Characterization of recombinant GRIP32 as a novel haze protein for protein-polyphenol haze models and prevention of haze formation with polysaccharides in the models

Abstract Haze-active proteins are an important factor in haze formation in wine and clear beverages. The aim of this study was to understand the role of haze proteins from pland origin, which are rich in basic amino acids from the plants, in the haze formation involving proteins and polyphenols, as well as to develop strategies to prevent haze formation. The grape ripening-related protein-like (GRIP) containing 12.50% histidine, 28.13% glycine, 16.41% glutamic acid and 1.56% arginine was screened as a potential haze protein from grape by bioinformatics, and recombinantly expressed, purified and characterized. The recombinant GRIP32 (rGRIP32) could be effectively removed by the adsorbents (i.e. bentonite and kaolin), or hydrolyzed by proteases (i.e. papain, neutral protease, and alkaline protease). Procyanidins (PCs) or epigallocatechin gallate (EGCG) were proved to form a haze with rGRIP32. Protein-polyphenol haze models were established using rGRIP32 at 25 °C and pH 5.0. Polysaccharides including pectin, xanthan gum, and guar gum were shown to suppress the haze caused by rGRIP32. The microscale thermophoresis (MST) assay provided new evidences for the binding of proteins and polyphenols to form haze, and demonstrated that the mechanisms of polysaccharide-based haze prevention include competitive binding of polyphenols and formation of ternary complexes with them.