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.