M . M AN S OU R, D . A MR I , A . B O UT TE F RO Y, M . L IN D ER AN D J. B. M IL LI E RE . 1999. 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 log 10 reduction at 10 h). Regrowth appeared between 10 and 24 h and reached a high population level (1·25 × 10 8 cfu ml −1 ) after 7 d. Monolaurin (250 mg ml) had a bacteriostatic effect during the first 10 h but thereafter, regrowth occurred slowly with a population level after 7 d (4 × 10 5 cfu ml) lower than that of nisin. Different combined effects of nisin (between 0 and 42 IU ml ) 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 ¾ 0·001), i.e. spore count decreased as the pH value increased in relation to germination. Sublethal concentrations of nisin (30 IU ml ) and monolaurin (100 mg 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.

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Strain C19, isolated from fermented cucumbers and identified as Lactobacillus plantarum, produced a bacteriocin. This bacteriocin, named plantaricin C19, was stable at acidic pH, was relatively thermostable, and had a molecular mass of about 3.5 kDa. It inhibited some pathogenic (i.e., Listeria spp.) and spoilage Gram-positive bacteria but had weak or no action against lactic acid bacteria. Its adsorption on sensitive and resistant bacteria was pH dependent and was reduced by pretreatment of cells with lipase or lysozyme. Curing treatments with acriflavine or novobiocin yielded nonproducing mutants sensitive to plantaricin C19.Key words: lactic acid bacteria, Lactobacillus plantarum, bacteriocin, protein.

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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.Some lactic acid bacteria, defined to be generally recognized as safe, produce bacteriocins that are industrially interesting antimicrobial substances. These small peptides have inhibitory activity against related bacterial species. In 1993, Klaenhammer (14) proposed a classification of lactic acid bacterial bacteriocins based on their modes of action and their structures. In this classification, class II bacteriocins are small, heat-stable, nonlanthionine peptides consisting of 30 to 60 amino acids (Ͻ10 kDa). Subclass IIa contains peptides with the consensus sequence YGNGV near the N terminus that are active against Listeria strains.The lethal activity of bacteriocins involves three steps (peptide binding, [ii] peptide insertion and association, and [iii] pore formation) leading to intracellular compound efflux (1,7,8,19). To investigate this mechanism of action, resistant strains were generated from susceptible wild-type strains by two methods: (i) culture of the wild-type strain in the presence of a large amount of bacteriocin (4, 18) and (ii) culture in media with increasing bacteriocin concentrations (11,16,28). Some induced resistance provides cross-resistance to other bacteriocins (4, 21). Some authors have therefore concluded that resistance to a class IIa bacteriocin protects against the lethal activities of other bacteriocins of this class (6,22,27). The resistance mechanism seems to be complex. In 1998, Crandall and Montville ...

) 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.