Olivier Claisse

Inventory and monitoring of wine microbial consortia

The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.

Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006

ABSTRACT Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.

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.

Multilocus Sequence Typing of Oenococcus oeni: Detection of Two Subpopulations Shaped by Intergenic Recombination

ABSTRACT Oenococcus oeni is the acidophilic lactic acid bacterial species most frequently associated with malolactic fermentation of wine. Since the description of the species (formerly Leuconostoc oenos), characterization of indigenous strains and industrially produced cultures by diverse typing methods has led to divergent conclusions concerning the genetic diversity of strains. In the present study, a multilocus sequence typing (MLST) scheme based on the analysis of eight housekeeping genes was developed and tested on a collection of 43 strains of diverse origins. The eight targeted loci were successfully amplified and sequenced for all isolates. Only three to 11 different alleles were detected for these genes. The average nucleotide diversity also was rather limited (0.0011 to 0.0370). Despite this limited allelic diversity, the combination of alleles of each strain disclosed 34 different sequence types, which denoted a significant genotypic diversity. A phylogenetic analysis of the concatenated sequences showed that all strains form two well distinct groups of 28 and 15 strains. Interestingly, the same groups were defined by pulsed-field gel electrophoresis, although this method targets different genetic variations. A minimum spanning tree analysis disclosed very few and small clonal complexes. In agreement, statistical analyses of MLST data suggest that recombination events were important during O. oeni evolution and contributed to the wide dissemination of alleles among strains. Taken together, our results showed that MLST is more efficient than pulsed-field gel electrophoresis for typing O. oeni strains, and they provided a picture of the O. oeni population that explains some conflicting results previously obtained.

Nitrogen catabolic repression controls the release of volatile thiols by Saccharomyces cerevisiae during wine fermentation

Volatile thiols such as 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) are aromatic molecules having an important organoleptic impact on white wines. These components are produced from inodorous nonvolatile cysteinylated precursors by Saccharomyces cerevisiae metabolic activity during alcoholic fermentation. Here we provide a new insight into the genetic determinism of the production of volatile thiols by yeast. Using a gene deletion approach, we investigated the role of three yeast beta-lyases and demonstrate that Irc7p, a putative cystathionine beta-lyase, is one of the main proteins catalyzing the 4MSP and 3SH release under enological conditions. Moreover, we demonstrate that Ure2p/Gln3p proteins mainly control the bioconversion of volatile thiols by the transcriptional regulation of the IRC7 gene through the general mechanism of nitrogen catabolic repression. Finally, our findings suggest that the enantiomer balance of 3SH may be modulated by activating specifically stereoselective enzymes such as Irc7p.

High frequency of histamine-producing bacteria in the enological environment and instability of the histidine decarboxylase production phenotype.

ABSTRACT Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the genera Oenococcus, Lactobacillus, and Pediococcus. All carry an hdcA gene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targeting hdcA was developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 107 CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 103 CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains of Oenococcus oeni that grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover, hdcA was detected on a large and possibly unstable plasmid in these strains of O. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 103 per ml. Strains of O. oeni producing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.

Probiotic properties of non-conventional lactic acid bacteria: immunomodulation by Oenococcus oeni.

The widely used probiotic bacteria belong to the genera Lactobacillus and Bifidobacterium and have in most cases been isolated from the human gastrointestinal tract. However, other less conventional bacteria, from allochthonous or extremophilic origin, sharing similar structural or functional features, may also confer specific health benefits to a host. Firstly, we explored the in vitro immuno-modulatory or immune-stimulatory activities of 25 wine lactic acid bacteria belonging to Oenococcus oeni and Pediococcus parvulus. While cytokines released by peripheral blood mononuclear cells (PBMCs) stimulated by P. parvulus strains, showed little variation, O. oeni strains induced strain-specific cytokine patterns. Some O. oeni strains were then further analyzed under various conditions for growth, dose and culture medium. In a second phase, we evaluated the oral tolerance and safety of two strains of O. oeni in mice fed a high dose of bacteria for a week. Finally, evidence was gathered on the in vivo anti-inflammatory potential of a selected O. oeni strain using an experimental 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis mouse model. Although results did not match the anti-inflammatory levels obtained with certain conventional probiotics, strain IOEB 9115 significantly lowered colonic injury and alleviated colitis symptoms. The natural tolerance towards acid, ethanol, and phenolic compounds of O. oeni strains combined with a measureable immunomodulatory potential, suggest a possible use of selected strains isolated from wine as live probiotics.

Evidence of Distinct Populations and Specific Subpopulations within the Species Oenococcus oeni

ABSTRACT Among the lactic acid bacteria (LAB) present in the oenological microbial ecosystem, Oenococcus oeni, an acidophilic lactic acid bacterium, is essential during winemaking. It outclasses all other bacterial species during malolactic fermentation (MLF). Oenological performances, such as malic acid degradation rate and sensorial impact, vary significantly according to the strain. The genetic diversity of the O. oeni species was evaluated using a multilocus sequence typing (MLST) scheme. Seven housekeeping genes were sequenced for a collection of 258 strains that had been isolated all over the world (particularly Burgundy, Champagne, and Aquitaine, France, Chile, South Africa, and Italy) and in several wine types (red wines, white wines, and champagne) and cider. The allelic diversity was high, with an average of 20.7 alleles per locus, many of them being rare alleles. The collection comprised 127 sequence types, suggesting an important genotypic diversity. The neighbor-joining phylogenetic tree constructed from the concatenated sequence of the seven housekeeping genes showed two major phylogenetic groups, named A and B. One unique strain isolated from cider composed a third group, rooting the phylogenetic tree. However, all other strains isolated from cider were in group B. Eight phylogenetic subgroups were statistically differentiated and could be delineated by the analysis of only 32 mutations instead of the 600 mutations observed in the concatenated sequence of the seven housekeeping genes. Interestingly, in group A, several phylogenetic subgroups were composed mostly of strains coming from a precise geographic origin. Three subgroups were identified, composed of strains from Chile, South Africa, and eastern France.

Leucoanthocyanidin reductase and anthocyanidin reductase gene expression and activity in flowers, young berries and skins of Vitis vinifera L. cv. Cabernet-Sauvignon during development

Proanthocyanidins, or condensed tannins, are crucial polyphenolic compounds for grape and wine quality. Recently, significant advances were achieved in understanding the biosynthesis of their main subunits: (+)-catechin and (-)-epicatechin, produced by catalysis of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), respectively. Expression studies had been published but no data were available on enzyme activity. In our work, we devised assays to measure LAR and ANR activity and determine their development throughout the growth of flowers, young berries, and skins of Vitis vinifera L. cv. Cabernet-Sauvignon. We also investigated the accumulation of compounds in these tissues and focused on the expression of both the structural genes and the transcription factors involved in regulating them: VvMYB5a and VvMYBPA1. Biosynthetic genes were expressed early and LAR and ANR were already active during flowering and at the beginning of berry growth, as well as during colour-change in skins. The profiles we determined correlated with total tannin, catechin, and epicatechin concentrations. The involvement of VvMYB5a and VvMYBPA1 was confirmed and specific expression patterns were also established for VvLAR transcripts.

High‑throughput sequencing of amplicons for monitoring yeast biodiversity in must and during alcoholic fermentation.

We compared pyrosequencing technology with the PCR-ITS-RFLP analysis of yeast isolates and denaturing gradient gel electrophoresis (DGGE). These methods gave divergent findings for the yeast population. DGGE was unsuitable for the quantification of biodiversity and its use for species detection was limited by the initial abundance of each species. The isolates identified by PCR-ITS-RFLP were not fully representative of the true population. For population dynamics, high-throughput sequencing technology yielded results differing in some respects from those obtained with other approaches. This study demonstrates that 454 pyrosequencing of amplicons is more relevant than other methods for studying the yeast community on grapes and during alcoholic fermentation. Indeed, this high-throughput sequencing method detected larger numbers of species on grapes and identified species present during alcoholic fermentation that were undetectable with the other techniques.