLANTARICINS S AND T, 2 NEW BACTERIOCINS PRODUCED BY LACTOBACILLUS-PLANTARUM LPCO10 ISOLATED FROM A GREEN OLIVE FERMENTATION

Twenty-six strains of Lactobacillus plantarum isolated from green oliv e fermentations were tested for cross-antagonistic activities in an ag ar drop diffusion test. Cell-free supernatants from four of these stra ins were shown to inhibit the growth of at least one of the L. plantar um indicator strains. L. plantarum LPCO10 provided the broadest spectr um of activity and was selected for further studies. The inhibitory co mpound from this strain was active against some gram-positive bacteria , including clostridia and propionibacteria as well as natural competi tors of L. plantarum in olive fermentation brines. In contrast, no act ivity against gram-negative bacteria was detected. Inhibition due to t he effect of organic acids, hydrogen peroxide, or bacteriophages was e xcluded. Since the inhibitory activity of the active supernatant was l ost after treatment with various proteolytic enzymes, this substance c ould be classified as a bacteriocin, designated plantaricin S. Plantar icin S was also sensitive to glycolytic and lipolytic enzymes, suggest ing that it was a glycolipoprotein. It exhibited a bactericidal and no nbacteriolytic mode of action against indicator cells. This bacterioci n was heat stable (60 min at 100-degrees-C), active in a pH range of 3 .0 to 7.0, and also stable in crude culture supernatants during storag e. Ultrafiltration studies indicated that plantaricin S occurred as mu ltimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels , plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum producti on of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated pl antaricin T) was produced in late-stationary-phase cultures of L. plan tarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plan taricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel el ectrophoresis, plantaricin T appeared different from plantaricin S. Cu ring experiments with L. plantarum LPCO10 resulted in the appearance o f variants that no longer produced either of the two bacteriocins but that were still immune to both of them.