Rapid Determination of Primary Amino Acids in Grape Juice Using an o-Phthaldialdehyde/N-Acetyl-L-Cysteine Spectrophotometric Assay

Abstract

Low levels of yeast assimilable nitrogenous compounds (YANC) in grape juice have been associated with sluggish or stuck fermentations. In contrast, excessive levels may lead to the increased formation of ethyl carbamate. Wine yeasts mainly consume primary amino acids and ammonium during vinification. While the ammonium fraction is easily determined by the winemaker, no winery-suitable analytical method has been available to estimate the primary amino nitrogen fraction. This research evaluated a spectrophotometric procedure to measure the primary amino nitrogen fraction. The assay was based on the derivatization of primary amino groups with an o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC) reagent. The resulting isoindole derivatives formed rapidly and were stable at a wavelength of 335 nm. The procedure used a juice blank to account for the absorbance of non-derivative forming compounds in the juice. The assay was insensitive to proline, showed a 3.5% response to ammonium, and correlated with HPLC estimates (R²= 0.988). The relative standard deviations for replicated analyses ranged from 0.27 to 3.20%. Estimates made using this procedure were within the systematic errors for model solutions, and known additions of isoleucine to grape juices had recoveries of 97.6 to 101.3%. The relative accuracy, precision, rapid analysis times and use of low-toxic reagents suggest that this procedure, dubbed NOPA for `nitrogen by OPA', is appropriate for use in wineries.