Use of Alkaline Phosphatase for the Analysis of Tannins in Grapes and Red Wines

Abstract

Tannins are the most abundant class of phenolics in grape berries and are the predominant determinants of astringency in red wines. We have adapted a microtiter plate assay that was first described for persimmon tannin, so that it can be used for analysis of tannins in grapes and wines. Our modification incorporates a high salt wash step that is thought to remove non-specifically bound alkaline phosphatase enzyme that is used to detect tannin in the analysis. Application of the plate binding assay to fractions collected from a normal phase HPLC separation of seed tannins indicates that the assay only detects tannins having more than three flavan-3-ol subunits. The standard assay uses bovine serum albumin (BSA) as the protein bound to the microtiter plate, but we have found that casein or gelatin can be substituted for BSA in the assay. Results show that the microtiter plate assay can be used to monitor extraction of tannin from grape skins and seeds into a model wine solution. We have developed a new protein precipitation assay for grape and wine tannin that is based on the tannin's ability to co-precipitate alkaline phosphatase and BSA from a mixture of the two proteins. The tannin-protein precipitate is pelleted by centrifugation and washed to remove residual unprecipitated alkaline phosphatase. The tannin-protein complex is then dissolved in a 1 M diethanolamine buffer pH 9.4 and the amount of alkaline phosphatase activity in the dissociated precipitate is determined by addition of p-nitrophenylphosphate substrate. The amount of alkaline phosphatase activity in the redissolved pellet was shown to be proportional to the amount of seed tannin used to form the precipitate. Because the solution assay is easy to perform and requires only a spectrophotometer, it should be suitable for use even in small winery laboratories.