Characterization of Aroma Volatiles and their Glycosidic Precursors in Grapes and Wines

The complex aroma of wine is derived from many sources, with grape-derived components being responsible for the varietal character. The ability to monitor grape aroma compounds would allow for better understanding of how vineyard practices and winemaking processes influence the final volatile composition of the wine. Previously we developed a procedure using GC-MS combined with solid-phase microextraction (SPME) for profiling the free volatile compounds in grapes and wines. We have also recently developed a method for monitoring the ‘aroma potential’ of grapes and wines without the need for initial isolation of the glycoside precursor fraction. However, this method still depends on indirect measurement of the glycosides and acid or enzymatic hydrolysis is needed to release the volatile aglycone which can result in artefact formation. In the current project we validated a novel method using UHPLC-qTOF MS/MS for direct analysis of intact aroma glycosides in grapes with minimal artifactual changes in composition. Eighteen monoterpene glycosides were identified including a monoterpene trisaccharide glycoside, which is tentatively identified here for the first time in any plant. Additionally, while previous studies have identified monoterpene malonylated glucosides in other grapevine tissue, we tentatively identify them for the first time in grape berries. Finally, we observed that depending on the glycoside monitored, there is differential accumulation of monoterpene glycosides during maturation of Muscat of Alexandria berries. This work sheds important insight into possible biochemical changes in glycosylation during grape berry maturation. In addition, this research will allow us to better understand the effects of viticultural and winemaking practices on grape and wine components that affect flavor.

Extended Maceration

In this study we looked at the effect of extended maceration (up to 8 weeks) with daily pumpovers or submerging the cap (also up to 8 weeks) on the sensory and chemical attributes of the resultant Merlot wines.  Chemical measures of polyphenols and basic wine chemistry, along with Descriptive Analysis (DA) comprised three data sets.  Principal component analysis (PCA) applied to the individual data sets discriminated the wines by treatment, with each of the three PCAs capturing 90 – 97{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the treatment variability with in the first three components.  We also used Temporal Dominance of Sensation (TDS) a relatively new sensory technique to evaluate the time component of mouthfeel and taste attributes.

  • Nine descriptive attributes were significantly different.  Maceration was associated with increases in red fruit, bitterness, astringency, drying, and astringent texture, along with a decrease in pepper spice.
  • Descriptive analysis results showed that 8 week extended maceration, whether by submerging the cap (Su8) or with daily pumpovers (Pu8), had similar sensory profiles. Also, the control wines with no extended maceration but with different cap management methods (Po0, Su0 and Punch down (PD0)) were grouped together as similar in profile.
  • The TDS profiles of Po8 and Su8 were different despite each having a similar chemical and descriptive profile, this shows that TDS can pick out sensory nuances that occur over the time course of the wines residence in the mouth that single point methods, such as DA.
  • For all 9 treatments, astringency became the dominant sensation at approximately the same time point.
  • Po0 and PD0 showed a clear temporal progression when compared to the Su0 treatment.
  • Maceration showed a decrease in anthocyanidin concentration that leveled after two weeks, while tannin concentration continued to increase.  The Su0 treatment had the highest anthocyanin measurements.
  • In general, maceration tended to increase compounds associated with apple aroma and decrease fruit/ floral aromas.
  • Maceration gave similar tannin measurements as submerged cap, but a different sensory profile

Oxidation of Wine: Control for Quality

This report presents, first, the electrophilic nature of Q4MeC toward wines antioxidants and polyfunctional odoriferous desirable volatile thiols. 1H, 13C and 2D NMR analyses were used to identify the reaction products between 4-methyl-1,2-benzoquinone (Q-4MeC) and wine relevant nucleophiles like SO2, glutathione, ascorbic acid, phloroglucinol and a key odoriferous volatile thiol the 3-sulfanylhexanol. The formation of these reaction products was studied by HPLC-UV-MS, in wine like medium where the different nucleophiles were present in to two to four compounds mixtures. SO2, ascorbic acid and glutathione appear to provide a protection against 3SH consumption by the quinone, by acting as sacrificial nucleophiles. The antioxidant powers of SO2, AA and GSH were very similar and no antioxidant synergic effect was observed between these compounds when they tested in equimolar concentration.

Second we observed that the presence of carbonyl compounds in the matrix appear to decrease the SO2 antioxidant activity in a compound dependent manner. Acetaldehyde and pyruvate are very strong and fast SO2 binders, resulting in limited 3SH protection via their addition reaction with quinones. Ketoglutarate and galacturonate appeared less reactive with SO2, but their SO2 binding power needs further investigation under enological conditions. Finally, our NMR metabolomic approach of measuring the nucleophilic potential of wines reveled the importance of both SO2 and glutathione in controlling oxidation in wines, and opened new horizons for the development of powerful analytical tools for controlling oxidation during the vinification process and aging.

Barrel Maturation, Oak Alternatives and Micro-Oxygenation: Influence on Red Wine Aging and Quality

The impact of micro-oxygenation (MOX) in conjunction with a variety of oak alternatives on phenolic composition and red wine aging was investigated and compared with traditional barrel aging.  Although several studies concluded that MOX give similar results to barrel aging, few have compared them directly and none directly compared MOX with and without wood alternatives and barrel aging.  Results confirmed that MOX had a positive effect on color density, similar to barrel aging, even after 5 months of bottle aging.  This is supported by an increase in polymeric phenol and pigment content not only with aging but in the MOX compared to non-MOX wine treatments.  Descriptive analysis results indicate that the wood treatment dominated differences among treatments.  This study showed that MOX in combination with wood alternatives such as oak chips and staves could mimic short term (six months) barrel aging in new American and French oak barrels.

Oxidation of Wine: Control for Quality

This report presents first evidence of managing wine flavor evolution during oxidation (including aging), by altering the reactions of quinones. O-quinone of 4-methylcatechol has been produced in acetonitrile by periodate resin. Michael addition reactions of o-quinone 4-methylcatechol to various nucleophiles, including varietal volatile thiols, hydrogen sulfide, glutathione, sulfur dioxide, ascorbic acid and amino acids like methionine and phenylalanine have been carried out, obtaining alkylated adducts in fairly good to quantitative yields. The reaction rate and selectivity of o-quinone 4-methylcatechol toward the studied nucleophiles have been investigated by UV-Vis spectrometry by following the loss of quinone chromophore. The observed reactivity spans 3 orders of magnitude on passing from amino acid (methionine and phenylalanine) (KNu = 0.0002 s-1) to the most reactive nucleophiles, the hydrogen sulfide (KNu = 0.4188 s-1). These are the first direct reaction rate measurements of nucleophilic addition to the oxidized catechol. The classification of the studied nucleophiles revealed the existence of three categories. The first group consisted of amino acids (methionine and phenylalanine) having rates of essentially zero. Next, phloroglucinol, has a very low rate (K = 0.0064). Despite the slow rate of this reaction, this data suggests phenolics may be able provide a protection for varietal thiol aroma under some circumstances. The next group of compounds includes the volatile thiols having increasing reactions rates K as steric inhibition declines. The tertiary thiol, 4MSP, was much less reactive with quinones than the secondary (3SH) and primary thiols (2FMT), and quite close to the rate of phloroglucinol. The odoriferous volatile thiols (4MSP, 3SH, 2FMT) showed lower K values than those of the third group of the wines antioxidant compounds (SO2, GSH, AA) and H2S. The result indicates that both the antioxidant compounds (SO2, GSH, AA) and H2S would react preferentially with oxidation induced quinones, resulting the preservation of varietal aromas. The characterization of the reaction products between the studied nucleophiles and ortho-quinones derived 4-methylcatechol, was performed by using HPLC-MS analyse. Three o-quinone 4-methylcatechol/H2S; phloroglucinol adducts, two o-quinone 4-methylcatechol/3SH; 2FMT; 4MSP adducts, and one o-quinone 4-methylcatechol/SO2 were detected. No adduct was detected into the reaction medium where o-quinone 4-methylcatechol was incubated with both methionine and phenylalanine explaining why there observed reaction rates were essentially zero. Competition experiments provided strong support to involvement of antioxidants such as such as SO2, glutathione and ascorbic acid should to the preservation of wine?s varietal flavors.

Evaluating the Effects of Sterile Filtration on the Sensory and Chemical Properties of Wine

The goal of our project is to evaluate the effects of sterile filtration on the sensory and chemical characteristics of wine. To do this, we have filtered two red wines, a Cabernet Sauvignon and a Merlot through 0.45 ?m PVDF and PES membrane filters and compared the sensory and chemical characteristics of these wines to unfiltered control wines. Treatments were expanded with the Merlot to also examine the effects of a pad filter and cartridge depth filter used as prefilters. We have examined changes in dissolved oxygen content, tannin content, and color during the course of filtration and found only minor changes. Sensory panels were trained for each of these wines and each of the treatments evaluated immediately after filtering and then on a regular basis for 9 weeks (for the Cabernet) and 4 weeks (for the Merlot) so far, with analysis ongoing. While both wines changed significantly over time in the bottle, very few significant differences were observed in aroma or mouthfeel between filtration treatments. In other words, our results thus far indicate limited impact of sterile filtration on the sensory or chemical properties of the wine, regardless of the type of filter material used. Sensory analysis of the treatments will continue to longer time in bottle for both wines. In addition, we are preparing to evaluate filtration of an aromatic white wine in the same manner over the next three months.

Development of GC Olfactometry for the Analysis of Wines: Investigation of Sur Lie and Smoke Wine Taints

Summary of Major Research Accomplishments and Results: Chardonnay juice from grapes harvested in the UC Davis vineyard in the fall of 2009 was fermented in duplicate in 3 gallon carboys using three different yeast strains: 522, 940, 950. For each strain, three different levels of grape solids were evaluated: 0{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}, 6.25{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}, 12.5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} by volume. At the end of the fermentation, samples were collected (day 0 samples) and the wines from the carboys were divided in two; one half was allowed to age sur lie and the other half was cold settled to separate the solids and used as controls. Samples were screened weekly, by project personnel, to identify samples that had veggie or other off aromas. Samples identified so far include: strain 950 with 6.25{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} and 12.5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} solids at days 30 and 60 and strain 940 with 12.5{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} solids at day 50. The two wild isolates, UCD950 and UCD940, continued to show higher development of undesirable off-characters than did the commercial strain UCD522. Samples were taken at day 0 and at 3 months and stored in the freezer for further analysis. These samples were analyzed by gas chromatography with a sulfur chemilumenescence detector (GC-SCD) to specifically measure low molecular weight sulfur compounds in the samples. A second analysis using a GC attached to a mass spectrometer (GC-MS) and equipped with a sniff port was used for olfactometry measurements (GC-O) on the same samples. A panel of 5 volunteers (four female, one male) was identified to complete the GC-O analyses. The panel was trained to distinguish between fruity and veggie aromas, first by bench-sniffing solutions containing one standard of each aroma-type. The standards chosen were distinctively fruity or veggie; the veggie standards used were: dimethyl sulfide (DMS), dimethyl disulfide (DMDS), isobutyl methoxypyrazine (IMBP) and hexanal. The fruity standards were the ethyl esters of the valeric, caproic and caprylic acids.

Impact of malolactic fermentation on red wine color

The color of a red wine is an important sensory attribute that originates primarily from anthocyanins. However, development of stable red wine color is impacted by compounds such as p-coumaric acid, caffeic acid, catechin, and quercetin that are involved in copigmentation reactions as well as acetaldehyde and pyruvic acid. While it is known that yeast can alter the concentrations of some of these compounds, little is known regarding the impact malolactic bacteria may have on red wine color development. This project investigates the effect of the malolactic fermentation (MLF) on red wine color and the ability of malolactic bacteria to degrade compounds important to the development of stable red wine color. Pinot noir and Merlot wines produced in 2008 were stored at 13°C, sampled every three months for twelve months and analyzed for a number of different color parameters. Pinot noir wines that had undergone MLF, including wine produced with a simultaneous alcoholic and MLF, had the lowest color throughout storage. The pH adjusted control (pH 3.67) gave the highest color at each time point analyzed, and consistently had higher color than the control wine (pH 3.53). Pigmented polymer for the Pinot noir wines showed similar trends to the color analysis in that the control wines gave the highest values while the wines that had undergone MLF had lower values. Overall, pigmented polymer values increased overtime. For the Merlot, similar trends were noted as for the Pinot noir. Wines that underwent MLF had lower color values at each time point with the pH adjusted control giving the highest color. The control wines also had higher pigmented polymer values than wines that had undergone MLF with pigmented polymer values increasing over time. In addition to the analysis performed on 2008 wines, Pinot noir and Merlot wines were again produced in 2009. A portion of the Pinot noir and Merlot wines underwent a simultaneous alcoholic and malolactic fermentation (S. cerevisiae VQ15 + O. oeni VFO) while the remainder were only inoculated with S. cerevisiae VQ15. At dryness, all wines were pressed and sterile filtered (0.45 ?m). For both the Pinot noir and Merlot wines, three carboys were inoculated with either O. oeni strain VFO, Alpha (Lallemand), or VP- 41 (Lallemand) at approximately1 x 106 cfu/mL. The remaining carboys of wine were not inoculated with O. oeni. All wines were kept at 20°C. Some of the wine that had not undergone MLF was pH adjusted to the same final pH of wines that had completed MLF. Samples were taken before and after MLF for analysis and wines were sterile filtered, bottled, and stored at 13°C. Prior to MLF, all wines had very similar concentrations of acetaldehyde and pyruvic acid although wines that had undergone a simultaneous alcoholic and malolactic fermentation had lower acetaldehyde and pyruvic acid concentrations. In wines inoculated for MLF post alcoholic fermentation, acetaldehyde and pyruvic acid concentrations decreased during the course of the MLF. Strains VFO and Alpha more completely degraded acetaldehyde than VP41 while VFO degraded more pyruvic acid than did the other two strains tested. The same trends were also observed for the Merlot wines although lower initial concentrations of acetaldehyde were observed compared to the Pinot noir wines. These results show that all the O. oeni strains tested degraded both pyruvic acid and acetaldehyde during the MLF in both Pinot noir and Merlot wines including during a simultaneous fermentation. Future experiments aim to determine the mechanism by which red wine color is impacted by MLF. This will include investigating the role that bacterial degradation of acetaldehyde and pyruvic acid plays as well as whether adsorption of anthocyanins to ML bacteria cell walls is occurring. In addition, experiments varying the time when wines are inoculated for MLF may suggest the best time to inoculate for MLF in order to maximize color development and stability.

Oxidation of Wine: Control for Quality

Studies testing metal chelation showed significant but relatively small effects, so that it is not clear whether chelating agents would be worth pursuing as protective agents. Final results from prior years were published. A related project on oxidation revealed new oxidation reactions in wine that might help identify markers of oxidation.

FTIR Spectrometer

The investment provided by the American Vineyard Foundation in 2002 was used to purchase a Fourier Transform Infra Red (FTIR) Spectrometer from FOSS North America. We were able to use the money to leverage significant educational discounts from FOSS. We received the instrument in the summer, and were able to put it into operational condition with the help of FOSS personnel. We did experience one significant software problem that hindered progress. The cause was recently identified by FOSS personnel in Europe and relates to a software upgrade that has now been solved. In our case, FOSS North America moved swiftly to correct problems and spent significant periods of time training us in the use of the software. We are indebted to Constellation Wine Company for extensive support we received from their Principal Chemist, Mr. Steve Kupina. Mr. Kupina?s experience with the Winescan software and the reference chemistry he was able to provide on grape juices, were invaluable in the calibration of the Grapes can software. We also received significant reference chemistry assistance from Mr. Randy Asher of McCalls Winery & Distillery for Must under Fermentation software calibration. The instrument is now being used extensively by the Faculty on a number of research projects:(a) Evaluation of the effect of six different irrigation treatments on yield and quality of Cabernet Sauvignon grape produced in the San Joaquin Valley. (b) Evaluation of the effects of timing and differential nitrogen applications on the quality and nutritional status and wine quality of Cabernet Sauvignon grapes. © Comparison of fruit and wine quality characteristics of Cabernet Sauvignon grapes produced in three regions of California and one region in Washington State. (d) Effect of Messenger on Leaf Photosynthesis, Vine Performance, Fermentation Potential, and Wine Chemistry in Cabernet Sauvignon Grapevines. (e) Develop and implement control methods for Eutypa dieback disease. (f) Wine composition prior to and during study of micro oxidation. (g) Wine composition during study of utilization of fermentable nitrogen. (h) Effect of wine closures (cork and manufactured) on wine composition. (i) Differences in chemical composition of oleate versus non-oleate treated raisins. (j) Chemical analysis of juice fermentations by different strains of yeast.