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%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.

Improvement of Wine Quality: Tannin and Polymeric Pigment Chemistry

Accomplished Objectives (06/2012-01/2013)

Upon receipt of funding experimental design was finalized and a strategy for implementation was refined. Appropriate grape varieties were selected for winemaking. Meanwhile, instrumentation needs were satisfied and preliminary cocoa, preveraison skin, grapeseed extracts, and fractionated young wine samples were obtained. We are 4 months into the practical and laboratory experimentation portion of the project which began with September’s harvest. At the end of September and into the beginning of October fruit was harvested from the UC Davis Oakville Research vineyard. Production progressed through December with bottling January 28, 2013. Upon completion of secondary fermentation, instrument training, method development, and data acquisition took place.

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

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.

Identification of Factors that Influence the Level of Tannins and Polymeric Pigments in Grapes and Wines

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.

Identification of Factors that Influence the Level of Tannins and Polymeric Pigments in Grapes and Wines

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.

Identification of Factors that Influence the Level of Tannins and Polymeric

This project had only one objective; to conduct an experiment on a small experimental wine lot with the goal of accounting for all of the tannin in fruit at harvest, showing how it is partitioned during fermentation and taking into account tannin that might be tightly bound to cell wall material in the pomace and gross lees. This objective was accomplished and we were able to account for nearly all of the tannin from the fruit in the various fractions resulting from fermentation including, pomace, lees, seeds from the lees and the resulting wine. The major result from this research is that compared to fruit there appears to be an increase in the amount of tannin in the pomace and lees that is not extractable with acetone. It is thought that this increase in non-extractable tannin represents tannin from the skin and seeds that becomes attached to the insoluble matrix of the berry such that after fermentation it is no longer extractable with acetone. While the mechanism for this phenomenon is unknown, it represents a significant portion of the extractable tannin in fruit at harvest. Understanding the winemaking parameters that influence this phenomenon should enable winemakers to more carefully control tannin extraction during red wine fermentations.

Amino acid levels and phenolic biosynthesis in skin and pulp of grape berries during ripening

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.

Metabolic Profiling of Grape and Wine Aromas

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. We have developed a procedure using GC-MS combined with solid-phase microextraction SPME) for profiling the free volatile compounds in Cabernet Sauvignon grapes. Different sample preparation (SPME fiber type, extraction time, extraction temperature and dilution solvent) and GC-MS conditions were evaluated to optimize the method. For the final method, grape skins were homogenized with water and 8 mL of sample were placed in a 20 mL headspace vial with addition of NaCl; a polydimethylsiloxane (PDMS) SPME fiber was used for extraction at 40° C for 30 minutes with continuous stirring. Using this method, twenty-seven flavor compounds were monitored and used to profile the free volatile components in Cabernet Sauvignon grapes at different maturity levels. Ten compounds from the grapes, including 2-phenylethanol and β-damascenone, were also identified in the corresponding wines. Using this procedure it is possible to follow selected volatiles through the winemaking process.

Identification of Factors that Influence the Level of Tannins and Polymeric Pigments in Grapes and Wines

We measured tannin in five separate lots of Pinot noir fruit destined for commercial fermentations ranging from 1088 Kg to 517 Kg. All of the values we obtained for fruit tannin are similar to values we have seen for Pinot noir fruit in our previous studies, ranging from 3.6- 4.3 mg/gFW of fruit. We collected wine samples from each of the lots at pressing and recorded volume of each lot. We measured tannin in the wine from each lot and found that the values for wine tannin were similar to values we have seen from Pinot noir press wine in the past, from 441 to 863 mg/L. We measured tannin in the pomace, and the seeds from the pomace and the gross lees associated with each lot. Nevertheless, our calculations show that we can account for only 48-71%of the tannin we measured in the fruit. Thus 29-52%of the tannin in the fruit is designated as “missing tannin”.

We used a procedure referred to as “œthe acid butanol/ferrous sulfate” method in an attempt to detect tightly bound tannin in the insoluble material from the pomace. The samples used were ones that had previously been extracted with acetone to remove all of the easily extracted non-covalently bound tannin from the pomace. This material was suspended in an aliquot of 1-butanol with HCl and ferrous sulfate and heated for the time specified in the procedure (Waterman and Mole, 1994). The pomace from all of the lots tested gave a very strong reaction in the acid butanol system. The fact that the insoluble material from the pomace gave such a high absorbance after treatment in the acid butanol procedure suggests that there may be a considerable amount of tightly or covalently bound tannin in the insoluble matrix of the pomace. At this point we cannot estimate how much tannin this might be in terms of catechin equivalents, which are the units we use for the protein precipitation analysis of tannins. Thus we cannot estimate how much of the “missing tannin” this might represent.

We made cell wall preparations from skin and mesocarp tissue from fruit at harvest by extracting the material with acetone and then grinding to a fine slurry in a ground-glass homogenizer. We also made similar preparations from the pomace and the gross tank lees after fermentation. These samples will be used to compare the tannin binding capacity of cell wall material in the fruit to the binding capacity of the insoluble matrix after fermentation. This will give information as to how the tannin binding capacity of the insoluble matrix of the berry changes during fermentation.

The Chemical Evolution and Preservation of Color in Red Wine Aging

During the last year we have been able to synthesize and purify labeled malvidin 3-glucoside (the major pigment in red wine). Our report on the novel procedure to prepare this radiolabeled substance has been accepted for publication. The amount of material produced was enough to start a series of experiments that study the fate of malvidin-3-glucoside during wine aging. The variables that are being varied are pH, temperature, tannin concentration, co-pigmentation and oxygen. In order to analyze the new products we have developed an analytical technique that combines the fractionation of polymeric compounds with tritium quantification.

We have also taken advantage of labeled malvidin-3-glucoside to study the fate of malvidin-3-glucoside during wine fermentation. In this case we have conducted small-scale fermentation where the labeled material has been added when the maximum concentration of anthocyanins occurred.

PDF: The Chemical Evolution and Preservation of Color in Red Wine Aging