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The Sway of Specialty Malts and Mash pH on Iron

Ion Speciation and the Reducing Power of Wort

Thijs Van Mieghem, Filip Delvaux, Sven Dekleermaeker, Hedwig Neven &
Scott J. Britton

To cite this article: Thijs Van Mieghem, Filip Delvaux, Sven Dekleermaeker, Hedwig Neven
& Scott J. Britton (2023): The Sway of Specialty Malts and Mash pH on Iron Ion Speciation
and the Reducing Power of Wort, Journal of the American Society of Brewing Chemists, DOI:
10.1080/03610470.2023.2178231

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Journal of the American Society of Brewing Chemists
https://doi.org/10.1080/03610470.2023.2178231

The Sway of Specialty Malts and Mash pH on Iron Ion Speciation

and the Reducing Power of Wort
Thijs Van Mieghema, Filip Delvauxa, Sven Dekleermaekerb, Hedwig Nevenc,d and Scott J. Brittonc,e
Biercentrum Delvaux, Neerijse, Belgium; bBrouwerij De Koninck, Antwerp, Belgium; cResearch & Development, Puurs-Sint-Amands, Belgium;
a
d
Centre for Food and Microbial Technology (CLMT), Department M2S, KU Leuven, Leuven, Belgium; eInternational Centre for Brewing and
Distilling, Institute of Biological Chemistry, Biophysics, and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt
University, Edinburgh, United Kingdom

ABSTRACT KEYWORDS
In contrast to other fermented beverages, beer quality generally diminishes over time. This Specialty malt; iron; ion
diminishing quality hinges heavily on the oxidative degradation of beer compounds by reactive speciation; mashing; mash pH;
oxygen species (ROS), whose formation is in part catalyzed by Fe(II) ions via the Fenton and wort; oxidative stability; beer
Haber-Weiss reactions. Consequently, ROS accumulation throughout the brewing process results
in oxidative instability and accelerates numerous beer staling reactions, like those frequently
associated with the onset of unwanted flavors, aromas, and an unaesthetic appearance. However,
despite its critical importance to beer stability, the oxidative state of iron in wort and finished
beer continues to be poorly characterized. In this investigation, the influence of kilned specialty
malt utilization on total free iron and iron ion speciation in wort was determined by EBC Method
9.13.1. Further, the reducing power of each wort was determined via 2,2-diphenyl-1-picrylhydrazyl
(DPPH). Here, we demonstrate that kilned specialty malt utilization influences total iron concentration,
the balance between Fe(II) and Fe(III) ion species, and the reducing power of wort. Furthermore,
our results reveal a negative correlation between mash pH and total iron concentration in finished
wort. These results indicate that beer’s oxidative flavor stability may be improved by using lower
kilned malts and adjusting mash pH.

Introduction and aromas, originating from Maillard reactions. The

Flavor evolution occurs unavoidably throughout beer aging;
however, the extent of transformation is dictated by an
amalgamation of the beer’s inherent physical properties and
the production, packaging, and storage conditions.[1] In par-
ticular, stale flavor formation is a common sensorial attribute
occurring over time, whereby undesirable aroma-active car-
bonyl compounds, such as aldehydes and ketones, develop
from oxidative reactions and become increasingly pro-
nounced, contributing to perceived olfactory and gustatory
aged flavors.[2,3] Therefore, aging beer may develop aromas
akin to toffee, caramel, and burnt sugar or exhibit unwanted
flavors, such as those reminiscent of sherry, solvent, earth,
hay, bread, or cheese. However, the papery and cardboard
notes, due to increasing levels of (E)-2-nonenal produced
from the oxidation of fatty acids (e.g., linoleic acid, linolenic
acid), have been cited as one of the most significant staling
compounds perceived in beer, particularly lager beer.[3]
Consequently, the (E)-2-nonenal sensorial attribute has
become synonymous with aged beer, as its concentration
increases to levels above the flavor threshold (0.03 μgL−1)
in time.[3] Alternatively, ales often present a more complex
aged character, including caramel and Madeira-like flavors
increased occurrence of Maillard products in ales compared
to lager beer is a consequence of the increased wort strength
and the routine use of colored malts.[4]
Fe(II), and other transition metal species, can catalyze
the formation of reactive oxygen species (ROS) throughout
malting, wort production, and beer storage.[3,5,6] The pres-
ence of these chemical species accelerates the oxidation of
fatty acids, leading to the rapid creation of (E)-2-nonenal,
as well as ester and iso-α-acid degradation, ethanol oxida-
tion, and other reactions that result in a deviation from
the intended sensory profile.[3,5,6] The participation of iron
in oxidative reactions, at least in part, arises from their
catalytic role within the Fenton and Haber-Weiss reactions,
where Fe(II) ions promote the activation of stable
ground-state oxygen into ROS via the oxidation of Fe(II)
to Fe(III).[6–9] Since temperatures are high and oxygen is
present, the conditions are optimal for the Fenton and
Haber-Weiss reactions throughout mashing and boiling.
During these reactions, Fe(II) will function as an electron
donor and oxidize to Fe(III), preventing the ion from acting
as a catalyst in further reactions.[10,11] However, these reac-
tions are regenerative, where Fe(II) oxidized to Fe(III) can

CONTACT Scott J. Britton sjb7@hw.ac.uk

Supplemental data for this article can be accessed online at https://doi.org/10.1080/03610470.2023.2178231.
© 2023 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
2 T. VAN MIEGHEM ET AL.
be reduced once again to Fe(II) through interactions with
reductones, intermediate Maillard compounds containing
enediol structures, innately present in wort.[9] This redox
cycling allows Fe(II) regeneration, consequently promoting
ongoing ROS formation.
Moreover, Fe(II) ions also imbue a metallic off-flavor to
beer when present at a concentration greater than 1 mg/L.[12]
More precisely, the perception of metallic flavor is not
caused directly by the presence of Fe(II) ions, but rather
by their subsequent reaction with lipids present within the
buccal cavity to form volatile flavor-active carbonyls, such
as 1-octen-3-one, 4,5-epoxy-(E)-2-decenal, and
1,5-octadien-3-one, yielding a metallic flavor.[13–17] These are
perceived via retronasal olfaction, since occlusion of the
nose during tasting removes the metallic perception.[18]
Conversely, this effect is not observed with Fe(III), as Fe(III)
does not lead to the formation of these carbonyls, because
of its non-reactive nature with the precursor lipids.[13,19] This
outcome is consistent with the age-old practice of rubbing
beer foam on the back of the hand to confirm the presence
of metallic flavor in beer. If a metallic odor is perceived, it
results from the reaction between involatile Fe(II) in the
beer foam and epidermal surface lipids, producing the vol-
atile carbonyls responsible for the metallic odor.
Overwhelming, free Fe(II) is a significant player in accel-
erating flavor deterioration in beer, and the concentration
of the ion should be kept low to guarantee sufficient oxi-
dative stability. Previously, this was confirmed with electron
spin resonance (ESR) spectroscopy, which confirmed that
the addition of Fe(II) increased the radical formation in
wort and beer significantly.[10,20] The authors recommend
reviewing the publications by Van Mieghem et al. 2022 and
Mertens et al. 2021 for further information on iron and
other transition metals’ role in flavor deterioration.[5,21]
However, despite its damaging influence on oxidative insta-
bility, the oxidative state of iron existing in the wort and
throughout the production process continues to be poorly
understood.
In this investigation, we demonstrate that kilned specialty
malt utilization 1) significantly impacts the total iron con-
tent, the balance between Fe(II) and Fe(III) species, and the
wort’s reducing power and 2) a negative correlation between
mash pH and wort iron content. Together, these results
suggest improving oxidative flavor stability could be
improved by avoiding caramel malts for color and mash pH
adjustment.
Experimental
An amber-colored beer was selected as a reference, since
this style often contains different specialty malts and fre-
quently suffers from the metallic off-flavor. The beer had
an OG of 12.6°P, 5.4% ABV, and a color of 35 EBC. Malts
used were Pilsner, Cara50, Cara120, and Pealed Roasted
Barley (PRB) from Mouterij Dingemans (Stabroek, Belgium).
Chemical products used were (NH4)2 Fe(II)SO4.6H2O, L(+)-
ascorbic acid, Fe(III)Cl3.6H2O, 1,10-phenanthroline mono-
hydrate (phenanthroline), and sodium acetate from Carl
Roth GmbH (Karlsruhe, Germany). HCl (37%) and NaOH
(50%) from VWR International BVBA (Leuven, Belgium).
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical (95%)
used was from Alfa Aesar (Thermo Fischer), Ward Hill,
MA, USA. Acetic acid (99.8%) was from Sigma-Aldrich
Chemie GmbH (Steinheim, Germany). Filtered (RO) water
was obtained from a Reverse Osmosis 6 PLUS 2.0 unit from
Wilhelm Werner GmbH (Leverkusen, Germany).
Wort production
Malt (50 g), milled at 0.70 mm (DLFU disc mill from Bühler
Holding AG, Uzwil, Switzerland), was mixed with 200 mL
65 °C RO water in a stainless steel beaker. The malt-water
mixture was afterward placed into the LB8 Mashing Device
(Lochner Labor + Technik GmbH, Berching, Germany) fol-
lowing a temperature and rest profile comparable to the
industrial standard: 65 °C for 50 min, 74 °C for 10 min, and
78 °C for 2 min before cooling to 20 °C. At the 74 °C tem-
perature step, an additional 100 mL 74 °C RO water was
added. The temperature increased at a rate of 1.5 °C/min.
The agitator was set to 60 RPM. The worts incorporating
special malts were made to a color of 35 EBC through the
combination of specialty malt with Pilsner malt
(Supplementary Table I).
The beaker’s contents were brought to a weight of 450 g
by adding RO water. The mash was then separated over a
filter (MN 614 ¼ − 320 mm; Macherey-Nagel GmbH, Düren,
Germany) to achieve the finished wort. If required, mash
pH was adjusted at the start using lactic acid (acidification)
or sodium hydroxide (basification) to achieve multiple wort
pH values between pH 5.1 to 6.1.
Wort measurements
The pH of the wort was measured using the HI5522-02 pH
meter from Hanna Instruments (Woonsocket, RI, USA). The
color was determined following the EBC method 8.5 ‘Colour
of Wort: Spectrophotometric Method (IM)’, using a spectro-
photometer (Spectronic Genesys 10uv, Thermo Fischer
Scientific, Waltham, MA, USA) at 430 nm. The wort’s density
was measured using the DMA 4500 Density meter from
Anton Paar GmbH (Graz, Austria) per the manufacturer’s
instructions.
Iron determination by spectrophotometry with
1,10-phenanthroline
Iron determination was executed as described in EBC
method 9.13.1 “Iron in Beer by Spectrophotometry with
2,2-Bipyridyl or 1,10-Phenanthroline”.[22] Calibration samples,
ranging from 0 to 3 mg/L (ppm) of iron, were prepared
using a stock solution of (NH4)2 Fe(II)SO4.6H2O (7.024 g/L)
in RO water with 0.2 mL concentrated HCl to prevent oxi-
dation to Fe(III). The calibration samples, with a volume
of 25 mL, also received 25 mg of ascorbic acid, to further
prevent oxidation of the Fe(II) ions.

Beer samples were degassed using agitation or sonication.
Wort samples were filtered (MN 614 ¼ − 185 mm;
Macherey-Nagel GmbH, Düren, Germany) to remove par-
ticles. Plastic 50 mL tubes with screw caps were filled with
10 mL of sample and 0.8 mL of phenanthroline solution
(3 g/L). For each sample, a blank was included, containing
0.8 mL of water instead of phenanthroline. When total iron
was determined, samples and the blanks received 25 mg of
ascorbic acid. To measure Fe(II), ascorbic acid was left out.
Samples were subsequently incubated for 15 min in a water
bath at 60 °C before cooling and measuring absorption at
505 nm against an RO water blank, using a spectrophotom-
eter (Spectronic Genesys 10uv, Thermo Fischer Scientific,
Waltham, MA, USA). In this method, only free iron is
measured, since no digestion step is included to liberate
bound iron.
Fe(III) reduction by beer and wort
To quantify the reducing capacity of beer and wort, Fe(III)
ions were introduced into samples using a solution of Fe(III)
Cl3. The iron determination method, described in Section
(Iron determination by spectrophotometr y with
1,10-phenanthroline), was utilized to quantify the amount
of Fe(II) within each sample. The supplemented amount
ranged from 0 to 3 mg/L (ppm) of iron. The reduced Fe(III)
percentage was used to quantify the beer or wort sample’s
reducing power (%).
Reducing power using the DPPH assay
In this method, based on a procedure by Kaneda et al.
(1995),[23] a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free rad-
ical solution was prepared by dissolving 0.01294 g DPPH in
200 mL EtOH and 100 mL 0.1 M acetate buffer (pH 4.3).
Beer samples were degassed using agitation or sonication,
while wort samples were filtered (MN 614 ¼ − 185 mm;
Macherey-Nagel GmbH, Düren, Germany). A 0.2 mL aliquot
of each sample was added to 2.8 mL of DPPH solution in
a cuvette. After precisely 5 min of incubation in the dark,
the absorption was measured at 525 nm with a spectropho-
tometer. Additionally, a blank was measured to compensate
for sample color by adding 0.2 mL of sample to 2.8 mL of
RO water. The absorption of the DPPH solution was also
measured. The reducing power from the DPPH assay could
be calculated using the following formula:
ADPPH  ( A5min  Ablank )
Reducing power  %  
ADPPH
Results
Iron speciation in beer
The concentration of Fe(II) was measured without the addi-
tion of ascorbic acid, while the samples for total iron
included the addition of ascorbic acid in a single reference
Journal of the American Society of Brewing Chemists 3
beer. As per the method, only when the coloring agent
phenanthroline complexes with Fe(II) will a red color be
formed. Therefore, ascorbic acid is added to reduce free
Fe(III) to Fe(II) to determine each sample’s total iron. The
results demonstrated that in beer, no significant differences
between Fe(II) and total iron concentrations were measured,
indicating that all free iron in beer appears as Fe(II)
(Table 1). For the reference beer, the average concentration
of Fe(II) across 15 measurements was 51.7 1 μg/L (ppb),
while the average total iron concentration was 48.6 μg/L
(ppb). As all free iron in beer appears in the Fe(II) form,
the use of ascorbic acid during beer analysis is unnecessary,
particularly when the standard deviation of the method is
more than doubled when ascorbic acid is used.
Additionally, eight different beers obtained from the
Belgian market were analyzed for their iron content
(Table 2). These results substantiated that all unbound free
iron in beer appears as Fe(II). The iron content was addi-
tionally measured again after one year of storage at room
temperature (20 ± 2 °C). The results revealed that iron levels
did not change throughout storage and remained as Fe(II).
Next, Fe(III) was added to finished beer and subsequently
measured. Here, the addition of Fe(III) to finished beer did
not lead to a measurable difference in Fe(II) and total iron
levels (Table 3). This experiment was examined in fresh
finished beer (the reference amber beer at 2 months old)
and a 6-year-old blond ale. In both instances, the fresh and
aged beer reduced all supplemented Fe(III) to Fe(II); there-
fore, the results indicate that all unbound free iron in beer
exists as Fe(II).
Iron speciation in wort
Four amber-colored worts of 35 EBC were made from dif-
ferent special malts, while the reference wort was made
from 100% Pilsner malt, using the congress mash set-up
described in the Materials and Methods (Table 4). The Fe(II)
content and the total iron content of these worts were deter-
mined in triplicate. While beer only contained Fe(II), wort
appeared to contain a mixture of both Fe(II) and Fe(III).
The balance of Fe(II)/Fe(III) species was demonstrated to
be different between worts created from different special
malts. The wort from Pilsner malt showed almost equal
proportions of Fe(II) and Fe(III) ions, while the wort-derived
from caramel malt was almost entirely present in the
Fe(II) form.
A Fe(III) reduction experiment was performed to examine
the laboratory-scale worts’ reducing power. Fe(III) was added
to the worts at different concentrations, and Fe(II) was
measured using the colorant phenanthroline, as described
in the Methods section. The amount of Fe(III) reduced by
the wort was used to quantify the reducing power associated
with each wort (Figure 1). The outcomes indicate a clear
Table 1. Fe(II) and total iron levels in the reference beer.
Iron in the reference beer Iron level (n = 15)
Total free iron 48.6 ± 7.2 μg/L (ppb)
Fe(II) 51.7 ± 3.3 μg/L (ppb)
4 T. VAN MIEGHEM ET AL.

Table 2. Overview of physical-chemical properties and the iron content of eight commercial beers obtained from the Belgian
market. Measurements were carried out in triplicate for iron content.
Fresh beer Aged beer
Fe(II) Total iron μg/L Fe(II) Total iron μg/L
μg/L (ppb) (ppb) μg/L (ppb) (ppb)
Beer Beer Style OG (°P) ABV Color (EBC) pH Mean + STD Mean + STD Mean + STD Mean + STD
1 Amber 12.63 5.48 31.05 4.29 57.4 ± 4.7 58.8 ± 6.6 55.8 ± 2.2 56.0 ± 4.6
2 Tripel 16.50 7.60 7.75 4.65 266.0 ± 3.6 265.1 ± 5.0 276.1 ± 4.0 272.0 ± 6.6
3 Pilsner 11.62 5.27 4.65 4.74 37.2 ± 4.4 37.0 ± 4.5 39.0 ± 1.6 38.3 ± 3.1
4 IPA 12.89 5.59 13.18 4.46 88.0 ± 6.5 89.4 ± 7.1 84.9 ± 5.2 82.5 ± 8.4
5 Blond 16.76 8.33 6.45 4.24 32.2 ± 7.2 30.6 ± 6.3 28.9 ± 4.3 30.0 ± 5.3
6 Blond 16.77 8.24 13.73 4.26 63.8 ± 4.1 63.7 ± 3.9 67.2 ± 2.8 66.0 ± 5.9
7 Amber 12.54 5.19 25.98 4.24 111.6 ± 8.3 114.3 ± 10.0 106.7 ± 9.3 106.3 ± 13.1
8 Amber 12.75 5.41 23.45 4.26 42.5 ± 4.5 42.0 ± 7.2 45.5 ± 2.0 42.8 ± 3.7

Table 3. Iron content in fresh and aged beer following Fe(III) addition. Iron measurements were carried out in triplicate.
Fresh beer, Total free iron Aged beer, Total free iron
Fe(III) added μg/L Fresh beer, Fe(II) μg/L (ppb) μg/L (ppb) Aged beer, Fe(II) μg/L (ppb) μg/L (ppb)
(ppb) Mean + STD Mean + STD Mean + STD Mean + STD
0 44.2 ± 2.1 43.9 ± 3.8 83.4 ± 3.7 94.1 ± 5.0
1000 972.4 ± 14.3 978.8 ± 10.9 1006.3 ± 12.2 1028.6 ± 14.3
2000 2011.2 ± 16.4 1989.0 ± 16.3 2039.4 ± 15.0 2028.7 ± 21.3
3000 2911.2 ± 14.9 2928.3 ± 17.5 3044.4 ± 16.7 2994.2 ± 11.5

Table 4. Analytical results from the amber-colored worts produced on a laboratory scale. Iron measurements were performed
in triplicate.
Total Iron μg/L
Fe(II) μg/L (ppb) (ppb)
Malt Grain Bill Color (EBC) pH OG (°P) Mean + STD Mean + STD
Pilsner 100% 10.4 6.04 12.24 51.3 ± 5.3 98.0 ± 6.9
Cara50 50% + 50% Pilsner 39.6 5.71 12.05 365.1 ± 7.6 372.7 ± 8.0
Cara120 20% + 80% Pilsner 36.3 5.82 12.10 220.8 ± 4.3 237.9 ± 6.6
Pealed Roasted Barley (PRB) 2.3% + 97.7% Pilsner 37.2 6.01 12.33 101.3 ± 5.3 160.2 ± 4.8
Mix 9.3% Cara120 + 1.1% PRB 39.6 5.93 12.31 183.7 ± 4.8 221.0 ± 7.1
+ 89.6% Pilsner

Reducing Power of Wort

(35 EBC)
3.5

3.0
Fe(II) Measured (ppm)

2.5
Water y = 0.0111x + 0.0115
2.0 Pils y = 0.5168x + 0.1585

Cara120 y = 0.8714x + 0.1934

1.5
Pealed Roasted Barley y = 0.6272x + 0.1693
1.0
Mix y = 0.6842x + 0.2166

0.5 Cara50 y = 0.8938x + 0.3168

0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Fe(III) Added (ppm)

Figure 1. Reducing power of wort, quantified by the reduction of Fe(III).

difference in reducing power between the various worts, This difference in wort-reducing power was also tested
which was determined from the linear slope of the data utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH). DPPH is a
points. The lowest reducing power was observed in the wort stable free radical molecule with an intense purple color
created solely from Pilsner malt, while the Cara50 and when dissolved. After adding the wort sample, reducing
Cara120 derived worts demonstrated the highest reducing compounds or antioxidants in the sample will react with
power. Furthermore, it was also demonstrated that the pro- the radical, resulting in a loss of color intensity. The color
portions of Fe(II)/Fe(III) remained constant despite increas- loss directly correlates to the amount of reducing compounds
ing iron concentrations. in the sample.
 Journal of the American Society of Brewing Chemists 5

Results from the DPPH assay correlate linearly (R2 =
0.8905) with the Fe(III)-reduction method, which substan-
tiates that worts with caramel malt have the highest reducing
power (Table 5 and Figure 2).
Mapping a broad range of special malts
Next, selected specialty malts were assessed for their influ-
ence on the iron concentration and reducing power in wort.
To make meaningful comparisons, congress worts with a
color of around 35 EBC were created by supplementing the
specialty malt with Pilsner malt (Supplementary Table I).
Overall, total iron levels in the congress worts ranged from
0.045 mg/L to 0.345 mg/L, while the reducing power mea-
sured with the DPPH method ranged from 39.3% to 81.3%.
The pH values of the worts ranged from 5.55 to 6.04. In
general, the reducing power and iron concentration detected
in wort were highest when caramel malts (~100 EBC) were
utilized to increase wort color (Figure 3). These worts also
had the lowest pH values (~5.6 pH) compared to the pale
worts and amber worts, containing roasted malts, which are
closer to pH 6.0. Overall, negative correlations were observed
between wort pH and iron content and between wort pH
and reducing power, while conversely, the iron content and
reducing power demonstrated a positive correlation.
Influence of mash pH on iron content and reducing
power of wort
Worts from seven specialty malts, plus a Pilsner wort as a
reference, were produced at three or four different pH values
Table 5. Reducing power for the worts measured with the Fe(III)-
reduction method and the DPPH-assay.
Reducing power Fe(III) reduction (%)
Water 0.011
Pilsner 0.517
Cara120 0.871
Pealed Roasted Barley 0.627
Cara50 0.894
Mix 0.684
Method Correlation
Reducing Power of Wort
70%
DPPH-Assay Method
ranging from pH 5.1 to 6.1 to unravel the impact of the
mash pH on the iron content and the reducing power of
final wort (Supplementary Table II). The results revealed
that increasing mash pH significantly lowered the total iron
content in the final wort and yielded a slight decrease in
reducing power (Figure 4). On average, when the mash pH
was increased from 5.1 to 6.1; the total free iron content
decreased by 40%, and the reducing power measured with
the DPPH method by 5%.
Discussion
Iron speciation in wort and beer
In finished beer, all free iron appears in the Fe(II) form
due to its inherent reductive character imposed by the yeast
during fermentation. During sugar metabolism, reductants
are formed by the yeast, the most prominent being NADH
and NADPH.[24,25] These compounds provide a strong reduc-
ing power to the medium. Even additions of Fe(III), at
concentrations twenty times higher than levels typically
found in beer are reduced. Our results demonstrate the
balance between Fe(III) and Fe(II) is maintained throughout
the beer aging process and continues to reduce Fe(III) suf-
ficiently despite aging. These results contradict the long-
standing previously held hypothesis that fresh beer contains
Fe(II), which will oxidize throughout beer aging to Fe(III),
resulting in a complete conversion to the Fe(III) form.[26]
Consequently, the constant reduction of Fe(III) to Fe(II)
throughout beer storage would readily ensure Fe(II) is con-
tinuously available for its catalytic activity in the formation
of reactive oxygen species (ROS), which leads to an increased
level of oxidation reactions such as the oxidation of fatty
acids, ester and iso-α-acid degradation, ethanol oxidation,
and more, that give rise to unwanted flavor, aroma, and
DPPH (%)
appearance of the beer, typically characterized as stale beer.
0.000
0.390
An important remark is that the method used to determine
0.507 iron in this study is limited to free Fe(II), as iron bound
0.422 to beer compounds (e.g., amino acids, polyphenols, Maillard
0.576
0.426
compounds) is not detectable by the coloring agent.[27,28]
Methods that can measure total iron (i.e., free and bound
iron), such as atomic emission spectroscopy and optical
emission spectrometry, require a sample digestion step to
y = 0.444x + 0.1451 free the bound iron. These methods however cannot make
R² = 0.8905
the distinction between Fe(II) and Fe(III).[29–31]
Conversely, Fe(II) and Fe(III) species were routinely
found to be present in wort. Here, the balance between

60%
Fe(II) and Fe(III) was demonstrated to be significantly influ-
50%
enced by the specialty malts, mainly due to their increased
concentration of Maillard compounds and their resulting
40% influence on the wort’s reducing power. Nevertheless, the
total level of iron in the wort did not impact the Fe(II)/
30% Fe(III) balance. These results demonstrate that employing
40% 50% 60% 70% 80% 90% 100%
caramel malts to increase wort color lead to the most potent
Fe(III) reduction method
reducing power and, therefore, the highest proportion of
Figure 2. Correlation between the reducing power of different Fe(II). Although not examined in this study, intermediate
worts measured with the Fe(III)-reduction method and the Maillard compounds containing an enediol structure, com-
DPPH-assay. monly referred to as reductones, could be responsible for
6 T. VAN MIEGHEM ET AL.

A Total Free Iron vs. Log Malt Color B pH vs. Log Malt Color
0.40 6.1
0.35
6.0

Total Free Iron (ppm)

0.30
5.9
0.25

pH
0.20 5.8
0.15
5.7
0.10
5.6
0.05
0.00 5.5
1 10 100 1000 10000 1 10 100 1000 10000
Malt Color (EBC) Malt Color (EBC)

C Reducing Power vs. Log Malt Color D Total Free Iron vs. pH
100% 0.40
90% 0.35

Total Free Iron (ppm)

80%
Reducing Power (%)

0.30
70%
60% 0.25
50% 0.20
40% 0.15
30%
0.10
20%
10% 0.05
0% 0.00
1 10 100 1000 10000 5.4 5.6 5.8 6 6.2
Malt Color (EBC) pH

E Reducing Power vs. pH F Reducing Power vs. Total Free Iron

100% 100%
90% 90%
80% 80%
Reducing Power (%)

Reducing Power (%)

70% 70%
60% 60%
50% 50%
40% 40%
30% 30%
20% 20%
10% 10%
0% 0%
5.4 5.6 5.8 6 6.2 0.00 0.10 0.20 0.30 0.40
pH Total Free Iron (ppm)

Figure 3. A, B, C: Analytical results of the amber-colored worts (around 35 EBC). The x-axis is the logarithmic scale of the special
malts’ color. D, E, F: The results were divided by the color of the specialty malt used; pale malt (2.5 – 20 EBC, yellow triangle), caramel
malts (20 – 300 EBC, orange circle), and roasted malts (300+ EBC, brown square). The highest iron values were found in the worts
with the lowest pH values (D). Also, the reducing power increased with decreasing wort pH-values (E). Finally, the highest reducing
power and iron content were found in worts made with caramel malts (F).

this phenomenon.[9,32] Then again, the use of highly roasted
malts to increase color yielded only a minimal impact on
reducing power compared to the Pilsner wort; this is likely
because less roasted malt was needed, compared to caramel
malt, to increase the wort color to 35 EBU, and the lower
reactivity of the terminal Maillard reaction products formed
during roasting.[33] The iron balance in wort, and the wort’s
reducing power, are important for the oxidative stability of
the resulting beer. For instance, if the Fe(II)/Fe(III) balance
is swung towards Fe(II), more Fe(II) is available to catalyze
the Fenton reaction, thus promoting an increased forma-
tion of ROS.
Impact of mash pH on iron and reducing power
A clear negative correlation was observed between mash
pH and iron content of the resultant wort, which aligns
with the previous literature. [21,34] This stems from the
decreased solubility of iron at higher pH.[35–37] In the pres-
ence of oxygen, soluble Fe(II) can undergo spontaneous
 Journal of the American Society of Brewing Chemists 7

A Influence of Mash pH on Free Iron in Wort B Influence of pH on the Reducing Power of Wort
0.50 100%

0.45 90%

0.40 80%

0.35 70%
Cafe (DC) Cafe (DC)
Total Free Iron (ppm)

Reducing Power (%)

0.30 Chocolat (DC) 60% Chocolat (DC)
Cara 120 (MD) Cara 120 (MD)
0.25 50%
Special B (MD) Special B (MD)
0.20 Aroma 150 (MD) 40% Aroma 150 (MD)
Pils (MD) Pils (MD)
0.15 30%
Mroost 900 (MD) Mroost 900 (MD)
0.10 Pealed Roasted Barley (MD) 20% Pealed Roasted Barley (MD)

0.05 10%

0.00 0%
5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2 5 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6 6.1 6.2
Mash pH Mash pH

Figure 4. Influence of mash pH on iron (A) and reducing power (B) for different amber-colored worts. The specialty malt is supple-
mented by Pilsner malt to achieve a wort color of around 35 EBC (Supplementary Table II). The selected specialty malts are from
Castle Malting (DC) and Mouterij Dingemans (MD): brown, Cafe (DC); beige, Chocolat (DC); blue, Cara120 (MD); orange, Special B (MD);
grey, Aroma 150 (MD); yellow, Pilsner (MD); green, Mroost900 (MD); red, Pealed Roasted Barley (MD).

Wort Spent grains

Iron – soluble maillard compounds Iron – insoluble malt solids

Figure 5. Distribution of iron between the liquid and solid phase during the mash.

chemical oxidation to insoluble Fe(III) hydroxide species, in wort such as melanoidins, phytosiderophores, and phy-
and this rate increases with higher pH values. Since mashing tates also show affinity for iron (Figure 5).[46–48] This dis-
is not oxygen-free, precipitated Fe(III) hydroxide species tribution of iron between the solid and liquid phase of the
will largely be retained by the spent grains during lautering mash appears to be influenced by the malt bill.[44] Caramel
or mash filtration.[21] Additionally, an increasingly sour mash and roasted malts increase iron content of wort, because of
will contain more protons, which compete with metal cations the higher concentration of soluble iron chelating Maillard
for binding sites on soluble wort compounds, e.g., polyphe- compounds.[44] Therefore, compared to pale malts, more
nols, and subsequently lead to an increase of free metal iron stays in the liquid phase, and the iron absorption capac-
ions in wort.[21] ity of the spent grains is lowered. Iron increase is the highest
Moreover, the reducing power of the wort is to some when using caramel malts for color, since these malts add
extent decreased at higher mash pH. This could result from the highest amount of iron chelation Maillard compounds.
the higher precipitation rate of reducing compounds, such
as polyphenols and Maillard products, due to the smaller
Conclusion
concentration of protons in the mash. This allows for more
complex formation between unprotonated reducing com- Since the Fe(II) form of iron is detrimental to beer quality,
pounds, which can form insoluble complexes. Proteins and due to its ability to catalyze radical formation and to induce
metals can be included in these complexes.[38,39] It is also metallic off-flavor, one of the aims of this study was to
possible that at higher pH, the content of intermediate elucidate the balance of Fe(II) and Fe(III) in wort and beer.
Maillard compounds (e.g., reductones) is lower because of Our results demonstrate that in wort, both Fe(II) and Fe(III)
a lower formation rate due to lower proton concentration, ions are present. In addition, our data further demonstrates
and an increased reaction rate of the intermediates into that the Fe(II)/Fe(III)-balance in amber-colored worts is
terminal Maillard compounds.[40,41] influenced by the type of specialty malt used to provide
It has been shown that the Fenton reaction is color. Caramel malts show a stronger reducing power, and
pH-dependent. The optimal pH for the Fenton reaction is therefore a larger fraction of the total iron content exists
between pH 3 and pH 5, for higher values the reaction is as Fe(II). On the other hand, in finished beer all free iron
slowed down due to the lower availability of soluble exists as Fe(II), likely because of the yeast providing a strong
Fe(II).[42,43] Therefore, a higher mash pH could benefit the reducing environment to beer during fermentation. Likewise
beer’s oxidative stability due to the reduced iron content, in aged beer, all free iron appears to be in the Fe(II) form,
and lesser ROS formed via the Fenton reaction during which means that the Fe(II) ion is still available for its
mashing. catalytic activity in oxidation reactions.
It has been shown that the majority of the iron in the It was also shown that amber-colored worts with caramel
mash is retained by the spent grains during lautering or malts have the highest free iron content, as well as the
wort filtration, due to iron affinity of malt solids, e.g., func- strongest reducing power. This is likely due to the increased
tional groups of lignin, cellulose, hemicellulose, and insol- content of intermediate Maillard products, i.e., reductones,
uble proteins.[10,44,45] On the other hand, soluble compounds which are able to chelate iron and reduce Fe(III) to Fe(II).
8 T. VAN MIEGHEM ET AL.
Moreover, it was confirmed that the mash pH has a strong
influence on the iron levels of the resulting wort. Increasing
the mash pH reduces iron content, probably due to the
lower solubility of iron. Therefore, increasing mash pH and
avoiding the use of caramel malts and high-kilned malts
could be beneficial for the oxidative stability of beer.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Scott J. Britton http://orcid.org/0000-0002-0874-3699
Literature cited
[1] Bamforth, C. W.; Lentini, A. The Flavor Instability of Beer. In
Beer: A Quality Perspective (Volume in Handbook of Alcoholic
Beverages); Bamforth, C. W., Russell, I., Stewart, G. G., Eds.;
Elsevier: Cambridge, Massachusetts, 2009; pp 85–109.
[2] Wauters, R.; Britton, S. J.; Verstrepen, K. J. Old Yeasts, Young
Beer—The Industrial Relevance of Yeast Chronological Life Span.
Yeast. 2021, 38, 339–351. DOI: 10.1002/yea.3650.
[3] Baert, J. J.; De Clippeleer, J.; Hughes, P. S.; De Cooman, L.;
Aerts, G. On the Origin of Free and Bound Staling Aldehydes
in Beer. J. Agric. Food Chem. 2012, 60, 11449–11472. DOI:
10.1021/jf303670z.
[4] Vanderhaegen, B.; Delvaux, F.; Daenen, L.; Verachtert, H.;
Delvaux, F. R. Aging Characteristics of Different Beer Types.
Food Chem. 2007, 103, 404–412. DOI: 10.1016/j.food-
chem.2006.07.062.
[5] Van Mieghem, T.; Delvaux, F.; Dekleermaeker, S.; Britton, S. J.
Top of the Ferrous Wheel – The Influence of Iron Ions on Flavor
Deterioration in Beer. J. Am. Soc. Brew. Chem. 2022. DOI:
10.1080/03610470.2022.2124363.
[6] De Schutter, D. P.; Saison, D.; Delvaux, F.; Derdelinckx, G. The
Chemistry of Aging Beer. In Beer in Health and Disease
Prevention; Preedy, V. R., Ed.; Academic Press: Cambridge,
Massachusetts, 2008, pp 375–388.
[7] Jenkins, D.; James, S.; Dehrmann, F.; Smart, K.; Cook, D. Impacts
of Copper, Iron, and Manganese Metal Ions on the EPR
Assessment of Beer Oxidative Stability. J. Am. Soc. Brew. Chem.
2018, 76, 50–57. DOI: 10.1080/03610470.2017.1402585.
[8] Vanderhaegen, B.; Neven, H.; Verachtert, H.; Derdelinckx, G.
The Chemistry of Beer aging - A Critical Review. Food Chem.
2006, 95, 357–381. DOI: 10.1016/j.foodchem.2005.01.006.
[9] Kunz, T.; Strähmel, A.; Cortés, N.; Kroh, L. W.; Methner, F. J.
Influence of Intermediate Maillard Reaction Products with
Enediol Structure on the Oxidative Stability of Beverages. J. Am.
Soc. Brew. Chem 2013, 71, 114–123. DOI: 10.1094/
ASBCJ-2013-0429-01.
[10] Frederiksen, A. M.; Festersen, R. M.; Andersen, M. L. Oxidative
Reactions during Early Stages of Beer Brewing Studied by
Electron Spin Resonance and Spin Trapping. J. Agric. Food Chem.
2008, 56, 8514–8520. DOI: 10.1021/jf801666e.
[11] Arts, M. J. T. J.; Grun, C.; De Jong, R. L.; Voss, H.-P.; Bast, A.;
Mueller, M. J.; Haenen, G. R. M. M. Oxidative Degradation of
Lipids during Mashing. J. Agric. Food Chem. 2007, 55, 7010–7014.
DOI: 10.1021/jf070505+.
[12] Meilgaard, M. C.; Reid, D. S.; Wyborski, K. A. Reference
Standards for Beer Flavor Terminology System. J. Am. Soc. Brew.
Chem. 1982, 40, 119–128. DOI: 10.1094/ASBCJ-40-0119.
[13] Glindemann, D.; Dietrich, A.; Staerk, H. J.; Kuschk, P. The Two
Odors of Iron When Touched or Pickled: (Skin) Carbonyl
Compounds and Organophosphines. Angew. Chem. Int. Ed. Engl.
2006, 45, 7006–7009. DOI: 10.1002/anie.200602100.
[14] Lawless, H. T.; Schlake, S.; Smythe, J.; Lim, J.; Yang, H.; Chapman,
K.; Bolton, B. Metallic Taste and Retronasal Smell. Chem. Senses.
2004, 29, 25–33. DOI: 10.1093/chemse/bjh003.
[15] Lubran, M. B.; Lawless, H. T.; Lavin, E.; Acree, T. E. Identification
of Metallic-Smelling 1-Octen-3-One and 1-Nonen-3-One from
Solutions of Ferrous Sulfate. J. Agric. Food Chem. 2005, 53,
8325–8327. DOI: 10.1021/jf0511594.
[16] Doi, N.; Kobayashi, M.; Masuda, S.; Aizawa, M. Identifying and
Controlling Formation of Compounds That Affect Metallic
Flavor of Beer. World Brewing Congress 2016, 170. https://
w w w. a s b c n e t . o r g / e v e n t s / a r c h i v e s / 2 0 1 6 / p r o c e e d i n g s /
Documents/170_Doi.pdf.
[17] Doi, N.; Kobayashi, M.; Masuda, S.; Aizawa, M. What Compound
is Primarily Responsible for the Metallic Flavor in Beer? in 2015
ASBC Annual Meeting 2015, https://www.asbcnet.org/events/
archives/2015Meeting/proceedings/2015Presentations/13_Doi.pdf.
[18] Epke, E. M.; Lawless, H. T. Retronasal Smell and Detection
Thresholds of Iron and Copper Salts. Physiol. Behav. 2007, 92,
487–491. DOI: 10.1016/j.physbeh.2007.04.022.
[19] Ömür-Özbek, P.; Dietrich, A. M.; Duncan, S. E.; Lee, Y. W. Role
of Lipid Oxidation, Chelating Agents, and Antioxidants in
Metallic Flavor Development in the Oral Cavity. J. Agric. Food
Chem. 2012, 60, 2274–2280. DOI: 10.1021/jf204277v.
[20] Andersen, M. L.; Skibsted, L. H. Electron Spin Resonance Spin
Trapping Identification of Radicals Formed during Aerobic Forced
Aging of Beer. J. Agric. Food Chem. 1998, 46, 1272–1275. DOI:
10.1021/jf9708608.
[21] Mertens, T.; Kunz, T.; Wietstock, P. C.; Methner, F. J. Complexation
of Transition Metals by Chelators Added during Mashing and
Impact on Beer Stability. J. Inst. Brew. 2021, 127, 345–357. DOI:
10.1002/jib.673.
[22] European Brewery Convention. Analytica-EBC. Fachverlag Hans
Carl: Nürnberg, 2010.
[23] Kaneda, H.; Kobayashi, N.; Furusho, S.; Sahara, H.; Koshino, S.
Reducing Activity and Flavor Stability of Beer. Tech. Q. Master
Brew. Assoc. Am. 1995, 32, 90–94.
[24] Dijken, J. P.; Scheffers, W. Redox Balances in the Metabolism of
Sugars by Yeasts. FEMS Microbiol. Rev. 1986, 32, 199–224. DOI:
10.1111/j.1574-6968.1986.tb01194.x.
[25] Frick, O.; Wittmann, C. Characterization of the Metabolic Shift
between Oxidative and Fermentative Growth in Saccharomyces
cerevisiae by Comparative 13C Flux Analysis. Microb. Cell Fact.
2005, 4, 1–16.
[26] Kaneda, H.; Kano, Y.; Koshino, S.; Ohya-Nishiguchi, H. Behavior
and Role of Iron Ions in Beer Deterioration. J. Agric. Food Chem.
1992, 40, 2102–2107. DOI: 10.1021/jf00023a013.
[27] Pohl, P. Metals in Beer. In Beer in Health and Disease Prevention;
Preedy, V. R., Ed.; Academic Press: Cambridge, Massachusetts,
2008; pp 349–358.
[28] Svendsen, R.; Lund, W. Speciation of Cu, Fe and Mn in Beer
Using Ion Exchange Separation and Size-Exclusion
Chromatography in Combination with Electrothermal Atomic
Absorption Spectrometry. Analyst 2000, 125, 1933–1937. DOI:
10.1039/b005187j.
[29] Pires, L. N.; de, S.; Dias, F.; Teixeira, L. S. G. Assessing the
Internal Standardization of the Direct Multi-Element
Determination in Beer Samples through Microwave-Induced
Plasma Optical Emission Spectrometry. Anal. Chim. Acta. 2019,
1090, 31–38. DOI: 10.1016/j.aca.2019.09.033.
[30] Asfaw, A.; Wibetoe, G. Direct Analysis of Beer by ICP-AES: A
Very Simple Method for the Determination of Cu, Mn and Fe.
Microchim. Acta. 2005, 152, 61–68. DOI: 10.1007/s00604-
005-0424-6.
[31] Bellido-Milla, D.; Oñate-Jaén, A.; Palacios-Santander, J. M.;
Palacios-Tejero, D.; Hernández-Artiga, M. P. Beer Digestions for
Metal Determination by Atomic Spectrometry and Residual
Organic Matter. Microchim. Acta. 2004, 144, 183–190. DOI:
10.1007/s00604-003-0082-5.

[32] Hoff, S.; Lund, M. N.; Petersen, M. A.; Jespersen, B. M.; Andersen,
M. L. Influence of Malt Roasting on the Oxidative Stability of
Sweet Wort. J. Agric. Food Chem. 2012, 60, 5652–5659. DOI:
10.1021/jf300749r.
[33] Echavarría, A. P.; Pagán, J.; Ibarz, A. Melanoidins Formed by
Maillard Reaction in Food and Their Biological Activity. Food
Eng. Rev. 2012, 4, 203–223. DOI: 10.1007/s12393-012-9057-9.
[34] Holzmann, A.; Piendl, A. Malt Modification and Mashing
Conditions as Factors Influencing the Minerals of Wort. J. Am.
Soc. Brew. Chem. 1977, 35, 1–8. DOI: 10.1094/ASBCJ-35-0001.
[35] Morgan, B.; Lahav, O. The Effect of pH on the Kinetics of
Spontaneous Fe(II) Oxidation by O2 in Aqueous Solution - Basic
Principles and a Simple Heuristic Description. Chemosphere 2007,
68, 2080–2084. DOI: 10.1016/j.chemosphere.2007.02.015.
[36] Stefánsson, A. Iron (III) Hydrolysis and Solubility at 25 Degrees
C. Environ. Sci. Technol. 2007, 41, 6117–6123. DOI: 10.1021/
es070174h.
[37] Jones, A. M.; Griffin, P. J.; Collins, R. N.; Waite, T. D. Ferrous
Iron Oxidation under Acidic Conditions – The Effect of Ferric
Oxide Surfaces. Geochim. Cosmochim. Acta 2014, 145, 1–12. DOI:
10.1016/j.gca.2014.09.020.
[38] Habschied, K.; Košir, I. J.; Krstanovi, V.; Kumri, G.; Mastanjevic,
K. Beer Polyphenols—Bitterness, Astringency, and Off-Flavors.
Beverages 2021, 7, 38. DOI: 10.3390/beverages7020038.
[39] Lewis, M. J.; Serbia, J. W. Aggregation of Protein and Precipitation
by Polyphenol in Mashing. J. Am. Soc. Brew. Chem. 1984, 42,
40–43. DOI: 10.1094/ASBCJ-42-0040.
[40] Ajandouz, E. H.; Tchiakpe, L. S.; Dalle Ore, F.; Benajiba, A.;
Puigserver, A. Effects of pH on Caramelization and Maillard
Reaction Kinetics in Fructose-Lysine Model Systems. J. Food Sci.
2001, 66, 926–931. DOI: 10.1111/j.1365-2621.2001.tb08213.x.
Journal of the American Society of Brewing Chemists 9
[41] Martins, S. I. F. S.; Van Boekel, M. A. J. S. Kinetics of the
Glucose/Glycine Maillard Reaction Pathways: Influences of pH
and Reactant Initial Concentrations. Food Chem. 2005, 92, 437–
448. DOI: 10.1016/j.foodchem.2004.08.013.
[42] Luo, W.; Abbas, M. E.; Zhu, L.; Deng, K.; Tang, H. Rapid
Quantitative Determination of Hydrogen Peroxide by Oxidation
Decolorization of Methyl Orange Using a Fenton Reaction
System. Anal. Chim. Acta. 2008, 629, 1–5. DOI: 10.1016/
j.aca.2008.09.009.
[43] Katsumata, H.; Kawabe, S.; Kaneco, S.; Suzuki, T.; Ohta, K.
Degradation of Bisphenol A in Water by the photo-Fenton
Reaction. J. Photochem. Photobiol. A Chem. 2004, 162, 297–305.
DOI: 10.1016/S1010-6030(03)00374-5.
[44] Pagenstecher, M.; Maia, C.; Andersen, M. L. Retention of Iron
and Copper during Mashing of Roasted Malts. J. Am. Soc. Brew.
Chem 2021, 79, 138–144. DOI: 10.1080/03610470.2020.179
5609.
[45] Piggott, C. O.; Connolly, A.; Fitzgerald, R. J. Application of
Ultrafiltration in the Study of Phenolic Isolates and Melanoidins
from Pale and Black Brewers’ Spent Grain. Int. J. Food Sci.
Technol. 2014, 49, 2252–2259. DOI: 10.1111/ijfs.12540.
[46] Ma, J. F.; Higashitani, A.; Sato, K.; Takeda, K. Genotypic Variation
in Fe Concentration of Barley Grain. Soil Sci. Plant Nutr. 2004,
50, 1115–1117. DOI: 10.1080/00380768.2004.10408583.
[47] Morales, F. J.; Fernández-Fraguas, C.; Jiménez-Pérez, S.
Iron-Binding Ability of Melanoidins from Food and Model
Systems. Food Chem. 2005, 90, 821–827. DOI: 10.1016/j.food-
chem.2004.05.030.
[48] Minihane, A. M.; Rimbach, G. Iron Absorption and the Iron
Binding and Anti-Oxidant Properties of Phytic Acid. Int. J. Food
Sci. Tech. 2002, 37, 741–748. DOI: 10.1046/j.1365-2621.2002.00619.x.