Short communication: impact of pH and temperature on the acidifying activity of Carnobacterium maltaromaticum.

The acidifying activity of Carnobacterium maltaromaticum LMA28, a strain isolated from French soft cheese, was studied in trypticase soy broth with yeast extract (TSB-YE) medium and in milk. In TSB-YE supplemented with lactose, glucose, or galactose, lactose and glucose were metabolized with a maximum growth rate of 0.32 h(-1) and galactose was not metabolized. During hydrolysis of lactose, the galactose moiety was not excreted. The major product was l(+) lactic acid, with no significant difference in the lactic acid yield. Glucose was not completely metabolized because cell growth stopped when pH values reached an average of 5.0. In sterilized UHT milk, the addition of 1 g/L of YE enhanced its coagulation. Compared with commercial starter lactic acid bacteria such as Lactococcus lactis DSMZ 20481 or Streptococcus thermophilus INRA 302, Carnobacterium maltaromaticum LMA 28 was shown to be a slow acidifying strain. However, in spite of this weak acidifying ability, C. maltaromaticum LMA 28 can sustain low pH values in coculture with Lc. lactis DSMZ 20481 or S. thermophilus INRA 302. The individual and interactive effects of initial pH values (5.2 to 8.0) and incubation temperatures (23 to 37 degrees C) on acidifying activity were studied by response surface methodology. The 3 strains displayed different behaviors depending on pH and temperature. The psychrotrophic lactic acid strain C. maltaromaticum LMA 28 was able to grow at alkaline pH values and during storage conditions. It could be used as a potential ripening flora in soft cheese.

Short communication: Carnobacterium maltaromaticum: the only Carnobacterium species in French ripened soft cheeses as revealed by polymerase chain reaction detection.

The PCR technique using Cb1-Cb2R and species-specific primers was applied to various French soft flowered or washed rind cheeses to identify Carnobacterium species. Thirty cheeses made from cow's, ewe's, or goat's milk (raw or pasteurized), of which 20 were Appellation d'Origine Contrôlée, were analyzed in the autumn and spring. The results revealed that, irrespective of the season, the Carnobacterium genus was initially detected in 5 cheeses and was detected in 5 others after an enrichment period. Polymerase chain reaction results using species-specific primers of Carnobacterium showed that these 10 cheeses contained only the species Carnobacterium maltaromaticum. Six different patterns of fermentation were found, and 3 of the 10 cheeses contained C. maltaromaticum isolates with anti-Listeria activity.

Optimization of the components concentrations of the lactoperoxidase system by RSM.

AIMS: The aim of this work was to use response surface methodology (RSM) approach, a statistical mathematical tool, to model effects and interactions of glucose oxidase (GOD), glucose, lactoperoxidase (LPO) and pH-values on the thiocyanate (SCN-) peroxidation, to determine the best concentrations of lactoperoxidase system (LP-s) components in order to obtain maximal SCN- peroxidation and so to enhance the LP-s antibacterial effects. METHODS AND RESULTS: Experimental design using RSM was used for modelling effects and interactions of GOD (28.5-142.5 IU l(-1)), glucose (0.55-11.11 mmol l(-1)), LPO (0-6284 IU l(-1)) concentrations, and pH-values (6.0-7.4) on thiocyanate peroxidation. A fixed SCN- concentration of 0.5 mmol l(-1) was used. Experiments were carried out at 4 or at 25 degrees C in 0.1 mol l(-1) phosphate buffer. Optimized concentrations for both temperatures (4 and 25 degrees C) were quite similar and were 85.5 IU l(-1) for GOD, 8 mmol l(-1) for glucose and 3927.5 IU l(-1) for LPO at an initial pH-value of 6.5. SCN- peroxidation was more efficient at 25 than at 4 degrees C. At 4 degrees C, no interaction between factors occurred. At 25 degrees C, thiocyanate peroxidation was affected by GOD/glucose, GOD/pH and LPO/pH. Thiocyanate peroxidation was mainly increased by glucose and LPO factors. The optimized system had a bacteriostatic effect on Listeria monocytogenes CIP 82110(T) and a strong bactericidal effect on Pseudomonas fluorescens CIP 6913(T). CONCLUSIONS: Appropriate combinations of LPO, GOD, glucose concentrations and pH-values allowed maximal thiocyanate peroxidation and enhanced the antibacterial effect of the LP-s. SIGNIFICANCE AND IMPACT OF THE STUDY: This optimization by RSM approach allowed a better understanding of the LP-s functioning, the description of the component impacts on the SCN- peroxidation, and the observation of different interactions between the factors. The antimicrobial efficiency of LP-s can be enhanced by better concentration ratios of the LP-s components.

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.

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 degrees 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 degrees 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.

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.

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.

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 i.u. 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 degrees 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.

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 degrees 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 x 10(8) cfu ml-1) after 7 d. Monolaurin (250 micrograms ml-1) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 x 10(5) 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 micrograms ml-1), pH values (between 5.0 and 7.0) and spore loads (10(3), 10(4), 10(5) 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 < or = 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 micrograms 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 degrees C.

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

Brevicin 27, a bacteriocin produced by Lacto bacillus 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.

Mesenterocin 52, a bacteriocin produced by Leuconostoc mesenteroides ssp. mesenteroides FR 52.

One hundred and sixty-five isolates of Leuconostoc spp. were tested for bacteriocin production. Only one strain, Leuc. mesenteroides ssp. mesenteroides FR 52, isolated from a raw milk, produced a bacteriocin which was named Mesenterocin 52. This bacteriocin inhibited other Leuconostoc strains and several strains of Enterococcus and Listeria spp. No activity was found against lactococci and lactobacilli. The antibacterial spectrum differed from that of previously described Leuconostoc bacteriocins. Mesenterocin 52 was secreted into the medium during the growth phase. It was inactivated with protease treatments. At pH 7.0 it had a relative stability after heating at 100 degrees C (15 min), but it had a greater stability at pH 4.5 than at pH 7.0 after 6 h at 80 degrees C. The apparent molecular mass was estimated to be less than 10 kDa by ultrafiltration. Mesenterocin 52 showed a bactericidal effect on Leuconostoc paramesenteroides DSM 20288.

Isolation and some properties of an arylamidase from Flavobacterium IIb.

A constitutive L-leucylarylamidase (EC 3.4.11) hydrolase able to cleave L-aminoacyl-beta naphthylamide and L-aminoacyl-4 nitroanilide substrates, was isolated from sonicated cells of Flavobacterium IIb and partially purified with a 0.9% yield and a 159-fold recovery. Its molecular weight was estimated to be about 170,000 +/- 10%. This arylamidase exhibited optimum activity at pH 7.0 and 28 degrees C for the hydrolysis of L-leucine-4NA and is inhibited strongly by metal chelating agents, and to a weaker extent, by some sulfhydryl and reducing agents. Heavy metal ions: Cd2+, Zn2+, Cu2+, Hg2+ and Co2+, markedly inhibit it, and Zn2+ is a competitive inhibitor. This metalloenzyme, free of carboxypeptidase, proteinase and L-leucine aminopeptidase (L-leucylglycine substrate) activities, hydrolyzes aminoacyl-beta NA, aminoacyl-4NA and some dipeptides with unsubstituted amino groups of the L-configuration. The lowest Km values are associated with substrates having neutral or basic residues, with large side chains.