The Sway of Specialty Malts and Mash pH on Iron Ion Speciation and the Reducing Power of Wort

Abstract In contrast to other fermented beverages, beer quality generally diminishes over time. This diminishing quality hinges heavily on the oxidative degradation of beer compounds by reactive oxygen species (ROS), whose formation is in part catalyzed by Fe(II) ions via the Fenton and 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
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 particular, stale flavor formation is a common sensorial attribute occurring over time, whereby undesirable aroma-active carbonyl compounds, such as aldehydes and ketones, develop from oxidative reactions and become increasingly pronounced, 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]onsequently, 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 and aromas, originating from Maillard reactions.The 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]e(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 presence 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 oxidation, and other reactions that result in a deviation from the intended sensory profile. [3,5,6][8][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 reactions are regenerative, where Fe(II) oxidized to Fe(III) can 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][15][16][17] These are perceived via retronasal olfaction, since occlusion of the nose during tasting removes the metallic perception. [18]onversely, 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 volatile carbonyls responsible for the metallic odor.
Overwhelming, free Fe(II) is a significant player in accelerating flavor deterioration in beer, and the concentration of the ion should be kept low to guarantee sufficient oxidative 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]owever, despite its damaging influence on oxidative instability, 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 content, 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.

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) following 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 temperature 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 spectrophotometer (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 (NH 4 ) 2 Fe(II)SO 4 .6H 2 O (7.024 g/L) in RO water with 0.2 mL concentrated HCl to prevent oxidation 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 particles.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 spectrophotometer (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) Cl 3 .The iron determination method, described in Section (Iron determination by spectrophotometry 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 radical 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 spectrophotometer.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:

Iron speciation in beer
The concentration of Fe(II) was measured without the addition of ascorbic acid, while the samples for total iron included the addition of ascorbic acid in a single reference 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 additionally 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); therefore, 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 different 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 determined 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 difference in reducing power between the various worts, which was determined from the linear slope of the data points.The lowest reducing power was observed in the wort created solely from Pilsner malt, while the Cara50 and Cara120 derived worts demonstrated the highest reducing power.Furthermore, it was also demonstrated that the proportions of Fe(II)/Fe(III) remained constant despite increasing iron concentrations.
This difference in wort-reducing power was also tested utilizing 2,2-diphenyl-1-picrylhydrazyl (DPPH).DPPH is a stable free radical molecule with an intense purple color when dissolved.After adding the wort sample, reducing compounds or antioxidants in the sample will react with the radical, resulting in a loss of color intensity.The color loss directly correlates to the amount of reducing compounds in the sample.Results from the DPPH assay correlate linearly (R 2 = 0.8905) with the Fe(III)-reduction method, which substantiates 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 influence 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 measured 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 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%.

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 reducing 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) sufficiently despite aging.These results contradict the longstanding 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]onsequently, the constant reduction of Fe(III) to Fe(II) throughout beer storage would readily ensure Fe(II) is continuously 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 appearance of the beer, typically characterized as stale beer.An important remark is that the method used to determine iron in this study is limited to free Fe(II), as iron bound to beer compounds (e.g., amino acids, polyphenols, Maillard compounds) is not detectable by the coloring agent. [27,28]ethods 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 free the bound iron.31] Conversely, Fe(II) and Fe(III) species were routinely found to be present in wort.Here, the balance between Fe(II) and Fe(III) was demonstrated to be significantly influenced by the specialty malts, mainly due to their increased concentration of Maillard compounds and their resulting influence on the wort's reducing power.Nevertheless, the total level of iron in the wort did not impact the Fe(II)/ Fe(III) balance.These results demonstrate that employing caramel malts to increase wort color lead to the most potent reducing power and, therefore, the highest proportion of Fe(II).Although not examined in this study, intermediate Maillard compounds containing an enediol structure, commonly referred to as reductones, could be responsible for  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 formation 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][37] In the presence of oxygen, soluble Fe(II) can undergo spontaneous chemical oxidation to insoluble Fe(III) hydroxide species, and this rate increases with higher pH values.Since mashing is not oxygen-free, precipitated Fe(III) hydroxide species will largely be retained by the spent grains during lautering or mash filtration. [21]Additionally, an increasingly sour mash will contain more protons, which compete with metal cations for binding sites on soluble wort compounds, e.g., polyphenols, and subsequently lead to an increase of free metal ions in wort. [21]oreover, the reducing power of the wort is to some extent decreased at higher mash pH.This could result from the higher precipitation rate of reducing compounds, such as polyphenols and Maillard products, due to the smaller concentration of protons in the mash.This allows for more complex formation between unprotonated reducing compounds, which can form insoluble complexes.Proteins and metals can be included in these complexes. [38,39]It is also possible that at higher pH, the content of intermediate Maillard compounds (e.g., reductones) is lower because of a lower formation rate due to lower proton concentration, and an increased reaction rate of the intermediates into terminal Maillard compounds. [40,41]t has been shown that the Fenton reaction is pH-dependent.The optimal pH for the Fenton reaction is between pH 3 and pH 5, for higher values the reaction is slowed down due to the lower availability of soluble Fe(II). [42,43]Therefore, a higher mash pH could benefit the beer's oxidative stability due to the reduced iron content, and lesser ROS formed via the Fenton reaction during mashing.
It has been shown that the majority of the iron in the mash is retained by the spent grains during lautering or wort filtration, due to iron affinity of malt solids, e.g., functional groups of lignin, cellulose, hemicellulose, and insoluble proteins. [10,44,45][48] This distribution of iron between the solid and liquid phase of the mash appears to be influenced by the malt bill. [44]Caramel and roasted malts increase iron content of wort, because of the higher concentration of soluble iron chelating Maillard compounds. [44]Therefore, compared to pale malts, more iron stays in the liquid phase, and the iron absorption capacity of the spent grains is lowered.Iron increase is the highest when using caramel malts for color, since these malts add the highest amount of iron chelation Maillard compounds.

Conclusion
Since the Fe(II) form of iron is detrimental to beer quality, due to its ability to catalyze radical formation and to induce metallic off-flavor, one of the aims of this study was to elucidate the balance of Fe(II) and Fe(III) in wort and beer.Our results demonstrate that in wort, both Fe(II) and Fe(III) ions are present.In addition, our data further demonstrates that the Fe(II)/Fe(III)-balance in amber-colored worts is influenced by the type of specialty malt used to provide color.Caramel malts show a stronger reducing power, and therefore a larger fraction of the total iron content exists as Fe(II).On the other hand, in finished beer all free iron exists as Fe(II), likely because of the yeast providing a strong reducing environment to beer during fermentation.Likewise in aged beer, all free iron appears to be in the Fe(II) form, which means that the Fe(II) ion is still available for its catalytic activity in oxidation reactions.
It was also shown that amber-colored worts with caramel malts have the highest free iron content, as well as the strongest reducing power.This is likely due to the increased content of intermediate Maillard products, i.e., reductones, which are able to chelate iron and reduce Fe(III) to Fe(II).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.

Figure 1 .
Figure 1.reducing power of wort, quantified by the reduction of fe(iii).

Figure 2 .
Figure 2. correlation between the reducing power of different worts measured with the fe(iii)-reduction method and the DPPh-assay.

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

Table 1 .
fe(ii) and total iron levels in the reference beer.

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.

Table 3 .
iron content in fresh and aged beer following fe(iii) addition.iron measurements were carried out in triplicate.

Table 4 .
analytical results from the amber-colored worts produced on a laboratory scale.iron measurements were performed in triplicate.

Table 5 .
reducing power for the worts measured with the fe(iii)reduction method and the DPPh-assay.

reduction method Method Correlation Reducing Power of Wort
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).