Two subpopulations of Listeria monocytogenes occur at subinhibitory concentrations of leucocin 4010 and nisin.

In situ analyses of single Listeria monocytogenes cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (DeltapH), and the other consisted of cells that maintained DeltapH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate DeltapH for 90% of the cell population (ED90) was predicted. ED90 increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED90 decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED90. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.

Global transcription profiles and intracellular pH regulation measured in Bacillus licheniformis upon external pH upshifts.

For optimization of propagation conditions for an industrially used Bacillus licheniformis, this study examines the effect of transferring cells at the early-stationary growth phase (pH 5.3) to fresh growth medium at pH 5.0-8.0. Intracellular pH (pH(i)) was measured on a single-cell level, using fluorescence ratio imaging microscopy after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. Transcription profiles were determined using a genome DNA microarray. The optimum extracellular pH (pH(ex)) value for growth of B. licheniformis was found to be pH 7.0, resulting in the shortest lag phase, highest maximum specific growth rate and maximum biomass formation. An average pH gradient (Delta pH = pH(i) - pH(ex)) of approx. 1.0 was found in B. licheniformis 15 min after transfer to pH(ex) 5.0-8.0. Up-regulation of genes involved in sucrose uptake at pH 7.0 could be related to the optimum growth observed. Transcription profiles indicated that the organism was experiencing phosphate starvation upon transfer to pH 7.0 and pH 8.0. Mechanisms involved in pH(i) regulation appeared to include changes in fatty acid synthesis to yield a more rigid cell membrane structure at low pH(ex) values and conversion of pyruvate to acetoin instead of acetate for neutralization of low pH(ex) values.

The effect of inoculum age and solid versus liquid propagation on inoculum quality of an industrial Bacillus licheniformis strain.

Shorter lag phases were obtained in cultivations of Bacillus licheniformis using early-compared to late-stationary growth phase inocula and using liquid versus solid propagation medium. Flow cytometry and fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), confirmed that liquid early-stationary growth phase inoculum had a higher vitality and was more homogeneous than solid late-stationary growth phase inoculum. DNA-microarray analyses indicated that liquid early-stationary growth phase inoculum was in a more active state in terms of cell multiplication whereas solid late-stationary growth phase inoculum was induced to some spore formation potentially causing delayed growth initiation.

The antilisterial effect of Leuconostoc carnosum 4010 and leucocins 4010 in the presence of sodium chloride and sodium nitrite examined in a structured gelatin system.

To further enhance biopreservation of meat products, the antilisterial effect of the newly described protective culture Leuconostoc carnosum 4010 and its bacteriocins, leucocins 4010, was examined in the presence of sodium chloride and sodium nitrite in a solid matrix using a structured gelatin system. Interaction between Listeria monocytogenes 4140 and Leuc. carnosum 4010 or the leucocins 4010-resistant mutant L. monocytogenes 4140P showed that the inhibitory effect of Leuc. carnosum 4010 in the gelatin system was caused by the production and activity of leucocins 4010. The presence of sodium chloride (2.5% w/v) and sodium nitrite (60 mg/l) reduced the antilisterial effect of Leuc. carnosum 4010 in the structured gel system compared to the use of Leuc. carnosum 4010 alone. Investigations carried out at 10 degrees C showed that the lag phase of L. monocytogenes 4140 in the presence of Leuc. carnosum 4010 was reduced from 71 to 58 h by the addition of sodium chloride and to 40 h by the addition of sodium nitrite. Addition of sodium chloride increased the maximum specific growth rate of L. monocytogenes 4140 in the presence of Leuc. carnosum 4010 from 0.02 to 0.06 h(-1), whereas no change was observed by the addition of sodium nitrite. Compared to the antilisterial effect of leucocins 4010 alone, the addition of sodium chloride (2.5%, w/v) decreased the antilisterial effect at high concentrations of leucocins 4010 (5.3 and 10.6 AU/ml) as measured after 11 days of incubation at 10 degrees C. In gels with added leucocins 4010, the most pronounced reduction in growth of L. monocytogenes 4140 was observed at the highest concentration of leucocins 4010 (10.6 AU/ml) together with sodium nitrite (60 mg/l). More detailed information on the lag phase and the maximum specific growth rate of single colonies of L. monocytogenes 4140 in the presence of leucocins 4010 was obtained using microscopy and image analysis. No pronounced difference in the growth of single colonies was observed in the gel system. Real-time measurements of colony growth at 10 degrees C in the gelatin matrix showed that the growth inhibiting effect of leucocins 4010, including a longer lag phase as well as a lower maximum specific growth rate for L. monocytogenes 4010, was negated in the presence of 2.5% (w/v) sodium chloride.

Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification, and meat application experiments.

A new culture, Leuconostoc carnosum 4010, for biopreservation of vacuum-packed meats is described. The culture originated from bacteriocin-producing lactic acid bacteria (LAB) naturally present in vacuum-packed meat products. Approximately, 72,000 colonies were isolated from 48 different vacuum-packed meat products and examined for antibacterial activity. Bacteriocin-producing colonies were isolated from 46% of the packages examined. Leuc. carnosum was the predominant bacteriocin-producing strain and Leuc. carnosum 4010 was selected for further experiments because it showed strong antilisterial activity without producing any undesirable flavour components in meat products. For identification of the bacteriocins produced, partial purification was carried out by ammonium sulphate precipitation, dialysis, and cation exchange chromatography. SDS-PAGE analysis revealed two bands with inhibitory activity corresponding to molecular sizes of 4.6 and 5.3 kDa. N-terminal amino acid sequencing showed that Leuc. carnosum 4010 produced two bacteriocins highly similar or identical to leucocin A and leucocin C. Application experiments showed that the addition of 10(7) cfu/g Leuc. carnosum 4010 to a vacuum-packaged meat sausage immediately reduced the number of viable Listeria monocytogenes cells to a level below the detection limit and no increase of L. monocytogenes was observed during storage at 5 degrees C for 21 days. The results presented demonstrate that Leuc. carnosum 4010 is suitable as a new protective culture for cold-stored, cooked, sliced, and vacuum-packed meat products.

Use of fluorescence ratio imaging microscopy and flow cytometry for estimation of cell vitality for Bacillus licheniformis.

For Bacillus licheniformis SJ4628, an organism widely used in the enzyme industry, methods for determination of cell vitality at a single cell level using 5(6)-carboxyfluorescein diacetate succinimidyl ester in combination with fluorescence ratio imaging microscopy and flow cytometry were developed. Immediately after inoculation and during growth, changes in intracellular pH values determined by fluorescence ratio imaging microscopy and in green fluorescence intensities determined by flow cytometry were observed. Correlations between the capacity to multiply and intracellular pH or green fluorescence intensity were demonstrated. Populations of cells not having a pH gradient or exhibiting low fluorescence intensities had significantly longer lag phases than populations of cells with a pH gradient and high fluorescence intensities.