Significance of Oak Ellagitannin Chemical Structure to Wine Oxidation

The pathway of wine oxidation, as currently understood, encompasses the cascade of reactions incited by the oxidation of phenols in the presence of oxygen, eventually coming to the conversion of ethanol into acetaldehyde. While it is evident that wines consume oxygen over time and acetaldehyde becomes increasingly apparent with age, the rate of oxidation can vary unpredictably among different wines, and it was hypothesized that different phenolic structural features, particularly those of oak ellagitannins, are the reason for such variability in oxidation. It was originally proposed that ellagitannins and other phenols with distinct functionalities be studied for their effects on oxygen consumption and acetaldehyde production. However, in light of recent studies conducted by our laboratory demonstrating that the input of oxygen does not guarantee the output of acetaldehyde, it was decided that attempting to study the pathway of oxidation in its entirety, from oxygen to acetaldehyde, would not be an effective approach.

Given the complexity of wine oxidation, a more sensible strategy would be to study the effects of phenolic structure on individual reactions rather than the pathway as a whole. In the first step of wine oxidation, the oxidation of phenols is coupled to the reduction of oxygen by iron, which acts as a shuttle for electrons between phenols and oxygen. A more specific hypothesis now is phenolic structure affects their reactivity with iron, subsequently affecting oxygen consumption and the remainder of the wine oxidation pathway. The initial reactions of wine oxidation may be characterized by the redox cycling of iron between its two oxidation states: Fe(II) and Fe(III). The addition of electrons to oxygen occurs with the oxidation of Fe(II) to Fe(III), and in the opposite direction, the loss of electrons from phenols takes place with the reduction of Fe(III) to Fe(II). The ratio of Fe(II) to Fe(III) should thus depend on the relative reaction rates of Fe(II) with oxygen and that of Fe(III) with phenols.

A quick and simple spectrophotometric method for iron speciation, employing the complexing agent Br-PADAP, is currently being optimized and validated, to be used not only to assess differential rates of iron reduction by structurally diverse phenols, but also by the industry to more generally measure the “redox status” of their wines. Our laboratory’s modified version of the Br-PADAP assay is simple and inexpensive, requiring a sample volume of only 200 μL and a reaction time of 10 min, and is done directly in a cuvette. Validation of the assay is currently underway; difficulty lies in the fact that there does not exist a standard method for iron speciation to compare, thus alternative methods of validation are being considered. This research would not only improve management of oxidation, but also furnish a more complete understanding of phenolic oxidation, with the ultimate goal being the prediction of wine aging based on phenolic content and composition.

Red Wine Tannin Interaction with Polysaccharides

The objectives of this proposal are to do the following:

  1. Investigate polysaccharide interaction with tannins in wine.
  2. Determine how intermolecular interactions between macromolecules in wine influence wine mouthfeel.

Following the first two years of activities, excellent progress has been made. Ten wineries were recruited and ten wines were provided with all different winemaking processes. Laboratory activities have made significant progress, with polysaccharides and tannins extraction from wine and chemical characterization. Interactions between tannins from red wines and polysaccharides have been characterized by dynamic light scattering. The effect of pH and ethanol content on the interactions between tannin and polysaccharide has been investigated.

Tannin Structure-Activity Relation to Red Wine Astringency

The objectives of this proposal have been to do the following:

In a companion project being submitted to the Agricultural Research Institute (California State University research initiative)

II. Conduct extended sensory studies with the University of California, Davis, on a subset of the above wines to determine the relationship between sensory and instrumental analysis of red wine mouthfeel.

These objectives are consistent with the highest priority research objective as outlined by the American Vineyard Foundation.

The overall purpose of this proposal is to determine the role of grape and wine production practices on tannin structure and perception.  In combination, tannin activity will be monitored by high performance liquid chromatography (HPLC) so that a comparison can be made between human perception and HPLC measurement.

Following the second year of activities, excellent progress has been made.  The major activity during the second year of this study has been to monitor the development of berries in approximately 75 Napa Valley blocks.  Monitoring the tannin activity in these blocks is expected to provide information on the role that grape production practices can have in overall grape and corresponding wine tannin.

Red Wine Tannin Interaction with Polysaccharides

The objectives of this proposal are to do the following:

I. Determine variation in tannin activity as a function of polysaccharides structure from yeast and/or grape in red wine.

II. Relate the polysaccharide-tannin interaction variation to the wine mouthfeel.

In a companion project funded by the Agricultural Research Institute (California State University research initiative)

III. Improve the understanding of tannin-polysaccharide interactions consequences on red wine stability.

These objectives are consistent with the highest priority research objective as outlined by the American Vineyard Foundation.
The overall purpose of this proposal is to determine the role of polysaccharides from yeast and/or grape in finished red wine on tannin structure and activity. In combination, competitive interactions between tannins and proteins or polysaccharides will be elucidated by spectrophotometry after variation of matrix parameters, so that an elaboration of a functioning interactions model can be made.

Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry

Seven vintages of UC Davis Oakville Cabernet Sauvignon spanning 35 years was selected for analysis by a complimentary suite of mass spectrometric techniques. The vintages from 1974 to 2009 selected by informal sensory evaluation and basic wine chemistry to be of a representative vertical style were: 1974, 1981, 1988, 1994, 2001, 2003 and 2009. All wines were produced at UC Davis for research purposes and stored in the UC Davis cellar together. This sample was determined to be the most controlled and uniform sample of wines varying by vintage only as could be obtained for the project. Al basic wine chemistry was obtained, pH, ethanol concentration etc. as were various assays for comparison to mass spectrometric data.

The following objective were submitted for the grant proposal under which this work was peformed.

1) Observe the evolution of pigmented tannin throughout aging

a. Employ our method of complimentary mass spectrometric techniques (ICR, QTOF, QTrap) for comprehensive identification of wine matrix compounds.

b. Observe the changes in relative abundance, depletion and accumulation in pigmented tannin composition (35 Year Vertical)

c. Correlate pigmented tannin structural analysis with well-known molecular characteristics (mDP, mass recovery) and newly developed sensory representative analyses (“grippiness”) to better convey the impact of these discoveries

2) Apply existing synthetic technologies.

a. Development of a library of standards for quantitation and calibration.

b. Postulate wine pigment precursors for examination of mechanistic pathways.

c. Employ the standards to quantitate the classes of polymeric pigment in wine.

Wine Research Assays

Phloroglucinolysis was performed on the wines used for the study (Table 1) as well as their extracts (Table 2). The mass recovery demonstrates typical value dropping off well below 80{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} after 7 years. Molecular mass after 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} elution also follows expected patterns of increase with age. This indicates overall larger tannin in the older wines as supported by McRae et al 2012. The molecular mass is a measure of intact tannins, as opposed to the mDP which measures only those tannins which could be cleaved by phloroglucinol. The inconsistency between these two values is reconciled by the mass recovery, indicating that more complexity of the tannin structure is a factor in the older wines, for instance additional interflavan bonds preventing phloroglucinol attack.

Tannin Structure-Activity Relation to Red Wine Astringency

Optimizing wine quality with regard to mouth feel is a quest for wineries that are trying to fine tune the astringency of their wines to a desirable level. Consistent with that, various wineries had agreed and participated in the extended maceration project in order to understand the driving force behind astringency. With that in mind, this project was born to comprise different analytical techniques, which are necessary to construct a conclusive understanding of tannin’s activity. During the 2014 vintage, maceration trials were conducted in five Napa Valley wineries. Winery selection was based upon winemaker interest. For each winery, fermentations were set up according the the specific cooperators protocol. At various times during the course of fermentation/maceration, samples were collected following pumpover operations. Following collection, samples were transported and stored at 7 degrees Centigrade until processed. For tannin isolation, each sample was filtered using a 9.0cm Whatman filter paper (20-25 μm pore size), diluted with milli-Q water (50:50 v/v) and then loaded onto a preconditioned column containing Toyopearl chromatography resin (HW 40C). Purification of tannins was conducted according to Aron et al.1 Briefly, and following application to the column, the column was washed with water followed by 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} v/v aqueous methanol and then tannins were eluted with 66{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} v/v acetone in water. The acetone was removed by rotary evaporation and the aqueous portion containing the tannin was lyophilized to a powder. Gravimetric yields were determined and the tannin powders were stored in glass, air tight, vials after being sparged with N2 and stored under -20°C. Tannin isolates underwent analysis using a variety of analytical techniques including gel permeation chromatography, phloroglucinolysis and stickiness. The analysis of tannins’ stickiness has recently been introduced by Barak et al.2 and further developed by Revelette et al.3. Samples were prepared according to the above methods and the enthalpy of interaction was determined for tannin polymers absorbing at 280 and 520 nm. Secondly, tannin powders underwent acid catalyzed degradation in the presence of excess phloroglucinol (phloroglucinolysis) to determine the subunit composition, average degree of polymerization and conversion yield following the method of Kennedy and Jones.

Following phloroglucinolysis, tannins were analyzed by gel permeation chromatography which provided information on size distribution5. Across all experiments, a total of 104 tannin isolates were prepared. Given that these isolates represent tannins collected at various points during fermentation and maceration and across different blocks and varieties, it is believed that a realistic picture of commercial structure variation for Napa Valley has been achieved. Results to Date are as follows: Winery 1 investigated the effect of extended maceration on tannin composition and activity. One Cabernet sauvignon block was harvested and equal portions of fruit were contained in two identical fermentation vessels. The must underwent a prefermentation soak for four days before inoculation. Samples for the control tank were collected on a semi-daily basis until press day (total soak time was 15 days). On the last day (day 15), the first extended maceration sample was collected. Similarly, extended maceration samples were taken on a semi-daily basis until press day (total soak time was 26 days).

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.

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.

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.