Formation of Volatile Sulfur Compounds in Pinot Noir Post-Fermentation -Part 2: Lees Level and Contact Time on Volatile Sulfur Compounds in Wine

During the first year of this project, wine fermentation and aging at various amounts of lees were conducted by Dr. James.P.Osborne as a part of a separate project “Formation of volatile sulfur compounds in Pinot noir post-fermentation. Part 1: Role of grape amino acid content and wine lees composition”. Two yeast strains, and three lee levels for used in the study. Initial samples were collected for amino acids and volatile sulfur analysis. Additional samples were taken after 2 and 4 weeks and 2 months of storage and assessed for the same parameters. The project is still at very early stage, and limited results showed:

  1. The concentrations of volatile sulfur compounds in the wines fermented with both two yeasts were low after fermentation and pressing. All of the major volatile sulfur compounds were analyzed including H2S, mercaptans, disulfides, thiol esters. Saccharomyces cerevisiae P1Y2 produced lower levels of methyl thioacetate and methionol than Saccharomyces cerevisiae RC212.
  2. Heavy lee loading resulted higher level of H2S initially, but the difference diminished after aging. No significant differences were observed for other volatile sulfur compounds at different levels of lees.
  3. After 2 months of aging, some sulfur compounds such as methionol decreased while some sulfur compounds like methyl thioacetate kept consistent.
  4. Some industrial samples were analyzed, and high levels of H2S were detected in some of the wine samples, and industrial partners responded with cupper treatment.

Understanding Pinot Noir Grape and Wine Aroma Composition as a result of changes in Vine Balance, year 1 of a 3

Vine balance is important in determining fruit and wine composition. Excessive canopy density is known to produce unbalanced musts, resulting in poor wine quality. In Oregon, crop thinning is normally conducted between fruit set and lag phase to increase the leaf area: fruit weight ratio in order to prevent over cropping, as well as to improve fruit size and composition. Earlier studies have shown that cluster thinning reduces fruit yield and increases the berry weight, soluble solids, and color of table grapes. However, the impacts of the timing and severity of cluster thinning on subsequent berry growth and fruit flavor composition has not been widely investigated. The main objective of the present study was to evaluate such effects in association with grape and wine aroma composition in Pinot noir grapes and wine. Preliminary results indicated that crop thinning had very limited impact on the grape major volatile composition analyzed in 2011 and 2012. However, the composition of other minor and important compounds, such as methoxypyrizes, are still under investigation.

Chiral Terpenes – Quantitation, Threshold Determination and Sensory Impact on Aromatic White Wines

Objective 1 – Development of methodology using multi-dimensional gas chromatography-mass spectrometery to measure chiral terpenes (limonene, linalool, citronellol, α-terpineol and α-pinene) in aromatic white wines.  

Since September 2013 method development has been ongoing. Specifically chemical synthesis of dueterated-limonene and α-pinene has been conducted. Synthesis of d2-α-pinene was successful. D2-limonene is proving more problematic and this synthesis is ongoing. These deuterated compounds are used for internal standards, and more accurate measurements are achieved when the internal standards have similar structure to the target compounds. Preliminary work has shown that the internal standards that can be purchased do not work well for limonene and pinene, therefore we are attempting chemical synthesis of these compounds. For all other compounds and standards have been composed into a working standard for the method and method development is ongoing. At the present time, method development is proceeding on schedule and is anticipated to finish the end of February.

Additional wines have been collected over the last few months to build up a substantial collection of aromatic white wines from regions around the globe. Areas in which aromatic white wines have been obtained include Washington, Oregon, New York, California, New Zealand, Australia, Germany, Austria and Italy.

Objective 2 –  Once quantitation of enantiomers is achieved the impact of the enantiomers will be investigated through a series of sensory testsThis objective is slated to begin during Spring/summer 2014.

Oxidation of Wine: Control for Quality?Understanding Effective Sulfur Dioxide and the Role of Glutathione

To date, the project has completed two goals, and is making progress on two others. We have analyzed the thermodynamics and kinetics for the sulfur dioxide binding of four different aldehydes and ketones, the major binders of SO2. This was carried out using a totally new approach, NMR spectroscopy, which allowed us to analyze the reactions under wine-like conditions. Two-dimensional (1H-1H) homonuclear and heteronuclear (13C-1H) single quantum correlations (COSY and HSQC) nuclear magnetic resonance experiments of wine samples were used for the simultaneous identification and quantification of free and, for the first time, sulfite bound acetaldehyde, pyruvic acid, acetoin, methylglyoxal and α-ketoglutaric acid. This new technological approach opens the door to possible new approaches to measuring free and bound sulfur dioxide. The effect of varying levels of SO2 and glutathione on micro-oxygenation was also investigated and it was surprising to see that both antioxidants suppress oxygen consumption. The cause for this effect may be related to suppression of the free radical formation by the Fenton reaction. When wines were depleted of these antioxidants, it appears that the formation of aldehydes rapidly increased, along with the formation of stabilized pigments. A new method to analyze glutathione, a possible new antioxidant for protecting wine, is also underway.

Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry

Our last update demonstrated the usefulness of MALDI-FTICR (Matrix Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance), the efficiency of QTOF (Quadrupole Time of Flight) tandem mass spectrometric analysis, and the successful fractionation of wine samples. Having demonstrated our methods adequate we set about tailoring them to our experimental needs and adapting our instrumentation and methodology. We have since discovered even better FTICR results with an electrospray ionization source (ESI), and created a method of analysis for pigmented tannin and wine polymers using nano-HPLC QTOF.

ESI-FTICR results have demonstrated resolution greater than 50,000 and tremendous mass accuracy with error less than 1ppm. The QTOF method was modeled originally on the diol stationary phase cocoa extract separation by Robbins (Kelm et al 2006). We have since made significant alterations so as to provide a nano-scale elution with total column volume around 50 μL. Unfortunately, nano columns comprised of diol stationary phases are not in production. We partnered with the manufacturer of our best performing traditional column as well as Agilent Technologies to fabricate a nano-LC chip made from Develosil Diol 100-5. The results we have obtained would not have been possible otherwise.

So far, we believe we have identified over one hundred ions by ESI-FTICR which have never before been published. With those same samples we refined our QTOF method to isolate and fragment those ions providing fragmentation data for structural identification. Unfortunately, the fundamental nature of the project requires that these ion fragmentation spectra be analyzed by hand for neutral mass loss functional assignments. The work is still ongoing. Soon we will have enough fragmentation spectra to verify their identities. We anticipate presentation of new compounds in time for the ASEV national conference 2014 in Austin, TX.

Influence of Grape and Wine Production Practices on Tannin Extractability and Activity

The primary objective of this proposal has been to develop an analytical method that predicts tannin interaction with salivary protein. This method is unique in that it moves away from tannin concentration as a predictor of astringency so that the impact of tannin structure variation (e.g.: color incorporation, oxidation of tannin structure) on interaction, can be measured. This analytical approach follows previous work which found that tannin structure variation related to grape maturity and wine age, could be related to thermodynamics of interaction.

Coupled to the development of an analytical method, this project also focused on the development of a rapid reproducible method for preparing extracts from grape berries. This method deviates from many extraction methods developed to date in that it does not rely on the addition of solvents to mimic a wine-like system. Instead, this extraction method imposes a mechanical stress on berries for a short period of time (5 min), thereby testing the robustness of plant cells and hypothesized durability of diffusional barriers. The two methods above are expected to provide new and novel information on tannin development, from grapes to wine. The objectives of this proposal are consistent with the highest priority research objective as outlined by the American Vineyard Foundation. The results to date have been very positive.

First, a new analytical method has been developed and is now being applied to grape extracts and wines. The analytical method has the ability to measure tannin interaction variation that would be consistent with “softening” and therefore has significant potential in managing grape and wine production operations. Importantly, the analytical method is able to measure the impact of tannin modification on the “stickiness” of tannins. With regard to the new extraction method, the results have also been successful in that extracts prepared from the developed extraction procedure have been associated with predicted differences based upon growing region and historical block differences in tannin quality.

The results from the first two years have led to the development of analytical methods for tannin assessment in grapes and wine. In year three of this project, research efforts are being directed to the development a more complete understanding of the utility of these new methods.

Metabolic Profiling of Grape and Wine Aromas

Most aroma compounds exist in grapes as glycosidically bound precursors and the aglycones (“free” aroma compounds) are released by enzymatic or acidic hydrolysis during crushing, fermentation and wine aging. We evaluated procedures for measuring the glycosidically bound volatiles using both acid and enzyme hydrolysis conditions followed by analysis of the free volatiles by Head. -Phase-Microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC-MS). Acid hydrolysis of a mixture of standard compounds at pH 1 and 100°C for 1 hour released 20-60{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of the bound volatiles, however, these conditions also resulted in significant degradation (>50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23}) of the free volatiles. Enzyme hydrolysis was generally more effective at releasing glycosidically bound compounds with minimal artifactual changes in the concentrations of the free volatiles. However, esterase and oxidase activity was still observed resulting in artifactual degradation of the free volatiles. Most previous procedures for evaluating volatile glycosides employ an initial sample preparation step using column chromatography to isolate the glycoside fraction from the grapes prior to hydrolysis and analysis of the free volatiles. We observed that different types of sample preparation columns were not equally effective at retaining the glycosides and no column-type was effective for all the glycosides in our mixture. Using optimized conditions obtained from the previous studies with model systems, we compared direct enzyme hydrolysis of Cabernet Sauvignon and Chardonnay grape homogenates with and without prior isolation of the glycoside fraction. Both procedures resulted in new “free” volatiles. However, results varied depending on the type of treatment. This research is providing important insight into approaches for rapid estimation of “aroma potential” of grapes.

Judging Wine Quality: Do We Need Experts, Consumers or Trained Panelists?

The overall objective is to determine if quality can be reliably evaluated by wine consumers, wine aficionados and/or wine experts and what their quality scores actually mean relative to the intensities of the sensory attributes we get from trained panels, and the liking scores we get from wine consumers. To choose wines with different quality levels we used the judging scores assigned to 27 Cabernet sauvignon wines in the 2012 California State fair judging competition.

At this competition Cabernet wines are divided into 9 regions. Within each region we picked the wine with the highest score (usually a gold medal wines), the wine with the lowest score (a wine that did not medal) and then a wine with a score as close to the mean between the highest and lowest score. The 15 descriptive analysis panelists (10 males, 22-72 years old, average age 37 yrs, median 31 yrs, standard deviation 17 yrs) were recruited from the students, staff and faculty of the Departments of Viticulture & Technology, Food Science & Technology, and Aerospace Engineering. They participated in 6 1-hr training sessions to collect, generate and obtain consensus on 21 aroma, 3 taste and 3 mouthfeel attributes. Their evaluation of the wines, in triplicate will be complete on 2/7/13. The consumer hedonic evaluation, using the 9-point hedonic scale has been scheduled for February 2, 2013 – we are aiming to have about 192 participants and each participant would evaluate 6 wines. Thus each of the 27 wines would be evaluated by 42 or 43 consumers.

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.

The Fate of Anthocyanins Under Warm Growing Conditions

In the proposal for the 2012, we outlined three objectives: 1. Compare the rate of anthocyanin degradation in warm climate grapes vs cool climate grapes 2. Determine if other phenolic compounds also experience degradation in warm climates 3. Identify by-products of degradation and potential mechanisms for their formation We have continued to make progress on developing the methods needed to make the desired measurements and those results are outlined in the attached proposal. We have clarified the accumulation of anthocyanins in Cabernet Sauvignon over two vintages and believe we have discovered a hidden pool of metabolites in the phenolic synthesis pathway in Vitis vinifera.

Towards objectives 1 and 2, we have collected all the grape samples from two vineyards, one at the UC Davis Oakville vineyard and the other at the UC Kearney Agricultural Center between July and October 2012. We have cluster temperature data from each site, collected hourly. Six berries were collected from 3 vines treated with tracer and 2 control vines at each site approximately 8 times during the growing season. The berries will be extracted during February, and student assistants are currently being trained to undertake this exercise. We extract the skin of each berry separately to improve the statistical significance of our data. We have the instrumentation ready to analyze the extracts once they are prepared. Towards objective 3, we have collected data from berries incubated in the lab, at 25?C versus 45?C, with high levels of the 13C6 phenylalanine (Phe) with an M+6 mass. This provided anthocyanin metabolites with approximately 35{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} containing the M+6 label. This allows for facile detection of the anthocyanins and other metabolites. Berry skins from the 8 biological replicate pairs of samples were all extracted and the extracts analyzed by Chip-LC-MS, using a QToF detector. This detector has a very high resolution and mass accuracy, so that detected substances can be identified with more certainty.

The large and complex dataset was transferred to Austrian collaborators we identified at the Grape Research Coordination Network meeting. Using this technique, lists of potentially labeled molecular features were found in positive and negative mode analyses. Of these candidate ions, 42 positive and 11 negative molecular features were found to be statistically different between 25°C and 45°C treatments. Only a select few molecular features were found to be significantly higher in the 45°C treatment; these compounds present our best candidates for the degradation products of anthocyanins. From the list of molecular features believed to be derived from Phe13 metabolism, a pair-comparison t-test was performed over the 8 biological replicates to determine significant differences in the concentrations of labeled features. As expected, many phenolic compounds are susceptible to degradation under high temperatures. At least one of each anthocyanidin moiety appeared to be degraded under 45?C temperatures, although every single anthocyanin did not vary significantly between treatments.

Most interestingly, 3 features were found to be in greater quantity in the 45?C than in 25?C grape treatments. These features had m/z values of 433.113, 347.076, and 585.170. The molecular feature of mass 433.113 was tentatively identified as a benzyl alcohol dihexose. A similar molecular feature was found when researchers probed the degradation of anthocyanins in flower petals (Bar-Akiva et al. 2010). The feature with m/z 347.076 was tentatively identified as a syringetin aglycone. Syringetin, while present in grapes at low levels, is most likely an artifact of malvidin-3-glycoside created during ESI, since their molecular structures only vary in the oxidation states of the flavonoid ?C? ring and their retention times are nearly identical. This could easily be caused in conditions in which the pH is above 2, when anthocyanins undergo structural transformations. The final molecular feature identified 585.170 has not yet been identified, although if the molecular feature represents a product of anthocyanin degradation, it would have to be an adduct of some kind since the m/z is higher than many of the degrading anthocyanins. We were expecting more compounds that would increase at higher temps, but have found background noise is limiting our sensitivity, most likely due to the type of LC separation used (Chip-LC). At the present time, we are trying various methods to reduce that noise, through complicated data analyses, in hopes that the signals of the temperature sensitive and rising metabolites will be more evident.