Jean-Bernard Millière

Nisin–curvaticin 13 combinations for avoiding the regrowth of bacteriocin resistant cells of Listeria monocytogenes ATCC 15313

Nisin (25-100 IU/ml) and curvaticin 13 (160 AU/ml), a bacteriocin produced by Lactobacillus curvatus SB13, were shown to have a bactericidal effect against Listeria monocytogenes ATCC 15313 in TSB-YE broth (pH 6.5), but it was only transitory. Regrowth was not due to the loss of bacteriocin activity. Cells surviving nisin or curvaticin 13 were more resistant to the respective bacteriocin than the parental strain. Survivors to curvaticin 13 were resistant to the class IIa bacteriocins (camocin CP5, pediocin AcH) but remained sensitive to nisin. The frequencies of spontaneous nisin resistants decreased with increasing bacteriocin concentration and the presence of salts (NaCl, K2HPO4). The behaviour of nisin (1000 IU/ml) or curvaticin 13 (640 AU/ml) resistant variants (Nis1000, Curv645) was investigated in the presence of nisin (100 IU/ml) or curvaticin 13 (320 AU/ml) at 22 and 37 degrees C, and compared with that of the parental strain. The effectiveness of nisin was the same at both temperatures, whereas curvaticin 13 displayed a faster bactericidal action at 37 degrees C. Nis1000 cells were less sensitive to curvaticin 13 than the parental strain, whereas Curv640 cells were more sensitive to nisin than the parental strain. Simultaneous or sequential additions of nisin (50 IU/ml) and curvaticin 13 (160 AU/ml) were performed at 22 degrees C in broth inoculated with the parental strain. All combinations induced a greater inhibitory effect than the use of a single bacteriocin. Simultaneous addition of bacteriocins at t0 led to the absence of viable cells in the broth after 48 h.

Inhibitory combinations of nisin, sodium chloride, and pH on Listeria monocytogenes ATCC 15313 in broth by an experimental design approach.

The influence of pH (5.0-8.2), NaCl concentrations (0-6% w/v), and incubation time (0-24 h) on the inhibitory activity of nisin (0-100 I.U./ml) against Listeria monocytogenes (10(3) cfu/ml) was studied using the Doehlert experimental design and was confirmed by kinetic experiments. Predicted values were in agreement with experimental values. Experiments were carried out at 22 degrees C in reconstituted TSB-YE1 broth with or without NaCl. Nisin had an immediate pH-dependent bactericidal effect, which increased with decreasing pH values. In modified TSB-YE1 broth without NaCl, the bactericidal efficacy of nisin (50 I.U./ml) was maximum at pH 6.6, with no L. monocytogenes survivors until 120 h at 22 degrees C. Nisin (50 I.U./ml) action decreased in the presence of NaCl, with a minimal inhibitory effect between 2 and 4%. This partially protective effect was cancelled at higher levels of nisin.

Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations.

The effects of nisin and monolaurin, alone and in combination, were investigated on Bacillus licheniformis spores in milk at 37 °C. In the absence of inhibitors, germinated spores developed into growing vegetative cells and started sporulation at the end of the exponential phase. In the presence of nisin (25 IU ml−1), spore outgrowth was inhibited (4 log10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1·25 × 108 cfu ml−1) after 7 d. Monolaurin (250 μg ml−1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 × 105 cfu ml−1) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml−1), monolaurin (ranging from 0 to 300 μg ml−1), pH values (between 5·0 and 7·0) and spore loads (103, 104, 105 spores ml−1) were investigated using a Doehlert matrix in order to study the main effects of these factors and the different interactions. Results were analysed using the Response Surface Methodology (RSM) and indicated that nisin and monolaurin had no action on spores before germination; only pH values had a significant effect (P ≤ 0·001), i.e. spore count decreased as the pH value increased in relation to germination. Sublethal concentrations of nisin (30 IU ml−1) and monolaurin (100 μg ml−1) in combination acted synergistically on outgrown spores and vegetative cells, showing total inhibition at pH 6·0, without regrowth, within 7 d at 37 °C.

Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC 15313 in skim milk

Individual or combined effects of nisin (100 or 200 IU/ml) and the lactoperoxidase system (LPS) were analysed against 1 x 10(4) cfu/ml Listeria monocytogenes ATCC 15313 cells in skim milk, at 25 degrees C for 15 days. Nisin induced an immediate bactericidal effect and LPS a 48 h bacteriostatic phase which in both cases was followed by re-growth of L. monocytogenes. LPS and nisin added together at t0 showed a synergistic and lasting bactericidal effect which after 8 days and until 15 days resulted in no detectable cells in 1 ml of milk. When LPS was added to cells already in contact with 100 or 200 IU/ml nisin for a period of 4 h, the inhibitory activity was enhanced with no L. monocytogenes detectable after 72 or 48 h, respectively, and until 15 days. When LPS was added after 12 h, the nisin bactericidal phase was followed by re-growth. When nisin, 100 or 200 UI/ml, was added to cells already in contact with LPS over 24 h, L. monocytogenes was not detectable after 196 and 244 h, respectively, without any re-growth. For nisin addition after 72 h, cell counts were 8 log10 cycles lower than in the control milk after 196 h, but population levels were similar to the control within 15 days. The best combination to inhibit L. monocytogenes ATCC 15313 was nisin present at t0 followed by the LPS addition 4 h later, when the maximum inhibitory effect of nisin was reached.

Characterization of Anti-Listeria monocytogenes Bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis Strains Isolated from Raïb, a Moroccan Traditional Fermented Milk

Seventy-four samples of raïb, a Moroccan traditional fermented milk, were screened for their anti-Listeria monocytogenes activity. Nine lactic acid bacteria with antilisterial activity were isolated and identified as Lactococcus lactis[4], Enterococcus faecium[4], and E. faecalis[1]. Antibacterial spectra, determined against 45 target strains, led to the selection of four antibacterial-producing strains, which were further characterized. Their anti-microbial agents, inactivated by one or more proteases, were designed as bacteriocins. Lactococcin R9/2 and R10/1 showed the broadest range of inhibitory action. Anti-bacterial spectra and physico-chemical properties suggest that these bacteriocins were similar to nisin. Enterocin R69 had a specificity of action against Listeria spp., whereas Enterocin R18 had a broad spectrum of activity. Lc. lactis R9/2 and E. faecalis R18 were able to coagulate sterilised UHT milk at 30°C in 24 h and induced a 2 log reduction in L. monocytogenes ATCC 15313 population.

Response Surface Methodology, an approach to predict the effects of a lactoperoxidase system, Nisin, alone or in combination, on Listeria monocytogenes in skim milk

Experimental designs using Response Surface Methodology (RSM) were used to determine effects and interactions of Nisin (0–200 IU ml−1), pH values (5·4–6·6), incubation time (0–36 h or 0–144 h) and the lactoperoxidase‐thiocyanate‐hydrogen peroxide system (LPS) on Listeria monocytogenes CIP 82110 in skim milk, at 25 °C. The LPS varied from level 0–2; LPS at level 1 consisted of lactoperoxidase (35 mg l−1), thiocyanate (25 mg l−1) and H2O2, which was supplied exogenously by glucose‐oxidase (1 mg l−1) and glucose (0·2 g l−1); LPS activity was dependent on LPS level and incubation time. In the presence of LPS at level 1, a bacteriostatic phase was followed by growth, whereas at a higher level, a bactericidic phase was observed. Nisin response was time‐ and pH‐dependent. Nisin was bactericidic at acidic pH values and for a short incubation time (12 h) only; then, a re‐growth phase was observed. Nisin and LPS in combination gave an original response which lacked the transitory bactericidal effect of Nisin and had a continuously bactericidal affect, leading to 10 cfu ml−1 of L. monocytogenes at 144 h; the response was greatly affected by incubation time. Predicted values were in good agreement with experimental values. Response Surface Methodology is a useful experimental approach for rapid testing of the effects of inhibitors.

Variations in the membrane fatty acid composition of resistant or susceptible Leuconostoc or Weissella strains in the presence or absence of Mesenterocin 52A and Mesenterocin 52B produced by Leuconostoc mesenteroides subsp. mesenteroides FR52.

ABSTRACT Mesenterocins 52A (Mes52A) and 52B (Mes52B) are antimicrobial peptides produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52. Mes52A is a class IIa bacteriocin of lactic acid bacteria with a broad spectrum of activity. Mes52B is an atypical class II bacteriocin with a narrow spectrum of activity. Four Leuconostoc and Weissella wild-type strains were selected for their susceptibility or insensitivity to these mesenterocins. Four strains resistant to Mes52A or Mes52B were generated from the three susceptible wild-type strains by increasing bacteriocin concentrations in culture media. These resistant strains were at least 30 times more resistant than the wild-type strains. No cross-resistance to Mes52A and Mes52B was observed in these strains. No significant differences in membrane fatty acid composition were observed among the three susceptible wild-type strains and the four resistant strains cultured in MRS broth. Thus, the mesenterocin resistance is unlikely to be due to changes in membrane fatty acid composition. When cultured with Mes52A or Mes52B, the membranes of insensitive and resistant strains contained more saturated fatty acids (1 to 10% more) and less unsaturated fatty acids (3 to 6% less), resulting in a more rigid membrane. Thus, the presence of mesenterocin in the culture media of insensitive or resistant strains induced a significant increase in saturated fatty acid contents and a decrease in unsaturated fatty acid contents. Weissella paramesenteroides DSM 20288BR, resistant to Mes52B, responded atypically, probably due to the production of an inhibitor.

Predictive models of the combined effects of curvaticin 13, NaCl and pH on the behaviour of Listeria monocytogenes ATCC 15313 in broth

Thirty‐three strains of Listeria monocytogenes belonging to different serotypes were tested for their sensitivity to curvaticin 13, an antilisterial bacteriocin produced by Lactobacillus curvatus SB13, using the well diffusion method in Institut Pasteur agar plates at 37 °C. No relationship between serotype and sensitivity was observed. The sensitivity of this species was strain‐dependent and a large variation in tolerance to curvaticin 13 was observed. The combined effects of curvaticin 13 (0–160 AU ml−1), NaCl (0–6% w/v), pH values (5·0–8·2) and incubation time (0–24 h) were investigated on L. monocytogenes ATCC 15313 in trypcase soy–yeast extract broth at 22 °C. For this study, two Doehlert matrices were used in order to investigate the main effects of these factors and their different interactions. The results were analysed using the Response Surface Methodology. Curvaticin 13 had a major inhibitory effect and the response was NaCl concentration‐, time‐ and pH‐dependent. This inhibitory activity was the same at pH values between 6·6 and 8·2. Curvaticin 13 was bactericidic at acidic pH values, but the surviving cells resumed growth. For a short incubation time (12 h), the effectiveness of curvaticin 13 was maximal in the absence of NaCl. For longer incubation times (12–48 h), with high NaCl (6%) and curvaticin 13 concentrations (160 AU ml−1), the inhibition of L. monocytogenes was greater than that observed with NaCl or curvaticin 13 alone.

Synergistic mode of action of mesenterocins 52A and 52B produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52.

Few studies have been published on the effects of two bacteriocins combinations and particularly on combinations of two bacteriocins with different structures produced by the same strain. In this work, the actions of mesenterocin 52A (class IIa) and mesenterocin 52B (class II), produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52, were studied on strains susceptible to only one bacteriocin or to both. In broth, combination of mesenterocins enhanced the adaptation time of the strain susceptible to the both mesenterocins (48 h vs 17 h with only one bacteriocin). In agar medium, mesenterocins displayed, as expected, a synergistic effect on this strain (FICindex < 1), but also on the two strains susceptible to only one mesenterocin. This original result was probably due to membrane composition modifications induced by the mesenterocin that enhanced bacteriocin action. Thus, this hurdle technique seems to be interesting in food preservation in terms of minimizing bacteriocin concentrations.

Purification and partial amino acid sequence of brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27.

Abstract. Brevicin 27, a bacteriocin produced by Lactobacillus brevis SB27, is inhibitory mainly against closely related Lactobacillus brevis and Lactobacillus büchneri strains. It was purified from the culture supernatant by a four-step purification procedure including ammonium sulfate precipitation, cation exchange, hydrophobic interaction, and reverse-phase, high performance liquid chromatographies. The purified bacteriocin was subjected to mass spectrometry, amino acid composition analysis, and sequencing by Edman degradation. It was shown to be an about 5200-Da basic protein containing a high proportion of lysine and of hydrophobic amino acids. The partial N-terminal amino acid sequence (25 residues) was unique when compared with the Protein Data Bank (PDB), Swiss Prot, and Protein Information Resource (PIR) data banks and to the translated Gen Bank.