Thomas J. Montville

Bacteriocins: safe, natural antimicrobials for food preservation

Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of other bacteria. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Though these bacteriocins are produced by LAB found in numerous fermented and non-fermented foods, nisin is currently the only bacteriocin widely used as a food preservative. Many bacteriocins have been characterized biochemically and genetically, and though there is a basic understanding of their structure-function, biosynthesis, and mode of action, many aspects of these compounds are still unknown. This article gives an overview of bacteriocin applications, and differentiates bacteriocins from antibiotics. A comparison of the synthesis. mode of action, resistance and safety of the two types of molecules is covered. Toxicity data exist for only a few bacteriocins, but research and their long-time intentional use strongly suggest that bacteriocins can be safely used.

Temperature- and Surfactant-Induced Membrane Modifications That Alter Listeria monocytogenes Nisin Sensitivity by Different Mechanisms

ABSTRACT Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation. Alterations in membrane composition might influence any of these steps. We hypothesized that cold temperatures (10°C) and surfactant (0.1% Tween 20) in the growth medium would influence Listeria monocytogenes membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin. Compared to the membranes of cells grown at 30°C, those of L. monocytogenes grown at 10°C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity. When 0.1% Tween 20 was included in the medium and the cells were cultured at 30°C, there were complex changes in lipid composition. They did not influence membrane fluidity but nonetheless increased nisin sensitivity. Further investigation found that these cells had an increased ability to bind radioactively labeled nisin. This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.

Mechanistic action of pediocin and nisin: recent progress and unresolved questions

Abstract Nisin and pediocin PA-1 are examples of bacteriocins from lactic acid bacteria (LAB) that have found practical applications as food preservatives. Like other natural antimicrobial peptides, LAB bacteriocins act primarily at the cytoplasmic membranes of susceptible microorganisms. Studies with in vivo as well as in␣vitro membrane systems are directed toward understanding how bacteriocins interact with membranes so as to provide a mechanistic basis for their rational applications. The dissipation of proton motive force was identified early on as the common mechanism for the lethal activity of LAB bacteriocin. Models for nisin/membrane interactions propose that the peptide forms poration complexes in the membrane through a multi-step process of binding, insertion, and pore formation. This review focuses on the current knowledge of: (1) the mechanistic action of nisin and pediocin-like bacteriocins, (2) the requirement for a cell factor such as a membrane protein, (3) the influence of membrane potential, pH, and lipid composition on the of specificity and efficacy of bacteriocins, and (4) the roles of specific amino acids and structural domains of the bacteriocins in their action.

Detection of bacteriocins produced by lactic acid bacteria

22 strains of lactic acid bacteria were examined for bacteriocin production and sensitivity using three screening techniques and two media. Distinct inhibition zones were obtained when 9 of 10 strains previously identified in other laboratories as bacteriocin producers were spotted onto yeast extract-supplemented trypticase soy agar without glucose and overlaid with agar seeded with an indicator strain. Of the 12 ATCC strains screened, only Lactobacillus brevis B155 produced bacteriocin. Lactobacillus MRS was not a satisfactory screening medium because the large amount of acid produced from glucose gave inhibition zones around all strains. The flip-streak deferred-antagonism method was cumbersome and of limited value. The well-diffusion method gave a large number of false-negative results compared to the spot-on-the-lawn method. Lactobacillus sake ATTC 15521 was sensitive to all of the bacteriocins and was chosen as the best indicator strain for further screening studies.

Carbon Dioxide and Nisin Act Synergistically on Listeria monocytogenes

ABSTRACT This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nisr) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nisrcells. Wild-type cells grown in 100% CO2 were two to five times longer than cells grown in air. Nisin (2.5 μg/ml) did not decrease the viability of Nisr cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO2. There was a quantifiable synergistic action between nisin and CO2 in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 μg of nisin per ml increased from 3.1 to 12.5 μg/ml over 35 days, but this increase was markedly delayed for cultures in CO2. This synergism between nisin and CO2 was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO2 and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO2 atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO2 and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO2 is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.

Bacteriocin inhibition of Clostridium botulinum spores by lactic acid bacteria

Twenty-three strains of lactic acid bacteria were tested by deferred antagonism methods for bacteriocin-like activity against types A and B spores from 11 proteolytic and nonproteolytic Clostridium botulinum strains. Pediococcus pentosaceus ATCC 43200, Pediococcus pentosaceus ATCC 43201, Lactococcus lactis subsp. lactis ATCC 11454, Lactobacillus acidophilus N2, Lactobacillus plantarum Lb75, Lactobacillus plantarum Lb592, and Lactobacillus plantarum BN exhibited bacteriocin-like inhibition of all C. botulinum strains tested. By excluding inhibition due to hydrogen peroxide, acid, and lytic phage and confirming their proteinaceous nature, the inhibitors were confirmed as bacteriocins. The minimum inhibitory cell concentrations (MICC) required to produce 1 mm radius inhibition zones were determined by direct antagonism testing. Only strains 43200, 43201, 11454, and N2 were inhibitory when cultured simultaneously with the botulinal spores. The MICCs of strains antagonistic to C. botulinum spores by simultaneous testing ranged between 1.6 × 105and 4.7 × 107CFU/ml. Based on the MICCs, P. pentosaceus 43200 was most inhibitory to C. botulinum .

Evidence that dissipation of proton motive force is a common mechanism of action for bacteriocins and other antimicrobial proteins

While bacteriocins from lactic acid bacteria (LAB) have generated tremendous interest among food microbiologists, they are not unique. The biosphere is awash with antimicrobial proteins such as colicins, defensins, cecropins, and magainins. These proteins share many characteristics. They are low molecular weight, cationic, amphiphilic, tend to aggregate and are benign to the producing organism. In cases where the mode of action has been investigated, the cell membrane appears to be the site of action. There is increasing evidence that bacteriocins from many bacterial genera also share these characteristics. After a brief introduction on the significance of LAB bacteriocins, this review provides some background on proton motive force. Current studies of mechanisms for various bacteriocins are reviewed. Evidence is then introduced that bacteriocins produced by lactic acid bacteria act by the common mechanism of depleting proton motive force. The role and importance of energized membranes in this process is examined. These observations are linked to literature which demonstrates that many other classes of antimicrobial proteins act by the same mechanism. Questions regarding the role of receptor proteins and the physical mechanism by which PMF is depleted remain unresolved.

Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136

ABSTRACT Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (ΔΨ) and the intracellular ATP pool of the indicator organism, the pH gradient (ΔpH) component of the proton motive force (Δp) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.

Improved agar diffusion assay for nisin quantification

Abstract Nisin activity is usually quantified using agar diffusion methods. The assay sensitivity (increase in zone size/increase in nisin concentration) for nisin was greatly increased by using Lactobacillus sake ATCC 15521 as the indicator organism, rather than Micrococcus luteus ATCC 10420, the previous indicator organism of choice.

Inhibition of Listeria monocytogenes in cold-smoked salmon by Carnobacterium piscicola CS526 isolated from frozen surimi

Strain CS526 was isolated from frozen surimi and identified as a bacteriocin producer that had strong inhibitory activity against Listeria monocytogenes. Strain CS526 was identified as Carnobacterium piscicola by partial 16S rDNA sequence similarity. The ability of this bacteriocinogenic strain and nonbacteriocinogenic C. piscicola JCM5348 to inhibit the growth of L. monocytogenes was examined in culture broth incubated at 12 degrees C and cold-smoked salmon stored at 4, 12, and 20 degrees C. L. monocytogenes viable counts in the culture broth rapidly declined from 10(6) colony-forming units per ml to less than 10 colony-forming units per ml within 1 day at 12 degrees C in the presence of C. piscicola CS526. At 4 and 12 degrees C, inhibition of L. monocytogenes on salmon depended on the initial inoculum level of C. piscicola CS526. However, C. piscicola CS526 was bactericidal to L. monocytogenes within 21 and 12 days at 4 and 12 degrees C in cold-smoked salmon, respectively, even when the initial inoculum levels were low. C. piscicola CS526 suppressed the maximum cell number of L. monocytogenes by two and three log cycles, even at 20 degrees C. However, C. piscicola JCM5348 did not prevent the growth of the pathogen, except at 4 degrees C. Bacteriocin was detected in the samples coinoculated with C. piscicola CS526. The study shows that C. piscicola CS526 might have potential for biopreservation of refrigerated foods against L. monocytogenes.