La biosynthèse d'exopolysaccharide par des souches de pediococcus damnosus isolées du vin : mise au point d'outils moléculaires de détection

Abstract

Exopolysaccharide biosynthesis by Pediococcus damnosus strains isolated from wine: elaboration of molecular detection tools. Lactic acid bacteria synthesize exopolysaccha- rides (EPS). These carbohydrate polymers excreted or produced at the cell membrane can drasti- cally increase viscosity of the medium. In dairy industry, this property is used to improve the texture of yoghurts for example. Other microbial EPS are currently under study for their possible use in food processes or in medicine. However, cider, beer and wine are spoiled by the presence of EPS. In particular strains of Pediococcus damnosusare responsible for the production of a β-D-glucan. For P. damnosusstrains isolated from ropy wines, glucan production is linked to a plas- mid of approximately 5 500 bp. The ropy phenotype disappears when the plasmid is lost. This plas- mid is currently being sequenced and several open reading frames (ORF) have already been revealed. The amino acid sequence of the first ORF has high identity scores with a plasmid mobilization pro- tein of Lactobacillus casei. The second ORF contains several motifs characterized on a glucosyl- transferase from Streptococcus pneumoniaetype 37 which produces the same polysaccharide as the Pediococcus strains isolated from ropy wines. From the partial sequence of the plasmid, molecular tools for detection of spoiling strains in wine were obtained. They involve PCR, Southern blot and colony hybridization. A new called GT probe has been isolated, making it possible to dif- ferentiate ropy from non-ropy strains. This could be used directly in colony hybridization. Two oligonucleotides (PF5 and PF6) were also selected for PCR experiments. A plasmid fragment from the ropy strains was specifically amplified. PCR was performed using whole cells from labo- ratory cultures and preliminary results obtained with a protocol for direct PCR on wine samples are presented. Wine quality depends on the availability of such detection tools. In fact, wines generally become ropy during storage in tanks, barrels and most of the time in bottles. In the latter case, no fur- ther treatment is possible. Therefore, detection of ropy strains before bottling can help the wine- maker predict the risks of spoilage with ropy P. damnosusand treat the wine in time.