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.

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.

Time course accumulation of polysulfides in Chardonnay and model juice fermentations.

Over the last five years, polysulfides in wine have become a topic of interest. The finding that both yeast and the wine composition could contribute to the formation and evolution of these compounds has prompted several studies, further corroborated by the discovery of a link between polysulfides and H2S release during ageing. In the present study, the accumulation of cysteinylated and glutathionylated polysulfides was followed during fermentation for the first time. Synthetic grape media and Chardonnay juice treated with CuSO4, elemental sulfur or both were fermented, and subsequently analysed using an in-house UHPLC-HRMS method. Differences in polysulfide accumulation were observed between the two media, highlighting the role of the matrix composition. Elemental sulfur, especially when combined with CuSO4, resulted in significantly increased accumulations of polysulfides compared to controls in both media. Polysulfides with longer S-chains generally appeared later in the fermentation, an observation that prompted further questions on these metabolites' formation.

A novel LC-HRMS method reveals cysteinyl and glutathionyl polysulfides in wine.

A novel ultra high pressure liquid chromatography combined with high resolution mass spectrometry (UHPLC-HRMS) method was developed to study glutathionyl and cysteinyl polysulfides in wine. Different HPLC columns were investigated in order to optimise the chromatographic resolution of the polysulfide standard mixtures synthesised in-house. The optimisation of the chromatographic conditions when trying to separate glutathionylated and cysteinylated species containing from 3 to 5 sulfur atoms proved particularly challenging, with the cationic exchange column IonPac CS12A-MS resulting to be the best column for this task.The synergistic application of the newly developed methods together with the synthesised reference standard mixtures allowed the identification and the detection of 11 different glutathionyl and cysteinyl polysulfides. Moreover, analysing 15 young white wines was possible to confirm the presence of GSSSG in wine (GS = glutathione). More importantly, this study allowed for the first identification of several symmetric and asymmetric new polysulfides, namely: GSSSSG, CSSSC (CS = cysteine), CSSSSC, CSSSG, and CSSSSG. These molecules have not previously been identified in wine, raising the question on their biogenesis and role on wine quality.

Bulk and compound-specific stable isotope ratio analysis for authenticity testing of organically grown tomatoes.

Until now, there has been a lack of analytical methods that can reliably verify the authenticity of organically grown plants and derived organic food products. In this study, stable isotope ratio analysis of hydrogen (H, δ2H), carbon (C, δ13C), nitrogen (N, δ15N), oxygen (O, δ18O) and sulfur (S, δ34S) was conducted along the tomato passata production process using organic and conventionally grown tomatoes from two Italian regions over two years. A gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) based method was developed and applied for analysis of C and N isotope ratios in amino acids derived from tomatoes. Of the bulk isotope ratios, δ15N was the most significant parameter for discriminating organic from conventional products. The classification power was improved significantly by compound-specific isotope analysis regardless of the production years and regions. We conclude that isotope analysis of amino acids is a novel analytical tool for complementing existing certification and control procedures in the organic tomato sector.

Differentiation of wood-derived vanillin from synthetic vanillin in distillates using gas chromatography/combustion/isotope ratio mass spectrometry for δ13 C analysis.

RATIONALE: Typical storage in oak barrels releases in distillates different degradation products such as vanillin, which play an important role in flavour and aroma. The addition of vanillin, as well as other aroma compounds, of different origin is prohibited by European laws. As vanillin samples from different sources have different δ13 C values, the δ13 C value could be used to determine whether the vanillin is authentic (lignin-derived), or if it has been added from another source (e.g. synthetic). METHODS: The δ13 C values for vanillin derived from different sources, including natural, synthetic and tannins, were measured by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), after diethyl ether addition and/or ethanol dilution. A method for analysing vanillin in distillates after dichloromethane extraction was developed. Tests were undertaken to prove the reliability, reproducibility and accuracy of the method with standards and samples. Distillate samples were run to measure the δ13 C values of vanillin and to compare them with values for other sources of vanillin. RESULTS: δ13 C values were determined for: natural vanillin extracts (-21.0 to -19.3‰, 16 samples); vanillin ex-lignin (-28.2‰, 1 sample); and synthetic vanillin (-32.6 to -29.3‰, 7 samples). Seventeen tannin samples were found to have δ13 C values of -29.5 to -26.7‰, which were significantly different (p < 0.05) from those of the natural and synthetic vanillins. The vanillin δ13 C values measured in distillates (-28.9 to -25.7‰) were mainly in the tannin range, although one spirit (-32.5‰) was found to contain synthetic vanillin. CONCLUSIONS: The results show that synthetic vanillin added to a distillate could be differentiated from vanillin derived from oak barrels by their respective δ13 C values. The GC/C/IRMS method could be a useful tool in the determination of adulteration of distillates.

Dietary Effects on Stable Carbon Isotope Composition of Fatty Acids in Polar and Neutral Fractions of Intramuscular Fat of Lambs.

In this study we measured δ13C values of the main fatty acids (FA) present in neutral and polar intramuscular lipids of meat samples from 24 lambs, fed with four different diets supplemented with sunflower and linseed oil and the tanniferous shrub Cistus ladanifer L. The objective was to understand if the increase in intramuscular fat observed in lambs fed simultaneously C. ladanifer and oil was explained mostly by incorporation of diet derived FA or by increased de novo FA synthesis. De novo FA synthesis was evaluated by 13C enrichment (‰) of 16:0 in intramuscular lipids compared to bulk diet or compared to dietary 16:0. Oil reduced 13C enrichment of 16:0 in muscle lipid but had no effect when the diet included C. ladanifer (P value <0.01). Thus, dietary C. ladanifer blocked the inhibitory effects of lipid supplementation on de novo FA synthesis.

Gas chromatography combined with mass spectrometry, flame ionization detection and elemental analyzer/isotope ratio mass spectrometry for characterizing and detecting the authenticity of commercial essential oils of Rosa damascena Mill.

RATIONALE: The essential oil of Rosa damascena Mill. is known for its fine perfumery application, use in cosmetic preparations and for several pharmacological activities. Due to its high value, it can be easily adulterated with flavors or cheaper oils. This study is aimed at a detailed phytochemical characterization of commercial samples of R. damascena essential oil and at their authenticity assessment. METHODS: Nineteen commercial samples of R. damascena essential oil of different geographic origin and an additional authentic one, directly extracted by hydro-distillation from fresh flowers, were considered. GC/MS and GC/FID techniques were applied for the phytochemical analysis of the samples. EA/IRMS (Elemental Analyzer/Isotope Ratio Mass Spectrometry) and GC/C (Combustion)/IRMS were used to determine the δ(13)C composition of bulk samples and of some specific components. RESULTS: Citronellol (28.7-55.3%), geraniol (13.5-27.3%) and nonadecane (2.6-18.9%) were the main constituents of Bulgarian and Turkish essential oils, while those from Iran were characterized by a high level of aliphatic hydrocarbons (nonadecane: 3.7-23.2%). The δ(13)C values of bulk samples were between -28.1 and -26.9‰, typical for C3 plants. The δ(13)C values of specific components were in the usual range for natural aromatic substances from C3 plants, except for geranyl acetate, which displayed higher values (up to -18‰). These unusual δ(13)C values were explained by the addition of a natural cheaper oil from a C4 plant (Cymbopogon martinii, palmarosa), which was found to occur in most of the essential oils. CONCLUSION: GC/C/IRMS, in combination with GC/MS and GC/FID, can be considered as an effective and reliable tool for the authenticity control of R. damascena essential oil.

Evolution and occurrence of 1,8-cineole (eucalyptol) in Australian wine.

A new method has been developed for the quantitation of 1,8-cineole in red and white wines using headspace solid-phase microextraction (SPME) combined with stable isotope dilution analysis (SIDA) and gas chromatography-mass spectrometry (GC-MS). An extensive survey of Australian wines (44 white and 146 red) highlighted that only red wines contained significant amounts of 1,8-cineole (up to 20 µg/L). Hydrolytic studies with limonene and α-terpineol, putative precursors to 1,8-cineole, showed a very low conversion into 1,8-cineole (< 0.6%) over a 2 year period, which does not account for the difference between white and red wines. 1,8-Cineole was chemically stable in model wine solution over 2 years, and absorption from a Shiraz wine by bottle closures was most evident for a synthetic closure only (14% absorption after 1 year). Two commercial ferments at two different locations were monitored daily to investigate the evolution of 1,8-cineole throughout fermentation. Both ferments showed daily increases in 1,8-cineole concentration while in contact with grape solids, but this accumulation ceased immediately after pressing. This observation is consistent with the extraction of 1,8-cineole into the ferment from the solid portions of the grape berries.

Odor detection thresholds and enantiomeric distributions of several 4-alkyl substituted gamma-lactones in Australian red wine.

The individual enantiomers of gamma-octalactone (1), gamma-nonalactone (2), gamma-decalactone (3) and gamma-dodecalactone (4) have been synthesized. The (R) series of enantiomers was prepared from L-glutamic acid by a strategy involving deamination and reduction to (S)-5-oxo-2-tetrahydrofurancarboxaldehyde (S)-7. The different length side chains were introduced by a series of Wittig reactions, varying in the choice of phosphorane used. Hydrogenation then gave the final gamma-lactones 1-4. The (S) series of enantiomers was prepared in an analogous fashion beginning with d-glutamic acid. Aroma detection thresholds for all eight enantiomers were determined in a "bag in a box" dry red wine by the application of ASTM method E 679, employing a panel of 25 members. The lowest threshold determined was 8 microg/L for (R)-dodecalactone (4) while the highest threshold was 285 microg/L for (R)-nonalactone (2). With the exception of gamma-decalactone (3) there were statistically significant differences (at the 5% level) in aroma detection thresholds between the two enantiomers of the same lactone. A stable isotope method developed for quantification of the lactones 1-4 has been extended for use with chiral phase GC (Rt-betaDEXcst capillary column) allowing quantification of the individual enantiomers. The enantiomeric distribution of gamma-octalactone (1) and gamma-nonalactone (2) in seven botrytized wines and of 2 in a total of 34 red wines were thus determined; with few exceptions, the (R) enantiomer of gamma-nonalactone (2) was found to be more prevalent than its (S) counterpart in the dry red and botrytized white wines analyzed. The same was true for gamma-octalactone (1) in the botrytized white wines.

Quantification of several 4-alkyl substituted gamma-lactones in Australian wines.

Stable isotope dilution assays have been developed for gamma-octalactone (1), gamma-nonalactone (2), gamma-decalactone (3) and gamma-dodecalactone (4) in both white and red wines for the first time. (2)H(7)-analogues of each lactone were prepared for use as internal standards via a strategy employing ring-opening, esterification and oxidation of the respective starting lactones. The methods were shown to be highly accurate and reproducible (R(2) > or = 0.999; SD < or = 1%). A large selection of Australian wines (n = 178) were analyzed for the presence of lactones 1-4. Fifty-eight white wines covering the varieties Chardonnay, Riesling, Sauvignon Blanc, Semillon and Viognier, as well as Botrytis style wines, were analyzed and showed broadly that gamma-octalactone (1) was the most common lactone, being observed above its limit of detection in 28 of the wines, followed by gamma-nonalactone (2) in 23 wines. The Botrytis style white wines had the highest concentrations of 1 and 2 (maximum concentrations 8.5 and 59 microg/L respectively). A total of 120 red wines covering the varieties Cabernet Sauvignon, Durif, Merlot, Pinot Noir and Shiraz were also studied and showed gamma-octalactone (1) and gamma-nonalactone (2) to be the most common lactones present, in 56 and 57 of the wines, respectively. gamma-Decalactone (3) was observed in only a small number (13) of red wine samples and not at all in the white varieties. gamma-Dodecalactone (4) was absent from all 178 samples studied. The highest concentrations of lactones 1, 2 and 3 in the red wines were 4.2, 39.7 and 4.0 microg/L respectively.