Mechanistic information about the bacteriocin nisin was obtained by examining the efflux of 5(6)-carboxyfluorescein from Listeria monocytogenes-derived liposomes. The initial leakage rate (percentage of efflux per minute) of the entrapped dye was dependent on both nisin and lipid concentrations. At all nisin concentrations tested, 5(6)-carboxyfluorescein efflux plateaued before all of the 5(6)-carboxyfluorescein was released (suggesting that pore formation was transient), but efflux resumed when more nisin was added. Isotherms for the binding of nisin to liposomes constructed on the basis of the Langmuir isotherm gave an apparent binding constant of 6.2 ؋ 10 5 M ؊1 at pH 6.0. The critical number of nisin molecules required to induce efflux from liposomes at pH 6.0 was Ϸ7,000 molecules per liposome. The pH affected the 5(6)-carboxyfluorescein leakage rates, with higher pH values resulting in higher leakage rates. The increased leakage rate observed at higher pH values was not due to an increase in the binding affinity of the nisin molecules towards the liposomal membrane. Rather, the critical number of nisin molecules required to induce activity was decreased (Ϸ1,000 nisin molecules per liposome at pH 7.0). These data are consistent with a poration mechanism in which the ionization state of histidine residues in nisin plays an important role in membrane permeabilization.
In Listeria monocytogenes, nisin induced ATP efflux, reduced the intracellular ATP concentration within 1 min, and dissipated the proton motive force within 2 min. Efilux accounted for only 20% of the ATP depletion, suggesting that ATP hydrolysis also occurred. ATP efflux depended on nisin concentration and followed saturation kinetics. These results suggest that nisin breaches the membrane permeability barrier in a manner more consistent with pore formation than with a nonspecific detergent-like membrane destabilization.
The ability of LactobaciUus bavaricus, a meat isolate, to inhibit the growth of three Listeria monocytogenes strains was examined in three beef systems: beef cubes, beef cubes in gravy, and beef cubes in gravy containing glucose. The beef was minimally heat treated, inoculated with L. bavaricus at 10' or 103 CFU/g and L. monocytogenes at 102 CFU/g, vacuum sealed, and stored at 4 or 10°C. The meat samples were monitored for microbial growth, pH, and bacteriocin production. The pathogen was inhibited by L. bavaricus MN. At 4°C, L. monocytogenes was inhibited or killed depending on the initial inoculum level of L. bavaricus. At 10°C, at least a 10-fold reduction of the pathogen occurred, except in the beef without gravy. This system showed a transient inhibition of the pathogen during the first week of storage followed by growth to control levels by the end of the incubation period. Bacteriocin was detected in the samples, and inhibition could not be attributed to acidification. Low refrigeration temperatures significantly (P c 0.05) enhanced L. monocytogenes inhibition. Moreover, the addition of glucose-containing gravy and the higher inoculum level of L. bavaricus were significantly (P < 0.05) more effective in reducing L. monocytogenes populations in most of the systems studied. Minimally processed, vacuum-packaged, refrigerated meat products have become increasingly popular. To fulfill consumer demands for "natural" foods, many of these products contain no preservatives (9). However, the microbial safety of these products is being questioned (33). They are susceptible to growth by psychrotrophic food-borne pathogens (37). Notably, Listeria monocytogenes, because of its ability to grow actively at refrigeration temperatures (7, 13, 42), poses a serious health threat to high-risk populations such as the unborn, newborn, or immunocompromised (28). Because of its ubiquitous distribution and its association with domestic livestock, L. monocytogenes is likely to occur in raw meats (4, 23). Furthermore, its prevalence both at the slaughterhouse (17) and in the processing environment (12) increases the potential for postprocessing contamination. The incidence and growth of L. monocytogenes in processed meat products are well documented (12, 19, 20, 23). Its prevalence ranges from 5 to 13% in ready-to-eat meat products, in which typical plate counts ranged from < 10 to 1,000 CFU/g (23). Although most cases of human listeriosis associated with the consumption of ready-to-eat meat products appear to be sporadic (32, 38), the high mortality rate (30%) (27) has led the Food and Drug Administration to impose a zero-tolerance policy for L. monocytogenes in ready-to-eat meat products (14). Novel strategies, such as biopreservation systems, have gained increasing attention as a means of "naturally" controlling the growth of pathogenic and spoilage organisms in ready-to-eat foods. Some lactic acid bacteria, such as those commonly associated with vacuum-packaged meats, produce antimicrobial proteins known as bacteriocins (24, 29)....
The ability of Lactococcus lactis 11454, Pediococcus pentosaceus 43200 and Lactobacillus bavaricus MN, originally isolatedfrom dairy, vegetable, and meat products, respectively, to inhibit growth of Listeria monocytogenes Scott A in a model beef gravy was examined. In thejrst series of experiments, where the lactic acid bacteria and L. monocytogenes were inoculated at levels of lo5 CFU/mL and lo3 CFU/mL, respectively only L. bavaricus inhibited listerial growth at 1OC. Subsequent experiments using L. bavaricus MN conjrmed that the inhibition was caused by a bacteriocin, occurred at temperatures at low as 4C, and could be initiated by 1 8 CFU/mL L. bavaricus in the presence of L. monocytogenes at levels IO-fold higher. Although the inhibitory agent was protease-sensitive and inhibition occurred in the absence of a fermentable carbohydrate, the presence of acid enhanced efJicacy of the bacteriocin.
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