Covalent Protein-Polyphenol Bonding as Initial Steps of Haze Formation in Beer

Beer subjected to forced aging by five (Medium) or ten (High) heat/chill cycles (60 degrees C 48 h/0 degrees C 24 h) showed increased turbidity from 5.4 +/- 0.4 EBC units without forced aging to 12.9 +/- 0.7 and 48 +/- 2 EBC units for Medium and High forced aged beers, respectively. The particle size diameter increased from 100-200 nm to 10-30 mu m. The colloidal changes were associated with modifications of the low molecular weight fraction (similar to 1 kDa), which were found to increase in the soluble part of the beer as identified by size exclusion chromatography. Polyphenol analyses by liquid chromatography and mass spectrometry showed significant losses of gallic acid, hydroxyphenyllactic acid, salicylic acid, chlorogenic acid, vanillic acid, epicatechin, pyrocatechuic acid, ferulic acid, and luteolin during the forced aging, indicating that these phenolic compounds took part in the colloidal changes, proposedly upon polymerization into tannins. Gel electrophoresis coupled with staining by nitroblue tetrazolium indicated polyphenol modification of all proteins and especially LTP1 (lipid transfer protein 1). It is proposed that the di- and tri-hydroxyl phenolic compounds upon oxidation to quinones react with protein nucleophiles of especially LTP1 to generate the initial covalent bonds, which eventually leads to larger insoluble permanent haze particles.