Les substances antagonistes (i.e Bactériocines) produites par une souche de Lactobacillus rhamnosus et une souche de Lactobacillus plantarum: caractérisation biochimique et effets in vitro attendus chez quelques germes responsables de toxi-infections

Abstract

The aim of this work was the isolation and identification of two new strains of lactic acid from infant stool, healthy, exclusively breastfed and receiving no antibiotics. The characterization of these two strains by biochemical tests and 16S rDNA gene sequences revealed their belonging to the genus Lactobacillus species plantarum (LbM2a) and rhamnosus (LBRE-LSAS). The antagonistic activity of these two strains against a wide range of Gram-positive and Gram-negative bacteria was measured using three different methods: Agar spot, the well diffusion and cell contact. These measurements confirmed a strong inhibitory action of the isolated strains against 18 strains belonging to Staphylococcus aureus, Listeria innocua, Micrococcus luteus, Shigella sonnei, Shigella dysenteria, E. coli, Enterococcus faecalis, Bacillus cereus and Carnobacterium maltaromaticum. The bacterial supernatants with neutralized acidity (SBAN) also exerted a strong inhibitory activity against the tested pathogenic strains. This activity proved stablity at pH ranging from 2 to 6, sensitive to proteolytic enzymes, relatively heat-stable (20 min at 100 ° C or 120 days at -80 ° C) and insensitive to ultraviolet radiation or treatment with mycomycine C. Screening of genes encoding plantaricin bacteriocins based on the PCR was performed for the LbM2a strain using specific primers which allowed to detect the presence of plantaricines EF and K. Proteins Fractionation of SBAN supernatants of the two strains LbM2a and LBRE-LSAS by reverse HPLC allowed the isolation of a single active peak by supernatant. These peaks were concentrated by ultrafiltration and assayed positively for their antagonist activity against an indicator strain, Micrococcus luteus ATCC 10420. These molecules have a molecular weight between 3 and 10 kDa for both strains LBRE-LSAS and LbM2.