Hydrogen sulfide production during early yeast fermentation correlates with volatile sulfur compound biogenesis but not thiol release.

Yeasts undergo intensive metabolic changes during the early stages of fermentation. Previous reports suggest the early production of hydrogen sulfide (H2S) is associated with the release of a range of volatile sulfur compounds (VSCs), as well as the production of varietal thiol compounds 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) from six-carbon precursors, including (E)-hex-2-enal. In this study, we investigated the early H2S potential, VSCs/thiol output, and precursor metabolism of 11 commonly used laboratory and commercial Saccharomyces cerevisiae strains in chemically defined synthetic grape medium (SGM) within 12 h after inoculation. Considerable variability in early H2S potential was observed among the strains surveyed. Chemical profiling suggested that early H2S production correlates with the production of dimethyl disulfide, 2-mercaptoethanol, and diethyl sulfide, but not with 3SH or 3SHA. All strains were capable of metabolizing (E)-hex-2-enal, while the F15 strain showed significantly higher residue at 12 h. Early production of 3SH, but not 3SHA, can be detected in the presence of exogenous (E)-hex-2-enal and H2S. Therefore, the natural variability of early yeast H2S production contributes to the early output of selected VSCs, but the threshold of which is likely not high enough to contribute substantially to free varietal thiols in SGM.

Mixed yeast communities contribute to regionally distinct wine attributes.

There is evidence that vineyard yeast communities are regionally differentiated, but the extent to which this contributes to wine regional distinctiveness is not yet clear. This study represents the first experimental test of the hypothesis that mixed yeast communities-comprising multiple, region-specific, isolates, and species-contribute to regional wine attributes. Yeast isolates were sourced from uninoculated Pinot Noir fermentations from 17 vineyards across Martinborough, Marlborough, and Central Otago in New Zealand. New methodologies for preparing representative, mixed species inoculum from these significantly differentiated regional yeast communities in a controlled, replicable manner were developed and used to inoculate Pinot Noir ferments. A total of 28 yeast-derived aroma compounds were measured in the resulting wines via headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. Yeast community region of origin had a significant impact on wine aroma, explaining ∼10% of the observed variation, which is in line with previous reports of the effects of region-specific Saccharomyces cerevisiae isolates on Sauvignon Blanc ferments. This study shows that regionally distinct, mixed yeast communities can modulate wine aroma compounds in a regionally distinct manner and are in line with the hypothesis that there is a microbial component to regional distinctiveness, or terroir, for New Zealand Pinot Noir.

Characterization of polysulfides in Saccharomyces cerevisiae cells and finished wine from a cysteine-supplemented model grape medium.

Polysulfide degradation in wine can result in hydrogen sulfide (H2S) release, imparting a rotten-egg smell that is detrimental to wine quality. Although the presence of wine polysulfides has been demonstrated, their biogenesis remains unclear. This study investigated the role of Saccharomyces cerevisiae in polysulfide formation during fermentation, with and without 5 mM cysteine supplementation as an H2S source. Using an established liquid chromatography-tandem mass spectrometry method, monobromobimane derivatives of hydropolysulfides, including CysSSSH, CysSSSSH and GSSSSH, and two oxidized polysulfides, GSSG and GSSSSG, were detected in yeast cells at the end of fermentation in a grape juice-like medium. Polysulfide production by four S. cerevisiae single deletion mutants (BY4743 Δcys3, Δcys4, Δmet17 and Δtum1) showed no significant differences compared to BY4743, suggesting that uncharacterized pathways maintain cellular polysulfide homeostasis. Five mM cysteine addition increased the formation of shorter sulfur chain species, including GSS-bimane and GSSG, but did not elevate levels of longer sulfur chain species. Additionally, polysulfides with even numbers of sulfur atoms tended to predominate in cellular lysates. Oxidized polysulfides and longer chain hydropolysulfides were not detected in finished wines. This evidence suggests that these polysulfides are unstable in wine-like environments or not transported extracellularly. Collectively, our data illustrate the complexity of yeast polysulfide metabolism under fermentation conditions.

Enantioselective Total Synthesis of (R,R)-Blumenol B and d9-(R,R)-Blumenol B.

C13-norisoprenoids are of particular importance to grapes and wines, as these molecules influence wine aroma and have been shown to significantly contribute to the distinct character of various wine varieties. Blumenol B is a putative precursor to a number of important wine aroma compounds, including the well-known compounds theaspirone and vitispirane. The enantioselective synthesis of (R,R)-blumenol B from commercially available 4-oxoisophorone was achieved using a short and easily scaleable route, which was then successfully applied to the synthesis of poly-deuterated d9-blumenol B.

Synthesis, Antiproliferative Activity and Radical Scavenging Ability of 5-O-Acyl Derivatives of Quercetin.

Quercetin is a flavonoid that is found in many plant materials, including commonly eaten fruits and vegetables. The compound is well known for its wide range of biological activities. In this study, 5-O-acyl derivatives of quercetin were synthesised and assessed for their antiproliferative activity against the HCT116 colon cancer and MDA-MB-231 breast cancer cell lines; and their radical scavenging activity against the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical species. Four derivatives were found to have improved the antiproliferative activity compared to quercetin whilst retaining radical scavenging activity.

Inter-regional survey of the New Zealand Pinot noir fermentative sulfur compounds profile.

BACKGROUND: New Zealand Pinot noir is gaining increasing attention both in New Zealand and internationally, becoming the second largest grape variety for both plantings and export. Despite the growing furore around this variety, the current coverage of the volatile chemical profile remains limited, with a lack of information on the fermentative sulfur compounds content in New Zealand Pinot noir wines. RESULTS: Thirty-five Pinot noir wines from three different vintages (i.e. 2016, 2017 and 2018) form five different grape growing regions were analysed for their fermentative sulfur compounds contents. Six fermentative sulfur compounds (i.e. methanethiol, ethanethiol, dimethyl sulfide, carbon disulfide, methionol and benzothiazol) were detected and measured for the first time in New Zealand Pinot noir wines. Their concentrations were compared against previously measured Pinot noir wines from other countries, and some preliminary evidence about inter-regional and ageing effects was obtained. CONCLUSION: The present study reports the first survey of the inter-regional differences in fermentative sulfur compounds contents in 35 New Zealand Pinot noir wines. Preliminary inter-regional and vintage trends prompt further research on the role of these molecules on this wine variety. © 2020 Society of Chemical Industry.

Addition of volatile sulfur compounds to yeast at the early stages of fermentation reveals distinct biological and chemical pathways for aroma formation.

Volatile sulfur compounds (VSCs) greatly influence the sensory properties and quality of wine and arise via both biological and chemical mechanisms. VSCs formed can also act as precursors for further downstream VSCs, thus elucidating the pathways leading to their formation is paramount. Short-term additions of exogenous hydrogen sulfide (H2S), ethanethiol (EtSH), S-ethylthio acetate (ETA), methanethiol (MeSH) and S-methylthio acetate (MTA) were made to exponentially growing fermentations of synthetic grape medium. The VSC profiles produced from live yeast cells were compared with those from dead cells and no cells. Interestingly, this experiment allowed the identification of specific biochemical and/or chemical pathways; e.g. most of the conversion of H2S to EtSH, and the further step from EtSH to ETA, required the presence of live yeast cells, as did the conversion of MeSH to MTA. In contrast, the reaction from MTA to MeSH and ETA to EtSH was due primarily to chemical degradation. Ultimately, this research unravelled some of the complex interactions and interconversions between VSCs, pinpointing the key biochemical and chemical nodes. These pathways are highly interconnected and showcase the complexity of both the sulfur pathways in yeast and the reactive chemistry of sulfur-containing compounds.

The role of yeast ARO8, ARO9 and ARO10 genes in the biosynthesis of 3-(methylthio)-1-propanol from L-methionine during fermentation in synthetic grape medium.

3-(methylthio)-1-propanol (methionol), produced by yeast as an end-product of L-methionine (L-Met) catabolism, imparts off-odours reminiscent of cauliflower and potato to wine. Saccharomyces cerevisiae ARO genes, including transaminases Aro8p and Aro9p, and decarboxylase Aro10p, catalyse two key steps forming methionol via the Ehrlich pathway. We compared methionol concentrations in wines fermented by single Δaro8, Δaro9 and Δaro10 deletants in lab strain BY4743 versus wine strain Zymaflore F15, and F15 double- and triple-aro deletants versus single-aro deletants, using headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry.Deletion of two or more aro genes increased growth lag phase, with the greatest delay exhibited by F15 Δaro8 Δaro9. The single Δaro8 deletion decreased methionol by 44% in BY4743 and 92% in F15, while the Δaro9 deletion increased methionol by 46% in F15 but not BY4743. Single deletion of Δaro10 had no effect on methionol.Unexpectedly, F15 Δaro8 Δaro9 and F15 Δaro8 Δaro9 Δaro10 produced more methionol than F15 Δaro8. In the absence of Aro8p and Aro9p, other transaminases may compensate or an alternative pathway may convert methanethiol to methionol. Our results confirm that Ehrlich pathway genes differ greatly between lab and wine yeast strains, impacting downstream products such as methionol.

A new analytical method to measure S-methyl-l-methionine in grape juice reveals the influence of yeast on dimethyl sulfide production during fermentation.

BACKGROUND: Dimethyl sulfide (DMS) is a small sulfur-containing impact odorant, imparting distinctive positive and / or negative characters to food and beverages. In white wine, the presence of DMS at perception threshold is considered to be a fault, contributing strong odors reminiscent of asparagus, cooked cabbage, and creamed corn. The source of DMS in wine has long been associated with S-methyl-l-methionine (SMM), a derivative of the amino acid methionine, which is thought to break down into DMS through chemical degradation, particularly during wine ageing. RESULTS: We developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with a stable isotope dilution assay (SIDA) to measure SMM in grape juice and wine. The application of this new method for quantitating SMM, followed by the quantitation of DMS using headspace-solid phase micro-extraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS), confirmed that DMS can be produced in wine via the chemical breakdown of SMM to DMS, with greater degradation observed at 28 °C than at 14 °C. Further investigation into the role of grape juice and yeast strain on DMS formation revealed that the DMS produced from three different Sauvignon blanc grape juices, either from the SMM naturally present or SMM spiked at 50 mmol L-1 , was modulated depending on each of the four strains of Saccharomyces cerevisiae wine yeast used for fermentation. CONCLUSION: This study confirms the existence of a chemical pathway to the formation of DMS and reveals a yeast-mediated mechanism towards the formation of DMS from SMM during alcoholic fermentation. © 2019 Society of Chemical Industry.

Hydrogen sulfide and its roles in Saccharomyces cerevisiae in a winemaking context.

The rotten-egg odour of hydrogen sulfide (H2S) produced by the yeast Saccharomyces cerevisiae has attracted considerable research interest due to its huge impact on the sensory quality of fermented foods and beverages. To date, the yeast genetic mechanisms of H2S liberation during wine fermentation are well understood and yeast strains producing low levels of H2S have been developed. Studies have also revealed that H2S is not just a by-product in the biosynthesis of the sulfur-containing amino acids, but indeed a vital molecule involved in detoxification, population signalling and extending cellular life span. Moreover, polysulfides have recently emerged as key players in signalling and the sensory quality of wine because their degradation leads to the release of H2S. This review will focus on the recent findings on the production of H2S and polysulfides in S. cerevisiae and summarise their potential roles in yeast survival and winemaking. Recent advances in techniques for the detection of H2S and polysulfides offer an exciting opportunity to uncover the novel genes and pathways involved in their formation from different sulfur sources. This knowledge will not only provide further insights into yeast sulfur metabolism, but could potentially improve the sensory quality of wine.

Yeast genes involved in regulating cysteine uptake affect production of hydrogen sulfide from cysteine during fermentation.

An early burst of hydrogen sulfide (H2S) produced by Saccharomyces cerevisiae during fermentation could increase varietal thiols and therefore enhance desirable tropical aromas in varieties such as Sauvignon Blanc. Here we attempted to identify genes affecting H2S formation from cysteine by screening yeast deletion libraries via a colony colour assay on media resembling grape juice. Both Δlst4 and Δlst7 formed lighter coloured colonies and produced significantly less H2S than the wild type on high concentrations of cysteine, likely because they are unable to take up cysteine efficiently. We then examined the nine known cysteine permeases and found that deletion of AGP1, GNP1 and MUP1 led to reduced production of H2S from cysteine. We further showed that deleting genes involved in the SPS-sensing pathway such as STP1 and DAL81 also reduced H2S from cysteine. Together, this study indirectly confirms that Agp1p, Gnp1p and Mup1p are the major cysteine permeases and that they are regulated by the SPS-sensing and target of rapamycin pathways under the grape juice-like, cysteine-supplemented, fermentation conditions. The findings highlight that cysteine transportation could be a limiting factor for yeast to generate H2S from cysteine, and therefore selecting wine yeasts without defects in cysteine uptake could maximise thiol production potential.

The effect of linoleic acid on the Sauvignon blanc fermentation by different wine yeast strains.

The level of linoleic acid in the Sauvignon blanc (SB) grape juice affects the development of different aroma compounds during fermentation by Saccharomyces cerevisiae EC1118, including key varietal thiols such as 3-mercaptohexanol (3MH) and 3-mercaptohexyl acetate (3MHA). However, it is still unknown if linoleic acid would affect in a similar way other commonly used S. cerevisiae wine strains. Here we investigated the effect of grape juice linoleic acid on the development of aroma compounds and other metabolites of SB wines using different wine yeast strains: EC1118, AWRI796 and VIN13. Linoleic acid clearly affected the levels of acetylated aroma compounds, several amino acids, and antioxidant molecules, independent of yeast strain, but the production of 3MH was affected by linoleic acid in a strain-specific manner. Moreover, the supplementation of deuterium-labelled 3MH also affected the production of varietal thiols in a strain-specific way. Linoleic acid reduced the acetylation process probably by inhibiting an acetyltransferase, an effect that was independent of the yeast strain. However, regulation of the 3MH biosynthesis is strain-specific, which suggests a mindful consideration not only towards the wine yeast but also to the linoleic acid concentration in the grape juice in order to obtain the desired wine aroma characteristics.

The yeast TUM1 affects production of hydrogen sulfide from cysteine treatment during fermentation.

The undesirable rotten-egg odour of hydrogen sulfide (H2S) produced by yeast shortly after yeast inoculation of grape musts might be an important source of desirable varietal thiols, which contribute to tropical aromas in varieties such as Sauvign-on Blanc. In this study, we observed that Saccharomyces cerevisiae strains produce an early burst of H2S from cysteine. Both Δmet2 and Δmet17 strains produce a larger burst, likely because they are unable to utilise the H2S in the sulfate assimilation pathway. For the first time, we show that TUM1 is partly responsible for the early production of H2S from cysteine. Overex-pressing TUM1 elevated production of H2S, whilst its deletion yields only half of the H2S. We further confirmed that yeast convert cysteine to H2S by analysing growth of mutants lacking components of the transsulfuration pathway. High concent-rations of cysteine overcame this growth block, but required TUM1 Collectively, the data indicate that S. cerevisiae does not convert cysteine to sulfate or sulfite, but rather to sulfide via a novel pathway that requires the action of Tum1p. The findi-ngs of this study may allow the improvement of commercial yeasts through the manipulation of sulfur metabolism that are better suited towards production of fruit-driven styles.

Exploring the Equilibrium between Glut3SHal and Glut3SH-SO3: A Method for the Quantification of These Compounds in Wine.

3S-Gluthathionylhexanal (glut3SHal) is an early precursor to the important wine aroma compound 3-sulfanylhexan-1-ol (3SH), imparting tropical passion fruit aromas, even at trace concentrations. In wine, glut3SHal occurs in equilibrium with its bisulfite adduct (glut3SH-SO3), challenging its quantification. To circumvent the issues encountered when attempting to describe the equilibrium between these compounds, a method for their quantification in wine samples was developed. Separation of glut3SHal and glut3SH-SO3 using solid-phase extraction followed by oxime derivatization and analysis via liquid chromatography-mass spectrometry allowed for measurement of both compounds in wine samples. Analysis of commercial Sauvignon Blanc wines using the developed method confirmed that glut3SH-SO3 is the major species in the wine matrix. The method developed in this work will enable further exploration of the relationship between glut3SHal and glut3SH-SO3 and their contribution to production of 3SH in wines. There is potential to extrapolate this work to explore other aldehyde-sulfonic acid equilibria in foods and beverages.

Insights into the relative contribution of four precursors to 3-sulfanylhexan-1-ol and 3-sulfanylhexylacetate biogenesis during fermentation.

The desirable wine aroma compounds 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) are released during fermentation from non-volatile precursors present in the grapes. This work explores the relative contribution of four precursors (E-2-hexenal, 3-S-glutathionylhexan-1-ol, 3-S-glutathionylhexanal, and 3-S-cysteinylhexan-1-ol) to 3SH and 3SHA. Through the use of isotopically labelled analogues of these precursors in defined fermentation media, new insights into the role of each precursor have been identified. E-2-Hexenal was shown to contribute negligible amounts of thiols, while 3-S-glutathionylhexan-1-ol was the main precursor of both 3SH and 3SHA. The glutathionylated precursors were both converted to 3SHA more efficiently than 3-S-cysteinylhexan-1-ol. Interestingly, 3-S-glutathionylhexanal generated 3SHA without detectable concentrations of 3SH, suggesting possible differences in the way this precursor is metabolised compared to 3-S-glutathionylhexan-1-ol and 3-S-cysteinylhexan-1-ol. We also provide the first evidence for chemical conversion of 3-S-glutathionylhexan-1-ol to 3-S-(γ-glutamylcysteinyl)-hexan-1-ol in an oenological system.

Integration and holistic analysis of multiple multidimensional soil data sets.

Complex matrices such as soil have a range of measurable characteristics, and thus data to describe them can be considered multidimensional. These characteristics can be strongly influenced by factors that introduce confounding effects that hinder analyses. Traditional statistical approaches lack the flexibility and granularity required to adequately evaluate such matrices, particularly those with large dataset of varying data types (i.e. quantitative non-compositional, quantitative compositional). We present a statistical workflow designed to effectively analyse complex, multidimensional systems, even in the presence of confounding variables. The developed methodology involves exploratory analysis to identify the presence of confounding variables, followed by data decomposition (including strategies for both compositional and non-compositional quantitative data) to minimise the influence of these confounding factors such as sampling site/location. These data processing methods then allow for common patterns to be highlighted in the data, including the identification of biomarkers and determination of non-trivial associations between variables. We demonstrate the utility of this statistical workflow by jointly analysing the chemical composition and fungal biodiversity of New Zealand vineyard soils that have been managed with either organic low-input or conventional input approaches. By applying this pipeline, we were able to identify biomarkers that distinguish viticultural soil from both approaches and also unearth links and associations between the chemical and metagenomic profiles. While soil is an example of a system that can require this type of statistical methodology, there are a range of biological and ecological systems that are challenging to analyse due to the complex interplay of global and local effects. Utilising our developed pipeline will greatly enhance the way that these systems can be studied and the quality and impact of insight gained from their analysis.

Monitoring hydrogen sulfide de-novo formation from polysulfides present in wine using ion chromatography and ultra high-pressure liquid chromatography combined with fraction collection and high-resolution mass spectrometry.

The study of polysulfides has been a recent topic of interest for wine research due to the possibility of these compounds to release hydrogen sulfide (H2S) during storage. However, studying these compounds has been challenging for several reasons. Polysulfides are low in concentration in natural samples, they are chemically unstable and pure standards of the single compounds (RSnR with n > 2) are not commercially available. In the present study, a method was developed in order to collect a single polysulfide and study its degradation and the consequent formation of H2S. For this approach, ultra-high pressure liquid chromatography was used with an integrated fraction collector and subsequently coupled to high resolution mass spectrometry. After fractionation, the degradation of the di-cysteinyl pentasulfide (CS5C) was induced by exposure to 30 °C and the H2S formation was measured in parallel using ion-exchange chromatography. This method showed the evolutions of different polysulfides and the H2S release originating from the target compound, an observation that to the best of our knowledge has never been made before. The method in the present study demonstrated promising applications for polysulfide studies and brought us a step closer to the understanding of the chemistry of polysulfides in wine.

A green liquid chromatography-tandem mass spectrometry method for the simultaneous analysis of volatile thiols and their precursors in oenological samples.

Improvements to the quantification of three white wine impact odorants 3-sulfanylhexan-1-ol, 3-sulfanylhexyl acetate, and 4-sulfanyl-4-methylpentan-2-one, and the non-volatile precursors from which they are released during fermentation, is of great interest to the wine science community. Recent reports of a "Quick, Easy, Cheap, Effective, Rugged and Safe" (QuEChERS) based method for the concurrent analysis of these thiols and their precursors via liquid chromatography tandem mass spectrometry (LC-MS/MS) has enabled the development of far simpler methods, as well as aligning these analyses with principles of green analytical chemistry. This current work reports the development and validation of a QuEChERS based LC-MS/MS method utilising a safer derivatising agent, 4,4'-dithiodipyridine, while greatly minimising the reagents involved and waste produced. We demonstrate that this new method compares favourably to the previously reported method with repeatability of 0.2-1.3%RSD and 0.4-5.2%RSD for precursors and free thiols. Further, the commercially available internal standard, 1-hexanethiol, used in previous analytical methods was compared to stable isotope labelled analogues of the analytes, with results suggesting that it may not be a reliable internal standard.

D-optimal design of an untargeted HS-SPME-GC-TOF metabolite profiling method.

In recent times we have seen the development of many "-omics" technologies. One of the youngest is undoubtedly metabolomics, which aims to define the whole chemical fingerprint unique to each specific organism. The development and optimisation of an untargeted high-throughput method capable of investigating the volatile fraction of a biological system represents a crucial step for the success of such holistic approaches, and specific optimisation criteria must be developed in connection with suitable experimental designs. In this paper experimental designs (D-optimal) were applied for the first time as an automatic optimisation tool to an untargeted HS-SPME-GC-TOF method. In this case, optimal conditions correspond to a maximal number of detected features, in order to provide a fingerprint that is as complete as possible. The system under study is the grape berry. Four variables were considered: the type of fibre, extraction time, equilibration time and temperature. The results show that the D-optimal design methodology provides an easily interpretable assessment of experimental settings. This and other specific properties of the D-optimal design, such as the possibility to explicitly exclude certain experimental conditions, make it an extremely suitable strategy for method optimisation in untargeted metabolomics.

Attempts to Create Products with Increased Health-Promoting Potential Starting with Pinot Noir Pomace: Investigations on the Process and Its Methods.

A process for using grape (Pinot noir) pomace to produce products with improved health-promoting effects was investigated. This process integrated a solid-liquid extraction (SLE) method and a method to acylate the polyphenolics in the extract. This report describes and discusses the methods used, including the rationale and considerations behind them, and the results obtained. The study begins with the work to optimize the SLE method for extracting higher quantities of (+)-catechin, (-)-epicatechin and quercetin by trialing 28 different solvent systems on small-scale samples of Pinot noir pomace. One of these systems was then selected and used for the extraction of the same flavonoids on a large-scale mass of pomace. It was found that significantly fewer quantities of flavonoids were observed. The resultant extract was then subject to a method of derivatization using three different fatty acylating agents. The antiproliferative activities of these products were measured; however, the resulting products did not display activity against the chosen cancer cells. Limitations and improvements to the methods in this process are also discussed.