Effect of abiotic and biotic factors on Brettanomyces bruxellensis bioadhesion properties.

Biofilms are central to microbial life because of the advantage that this mode of life provides, whereas the planktonic form is considered to be transient in the environment. During the winemaking process, grape must and wines host a wide diversity of microorganisms able to grow in biofilm. This is the case of Brettanomyces bruxellensis considered the most harmful spoilage yeast, due to its negative sensory effect on wine and its ability to colonise stressful environments. In this study, the effect of different biotic and abiotic factors on the bioadhesion and biofilm formation capacities of B. bruxellensis was analyzed. Ethanol concentration and pH had negligible effect on yeast surface properties, pseudohyphal cell formation or bioadhesion, while the strain and genetic group factors strongly modulated the phenotypes studied. From a biotic point of view, the presence of two different strains of B. bruxellensis did not lead to a synergistic effect. A competition between the strains was rather observed during biofilm formation which seemed to be driven by the strain with the highest bioadhesion capacity. Finally, the presence of wine bacteria reduced the bioadhesion of B. bruxellensis. Due to biofilm formation, O. oeni cells were observed attached to B. bruxellensis as well as extracellular matrix on the surface of the cells.

High intraspecific variation of the cell surface physico-chemical and bioadhesion properties in Brettanomyces bruxellensis.

Brettanomyces bruxellensis is the most damaging spoilage yeast in the wine industry because of its negative impact on the wine organoleptic qualities. The strain persistence in cellars over several years associated with recurrent wine contamination suggest specific properties to persist and survive in the environment through bioadhesion phenomena. In this work, the physico-chemical surface properties, morphology and ability to adhere to stainless steel were studied both on synthetic medium and on wine. More than 50 strains representative of the genetic diversity of the species were considered. Microscopy techniques made it possible to highlight a high morphological diversity of the cells with the presence of pseudohyphae forms for some genetic groups. Analysis of the physico-chemical properties of the cell surface reveals contrasting behaviors: most of the strains display a negative surface charge and hydrophilic behavior while the Beer 1 genetic group has a hydrophobic behavior. All strains showed bioadhesion abilities on stainless steel after only 3 h with differences in the concentration of bioadhered cells ranging from 2.2 × 102 cell/cm2 to 7.6 × 106 cell/cm2. Finally, our results show high variability of the bioadhesion properties, the first step in the biofilm formation, according to the genetic group with the most marked bioadhesion capacity for the beer group.

Brettanomyces bruxellensis: Overview of the genetic and phenotypic diversity of an anthropized yeast.

Human-associated microorganisms are ideal models to study the impact of environmental changes on species evolution and adaptation because of their small genome, short generation time, and their colonization of contrasting and ever-changing ecological niches. The yeast Brettanomyces bruxellensis is a good example of organism facing anthropogenic-driven selective pressures. It is associated with fermentation processes in which it can be considered either as a spoiler (e.g., winemaking, bioethanol production) or as a beneficial microorganism (e.g., production of specific beers, kombucha). In addition to its industrial interests, noteworthy parallels and dichotomies with Saccharomyces cerevisiae propelled B. bruxellensis as a valuable complementary yeast model. In this review, we emphasize that the broad genetic and phenotypic diversity of this species is only beginning to be uncovered. Population genomic studies have revealed the coexistence of auto- and allotriploidization events with different evolutionary outcomes. The different diploid, autotriploid and allotriploid subpopulations are associated with specific fermented processes, suggesting independent adaptation events to anthropized environments. Phenotypically, B. bruxellensis is renowned for its ability to metabolize a wide variety of carbon and nitrogen sources, which may explain its ability to colonize already fermented environments showing low-nutrient contents. Several traits of interest could be related to adaptation to human activities (e.g., nitrate metabolization in bioethanol production, resistance to sulphite treatments in winemaking). However, phenotypic traits are insufficiently studied in view of the great genomic diversity of the species. Future work will have to take into account strains of varied substrates, geographical origins as well as displaying different ploidy levels to improve our understanding of an anthropized yeast's phenotypic landscape.

Evaluating the influence of operational parameters of pulsed light on wine related yeasts: focus on inter- and intra-specific variability sensitivity.

In oenology, there is a growing demand by consumers for wines produced with less inputs (such as sulphite, frequently used for microbial control). Emerging control methods for managing microorganisms in wine are widely studied. In this study, the efficiency of pulsed light (PL) treatment was investigated. A drop-platted system was used to evaluate the impact of three PL operational parameters: the fluence per flash, the total fluence and the flash frequency. Fluence per flash appeared to be a key parameter prior to total fluence, thus demonstrating the importance of the effect of peak voltage during PL treatments. The efficiency of PL treatment was assessed on 198 strains distributed amongst fourteen yeast species related to wine environment, and an important variability in PL response was observed. Brettanomyces bruxellensis strains were strongly sensitive to PL, with intraspecific variation. PL was then applied to red wines inoculated with 9 strains of B. bruxellensis, Saccharomyces cerevisiae and Lachancea thermotolerans. Results confirmed interspecific response variability and a higher sensitivity of B. bruxellensis species to PL. Wine treatments with a total fluence of 22.8 J cm-2 resulted in more than 6 log reduction for different B. bruxellensis strains. These results highlight the potential of PL for wine microbial stabilization.

Assessment of chitosan antimicrobial effect on wine microbes.

Chitosan is an active highly charged polysaccharide that has initially been developed in oenology to eliminate the spoilage yeast B. bruxellensis. However, different forms of chitosan exist, some complying with EU regulation for their use in wines, others not. Moreover, with the trend in oenology of limiting SO2, more and more questions arise as to the impact of chitosan on other microorganisms of the grape and wine environment. We investigated the antimicrobial efficiency of chitosan on a large oenological microbial collection, englobing technological as well as spoilage microorganisms. Results show that most species are affected at least transiently. Furthermore, a high variability prevails within most species and sensitive, intermediate and tolerant strains can be observed. This study also highlights different efficiencies depending on the wine parameters or the winemaking stage, giving important indications on which winemaking issues can be solved using chitosan. Chitosan treatment does not seem to be appropriate to limit the musts microbial pressure and Saccharomyces cerevisiae cannot be stopped during alcoholic fermentation, especially in sweet wines. Likewise, acetic acid bacteria are poorly impacted by chitosan. After alcoholic fermentation, chitosan can efficiently limit non-Saccharomyces yeast and lactic acid bacteria but special care should be given as to whether malolactic fermentation is wanted or not. Indeed, O. oeni can be severely impacted by chitosan, even months after treatment. Finally, this study highlights the crucial importance of the chitosan type used in its efficiency towards microbial stabilization. While a high molecular weight chitosan has limited antimicrobial properties, a chitosan with a much lower one, complying with EU and OIV regulation and specifications for its use in wine is much more efficient.

Impact of Grape Maturity on Ester Composition and Sensory Properties of Merlot and Tempranillo Wines.

The goal of this study was to evaluate how grape composition modifications linked to maturity level could affect the wine ester composition and aromatic expression. An experimental design has been developed from grapes of Vitis vinifera cv Merlot and cv Tempranillo. On each vine plot, grapes have been harvested at two maturity levels and have been fermented using a commercial yeast strain under standardized conditions, specifically after having the sugar and nitrogen concentrations adjusted to the same target values. Tempranillo wine ester content was not impacted by the maturity level, whereas Merlot wines from the highest maturity level showed lower concentrations for fatty acid ethyl esters and higher alcohol acetates but higher concentrations for substituted ethyl esters. Sensory analysis corroborated these analytical results: when Merlot maturity increased, wine fruity aromatic expression decreased (particularly its global intensity and the fresh, red-berry, and fermentative fruit characters). In addition, aromatic reconstitution experiments showed that esters were not, alone, responsible for the sensory differences linked to grapes' maturity. Globally, our results highlight the role of esters in the overall wine fruity aromatic expression associated to Merlot ripeness and show that their levels are impacted by other parameters than the grape content in sugars and amino acids, well known as being their precursors.

Impact of Lachancea thermotolerans on Chemical Composition and Sensory Profiles of Viognier Wines.

Viognier is a warm climate grape variety prone to loss of acidity and accumulation of excessive sugars. The yeast Lachancea thermotolerans can improve the stability and balance of such wines due to the partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in co-inoculations and sequential inoculations with Saccharomyces cerevisiae in high sugar/pH Viognier fermentations. The results highlighted the dichotomy between the non-acidified and the bio-acidified L. thermotolerans treatments, with either comparable or up to 0.5 units lower pH relative to the S. cerevisiae control. Significant differences were detected in a range of flavour-active yeast volatile metabolites. The perceived acidity mirrored the modulations in wine pH/TA, as confirmed via "Rate-All-That-Apply" sensory analysis. Despite major variations in the volatile composition and acidity alike, the varietal aromatic expression (i.e., stone fruit aroma/flavour) remained conserved between the treatments.

Different trajectories of polyploidization shape the genomic landscape of the Brettanomyces bruxellensis yeast species.

Polyploidization events are observed across the tree of life and occur in many fungi, plant, and animal species. During evolution, polyploidy is thought to be an important source of speciation and tumorigenesis. However, the origin of polyploid populations is not always clear, and little is known about the precise nature and structure of their complex genome. Using a long-read sequencing strategy, we sequenced 71 strains from the Brettanomyces bruxellensis yeast species, which is found in anthropized environments (e.g., beer, contaminant of wine, kombucha, and ethanol production) and characterized by several polyploid subpopulations. To reconstruct the polyploid genomes, we phased them by using different strategies and found that each subpopulation had a unique polyploidization history with distinct trajectories. The polyploid genomes contain either genetically closely related (with a genetic divergence <1%) or diverged copies (>3%), indicating auto- as well as allopolyploidization events. These latest events have occurred independently with a specific and unique donor in each of the polyploid subpopulations and exclude the known Brettanomyces sister species as possible donors. Finally, loss of heterozygosity events has shaped the structure of these polyploid genomes and underline their dynamics. Overall, our study highlights the multiplicity of the trajectories leading to polyploid genomes within the same species.

Wine yeast species show strong inter- and intra-specific variability in their sensitivity to ultraviolet radiation.

While the trend in winemaking is toward reducing the inputs and especially sulphites utilization, emerging technologies for the preservation of wine is a relevant topic for the industry. Amongst yeast spoilage in wine, Brettanomyces bruxellensis is undoubtedly the most feared. In this study, UV-C treatment is investigated. This non-thermal technique is widely used for food preservation. A first approach was conducted using a drop-platted system to compare the sensitivity of various strains to UV-C surface treatment. 147 strains distributed amongst fourteen yeast species related to wine environment were assessed for six UV-C doses. An important variability in UV-C response was observed at the interspecific level. Interestingly, cellar resident species, which are mainly associated with wine spoilage, shows higher sensitivity to UV-C than vineyard-resident species. A focus on B. bruxellensis species with 104 screened strains highlighted an important effect of the UV-C, with intra-specific variation. This intra-specific variation was confirmed on 6 strains in liquid red wine by using a home-made pilot. 6624 J.L-1 was enough for a reduction of 5 log10 of magnitude for 5 upon 6 strains. These results highlight the potential of UV-C utilization against wine yeast spoiler at cellar scale.

Untargeted LC-HRMS profiling followed by targeted fractionation to discover new taste-active compounds in spirits.

Taste is a key driver of food and beverage acceptability due to its role in consumers' pleasure. The great interest that natural food and beverages now arouse lies notably in the complexity of their taste, which in turn is related to a wide range of taste-active compounds. Going beyond the classic divide between targeted and untargeted strategies, an integrative methodology to spirits was applied. Untargeted profiling of several cognac spirits was implemented by LC-HRMS to identify compounds of interest among hundreds of ions. A targeted fractionation protocol was then developed. By using HRMS and NMR, dihydrodehydrodiconiferyl alcohol was identified and described for the first time in spirits and oak wood. It was characterized as sweet at 2 mg/L in two matrices and was quantified in spirits up to 4 mg/L. These findings demonstrated how this methodology is relevant and effective to discover new taste-active compounds.

Metabolic, Organoleptic and Transcriptomic Impact of Saccharomyces cerevisiae Genes Involved in the Biosynthesis of Linear and Substituted Esters.

Esters constitute a broad family of volatile compounds impacting the organoleptic properties of many beverages, including wine and beer. They can be classified according to their chemical structure. Higher alcohol acetates differ from fatty acid ethyl esters, whereas a third group, substituted ethyl esters, contributes to the fruitiness of red wines. Derived from yeast metabolism, the biosynthesis of higher alcohol acetates and fatty acid ethyl esters has been widely investigated at the enzymatic and genetic levels. As previously reported, two pairs of esterases, respectively encoded by the paralogue genes ATF1 and ATF2, and EEB1 and EHT1, are mostly involved in the biosynthesis of higher alcohol acetates and fatty acid ethyl esters. These esterases have a moderate effect on the biosynthesis of substituted ethyl esters, which depend on mono-acyl lipases encoded by MGL2 and YJU3. The functional characterization of such genes helps to improve our understanding of substituted ester metabolism in the context of wine alcohol fermentation. In order to evaluate the overall sensorial impact of esters, we attempted to produce young red wines without esters by generating a multiple esterase-free strain (Δatf1, Δatf2, Δeeb1, and Δeht1). Surprisingly, it was not possible to obtain the deletion of MGL2 in the Δatf1/Δatf2/Δeeb1/Δeht1 background, highlighting unsuspected genetic incompatibilities between ATF1 and MGL2. A preliminary RNA-seq analysis depicted the overall effect of the Δatf1/Δatf2/Δeeb1/Δeht1 genotype that triggers the expression shift of 1124 genes involved in nitrogen and lipid metabolism, but also chromatin organization and histone acetylation. These findings reveal unsuspected regulatory roles of ester metabolism in genome expression for the first time.

Impact of Lachancea thermotolerans on chemical composition and sensory profiles of Merlot wines.

Wines from warm(ing) climates often contain excessive ethanol but lack acidity. The yeast Lachancea thermotolerans can ameliorate such wines due to partial conversion of sugars to lactic acid during alcoholic fermentation. This study compared the performance of five L. thermotolerans strains in two inoculation modalities (sequential and co-inoculation) to Saccharomyces cerevisiae and un-inoculated treatments in high sugar/low acidity Merlot fermentations. The pH and ethanol levels in mixed-culture dry wines were either comparable, or significantly lower than in controls (decrease of up to 0.5 units and 0.90% v/v, respectively). The analysis of volatile compounds revealed marked differences in major flavour-active yeast metabolites, including up to a thirty-fold increase in ethyl lactate in certain L. thermotolerans modalities. The wines significantly differed in acidity perception, alongside 18 other sensory attributes. Together, these results highlight the potential of some L. thermotolerans strains to produce 'fresher' wines with lower ethanol content and improved flavour/balance.

Quantifying the effect of human practices on S. cerevisiae vineyard metapopulation diversity.

Saccharomyces cerevisiae is the main actor of wine fermentation but at present, still little is known about the factors impacting its distribution in the vineyards. In this study, 23 vineyards and 7 cellars were sampled over 2 consecutive years in the Bordeaux and Bergerac regions. The impact of geography and farming system and the relation between grape and vat populations were evaluated using a collection of 1374 S. cerevisiae merlot grape isolates and 289 vat isolates analyzed at 17 microsatellites loci. A very high genetic diversity of S. cerevisiae strains was obtained from grape samples, higher in conventional farming system than in organic one. The geographic appellation and the wine estate significantly impact the S. cerevisiae population structure, whereas the type of farming system has a weak global effect. When comparing cellar and vineyard populations, we evidenced the tight connection between the two compartments, based on the high proportion of grape isolates (25%) related to the commercial starters used in the cellar and on the estimation of bidirectional geneflows between the vineyard and the cellar compartments.

+Brettanomyces bruxellensis Displays Variable Susceptibility to Chitosan Treatment in Wine.

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 B. bruxellensis 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.

SSU1 Checkup, a Rapid Tool for Detecting Chromosomal Rearrangements Related to the SSU1 Promoter in Saccharomyces cerevisiae: An Ecological and Technological Study on Wine Yeast.

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological Saccharomyces cerevisiae strains, CR involving the promoter region of the gene SSU1 lead to a higher sulfite tolerance by enhancing the SO2 efflux. To date, three different SSU1 associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method (SSU1 checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 S. cerevisiae strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the SSU1 promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged SSU1 promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of SSU1 promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO2 concentrations. The SSU1 checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers.

Brettanomyces bruxellensis phenotypic diversity, tolerance to wine stress and wine spoilage ability.

Brettanomyces bruxellensis is a yeast species found in many fermented matrices. A high level of genetic diversity prevails in this species and was recently connected with tolerance to sulfur dioxide, the main preservative used in wine. We therefore examine other phenotypes that may modulate the ability of the species to spoil wine, in a selection of representative strains. The species shows a fairly high homogeneity with respect to the carbohydrates that can support growth, but more diverse behaviors regarding tolerance to low pH or ethanol. Thought no clear link can be drawn with genotype, some strains appear more tolerant than the others, mainly in the AWRI1499 like genetic group. Volatile phenol production is ubiquitous within the species, independent from yeast growth profile and not affected by the nature of the growth substrate. The specific production. n rate of volatile phenol production raises in case of increased aeration. It is little affected by pH decrease until 3.0 or by ethanol concentration increase up to 12% vol, but it decreased in case of increased constraint (pH < 3.0, Ethanol ≥14% vol) or combination of constraints. All the strain studied have thus the ability to spoil wine but some outstanding dangerous strains can even spoil the wine with high level of constrainst.

Non-Saccharomyces Commercial Starter Cultures: Scientific Trends, Recent Patents and Innovation in the Wine Sector.

For 15 years, non-Saccharomyces starter cultures represent a new interesting segment in the dynamic field of multinationals and national companies that develop and sell microbial-based biotechnological solutions for the wine sector. Although the diversity and the properties of non- Saccharomyces species/strains have been recently fully reviewed, less attention has been deserved to the commercial starter cultures in term of scientific findings, patents, and their innovative applications. Considering the potential reservoir of biotechnological innovation, these issues represent an underestimated possible driver of coordination and harmonization of research and development activities in the field of wine microbiology. After a wide survey, we encompassed 26 different commercial yeasts starter cultures formulated in combination with at least one non-Saccharomyces strain. The most recent scientific advances have been explored delving into the oenological significance of these commercial starter cultures. Finally, we propose an examination of patent literature for the main yeasts species commercialised in non-Saccharomyces based products. We highlight the presence of asymmetries among scientific findings and the number of patents concerning non-Saccharomyces-based commercial products for oenological purposes. Further investigations on these microbial resources might open new perspectives and stimulate attractive innovations in the field of wine-making biotechnologies.

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.

Brettanomyces bruxellensis wine isolates show high geographical dispersal and long persistence in cellars.

Brettanomyces bruxellensis is the main wine spoiler yeast all over the world, yet the structure of the populations associated with winemaking remains elusive. In this work, we considered 1411 wine isolates from 21 countries that were genotyped using twelve microsatellite markers. We confirmed that B. bruxellensis isolates from wine environments show high genetic diversity, with 58 and 42% of putative triploid and diploid individuals respectively distributed in 5 main genetic groups. The distribution in the genetic groups varied greatly depending on the country and/or the wine-producing region. However, the two possible triploid wine groups showing sulfite resistance/tolerance were identified in almost all regions/countries. Genetically identical isolates were also identified. The analysis of these clone groups revealed that a given genotype could be isolated repeatedly in the same winery over decades, demonstrating unsuspected persistence ability. Besides cellar residency, a great geographic dispersal was also evidenced, with some genotypes isolated in wines from different continents. Finally, the study of old isolates and/or isolates from old vintages revealed that only the diploid groups were identified prior 1990 vintages. The putative triploid groups were identified in subsequent vintages, and their proportion has increased steadily these last decades, suggesting adaptation to winemaking practices such as sulfite use. A possible evolutionary scenario explaining these results is discussed.

Genetic and Phenotypic Characterisation of a Saccharomyces cerevisiae Population of 'Merwah' White Wine.

The study of yeast biodiversity represents an important step in the preservation of the local heritage, and this work in particular has an innovative character since no further studies have investigated 'Merwah', one of the main grape varieties used in winemaking in Lebanon. To gain deeper knowledge of the genetic diversity and population structure of native Saccharomyces cerevisiae wine strains, 202 isolates were collected during spontaneous alcoholic fermentation of eight must/wine samples of cultivar 'Merwah', over two consecutive years (2016, 2017) in a traditional winery in Mount Lebanon (1400 m a.s.l.). The isolates were identified as S. cerevisiae on the basis of their morphology and preliminary sequence analysis of their internal transcribed spacer (ITS) PCR. They were then characterised at the strain level by interdelta PCR and genotyped using multiplex PCR reactions of 12 microsatellite markers. High genetic diversity was observed for the studied population. To select potential yeast starter strains from this population, micro-fermentations were carried out for 22 S. cerevisiae strains that were selected as representative of the 'Merwah' wine yeast population in order to determine their technological and oenological properties. Three indigenous yeast strains might represent candidates for pilot-scale fermentation in the winery, based on relevant features such as high fermentation vigour, low production of volatile acidity and H2S and low residual sugar content at the end of alcoholic fermentation.