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 (k(H)/k(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.

Evolution of oxidative and structural characteristics of proteins, especially lipid transfer protein 1 (LTP1) in beer during forced‐ageing

“…After that, the isolation and purification of LTP1 was carried out according to the method of de Almeida et al . (), and the resulting solution containing LTP1 was dialysed with molecular weight cutoff 1000 Da overnight and lyophilised for use. SDS‐PAGE was performed to identify the purified LTP1 (Figure S1).…”

“…One litre of beer sample was concentrated and lyophilised, which was dissolved in 30 mL Tris-HCl buffer and then centrifuged at 10 000 g and 4°C for 30 min. After that, the isolation and purification of LTP1 was carried out according to the method of de Almeida et al (2013), and the resulting solution containing LTP1 was dialysed with molecular weight cutoff 1000 Da overnight and lyophilised for use. SDS-PAGE was performed to identify the purified LTP1 ( Figure S1).…”

“…de Almeida et al . () reported that the concentration of thiol‐containing peptides and proteins was relevant to the oxidative stability of beer, and the higher its content in beer, the stronger the oxidative stability of the beer. Moreover, lipid transfer protein 1 (LTP1) containing eight cysteine residues is the second most abundant protein in beer after protein Z with only two cysteine residues.…”

“…Jiang et al (2018) examined the antioxidant capacity of eight thiol-containing compounds by electron spin resonance spectroscopy, and found that the activity of these compounds was significantly correlated with the amount of free sulfhydryl bonds presenting in the molecules. de Almeida et al (2013) reported that the concentration of thiol-containing peptides and proteins was relevant to the oxidative stability of beer, and the higher its content in beer, the stronger the oxidative stability of the beer. Moreover, lipid transfer protein 1 (LTP1) containing eight cysteine residues is the second most abundant protein in beer after protein Z with only two cysteine residues.…”

Increased protein-thiol solubilization in sweet wort by addition of proteases during mashing

“…The protein structure of native barley LTP1 is stabilized by four disulphide bonds, but during fermentation these disulphides are reduced to thiols (14). LTP1 isolated or purified from beer has been found to exhibit high antioxidative activity in two different antioxidant assays (5), as well as being a highly efficient scavenger towards the 1-hydroxylethyl radical (9). These results suggest that LTP1 is an important protein in relation to the hypothesis about protein thiols being antioxidants in beer.…”