Composition of Tannins Extracted into Red Wine Emphasizing the Relative Amount of Skin and Seed Tannin

The goal of this proposal is to develop an analytical method to measure seed and skin tannin extraction during red wine fermentations, and to use this method to explore the relative significance of seed and skin tannins in red wine. With this technique, an improved understanding of the influence of viticultural and winemaking practices on wine composition and flavor will be achieved. To date, development of the analytical method is nearing completion. The method utilizes high performance liquid chromatography to analyze the composition of the grape source material as well as the finished wine. Based upon this information, the amount and proportion of skin and seed tannin can be determined. The method is reproducible and accurate. The final method will be used to monitor several experiments. These experiments include both vineyard and winery experiments designed to potentially affect the proportion of skin and seed tannins in wine. Experiments are being conducted on Pinot noir grapes and wine, with plans to include other varieties as well. Viticulture experiments include: a clonal variation experiment, ethanol application and maturity effects on tannin extraction in red wine. Enology experiments include: variations in cold soak time and enzyme addition.

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

During the 2001 season we set out to identify the most important factors that influence the level of large and small polymeric pigments in wines. Polymeric pigments are important because they are the stable color compounds that form during fermentation and wine aging. We found that Pinot noir wines made after cold soak had more polymeric pigment at the time of pressing but that they had less after a period of barrel aging. All wines in our experiments exhibited dramatic compositional changes from pressing until the first racking, showing a decline in tannin and a large increase in both large and small polymeric pigments. Experiments with maximum fermentation temperatures showed that temperature probably plays the biggest role in tannin extraction and polymeric pigment formation. Three temperatures were used, 25C, 30C and 33C. Tannin, large polymeric pigment and small polymeric pigment were all greater at 33C than 25C with 30C intermediate. In all cases there were declines in tannin levels during barrel aging but a concomitant increase in the amount of polymeric pigments that formed. It is clear that important compositional changes occur during barrel aging with regard to tannins and polymeric pigments. It will be important to follow all of the wines made this season through to the level of finished wine in order to adequately evaluate all of the treatment effects imposed on the experimental wines.

Preliminary work indicated that polymeric pigments bleach to some extent with SO2. It was important to determine the extent to which they bleach so that correction factors could be derived for calculating the actual amount of polymeric pigments in wines. This is particularly important in aged wines (3 years or greater) where nearly all of the pigments can be present in the polymeric form. Using aged Pinot noir and Cabernet Sauvignon wines (8 to 28 years old) that contained no monomeric pigments we performed experiments using column purified polymeric pigments to show that they were indeed bleached extensively by SO2. Using combined protein precipitation and SO2 bleaching we were able to derive correction factors that we can now use to more accurately calculate the amount of polymeric pigments present in grape extracts and wines.

PDF: Identification of Factors that Influence the Level of Large and Small Polymeric Pigments in Grapes and Wines

The Extraction of Condensed Tannins in Red Wine Production

In order to validate the oxidation hypothesis of tannin development, and address our goal: Measure the presence of oxidized tannins in seeds, we are investigating methods to directly measure the initial oxidation produce of phenols, quinones. One method using a redox titration did not yield any oxidation product, and an attempt to directly observe quinones by NMR spectroscopy did not show any quinones present. The lack of response may be due to the low sensitivity of these two methods, so a third, and much more sensitive method is now being tested. It involves the production of a reaction product, phenazine, which is totally specific for the presence of ortho-phenols, the expected phenol in seed tannins. We are synthesizing a standard phenazine and will apply this method to testing for quinones in tannins. This method should be simple enough to apply in winery laboratories.

PDF: The Extraction of Condensed Tannins in Red Wine Production

Chemical Characterization of Small Polymeric Pigments in Wines and Red Grape Skin Extracts

Polymeric pigments are important because they are the stable form of color in wines. They are thought to be formed by reaction of monomeric anthocyanin pigments with tannins or flavan-3-ols, such as catechin or epicatechin. During the 1999 season we observed a class of low molecular weight polymeric anthocyanin pigments in wines. In new wines these pigments were a large percentage of the anthocyanin color not bleached by SO2, thus classifying them as polymeric pigments. On the other hand they were not precipitated by protein, which suggested that they had a low molecular weight compared to typical tannins. This observation raised many practical questions, but also brought up several important questions regarding the chemical nature of the small polymeric pigments (SPP), which we addressed in this project during the 2000 season.

One of the objectives of the work was to devise a purification scheme that would permit separation of small polymeric pigment (SPP) from monomeric anthocyanins and large polymeric pigment (LPP). We were successful in developing a procedure to purify SPP based on column chromatography on a Toyopearl HW-40(F) column and eluting the monomeric anthocyanins and the tannin fraction separately with different solvents. Using standard SO2 bleaching to assay polymeric pigments, we were able to show that the SPP actually resides in the monomeric fraction from the column. In the course of this work we discovered that the SPP could be partially bleached by SO2. This was an unexpected result, but indicates that further work needs to be done with polymeric pigments to determine the extent to which they are affected by SO2 bleaching. This is important because most assays for polymeric pigment rely on SO2 bleaching to distinguish them from monomeric anthocyanins. If polymeric pigments are indeed bleached by SO2, it means that such assays give an underestimate of the amount present, and that monomeric anthocyanins are overestimated.

A major accomplishment in this work during the past year has been the synthesis of an SPP dimer containing catechin as the extension unit and malvadin-3-glucoside as the terminal unit. The availability of this dimer by an unambiguous chemical synthetic route will enable us to confirm the structure of the naturally occurring dimer found in grape skin extracts and wine. The availability of the synthetic dimer will also enable us to determine if anthocyanin pigments having that configuration (catechin-malvadin-3-glucoside) are affected by SO2 bleaching. The chemical reaction by which we created the dimer may also point the way to understanding how polymeric pigments are formed in wines during aging.

PDF: Chemical Characterization of Small Polymeric Pigments in Wines and Red Grape Skin Extracts

The Chemical Evolution and Preservation of Color in Red Wine Aging

During the last year we have been working on the production of labeled malvidin 3-glucoside. Using unlabeled material, we have been able to optimize the conditions of enzymatic synthesis as well as the purification of malvidin-3-glucoside from starting material and side-products so that the reaction will yield adequate material to continue the project. Labeled malvidin-3-glucoside is not commercially available and there is no published method to produce it, which made the synthesis particularly difficult. An alternative procedure based on chemical labeling, which was tried first, did not work due to the ionization of anthocyanins, but it has produced other anthocyanins not described in the literature, and so these products may be interesting to analyze at some point later on.

We have also advanced the understanding of red colored polymeric structures and the analytical procedures to measure them. We have found a poor agreement between two analytical procedures, the traditional method of SO2 bleaching and the newly developed Normal Phase ? HPLC methodology. It is necessary to resolve the causes of these differences and also to compare them with the Adams? assay (based on tannin precipitation). This wine study will unequivocally show the origin of the pigmented polymers and clarify the best method to measure them.

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

Biological Effects of Phenolic Compounds on Saccharomyces Cerevisiae

The goal of this project in the 2000-01 funding cycle was to further define the conditions under which phenolic compounds stimulate or inhibit fermentations. Phenolic extracts prepared from Cabernet Sauvignon grape skins were found to be stimulatory to yeast fermentation, particularly accelerating the rate of late fermentation. In contrast, seed phenolic extracts were inhibitory, again with the stronger effect being observed later in fermentation. Purified phenolic compounds were also evaluated for their effects. As reported last year, the effect of the individual phenolic compounds was influenced by media composition. In addition we discovered that there is significant strain variation in response to phenolic compounds.

PDF: Biological Effects of Phenolic Compounds on Saccharomyces Cerevisiae

Studies on the Interaction of Flavor Compounds with Non-Volatile Components of Wine

Flavor and aroma are important factors in influencing food and beverage (i.e., wine) choices. However, knowledge of flavor concentration alone is not sufficient to determine the perceived sensory aroma intensity. This is due to the presence of interactions between flavors and nonvolatile food components which can alter flavor volatility and release. We used NMR techniques to study the mechanisms of binding between flavor compounds and polyphenols, the main nonvolatile constituents of wine. Our results showed that the interactions are dependent on the structure of both the flavor compounds and the polyphenols. The interactions are principally due to hydrophobic interactions between the aromatic rings of the flavors and the polyphenols. However hydrogen-bonding effects help to stabilize the complex and enhance the specificity. By understanding the mechanisms of these interactions and their effects on flavor perception we will be better able to optimize grape and wine composition and winemaking procedures to improve wine flavor.

PDF: Studies on the Interaction of Flavor Compounds with Non-Volatile Components of Wine

Investigation of Mechanisms for Perception of Astringency

The mechanism by which astringency is perceived is unknown. Two different mechanisms have been proposed. 1. The ASTM defines astringency as the puckering or constricting of the oral epithelial tissue, suggesting that a morphological change occurs when an astringent system is evaluated. 2. In contrast, others hypothesize that the rough feeling of astringency occurs when salivary proteins bind to tannins, oral lubrication decreases and friction (roughness) is perceived. Preliminarily, from our study of the morphological changes in the epithelial mucosa, it appears that astringency does not result from “constriction of the oral tissue”. No obvious differences in distances between epithelial cells were observed after application of astringents. However, it is possible that the loose cells on the surface slough off after binding with tannins, perhaps resulting in perception of astringency, but this has not been substantiated. Consistent with the second hypothesis, in previous studies, it has been shown that individuals with low flow rates of saliva perceive astringency more intensely and longer than high-flow subjects. To confirm this effect of salivary flow rate independent of variation in use of the intensity rating scale, artificial saliva was introduced to the judge’s mouth at different flow rates. Astringency was rated lower at high flow rates of application than when lower rates of application were used. In both cases, comparison of individuals with different flow rates and with the application of artificial saliva at varying flow rates, the presumption has been that astringency decreases at a higher flow rate due to restoration of lubrication. However, no difference in astringency was found between application of artificial saliva with protein vs. an artificial saliva containing no protein. These results suggest that higher salivary flow rates may dilute or more thoroughly flush the mouth, and suggest that further research into the mechanism of astringency is needed. The persistence of astringency has been recognized but only recently has the carry-over effect been documented in which each sip of wine influences the astringency perceived in subsequent sips or wines. For meaningful evaluations of astringent red wines during winemaking, blending or in competitions a tasting protocol to remove or reduce the buildup of astringency must be developed. To do this, astringency was rated continuously while red wines were sipped, spit, and judges rinsed with one of 5 solutions. A pectin rinse reduced astringency intensity significantly more than the other rinses. We are presently investigating the most effective way to prevent the increase in intensity for wines varying in astringency level and to define the minimum time to recommend between wines.

Studies on the Interaction of Flavor Compounds with Non-Volatile

Flavor and aroma are important factors in influencing food and beverage (i.e., wine) choices. However, knowledge of flavor concentration alone is not sufficient to determine the perceived sensory aroma intensity. This is due to the presence of interactions between flavors and nonvolatile food components which can alter flavor volatility and release. We used NMR techniques to study the mechanisms of binding between flavor compounds and polyphenols, the main nonvolatile constituents of wine. Our results showed that the interactions are dependent on the structure of both the flavor compounds and the polyphenols. The interactions are principally due to hydrophobic interactions between the aromatic rings of the flavors and the polyphenols. However hydrogen-bonding effects help to stabilize the complex and enhance the specificity. By understanding the mechanisms of these interactions and their effects on flavor perception we will be better able to optimize grape and wine composition and winemaking procedures to improve wine flavor.

Biological Effects of Phenolic Compounds on Saccharomyces Cerevisiae

In this current grant year, it was discovered that some of the variability in response to phenolic compounds displayed by different commercial strains was possibly due to subtle differences in preparation of the synthetic juice medium, Triple M. This was confirmed using slightly different protocols and the Premier Cuvee yeast strain. Therefore, the preparation and composition of this Medium has been redesigned this year in order to avoid this problem. Analysis of the impact of phenolic compounds is continuing. To date, gallic acid appears to be uniformly stimulatory at concentrations found in wine while ferulic acid may be neutral, stimulatory or inhibitory to sugar consumption under laboratory conditions mimicking natural fermentations. The hexose transporters encoded by the FDTT9/HXT11 genes that are under multidrug resistance control were shown to be expressed under enological conditions. The presence of these genes has been shown to be stimulatory for the uptake of inhibitory drugs. They may therefore play a role in uptake or release of phenolic compounds inside of the cell by coupling such movements to the uptake of glucose. In this current grant year, we launched the comparative analysis of the impact of phenolic compounds on different yeast strains (conducted by Laura Lange). She initially found that the effects were quite variable and there appeared to be no consensus as to whether the effects of a given compound were stimulatory or inhibitory with the exception of gallic acid. In the course of her work, we discovered that the inherent variability in the composition of the Triple M medium led to variation in the response of the yeast. The pH of this medium is adjusted using ammonium hydroxide. We found that since the pH of our water supply varied, so did the ammonium content of the medium. Further analysis revealed that this difference in nitrogen content was not impacting the results. However, the medium has been redesigned to contain a constant concentration of nitrogen. Another problem with the medium was the low concentration of potassium. Previous work in my laboratory indicated that potassium deficiency could lead to a sluggish fermentation and that this effect was somewhat strain dependent. We therefore decided to use potassium hydroxide to adjust the pH of the medium to assure a reasonably high concentration of potassium. Finally, the micronutrients were prepared as a stock solution and diluted when added to the medium. We noticed that a precipitate formed in the stock upon storage and that the age of the stock solution was correlated with variability in the effect of the phenolic compounds. This makes sound physiological sense since the cofactors derived from the vitamins and minerals in this mix are critical for respiratory activity of the yeast. To eliminate this problem, we now make the micronutrient cocktail fresh each time and use the Triple M medium within 48 hours of preparation. This has eliminated the variability in the response. This important optimization of the medium slightly delayed progress on the grant, but we are now conducting the comparative studies across strains. This will not be finished as originally hoped by the end of this grant year, so will continue into the first three months of the next grant year.