Food Research International 140 (2021) 110049

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Food Research International

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Monitoring the evolution of free and cysteinylated aldehydes from malt to

fresh and forced aged beer
P. Bustillo Trueba a, *, 1, B. Jaskula-Goiris a, 1, M. Ditrych a, W. Filipowska a, J. De Brabanter a,
G. De Rouck a, G. Aerts a, L. De Cooman a, J. De Clippeleer b, c
a
KU Leuven, Department of Microbial and Molecular Systems (M2S), Cluster for Bioengineering Technology (CBeT), Laboratory of Enzyme, Fermentation and Brewing
Technology (EFBT), Technology Campus Ghent, Gebroeders De Smetstraat 1, B-9000 Ghent, Belgium
b
Innovation centre for Brewing & Fermentation – IBF, Ghent University, Faculty of Bioscience Engineering, Department of Biotechnology, Valentin Vaerwyckweg 1, B-
9000 Ghent, Belgium
c
Innovation centre for Brewing & Fermentation – IBF, HOGENT University of Applied Sciences and Arts, Department of Life Sciences and Industrial Technology, Research
Centre AgroFoodNature, Valentin Vaerwyckweg 1, B-9000 Ghent, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: During storage, beer staling coincides with a gradual increase in the concentrations of aldehydes resulting in the
Cysteinylated aldehydes (2-substituted 1,3- appearance of undesirable flavours. Cysteinylated aldehydes, also referred to as 2-substituted 1,3-thiazolidine-4-
thiazolidine-4-carboxylic acids) carboxylic acids, have been proposed as potential precursors of this increase. This study aimed to further un­
Free aldehydes
derstand the origin of aldehydes in aged beer, by monitoring both free and cysteinylated aldehydes throughout
Quantification
Flavour instability
the brewing process, from the raw materials until the stored product. Quantification of free and cysteinylated
Malt aldehydes was performed for two different brews via headspace solid phase microextraction-gas chromatog­
Brewery samples raphy-mass spectrometry (HS-SPME-GC-MS) and ultra-high performance liquid chromatography-mass spec­
trometry (UHPLC-MS), respectively. All selected marker aldehydes were quantified in malt, wort, and the
resulting fresh and aged beer samples. Cysteinylated aldehydes were quantifiable in malt and up to the wort
boiling phase. The highest levels of free aldehydes were found in malt, whereas cysteinylated aldehydes showed
highest levels at mashing-in pointing to their formation during both malting and subsequent mashing-in. During
beer ageing, an increase in all free aldehydes was measured. In particular, a rise in 2-methylpropanal and furfural
is most striking. Although the presented experimental data obtained on malt and brewery samples do support the
concept of bound-state aldehydes, cysteinylated aldehydes cannot be consider as the cause of increasing levels of
staling aldehydes during beer ageing.

1. Introduction beer is characterised by a limited shelf life (Bamforth, 2011; Rodrigues

The majority of food and beverage products deteriorate in their
sensory properties over time, in particular flavour. Flavour instability is
a complex and highly relevant topic that has been affecting the food and
beverage industry for decades (Kilcast & Subramaniam, 2011; Weer­
awatanakorn et al., 2015). The flavour of food and beverages is expected
to be pleasant, characteristic for the given product and consistent within
its expected shelf life to meet consumers’ expectations (Paternoster
et al., 2020; Stewart et al., 2018). Determining flavour stability is of
utmost importance for the entire food and beverage sector. In particular,
et al., 2011; Saison et al., 2009; Stewart, 2016). The chemical changes
during beer ageing have been studied with regard to both the volatile
and non-volatile fractions, where changes in the concentrations of
different compounds have been identified, e.g. aldehydes, bitter acids,
proteins, esters, etc. (Baert et al., 2012; Liégeois et al., 2002; Malfliet
et al., 2008; Vanderhaegen et al., 2006; Yu et al., 2014). Nevertheless,
beer staling has mainly been described by the loss of its pleasant
bitterness, due to iso-α-acids degradation, and the appearance of staling
off-flavours attributed to free aldehydes, due to their very low flavour
threshold values (Caballero et al., 2012; Lehnhardt et al., 2018; Saison

* Corresponding author.
E-mail addresses: paula.bustillotrueba@kuleuven.be (P. Bustillo Trueba), barbara.jaskulagoiris@kuleuven.be (B. Jaskula-Goiris), maciej.ditrych@kuleuven.be
(M. Ditrych), weronika.filipowska@kuleuven.be (W. Filipowska), jos.debrabanter@kuleuven.be (J. De Brabanter), gert.derouck@kuleuven.be (G. De Rouck),
guido.aerts@kuleuven.be (G. Aerts), luc.decooman@kuleuven.be (L. De Cooman), jessika.declippeleer@ugent.be (J. De Clippeleer).
1
Shared co-first/equal authorship with Barbara Jaskula-Goiris.

https://doi.org/10.1016/j.foodres.2020.110049
Received 24 June 2020; Received in revised form 14 December 2020; Accepted 16 December 2020
Available online 8 January 2021
0963-9969/© 2020 Elsevier Ltd. All rights reserved.
P. Bustillo Trueba et al.
et al., 2009; Vanderhaegen et al., 2006). Although very low amounts of
aldehydes are already found in fresh beer, the appearance of charac­
teristic beer staling on storage coincides with their increase in levels
(Hashimoto, 1966). Several more recent studies also point to aldehydes
and their potential role as the major source of beer staling (Baert et al.,
2018; Gibson et al., 2018; Jaskula-Goiris et al., 2019; Lehnhardt et al.,
2018; Wietstock et al., 2016). Moreover, staling aldehydes still emerge
in beers despite of improved storage conditions, such as lightproof
packaging, exclusion of oxygen to a minimum and low temperature
preservation (Fratianni, 2001; Jaskula-Goiris et al., 2011; Malfliet et al.,
2008). Various pathways for the appearance of aldehydes have been
proposed (Baert et al., 2012), however, despite extensive previous
research, elucidation of the mechanism of the aldehyde increase during
beer ageing has not yet been accomplished.
Aldehydes are highly reactive molecules, thus, able to react with a
variety of compounds, e.g. amino acids, proteins, and sulphur com­
pounds (Solomons et al., 2013). In particular the reaction of aldehydes
with cysteine and/or bisulphite leading to the formation of bound-state
aldehydes has recently been proposed as a potential source of the
aldehyde increase during beer ageing as a result of subsequent aldehyde
release from their bound state(s) (Baert et al., 2018, 2015a, 2015b;
Kaneda et al., 1996; Lehnhardt et al., 2018). As the presence of cysteine
and bisulphite in intermediate brewery products is well-known (Baert
et al., 2015a; Guido, 2016; Matsui & Kitabatake, 1984), the formation of
a bound-state between the aldehyde and cysteine or bisulphite is feasible
(Baert et al., 2018, 2015a, 2015b, 2012). Due to their non-volatile
character, these bound-state aldehydes would neither be removed by
evaporation during wort boiling, nor be reduced by yeast during
fermentation (Saison et al., 2010), and, as a result, they may end up in
the final beer. Subsequently, during beer ageing, release of staling al­
dehydes from their bound-state can be expected, due to beer charac­
teristics, i.e. pH of the final beer (Bustillo Trueba et al., 2018a, 2018b),
or beer storage conditions, e.g. storage at increased temperature (Fra­
tianni, 2001; Jaskula-Goiris et al., 2019).
The aim of the present study is to assess for the first time the potential
relevance of bound-state aldehydes, in particular cysteinylated alde­
hydes, in relation to beer flavour instability by monitoring the evolution
of free and cysteinylated aldehydes in malt, throughout the brewing,
fermentation and maturation processes, as well as in the resulting fresh
and forced aged beer samples.
2. Material and methods
2.1. Chemicals
The carbonyl compounds 2-methylpropanal (2MP ≥ 99%, CAS 78-
84-2), 2-methylbutanal (2MB ≥ 95%, CAS 96-17-3), 3-methylbutanal
(3MB ≥ 98%, CAS 590-86-3), methional (METH ≥ 95%, CAS 3268-49-
3), phenylacetaldehyde (PHEN ≥ 98%, CAS 122-78-1), hexanal (HEX
≥ 98%, CAS 66-25-1), and furfural (FURF ≥ 99%, CAS 98-01-1) were
purchased from Acros Organics (Geel, Belgium). The amino acid L-
cysteine (CYS ≥ 98%, CAS 52-90-4) was acquired from Sigma Aldrich
(St.-Louis, MO, USA). The cysteinylated aldehydes, 2-isopropylthiazoli­
dine-4-carboxylic acid (2MP-CYS ≥ 99%, CAS 14347-75-2), 2-(sec-
butyl)thiazolidine-4-carboxylic acid (2MB-CYS ≥ 99%, CAS 1214831-
88-5), 2-isobutylthiazolidine-4-carboxylic acid (3MB-CYS ≥ 98%, CAS
215669-71-9), 2-(2-(methylthio)ethyl)thiazolidine-4-carboxylic acid
(METH-CYS ≥ 99%, CAS 53943-83-2), 2-benzylthiazolidine-4-carbox­
ylic acid (PHEN-CYS ≥ 94%, CAS 50739-30-5), 2-pentylthiazolidine-4-
carboxylic acid (HEX-CYS ≥ 92%, CAS 69588-05-2) and 2-(furan-2-yl)
thiazolidine-4-carboxylic acid (FURF-CYS ≥ 99%, CAS 72678-98-9)
were synthesized according to Ershov’s methodology (Ershov et al.,
2014) as described by (Bustillo Trueba et al., 2019). Table 1 shows the
molecular and structural formula of the compounds under study. Citric
acid monohydrate (≥99.5%, CAS 5949-29-1), boric acid (≥99.5%, CAS
10043-35-3), tri-sodium phosphate dodecahydrate (≥98%, CAS 10101-
2
Food Research International 140 (2021) 110049
89-0), ethanol (≥99.2%, CAS 64-17-5), dehydrated calcium chloride
(CaCl2 ≥ 95.0%, CAS 10043-52-4), and (L)-Lactic acid (≥90.0%, CAS
50-21-5) were obtained from Merck (Darmstad, Germany). Ultrapure
water was obtained by a Synergy 185 system from Millipore (mQ, 18.2
MΩ⋅cm at 25 ◦ C) (Molsheim, France). LCMS/MS grade acetonitrile was
obtained from Biosolve Chemie (Dieuze, France). Formic Acid (≥98%,
CAS 64-18-6) LCMS/MS grade was acquired from Merck (Darmstad,
Germany). Nitrogen (N28), helium (P0252), and methane gas (N46)
were obtained from Air Liquide (Liège, Belgium). The derivatization
reagent o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA, ≥ 99%,
CAS 57981-02-9) was obtained from Sigma-Aldrich (St.-Louis, MO,
USA). Deuterated 2-methylbutanal (2-methylbutanal-d10, 100 atom %D,
≈200 mg in 1.5 mL of CDCl3) was synthesized upon ordering by Mer­
caChem (Nijmegen, the Netherlands. Deuterated benzaldehyde (benz­
aldehyde-d6, ≥ 98%, 98 atom %D, CAS 17901-93-8) was purchased
from Sigma-Aldrich (St. Louis, MO, USA).
2.2. Standard malt analyses
Four industrial malts, two Pilsner malts (malt A (Fermentis, Marcq-
en-Barœul, France) and malt B (Holland malt, The Netherlands)), and
two specialty malts (Munich and Biscuit (Dingemans, Stabroek,
Belgium)) were used in this study. Evaluation of malt quality parameters
comprised different analyses according to the EBC (Analysis committee
of the EBC, 2018) and ASBC (ASBC Methods of Analysis, 2004): malt
moisture (EBC 4.1), extract (EBC 4.4), extract difference (EBC 4.5), pH
(EBC 4.6), visual colour (EBC 4.7), soluble nitrogen (EBC 4.9), total
protein (ASBC malt chapter 8A), photometric colour (EBC 8.3), boiling
wort colour (EBC 8.3.2), and free amino nitrogen (FAN, EBC 8.8). The
malt standard analyses were carried-out using the Anton Paar DMA
5000 Alcolyser (Anton Paar, Austria), Anton Paar Automated Micro
Viscometer (Anton Paar, Austria), UV–VIS spectrophotometer Cary 100
UV–Vis (Agilent Technologies, Santa Clara, USA), Kjeldahl Digest Sys­
tem K-437 (Buchi, Switzerland), and Kjeldahl Distillation Unit K-350
(Buchi, Switzerland). Malt quality parameters are summarised in
Table 2.
2.3. Preparation of pilot-scale beers
Two different experimental brews were prepared in the 5 hL pilot
brewery (Meura, Belgium) of KU Leuven Technology Campus Ghent
(Ghent, Belgium): the brew of a top fermented blond beer (beer A) and a
top fermented amber beer (beer B). The applied mashing ratio was 2.2 L
brewing water/kg milled malt for both beers. For the top fermented
beer, 87 kg of Pilsner malt A (Fermentis, Marcq-en-Barœul, France), and
for the amber beer, 54 kg of Pilsner malt B1 (Holland malt, The
Netherlands), 25 kg of Munich malt2 (Dingemans, Stabroek, Belgium)
and 8 kg of Biscuit malt (Dingemans, Stabroek, Belgium), were respec­
tively mixed with 1.9 hL of reverse osmosis water. Brewing water was
enriched with 80 mg/L Ca2+ (CaCl2). The same mashing protocol was
applied for both brews: 30 min at 63 ◦ C; 15 min at 72 ◦ C; 1 min at 78 ◦ C,
with a temperature rise of 1 ◦ C/min. The pH of the mash was adjusted to
pH 5.3 with lactic acid (30% v/v). The wort was filtered using a mem­
brane assisted thin bed filter (Meura 2001, Meura, Belgium); a sparging
rate of 2.33 L/kg was applied. At the onset of boiling, the wort was
adjusted to 13 ◦ P (Beer A) and 19 ◦ P (Beer B), respectively. Boiling was
carried out for 60 min. Hopping of the beers was done with hop pellets.
Beer A was first hopped with the Magnum hop variety (α-acids 13.0%
(w/w); 50.5 g/hL, Geert Clarebout, Heuvelland, Belgium), and second
hopping in the whirlpool was done with Tettnanger (α-acids 3.0% (w/
w); 100 g/hL, Geert Clarebout, Heuvelland, Belgium) and Saaz (α-acids
2.5% (w/w); 120 g/hL, Geert Clarebout, Heuvelland, Belgium). For beer
2
Munich malt was not available for analysis due to the complete use during
the brewing process
P. Bustillo Trueba et al. Food Research International 140 (2021) 110049

Table 1
Molecular and structural formula of representative free and cysteinylated beer staling aldehydes (the latter also referred to as 2-substituted 1,3-thiazolidine-4-car­
boxylic acids).
Aldehyde Abbreviation Molecular Molecular Structure Cysteinylated aldehyde Abbreviation Molecular Molecular Structure
Formula Formula

2-Methylpropanal 2MP C4H8O 2-isopropylthiazolidine-4- 2MP-CYS C7H13NO2S

carboxylic acid

2-Methylbutanal 2MB C5H10O 2-(sec-butyl) thiazolidine-4- 2MB-CYS C8H15NO2S

carboxylic acid
3-Methylbutanal 3MB C5H10O 2-isobutylthiazolidine-4- 3MB-CYS C8H15NO2S
carboxylic acid

Methional METH C4H8OS 2-(2-(methylthio)ethyl) METH-CYS C7H13NO2S2

thiazolidine-4-carboxylic acid
Phenylacetaldehyde PHEN C8H8O 2-benzylthiazolidine-4- PHEN-CYS C10H11NO2S
carboxylic acid

Furfural FURF C5H4O2 2-(furan-2-yl) thiazolidine-4- FURF-CYS C8H9NO3S

carboxylic acid

Hexanal HEX C6H12O 2-pentylthiazolidine-4- HEX-CYS C9H17NO2S

carboxylic acid
trans-2-Nonenal T2N C9H16O (E)-2-(oct-1-en-1-yl) T2N-CYS C12H21NO2S
thiazolidine-4-carboxylic acid

B, Mosaic (α-acids 12.0% (w/w); 49.9 g/hL, harvest 2017, YCH hops,
Yakima, WA, United States) was used as first hopping, and second
hopping was done with Strisselspalt (α-acids 12.0% (w/w); 111.1 g/hL,
harvest 2017, Comptoir Commercial Agricole, Sombreffe, Belgium) at
the end of the boil and Aramis (α-acids 5.8% (w/w); 34.5 g/hL, harvest
2017, Comptoir Commercial Agricole, Sombreffe, Belgium) in the
whirlpool. Wort clarification was performed using a whirlpool. Freshly
propagated yeast and dry yeast (BE-256, Fermentis, Marcq-en-Barœul,
France) were used in a concentration of 80 g/hL for beer A and B,
respectively. Fermentation was carried out at 24 ◦ C for 7 days. Lagering
at 0 ◦ C was performed in kegs for 14 days for beer A, and 22 days for beer
B. The beers were filtered using 1 μm cellulose sheets (BECOPAD 350-
Eaton, Groot-Bijgaarden, Belgium). The beers were carbonated up to
5.6 g CO2/L and bottled in 25cL brown glass bottles using a 6 head
rotating counter pressure filler with double pre-evacuation and high
pressure injection of hot water before bottle sealing (CIMEC, Italy).
During the entire brewing process, samples were taken for the quanti­
fication of free and cysteinylated aldehydes at the following steps: onset
of mashing (sample n◦ 1), end of mashing (2), wort filtration (3), onset of
boiling (4), end of boiling (5), wort clarification (6), wort cooling (7),
onset of fermentation (after 24 h) (8), onset of maturation (9), end of
maturation (before beer filtration) (10), and freshly bottled beer (11).
2.4. Forced ageing of beer samples
Bottled beers were forced aged up to 3 months in the dark at 30 ◦ C in
a thermostatically controlled room. The following beer samples were
taken during storage at different time intervals: 1 week (sample n◦ 12), 2
weeks (13), 1 month (14), 2 months (15), and 3 months (16).
2.5. Standard beer analyses
Aiming at basic characterisation of the obtained pilot-scale beers,
alcohol, density, apparent, real and original extract, as well as apparent
and real degree of fermentation were determined with the DMA 5000
Alcolyser (Anton Paar, Austria). Moreover, the pH of the beers and the
oxygen levels were determined using a P600 pH-meter (Consort,
Turnhout, Belgium) and Haffmans Inpack TPO/CO2 Meter c-TPO (Pen­
tair, UK), respectively. Table 3 covers the beer analyses.
2.6. Quantification of free aldehydes via HS-SPME-GC-MS in malt, wort
and beer
The following aldehydes under study are considered markers for beer
ageing: 2-methylpropanal (2MP), 2-methylbutanal (2MB), 3-methylbu­
tanal (3MB), methional (METH), phenylacetaldehyde (PHEN), furfural
(FURF), hexanal (HEX), and trans-2-nonenal (T2N). Samples for free
aldehydes analysis were prepared under nitrogen gas in an anaerobic
cabinet (Whitley Anaerobic Workstation A100 model B, Don Whitley
Scientific Ltd., West Yorkshire, UK) in order to avoid oxidation. Malt
extract was prepared by weighing 1 g of finely milled malt in 100 g
N2–flushed Milli-Q water in a 100 mL Duran glass bottle, and by sub­
sequent agitation for 15 min (200 rpm) at ambient temperature. In order
to also avoid light-induced degradation, the bottles were covered with
aluminium foil. After sedimentation, an aliquot of 10 mL of supernatant
of malt extract was transferred to a 20 mL amber glass headspace vial
and sealed with a magnetic bimetal crimp cap prior to the analysis.
Considering brewery samples (wort, fresh and aged beer samples), an
aliquot of 4 mL was transferred to a 20 mL amber vial and sealed with a
magnetic bimetal crimp cap for analysis. Wort samples (from onset
mashing up to pitching wort (sample points 1–8, see section 2.3)) and
samples taken during fermentation (sample points 9–10, see section 2.3),
were 10-fold diluted with N2-flushed Milli-Q water prior to transfer.
Before transfer to the GC vials, fresh and aged beers were initially
degassed inside a nitrogen atmosphere cabinet by pouring out in a glass
recipient. Calibration curves based on N2 flushed Milli-Q water in the
case of malt and wort, and N2 flushed Milli-Q water with addition of
ethanol (in accordance with the alcohol percentage of the beer analysed)
in the case of beer, were used for quantification of all samples. Linear
calibration fittings with correlation coefficients R ≥ 0.9900 were ob­
tained for the assessment of free aldehydes concentrations. The free
aldehydes were then quantified according to Baert et al. (2015a, 2015b),
using headspace solid-phase microextraction gas chromatography-mass
spectrometry (HS-SPME-GC-MS).
2.7. Quantification of cysteinylated aldehydes via LC-MS in malt, wort
and beer
The cysteinylated aldehydes, as summarised in Table 1, were quan­
tified in malt, wort and beer samples according to our previously pub­
lished work (Bustillo Trueba et al., 2019), by applying ultra-high-

3
P. Bustillo Trueba et al. Food Research International 140 (2021) 110049

Table 2 Table 3
Malt quality parameters and concentrations of free and cysteinylated aldehydes Standard characterisation of fresh pilot-scale top fermented beers A (blond beer)
for two Pilsner malts, A and B1, and the specialty malt B2 Biscuit. and B (amber beer).
Pilsner - Pilsner - Biscuit - Beer A Beer B
malt A malt B1 malt B2
Alcohol (% v/v) 5.50 8.43
Moisture (%) 4.0 4.3 4.2 Density (g/cm3) 1.01 1.01
Extract (% D.M.) 80.9 81.0 78.6 Apparent extract (◦ P) 2.99 2.56
Extract difference 0.1 0.7 1.1 Real extract (◦ P) 5.03 5.48
(%) Original extract (◦ P) 13.33 17.86
Total protein (% D. 9.9 10.0 8.4 App. degree of fermentation (%) 77.61 85.65
M.) Real degree of fermentation (%) 64.32 71.32
Soluble N in wort 75.6 76.1 60.5 O2 (µg/L) 186 106
(mg/100 mL) pH 4.38 4.30
Kolbach index (%) 47.5 42.6 39.7
FAN (mg/L) 136.0 163.0 28.0 Beer quality parameters are expressed as mean values (n = 2).
pH 6.0 6.0 5.6
Visual colour (EBC) 7.0 4.4 47.0 performance liquid chromatography-mass spectrometry (UHPLC-MS).
Photometric colour 5.8 4.3 42.6
For the extraction of cysteinylated aldehydes from malt, 5 g of finely
(EBC)
Congress boiling 11.6 6.8 48.0 milled malt was weighed in 100 g of Milli-Q water, followed by agitation
wort colour (EBC) at 200 rpm for 5 min at ambient temperature (Grant-Bio Multifuncional
Viscosity 10% (cP) 1.5 1.6 1.5 PSU20-I Orbital Shaker, Cambridge, UK). The extraction bottles were
Mean ± Mean ± SD Mean ± SD covered with aluminium foil. Initially, centrifugation was performed at
SD
Aldehydes (µg/kg 2MP 945.8 ± 1003.8 ± 52521.9 ±
9000 rpm during 10 min at 10 ◦ C (Hettich Zentrifugen Universal 320R
DM) 131.5 182.8 3243.1 (Tuttlingen, Germany). Malt extract as well as brewery samples were
2MB 880.4 ± 940.0 ± 46181.0 ± centrifuged for a second time in an Eppendorf centrifuge 5424R (Aar­
89.7 161.4 867.0 schot, Belgium) for 5 min at 10 ◦ C and 11,000 rpm. Samples were
3MB 2143.9 ± 2432.8 ± 56174.1 ±
filtered with a Hamilton Gastight syringe #1001 of 1 mL (PTFE Luer
342.3 496.0 1576.8
METH 189.6 ± 256.7 ± 1018.0 ± Lock) and a SPARTAN HPLC Syringe filter (regenerated cellulose, 13
31.7 33.0 63.0 mm syringe filter, 0.2 μm pore size). An aliquot of 1 mL was transferred
PHEN 1011.6 ± 1139.3 ± 7795.1 ± into a 2 mL amber LC vial closed with a screw cap with PTFE septum.
82.8 150.9 341.3 Calibration curves were prepared by dissolving the authentic synthe­
FURF 316.7 ± 317.5 ± 6331.7 ±
2.1 134.1 616.5
sized cysteinylated aldehydes in an aqueous buffer solution at pH 9.0
HEX 268.3 ± 234.1 ± 3187.8 ± (Carmody, 1961) and by diluting to different concentrations ranging
70.7 4.4 32.4 from 1 to 1000 µg/L (Bustillo Trueba et al., 2019). For each cysteiny­
T2N 131.0 ± 122.5 ± 360.3 ± lated aldehyde linear calibration fittings were obtained. Accepted cor­
43.6 1.0 14.7
relation coefficients were of R ≥ 0.995.
Cysteinylated 2MP-CYS 206.5 ± 172.5 ± 148.0 ± 4.9
aldehydes (µg/kg 26.0 17.4
DM)
2MB-CYS 114.3 ± 99.9 ± 49.4 ± 4.0
2.8. Statistical analysis
10.5 17.0
3MB-CYS 552.5 ± 587.8 ± 122.9 ± Data analysis was carried out using the statistical software SPSS
96.5 140.9 36.8 (Statistical Package for Social Science, version 26, IBM Corp., Armonk,
METH-CYS 130.3 ± 85.2 ± –
NY, USA). For analysis of the statistical significance, the T-test was
7.9 14.9
PHEN-CYS 269.3 ± 251.2 ± 108.7 ± applied. Samples were considered significantly different when P-values
27.1 19.0 20.8 were lower than 0.05. All samples were analysed in triplicate, allowing
FURF-CYS 35.6 ± 2.8 12.8 ± 0.3 27.1 ± 8.1 the calculation of mean values and standard deviations.
HEX-CYS 76.6 ± 5.4 27.9 ± 3.0 17.6 ± 2.4
Sum Sum Sum
Aldehydes (µg/kg Strecker 5171.3 5772.5 163690.2 3. Results and discussion
DM) degradation
Maillard 316.7 317.5 6331.7 3.1. Free and cysteinylated aldehydes in malt samples
reaction
Lipid 399.3 356.5 3548.1
oxidation
One of the paramount challenges that the brewing industry faces, is
TOTAL 5887.3 6446.5 173570.0 to prolong the shelf life of beer. Malt, the major raw material used in
Cysteinylated Strecker 1272.9 1196.6 428.9 beer production, is known to have a positive impact on beer flavour
aldehydes (µg/kg degradation stability, for instance due to the presence of antioxidants (Boivin, 2001;
DM)
Coghe et al., 2004a, 2004b; Guido et al., 2007; Mikyška & Hrabak, 2002;
Maillard 35.6 12.8 27.1
reaction Vaughan et al., 2005; Yu et al., 2014). However, research has also shown
Lipid 76.6 27.9 17.6 a clear correlation between the rate of beer ageing and malt quality
oxidation parameters, e.g. free aldehyde levels, free amino nitrogen (FAN) and
TOTAL 1385.1 1237.3 473.6 malt filterability (De Clippeleer et al., 2010a, 2010b; Jaskula-Goiris
Malt quality parameters are expressed as mean values (n = 2). The individual et al., 2011), pointing to this raw material as a critical factor in rela­
aldehyde levels are expressed as mean values (µg/kg DM) ± standard deviation; tion to beer flavour stability. Therefore, in this paper we aimed at a
(n = 3). Classification of free aldehydes under study by their nature: I) Strecker detailed evaluation of different industrial malt samples by comparison
degradation aldehydes: 2MP (2-methylpropanal), 2MB (2-methylbutanal), 3MB of standard malt quality parameters and their content in free and cys­
(3-methylbutanal), METH (methional) and PHEN (phenylacetaldehyde); II) teinylated aldehydes (Table 2).
Maillard reaction aldehydes: FURF (furfural); and III) Lipid oxidation aldehydes:
Comparing the standard malt quality characteristics, we can pinpoint
HEX (hexanal) and T2N (trans-2-nonenal). Cysteinylated aldehydes (-CYS) are
colour and FAN as the parameters differing the most between the Pilsner
indicated in the table by using the same abbreviations for free aldehydes.
malt samples A and B1 versus the specialty malt B2 (Table 2). A well-

4
P. Bustillo Trueba et al.
known consequence of the application of higher temperatures (during
malt kilning and wort boiling) and resulting Maillard reactions is a
higher degree of malt colour and wort boiled colour, respectively (Coghe
et al., 2004a, 2004b). The used Biscuit malt B2 is characterised by a
visually determined malt colour of 47.0 EBC, a photometrically deter­
mined malt colour of 42.6 EBC, and a colour of 48.0 EBC for malt-
derived Congress boiled wort, due to exposure to a considerable de­
gree of malt roasting. Conversely, due to less heat-load during kilning
and therefore less Maillard reactions, the Pilsner malts A and B1 are only
moderately coloured with a visual malt colour of 7.0 EBC, a photometric
malt colour of 5.8 EBC and a colour of 11.6 EBC for Congress boiled wort
for malt A, versus 4.4 EBC, 4.3 EBC, and 6.8 EBC for malt B1, respec­
tively. The difference in colour between the Pilsner malts A and B1 re­
flects the somewhat higher kilning-off temperature to which malt A was
exposed to during the malting process compared to malt B1. A high FAN
value can indicate strong malt modification, i.e. proteolysis (Jaskula-
Goiris et al., 2011). However, as a consequence of high kilning and
roasting temperatures, more Maillard reactions involving free amino
acids and peptides are expected, resulting in a decreased FAN level. This
is in particular observed for malt B2, but also for Pilsner malt A
compared to malt B1, which corresponds to the applied kilning tem­
peratures and the observed colour of these malt samples. Specialty malt
B2 is characterised by a FAN level of 28.0 mg/L in contrast to the
significantly higher FAN levels of Pilsner malts A and B1, with 136.0
mg/L and 163.0 mg/L, respectively. Conversion of amino acids and
peptides, with the concomitant lower FAN values, results in Strecker
degradation aldehydes and Maillard reaction products. In this respect,
analysis of free and cysteinylated aldehydes was carried out (Table 2).
High concentrations of free aldehydes were observed in the malt sam­
ples under study (Table 2), which is in agreement with previous data
from literature (Cramer et al., 2005; De Clippeleer et al., 2010a, 2010b;
Ditrych et al., 2019; Liégeois & Collin, 2003). In particular, specialty
malt B2 showed the highest concentration of free aldehydes, probably
due to the application of higher kilning temperatures and longer heating
periods, with a total marker aldehydes level of 173,570 µg/kg malt. In
comparison, Pilsner malts A and B1 show in total 5887 and 6446 mg of
marker aldehydes per kg of malt, respectively. All quantified aldehydes
are present to a larger extent in specialty malt B2, in particular the
Strecker aldehydes 2MP, 2MB and 3MB, for which the highest levels
were noted (Table 2). These levels of 2MP and 2MB are up to 50 times
higher, and for 3MB up to 23 times higher than what is present in the
Pilsner malts. Also furfural is present at a 20 times higher level in malt
B2. When comparing the levels of free aldehydes between the Pilsner
malts A and B1, slightly higher concentrations of free aldehydes are
found in malt B1, however, no significant differences were observed (P-
value ≥ 0.05). In these Pilsner malt samples, 3MB was found to be pre­
sent in the highest concentration, followed by PHEN, 2MP, and 2MB.
Similar findings regarding the levels of the Strecker degradation alde­
hydes 3MB, PHEN, 2MP and 2MB in pilsner malt were previously ob­
tained by Jaskula-Goiris et al. (2011). For the cysteinylated aldehydes in
the Pilsner malts, the same trend was observed as for the free aldehydes,
i.e. 3MB-CYS is present in the highest concentration, followed by PHEN-
CYS, 2MP-CYS, and 2MB-CYS (Table 2). The specialty malt B2 showed
lower amounts of cysteinylated aldehydes compared to the Pilsner malts
A and B1 with the highest level for 2MP-CYS, followed by 3MB-CYS,
PHEN-CYS, and 2MB-CYS (Table 2). Clearly, the lower amounts of
cysteinylated aldehydes in malt B2 are in agreement with its lower level
of FAN.
When the levels of both free and cysteinylated aldehydes are
compared for the respective malt samples A, B1, and B2 (as presented in
the Supplementary material 1), malt B2 clearly shows the highest levels
of free but the lowest levels of corresponding cysteinylated aldehydes,
compared to Pilsner malts A and B1. This behaviour was expected
because of the high kilning temperatures applied during the malting
process for malt B2 and, therefore, the lower availability of residual
amino acids, including cysteine. Furthermore, malts A and B1 show a
5
Food Research International 140 (2021) 110049
similar profile in that the aldehydes that exhibit higher concentrations in
their free form also appear at higher concentrations in their cysteiny­
lated form.
3.2. Free and cysteinylated aldehydes during brewing and beer ageing
3.2.1. Brew A
3.2.1.1. Free and cysteinylated aldehydes: From malt to onset mashing. As
discussed in section 3.1., high concentrations of free aldehydes were
observed in the malt samples. Comparison of the concentrations of free
aldehydes quantified in malt (µg/kg DM) and the recalculated concen­
trations at the onset of mashing (based on 2.2 L/kg mashing ratio,
expressed as µg/kg DM) is shown in Fig. 1A. In comparison with malt A,
significantly lower concentrations of PHEN, HEX, T2N and 2MB were
present at the onset of mashing. On the other hand, METH and FURF
showed higher concentrations at the onset of the mash. No significant
differences were observed for 2MP and 3MB. Differences in the aldehyde
levels observed between the malt and at onset of mashing, could be
related to adsorption to insoluble material, evaporation (depending on
the nature of the compound) or even binding to cysteine or the forma­
tion of another type of bound-state aldehydes. When comparing the
cysteinylated aldehydes (or non-volatile fraction) in malt and at the
onset of mashing (Fig. 1B), the levels of most cysteinylated aldehydes
(2MP-CYS, 2MB-CYS, 3MB-CYS, PHEN-CYS, and HEX-CYS) were
significantly higher at the onset of mashing in comparison to the levels
quantified in malt A. However, METH-CYS and FURF-CYS showed lower
concentrations at the onset of mashing compared to the malt. These
findings suggest that both free and cysteinylated aldehydes from malt
are extracted into the brewing water at the onset of mashing, and that
free aldehydes, either extracted or produced de novo from e.g. amino
acids via the well-known Strecker degradation, are bound to cysteine at
mashing-in.
3.2.1.2. Free and cysteinylated aldehydes: During brewing until aged beer.
As explained in section 2.3. and 2.4., samples from the onset of mashing
until the final beer as well as during forced ageing of the beer samples,
were analysed for their free and cysteinylated aldehyde content. This
evolution is presented in Fig. 2 for brew A. All free aldehydes were
detected at all the sampling points under study (Fig. 2A). The highest
concentration levels were observed at the onset of mashing, suggesting
that malt is the major source of aldehydes introduced into the brewing
process. In particular, the Strecker degradation aldehydes 3MB, fol­
lowed by 2MP and 2MB, showed the highest concentrations at the
beginning of wort production, i.e. onset of mashing. During mashing,
some increase in 2MP is noticed in addition to a remarkable decrease in
T2N, (most probably due to binding to insoluble material). When
comparing the behaviour of free aldehydes till the end of boiling, a
general trend shows a clear decrease in all Strecker degradation alde­
hydes (2MP, 2MB, 3MB, METH and PHEN) and the lipid oxidation
aldehyde HEX. The Strecker aldehydes in the highest concentrations
(2MP, 2MB and 3MB) decreased more rapidly, when compared to other
aldehydes, which is in accordance with their relatively low boiling
points (Ditrych et al., 2019). The lipid oxidation aldehyde T2N clearly
increased during wort filtration, possibly due to lipoxygenase activity in
the spent grains. During the subsequent wort boiling step, the concen­
tration of T2N remained stable, likely due to its relatively high boiling
point (Lide, 2005). A different behaviour is observed for FURF, for
which the concentration at the onset of mashing initially remained
stable until the onset of boiling, after which levels increased as a result of
the applied heat load during wort boiling. This is not surprising since
this aldehyde is a Maillard reaction product, as described in literature
(Baert et al., 2012; De Schutter et al., 2008, 2009; Vanderhaegen et al.,
2006). The concentration of FURF also further increased during the
following wort clarification step where the heat load remains high. After
P. Bustillo Trueba et al. Food Research International 140 (2021) 110049

Fig. 1. Comparison of free and cysteinylated aldehydes in Pilsner malt A, and in onset mashing samples for brew A: 2MP (2-methylpropanal), 2MB (2-methyl­
butanal), 3MB (3-methylbutanal), METH (methional) and PHEN (phenylacetaldehyde), FURF (furfural), HEX (hexanal) and T2N (trans-2-nonenal), 2-isopropylthia­
zolidine-4-carboxylic acid (2MP-CYS), 2-(sec-butyl) thiazolidine-4-carboxylic acid (2MB-CYS), 2-isobutylthiazolidine-4-carboxylic acid (3MB-CYS), 2-(2-(methylthio)
ethyl) thiazolidine-4-carboxylic acid (METH-CYS), 2-benzylthiazolidine-4-carboxylic acid (PHEN-CYS), 2-(furan-2-yl) thiazolidine-4-carboxylic acid (FURF-CYS), and
2-pentylthiazolidine-4-carboxylic acid (HEX-CYS). Results are expressed as mean values (n = 3). Error bars represent standard deviation. (NS: no significant
differences).

24 h of fermentation, FURF disappeared almost completely, most
probably due to the reducing capacity of the yeast (Saison, 2009; Saison
et al., 2010; Vanderhaegen et al., 2004). In contrast, a clear increase for
PHEN, 3MB, and 2MB was observed after the first 24 h of fermentation,
which could be related to yeast metabolism of amino acids.
During further maturation, free aldehydes were found at marginal
levels, below their limit of detection (LOD). When beer A, a top fer­
mented blond beer with 5.5% (v/v) alcohol (Table 3), was subsequently
subjected to forced ageing, levels of all free aldehydes (Fig. 2D, 2E and
2F) proved to increase, but in particular FURF and 2MP (the most typical
marker aldehydes of beer ageing) showed a noticeable increase. The
increase in free aldehydes during beer ageing can be explained by de
novo formation and/or release from a bound-state precursor form (Baert
et al., 2018, 2015a, 2015b, 2012).
Cysteinylated aldehydes were quantified for the first time during the
entire brewing process and subsequent beer ageing (Fig. 2B and 2C). The
highest concentrations of all cysteinylated aldehydes were observed at
the beginning of wort production, i.e. the onset of mashing. In addition,
3MB-CYS followed by 2MP-CYS showed the highest concentrations of all
cysteinylated aldehydes under study. These results can be explained by
the equilibrium between the cysteinylated form of these aldehydes and
their corresponding free form (Bustillo Trueba et al. 2019, 2018a,
2018b), since also the highest concentrations of free aldehydes were
observed for 3MB and 2MP (Fig. 2A), at the onset of mashing. A decrease
in all cysteinylated aldehydes was found from the onset of mashing until
the onset or the end of wort boiling, depending on the nature of the 2-
substitution in the thiazolidine ring. In particular, the compounds
showing the highest concentrations (3MB-CYS and 2MP-CYS) decreased
more rapidly compared to those present at a lower range. As described
previously, levels in free aldehydes were relatively low from the end of
wort boiling on, except for FURF. Therefore, lower amounts of alde­
hydes are available for binding with cysteine. Moreover, due to heat,
degradation of the cysteinylated forms may occur (Bustillo Trueba et al.,
2018a, 2018b), followed by evaporation of the free aldehyde when
applying increased temperatures from mashing-in until the end of
boiling and wort clarification. Unambiguous detection of most of the
cysteinylated aldehydes was not achieved from the end of wort boiling
throughout fermentation and maturation until the final fresh beer.
However, in forced aged beer samples, an increase in 2MP-CYS was
observed (up to 10 µg/L), which is in line with the significant increase in
free 2MP found during beer ageing. Furthermore, 3MB-CYS was found in
all aged beer samples at relatively constant concentrations (0.7–0.9 µg/
L). These findings are supported by data presented by Baert et al.
(2015b) in beer pH model systems which describe the relative stability
of cysteinylated aldehydes, e.g. 3MB-CYS, due to the aliphatic nature of
the 2-substitution. Although increasing levels of FURF were found dur­
ing beer ageing, FURF-CYS appeared only in limited amounts after 2
months of forced ageing (1.5 µg/L). This observation suggests that the
FURF increase over beer storage is not related to its bound form with
cysteine, but more likely to the presence and conversion of Maillard
reaction intermediates such as 3-deoxyosones, as suggested earlier by
Baert et al. (2012). Moreover, these results are also supported by the
instability of the cysteinylated form of furfural (FURF-CYS), caused by
the aromatic character of furfural, presented in the work of Baert and co-
workers in model solutions at brewing pH values (Baert et al., 2015b).
All of the other cysteinylated aldehydes were below their limit of
detection in forced aged beer samples.
3.2.2. Brew B
3.2.2.1. Free and cysteinylated aldehydes: During brewing until aged beer.
Beer B is a top fermented amber beer of 8.5% (v/v) alcohol (Table 3).
Results on free and cysteinylated aldehydes in wort and fresh and forced
aged beers are presented in Fig. 3. Free aldehydes were quantifiable
throughout the entire process, i.e. from the onset of mashing until the
forced aged beer samples (Fig. 3A, 3D, 3E and 3F). Overall, compared to
brew A, in brew B higher levels of free aldehydes were found, which is in
accordance with the significant proportion of specialty malt B2 used in
this brew, and its high content in aldehydes. In analogy with brew A, the
highest concentrations of free aldehydes were measured at the initial
stage of wort production, i.e. the onset of mashing, and, typically, a
decrease throughout the brewing process until the end of beer matura­
tion is again observed for all free aldehydes under study. The Strecker
aldehyde 2MP was found to be present in the highest concentration,
followed by 2MB and 3MB, i.e. the same Strecker aldehydes as previ­
ously observed in brew A (Fig. 2A). In brew B, relatively high levels of
HEX were noticed during the process, which corresponds to the high
levels quantified in the applied specialty malt. Furthermore, the Mail­
lard reaction product FURF again showed a remarkable increase in
concentration when heat load was applied. Interestingly, an increase
was again observed during the first 24 h of fermentation for the alde­
hydes 3MB, 2MB, and PHEN, which is related to the amino acid

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P. Bustillo Trueba et al. Food Research International 140 (2021) 110049

Fig. 2. Evolution of free and cysteinylated staling aldehydes for brew A, from the onset of mashing until the fresh and forced aged beer (up to 3 months at 30 ◦ C): 2-
methylpropanal (2MP), 2-methylbutanal (2MB), 3-methylbutanal (3MB), methional (METH), phenylacetaldehyde (PHEN), furfural (FURF), hexanal (HEX), trans-2-
nonenal (T2N), 2-isopropylthiazolidine-4-carboxylic acid (2MP-CYS), 2-(sec-butyl) thiazolidine-4-carboxylic acid (2MB-CYS), 2-isobutylthiazolidine-4-carboxylic
acid (3MB-CYS), 2-(2-(methylthio)ethyl) thiazolidine-4-carboxylic acid (METH-CYS), 2-benzylthiazolidine-4-carboxylic acid (PHEN-CYS), 2-(furan-2-yl)
thiazolidine-4-carboxylic acid (FURF-CYS), and 2-pentylthiazolidine-4-carboxylic acid (HEX-CYS). Results are expressed as mean values (n = 3). Error bars represent
standard deviation.

metabolism of yeast. As for beer A, the lowest concentrations of free
aldehydes were observed in the final fresh beer B. However, levels of
free aldehydes were somewhat higher in the fresh beer B compared to
beer A, which may be the result of higher levels of aldehydes introduced
by the specialty malt B2. Forced beer ageing showed the expected in­
crease in all free aldehydes (Fig. 3D, 3E and 3F). Most apparent was the
increase in both FURF and 2MP as also observed for brew A. In partic­
ular, FURF showed a high increase up to 600 µg/L after 3 months of
storage at 30 ◦ C.
In accordance with brew A, the corresponding cysteinylated alde­
hydes were detected and quantified in the first steps of the brewing
process (Fig. 3B). The compounds present in the highest concentrations
at the onset of mashing were the cysteinylated forms of the Strecker
degradation aldehydes 2MP, 2MB and 3MB. All three aldehydes were
also most abundant in their free form, and, as a consequence, results are
in accordance with the equilibrium mechanism proposed by Bustillo
Trueba et al. (2019, 2018a, 2018b). Furthermore, the same trend as for
brew A was noticed, i.e. a decrease throughout the brewing process for
all cysteinylated aldehydes under study, with quantification achievable
till the onset or the end of wort boiling, depending on the nature of the
cysteinylated aldehyde. For cysteinylated aldehydes, unambiguous
detection was not accomplished for all compounds in samples taken
after wort boiling. In the fresh beer B, however, the following cys­
teinylated aldehydes were found: PHEN-CYS (which was detected but

7
P. Bustillo Trueba et al. Food Research International 140 (2021) 110049

Fig. 3. Evolution of free and cysteinylated staling aldehydes for brew B, from the onset of mashing until the fresh and forced aged beer (up to 3 months at 30 ◦ C): 2-
methylpropanal (2MP), 2-methylbutanal (2MB), 3-methylbutanal (3MB), methional (METH), phenylacetaldehyde (PHEN), furfural (FURF), hexanal (HEX), trans-2-
nonenal (T2N), 2-isopropylthiazolidine-4-carboxylic acid (2MP-CYS), 2-(sec-butyl) thiazolidine-4-carboxylic acid (2MB-CYS), 2-isobutylthiazolidine-4-carboxylic
acid (3MB-CYS), 2-(2-(methylthio)ethyl) thiazolidine-4-carboxylic acid (METH-CYS), 2-benzylthiazolidine-4-carboxylic acid (PHEN-CYS), 2 (furan-2-yl)
thiazolidine-4-carboxylic acid (FURF-CYS), and 2-pentylthiazolidine-4-carboxylic acid (HEX-CYS). Results are expressed as mean values (n = 3). Error bars represent
standard deviation.

below the limit of quantification), METH-CYS and HEX-CYS at 6.2 µg/L
and 3.7 µg/L, respectively. Furthermore, some increase in 2MP-CYS was
observed as a function of forced beer ageing (Fig. 3C). As mentioned
previously, this data is in agreement with the increase observed for free
2MP over beer ageing and, supported by the data on the stability of 2MP-
CYS as reported by Baert et al. (2015a, 2015b). In contrast, the con­
centrations of HEX-CYS, METH-CYS and PHEN-CYS did not change
during beer ageing.
4. Conclusions
For the first time, the behaviour of both free aldehydes and their
corresponding cysteinylated forms, also referred to as 2-substituted 1,3-
thiazolidine-4-carboxylic acids, was monitored throughout the entire
brewing process, from the malt until the final beer, including beer
ageing. In malt, quantification of all selected free and cysteinylated al­
dehydes was achieved. Clearly, the presence of high levels of free al­
dehydes in malt results in higher concentrations of the corresponding
cysteinylated aldehydes. Comparison between malt samples and brew­
ery samples taken at the onset of mashing showed higher concentrations

8
P. Bustillo Trueba et al.
of free aldehydes in malt samples. Conversely, cysteinylated aldehydes
appeared to be present in higher concentrations in samples taken at the
onset of mashing when compared to the malt samples, pinpointing to
additional binding of free aldehydes to cysteine as a result of mashing.
Free aldehydes could be quantified in all intermediate brewery samples
and aged beer samples. For all aldehydes, a decrease along the brewing
process was observed, resulting in fresh beer samples showing the lowest
levels in free aldehydes. Aged beer samples showed an increase in all
free aldehydes (in particular in furfural and 2-methylpropanal) for both
brews. The use of specialty malt in the brewing process is reflected in
higher concentrations of free aldehydes, both in wort samples as well as
in fresh beers. In particular, furfural was highly abundant in the roasted
malt B2, and strong increases in furfural were found in the beer derived
thereof upon ageing. Analogist to the free aldehydes, a decrease in
cysteinylated aldehydes was noticed during the brewing process. Un­
equivocal quantification of cysteinylated aldehydes in brewery samples
was only achieved until either the start or the end of wort boiling
(depending on the nature of the cysteinylated aldehyde). Throughout
beer ageing, only the cysteine adduct of 2-methylpropanal showed an
increase in levels. Even though cysteinylated aldehydes could not be
identified as the direct cause of increase in free aldehydes during beer
ageing, the results presented in this study support the concept of for­
mation of bound-state aldehydes in malting and brewing.
Funding
The authors wish to thank “Internal Funds KU Leuven” (project: C24/
15/020) for financial support.
CRediT authorship contribution statement
P. Bustillo Trueba: Conceptualization, Methodology, Investigation,
Writing - review & editing. B. Jaskula-Goiris: Supervision, Writing -
review & editing. M. Ditrych: Conceptualization, Methodology, Inves­
tigation. W. Filipowska: Conceptualization, Methodology, Investiga­
tion. J. De Brabanter: Supervision. G. De Rouck: Supervision. G.
Aerts: Supervision. L. De Cooman: Supervision, Writing - review &
editing. J. De Clippeleer: Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors wish to thank Jasper De Buyse for his support with the
brewing trials, and Agata Soszka and Johanna Schlich for their technical
support.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodres.2020.110049.
References
Analysis committee of the EBC. (2018). Analytica-EBC (4th ed.). Brauerei-und Getränke.
American Society of Brewing Chemists (2004). ASBC Methods of Analysis (14th ed.). St.
Paul, MN, U.S.A.
Baert, J. J., De Clippeleer, J., Bustillo Trueba, P., Jaskula-Goiris, B., De Rouck, G.,
Aerts, G., & De Cooman, L. (2018). Exploring aldehyde release in beer by 4-vinyl­
pyridine and the effect of cysteine addition on the beer’s pool of bound aldehydes.
Journal of the American Society of Brewing Chemists, 76(4), 257–271. https://doi.org/
10.1080/03610470.2018.1518639.
Baert, J. J., De Clippeleer, J., De Cooman, L., & Aerts, G. (2015a). Exploring the binding
behavior of beer staling aldehydes in model systems. Journal of the American Society
9
Food Research International 140 (2021) 110049
of Brewing Chemists, 73(1), 100–108. https://doi.org/10.1094/ASBCJ-2015-0109-
01.
Baert, J. J., De Clippeleer, J., Hughes, P. S., De Cooman, L., & Aerts, G. (2012). On the
origin of free and bound staling aldehydes in beer. Journal of Agricultural and Food
Chemistry, 60(46), 11449–11472. https://doi.org/10.1021/jf303670z.
Baert, J. J., De Clippeleer, J., Jaskula-Goiris, B., Van Opstaele, F., De Rouck, G., Aerts, G.,
& De Cooman, L. (2015b). Further elucidation of beer flavor instability: The
potential role of cysteine-bound aldehydes. Journal of the American Society of Brewing
Chemists, 73(3), 243–252. https://doi.org/10.1094/ASBCJ-2015-0531-01.
Bamforth, C. W. (2011). 125th Anniversary Review: The non biological instability of
beer. Journal of the Institute of Brewing, 57(3), 81–90. https://doi.org/10.1002/
j.2050-0416.2011.tb00496.x.
Boivin, P. (2001). Pro- and anti-oxidant enzymatic activity in malt. Cerevisia, 26(2),
109–115.
Bustillo Trueba, P., De Clippeleer, J., De Rouck, G., Aerts, G., & De Cooman, L. (2018a).
Study of cysteine-bound aldehydes in model solutions as a cause of flavour
instability: Influence of pH and temperature. Poster presentation session at the 13th
trends in brewing symposium.
Bustillo Trueba, P., De Clippeleer, J., Van der Eycken, E., Guevara Romero, J. S., De
Rouck, G., Aerts, G., & De Cooman, L. (2018b). Influence of pH on the stability of 2-
substituted 1,3-thiazolidine-4-carboxylic acids in model solutions. Journal of the
American Society of Brewing Chemists, 76(4), 272–280. https://doi.org/10.1080/
03610470.2018.1546094.
Bustillo Trueba, P., Jaskula-Goiris, B., De Clippeleer, J., Goiris, K., Praet, T.,
Sharma, U. K., Van der Eycken, E., Sanders, M. G., Vincken, J.-P., De Brabanter, J.,
De Rouck, G., Aerts, G., & De Cooman, L. (2019). Validation of an ultra-high-
performance liquid chromatography-mass spectrometry method for the
quantification of cysteinylated aldehydes and application to malt and beer samples.
Journal of Chromatography A, 1604, Article 460467. https://doi.org/10.1016/j.
chroma.2019.460467.
Caballero, I., Blanco, C. A., & Porras, M. (2012). Iso-α-acids, bitterness and loss of beer
quality during storage. Trends in Food Science and Technology, 26(1), 21–30. https://
doi.org/10.1016/j.tifs.2012.01.001.
Carmody, W. R. (1961). An easily prepared wide range buffer series. Journal of Chemical
Education, 38(11), 559–560.
Coghe, S., Derdelinckx, G., & Delvaux, F. R. (2004a). Effect of non-enzymatic browning
on flavour, colour and antioxidative activity of dark specialty malts - A review.
Monatsschrift Für Brauwissenschaft, 57(5–6), 25–38.
Coghe, S., Martens, E., D’Hollander, H., Dirinck, P. J., & Delvaux, F. R. (2004b). Sensory
and instrumental flavour analysis of wort brewed with dark specialty malts. Journal
of the Institute of Brewing, 110(2), 94–103. https://doi.org/10.1002/j.2050-
0416.2004.tb00188.x.
Cramer, A.-C.-J., Mattinson, D. S., Fellman, J. K., & Baik, B.-K. (2005). Analysis of
volatile compounds from various types of barley cultivars. Journal of Agricultural and
Food Chemistry, 53, 7526–7531. https://doi.org/10.1021/jf0506939.
De Clippeleer, J., De Rouck, G., De Cooman, L., & Aerts, G. (2010a). Influence of the
hopping technology on the storage-induced appearance of staling aldehydes in beer.
Journal of The Institute of Brewing, 116(4), 381–398.
De Clippeleer, J., Van Opstaele, F., Vercammen, J., Francis, G. J., De Cooman, L., &
Aerts, G. (2010b). Real-time profiling of volatile malt aldehydes using selected ion
flow tube mass spectrometry. LCGC North America, 28(5), 386–395.
De Schutter, D. P., Saison, D., Delvaux, F., Derdelinckx, G., Rock, J. M., Neven, H., &
Delvaux, F. R. (2008). Release and evaporation of volatiles during boiling of
unhopped wort. Journal of Agricultural and Food Chemistry, 56(13), 5172–5180.
https://doi.org/10.1021/jf800610x.
De Schutter, D. P., Saison, D., Delvaux, F. R. F., Derdelinckx, G., & Delvaux, F. R. F.
(2009). The chemistry of aging beer. In Beer in Health and Disease. Prevention, Issue
36, 375–388.
Ditrych, M., Filipowska, W., De Rouck, G., Jaskula-Goiris, B., Aerts, G., Andersen, M. L.,
& De Cooman, L. (2019). Investigating the evolution of free staling aldehydes
throughout the wort production process. BrewingScience, 72, 10–17. https://doi.
org/10.23763/BrSc18-21ditrych.
Ershov, A. Y., Nasledov, D. G., Lagoda, I. V., & Shamanin, V. V. (2014). Synthesis of 2-
substituted (2R,4R)-3-(3-mercapto-propionyl)thiazolidine-4-carboxylic acids.
Chemistry of Heterocyclic Compounds, 50(7), 1119–1126. https://doi.org/10.1002/
cbic.200800164.
Fratianni, A. J. (2001). Results of the effect of storage temperature on packaged beer
flavour. Master Brewers Association of the Americas Technical Quarterly, 38(3),
159–161.
Gibson, B., Aumala, V., Heiniö, R. L., Mikkelson, A., & Honkapää, K. (2018). Differential
evolution of Strecker and non-Strecker aldehydes during aging of pale and dark
beers. Journal of Cereal Science, 83(August), 130–138. https://doi.org/10.1016/j.
jcs.2018.08.009.
Guido, L. F., Curto, A. F., Boivin, P., Benismail, N., Gonçalves, C. R., & Barros, A. A.
(2007). Correlation of malt quality parameters and beer flavor stability: Multivariate
analysis. Journal of Agricultural and Food Chemistry, 55(3), 728–733. https://doi.org/
10.1021/jf0623079.
Guido, L. F. (2016). Sulfites in beer: Reviewing regulation, analysis and role. Scientia
Agricola, 73(2), 189–197. https://doi.org/10.1590/0103-9016-2015-0290.
Hashimoto, N. (1966). Studies on volatile carbonyl compounds in beer. Report of the
Research Laboratories of Kirin Brewery Co., Ltd.
Jaskula-Goiris, B., De Causmaecker, B., De Rouck, G., Aerts, G., Paternoster, A., Braet, J.,
& De Cooman, L. (2019). Influence of transport and storage conditions on beer
quality and flavour stability. Journal of the Institute of Brewing, 125(1), 60–68.
https://doi.org/10.1002/jib.535.
P. Bustillo Trueba et al.
Jaskula-Goiris, B., De Causmaecker, B., De Rouck, G., De Cooman, L., & Aerts, G. (2011).
Detailed multivariate modeling of beer staling in commercial pale lagers. Brewing
Science, 64(11–12), 119–139.
Kaneda, H., Takashio, M., Osawa, T., Kawakishi, S., Koshino, S., & Tamaki, T. (1996).
Analysis of aldehyde-bisulfites in beer by HPLC-fluorescence detection with post-
column derivatization. Journal of Food Science, 61(1), 105–108. https://doi.org/
10.1111/j.1365-2621.1996.tb14736.x.
Kilcast, D., & Subramaniam, P. (2011). Food and beverage stability and shelf life. Food
and Beverage Stability and Shelf Life. https://doi.org/10.1533/9780857092540.1.3.
Lehnhardt, F., Gastl, M., & Becker, T. (2018). Forced into aging: Analytical prediction of
the flavor-stability of lager beer. A review. Critical Reviews in Food Science and
Nutrition, 59, 1–12. https://doi.org/10.1080/10408398.2018.1462761.
Lide, D. R. (2005). CRC Handbook of Chemistry and Physics (W. M. Haynes (Ed.); 97th
ed.).
Liégeois, C., & Collin, S. (2003). Contribution of malt kilning to the cardboard flavour of
aged beers. 29th European Brewery Convention.
Liégeois, C., Meurens, N., Badot, C., & Collin, S. (2002). Release of deuterated (E)-2-
nonenal during beer aging from labeled precursors synthesized before boiling.
Journal of Agricultural and Food Chemistry, 50(26), 7634–7638. https://doi.org/
10.1021/jf020617v.
Malfliet, S., Van Opstaele, F., De Clippeleer, J., Syryn, E., Goiris, K., De Cooman, L., &
Aerts, G. (2008). Flavour instability of pale lager beers: Determination of analytical
markers in relation to densory ageing. Journal of the Institute of Brewing, 114(2),
180–192. https://doi.org/10.1002/j.2050-0416.2008.tb00324.x.
Matsui, S., & Kitabatake, K. (1984). Fluorimetric determination of cysteine in beer by
High-Performance Liquid Chromatography with precolumn derivatisation. Journal of
the Institute of Brewing, 90, 20–23.
Mikyška, A., & Hrabak, M. (2002). The role of malt and hop polyphenols in beer quality,
flavour and haze stability. Journal of the Institute of Brewing, 108(1), 78–85. https://
doi.org/10.1002/j.2050-0416.2002.tb00128.x.
Paternoster, A., Jaskula-Goiris, B., Buyse, J., Perkisas, T., Springael, J., Braet, J., De
Rouck, G., & De Cooman, L. (2020). The relationship between flavour instability,
preference and drinkability of fresh and aged beer. Journal of the Institute of Brewing,
126, 59–66. https://doi.org/10.1002/jib.582.
Rodrigues, J. A., Barros, A. S., Carvalho, B., Brandão, T., & Gil, A. M. (2011). Probing
beer aging chemistry by nuclear magnetic resonance and multivariate analysis.
Analytica Chimica Acta, 702(2), 178–187. https://doi.org/10.1016/j.
aca.2011.06.042.
10
Food Research International 140 (2021) 110049
Saison, D. (2009). Aged beer flavour: Role of carbonyl compounds and the impact of
yeast reducing activity. In PhD Dissertation.
Saison, D., De Schutter, D. P., Delvaux, F., & Delvaux, F. R. (2009). Determination of
carbonyl compounds in beer by derivatisation and headspace solid-phase
microextraction in combination with gas chromatography and mass spectrometry.
Journal of Chromatography A, 1216(26), 5061–5068. https://doi.org/10.1016/j.
chroma.2009.04.077.
Saison, D., De Schutter, D. P., Vanbeneden, N., Daenen, L., Delvaux, F., & Delvaux, F. R.
(2010). Decrease of aged beer aroma by the reducing activity of brewing yeast.
Journal of Agricultural and Food Chemistry, 58(5), 3107–3115. https://doi.org/
10.1021/jf9037387.
Solomons, T. W. G., Fryhle, C., & Snyder, S. (2013). Organic chemistry (11th ed.).
Stewart, G. G. (2016). Beer shelf life and stability. In The Stability and Shelf Life of Food
(2nd ed.). Food Science, Technology and Nutrition. https://doi.org/10.1016/b978-
0-08-100435-7.00010-1.
Stewart, G. G., Russell, I., & Anstruther, A. (2018). Handbook of brewing (3rd ed.). CRC
Press. https://doi.org/10.1038/213765a0.
Vanderhaegen, B., Neven, H., Verachtert, H., & Derdelinckx, G. (2006). The chemistry of
beer aging - A critical review. Food Chemistry, 95(3), 357–381. https://doi.org/
10.1016/j.foodchem.2005.01.006.
Vanderhaegen, B., Neven, H., Verstrepen, K. J., Delvaux, F. R., Verachtert, H., &
Derdelinckx, G. (2004). Influence of the brewing process on furfuryl ethyl ether
formation during beer aging. Journal of Agricultural and Food Chemistry, 52(22),
6755–6764. https://doi.org/10.1021/jf0490854.
Vaughan, A., O’Sullivan, T., & Sinderen, D. (2005). Enhancing the microbiological
stability of malt and beer - A review. Journal of the Institute of Brewing, 111(4),
355–371. https://doi.org/10.1002/j.2050-0416.2005.tb00221.x.
Weerawatanakorn, M., Wu, J. C., Pan, M. H., & Ho, C. T. (2015). Reactivity and stability
of selected flavor compounds. Journal of Food and Drug Analysis, 23(2), 176–190.
https://doi.org/10.1016/j.jfda.2015.02.001.
Wietstock, P. C., Kunz, T., & Methner, F. J. (2016). Relevance of oxygen for the formation
of Strecker aldehydes during beer production and storage. Journal of Agricultural and
Food Chemistry, 64(42), 8035–8044. https://doi.org/10.1021/acs.jafc.6b03502.
Yu, J., Huang, S., Dong, J., Fan, W., Huang, S., Liu, J., Chang, Z., Tian, Y., Hao, J., &
Hu, S. (2014). The influence of LOX-less barley malt on the flavour stability of wort
and beer. Journal of the Institute of Brewing, 120(2), 93–98. https://doi.org/10.1002/
jib.122.