thesis

A preliminary study showed that the appearance of Listeria monocytogenes in a smear-surface cheese (Munster cheese) did not occur before one week of ripening. Furthermore L. monocytogenes appeared exclusively in the crust and was not detected in the core of cheese even in heavily contaminated samples (up to 106 CFU/g). These characteristics of Munster cheese contamination with L. monocytogenes and its ripening process (washes) allow the inoculation of an antilisterial bacteriocinogenic bacterial strain at the beginning of the ripening period during a humidification operation and not as a starter. Provided that the strain grows in the cheese surface, produces its bacteriocin and prevent the appearance of L. monocytogenes, this methodology might be interesting as it would not affect the manufacturing and the sensory characteristics of the food.
During this same study, it was also shown, unexpectedly, that a group of Munster cheeses of farm origin, ripened in the same cellar as preceding groups of industrial manufacture, demonstrated a much lower contamination with L. monocytogenes. Such a result could be explained by the presence of lactic acid bacteria, capable of producing antilisterial bacteriocins, in the rind of these cheeses. Among 1962 bacterial isolates from farm-produced cheeses screened for activity against L. monocytogenes, 6 lactic acid bacteria produced antilisterial compounds other than organic acids. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compounds produced by these bacteria suggested that they were bacteriocins.
The bacterial strain (WHE 92) displaying the strongest antilisterial effect (17000 UA/ml by the well diffusion method) was identified at the DNA level as Lactobacillus plantarum. Kinetic studies of the bacteriocin production, activity and stability in fermentor in pH and temperature conditions of cheese ripening showed that this strain was also the most efficient. Lb. plantarum WHE 92 was seeded on the cheese surface after manufacture (at the beginning of the ripening period). No installation or growth of L. monocytogenes happened, with artificial or natural addition of this bacterium, in the treated samples till the end of ripening, while it often reached 105 CFU/g in control samples (publication is in preparation). Moreover, the rinsing of contaminated cheese samples with culture extracts containing the bacteriocin succeeded in reducing notably L. monocytogenes populations.
The use of Lb. plantarum WHE 92 in soft cheeses has been successfully introduced in some cheeses manufactures in the Alsace Region.
Purification to homogeneity (using ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic interaction and reverse-phase FPLC) and sequencing of the bacteriocin produced by Lb. plantarum WHE 92 showed that it was a 4.6 kDa, 44 amino acid peptide, the primary structure of which being identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. This is the first case of the same bacteriocin appearing naturally with bacteria of different species and genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in this production from Lb. plantarum WHE 92 was observed up to pH 6.0. This fact is important from an industrial angle. As the pH of food products contaminated with L. monocytogenes is often higher than 5.0, Lb. plantarum WHE 92 could constitute an effective means of biological combat against this pathogen in foodstuff.

Claude Hasselmann Professor
                               Département des Sciences de l'Aliment
                               Université Louis Pasteur, Faculté de Pharmacie, Strasbourg, France

Jean-Claude Hubert Professor
                              Dynamique de génomes de microorganismes
                              Institut de botanique, Université Louis Pasteur, Strasbourg, France

Patrice Bracquart     Professor
                              Laboratoire des Biosciences de l'Aliment,
                              Unité Associée à l'INRA, Université Henri Poincaré, Nancy, France

Henry Delincée        Professor
                              Federal Research Centre for Nutrition, Karlsruhe, Germany

Dalal Werner           Ph.D.,
                              AERIAL CRITT, Shiltigheim, France