Variety and variability of glycosidase activities in an Oenococcus oeni strain collection tested with synthetic and natural substrates.

AIMS: To evaluate the capacity of Oenococcus oeni strains to release aroma compounds from glycosylated precursors by measuring glycosidase activities with both synthetic and natural substrates. METHODS AND RESULTS: Five glycosidase activities were investigated in 47 O. oeni strains using synthetic substrates. This screening revealed that activity levels vary considerably, not only for each strain (depending on the substrate tested), but also between strains. Fifteen strains exhibiting different activity profiles were further analysed using natural substrates extracted from both untoasted and toasted oak. In the latter, various amounts of aromatic compounds were measured, thus confirming the specific potentials of the selected strains, but the results were different from those obtained using synthetic substrates. In addition, the use of toasted wood extracts significantly increased the release of wood aromas, which minimized differences between strains. CONCLUSIONS: The capability of O. oeni to hydrolysate glycoconjugate aroma precursors is strain-dependent and variable, depending on the substrate. SIGNIFICANCE AND IMPACT OF THE STUDY: Instead of synthetic substrates, natural aroma precursors should be used for an adequate evaluation of the glycosidase potential of O. oeni.

In vitro anti-bacterial and anti-adherence effects of natural polyphenolic compounds on oral bacteria.

AIMS: To investigate the action of different polyphenolic compounds, extracted from red wine, grape marc and pine bark, on oral bacteria. METHODS AND RESULTS: The anti-microbial activity of extracts was examined by determining the Minimal Inhibitory Concentration and Minimal Bactericidal Concentration using the macro dilution broth technique. Their effect on the adherence was tested on growing cells of Streptococcus mutans on a glass surface and on a multi-species biofilm grown on saliva-coated hydroxyapatite discs. The effect on glucosyltransferase activity was analysed through the reductions in the overall reaction rate and the quantity of insoluble glucan (ISG) synthesized. Pine bark and grape marc extracts were the most effective inhibitors of the multi-species biofilm formation and of the ISG synthesis. CONCLUSION: The tested components showed an interesting anti-plaque activity in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is, to our knowledge, the first and the most complete report on the properties of wine and pine bark extracts that could be used for oral disease prevention purpose.

In Vivo PCR-DGGE analysis of Lactobacillus plantarum and Oenococcus oeni populations in red wine.

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety "Primitivo di Puglia"), a polymerase chain reaction- denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30-60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.

The arcABC gene cluster encoding the arginine deiminase pathway of Oenococcus oeni, and arginine induction of a CRP-like gene.

Oenococcus oeni, the main species which induces malolactic fermentation in wine, uses arginine via the arginine deiminase (ADI) pathway. Using degenerated primers, two specific probes, one for ornithine transcarbamoylase (OTC) and the other for carbamate kinase (CK), were synthesized. These made it possible to clone and sequence a cluster containing genes encoding ADI (arcA), OTC (arcB) and CK (arcC). In addition, sequence analysis upstream of the arcA gene revealed the presence of an open reading frame (orf229) whose 3'-end was only 101 bp-distant from the start codon of the arcA gene and showed similarity with members of the FNR (regulation for fumarate and nitrate reduction) and CRP (cAMP receptor protein) family of transcriptional regulators. Moreover, a putative binding site for such regulators lies in the promoter region of the arcA gene. Induction of the arc cluster by arginine was studied first at the enzymatic level. The activities of the three enzymes strongly increased when cells were grown in the presence of the amino acid. In addition, the influence of arginine on gene transcription was monitored by RT-PCR (reverse transcriptase-polymerase chain reaction). Expression of the three arc genes, and particularly that of arcA, was positively affected by arginine supplementation and thus confirmed the enzymatic results. Moreover, transcription of the putative CRP-like gene orf229 was also stimulated by arginine. These data suggest that the protein encoded by orf229 could be a CRP-like regulator involved in the metabolism of O. oeni.