Comparative Mass Spectrometric Analysis of Proteolytic Activity in Wine and Wine Vinegars

Plant-derived proteolytic enzymes are widely used in biochemistry and food processing. For example, bromelain, ficin, and papain serve as meat tenderizers, while cardosin A is used as a plant-based rennet in cheese production. A cysteine protease has been identified in grapevine products such as fresh juice, wine, and wine vinegar. The enzyme (CYSP) shares sequence similarity with RD21A from Arabidopsis and other plant cysteine endopeptidases. This mass spectrometry-based study investigated the proteolytic activity of wine and wine vinegars, including both commercial and laboratory-prepared samples. Vinegar types examined included white wine vinegar, balsamic vinegar, and red wine vinegar produced through grape juice fermentation and spontaneous acetification. Protease activity and specificity were assessed using substrates such as pure protein standards, casein, and minced beef proteins. The activity assay also included spectrophotometry with azocasein and electrophoresis followed by gelatin zymography. Results confirmed the presence of CYSP and indicated aspartic protease involvement. Digestion experiments coupled with mass spectrometry identified peptide cleavage sites, with C-terminal residues frequently being L, F, R, Y, K, and D/E. This pattern reflects the combined specificity of CYSP and pepsin-like proteases. Notably, CYSP activity was higher in wine, whereas aspartic protease activity predominated in vinegar.