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%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.

The objectives of this proposal are to do the following: I. Develop optimized analytical methods for the measurement of: A. Grape skin tannin extractability and concentration potential in red wine. B. Predicted concentration-independent interaction of red wine tannin with protein. II. Explore the influence of grape and wine production practices on developed analytical methods These objectives are consistent with the highest priority research objective as outlined by the American Vineyard Foundation as well as the National Grape and Wine Initiative Part A of the first objective addresses observations that higher quality wines are associated with an increase in skin tannin concentration. Presumably this is a vineyard-derived aspect of wine quality in that the data collected to date were designed to understand vineyard management practices. Historically it has been difficult to predict tannin extraction into wine from grape-based data. Part of this difficulty can be attributed to the lack of accounting for the dependence of extraction on cellular integrity. In order to take into consideration the cellular integrity of the grape berry, an analytical approach which exposes grape berry samples to mild mechanical force is being employed. It is expected that incorporating an analytical component that accounts for cell integrity will provide an improved ability to predict red wine tannin extraction and quality, from fruit samples. Part B of the first objective is designed to improve our understanding of how tannins are perceived and address the generally held perception that tannin quality has a concentration-independent component. Recently published research has found that the enthalpy change of a protein-tannin interaction experiment is dependent in part on the “age” of the tannin in that apparent interaction between tannin and protein declines as tannin structure modification increases. This research was performed using Isothermal Titration Calorimetry on previously purified tannins. This portion of the project will focus on the development of an analytical method that can predict protein-tannin interaction from wines directly. In the second objective, optimized methods will be used to understand experiments in progress by other research programs, vineyards, and wineries. IV. Objective(s) and Experiments Conducted to Meet Stated Objective(s): Part A, Objective #1 The objective is to develop an extraction method that is mild yet predictive of potential skin tannin extraction. A successful method will compare favorably with wines made consistently from the same fruit. A further criterion for success will be that the analytical approach is as rapid as practically possible. Fruit samples have been collected from the Fresno State vineyard and have been subjected to readily-available equipment designed to introduce various degrees of sheer to the sample. The specific target was the introduction of sufficient sheer to differentiate between berries having variable cellular integrity. We initially investigated a Kitchenaid kitchen mixer equipped with various implements. After exploring this mixer with and without added ethanol, a decision was made to test a new instrument due to insufficient extraction. We next transitioned to a Cuisinart food processor. The food processor with a dough kneader worked well for our purposes and all extracts prepared were found to be sufficiently extracted. E&J Gallo was approached and they expressed interest in cooperating on this project. To provide a meaningful data set, they offered to provide us with berry samples and wines from Cabernet Sauvignon vineyards from warm interior valley sites, to cool north coast sites. Wines were also prepared from this fruit to provide comparison. To date, extracts have been prepared and have been analyzed. Also to be analyzed by May 2012 will be exhaustive skin extracts and the red wines produced from samples. Extracts prepared with the food processor and preliminary comments from E&J Gallo indicate that the results are positive. Extracts were analyzed by acid-catalyzed degradation in the presence of excess phloroglucinol. This analytical approach was used because it could provide information on the composition of tannins in the extracts and hence the tissue of origin. Of specific interest was determining the prodelphinidin content which is skin tissue-derived.

The objectives during the 2010 season were to 1) Conduct a small-scale experiment to determine when during fermentation the increase in bound tannin occurs. 2) Conduct experiments using alcohol free tannin and cell wall material obtained from mesocarp tissue to study the mechanism by which tannins become tightly bound to the insoluble matrix during fermentation. 3) Measure free and bound tannin in fruit and pomace samples from larger scale fermentations to confirm our observation that there is a significant increase in bound tannin attached to the insoluble matrix after fermentation.

With regard to objective one, our results show that there is a steady increase in the amount of tannin immobilized onto the insoluble matrix during red wine fermentation. This result is of practical interest because it suggests that interventions intended to alter the adsorption of tannins to cell wall material must begin early in the fermentation. In studying the effect of particle size on tannin adsorption to cell wall material we found that more tannin could be adsorbed to smaller particles than to large ones. This result is interesting because is seems to suggest that adsorption could be a surface phenomenon and that the size of the fragments into which the cell wall material is cleaved during winemaking can influence the amount of tannin bound. This could have practical implications for handling skins and gross lees during fermentation. We found that temperature has an inhibitory effect on tannin binding to cell wall material, the amount removed decreased as temperature increased. Interestingly, however, the amount of tannin immobilized, i.e. not extractable with acetone after incubation increased with increasing temperature. We studied the effect of sugar concentration on tannin binding to cell wall material and found that increasing the amount of sugar present during the incubation of tannin with the insoluble matrix reduced both the amount of tannin adsorbed to the cell wall material and the amount immobilized.

This result may help explain our observation that bound tannin steadily increased during fermentation (see above). We studied the effect of tannin concentration on the binding and immobilization of tannin to cell wall material. We found that tannin adsorbed to the insoluble matrix increases along with increasing tannin concentration but that the level of tannin immobilized to the cell wall material reached a maximum around 750 mg/L. While this result is difficult to interpret without further experiments, it may suggest that at some level tannin binding actually inhibits tannin immobilization to the cell wall material. We also studied the effect of pH, ionic strength and bisulfite addition on the binding and immobilization of tannins to cell wall material. None of these were found to have any influence on tannin binding.

We have accomplished all of the objectives we proposed for the 2008 work. We studied Merlot at a site we sampled in 2007 and found substantially the same results in both years. Fruit from Merlot on 1103-P had 1.7 times as much total amino acids as fruit from 101-14. This result held for Merlot from a second site at Oakville, but not in Merlot from a rootstock trial in Washington. In studying other varieties we found that in some cases (Sauvignon Blanc and Pinot noir) there was a similar effect as seen in Merlot from Oakville, but in other cases the effect was not observed (Monterey Chardonnay) or even reversed (Oakville Cabernet Sauvignon, Washington Chardonnay and Syrah). Our work with Pinot noir from Edna Valley and Sauvignon blanc from Monterey demonstrated that these arginine accumulators can show the same effect as was observed in the proline accumulator Merlot.