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.
