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.

Characterization of the yeast ecosystem in grape must and wine using real-time PCR.

The complex microbial ecosystem of grape must and wine harbours a wide diversity of yeast species. Specific oligonucleotide primers for real-time quantitative PCR(QPCR) were designed to analyse several important non-Saccharomyces yeasts (Issatchenkia orientalis, Metschnikowia pulcherrima, Torulaspora delbrueckii, Candida zemplinina and Hanseniaspora spp.) and Saccharomyces spp. in fresh wine must, during fermentation and in the finished wine. The specificity of all primer couples for their target yeast species were validated and the QPCR methods developed were compared with a classic approach of colony identification by RFLP-ITS-PCR on cultured samples. Once the methods had been developed and validated, they were used to study these non-Saccharomyces yeasts in wine samples and to monitor their dynamics throughout the fermentation process. This study confirms the usefulness and the relevance of QPCR for studying non-Saccharomyces yeasts in the complex yeast ecosystem of grape must and wine.

Glycerol metabolism and bitterness producing lactic acid bacteria in cidermaking.

Several lactic acid bacteria were isolated from bitter tasting ciders in which glycerol was partially removed. The degradation of glycerol via glycerol dehydratase pathway was found in 22 out of 67 isolates. The confirmation of glycerol degradation by this pathway was twofold: showing their glycerol dehydratase activity and detecting the presence of the corresponding gene by a PCR method. 1,3-propanediol (1,3-PDL) and 3-hydroxypropionic acid (3-HP) were the metabolic end-products of glycerol utilization, and the accumulation of the acrolein precursor 3-hydroxypropionaldehyde (3-HPA) was also detected in most of them. The strain identification by PCR-DGGE rpoB showed that Lactobacillus collinoides was the predominant species and only 2 belonged to Lactobacillus diolivorans. Environmental conditions conducting to 3-HPA accumulation in cidermaking were studied by varying the fructose concentration, pH and incubation temperature in L. collinoides 17. This strain failed to grow with glycerol as sole carbon source and the addition of fructose enhanced both growth and glycerol degradation. Regarding end-products of glycerol metabolism, 1,3-PDL was always the main end-product in all environmental conditions assayed, the only exception being the culture with 5.55 mM fructose, where equimolar amounts of 1,3-PDL and 3-HP were found. The 3-HPA was transitorily accumulated in the culture medium under almost all culture conditions, the degradation rate being notably slower at 15 degrees C. However, no disappearance of 3-HPA was found at pH 3.6, a usual value in cider making. After sugar exhaustion, L. collinoides 17 oxidated lactic acid and/or mannitol to obtain energy and these oxidations were accompanied by the removal of the toxic 3-HPA increasing the 1,3-PDL, 3-HP and acetic acid contents.

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods.

AIMS: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. METHODS AND RESULTS: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10(6) cells ml(-1)) during spontaneous malolactic fermentation. A minimum of 10(3) ml(-1) producing cells was required for production of more than 1 mg l(-1) of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. CONCLUSION: Methods using quantitative PCR are efficient to enumerate biogenic amines-producing cells in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.

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.

Detection and quantification of Brettanomyces bruxellensis and 'ropy' Pediococcus damnosus strains in wine by real-time polymerase chain reaction.

AIMS: Brettanomyces bruxellensis is a well-known wine spoilage yeast that causes undesirable off-flavours. Likewise, glucan-producing strains of ropy Pediococcus damnosus are considered as spoilage micro-organisms because the synthesis of glucan leads to an unacceptable viscosity of wine. METHODS AND RESULTS: We developed a real-time PCR method to detect and quantify these two spoilage micro-organisms in wine. It is based on specific primer pairs for amplification of target DNA, and includes a melting-curve analysis of PCR products as a confirmatory test. CONCLUSIONS: The detection limit in wine was 10(4) CFU ml(-1) for B. bruxellensis and 40 CFU ml(-1) for ropy Pediococcus damnosus. The real-time PCR proved to be reliable for the early, sensitive detection and quantification of B. bruxellensis and ropy P. damnosus in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR-based method described in this study provides a new tool for monitoring spoilage micro-organisms in wine. Time-consuming culture and colony isolation steps are no longer needed, so winemakers can intervene before spoilage occurs.

Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders.

Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1.3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider.