The establishment of a fungal consortium in a new winery.

The biodiversity and evolution of fungal communities were monitored over a period of 3 vintages in a new winery. Samples were collected before grape receipt and 3 months after fermentation from 3 different wine related environments (WRE): floor, walls and equipment and analyzed using Illumina Mi-Seq. Genera of mold and filamentous fungi (294), non-enological (10) and wine-associated yeasts (25) were detected on all WREs before the arrival of the first harvest. Among them, genera like Alternaria and Aureobasidium persisted during two vintages. Therefore, these genera are not specific to winery environment and appear to be adapted to natural or anthropic environments due to their ubiquitous character. Some genera like Candida were also detected before the first harvest but only on one WREs, whereas, on the other WREs they were found after the harvest. The ubiquitous character and phenotypic traits of these fungal genera can explain their dynamics. After the first harvest and during 3 vintages the initial consortium was enriched by oenological genera like Starmerella introduced either by harvest or by potential transfers between the different WREs. However, these establishing genera, including Saccharomyces, do not appear to persist due to their low adaptation to the stressful conditions of winery environment.

New advances on the Brettanomyces bruxellensis biofilm mode of life.

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.

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.

The sensitivity of yeasts and yeasts-like fungi to copper and sulfur could explain lower yeast biodiversity in organic vineyards.

Although differences in yeast biodiversity have often been found between vineyards subjected to organic protection or conventional protection, little is known about the effect of copper and sulfur fungicides (the only fungicides allowed in organic farming) on yeast populations. The sensitivity to copper and sulfur of 158 yeast isolates of seven different species (Aureobasidium pullulans, Hanseniaspora guilliermondii, H. uvarum, Metschnikowia sp., Pichia membranifaciens, Saccharomyces cerevisiae and Starmerella bacillaris) was evaluated. The species A. pullulans and St. bacillaris appeared to be more resistant to copper than the other species tested. The species A. pullulans, H. guilliermondii and Metschnikowia sp. had the highest sulfur resistance. Thus, only isolates of the species A. pullulans exhibited high resistance to both antifungal agents. These results may explain the lower diversity of yeasts present on berries and the strong dominance of the species A. pullulans for vineyards protected by copper or sulfur-based fungicides compared to other vineyards reported by several studies.

PCR ITS-RFLP for Penicillium Species and Other Genera.

Among numerous molecular methodologies developed for highly specific identification of filamentous fungi isolates, here we describe restriction digestion analysis of the ITS products as an easy method to identify isolates of filamentous fungi. This technique is a rapid and reliable method appropriate for routine identification of filamentous fungi. This can be used to screen large numbers of isolates from various environments in a short time. The use of different endonucleases allowed generating individual restriction profiles. The individual profiles obtained were combined into composite restriction patterns characteristic of a species. Eleven different genera can be differentiated and among them 41 different species.

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.

Application of flow cytometry to wine microorganisms.

Flow cytometry (FCM) is a powerful technique allowing detection and enumeration of microbial populations in food and during food process. Thanks to the fluorescent dyes used and specific probes, FCM provides information about cell physiological state and allows enumeration of a microorganism in a mixed culture. Thus, this technique is increasingly used to quantify pathogen, spoilage microorganisms and microorganisms of interest. Since one decade, FCM applications to the wine field increase greatly to determine population and physiological state of microorganisms performing alcoholic and malolactic fermentations. Wine spoilage microorganisms were also studied. In this review we briefly describe FCM principles. Next, a deep revision concerning enumeration of wine microorganisms by FCM is presented including the fluorescent dyes used and techniques allowing a yeast and bacteria species specific enumeration. Then, the last chapter is dedicated to fluorescent dyes which are used to date in fluorescent microscopy but applicable in FCM. This chapter also describes other interesting "future" techniques which could be applied to study the wine microorganisms. Thus, this review seeks to highlight the main advantages of the flow cytometry applied to wine microbiology.

Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

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.

FT-IR spectroscopy: A powerful tool for studying the inter- and intraspecific biodiversity of cultivable non-Saccharomyces yeasts isolated from grape must.

The efficiency of the FT-IR technique for studying the inter- and intra biodiversity of cultivable non-Saccharomyces yeasts (NS) present in different must samples was examined. In first, the capacity of the technique FT-IR to study the global diversity of a given sample was compared to the pyrosequencing method, used as a reference technique. Seven different genera (Aureobasidium, Candida, Cryptococcus, Hanseniaspora, Issatchenkia, Metschnikowia and Pichia) were identified by FT-IR and also by pyrosequencing. Thirty-eight other genera were identified by pyrosequencing, but together they represented less than 6% of the average total population of 6 musts. Among the species identified, some of them present organoleptic potentials in winemaking, particularly Starmerella bacillaris (synonym Candidazemplinina). So in a second time, we evaluated the capacity of the FT-IR technique to discriminate the isolates of this species because few techniques were able to study intraspecific NS yeast biodiversity. The results obtained were validated by using a classic method as ITS sequencing. Biodiversity at strain level was high: 19 different strains were identified from 58 isolates. So, FT-IR spectroscopy seems to be an accurate and reliable method for identifying major genera present in the musts. The two biggest advantages of the FT-IR are the capacity to characterize intraspecific biodiversity of non-Saccharomyces yeasts and the possibility to discriminate a lot of strains.

Efficiency of population-dependent sulfite against Brettanomyces bruxellensis in red wine.

Brettanomyces bruxellensis is considered as a spoilage yeast encountered mainly in red wine. It is able to reduce vinylphenols from phenolic acids to ethylphenols. These volatiles are responsible for the phenolic "Brett character" described as animal, farm, horse sweat and animal leather odors. Other molecules are responsible for organoleptic deviations described as "mousiness taint". SO2 is the product most often used by winemakers to prevent B. bruxellensis growth. Usually, the recommended molecular dose of SO2 (active SO2, mSO2) is highly variable, from 0.3 to 0.8mg/L. But these doses do not take into account differences of strain resistance to sulfites or population levels. Moreover, SO2 is known as a chemical stressor inducing a viable but nonculturable (VBNC) state of B. bruxellensis. These cells, which are non-detectable by plate counting, can lead to new contamination when the amount of sulfite decreases over time. Consequently, we first assessed the effect of SO2 levels in red wine on two strains with phenotypically different sulfite resistances. Then, we studied the relationship between amounts of SO2 (0, 0.5, 0.9 and 1.1mg/L active SO2) and population levels (103, 104 and 105cells/mL) in red wine. Yeasts were enumerated by both plate counting and flow cytometry over time using viability dye. Our results showed different SO2 resistances according to the strain used. A relationship between yeast population level and SO2 resistance was demonstrated: the higher the yeast concentration, the lower the efficiency of SO2. Under certain conditions, the VBNC state of B. bruxellensis was highlighted in red wine. Yeasts in this VBNC state did not produce 4-EP. Moreover, cells became culturable again over time. All these results provide new information enabling better management of sulfite addition during wine aging.

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.

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.

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.

Partial vinylphenol reductase purification and characterization from Brettanomyces bruxellensis.

Brettanomyces is the major microbial cause for wine spoilage worldwide and causes significant economic losses. The reasons are the production of ethylphenols that lead to an unpleasant taint described as 'phenolic odour'. Despite its economic importance, Brettanomyces has remained poorly studied at the metabolic level. The origin of the ethylphenol results from the conversion of vinylphenols in ethylphenol by Brettanomyces hydroxycinnamate decarboxylase. However, no information is available on the vinylphenol reductase responsible for the conversion of vinylphenols in ethylphenols. In this study, a vinylphenol reductase was partially purified from Brettanomyces bruxellensis that was active towards 4-vinylguaiacol and 4-vinylphenol only among the substrates tested. First, a vinylphenol reductase activity assay was designed that allowed us to show that the enzyme was NADH dependent. The vinylphenol reductase was purified 152-fold with a recovery yield of 1.77%. The apparent K(m) and V(max) values for the hydrolysis of 4-vinylguaiacol were, respectively, 0.14 mM and 1900 U mg(-1). The optimal pH and temperature for vinylphenol reductase were pH 5-6 and 30 degrees C, respectively. The molecular weight of the enzyme was 26 kDa. Trypsic digest of the protein was performed and the peptides were sequenced, which allowed us to identify in Brettanomyces genome an ORF coding for a 210 amino acid protein.

Effects of yeast proteolytic activity on Oenococcus oeni and malolactic fermentation.

Alcoholic fermentation of synthetic must was performed using either Saccharomyces cerevisiae or a mutant Deltapep4, which is deleted for the proteinase A gene. Fermentation with the mutant Deltapep4 resulted in 61% lower levels of free amino acids, and in 62% lower peptide concentrations at the end of alcoholic fermentation than in the control. Qualitative differences in amino acid composition were observed. Changes observed in amino acids in peptides were mainly quantitative. After alcoholic fermentation, each medium was inoculated with Oenococcus oeni. Malolactic fermentation in the medium with the Deltapep4 strain took 10 days longer than the control. This difference may have been due to a difference in the nitrogen composition of the two media. Free amino acids and amino acids in peptides were poorly consumed by O. oeni. Thus, the qualitative aspects of nitrogen composition, which depend in part on yeast metabolism, may be a determinant for the optimal growth of O. oeni in wine.

Oenococcus oeni preference for peptides: qualitative and quantitative analysis of nitrogen assimilation.

Optimization of malolactic fermentation in wine depends mainly on better understanding of nitrogen nutritional requirements of Oenococcus oeni. Four widely used starter strains and the reference ATCC BAA-1163 strain were grown in media containing different N sources: free amino acids, oligopeptides (0.5-10 kDa) or polypeptides (> 10 kDa). Amino acid auxotrophies were determined by the single omission technique. The tested strains were indifferent to only two to four amino acids and two of the starter strains appeared to be particularly demanding. Nitrogen consumption was investigated and a significant level of nitrogen was consumed by O. oeni only in the free amino acid medium. In media containing complex nitrogen sources, a global balance above 5 mg N l(-1) was enough to ensure biomass formation of all tested strains. Moreover, for all strains, bacterial growth yield was higher in the presence of nitrogen from peptides than that from free amino acids. However, no direct relationship between the bacterial growth level and the amount of nitrogen metabolized could be established. These findings were discussed in relation to the physiology of wine malolactic bacteria.

Saccharomyces cerevisiae-Oenococcus oeni interactions in wine: current knowledge and perspectives.

Winemaking can be summarized as the biotransformation of must into wine, which is performed principally by Saccharomyces cerevisiae strains during the primary or alcoholic fermentation. A secondary fermentation, the so-called malolactic fermentation (MLF) is a biodeacidification that is often encouraged, since it improves wine stability and quality. Malolactic fermentation usually occurs either spontaneously or after inoculation with selected bacteria after alcoholic fermentation. The main organism responsible for MLF, the lactic acid bacterium Oenococcus oeni, develops in physicochemically harsh conditions, which may lead to MLF failure. Furthermore, yeast that ferment must before or together with O. oeni can prevent or stimulate the progress of MLF. These phenomena are part of the interactions observed between yeast and bacteria. The mechanisms that govern yeast bacteria interaction are reviewed and the consequences for winemaking are discussed. In the light of recent advances, future prospects are also presented.

Comparison of components released by fermented or active dried yeasts after aging on lees in a model wine.

Comparison of different components released during autolysis with fermented or active dried yeast, which has never been reported previously, is related in this paper. Three autolysates were elaborated with Saccharomyces cerevisiae in a model wine (pH 3.5) at 30 or 18 degrees C. Composition of the autolysate appears to depend on both the growth medium and the physiological state of the yeast. The autolysate obtained from active dried yeast presents a higher total nitrogen concentration (a factor of 1.5-1.9 for the fermented yeast autolysate), a greater proportion of free amino acids (42 vs 16-25%), the lowest proportion of oligopeptides (25 vs 31-37%) and polypeptides (27 vs 45-34%), and an absence of nucleosides. Distribution of amino acids in peptides and proteins is relatively homogeneous in the autolysates but shows significant differences in free amino acids. Parietal macromolecules (mannoproteins and glucans) are present in greater quantities in autolysates elaborated from fermented yeast. The influence of the different fractions of these autolysates (<0.5, 0.5-1, 1-10, and >10 kDa) on bacterial growth has been investigated.