Population dynamics and yeast diversity in early winemaking stages without sulfites revealed by three complementary approaches

Nowadays, the use of sulfur dioxide (SO2 ) during the winemaking process is a controversial societal issue. In order to reduce its use, various alternatives are emerging, in particular bioprotection by adding yeasts, with different impacts on yeast microbiota in early winemaking stages. In this study, quantitative-PCR and metabarcoding high-throughput sequencing (HTS) were combined with MALDI-TOF-MS to monitor yeast population dynamic and diversity in the early stages of red winemaking process without sulfites and with bioprotection by Torulaspora delbrueckii and Metschnikowia pulcherrima addition. By using standard procedures for yeast protein extraction and a laboratory-specific database of wine yeasts, identification at species level of 95% of the isolates was successfully achieved by MALDI-TOF-MS, thus confirming that it is a promising method for wine yeast identification. The different approaches confirmed the implantation and the niche occupation of bioprotection leading to the decrease of fungal communities (HTS) and Hanseniaspora uvarum cultivable population (MALDI-TOF MS). Yeast and fungi diversity was impacted by stage of maceration and, to a lesser extent, by bioprotection and SO2, resulting in a modification of the nature and abundance of the operational taxonomic units (OTUs) diversity

L’identification des levures et bactéries œnologiques par spectrométrie de masse de type MALDI-TOF

La spectrométrie de masse de type MALDI-TOF a été adaptée afin d’être utilisée comme outil innovant d’identification au niveau de l’espèce des levures et bactéries isolées d’échantillons variés (moûts, vins, boissons). L’analyse d’un grand nombre de clones permet d’apprécier la diversité des espèces de levures, bactéries acétiques et lactiques présentes dès les phases pré-fermentaires, au cours des fermentations, pendant l’élevage ou après conditionnement. Dans le cas d’altération de produits, cet outil innovant participera à une meilleure maitrise des risques microbiologiques

Front Microbiol

Brettanomyces bruxellensis is the main spoilage microbial agent in red wines. The use of fungal chitosan has been authorized since 2009 as a curative treatment to eliminate this yeast in conventional wines and in 2018 in organic wines. As this species is known to exhibit great genetic and phenotypic diversity, we examined whether all the strains responded the same way to chitosan treatment. A collection of 53 strains of was used. In the conditions of the reference test, all were at least temporarily affected by the addition of chitosan to wine, with significant decrease of cultivable population. Some (41%) were very sensitive and no cultivable yeast was detected in wine or lees after 3 days of treatment, while others (13%) were tolerant and, after a slight drop in cultivability, resumed growth between 3 and 10 days and remained able to produce spoilage compounds. There were also many strains with intermediate behavior. The strain behavior was only partially linked to the strain genetic group. This behavior was little modulated by the physiological state of the strain or the dose of chitosan used (within the limits of the authorized doses). On the other hand, for a given strain, the sensitivity to chitosan treatment was modulated by the chitosan used and by the properties of the wine in which the treatment was carried out.Recherches sur l’origine et les effets secondaires des propriétés stabilisantes du chitosane fongique dans le vi

Int J Food Microbiol.

Oenophages have so far been mostly isolated from red wines under malolactic fermentation (MLF), and correspond to temperate or ex-temperate phages of Oenococcus oeni. Their genomes are clustered into 4 integrase gene sequence groups, which are also related to the chromosomal integration site. Our aims were to survey the occurrence of oenophages in a broader and more diverse collection of samples than those previously explored. Active phages were isolated from 33 out of 166 samples, which mostly originated from must and MLF. Seventy one phage lysates were produced and 30% were assigned to a novel group with unusual genomic characteristics, called unk. All unk members produced similar RAPD and DNA restriction patterns, were negative by PCR for the signature sequences previously identified in the integrase and endolysin genes of oenophages, and lacked any BamHI restriction site in their genome. The data support that development of additional and novel signature genes for assessing oenophage diversity is now required

Quantification des Brettanomyces par qPCR : Étude de la fiabilité, répétabilité et reproductibilité des kits

Article de revue professionnelleBrettanomyces bruxellensis est une levure d’altération du vin avec de faibles besoins nutritionnels, résistante à l’éthanol et aux faibles pH, lui permettant une implantation en vin durant ou après la fermentation alcoolique (Conterno et al., 2006). B. bruxellensis est capable de produire des phénols volatils (éthyl-4-phénol, éthyl-4-gaïacol et éthyl-4-catéchol) (Oelofse et al., 2008). Ces molécules volatiles odorantes amènent un caractère phénolé et animal au vin connu sous le nom de « caractère Brett ». D’autres molécules (2-acétyltétrahydropyridine et 2-éthyltétrahydropyridine) produites par B. bruxellensis sont également responsables d’une déviation organoleptique appelée communément « goût de souris ». La détection et la quantification de cette levure sont donc nécessaires afin d’éviter l’altération du vin. L’isolement de cette levure sur milieu gélosé est utilisé en routine par les laboratoires spécialisés en œnologie. Cette méthode nécessite un temps d’incubation très long (> 7 jours) mais ne permet pas la quantification des cellules viables non cultivables. Des cultures en milieu liquide contenant les précurseurs des phénols volatils sont parfois effectuées afin de déterminer si les vins testés contiennent ou non B. bruxellensis. L’apparition de l’odeur « Brett » plus ou moins rapide permet d’avoir une estimation sur la quantité de cellules présentes. La cytométrie en flux est également un outil utilisé par les laboratoires afin de quantifier de manière spécifique B. bruxellensis : sonde spécifique fluorescente (Serpaggi et al., 2010), anticorps. En France comme à l’étranger, la principale méthode moléculaire utilisée est la PCR quantitative (qPCR). De nombreuses études portant sur cette dernière ont permis le développement de kits commerciaux afin de dénombrer spécifiquement B. bruxellensis dans le vin. Cependant, aucune étude n’a été effectuée sur la fiabilité, la répétabilité et la reproductibilité de ces kits. C’est pourquoi, dans cette étude, nous avons comparé les résultats obtenus en utilisant trois kits qPCR commerciaux permettant d’extraire l’ADN des cellules et de quantifier B. bruxellensis en vin rouge. Les analyses ont été effectuées par trois laboratoires sélectionnés, spécialisés en analyses œnologiques et sur trois vins rouges élaborés dans 3 régions différentes

Quantification des Brettanomyces par qPCR

International audienceBrettanomyces bruxellensis est une levure d’altération du vin avec de faibles besoins nutritionnels, résistante à l’éthanol et aux faibles pH, lui permettant une implantation en vin durant ou après la fermentation alcoolique (Conterno et al., 2006). B. bruxellensis est capable de produire des phénols volatils (éthyl-4-phénol, éthyl-4-gaïacol et éthyl-4-catéchol) (Oelofse et al., 2008). Ces molécules volatiles odorantes amènent un caractère phénolé et animal au vin connu sous le nom de « caractère Brett ». D’autres molécules (2-acétyltétrahydropyridine et 2-éthyltétrahydropyridine) produites par B. bruxellensis sont également responsables d’une déviation organoleptique appelée communément « goût de souris ».La détection et la quantification de cette levure sont donc nécessaires afin d’éviter l’altération du vin. L’isolement de cette levure sur milieu gélosé est utilisé en routine par les laboratoires spécialisés en œnologie. Cette méthode nécessite un temps d’incubation très long (> 7 jours) mais ne permet pas la quantification des cellules viables non cultivables. Des cultures en milieu liquide contenant les précurseurs des phénols volatils sont parfois effectuées afin de déterminer si les vins testés contiennent ou non B. bruxellensis. L’apparition de l’odeur « Brett » plus ou moins rapide permet d’avoir une estimation sur la quantité de cellules présentes. La cytométrie en flux est également un outil utilisé par les laboratoires afin de quantifier de manière spécifique B. bruxellensis : sonde spécifique fluorescente (Serpaggi et al., 2010), anticorps. En France comme à l’étranger, la principale méthode moléculaire utilisée est la PCR quantitative (qPCR). De nombreuses études portant sur cette dernière ont permis le développement de kits commerciaux afin de dénombrer spécifiquement B. bruxellensis dans le vin. Cependant, aucune étude n’a été effectuée sur la fiabilité, la répétabilité et la reproductibilité de ces kits.C’est pourquoi, dans cette étude, nous avons comparé les résultats obtenus en utilisant trois kits qPCR commerciaux permettant d’extraire l’ADN des cellules et de quantifier B. bruxellensis en vin rouge. Les analyses ont été effectuées par trois laboratoires sélectionnés, spécialisés en analyses œnologiques et sur trois vins rouges élaborés dans 3 régions différentes

Evaluation of three Brettanomyces qPCR commercial kits: results from an interlaboratory study

Aim: Brettanomyces bruxellensis is well adapted to high ethanol concentrations and low pH which allows it to develop in difficult environments, such as wine. B. bruxellensis is mainly found in red wine and is regarded as a spoilage yeast due to its production of ethylphenols and other compounds responsible for organoleptic defects. The detection and quantification of this yeast is essential to preventing wine spoilage. Several specific detection and quantification kits based on real time quantitative PCR (qPCR) are commercially available. Although these kits are frequently used by private enological and research laboratories, no scientific reports on the reliability and performance of these kits, including interlaboratory and interassay comparisons, have been published. The aim of this work was to compare commercially available kits for the quantification of B. bruxellensis in red wine to classical method (plate counting on selective medium) in an interlaboratory study. Methods and results: Three different commercial kits were tested on three different wines from Bordeaux, Cotes du Rhone, and Burgundy inoculated with B. bruxellensis at four different concentrations. Five naturally contaminated wines from different French wine regions were also tested. Our results suggest that all the kits tested probably over or underestimate the quantity of B. bruxellensis in red wine and, under specific conditions, give false positives. Conclusion: Quantification may be very heterogeneous depending on the wine, laboratory, or population level. Underestimations or false negative results may have serious consequences for winemakers. Overestimation may be partly due to the quantification of dead cells by qPCR. Significance and impact of the study: This study highlights that quantification of B. bruxellensis in red wine using commercial kits requires a high level of expertise in molecular biology. We recommend that all users use a microbiological internal control to validate DNA extraction yield

Molecular Diagnosis of Brettanomyces bruxellensis’ Sulfur Dioxide Sensitivity Through Genotype Specific Method

The yeast species Brettanomyces bruxellensis is associated with important economic losses due to red wine spoilage. The most common method to prevent and/or control B. bruxellensis spoilage in winemaking is the addition of sulfur dioxide into must and wine. However, recently, it was reported that some B. bruxellensis strains could be tolerant to commonly used doses of SO2. In this work, B. bruxellensis response to SO2 was assessed in order to explore the relationship between SO2 tolerance and genotype. We selected 145 isolates representative of the genetic diversity of the species, and from different fermentation niches (roughly 70% from grape wine fermentation environment, and 30% from beer, ethanol, tequila, kombucha, etc.). These isolates were grown in media harboring increasing sulfite concentrations, from 0 to 0.6 mg.L-1 of molecular SO2. Three behaviors were defined: sensitive strains showed longer lag phase and slower growth rate and/or lower maximum population size in presence of increasing concentrations of SO2. Tolerant strains displayed increased lag phase, but maximal growth rate and maximal population size remained unchanged. Finally, resistant strains showed no growth variation whatever the SO2 concentrations. 36% (52/145) of B. bruxellensis isolates were resistant or tolerant to sulfite, and up to 43% (46/107) when considering only wine isolates. Moreover, most of the resistant/tolerant strains belonged to two specific genetic groups, allowing the use of microsatellite genotyping to predict the risk of sulfur dioxide resistance/tolerance with high reliability (>90%). Such molecular diagnosis could help the winemakers to adjust antimicrobial techniques and efficient spoilage prevention with minimal intervention

Int J Food Microbiol

The wine spoilage yeast Brettanomyces bruxellensis can be found at several steps in the winemaking process due to its resistance to multiple stress conditions. The ability to form biofilm is a potential resistance strategy, although it has been given little attention so far for this yeast. In this work, the capacity to form biofilm and its structure were explored in YPD medium and in wine. Using microsatellite analysis, 65 isolates were discriminated into 5 different genetic groups from which 12 strains were selected. All 12 strains were able to form biofilm in YPD medium on a polystyrene surface. The presence of microcolonies, filamentous cells and extracellular polymeric substances, constituting the structure of the biofilm despite a small thickness, were highlighted using confocal and electronic microscopy. Moreover, different cell morphologies according to genetic groups were highlighted. The capacity to form biofilm in wine was also revealed for two selected strains. The impact of wine on biofilms was demonstrated with firstly considerable biofilm cell release and secondly growth of these released biofilm cells, both in a strain dependent manner. Finally, B. bruxellensis has been newly described as a producer of chlamydospore-like structures in wine, for both planktonic and biofilm lifestyles.European Union (FEDER