Investigations into protein reduction in grape must and wine: Screening the efficacy of 21 peptidases and the effects of thermal treatments, ultrasound, and reducing agents

Protein denaturation and aggregation during storage of wine often lead to undesired haze formation, causing the wine to lose its marketability. To prevent haze formation, proteins are removed from musts and wines primarily through bentonite fining or, less commonly, using proteolytic enzyme preparations. Both unit operations represent currently authorized enological procedures with their own drawbacks. To enhance our understanding of wine protein degradation by proteolytic treatments, this study aimed to provide deeper insights into the technical applications of protein removal by thermal treatments, being eventually complemented with experiments applying reducing agents, ultrasound and a selection of 21 peptidases, including novel insect-based preparations. First, wine proteins were subjected to variable heat treatments (5–60 min, 30–80 °C; equivalent to 0.0005–60 pasteurization units [PU]) without peptidase addition in a citrate buffer (pH 3.3), revealing that protein denaturation and immediate aggregation occurred even during mild heat treatments at 0.01 PU. Ultrasound and reducing agents were ineffective for modulating protein denaturation and aggregation. An inhibiting effect of the reducing additives on the tested peptidases was not found. Efficient proteolysis was achieved only with five enzyme preparations, all of them aspergillopepsin-based ones and all requiring a thermal treatment with 0.1 PU or higher. Evaluating the efficacy of the peptidases in different matrices, effective proteolysis of must and wine proteins was achieved in buffer and grape must, but not in wine.