Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry


As we were breaking new ground from several perspectives, the project took some unexpected turns. At this point, we have completed several goals. First, we have created a table of expected wine pigments based on known reactions between anthocyanins and proanthocyanidins, goals 2a and 2b. These results were published in two papers, the first describing how to enumerate all possible proanthocyanidins that could be distinguished by mass spectrometry, up to a degree of polymerization (DP) of 10 (Cave and Waterhouse 2019), and the second, taking that list and “reacting” those tannins with the pigment to create a list of all possible wine pigments. This was a list of over 1 million structures. (Cave et al. 2019)

Having that list, we then compared the masses of those proposed structures with mass spectral data from two sources, QToF and ICR. In the case of the QToF, we could match about 10% of the several hundred observable compounds (Cave et al. 2020). However, the ICR is much more sensitive and precise, and here we could observe about 18,000 signals, and of those could match over 20% of the signals to entries in our table (Cave et al. 2020). Unfortunately, many signals could be represented by more than one structure, so additional study is needed to determine which of the options is present. These results addressed goals 1a, 1b and 2c. The data we now have will require further mass spectral analysis to discriminate between a number of redundant structures.

Development of a Tribology Method to Assess Mouth-feel Perceptions of Red Wines

We have successfully extracted and chemically characterized red wine with tannins, as well as the interactions between red wine tannins and salivary proteins by chemical methods. The effect of tannin-protein interactions on friction forces has been introduced. A tribology method has been developed to show an effect of red wine tannin on mouth lubrication. The formation of turbidity had the strongest relationship to the sensory effect of astringency.
This research has successfully chemically characterized tannins and the consequences of their interaction with saliva or proteins. The tribology experiments carried out on the two red wines with different lubricant (saliva or mucin) has also given promising results, and received much attention in the press. The research is continuing with Watrelot and Kuhl.

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

Summary: 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 also 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 artifact 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. Using this method we tentatively identified 27 monoterpene glycosides including two monoterpene trisaccharide glycosides, tentatively identified for the first time in any plant. We measured the terpene glycosides in six cultivars at three maturity time points and demonstrated differential profiles depending on cultivar and maturity. We also modified the method so that it can be used to monitor monoterpene glycosides in wines and during winemaking. We have analyzed the glycoside content during fermentation for wines made in fall 2016 and 2017 with different varieties (Chardonnay, Merlot, Cabernet Sauvignon) and winemaking/processing methods. Monoterpenyl glycoside profiles differed between the grapes and the first alcoholic fermentation samples. In red wines, malonylated monoterpenol glucosides and monoterpenol hexose-pentoses decreased after the completion of alcoholic fermentation. We also measured the volatile composition of the wines during fermentation and we have started to relate changes in terpene volatiles to changes in the glycoside profiles. 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.

Rapid Determination of Molecular and “Truly Free” Sulfur Dioxide by Headspace Gas Chromatography

We completed development of an analytical procedure using headspace gas chromatography (HS-GC) coupled with sulfur chemiluminescence detection (SCD) which can rapidly and precisely quantify molecular and free sulfur dioxide in wine. The method requires minimal sample preparation and involves no chemical reagents (with the exception of a trace internal standard). At room temperature the method can successfully detect levels of molecular sulfur dioxide at concentrations as low as 0.03 mg/L. The total chromatographic time for the method is 8 minutes and, provided that information on the alcohol concentration and pH is readily available, the molecular and free sulfur dioxide concentrations for the sample can be rapidly calculated using simple formulae. The HS-GC method offers a high degree of precision, with a reported coefficient of variation of 3.72%. Comparisons with standard A/O and Ripper results on a large set of wine samples showed large discrepancies for those wines with high anthocyanin levels, suggesting that SO2 bound to anthocyanins is released during those procedures, inflating the amount of free SO2 that is actually available to protect the wine. The characteristics of rapid analysis, good sensitivity, and high precision, demonstrate that the method could be applicable in a production environment, albeit a large scale operation where a Gas Chromatograph could be utilized and maintained.

Oxidation of Wine: Central Role of Iron for Polyphenol Oxidation

We were able to find a quick and simple method to discriminate between the two species of iron in wine, using only a spectrometer and very simple reagents, so the method is accessible in winemaking. We applied the method to measure iron in about 10 wines, and evaluated how the iron species in those wines changed in response to oxygen exposure. The observed responses suggested that the level of iron species in response to oxygen exposure could be used to evaluate a wine’s capacity or reactivity to oxidation. Separately, we also showed the products of reacting caffeic acid quinone with a number of flavonoids, including catechin. The observations help explain the basis of browning in white wines. And looking at quinone reacting with wines, we were able to find specific oxidation products.

Rapid Analysis of Wine Phenolics by Laser-induced Fluorescence

Under the current grant, we conducted an initial evaluation of wine fluorescence properties with a time-resolved (lifetime) fluorescence spectroscopy (TRFS) device that allows for rapid, in situ measurements of fluorescence intensity, spectra and lifetime upon UV laser light excitation and visible autofluorescence light detection. Below, we provide a synopsis of the main results along with an extended report.

First, we measured the excitation-emission matrices of model wine solutions (wine analogs with individual components typically found in wines) that may contribute to the overall fluorescence of finished wine products, together with grape seed extracts and proteins. This analysis will guide the configuration of the TRFS device, which spectral distribution can be adjusted for optimal performance in each application. Moving forward with wine applications, narrower optical filters along the main fluorescence peaks found in the EEMs of wines and their respective components have the potential to increase the detection sensitivity. Only a small subset of wine components were tested in this work, namely caffeic acid, gallic acid, rutin, catechin, and malvidin-3-glucose. Expanding the library of potential contributors to the fluorescence of the final wine product is expected to further guide the optimization procedure of the final device, which could be different depending on the specific application, i.e. it might be of interest to tune the spectral bandwidths to detect a particular contaminant instead of intrinsic wine properties.

Second, we tested the performance of the current lab configuration of the TRFS device to detect spectra (intensity ratio) and fluorescence lifetime of the wine models as well as a variety of wines. Consistently with the EEMs measured in the first place, we found a red shift of red wine with respect to most of the tested wine models, except for caffeic acid, which spectral properties closely resemble those of the finished product. Fluorescence lifetime of all tested wine models was shorter in spectral band 1 than that of red wine. However, for the rest of the spectral bands, fluorescence lifetime of all models except for caffeic acid was longer, where detectable. For caffeic acid, lifetime was always found shorter than for red wine. Commercial wine bottles were then tested. Tannin levels and fluorescence properties were measured to find that both intensity ratio and lifetime in spectral band 4 (570 – 650 nm) better correlate with tannin levels than fluorescence parameters in any other spectral range. This further confirmed some preliminary data that we had acquired previous to this award. Interestingly, the selection of wines for this analysis had a narrow range of tannin levels. Combining the results from the two experiments extends the range of tannins, and initial evaluations indicate a trend: as tannin concentration increases, fluorescence intensity increases, and fluorescence lifetime decreases. With a considerable increase of tested wines and further statistical analysis, these fluorescence parameters have the potential to be used as a proxy for tannin concentration, which would be a faster and economic assay to run compared to current methods. We also applied a multivariate analysis of fluorescence parameters to explore the potential of TRFS to identify or discriminate between different wine varieties. The presented analysis is a very simplified model, but already shows discrimination power. Applying more advanced computational methods and expanding the database with different wines could result in a classifier capable of identifying different wine types in a rapid and inexpensive manner. 2

Third, we evaluated how fluorescence parameters are affected by oxygen levels in wine. This could have potential implications in determining wine quality. For example, after opening a wine bottle, fluorescence parameters could establish when the wine gets spoiled. Our modest first trial showed that wine oxygenation changes some of the fluorescence parameters, but not all. Further measurements and analysis are required to understand these changes and establish an experimental model.

In summary, the studies enabled by this award yielded very promising results and our group plans to continue working on this space, which provides a new and exciting area of research for our time-resolved fluorescence spectroscopy technology that is complementary to our current biomedical applications.


Anthocyanin-Cell Wall Interactions Effect on Tannin Extraction

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

1) Perform extractions on white grape skins at 5 time points (13,17,20,22, and 25 °Brix) throughout maturity with varying levels of added anthocyanins.

• Extracted tannin will be analyzed for concentration, average molecular size, subunit composition, activity, and fraction extracted.
• Measure the levels of pectin methylation in the white grape skins

2) Conduct red-styled fermentations on Sauvignon blanc and Cabernet Sauvignon at 300 pounds per fermentation, with and without an anthocyanin addition. Measurements are the same as Objective 1.

The overall purpose of this proposal is to determine anthocyanins role on the extraction of procyanidin material due to interactions with cell wall material of grape skins. Activity to date has been the isolation and purification of color along with the sampling and extraction of skins with varying concentration of anthocyanins added. Furthermore, 300lb fermentations were conducted, in triplicate, under standard red wine making conditions with both Sauvignon Blanc and Cabernet Sauvignon with a 1.4% (v/v) addition of color concentrate.

Investigating Fruitiness Perception in Red and White Wines

This report details activities that occurred from February 2018 – January 2019. The final date of this project is August 2019 and the next 6 months will include completing the last of the sensory panels and combining all data analysis. A final report will be submitted in January 2020. We are still slightly behind on the timeline due to issues detailed in last year’s report and we also had to renew our IRB (human ethics approval) in June 2018. Once the renewal is submitted it is illegal to run sensory tests on the project until approval is given, which was obtained in September 2018. In January 2019 we completed the last of the Pinot noir sensory panels, although we have not yet done data analysis on the January 2019 panel. Also as stated previously, we have not been able to complete any predictive modeling, some initial reviewer comments said that this objective might have been too ambitious in the timeline and after the 1st year we have to agree and have since removed this objective. We plan on working on predictive modeling in the future but this would be after the current grant is completed.

To date we have investigated 80 different compound combinations and their impact to fruit aroma in Pinot noir wine. We have also completed a panel that shows the influences of phenolic content on fruity aromas in Pinot noir and one panel that shows the impact of ethanol content on fruity aroma in Pinot noir. We have 2 potential marker compounds for red fruit aroma in Pinot noir and 4 red fruit solution sets using fsQCA that show the cause of red fruit aroma in Pinot noir. We have also found 5 solution sets for dark fruit aroma in Pinot noir using fsQCA.

We have also investigated 49 compound combinations for fruitiness in white wine. We are still working on using fsQCA to analyze this data. Preliminary results suggest a combination of low thiols and high esters are responsible for tropical fruit aromas, low to no esters are needed for citrus aromas, and esters and terpenes cause pear, peach and apricot aromas.

We will be running the last 4 Pinot gris sensory panels from February2019-June 2019 and completing the final data analysis. We are in the process of writing the first paper for publication and have done 2 presentations at domestic conferences on the analytical data analysis. Spring /Summer 2019 we will be presenting at 4 different international conferences in Europe and have plans for at least 3 more peer-reviewed publications.

Effect of grapevine red blotch disease (GRBD) on flavor and flavor precursor formation in the grape and on wine quality

Two field experiments were established to investigate the effects of grapevine red blotch disease (GRBD) on flavor and flavor precursor formation in the grape berry and on resulting wine quality. The two objectives of the overall study were to 1) investigate the effect of GRBD on grape berry development with a specific focus on flavor and flavor precursor formation; and to 2) investigate the effect of GRBD on wine quality. Both experiments were located in the same vineyard located near the town of Jacksonville, OR. In both experiments, data vines were identified by visual disease symptoms (or lack thereof), and disease status was confirmed using PCR-based assays in Dr. Achala KC’s laboratory at SOREC.

To evaluate the response of flavor and flavor precursor compounds to GRBV infection during berry development (objective 1), clusters from GRBV+ and GRBV- vines were sampled weekly beginning from just before veraison through to commercial harvest. Vine water status, berry growth, and development were also monitored in those plots subjected to different irrigation treatments. Vine water status was monitored by measurements of midday stem water potential (Ψstem). Results showed that there was no significant interaction between irrigation treatment and disease status on Ψstem. However, there were significant effects of irrigation treatment and disease status on Ψstem independently. Berry size (fresh weight; FW) was consistently higher in GRBV+ vines, significant differences in TSS between GRBV+ and GRBV- vines were observed. There were no significant differences in berry pH between vines of different disease status over the entire course of berry development. Berry titratable acidity (TA; g L-1) were lower in GRBV+ fruit. These responses were only observed after veraison, but they are not as consistent. Flavor and flavor precursor analysis in the grapes is underway.

To evaluate the response of wine quality to GRBV infection (Objective 2), replicate wines were produced from field plots under the supervision of Dr. James Osborne using a standard protocol. Wines were analyzed for volatile aroma compounds using  different techniques including headspace-GC-FID, solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS) and stir bar sorptive extraction-gas chromatography-mass spectrometry method (SBSE-GC-MS), stale-isotope compounds were used as internal standards for accurate analysis. Monomeric anthocyanin and total phenolic contents in wine were also analyzed. Results showed that the RB negative wines under irrigation condition have the highest level of monomeric anthocyanin than other three groups. Total phenolic content varies in wines with different irrigation conditions. Fermentation derived aroma compounds did not show any difference between RB+ and RB-, nor free form grape-derived aroma compounds. Since the free form of grape derived aroma compounds only exist in a small portion, and the majority of these compounds exist in the bound form, analysis is underway for the bound form of volatile flavor compounds in the wine.

Utilizing malolactic fermentation as a tool to prevent Brettanomyces bruxellensis wine spoilage

Brettanomyces bruxellensis is considered one of the most problematic wine spoilage yeasts due to the difficulty of controlling it, the potential significant financial losses due to loss of wine quality, and the cost of prevention and remediation measures. Wine is particularly vulnerable to B. bruxellensis infection during and shortly after the malolactic fermentation (MLF) as SO2 cannot be added until this process is complete. It has been suggested that conducting a rapid MLF initiated by inoculation of Oenococcus oeni is a useful strategy to prevent B. bruxellensis spoilage as this minimizes the length of time the wine is not protected by SO2. This project investigates an additional benefit of conducting a rapid MLF, the prevention of B. bruxellensis growth due to inhibitory interactions with O. oeni. Pinot noir wine (no SO2 additions, no MLF) was produced and used to test the ability of a number of commercial O. oeni strains to inhibit B. bruxellensis growth at the end of MLF. Sterile filtered wine was inoculated with various O. oeni strains and growth and malic acid monitored. When MLF is complete, wines will be inoculated with a select strain of B. bruxellensis and growth and volatile phenol production monitored.

The sensitivity of a number of B. bruxellensis strains to O. oeni is also being determined. B. bruxellensis strains have been sourced representing B. bruxellensis isolates from a wide range of winemaking regions including Oregon. A model wine system was identified for use to improve the rate that B. bruxellensis strains can be tested for inhibition by O. oeni. Results from the model wine system will be used to select which strains will be used in wine experiments that take significantly longer to complete.