Bruno Fedrizzi

First Identification of 4-S-Glutathionyl-4-methylpentan-2-one, a Potential Precursor of 4-Mercapto-4-methylpentan-2-one, in Sauvignon Blanc Juice

The identification of 4-S-glutathionyl-4-methylpentan-2-one (glut-4-MMP) by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) experiments in a Sauvignon Blanc juice extract is described. Synthesis of an authentic reference compound enabled confirmation of the presence of glut-4-MMP in a Sauvignon Blanc juice for the first time. The juice extract was obtained by batch-wise percolation of 6 L of juice through a sintered glass funnel packed with C18 sorbent, followed by further purification using low-pressure chromatography on C18. Analysis of the juice extract revealed a chromatographic peak with the same retention time and mass spectrum as the synthetic reference compound, and spiking experiments verified the findings. The presence of glut-4-MMP in grape juice may be related to the biosynthesis of the relevant S-cysteinyl conjugate and, subsequently, to the formation of aroma-active 4-mercapto-4-methylpentan-2-one (4-MMP). This compound has a very low reported sensory threshold (3 ng/L) in wine and is partially responsible for the aromas that are important to the quality and style of some wine varieties.

Effect of nitrogen supplementation and saccharomyces species on hydrogen sulfide and other volatile sulfur compounds in Shiraz fermentation and wine

A Shiraz must with low yeast assimilable nitrogen (YAN) was supplemented with two increasing concentrations of diammonium phosphate (DAP) and fermented with one Saccharomyces cerevisiae and one Saccharomyces bayanus strain, with maceration on grape skins. Hydrogen sulfide (H(2)S) was monitored throughout fermentation, and a total of 16 volatile sulfur compounds (VSCs) were quantified in the finished wines. For the S. cerevisiae yeast strain, addition of DAP to a final YAN of 250 or 400 mg/L resulted in an increased formation of H(2)S compared to nonsupplemented fermentations (100 mg/L YAN). For this yeast, DAP-supplemented fermentations also showed prolonged formation of H(2)S into the later stage of fermentation, which was associated with increased H(2)S in the final wines. The S. bayanus strain showed a different H(2)S production profile, in which production was inversely correlated to initial YAN. No correlation was found between total H(2)S produced by either yeast during fermentation and H(2)S concentration in the final wines. For both yeasts, DAP supplementation yielded higher concentrations of organic VSCs in the finished wines, including sulfides, disulfides, mercaptans, and mercaptoesters. PCA analysis indicated that nitrogen supplementation before fermentation determined a much clearer distinction between the VSC profiles of the two yeasts compared to nonsupplemented fermentations. These results raise questions concerning the widespread use of DAP in the management of low YAN fermentations with respect to the formation of reductive characters in wine.

Regional microbial signatures positively correlate with differential wine phenotypes: evidence for a microbial aspect to terroir

Many crops display differential geographic phenotypes and sensorial signatures, encapsulated by the concept of terroir. The drivers behind these differences remain elusive, and the potential contribution of microbes has been ignored until recently. Significant genetic differentiation between microbial communities and populations from different geographic locations has been demonstrated, but crucially it has not been shown whether this correlates with differential agricultural phenotypes or not. Using wine as a model system, we utilize the regionally genetically differentiated population of Saccharomyces cerevisiae in New Zealand and objectively demonstrate that these populations differentially affect wine phenotype, which is driven by a complex mix of chemicals. These findings reveal the importance of microbial populations for the regional identity of wine, and potentially extend to other important agricultural commodities. Moreover, this suggests that long-term implementation of methods maintaining differential biodiversity may have tangible economic imperatives as well as being desirable in terms of employing agricultural practices that increase responsible environmental stewardship.

Development of reliable analytical tools for evaluating the influence of reductive winemaking on the quality of Lugana wines

This paper presents methods for the definition of important analytical tools, such as the development of sensitive and rapid methods for analysing reduced and oxidised glutathione (GSH and GSSG), hydroxycinnamic acids (HCA), bound thiols (GSH-3MH and Cys-3MH) and free thiols (3MH and 3MHA), and their first application to evaluate the effect of reductive winemaking on the composition of Lugana juices and wines. Lugana is a traditional white wine from the Lake Garda region (Italy), produced using a local grape variety, Trebbiano di Lugana. An innovative winemaking procedure based on preliminary cooling of grape berries followed by crushing in an inert environment was implemented and explored on a winery scale. The effects of these procedures on hydroxycinnamic acids, GSH, GSSG, free and bound thiols and flavanols content were investigated. The juices and wines produced using different protocols were examined. Moreover, wines aged in tanks for 1, 2 and 3 months were analysed. The high level of GSH found in Lugana grapes, which can act as a natural antioxidant and be preserved in must and young wines, thus reducing the need of exogenous antioxidants, was particularly interesting. Moreover, it was clear that polyphenol concentrations (hydroxycinnamic acids and catechins) were strongly influenced by winemaking and pressing conditions, which required fine tuning of pressing. Above-threshold levels of 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) were found in the wines and changed according to the winemaking procedure applied. Interestingly, the evolution during the first three months also varied depending on the procedure adopted. Organic synthesis of cysteine and glutathione conjugates was carried out and juices and wines were subjected to LC-MS/MS analysis. These two molecules appeared to be strongly affected by the winemaking procedure, but did not show any significant change during the first 3 months of post-bottling ageing. This supports the theory, already proposed in the literature, that there are other synthetic pathways for free thiol formation.

Ethyl propiolate derivatisation for the analysis of varietal thiols in wine

Varietal thiols [3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl acetate (3MHA) and 4-mercapto-4-methylpentan-2-one (4MMP)] have been extensively studied in the recent literature. Nonetheless the hardest obstacle for research focussing on this class of compounds is the lack of quick, user-friendly and sensitive analytical methods. The current paper presents the use of ethyl propriolate (ETP) as a novel derivatising agent to quantify varietal thiols and the first time quantification of the thiol-ETP adducts via gas chromatography-mass spectrometry. Method optimisation including choice of the best SPE cartridge, derivatisation pH and adducts stability is presented. Validation of the method via stable isotope dilution was carried out. Detection limits in both model wine (4MMP 7.2ng/L, 3MHA 40.0ng/L and 3MH 91.2ng/L) and white wine (4MMP 24.5ng/L, 3MHA 120.9ng/L and 3MH 194.6ng/L) for the novel ETP-based method were lower than those obtained with the p-HMB method. Finally, 14 New Zealand Sauvignon blanc were analysed with both the new method and the organo-mercury based procure: good correlations were obtained for 3MH and 3MHA. Detection limits obtained with the new methods, its rapidity and reproducibility make this protocol perfectly suitable for oenological purposes.

Model aging and oxidation effects on varietal, fermentative, and sulfur compounds in a dry botrytized red wine

From harvest until wine arrives to the consumer, oxygen plays a crucial role in the definition of the final aroma. In the present research, the effect of the model oxidative aging on a dry red Botrytis wine, such as Italian Amarone, was considered. Amarone wine was submitted to model oxidative aging and then analyzed with two different approaches (SPE-GC-MS and HS-SPME/GC-MS). The same sampling plan was adopted to study the model aging of the same Amarone wine in anaerobic conditions. The HS-SPME/GC-MS method was applied to investigate for the first time the effect of the oxidative aging on a vast number of fermentative sulfur compounds. This research highlighted peculiar evolutions for several volatile compounds. In particular, benzaldehyde showed a sensitive increment during the oxidative aging, with a rate much higher than that reported for non-Botrytis red wines. On the other hand, several sulfides (dimethyl sulfide, 3-(methylthio)-1-propanol, etc.) disappeared after just 15 days of oxidative aging. A wine oxidation marker such as 3-(methylthio)-propanal was not found in any of the oxidized wines; conversely methionol-S-oxide was tentatively identified. This evidence has not been mentioned in the literature. A possible involvement of grape withering process and Botrytis in these mechanisms was supposed: a dry red wine, produced from the same but without any grape withering process and Botrytis infection (e.g., Bardolino wine), was submitted to oxidative aging and analysis. This red wine showed an evolution similar to those reported in the literature for dry red wines but significantly different from the Amarone wine.

Variation of some fermentative sulfur compounds in Italian "millesime" classic sparkling wines during aging and storage on lees.

Fermentative sulfur compounds are recognized as strongly affecting wine aroma, mainly as off-flavors, but recently also as possible positive contributors to wine quality and typicity in still wines. Nevertheless, no evidence has been provided for the influence of these molecules on sparkling wine aroma, except for peculiar volatile thiols found in French Champagne. According to the traditional method, the second fermentation, occurring in sealed bottles, is the essence of the procedure. After this fermentation, sparkling wine is aged on yeast lees for a period ranging from a few months to several years so that yeast autolysis can take place. So far, no evidence is provided for the effect of yeast contact duration on the level of sulfur compounds. Following a HS-SPME/GC-MS method, 13 sulfur compounds, that is, ethylmercaptan, dimethyl sulfide, diethyl sulfide, dimethyl disulfide, diethyl disulfide, methyl thioacetate, ethyl thioacetate, 2-mercaptoethanol, 2-(methylthio)-1-ethanol, 3-(methylthio)-1-propanol, 4-(methylthio)-1-butanol, benzothiazole, and 5-(2-hydroxyethyl)-4-methylthiazole, were quantified in several Italian sparkling wines, produced according to the traditional method in two wineries from Trentino-South Tyrol, region. Additionally, the influence of winemaking technology differences and vintage effects on the evolution of the quoted sulfur compounds was considered. This investigation was carried out by coupling the HS-SPME data with those obtained by SPE method and relevant to other volatile compounds, which are considered as winemaking markers. This work furnished the first evidence of the effect of aging and lees contact at different storage temperatures on the levels of these analytes in sparkling wines. Significant increments were observed for dimethyl sulfide, diethyl sulfide, 2-(methylthio)-1-ethanol, 3-(methylthio)-1-propanol, and 4-(methylthio)-1-butanol during aging with a different variation slope possibly due to the remarkably different storage temperatures. No clear influence of lees contact duration was found for the majority of the sulfur compounds considered.

Evolution of Volatile Sulfur Compounds during Wine Fermentation

Volatile sulfur compounds (VSCs) play a significant role in the aroma of foods and beverages. With very low sensory thresholds and strong unpleasant aromas, most VSCs are considered to have a negative impact on wine quality. In this study, headspace solid phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS) was used to analyze the time course of the biosynthesis of 12 VSCs formed during wine fermentation. Two different strains of Saccharomyces cerevisiae, the laboratory strain BY4743 and a commercial strain, F15, were assessed using two media: synthetic grape media and Sauvignon Blanc juice. Seven VSCs were detected above background, with three rising above their sensory thresholds. The data revealed remarkable differences in the timing and evolution of production during fermentation, with a transient spike in methanethiol production early during anaerobic growth. Heavier VSCs such as benzothiazole and S-ethyl thioacetate were produced at a steady rate throughout grape juice fermentation, whereas others, such as diethyl sulfide, appear toward the very end of the winemaking process. The results also demonstrate significant differences between yeast strains and fermentation media.

Identification of intermediates involved in the biosynthetic pathway of 3-mercaptohexan-1-ol conjugates in yellow passion fruit (Passiflora edulis f. flavicarpa)

Yellow passion fruit is one of the most well-known tropical fruits and much of its success comes from its typical aroma. Key compounds in explaining yellow passion fruit scent are volatile thiols. These molecules are reported to be present in several fruits and originate from non-volatile precursors. Such free thiols are particularly appreciated in white wines and considerable efforts have been made to try to maximise their production and understand their biosynthesis. Two main precursors have been identified so far: S-glutathionylated and S-cysteinylated precursors, the latter originating in the breaking down of the glycyl and glutamyl moieties of the former. Improving knowledge about this pathway is currently one of the main challenges in the field of aroma chemistry. Only S-cysteinylated precursors have been reported in the literature for yellow passion fruit, thus much of the biochemical pathway remains unknown. In this paper a combination of organic synthesis, MS and NMR experiments was developed in order to investigate this pathway in yellow passion fruit. The three missing stages leading to the S-cysteinylated precursor were clearly identified. Both intermediate species between S-glutathionyl and S-cysteinyl 3-mercaptohexan-1-ol were found, suggesting that the plant is capable of activating both metabolic routes. The information gained would appear to be crucial for study of this important pathway and for potentially extending this knowledge to other plants, in particular the grapevine.

Hydrogen sulfide production during yeast fermentation causes the accumulation of ethanethiol, S-ethyl thioacetate and diethyl disulfide.

Hydrogen sulfide (H2S) is produced by yeast during winemaking and possesses off-flavors reminiscent of rotten eggs. The production of H2S during fermentation has also been associated in the finished wine with the rise of additional volatile sulfur compounds (VSCs) with strong aromas of cooked onions and vegetables. To characterize these more complex VSCs produced from H2S, we performed fermentations in synthetic grape juice. H2S production was manipulated experimentally by feeding increasing concentrations of sulfate to mutant strains that are unable to incorporate H2S efficiently as part of the sulfur assimilation pathway. In finished wines from these mutants, three VSCs - ethanethiol, S-ethyl thioacetate and diethyl disulfide - increased proportionally to H2S. (34)S-labeled sulfate fed to the MET17-deleted strain was incorporated into same three VSCs, demonstrating that they are formed directly from H2S.