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

This research project has been aimed at developing an assay for tannins that can be used in wineries. The first such assay we developed was based on tannin binding to a protein coated onto 96-well micortiter plates. This assay is easy to perform, it is sensitive to very low levels of tannins and can provide very high throughput for situations where large numbers of samples must be analyzed. One disadvantage of the plate-binding assay is the well-to-well variability which was minimized by introducing high salt washes into the procedure. A further disadvantage is that the procedure requires a specialized piece of equipment in the form of a microtiter plate reader which is uncommon in most winery laboratories. We used the principle of the plate-binding assay to develop a solution assay which requires only a spectrophotometer to perform. The variability seen in the plate binding assay is eliminated in the solution assay. The solution assay is less sensitive than the plate binding assay, but it has a much wider useful range, and is still sensitive enough to measure tannins even in light red wines. The solution assay is based on measuring the activity of an alkaline phosphatase enzyme (AP) which co-precipitates with bovine serum albumin (BSA) when tannin is added to a solution containing both proteins at a pH near the pi of BSA. The solution assay requires measuring the rate at which AP hydrolyzes a substrate and is thus a kinetic-based assay. In the current year we developed a third assay for tannins in grapes and wines. It takes advantage of protein precipitation by tannins, but is an end point assay rather than kinetic. Furthermore it measures the amount of tannin directly, by formation of a colored complex with ferric ion, rather than indirectly by the amount of protein precipitated by the tannin. This assay is even easier to perform than the solution assay with AP and is less expensive because the AP enzyme is replaced with a suitable ferric chloride solution. Throughput is also higher because it is an end point assay rather than a kinetic-based one. During the 1998 season we used the end point assay to determine the amount of tannin in skins and seeds of Cabernet Sauvignon and Pinot noir berries during development, from three weeks prior to veraison until harvest. In both varieties the amount of tannin in skins changed very little during this time, but the amount in skins of Cabernet Sauvignon was greater than in Pinot noir. Seed tannin was found to be highest shortly before veraison and showed a rapid decline just afterward. In Cabernet Sauvignon seeds the tannin level declined soon after veraison and remained constant during the four weeks prior to harvest, at which time the amount of tannin in skins and seeds was nearly the same. Comparison of tannins in berries with tannin levels in wines made from the fruit showed that less than 25%of the tannins in the berries at harvest was present in the finished wine.

New Parameters to Measure Ripeness

We have accomplished our primary goal of finding what phenolic components change as a result of ripening. We have developed new analytical methods to quantify grape tannins, in particular the skin tannins. The new method allows for the quantification of the polymeric tannins in grapes with a distinction between tannins of different sizes. Using this new method, our data shows that skin and seed tannins are very different, with skin tannins being of a larger size than seed. In addition, the average size of the grape seed tannins increases during the season, and this trend continues through the time of commercial harvest. Another result is that the seed tannins become harder to extract and thus a lower component in wines, while skin tannin levels increase. Thus wines made from more mature grapes should have lower levels of seed tannin and higher levels of skin tannins. While our methods cannot answer the question of “when” grapes are considered mature, as such a judgement requires an interpretation of data, it may be possible to use them to come up with guidelines for assessing relative grape maturity. In addition, it appears that water stress induces a slightly greater maturity based on our analysis.

New Parameters to Measure Ripeness

Grape samples were collected at two Oakville area vineyards during an 8 week period for analysis of phenolics. Wines were also made from these two vineyards at three different maturity stages. All samples are being analyzed for phenolic content using a relatively new procedure which separates phenols based on size. This year of the project was designed to replicate last year’s effort in order to test for year to year differences, and to expand the use of a new analytical method which is capable of measuring polyphenols from both skins and seeds. At this point, all the samples have been collected and extracted. The chromatographic analysis of the seeds is complete, but the skin and wine analyses are still underway. Preliminary analysis of sensory data on the wines prepared by Mondavi winery for both bitterness and astringency shows increasing astringency with ripeness, the same as last year’s wines, but also shows a trend for increasing bitterness. While these trends are interesting, further statistical analysis will establish the significance of the sensory data. The goals of this year of the project were to: 1) Repeat the measurement of the levels and polymer distribution of phenolic compounds during ripening in vineyards selected in consultation with NCVRG representatives. 2) Compare maturity with specific chemical levels. 3) Prepare, in consultation with NCVRG representatives, wines from the same vineyards at different stages of maturity. 4) Analyze the wines using the same procedure to see if the changes observed in the grapes follow through to the wine.

Impact of Fungicide Use on Wine Quality

The purpose of this continuing research project was to investigate the use of combinations of sulfur and commercial fungicides to determine, if control of powdery mildew could be achieved with minimum or no sulfur residues remaining on the grapes themselves or in wine produced from those grapes. The judging panel could distinguish all of the white wine lots and two of the red wine lots from the control indicating some “sulfur” problems. These results indicate that late season applications of sulfur dust, in conjunction with the use of a fermenting yeast known to produce larger amounts of hydrogen sulfide, have the potential to produce off aromas in both red and white wines.

Influence of nitrogen fertilization and other vineyard cultural practices on the

Sauvignon blanc and Chardonnay grapevines grown at the North Oakville Experimental Vineyard (OEV) generally responded favorably to increased rate of nitrogen fenilization up to 90 lbs/acre as demonstrated by higher yield, shoot growth, pruning weight and total leaf area per vine. Differences in fruit composition between low and high nitrogen fertilization were relatively small. However, high N generally increased the pH, TA, malate, K, and arginine levels of must of both cultivars. The field fertilization did certainly improve the ability of the juices to ferment. Without the nitrogen additions, the juices took twice as long to ferment. Otherwise, the routine must and wine analyses show little differences.

Shoot positioning as a means of improving fruit composition, wine quality

Two replicated shoot positioning field trials were conducted from 1987 to 1990; one with Chardonnay, Sauvignon blanc and Cabernet Sauvignon vines at the Oakville Experimental Vineyard and the other with Chenin blanc vines at Davis. Chardonnay. Sauvignon blanc and Cabernet Sauvignon trial: The main conclusions obtained from the Chardonnay, Sauvignon blanc and Cabernet Sauvignon shoot positioning-hedging trial conducted at the Oakville Experimental Vineyard over a period of five years (1987 to 1990) may be summarized as follows. Conclusions are based on five-year averages. 1. Shoot positioning/hedging (SP) of Chardonnay and Sauvignon blanc increased crop yield by 4 to 5%or 0.4 tons/acre compared to no shoot positioning (NSP) and hedging (Tables 1 and 2). Shoot positioning and hedging did not effect the crop yield of Cabernet Sauvignon. 2. SP reduced the amount of direct light and increased the amount of indirect light (diffuse light) in the fruiting zone.