Characterisation of protein-polyphenol interactions in beer during forced aging

Proteins and proteinaceous material were extracted by acetone precipitation of beer that had undergone forced aging through 0 (control), 5 (medium) or 10 (high) heat/chill cycles (60°C 48h/0°C 24h). Size exclusion chromatography analysis of the crude beer extract showed that forced ageing led to a significant increase in binding of phenolic compounds to Protein Z and especially to lipid transfer protein 1 (LTP1). Protein-polyphenol conjugates were also present in high molecular weight (> 100 kDa) and low molecular weight fractions (< 5 kDa), but these conjugates were already present in the fresh beer and were not affected by the forced aging. Treatment of the crude beer extract with sulphite (2 M) dissociated the protein-polyphenol bindings in LTP1 and Protein Z that had been generated during medium forced aging. Identification and quantification of the free, the non-covalently, and the covalently bound phenolic compounds were performed by UHPLC after extraction by methanol, acetic acid, and sulphite, respectively. The amounts of vanillic acid and caffeic acid decreased in the free polyphenol fraction, indicating binding to proteins during forced aging. Epicatechin and quercetin-3-O-glucoside were found to be non-covalently bound during forced aging. Finally, gallic acid, epicatechin, protocatechuic acid, and astragalin were found to be covalently bound already in the fresh beer. © 2020 The Institute of Brewing & Distilling