Development of a New Assay to Measure Tannins in Grapes and Wines

We studied pectin’s effect on the analysis of tannins in our precipitation assay, and found that the order in which the assay components were added determined whether or not precipitation was inhibited. If pectin was mixed with tannin before tannin was exposed to protein, then there was a marked inhibition of the amount of tannin precipitated. However, if the tannin was mixed with the protein prior to the addition of pectin, then there was no effect on the amount of tannin precipitated. We used citrus pectin and carboxymethylcellulose (CMC) and determined that the amount of citrus pectin required to inhibit the tannin assay by 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} was approximately 500 ng/ml whereas CMC showed 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} inhibition at about 70 ug/ml. We extracted water-soluble and EDTA soluble pectin from grape berries and found that water-soluble pectin does not inhibit tannin precipitation, but EDTA soluble fraction can. However, EDTA soluble grape pectin has a much higher 150 (1400 ug/ml) in the assay than either citrus pectin or the CMC. Thus, since the water-soluble grape pectin was ineffective at all levels tested, and the EDTA soluble pectin was only marginally effective, this result indicates that it is highly unlikely that grape pectin will interfere with the tannin solution assay. We studied tannin development in Cabernet Sauvignon, Pinot noir, Syrah and Zinfandel. Zinfandel looked very similar to Cabernet Sauvignon but compared to Cabernet Sauvignon, Syrah had more tannin in the seeds than the skins at harvest. Even though the Syrah seeds contained more than twice as much tannin as Cabernet seeds, the wine made from the Cabernet fruit had 2.4 times as much tannin (1414 mg/1) as the wine from the Syrah fruit (585 mg/1). This points to tannin extraction as being more important for the amount in the resulting wine than the amount in the fruit at harvest. In the course of experiments to determine if the “background” absorbance in our tannin assay represented polymeric pigments, we found that protein precipitation separated the polymeric pigments into two classes that we have designated LPP for large polymeric pigments, and SPP for small polymeric pigments. We devised a combined procedure based on protein precipitation and traditional SO2 bleaching methods to allow measurements of SPP and LPP in wines along with the determination of total tannin. Thus, we have extended the scope of the solution assay and we are able to use the assay to provide a direct measure of the polymeric pigments in wines.

Investigation of Mechanisms for Perception of Astringency

Astringency has been shown to have carry-over effects which influences the perception of astringency of red wine and astringent compounds. To quantitatively measure this effect, trained judges rated intensity of astringency continuously while repeatedly sipping wine. When red wines were sipped 4 times at 25 second intervals between sips, the astringency intensity increased significantly with each sip. At each sip, astringency increased, reached a maximum intensity about 15-16 seconds after the sip was taken, then decreased slowly until the next sip was taken and again astringency increased. For two of the wines the increase with each sip was significant, whereas the other two the increase in astringency between the 3rd and 4th sip was not significant. To see what happened when more sips were taken, astringency was continuously rated over 8 sips for alum and tannic acid. The 4th sip of tannic acid was not more significantly intense than the 3rd , although the astringency continued to slowly rise. The practical consequences of these results are that most evaluations of red wines are rendered invalid due to the carry-over phenomenon demonstrated in this study. Astringency of red wines evaluated either by a winemaker during winemaking or for blending or by wine show judges is influenced by the wines tasted before it. To quantify the effects of physiological differences on perception of astringency the saliva flow rates of the judges in the red wine and alum/tannic acid studies above were determined. Individuals with low flow rates of saliva perceived astringency of red wine and of the alum and tannic acid more intensely and longer than high-flow subjects. This physiological difference in perception of astringency is of great significance in understanding how astringency is perceived. Since saliva flow rate varies with the size of the person generally speaking, the practical value of this information may lie in marketing wine, where it may account for preferences of “smaller” consumers. In evaluations of the viscosity of saliva-tannin mixtures, it was shown that viscosity of saliva decreases when tannin is added, analogous to sipping red wine. This is consistent with the observations of the effect of salivary flow status. If astringency is felt as the friction resulting from precipitation of saliva proteins by tannins, than high-flow subjects can restore lubrication better and hence perceive lower levels of astringency than low-flow individuals.

Studies on the Interactions of Flavor Compounds with Nonvolatile

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. This proposal is a continuation of a previously funded project. During previous years we focused on developing analytical methods (Gas Chromatographic and NMR) and sensory procedures for measuring odorant interactions in model solutions (Objective b and c). Results have been published (Mialon and Ebeler, 1997) and presented at national meetings of the ASEV (June 1996 & June 1998). An oral presentation outlining recent progress was presented at the National Meeting of the American Chemical Society in New Orleans, LA in August, 1999. A manuscript has been accepted for publication as a result of this research.

The Extraction of Condensed Tannins in Red Wine Production

Over 25 wines were fermented on -20 scale using Cabernet Sauvignon grapes from four different vineyards in California. For each vineyard, a control fermentation was compared with treatments employing maceration treatments including oak extract addition, extended maceration, heat at the end, color enzyme addition, and rotary fermentation. All wines were analyzed for phenolic composition by several different assays. Many of the treatments had small or variable effects, but heat at the end appears to consistently increase phenolic levels. Wines from this and the previous years’ experiment will be offered for tasting at an event in the winter of 2001.

Biological Effects of Phenolic Compounds on Saccharomyces cerevisiae

Plant phenolic compounds are biologically active, affecting many functions of eukaryotic cells. Grapes are rich in a variety of phenolic compounds, many of which have been shown to possess activity in mammalian systems. The effects of phenolic compounds on the metabolic activities of the yeast Saccharomyces have not been thoroughly studied. The goal of this work is to investigate the effect of phenolic compounds on growth and fermentation rates in Saccharomyces cerevisiae under enological conditions and to evaluate the role of these compounds as antioxidants in yeast.

Hard and Soft Tannins: Sensory Definition and Chemical Interpretation

Twelve 1998 Cabernet Sauvignon wines have been obtained from wineries (with minimal oak contact) to select those perceived to be “hard” and “soft”. Winemakers have been interviewed in an attempt to define how each makes the judgement. Although ‘hard” or “soft” are customarily defined by mouthfeel, the contribution of other wine sensory properties is not known. These factors could include color, the aroma and flavor of the wine. More subtle, yet important, we do not know how each individual integrates the perception of taste and mouthfeel. Ten winemakers participated in a “sorting” test in which they were asked to taste the 12 wines and group them on the basis of their perceived similarity. The wines were served in dark beakers, so no information from smell or color was available for this task. Each winemaker used his/her own criteria for the grouping. (For these tests, dark glasses were used to remove color cues). Students at UCD did the same similarity sorting tasks and then were trained for time-intensity (TI) studies in which the intensity of each attribute was rated continuously over time. For each wine, the trained judges took one sip of wine and initiated rating intensity; at a prompt they swallowed the wine, then 25 sec after the first sip, they took a second sip, while continuing to rate the intensity. The procedure of rating for two successive sips was used to include the carryover effects that occur under standard tasting conditions in which several sips are taken, without rinsing. Bitterness, astringency and sourness were evaluated in duplicate, using blue glasses and testing under red light.

Investigation of Mechanisms for Perception of Astringency

Astringency intensity increases progressively with sips, a protocol for measuring astringency in the sensory laboratory was developed which would optimally mimic normal wine drinking behavior. Sip volumes and times between sips of typical consumers were quantified. On the average, 10 ml of wine were sipped at 25 sec intervals, with the wine held in the mouth 3 sec before being swallowed. Using this information, the protocol for rating astringency continuously through four sips was implemented. Judges sipped 10-ml of red wine, swallowed 6 sec later, and sipped three more sips at 25 sec intervals to mimic a realistic pattern of wine consumption by normal consumers. Upon successive sips, a consistent increase in astringency was found. After each sip, the intensity of astringency increased continuously to a maximum (FIG. 2.2), and then decreased slightly, but the initial intensity values before ingestion increased with each sip. The first two sips produced larger increases in astringency for the more astringent wines. However for sips 3 and 4, the absolute increase in astringency per sip becomes more similar. These results have important implications for wineries testing the effects of variables such as maturity on astringency. Sipping several samples in a row can not provide valid information about the effect of the treatment. Thorough rinsing between wines is essential or a time intensity method could be used. It has been hypothesized that sensory perception of astringency is the result of tannins binding to salivary proteins. As a consequence, the effectiveness of salivary lubrication is decreased, and astringency is perceived as the friction between two “unlubricated” surfaces. In previous sensory studies, we demonstrated that increasing viscosity lowers astringency. This was hypothesized to occur due to restoration of lubrication. In the research conducted this year, the change in viscosity of saliva upon addition of tannin was measured. The viscosity of saliva decreased after reacting with tannin. Future research is needed to determine if the observed small viscosity change corresponds to a significant change in perception of astringency.

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

The overall goal of this research is to understand how polyphenols, important nonvolatile constituents of grapes and wines, influence the volatility, intensity, and release of flavor aroma compounds in wines. Preliminary studies suggest that polyphenolics interact with flavor compounds in model solutions. In initial studies, we have shown that the structure of the polyphenol as well as the structure of the odorant significantly affect the interaction. The information obtained from this work will lead to new insights into the way in which polyphenol interactions influence wine aroma and taste as well as wine color, haze formation, and astringency perception.

The Extraction of Condensed Tannins in Red Wine Production

The winemaking portion of the experiment was completed on time on the commercial scale and on the small scale at UCD. Samples were collected and prepared for analysis. The various chemical analyses are still underway due to the large number of samples and the demand for analysis equipment by other projects. In summary, approximately 300 samples have been analyzed to date, all for phenolics by normal and reverse phase HPLC, as well as spectral measures of co-pigmentation. These have included all samples from the commercial wines, but not those from the small scale analyses. Since each of those procedures yields many results, the challenge now is to try an interpret the data to the point where it can be summarized in simple and understandable terms. Data collected so far shows some expected trends, but also some unexpected ones, in particular the increase in tannin levels during aging. With the large amount of data collected, additional analysis is needed to look for interactions and consistency. Additional data still must be collected and analyzed on the small scale wines. This data will be used to plan for the third year’s experiments during summer 1999.

Adaptation of a Simple Assay to Measure Tannins in Grapes and Wines

Premium red wine production is dependent upon proper extraction of pigments and tannins from grape berry skins and seeds during winemaking. While many methods are available to estimate the level of polymeric phenols in grapes and wines, none of the simple ones give an independent estimate of the amount of tannin present. Our aim was to provide a simple tannin assay that would be suitable for analysis of grape berries during maturation as well as for monitoring tannin extraction during fermentation and extended maceration. We have taken a tannin assay that was originally developed for measuring tannins in persimmon and adapted it so that it can be used for measuring tannins in grapes and wines. We studied many of the compounds in wine that could potentially interfere with this analysis and have found that while some can indeed inhibit the assay at very high concentrations, they do not interfere at the levels found in wines. Thus, the plate binding assay has been successfully adapted. In the course of working with the plate binding assay we realized that with some simple modifications we might be able to develop a much simpler solution assay that would not require a specialized piece of equipment like a microtiter plate reader. Although our initial research objectives were only related to adapting the plate binding assay, we were able to quickly demonstrate the feasibility of a solution assay, and we have now successfully developed a new assay method for tannins. The solution assay is simpler than the plate binding method we originally set out to adapt for grapes and wine. It is also much less variable than the plate-binding assay and is useful over a much wider range. Because it requires only a spectrophotometer and a small table-top centrifuge rather than a specialized piece of equipment like a microtiter-plate reader, this analysis will be suitable for use in many winery laboratories. One of our primary objectives in this project was to provide a simple tannin assay. The new analysis that we have developed meets that objective better than the plate binding assay ever could have.