41 March / April 2010 (Vol.

63) BrewingScience

Saison, D., Vanbeneden, N., De Schutter, D. P., Daenen, L., Mertens, T., Delvaux, F. and
Delvaux, F. R.

Characterisation of the Flavour and the Chemical

Composition of Lager Beer after Ageing in Varying
Conditions
Aged beer flavour was studied by ageing a lager beer in different conditions (varying temperature-time profiles, different
oxidative conditions and varying pH and ethanol concentrations). This led to beers with a varying aged flavour, which could
be explained by differences in the reaction rate of ageing reactions. High temperatures, oxidative conditions and to a lesser
extent, a lower pH, accelerated beer ageing. Enhanced (E)-2-nonenal formation probably led to the greater perception of
cardboard flavour after ageing at high temperatures. Madeira flavour was only perceived after ageing at 20 °C and ribes
flavour was mainly perceived in oxidatively aged beer. In beers with these flavour notes, high concentrations of acetal-
dehyde, Strecker aldehydes and diacetyl were found and in the Madeira flavoured beer, also of 2-furfuryl ethyl ether and
5-hydroxymethylfurfural. In the end, this study provides an overview of the different aged flavours that can develop in
different ageing conditions and the corresponding flavour compounds that make up the chemical composition of these
flavours.

Descriptors: beer, flavour, ageing, carbonyl compounds, flavour stability

1 Introduction conditions determine the type of flavour evolution considerably

[3, 4]. Furthermore, it is evident that different beers age in varying
During storage of beer, the ‘fresh’ flavour profile is disturbed ways depending on their raw materials and production process [5,
and typical aged flavour notes such as cardboard, solvent, ribes, 6]. This can be explained by the many different ageing reactions
Madeira and caramel, appear. These flavour notes can be found in that are influenced in their own specific way by the beer composi-
varying intensities in aged beer. Perception of beer flavour is the tion, several variables inherent to beer (especially pH and oxygen
result of the interaction of numerous chemical compounds with content [3, 7, 8]) and external conditions (e.g. temperature and
receptors in the olfactory organ and thus, this flavour evolution light [4, 9]). As a result of differences in reaction rates, concen-
during storage is the result of gradual changes in the chemical trations of flavour compounds resulting from these reactions will
composition of beer. Many chemical reactions such as oxidation, vary accordingly.
Strecker degradation, aldol condensation, furanic ether formation,
degradation of hop bitter acids, Maillard reactions and hydrolysis In this study, the flavour changes and the concentration evolution
of esters, occur during storage and result in the increase of aged of selected flavour compounds was studied after ageing of lager
flavour compound and the decrease of fresh flavour compound beer in varying conditions (varying temperature-time profiles,
concentrations. However, explaining aged flavour notes from the different oxidative conditions and varying pH and ethanol con-
increase and decrease of the concentration of flavour compounds centrations) in order to obtain better insights in aged beer flavour
remains difficult [1]. In a previous study, 26 compounds were and the contribution of aged flavour compounds to aged flavour.
tested for their contribution to aged flavour and 11 compounds
were suggested to play an important role [2]. Additionally, the
flavour of a beer that was thermally aged for 3 weeks at 40 °C, 2 Experimental
could be mimicked fairly well by addition of a combination of
these compounds to fresh beer. However, earlier studies show 2.1 Chemicals and beers
clearly that beer finishing and packaging operations and storage
All chemicals were purchased from Sigma (St. Louis, MO, USA)
with the highest purity available. A commercial lager beer with
an original gravity of 11.72 °P, an alcohol content of 5.12 v/v %,
Authors: a colour of 6.5 EBC and a bitterness of 26 EBU was used for the
Daan Saison, Nele Vanbeneden, David P. De Schutter, Luk Daenen, Tom ageing experiments.
Mertens, Filip Delvaux, Freddy R. Delvaux, Centre for Malting and
Brewing Science, Faculty of Bioscience Engineering, K.U.Leuven, 2.2 Preparation and ageing of lager beer
Heverlee, Belgium; corresponding author: [email protected].
be
Lager beers were aged in different conditions. An overview of
Tables and figures see Appendix the tested conditions is given in table 1. The temperature-time
BrewingScience March / April 2010 (Vol. 63) 42

profiles were 5 days at 60 °C, 3 weeks at 40 °C, 3 months at 28 °C, temperature for 3.5 min. The FID temperature was kept constant
6 months at 20 °C and 10 years at 20 °C. Oxidative reactions at 250 °C. Analyses were carried out in duplicate and results were
were initiated in two ways. Oxygen was introduced in the beer by evaluated with Perkin Elmer Turbochrom Navigator.
flushing the headspace of the bottle with oxygen for 20 seconds
after opening. Afterwards, the bottle was closed and was turned Carbonyl compounds were measured with HS solid-phase
several times. The total in-pack oxygen content of the resulting microextraction (SPME) coupled to gas chromatography and
beer was 12 ppm compared to 0.1 ppm in the initial beer. The mass spectrometry (MS). Three procedures were used that were
other oxidative condition was initiated by supplementing the bottle previously described by Saison et al. [10, 11]. Carbonyls were
with Fenton reagent (addition of 1.402 mM H2O2 and 0.00863 determined with on-fibre ((E)-2-nonenal and (E,E)-2,4-deca-
mM FeSO4.7H2O). Afterwards, the bottle headspace was made dienal) and in-solution (other carbonyls) derivatisation using
relatively oxygen free by overfoaming before closing. The pH pentafluorobenzyl hydroxylamine (PFBHA) as derivatisation
of beer was adjusted by adding H2SO4 (20 %) or NaOH (20 %) agent. The other compounds were measured after extraction of
for the low (pH 3.8) and the high pH (pH 4.6) respectively. The underivatised compounds.
reference beer had a pH of 4.2. Finally, the ethanol content was
adapted to 6.2 % (v/v) compared to 5.2 of the reference, by add- Ten minutes of pre-incubation was applied for the three proce-
ing pure ethanol. After the additions, overfoaming was performed dures. Subsequent SPME extraction was different for the three
before closing the bottles and bottles were turned several times. procedures. On-fibre derivatisation was performed after loading a
Oxidative beers and other beers to which additions were made PDMS-DVB fibre with PFBHA by exposing the fibre to the head-
were aged for 3 months at 28 °C. Beers to which additions were space of a PFBHA solution (1 mg/mL, 10 min, 45 °C, 250 rpm).
made, were compared to a reference beer that was aged in the The fibre was subsequently exposed to the HS of a vial containing
same temperature-time interval. Initially, 2 references were used: 10 mL beer, 50 µL of an ethanol solution with 100 mg/L p-fluoro-
bottles of beer to which no addition was made, but that were benzaldehyde, and 3.5 g NaCl (30 min, 45 °C, 250 rpm). In-solution
opened and overfoamed afterwards, and bottles of beer that were derivatisation was done by exposing a PDMS-DVB fibre to the
not opened before ageing. Since no noteworthy differences were HS of a vial containing 10 mL beer, 50 µL of an ethanol solution
found between both, the unopened beer bottles are described as with 100 mg/L p-fluorobenzaldehyde, and 0.375 mL of a PFBHA
the reference beer further in the text. solution (20 g/L) (40 min, 60 °C, 250 rpm). The underivatised
compounds were measured following extraction with a DVB-
2.3 Total in-pack oxygen CAR-PDMS fibre of the HS of a vial containing 10 mL beer, 50
µL of an ethanol solution with 200 mg/L 2-heptanol and 100 mg/L
The total in-pack oxygen content was determined by first shaking guaiacol, and 3.5 g NaCl (30 min, 40 °C, 250 rpm).
the bottles for 5 minutes on an orbital shaker, followed by measu-
ring the dissolved oxygen content with a Mettler Toledo Intap GC-MS analysis was carried out using a Trace GC Ultra coupled
4000 instrument. to a dual stage quadrupole MS, consisting of a curved, small
quadrupole as prefilter and a normal quadrupole (both from
2.4 Sensory analysis Thermo, Austin, TX, USA). A Rtx-5SilMS column (60 m x 0.25
mm I.D.) with a film thickness of 1 µm was used. The GC was
Sensory tests were carried out with a trained panel of at least 8 equipped with a split-splitless injector which was held at 250 °C.
members. Beers were always presented in black glasses. Besides Compounds were analysed following 2 min desorption and split-
an evaluation of some fresh flavour aspects and the general ageing less injection. During the GC run, a constant flow rate (1.5 mL/
character, stale flavour was evaluated for 9 aspects (cardboard, min) of the carrier gas (Helium) was maintained. The GC program
metal, solvent, old hops, ribes, Maillard (caramel, burnt, bread, for both derivatisation procedures was the same and started at
butter), stale-sulphury, acetaldehyde (green apple) and Madeira) 60 °C for 2 min, then increased in 4 steps: 60 to 165 °C at 50 °C/
by giving a score from 0 to 8. A score of 0 meant that the particular min; 165 to 200 °C at 2 °C/min, 200 to 260 °C at 4 °C/min and
flavour aspect was not present, while a score of 8 indicated that 260 to 290 °C at 5 °C/min and was held at 290 °C for 6 min. The
this aspect was extremely strong. Finally, an overall appreciation GC program for the underivatised compounds started at 30 °C,
score was given on a scale from 1 to 9. the oven temperature was raised in 3 steps after 2 minutes: 30 to
70 °C at 10 °C/min followed by 1 minute at 70 °C; 70 to 190 °C
2.5 Analysis of volatile flavour compounds at 4 °C/min and 220 to 270 °C at 25 °C/min and was finally held
at 270 °C for 6 minutes. The mass spectra were obtained by
Esters and higher alcohols were analysed with a calibrated Autosys- electron impact ionisation at 70 eV and the ion volume and the
tem XL gas chromatograph (GC) (HS40; Perkin Elmer, Norwalk, transfer line were held at 250 °C and 290 °C respectively. The
USA) coupled with flame ionization detection (FID). The GC was detector measured from m/z 35 to 400 and results were analysed
equipped with a Chrompack-Wax 52 CB column (length: 50 m; using Xcalibur software (Thermo, Austin, TX, USA). Analyses
0.32 mm ID; layer thickness: 1.2 µm; Varian, Palo Alto, CA). were performed in triplicate.
Filtered beer (5 mL) was transferred to vials and subsequently
heated for 16 min at 60 °C in the headspace (HS) autosampler 2.6 Statistical analysis
before injection (needle temperature: 70 °C). Helium was used
as the carrier gas. The oven temperature was kept at 50 °C for Sensory data were analysed for statistical significance by parametric
7.5 min, increased to 110 °C at 25 °C/min and was held at that t-tests for paired samples, using the software XLSTAT 2007.6 for
43 March / April 2010 (Vol. 63) BrewingScience

MS Excel. Partial Least Squares Regression (PLS2) was carried beers aged at higher temperature, but after a certain period, the
out using the Unscrambler 9.7 software. intensity declined [13, 14]. Furthermore, it appeared that solvent
flavour was more or less perceived evenly strong in all aged
beers, even in the beer that was aged for 10 years. Ageing at 20
3 Results and discussion °C resulted in a beer with a flavour profile consisting of similar
intensities for all 5 aged flavour notes presented. Especially
A lager beer was aged in various conditions (varying temperature- the Maillard-like flavour was intensely perceived compared to
time profiles, different oxidative conditions and varying pH and beers stored at higher temperatures, which is in accordance with
ethanol concentrations) and resulting beers were tasted and analysed results of Kaneda et al. [9]. Furthermore, it was apparent that
afterwards. Sensory results and the concentration evolution of a Madeira flavours were only formed in beers aged at 20 °C for a
selection of flavour compounds will first be discussed separately. longer storage period, especially after ageing for 10 years. This
Afterwards, correlations between sensory and analytical results is not unexpected, since this flavour is primarily known from
will be discussed in order to explain the sensory results from the its appearance in aged ports and Madeira wines. Finally, apart
chemical composition. from the Madeira flavour, ribes and Maillard-like flavour notes
were strongly perceived.
3.1 Sensory analysis of beers aged in different conditions
3.1.2 Oxidative conditions
3.1.1 Temperature-time profile
Beer flavour is known to be affected by reactive oxygen species
A lager beer was aged with varying temperature-time profiles: (ROS) (i.e. singlet oxygen (1O2), superoxide anion radical (O2•-),
5 days at 60 °C, 3 weeks at 40 °C, 3 months at 28°C, 6 months at hydroperoxyl radical (•OOH), hydroxyl radical (•OH), and hy-
20 °C and 10 years at 20 °C. Obviously, the latter beer was not drogen peroxide (H2O2)) [15]. These ROS can be activated from
produced in the same batch process and probably, in the 10 year molecular oxygen in its unreactive ground state (3O2) by catalytic
time period, changes have been applied to the production process. activity of electron donors (e.g. metal ions such as Fe and Cu),
Still, it was thought that including this beer can give added value energy or light [15]. In this study, the effect of oxidation on beer
to the study since extreme conditions may give more explicit ageing was examined by increasing the total in-pack oxygen
results. content (from 0.1 ppm in the reference beer to 12 ppm) on the one
hand, and adding Fenton reagent (a mixture of H2O2 and FeSO4)
The beers were tasted after ageing and sensory results are pre- on the other hand. Afterwards, beers were tasted and results are
sented in figure 1. The fresh beer had an ageing intensity of zero presented in figure 2.
and aged flavour notes were absent. Therefore, this beer was not
included in the figure. The overall appreciation score on the other Ageing was accelerated significantly in beers stored with oxygen
hand was 6.6. From the ageing intensity and overall appreciation and Fenton reagent. This is confirmed by many studies [3, 13, 16].
scores, it is clear that higher temperatures accelerate beer age- Oxidative ageing has long been regarded as the only reason for
ing enormously. The beer that was aged at 60 °C for 5 days was beer ageing. However, studies by Currie et al. [17] and Furusho
even quoted as the second most aged beer. This emphasises the et al. [13] which dosed low amounts of oxygen in the bottle (0.5
importance of storing beer as cold as possible. ppm, and 0.42 and 0.69 ppm respectively compared to the controls
of 0.05 and 0.16 ppm respectively) followed by ageing, observed
Next to differences in the general ageing score, it was obvious only minor effects on aged flavour. Currie et al. [17] concluded
that separate flavour notes were influenced completely different that low concentrations of oxygen (< 0.50 ppm) are desirable,
depending on the applied temperature. In general, reaction rates but that attempts to reduce oxygen to the ppb concentration range
will increase at higher temperatures. However, this increase de- appears unlikely to significantly increase flavour stability. In this
pends on the activation energy of a specific reaction which differs study, the dosage of oxygen in beer appeared to result in a slightly
between distinct chemical reactions. Consequently, reactions rates higher ageing intensity compared to the beer supplemented with
of separate reactions will not increase equally and ageing will Fenton reagent. However, the difference was small.
give rise to an alteration of the relative concentration of staling
compounds depending on the applied temperature [1]. In this Furthermore, separate flavour notes were influenced differently. In
study, it was clear that ageing at 60 °C predominantly gave rise the beer aged with oxygen, ribes flavour was especially apparent
to the perception of cardboard flavour. Other flavour notes hardly and also Madeira and Maillard-like flavour notes were enhanced
developed during the 5 days of ageing. Relatively, the cardboard substantially. The cardboard and solvent flavour were unaffected
flavour was also more prominent in the beers aged at 40 °C or 28 °C by the presence of oxygen. The addition of Fenton reagent resulted
compared to ageing at 20 °C. The greater appearance of cardboard in a more or less equal increase of ribes, Maillard, Madeira and
flavour during ageing at higher temperatures was already observed cardboard flavours. Solvent flavour was again unaffected. Earlier,
in previous studies [4, 9]. However, Greenhoff & Wheeler [12] samples stored with oxygen were described as sweet caramelised
claimed that the nature of flavour changes, in particular the [7] and a close correlation has been found between ribes flavour
cardboard character, displayed at 60 °C was similar to that at and headspace air [16]. However, ribes flavour could also de-
18 or 37 °C. In two other studies, the beers were not only tasted velop quite strong in a number of beers with low headspace-air
after ageing, but flavour evolution was also monitored. Both content. Oxygen thus enhances ribes flavour formation, but it is
studies showed a faster increase of the cardboard flavour in the not required.
BrewingScience March / April 2010 (Vol. 63) 44

3.1.3 pH Acetaldehyde was mainly influenced by increased oxidative

conditions, especially by the addition of Fenton reagent. It was
In order to study the effect of pH on beer ageing, the pH was adapted previously suggested that hydroxyl radicals react non-selectively
to 3.8 and 4.6 starting from a pH of 4.2 in the unmodified lager beer. with ethanol, the most abundant compound in beer after water,
From the overall score and general ageing intensity, it seems that resulting in the 1-hydroxyethyl radical [1]. This radical can further
the aged flavour decreases as the pH increases. These differences react to acetaldehyde. H2O2 is more stable, and also other reactions
were statistically compared with the paired t-test, and it appeared are more probable to occur. However, according to Kaneda et al.
that only a significant difference (α < 0.05) could be established [15], the hydroxyl radical can be generated by reaction of H2O2
between the pH 3.8 and the pH 4.6 beers, and to a lesser extent with Fe2+, called the Fenton reaction. The main effect of adding
(α < 0.1) between pH 3.8 and 4.2 for the overall ageing scores. Fenton reagent might thus be the formation of acetaldehyde. In
Grigsby et al. [7] were the first to show that a correlation exists be- this study, the formation of acetaldehyde was accelerated mod-
tween the pH of beer and its ‘oxidisability’. Beers aged at extremely erately at higher temperatures. The ethanol content appeared to
low pH (pH 2-3) were shown to have very poor flavour stability. have only a minor influence, which might be attributable to the
In contrast, beers with a high pH (4.5-4.8) have been reported to limited concentration increase compared to the reference, and the
possess better flavour stability [7]. Furthermore, Kaneda et al. [18] greater importance of ROS. Finally, formation of acetaldehyde
showed that lowering the pH from 4.3 to 4.1 or 3.8 significantly was the highest at pH 4.2. The formation of hexanal was mainly
accelerated staling. Currie et al. [17], on the other hand, who tasted influenced by the presence of oxygen, but a lower pH also led to
beers that were aged at pH 3.75, 4.06 and 4.25 could not ascertain a higher concentrations in the aged beer.
significant difference between the beer aged at pH 4.25 compared
to pH 4.06. They suggested that keeping the beer above pH 4.0 The formation of (E)-2-nonenal (T2N) was accelerated tremen-
may be desirable for flavour stability, but that small decreases will dously at higher temperatures as can be deduced from the high
only result in marginally inferior flavour stability. concentrations in the thermally beers. This indicates that T2N
may be specific for thermally aged beers, while it is less formed
Differences in the appearance of separate flavour notes were in naturally aged beers (20 °C). This assumption is confirmed by
small. Only cardboard flavour was perceived more at lower beer Van Eerde & Strating [21] who observed a substantial increase of
pH. Other flavour notes were more or less equally perceived in all T2N at 40 °C in several beers within a few days, whereas at 20 °C,
beers. Guyot-Declerck et al. [19] performed aroma extract dilution this was not seen, even after 4 months of storage. A similar result
analysis (AEDA) on beers aged at pH 4.2 and 4.6 and found out was obtained by Narziss et al. [22] and Nordlov & Winell [23]. This
that cardboard perception was higher after ageing at pH 4.2. might explain why T2N is indicated as very important in some
studies [24], while others observe only a limited or no increase
3.1.4 Ethanol content during ageing [25, 26]. Oxidative conditions also increased the
concentration of T2N during ageing. In contrast, other authors
Another parameter that was varied was the ethanol content, since observed an increase of T2N independent of the oxygen concen-
ethanol is involved in several ageing reactions. It appeared that the tration in bottled beer [22, 27] and they doubted the occurrence of
influence on beer ageing was small from the overall appreciation oxidative formation reactions leading to T2N in beer. Therefore,
and the ageing score (Fig. 2). Also when separate flavour notes non-oxidative mechanisms were suggested, such as aldol con-
were studied, only slight differences were observed. The only dif- densation of acetaldehyde and heptanal [28]. However, it is not
ference worth mentioning was the slightly increased perception clear whether the amounts formed in this reaction under normal
of Madeira flavour. Perpète & Collin [20] showed that ethanol storage conditions, are sufficiently high to contribute [6]. Nord-
significantly increases aldehyde retention in a study on low-al- lov & Winell [23] proposed the dissociation of nonenal-sulphite
cohol beers. Higher alcohol contents consequently lead to lower adducts at low pH values. However, Kaneda et al. [29] showed
perception of the worty character. This difference was however a very low activity of T2N with sulphites and Dufour et al. [30]
not observed in the obtained results and it is questionable if the showed the irreversible nature of the sulphite addition to the double
increased retention is substantial when the alcohol content is already bond. Another proposition for the formation of T2N, the release
quite high. Finally, it should be mentioned that a relatively small from imine adducts [31], seems more plausible. This hypothesis
amount of ethanol (1 v/v %) was added because of limitations of describes the formation of T2N during malting and mashing, and
the remaining headspace volume. subsequent binding by amino compounds leading to the presence
of T2N-imine adducts in beer. Acid hydrolysis at the lower beer
3.2 Evolution of staling compounds in beers aged in different pH (±4.2) compared to wort pH (±5.2) subsequently leads to the
conditions release of T2N. This reaction mechanism might also explain the
higher concentrations of T2N in the beer aged at pH 3.8 compared
The concentrations of a selection of staling compounds were de- to pH 4.2 and 4.6. Kaneda et al. [18] on the other hand, suggested
termined in the differently aged beers and are presented in table that T2N formation is independent of the dissociation from adducts,
2 and 3. Apart from the concentrations, the thresholds (TH) of but based on the fact that the decreasing pH accelerates flavour
the compounds are presented. These thresholds were determined staling reactions such as free radical reactions.
in a previous study in the same lager beer as the one studied here
[2]. The concentration evolutions will be discussed shortly in In this study, forced ageing enhanced the formation of all Strecker
order to obtain an overview of the origin and dependence of the aldehydes considerably. Oxygen even seemed to have a greater
compounds on the tested conditions. promoting effect. Methner et al. [32] confirmed the large effect of
45 March / April 2010 (Vol. 63) BrewingScience

temperature. Schieberle et al. [33] observed a large enhancement The formation of β-damascenone (β-DS) was highly dependent
of the concentration of Strecker aldehydes during ageing at 45 °C on the temperature. In beers aged at low temperatures, even after
with oxygen. When oxygen was omitted, but ageing was still a long storage time, concentrations were quite low. It can thus be
performed at 45 °C, this enhancement was considerably less. This proposed that a thermal degradation mechanism is responsible.
suggested the greater impact of oxygen compared to temperature. Low pH values also enhanced the level of β-DS greatly which may
The promoting effect of oxygen on Strecker aldehyde formation be attributed to a faster acid-catalysed glycoside hydrolysis [42].
has been observed in several other studies [22, 34] and might be Additionally, β-DS can also be formed by oxidative degradation
attributable to oxidation of the respective alcohols [1]. However, of carotenoids [1]. However, no increase of β-DS was observed
oxidative reactions may also lead to a greater release of Strecker in higher oxidative conditions.
aldehydes from precursors formed in the boiling kettle. Most likely,
these precursors are Amadori rearrangement products which can Although it is assumed that ethyl nicotinate and ethyl pyruvate
be oxidised to 2-hexosulose-imines in the presence of transition are formed similarly from the condensation of ethanol and the
metal ions and air oxygen [35]. In this study, the effect of pH and corresponding acid, they behaved differently and higher ethanol
ethanol content on the formation of Strecker aldehydes was neg- content seemed of little importance.
ligible in the studied ranges, except for 2-methylpropanal, which
was formed more at higher pH. Benzaldehyde showed a different γ-nonalactone can be formed by intramolecular esterification of
trend over the differently aged beers. This can be explained as 4-hydroxynonanoic acid and was only formed in low amounts in
benzaldehyde can be formed from phenyl acetaldehyde [36], but the thermally aged beers. An oxidative environment on the other
not by direct Strecker degradation. hand, accelerated its formation.

Furfural, 5-hydroxymethylfurfural (5-HMF) and 5-methylfur- Hydrolysis of isoamyl acetate occurred somewhat faster at hig-
fural behaved similar in the varying ageing conditions. Maillard her temperatures and also oxygen and a lower or higher beer pH
reactions are known for their correlation with high temperature induced hydrolysis. The presence of Fenton reagent and ethanol
processes. As in previous studies [37-39], this was confirmed by on the other hand, did not. These results are in accordance with
the presented results. Oxidative conditions and ethanol content Stenroos et al. [43] who observed a substantial decrease of isoamyl
on the other hand, seemed without any effect. Furthermore, the acetate during ageing at 38 °C compared to 20 °C and also high
lower the pH value, the more formation of furanic aldehydes was air contents led to a somewhat faster decrease.
observed as also observed previously by Shimizu et al. [37]. This
pH dependency was even more pronounced for acetylfuran. Al- 3.3 Impact of the selected staling compounds on aged
though the initiation of the Maillard reaction is improved at high flavour
pH, subsequent reactions are either acid- or base-catalysed [40].
The formation of diacetyl, was mainly enhanced in the presence In a previous study, the aged flavour of a lager beer aged at 40 °C
of oxygen, and to a much lesser extent in the presence of Fenton for 3 weeks could be explained relatively well [2]. In this study, it
reagent. Vanderhaegen et al. [1] suggested that the oxidation of was shown that aged beer flavour is variable depending on finishing
acetoin or 2,3-butanediol may be responsible. operations and storage conditions because of differences in the
relative concentrations of staling compounds. Therefore, it was
2-furfuryl ethyl ether (FEE) is formed from a condensation reaction attempted to explain the sensory results of the differently aged
of furfuryl alcohol and ethanol and increases linearly with the beers from the analytical data in order to obtain a better insight
concentration of its precursors [41]. Consequently, in this study, in aged flavour. In the discussion, the concept of the flavour unit
FEE formation was enhanced in the beer with the higher ethanol (FU), calculated as the ratio of the concentration of a compound
level and the conditions that increased the formation of furfural and its TH value [44] is used to indicate its contribution to fla-
(which can be considered as a good marker for the formation of vour. Previously, it was stressed that care should be taken when
furfuryl alcohol) (i.e. high temperature and low pH). Moreover, extrapolating TH values to other studies because of the influence
the condensation reaction is acid-catalysed [40]. Finally, it should of differing endogenous concentrations and masking flavours [2].
be mentioned that the FEE concentration was very high in the beer However, the same lager beer was used and TH values are thus
aged for 10 years at 20°C. very representative. Still, THs and FUs should be considered as
indicative rather than absolute since interactions can occur and
Methyl isobutyl ketone, ethyl-2-methylbutyrate and ethyl-3- because of large personal variations [2]. Since flavour compounds
methylbutyrate can directly or indirectly be obtained from the are not likely to contribute considerably to flavour when their
oxidative degradation of hop bitter acids and consequently, their FUs stay beyond 0.1 FU, only compounds that exceeded 0.1FU
concentrations were higher in the oxidatively aged beers. Additi- were considered.
onally, it was observed that a lower pH led to higher levels, which
suggests that bitter acids are oxidised more easily at lower pH. Two visualisations were made of the combination of analytical and
Ethyl-2-methylbutyrate and ethyl-3-methylbutyrate are actually sensory results to obtain a clear insight. In figure 3, FUs of the most
formed from a condensation reaction between the corresponding important compounds are presented in spider plots to get an insight
acids and ethanol [1]. Consequently, high ethanol levels resulted in the flavour active ageing compounds in the respective beers.
in higher concentrations of these compounds. Finally, it seemed The FUs of Strecker aldehydes were added up, since they showed
that increasing the storage temperature did not accelerate the a similar concentration profile in the differently aged beers, they
oxidation of hop bitter acids that much. can interact greatly and for clarity reasons. Because of the great
BrewingScience March / April 2010 (Vol. 63) 46

complexity and great amount of data, multivariate statistics are perceived. In these oxidatively aged beers, no increase of specific
useful to obtain a visualisation of the main variability of the data Maillard compounds such as 5-HMF was observed, but Maillard-
set. Additionally, an understanding of the relationship between two like flavour was definitely present. This may be attributed to the
data sets can be gained with partial least squares regression (PLS). combination of Strecker aldehydes and diacetyl as shown by
Therefore, PLS2 (sensory vs analytical results) was used in order Saison et al. [2]. In the beers aged for 3 months at 28 °C (pH and
to obtain insight in their correlations. For the analytical data, FUs ethanol variations), a modest Maillard-like flavour was apparent
were used and after centering the data, a PLS2 plot was created and ribes flavour was virtually absent. These beers showed very
as presented in figure 4. The scores plot containing the samples low acetaldehyde contents, while Strecker aldehydes were defi-
(not shown), showed the initial beer in the upper left corner and nitely present. The latter were already shown to cause a shift to
the beer aged at pH 4.6 quite close. The beers that were aged most sweet and bread-like off-flavours during storage [32]. In the end,
were spread at the opposite site according to their separate sensory it may be that a specific ratio of the levels of these compounds
ageing characteristics. The more distant X-variables (sensory) are may lead to ribes or Maillard-like flavours, but it is very likely
located from the centre, the more variability they explain and the that other compounds than the ones measured in this study also
more they contributed to the creation of the plot. The more distant contribute. Ribes-like flavour for example, was already linked to
Y-variables are from the centre, the more they might explain the the occurrence of polyfunctional thiols in beer [46].
sensory results. The plot largely confirms the greater importance
of T2N, Strecker aldehydes, FEE, acetaldehyde and diacetyl as The Madeira flavour was especially apparent in the beer aged for
compared to 5-HMF and β-DS, as proposed by Saison et al. [2]. 10 years at 20 °C. Also in this beer, an increase of 3 FUs acetalde-
hyde was seen. However, especially the very high concentration
As can be deduced from the plot, cardboard flavour correlated well of FEE catches the eye. Furthermore, high concentrations of
with T2N. The flavour of the beer aged for 5 days at 60 °C was Strecker aldehydes, 5-HMF, diacetyl, but not of T2N and β-DS,
mainly made up of cardboard. This may be explained by the incre- were observed. The outcome was a beer with less harsh and more
ase of about 4 FUs. However, although figure 3 clearly indicated warming sensory notes such as Madeira, Maillard and ribes. In this
that many compounds such as Strecker aldehydes, 5-HMF, FEE beer, acetylfuran also reached a FU of 0.11. Although this is still
and β-DS increased substantially, it is remarkable that other aged quite low, it indicates that flavour compounds that are thought to
flavours were not substantially perceived. They may be masked by be unimportant may contribute through interactions in this highly
the cardboard flavour. Moreover, Strecker aldehydes were already aged beer. In conclusion, Madeira flavour seems to be the results
shown to contribute to cardboard flavour [2]. The beer aged for 3 of a very complex combination of many compounds that also
weeks at 40 °C had lower concentrations of Strecker aldehydes, involves others than the ones presented.
FEE, diacetyl and 5-HMF, but the sensory characteristics, solvent,
ribes and Maillard-like were not perceived less. On the contrary, Solvent flavour was previously shown to be related to FEE for-
on average, higher scores were given for these flavour notes. The mation [2, 41]. In this study, the variability of the solvent flavour
beer aged at 20 °C for 6 months had a comparable FU profile in was relatively low between aged beers as can be deduced from the
figure 3, except for T2N. However, when its sensory profile was PLS plot and thus, not many assumptions can be made.
studied, ribes, Maillard and Madeira flavour notes were more
apparent. It may be that T2N and similar compounds such as Finally, beers aged at different pH or ethanol content did not vary
TT24DD, are quite prominent and may render beers more harsh, greatly and sensory results were not significantly different. Howe-
leathery and papery. Lower concentrations of T2N may allow ver, some tendencies were observed. Differences were observed
other flavour notes to become more apparent. in the cardboard flavour of beers aged at varying pH’s, which may
be explained by the higher T2N level with lower pH. Furthermore,
Although the concentration of acetaldehyde increased with about β-DS and FEE concentrations differed. They might also correlate
8 and 5 FUs for the Fenton and the oxygen beer respectively, none with cardboard flavour, but also with the varying solvent flavour.
of the tasters assigned a considerable intensity to the acetaldehyde The slightly higher ethanol content resulted in an increase of the
flavour (sour apple-like) (results not shown). This confirms previous same compounds (i.e. T2N, FEE and β-DS) and mainly caused
suggestions that acetaldehyde mainly contributes to aged flavour the beer to be slightly more Madeira-like. It is probable that not
as a supporting background for other aged flavours [2, 45], such as only increased concentrations of these compounds contributed,
ribes and Maillard-like flavours. Furthermore, diacetyl and Strecker but also ethanol itself may contribute considerably. This is not
aldehydes were also enhanced greatly in these beers. surprising since Madeira flavour is mainly known in beverages of
higher ethanol content such as Ports and Madeira wines.
From the PLS2 plot, it seems that ribes and Maillard-like flavours,
when given a high intensity, were regularly both given a high
intensity. It is therefore difficult to discuss them separately. Ribes 4 Conclusion
flavour was especially apparent in the beer that was spiked with
oxygen. The spider plot in figure 3 shows that high concentrations Aged flavour of beer was studied in more detail by ageing lager
of Strecker aldehydes, diacetyl, T2N and acetaldehyde were found beer in varying conditions. It was evident that different ageing
in this beer. Adding Fenton reagent resulted in lower concentrations conditions caused aged beer flavour to vary greatly. This could
of diacetyl, Strecker aldehydes and T2N, but a higher concentration be explained by differences in the formation rate of aged flavour
of acetaldehyde. In this beer, a lower ribes and a more balanced compounds. Ageing at higher temperatures, stronger oxidative
sensory profile concerning the 5 attributes shown in figure 2 was conditions and to a lesser extent, lower pH, clearly accelerated
47 March / April 2010 (Vol. 63) BrewingScience

beer ageing. Ageing at 60 °C resulted in a prominent cardboard and other beverages, Proc. 31th EBC Congr., Fachverlag Hans Carl,
flavour and at lower storage temperatures, cardboard gradually Nürnberg, Germany, (2007).
decreased. Especially the formation of T2N was enhanced greatly 9. Kaneda, H.; Kobayashi, N.; Furusho, S.; Sahara, H. and Koshino,
at higher temperatures and its concentration correlated strong with S.: Chemical evaluation of beer flavor stability, MBAA Technical
cardboard. Strecker degradation, Maillard reactions and β-DS Quarterly, 32 (1995), pp. 76-80.
formation were also enhanced at higher temperatures, but these 10. Saison, D.; De Schutter, D. P.; Delvaux, F. and Delvaux, F. R.:
compounds seemed to support cardboard flavour or were masked Determination of carbonyl compounds in beer by derivatisation
by higher concentrations of T2N. In contrast, at lower tempera- and headspace solid-phase microextraction in combination with gas
tures, ribes and Maillard-like flavour notes were more manifest. chromatography and mass spectrometry, Journal of Chromatography
Solvent flavour seemed to be influenced less markedly by storage A, 1216 (2009), no. 26, pp. 5061-5068.
temperature, and Madeira flavour was only perceived in beers aged 11. Saison, D.; De Schutter, D. P.; Delvaux, F. and Delvaux, F. R.: Opti-
at 20°C, especially after storage for 10 years. This flavour seemed misation of a complete method for the analysis of volatiles involved
to be especially correlated with FEE and also with acetaldehyde, in the flavour stability of beer by solid-phase microextraction in
diacetyl, Strecker aldehydes and 5-HMF. These results indicate combination with gas chromatography and mass spectrometry, Journal
that care should be taken when considering forcing tests which are of Chromatography A, 1190 (2008), no. 1-2, pp. 342-349.
necessary in order that research on flavour stability can be done 12. Greenhoff, K. and Wheeler, R. E.: Evaluation of stale flavour and
over realistic time frames. Oxidative conditions predominantly beer carbonyl development during normal and accelerated aging
accelerated the development of ribes flavour, but also of Maillard utilising liquid and high pressure liquid chromatography, Proc. 18th
flavours. Particularly the formation of acetaldehyde was enhanced EBC Congr., Fachverlag Hans Carl, Nürnberg, Germany (1981),
in these beers, but also Strecker degradation and diacetyl formation. pp. 405-412.
Lower beer pH resulted in slightly increased cardboard flavour, but 13. Furusho, S.; Kobayashi, N.; Nakae, N.; Takashio, M.; Tamaki, T.
differences were low and were mainly suggested to be caused by and Shinotsuka, K.: A developed descriptive sensory test reveals
a difference in T2N, FEE and β-DS content. Ageing beer with a beer flavor changes during storage, MBAA Technical Quarterly, 36
slightly higher ethanol level led to a higher perception of Madeira (1999), no. 2, pp. 163-166.
flavour and a slightly higher FEE content. 14. Eger, C.; Habich, N.; Akdokan, N.; Gossling, U. and Bellmer, H. G.:
Profiling of beers during ageing at different temperatures, Proc. 30th
EBC Congr., Fachverlag Hans Carl, Nürnberg, Germany (2005).
5 Acknowledgements 15. Kaneda, H.; Kobayashi, N.; Takashio, M.; Tamaki, T. and Shinotsuka,
K.: Beer Staling Mechanism, MBAA Technical Quarterly, 36 (1999),
We acknowledge the Research Fund (K. U. Leuven, Leuven, no. 1, pp. 41-47.
Belgium) for financial support. 16. Clapperton, J. F.: Ribes Flavor in Beer, Journal of the Institute of
Brewing, 82 (1976), no. 3, pp. 175-176.
17. Currie, B. R.; Kulandai, J.; FitzRoy, M. D.; Hawthorne, D. B. and
6 References Kavanagh, T. E.: Processing influences on the stability of packaged
beer, Proc. Conv. Inst. Brew. (1990), pp. 117−125.
1. Vanderhaegen, B.; Neven, H.; Verachtert, H. and Derdelinckx, G.: 18. Kaneda, H.; Takashio, M.; Tomaki, T. and Osawa, T.: Influence of
The chemistry of beer aging – a critical review, Food Chemistry, 95 pH on flavour staling during beer storage, Journal of the Institute of
(2006), no. 3, pp. 357-381. Brewing, 103 (1997), no. 1, pp. 21-23.
2. Saison, D.; De Schutter, D. P.; Uyttenhove, B.; Delvaux, F. and Del- 19. Guyot-Declerck, C.; Francois, N.; Ritter, C.; Govaerts, B. and Collin,
vaux, F. R.: Contribution of staling compounds to the aged flavour S.: Influence of pH and ageing on beer organoleptic properties. A
of lager beer by studying their flavour thresholds, Food Chemistry, sensory analysis based on AEDA data, Food Quality and Preference,
114 (2009), no. 4, pp. 1206-1215. 16 (2005), no. 2, pp. 157-162.
3. Vanderhaegen, B.; Neven, H.; Coghe, S.; Verstrepen, K. J.; Verachtert, 20. Perpète, P. and Collin, S.: Influence of beer ethanol content on the
H. and Derdelinckx, G.: Evolution of chemical and sensory properties wort flavour perception, Food Chemistry, 71 (2000), no. 3, pp. 379-
during aging of top-fermented beer, Journal of Agricultural and Food 385.
Chemistry, 51 (2003), no. 23, pp. 6782-6790. 21. Van Eerde, P. and Strating, J.: Trans-2-Nonenal, European Brewery
4. Fratianni, A. J.: Results of the effect of storage temperature on Convention – Flavour Symposium, (1981), pp. 117-121.
packaged beer flavor, MBAA Technical Quarterly, 38 (2001), no. 3, 22. Narziss, L.; Miedaner, H. and Graf, H.: Carbonyls and Aging of
pp. 159-161. Beer – Influence of Some Technical Parameters, Monatsschrift fur
5. Vanderhaegen, B.; Delvaux, F.; Daenen, L.; Verachtert, H. and Delvaux, Brauwissenschaft, 38 (1985), no. 12, pp. 472-477.
F. R.: Aging characteristics of different beer types, Food Chemistry, 23. Nordlov, H. and Winell, B.: Beer flavour stabilization by interaction
103 (2007), no. 2, pp. 404-412. between bisulfite and trans-2-nonenal, Proc. 19th EBC Congr., Fach-
6. Bamforth, C. W.: The science and understanding of the flavour stability verlag Hans Carl, Nürnberg, Germany, (1983), pp. 271-278.
of beer: a critical assessment, BRAUWELT, 2 (1999), pp. 98-110. 24. Jamieson, A. M. and Van Gheluwe, J. E. A.: Identification of a
7. Grigsby, J. H.; Palamand, S. R. and Hardwick, W. A.: Further studies compound Responsible for Cardboard Flavor in Beer, Journal of the
on the staling of beer, Journal of the American Society of Brewing American Society of Brewing Chemists, 28 (1970), pp. 192-197.
Chemists, 31 (1974), pp. 64-69. 25. Vesely, P.; Lusk, L.; Basarova, G.; Seabrooks, J. and Ryder, D.:
8. Methner, F. J.; Kunz, T. and Schön, T.: Application of optimized Analysis of aldehydes in beer using solid-phase microextraction with
methods to determine the endogenous anti-oxidative potential of beer on-fiber derivatization and gas chromatography/mass spectrometry,
BrewingScience March / April 2010 (Vol. 63) 48

Journal of Agricultural and Food Chemistry, 51 (2003), no. 24, cocoa and model systems: New insights into pathways of the Strecker
pp. 6941-6944. reaction, Journal of Agricultural and Food Chemistry, 54 (2006), no.
26. Narziss, L.; Miedaner, H. and Lustig, S.: The behaviour of volatile 5, pp. 1730-1739.
aromatic substances as beer ages, Monatsschrift fur Brauwissenschaft, 37. Shimizu, C.; Nakamura, Y.; Miyai, K.; Araki, S.; Takashio, M. and
52 (1999), no. 9-10, pp. 164-175. Shinotsuka, K.: Factors affecting 5-hydroxymethyl furfural formation
27. Noel, S.; Metais, N.; Bonte, S.; Bodart, E.; Peladan, F.; Dupire, S. and and stale flavor formation in beer, Journal of the American Society
Collin, S.: The use of Oxygen 18 in appraising the impact of oxidation of Brewing Chemists, 59 (2001), no. 2, pp. 51-58.
process during beer storage, Journal of the Institute of Brewing, 105 38. Madigan, D.; Perez, A. and Clements, M.: Furanic aldehyde analysis
(1999), no. 5, pp. 269-274. by HPLC as a method to determine heat-induced flavor damage to
28. Hashimoto, N. and Kuroiwa, Y.: Proposed pathways for the formation beer, Journal of the American Society of Brewing Chemists, 56 (1998),
of volatile aldehydes during storage of bottled beer, Journal of the no. 4, pp. 146-151.
American Society of Brewing Chemists, 33 (1975), pp. 104-111. 39. Carneiro, J. R.; Guido, L. F.; Almeida, P. J.; Rodrigues, J. A. and Barros,
29. Kaneda, H.; Takashio, M.; Osawa, T.; Kawakishi, S. and Tamaki, T.: A. A.: The impact of sulphur dioxide and oxygen on the behaviour of
Behavior of sulfites during fermentation and storage of beer, Journal 2-furaldehyde in beer: an industrial approach, International Journal
of the American Society of Brewing Chemists, 54 (1996), no. 2, of Food Science and Technology, 40 (2005), pp. 1-8.
pp. 115-120. 40. De Schutter, D. P.; Saison, D.; Delvaux, F.; Derdelinckx, G.; Rock,
30. Dufour, J. P.; Leus, M.; Baxter, A. J. and Hayman, A. R.: Characte- J. M.; Neven, H. and Delvaux, F. R.: Release and evaporation of
rization of the reaction of bisulfite with unsaturated aldehydes in a volatiles during boiling of unhopped wort, Journal of Agricultural
beer model system using nuclear magnetic resonance spectroscopy, and Food Chemistry, 56 (2008), no. 13, pp. 5172-5180.
Journal of the American Society of Brewing Chemists, 57 (1999), 41. Vanderhaegen, B.; Neven, H.; Daenen, L.; Verstrepen, K. J.; Verach-
no. 4, pp. 138-144. tert, H. and Derdelinckx, G.: Furfuryl ethyl ether: Important aging
31. Lermusieau, G.; Noel, S.; Liegeois, C. and Collin, S.: Nonoxidative flavor and a new marker for the storage conditions of beer, Journal of
mechanism for development of trans-2-nonenal in beer, Journal Agricultural and Food Chemistry, 52 (2004), no. 6, pp. 1661-1668.
of the American Society of Brewing Chemists, 57 (1999), no. 1, 42. Gijs, L.; Chevance, F.; Jerkovic, V. and Collin, S.: How low pH can
pp. 29-33. intensify beta-damascenone and dimethyl trisulfide production through
32. Methner, F. J.; Fritsch, H. and Stephan, A.: Influence of storage beer aging, Journal of Agricultural and Food Chemistry, 50 (2002),
conditions, raw materials and brewing technology on the formation no. 20, pp. 5612-5616.
of Strecker aldehydes during beer aging, Proc. 29th EBC Congr., 43. Stenroos, L. E.: The Effects of Time, Temperature, and Air on Va-
Fachverlag Hans Carl, Nürnberg, Germany, (2003). rious Finished Beer Components, Journal of the American Society
33. Schieberle, P. and Komarek, D.: Staling of beer aroma: a long-known, of Brewing Chemists, 31 (1973), pp. 50-56.
but still unresolved challenge in brewing science, Proc. 30th EBC 44. Meilgaard, M. C.: Flavor Chemistry of Beer: Part II: Flavor and
Congr., Fachverlag Hans Carl, Nürnberg, Germany, (2005). Threshold of 239 Aroma Volatiles, MBAA Technical Quarterly, 12
34. Miedaner, H.; Narziss, L. and Eichhorn, P.: Einige faktoren der ge- (1975), no. 3, pp. 151-168.
schmacksstabilität – sensorische und analytische bewertung, Proc. 45. Palamand, S. R. and Hardwick, W. A.: Studies on the relative flavor
23th EBC Congr., Fachverlag Hans Carl, Nürnberg, Germany, (1991), importance of some beer constituents, MBAA Technical Quarterly,
pp. 401-408. 6 (1969), no. 2, pp. 117-127.
35. Hofmann, T. and Schieberle, P.: Formation of aroma-active Strecker- 46. Schieberle, P.: Primary Odorants of Pale Lager Beer – Differences to
aldehydes by a direct oxidative degradation of Amadori compounds, Other Beers and Changes During Storage, Zeitschrift fur Lebensmit-
Journal of Agricultural and Food Chemistry, 48 (2000), no. 9, pp. tel-Untersuchung Und-Forschung, 193 (1991), no. 6, pp. 558-565.
4301-4305.
36. Granvogl, M.; Bugan, S. and Schieberle, P.: Formation of amines
and aldehydes from parent amino acids during thermal processing of Received 11 December, 2009, accepted 20 February, 2010

Appendix

Table 1 Overview of the conditions that were tested for their effect on beer ageing

Temperature-time 3 months 28 °C
profiles Oxidative conditions pH Ethanol content
5 days 60 °C Oxygen Flush 3.8 5.2 v/v % EtOH (Ref)
3 weeks 40 °C Fenton reagent 4.2 (Ref) 6.2 v/v % EtOH
3 months 28 °C Non-oxidative (Ref) 4.6
6 months 20 °C
10 years 20 °C
49 March / April 2010 (Vol. 63) BrewingScience

Table 2 Concentrations of a selection of staling compounds in beers that were aged according to varying time-temperature
profiles. Concentrations are expressed in ppb unless stated otherwise

TH Initial 5 days 3 weeks 3 months 6 months 10 years

60 °C 40 °C 28 °C 20 °C 20 °C
Acetaldehyde (ppm) 1.1 0.6 1.2 1.0 1.0 1.3 3.9
Hexanal 88 0.5 2.0 2.0 0.9 1.3 2.7
(E)-2-nonenal 0.03 0.03 0.16 0.11 0.05 0.08 0.09
2-methylpropanal 86 11 72 48 29 46 72
2-methylbutanal 45 2.9 16.7 6.2 3.6 4.9 17.4
3-methylbutanal 56 9 17 14 10 18 28
Benzaldehyde 515 1.2 1.6 1.8 1.5 1.8 13.4
Phenyl acetaldehyde 105 22 48 35 29 38 43
Methional 4.2 1.6 3.6 2.5 2.2 2.6 2.5
Furfural 15157 19 916 287 171 273 1094
5-HMF (ppm) 36 5 38 13 8 11 47
5-methylfurfural 1174 0.2 6.4 2.1 1.0 1.2 9.7
Acetylfuran 513 7 26 25 18 10 57
Diacetyl 17 6 20 12 11 20 40
Methyl isobutyl ketone 2560 4 4 4 5 5 12
Ethyl-2-methylbutyrate 27 0.4 0.4 0.5 0.4 0.6 2.0
Ethyl-3-methylbutyrate 91 0.7 1.0 1.1 0.9 1.5 5.3
Ethyl nicotinate 4555 4 18 18 11 – 164
Ethyl pyruvate 22525 39 50 66 86 112 61
2-Furfuryl ethyl ether 11 2 31 13 7 13 90
β-damascenone 203 123 380 303 242 195 88
γ-nonalactone 607 30 33 35 36 31 30
Isoamyl acetate 510 356 284 273 295 360 8

Table 3 Concentrations of a selection of staling compounds in beers that were aged for 3 months at 28 °C after varying several
parameters: oxidative conditions, pH or ethanol concentration. Concentrations are expressed in ppb unless stated
otherwise
Fenton Oxygen Ref 6.2 v/v
TH Initial reagent Flush (pH 4.2) pH 3.8 pH 4.6 % EtOH
Acetaldehyde (ppm) 1.1 0.6 9.5 5.9 1.0 0.6 0.8 1.0
Hexanal 88 0.5 2.4 3.2 0.9 1.3 0.9 1.2
(E)-2-nonenal 0.03 0.03 0.06 0.09 0.05 0.10 0.04 0.07
2-methylpropanal 86 11 41 102 29 24 44 35
2-methylbutanal 45 2.9 11.2 14.4 3.6 4.2 3.6 3.9
3-methylbutanal 56 9 24 39 10 11 12 13
Benzaldehyde 515 1.2 4.3 13.2 1.5 1.9 1.0 1.3
Phenyl acetaldehyde 105 22 57 70 29 26 29 24
Methional 4.2 1.6 2.4 4.1 2.2 2.0 1.8 1.6
Furfural 15157 19 165 214 171 292 91 154
5-HMF (ppm) 36 5 7 9 8 13 7 9
5-methylfurfural 1174 0.2 1.1 1.1 1.0 1.9 0.6 0.9
Acetylfuran 513 7 11 15 18 29 8 16
Diacetyl 17 6 30 76 11 7 7 10
Methyl isobutyl ketone 2560 4 3 10 5 6 2 5
Ethyl-2-methylbutyrate 27 0.4 0.7 0.9 0.4 0.6 0.4 0.5
Ethyl-3-methylbutyrate 91 0.7 1.6 1.8 0.9 1.2 0.8 1.2
Ethyl nicotinate 4555 4 19 20 11 7 13 6
Ethyl pyruvate 22525 39 23 70 86 242 30 83
2-Furfuryl ethyl ether 11 2 7 7 7 16 5 11
β-damascenone 203 123 267 259 242 337 215 297
γ-nonalactone 607 30 45 43 36 38 29 31
Isoamyl acetate 510 356 301 246 295 251 254 296
BrewingScience March / April 2010 (Vol. 63) 50

DAYS—# CARDBOARD YEARS—#

CARDBOARD
5 5
WEEKS—#
4 4
MONTHS—#
MONTHS—# 3 3

2 2
-ADEIRA SOLVENT -ADEIRA SOLVENT
1 1

0 0

-AILLARD RIBES -AILLARD RIBES

/VERALLSCORE AGEINGINTENSITY







DAYS WEEKS MONTHS MONTHS YEARS
—# —# —# —# —#

Fig. 1 Sensory results of beers that were aged according to varying temperature-time profiles
51 March / April 2010 (Vol. 63) BrewingScience

2EFERENCE
/XYGEN
&ENTONREAGENT CARDBOARD /VERALLSCORE AGEINGINTENSITY
5 
4 
3
2

-ADEIRA SOLVENT
1 
0



-AILLARD RIBES 2EFERENCE /XYGEN &ENTONREAGENS
A

P(
P( CARDBOARD
/VERALLSCORE AGEINGINTENSITY
P( 5 
4 
3

2
-ADEIRA SOLVENT 
1
0 


P( P( P(
B -AILLARD RIBES

CARDBOARD
"VV 5
/VERALLSCORE AGEINGINTENSITY
VV 4 
3

2
-ADEIRA SOLVENT
1 
0



-AILLARD RIBES VV VV
C

Fig. 2 Sensory results of beers that were aged for 3 months at 28 °C under varying conditions:
(A) oxidative conditions;
(B) pH;
(C) ethanol content
BrewingScience March / April 2010 (Vol. 63) 52

D— Y—

W— !CETALDEHYDE !CETALDEHYDE

M— 

M—
 
 $3 4.  $3 4.
 

 

 

&%% 3TRECKERS &%% 3TRECKERS

$IACETYL  (-& $IACETYL  (-&

2EFERENCE P(
!CETALDEHYDE !CETALDEHYDE
/XYGEN P( 
&ENTONREAGENT  P(

 $3  4.  $3 4.
 

 

 

&%% 3TRECKERS &%% 3TRECKERS

$IACETYL  (-& $IACETYL  (-&

VV
!CETALDEHYDE
VV



 $3 4.



&%% 3TRECKERS

$IACETYL  (-&

Fig. 3 Spiderplots of the flavour units of ageing compounds in differently aged beers
53 March / April 2010 (Vol. 63) BrewingScience

Fig. 4 PLS2 plot of beers aged in varying conditions (X: sensory data; Y: analytical data).

The plot presents the scores of the plot (sensory and analytical variables)