Beer Thiol-Containing Compounds and Redox Stability: Kinetic Study of 1‑Hydroxyethyl Radical Scavenging Ability

Abstract

The 1-hydroxyethyl radical is a central intermediate in oxidative reactions occurring in beer. The reactivity of thiol-containing compounds toward 1-hydroxyethyl radical was evaluated in beer model solutions using a competitive kinetic approach, employing the spin-trap 4-POBN as a probe and by using electron paramagnetic resonance to detect the generated 1-hydroxyethyl/4-POBN spin adduct. Thiol-containing compounds were highly reactive toward the 1-hydroxyethyl radical with apparent second-order rate constants close to the diffusion limit in water and ranging from 0.5 × 10⁹ L mol^–1 s^–1 for the His-Cys-Lys-Phe-Trp-Trp peptide to 6.1 × 10⁹ L mol^–1 s^–1 for the reduced lipid transfer protein 1 (LTP1) isolated from beer. The reactions gave rise to a moderate kinetic isotope effect (<i>k</i>_H/<i>k</i>_D = 2.3) suggesting that reduction of the 1-hydroxyethyl radical by thiol-containing compounds takes place by hydrogen atom abstraction from the RSH group rather than electron transfer. The content of reduced thiols in different beers was determined using a previously established method based on ThioGlo-1 as the thiol derivatization reagent and detection of the derivatized thiols by reverse-phase liquid chromatography coupled to a fluorescence detector. The total level of thiol in beer (oxidized and reduced) was determined after a reduction step employing 3,3′,3″-phosphanetriyltripropanoic acid (TCEP) as the disulfide reductant. A good correlation among total protein and total thiol content in different beers was observed. The results suggest a similar ratio between reduced thiols and disulfides in all of the tested beers, which indicates a similar redox state