Characterization of anti-Listeria innocua. F Bacteriocins Produced by Lactococcus lactis ssp raffinolactis Isolated from Algerian Camel Milk

Abstract

Aims: Our work is focused on the characterization of anti-Listeria innocua. F bacteriocins produced by lactic acid bacteria belonging to the genus Lactococcus isolated from camel milk. Methodology: We tested the bacteriocins activities by diffusion wells method, followed by protease inactivation. The antibacterial peptides are extracted by adsorption/desorption method and then separated on a PAGE-SDS and their activity is detected by the zymogram technique. On the other hand, genetic characterization of these molecules was realized by the plasmid curing using two antibiotics, Rifampicin and Novobiocin. The cure is checked by extraction of the plasmids followed by a migration on an agarose gel. The Bac-mutants obtained underwent testing activity by well diffusion method and by zymogram technique, using as a positive control, wild strains Bac+. Results: Lactococcus lactis ssp raffinolactis gave inhibition zones against  Listeria innocua F strain with a diameter of 16 mm as well as by the zymogram has inhibition zones between 5 and 10 Kda. Bacteriocins produced are sensitive to the proteases used. The disappearance of the zones of inhibition after the plasmid treatment confirms the plasmid location of the genetic clusters bacteriocins. After cure of the plasmids, it is indicated that the genes for immunity to the parental bacteriocins are also carried by the same plasmid and therefore co-transcribed with genes encoding the bacteriocin. Finally, our work is completed by the determination of CMI of bacteriocins extracts; the value found is 7.14 IU/ml. Conclusion: Bacteriocins produced are sensitive to trypsin and pepsin, two proteolytic enzymes most commonly used to confirm the protein nature of bacteriocins and gave a protein pattern and a zone of inhibition of 9.26 KDa. This MW is situated between 5 to 10 KDa and it is corresponding to sub-class IIa of bacteriocins. The Minimal inhibitory concentration of bacteriocin was 7.14 IU/ml.