Sandrine Rousseaux

Yeast–yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of non-Saccharomyces and Saccharomyces yeasts

There has been increasing interest in the use of selected non-Saccharomyces yeasts in co-culture with Saccharomyces cerevisiae. The main reason is that the multistarter fermentation process is thought to simulate indigenous fermentation, thus increasing wine aroma complexity while avoiding the risks linked to natural fermentation. However, multistarter fermentation is characterised by complex and largely unknown interactions between yeasts. Consequently the resulting wine quality is rather unpredictable. In order to better understand the interactions that take place between non-Saccharomyces and Saccharomyces yeasts during alcoholic fermentation, we analysed the volatile profiles of several mono-culture and co-cultures. Candida zemplinina, Torulaspora delbrueckii and Metschnikowia pulcherrima were used to conduct fermentations either in mono-culture or in co-culture with S. cerevisiae. Up to 48 volatile compounds belonging to different chemical families were quantified. For the first time, we show that C. zemplinina is a strong producer of terpenes and lactones. We demonstrate by means of multivariate analysis that different interactions exist between the co-cultures studied. We observed a synergistic effect on aromatic compound production when M. pulcherrima was in co-culture with S. cerevisiae. However a negative interaction was observed between C. zemplinina and S. cerevisiae, which resulted in a decrease in terpene and lactone content. These interactions are independent of biomass production. The aromatic profiles of T. delbrueckii and S. cerevisiae in mono-culture and in co-culture are very close, and are biomass-dependent, reflecting a neutral interaction. This study reveals that a whole family of compounds could be altered by such interactions. These results suggest that the entire metabolic pathway is affected by these interactions.

Development of a qPCR assay for specific quantification of Botrytis cinerea on grapes.

The aim of this study was to develop a system for rapid and accurate real-time quantitative PCR (qPCR) identification and quantification of Botrytis cinerea, one of the major pathogens present on grapes. The intergenic spacer (IGS) region of the nuclear ribosomal DNA was used to specifically detect and quantify B. cinerea. A standard curve was established to quantify this fungus. The qPCR reaction was based on the simultaneous detection of a specific IGS sequence and also contained an internal amplification control to compensate for variations in DNA extraction and the various compounds from grapes that inhibit PCR. In these conditions, the assay had high efficiency (97%), and the limit of detection was estimated to be 6.3 pg DNA (corresponding to 540 spores). Our method was applied to assess the effects of various treatment strategies against Botrytis in the vineyard. Our qPCR assay proved to be rapid, selective and sensitive and may be used to monitor Botrytis infection in vineyards.

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.

Characterization of the Viable but Nonculturable (VBNC) State in Saccharomyces cerevisiae

The Viable But Non Culturable (VBNC) state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to “resuscitate”. The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the “resuscitation” of VBNC cells during the VBNC state.

Non-Botrytis grape-rotting fungi responsible for earthy and moldy off-flavors and mycotoxins

The grape microflora is complex and includes filamentous fungi, yeasts and bacteria with different physiological characteristics and effects on wine production. Most studies have focused on the wine microbiota, but a few studies have reported the ecology of grape microorganisms. Some of these organisms - such as non-Botrytis bunch rotting fungi, which greatly influence the safety or sensory quality of wine, due to the production of mycotoxins and off-flavors, respectively - are considered to be spoilage agents. We review here the diversity of filamentous fungi on grapes and the factors influencing their development, such as grape ripening stage, environmental factors (climate, rain and cultivation practices), grape variety and grape health status. We also discuss the pathways by which mycotoxins and off-flavors are produced, the control of the population, the metabolites responsible for wine spoilage and the methods for detecting and characterizing the microorganisms involved.

Wine microbiome: A dynamic world of microbial interactions

ABSTRACT Most fermented products are generated by a mixture of microbes. These microbial consortia perform various biological activities responsible for the nutritional, hygienic, and aromatic qualities of the product. Wine is no exception. Substantial yeast and bacterial biodiversity is observed on grapes, and in both must and wine. The diverse microorganisms present interact throughout the winemaking process. The interactions modulate the hygienic and sensorial properties of the wine. Many studies have been conducted to elucidate the nature of these interactions, with the aim of establishing better control of the two fermentations occurring during wine processing. However, wine is a very complex medium making such studies difficult. In this review, we present the current state of research on microbial interactions in wines. We consider the different kinds of interactions between different microorganisms together with the consequences of these interactions. We underline the major challenges to obtaining a better understanding of how microbes interact. Finally, strategies and methodologies that may help unravel microbe interactions in wine are suggested.

Diversity of yeast strains of the genus Hanseniaspora in the winery environment: What is their involvement in grape must fermentation?

Isolated yeast populations of Chardonnay grape must during spontaneous fermentation were compared to those isolated on grape berries and in a winery environment before the arrival of the harvest (air, floor, winery equipment) and in the air through time. Two genera of yeast, Hanseniaspora and Saccharomyces, were isolated in grape must and in the winery environment before the arrival of the harvest but not on grape berries. The genus Hanseniaspora represented 27% of isolates in the must and 35% of isolates in the winery environment. The isolates of these two species were discriminated at the strain level by Fourier transform infrared spectroscopy. The diversity of these strains observed in the winery environment (26 strains) and in must (12 strains) was considerable. 58% of the yeasts of the genus Hanseniaspora isolated in the must corresponded to strains present in the winery before the arrival of the harvest. Although the proportion and number of strains of the genus Hanseniaspora decreased during fermentation, some strains, all from the winery environment, subsisted up to 5% ethanol content. This is the first time that the implantation in grape must of populations present in the winery environment has been demonstrated for a non-Saccharomyces genus.

Wine microbiology is driven by vineyard and winery anthropogenic factors

The effects of different anthropic activities (vineyard: phytosanitary protection; winery: pressing and sulfiting) on the fungal populations of grape berries were studied. The global diversity of fungal populations (moulds and yeasts) was performed by pyrosequencing. The anthropic activities studied modified fungal diversity. Thus, a decrease in biodiversity was measured for three successive vintages for the grapes of the plot cultivated with Organic protection compared to plots treated with Conventional and Ecophyto protections. The fungal populations were then considerably modified by the pressing‐clarification step. The addition of sulfur dioxide also modified population dynamics and favoured the domination of the species Saccharomyces cerevisiae during fermentation. The non‐targeted chemical analysis of musts and wines by FT‐ICR‐MS showed that the wines could be discriminated at the end of alcoholic fermentation as a function of adding SO2 or not, but also and above all as a function of phytosanitary protection, regardless of whether these fermentations took place in the presence of SO2 or not. Thus, the existence of signatures in wines of chemical diversity and microbiology linked to vineyard protection has been highlighted.

Predominant mycotoxins, mycotoxigenic fungi and climate change related to wine

Wine is a significant contributor to the economies of many countries. However, the commodity can become contaminated with mycotoxins produced by certain fungi. Most information on mycotoxins in wine is from Spain, Italy and France. Grapes can be infected by mycotoxigenic fungi, of which Aspergillus carbonarius producing ochratoxin A (OTA) is of highest concern. Climate is the most important factor in determining contamination once the fungi are established, with high temperatures being a major factor for OTA contamination: OTA in wine is at higher concentrations in warmer southern Europe than northern. Contamination by fumonisins is a particular concern, related to Aspergillus niger producing these compounds and the fungus being isolated frequently from grapes. Aflatoxins can be present in wine, but patulin is seldom detected. Alternaria mycotoxins (e.g. alternariol) have been frequently observed. There are indications that T-2 toxin may be common. Also, the combined effects of mycotoxins in wine require consideration. No other mycotoxins are currently of concern. Accurate fungal identifications and mycotoxin detection from the fungi are important and a consideration of practical methods are required. There is a diversity of wines that can be contaminated (e.g. red, white, sweet, dry and fortified). The occurrence of OTA is higher in red and sweet than white wines. Steps to control mycotoxins in wine involve good agriculture practices. The effect of climate change on vines and mycotoxins in wine needs urgent consideration by well-constructed modelling studies and expert interpretation of existing data. Reliable models of the effect of climate change on vines is a priority: the health of vines affects mycotoxin contamination. A modelling study of OTA in grapes at higher temperatures over 100years is required. Progress has been made in reducing OTA in wine. The other mycotoxins require consideration and the effects of climate change will become crucial.

Persistence of Two Non-Saccharomyces Yeasts (Hanseniaspora and Starmerella) in the Cellar.

Different genera and/or species of yeasts present on grape berries, in musts and wines are widely described. Nevertheless, the community of non-Saccharomyces yeasts present in the cellar is still given little attention. Thus it is not known if the cellar is a real ecological niche for these yeasts or if it is merely a transient habitat for populations brought in by grape berries during the winemaking period. This study focused on three species of non-Saccharomyces yeasts commonly encountered during vinification: Starmerella bacillaris (synonymy with Candida zemplinina), Hanseniaspora guilliermondii and Hanseniaspora uvarum. More than 1200 isolates were identified at the strain level by FT-IR spectroscopy (207 different FTIR strain pattern). Only a small proportion of non-Saccharomyces yeasts present in musts came directly from grape berries for the three species studied. Some strains were found in the must in two consecutive years and some of them were also found in the cellar environment before the arrival of the harvest of second vintage. This study demonstrates for the first time the persistence of non-Saccharomyces yeast strains from year to year in the cellar. Sulfur dioxide can affect yeast populations in the must and therefore their persistence in the cellar environment.