Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry

The task we are undertaking is fundamental. There is currently no quantitative method for determining tannin and pigmented tannin. It is not understood to what extent certain compounds contribute to the overall color of aged (> 2 years old) wine, nor the relative abundances of those compounds. Furthermore, there are many compounds that comprise the molecular basis of pigmented tannin whose structures are unknown. This investigation aims at using mass spectrometry as a tool for assessing the extent of color contribution due to these compounds, identifying new compounds, and providing a means of identifying the relative abundances for determining which compounds are most highly associated with quality parameters. Once we better understand the molecular basis of pigmented tannin we can provide tools for winemakers to improve the quality of their product. Pursuant to our goals for this year of the project we have (1a) identified many compounds by mass spectrometry which comprise the wine matrix, and many yet which have not been observed. Our FT-ICR experiment for determination of the relative abundance, depletion and accumulation of particular compounds (1b) will be performed in April with our collaborator Professor Nikolai Kuhnert at his laboratories and the Bruker research laboratories in Bremen Germany. Development of synthetic standards (2abc) is in progress as we are still assessing the appropriate standards to synthesize. We have defined the compound classes which comprise the majority of molecular peaks and will be performing further iterative fragmentation of these compounds to determine their structural characteristics in March with fellow anthocyanin researcher Professor Colin Kay at the University of East Anglia. All in all we are on track to accomplish our objectives and maintain pace for the upcoming year of data analysis and subsequent experimentation.

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

Assessments of Difficult to Ferment Juices

The goal of this project is to uncover the causes of chronically difficult to ferment juices. These juices often derive from the same vineyard or block of a vineyard and other similarly managed vineyards and blocks display normal fermentation kinetics. These difficult to ferment juices do not appear to respond to nitrogen or other commercial nutrient addition, occur regardless of strain used, and are challenging to restart. Our aims for the first year of this project were to evaluate the nutritional content of these juices to determine if a nutritional deficiency or presence of toxic compound was the cause of inhibition of fermentation. In this first year we were able to narrow down the possible issues with these juices. Although low nitrogen is a factor, addition of nitrogen to the fermentation seems to partially address the nutritional limitation. In addition these juices appear to impose a high vitamin demand on the yeast which we will explore in detail in this second year. This past year we were able to demonstrate that the accumulation of mannitol observed from the metabolomics analysis of the yeast strains grown in difficult to ferment juices is an indicator of oxidative stress in the juice.

It is not clear what juice compositional factors are leading to the oxidative stress and why this is not alleviated by use of Sulfur dioxide, and this will be explored further in this coming year. The low arginine and high proline of these juices suggests a problem with development, activity or destruction of the fine roots of the vine. Addressing this problem by adjustment of either yeast strain or nutrient supplementation would prevent the need for more invasive vineyard management strategies.

In this past year several commercial wineries sent us examples of chronic to ferment juices and we discovered that these juices either resembled the J. Lohr juice in having a deficient nutrient profile or were characterized by the presence of four rare (for grape) acetic acid bacteria species that seem to retain viability throughout the winemaking process and that appear to be inhibitory in ways not due to simple acetic acid production. An added goal for this coming year’s grant is to evaluate the role(s) of these bacteria in fermentation progression and to assess their sulfite sensitivities and persistence during fermentation.

Understanding Alcohol Tolerance in Wine Yeasts

Understanding alcohol tolerance in wine yeasts is important in order to develop tools to rectify sluggish and stuck wine fermentations and new commercial yeast strains with desirable sensory or process characteristics. In previous work, we have identified two potential mechanisms for alcohol tolerance including ones related to the composition of the yeast cell membrane and to the nutrient utilization efficiency of the strain. To understand these mechanisms, we further examined both potential effects. Specifically, we were able to find that the composition of the cell membrane changes significantly as fermentation temperature is raised or lowered. Using our high resolution methods for measuring membrane composition, we were also able to identify specific types of lipids in the cell membrane that are associated with lower ethanol tolerance (e.g. several phosphatidylinositol lipids) and higher ethanol tolerance (e.g. several phosphatidylcholine lipids). In this period, we also developed and used a method for measuring a wide range of metabolites inside and outside of yeast cells during fermentation. Our analysis shows that certain metabolic pathways like the pentose phosphate pathway and glycerol secretion are negatively correlated with cell growth and ethanol tolerance of these strains. These major results have allowed us to begin to identify targets for genetic manipulation of wine yeasts that should increase or decrease alcohol tolerance. We hope to actively initiate this part of the project prior to the completion of funding this year. The work completed as part of this grant has resulted in two papers (one published and the other in final revisions after review) with two more in preparation and a number of presentations at local and national meetings.

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.