59. Sulfite and thioredoxin: An approach for potentially increasing the free thiol concentration in beer for improvement of flavor stability

Marianne N. Lund (1), Per Hägglund (2), Anne N. Murmann (1); (1) University of Copenhagen, Frederiksberg C, Denmark; (2) Technical University of Denmark, Kgs Lyngby, Denmark

Technical Session 17: Beer Aging
Tuesday, August 16  •  3:30–5:15 p.m.
Plaza Building, Concourse Level, Governor’s Square 14

Oxidation and Maillard reactions both influence flavor stability by decreasing flavor notes associated with a freshly brewed beer and by increasing stale flavors. Thiol-containing proteins have been found to play a role, together with sulfite, in the antioxidative mechanism controlling the oxidative stability of beer, but the free thiol concentration in beer has to be increased in order to have a significant effect on flavor stability. Analysis of the total amount of thiols (free and disulfides) shows that 60-80% of thiols in beer are in their oxidized disulfide form. Thus, one approach to increase the free thiol concentration could be to reduce these disulfides by exploiting the reducing capacity provided by the yeast during fermentation. Sulfite, which is a reductant secreted by the yeast during fermentation, has been tested for its disulfide-reducing capacity in boiled wort, but the concentrations required for efficient reduction were above the limits permitted by EU legislation. Thioredoxin is a small redox protein that reduces disulfide bonds in target proteins, and it has been reported to be secreted from yeast during fermentation. In this study the ability of thioredoxin to reduce disulfides in wort and thereby potentially increase the free thiol concentration in beer has been explored. The efficiency of the disulfide-reducing capacity of thioredoxin was investigated by incubating boiled wort with different combinations of thioredoxin, thioredoxin reductase, and NADPH in an anaerobic chamber for 10 min, 60 min, and 24 hr and analyzed for free thiols by derivatization with ThioGlo 1 and fluorescence detection of thiol-derivatives. When all components of the thioredoxin system were present the free thiol concentration was increased from 10 µM to 90 µM. A complete reduction of wort with tris-(2-carboxyethyl)phosphine (TCEP) resulted in 140 µM free thiols, so under the conditions and concentrations employed in the current study it was possible to obtain an ~60% reduction of the total pool of disulfides by the thioredoxin system at pH 7.0. The efficiency of thioredoxin in reducing disulfides decreased at pH values similar to wort (pH 5.7) and beer (pH 4.5), but an increase in free thiol was still observed at these pH values. The stability of NADPH was found to decrease at pH values similar to wort and beer, but the concentration of NADPH was still found to be in large excess (a factor of 10,000) to the concentration of thioredoxin reductase. The presence and activity of thioredoxin reductase and NADPH in beer is unknown, but we found that sulfite (at concentrations representative for beer) was capable of fully reducing, and thereby activating, thioredoxin. Thioredoxin in combination with sulfite, therefore, may be sufficient for a reduction of disulfides in beer and, thus, a feasible strategy for increasing the concentration of free thiols in beer in order to improve flavor stability.

Marianne N. Lund received an M.S. degree in food science and technology in 2003 from the University of Copenhagen and a Ph.D. degree in 2007 based on studies of protein oxidation in meat. In 2008 she began as a postdoc working with oxidation in beer and the influence of protein thiols and was appointed associate professor in 2012. She worked in collaboration with Novozymes on flavor stability of beer from 2011 to 2013. She has published 40 peer-reviewed scientific papers.