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Beer lightstruck flavor: The full story
Article in Cerevisia · January 2008
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Beer Lightstruck Flavor: The Full Story
Denis DE KEUKELEIRE1 Arne HEYERICK1, Kevin HUVAERE1,2,
Leif H. SKIBSTED2 and Mogens L. ANDERSEN2
1
Ghent University, Faculty of Pharmaceutical Sciences,
Laboratory of Pharmacognosy and Phytochemistry, Ghent, Belgium;
2
Royal Veterinary and Agricultural University, Department of Food Science,
Food Chemistry, Frederiksberg C, Denmark
Email: Denis.DeKeukeleire@UGent.be
ABSTRACT
The pronounced sensitivity of beer to light is well known
and leads irreversibly to the formation of lightstruck flavor
(LSF), the so-called “skunking” of beer. It is the cause of a
significant shelf-life problem for the brewing industry and
is the primary reason for the storage of beer in darkcolored containers. The light-sensitivity of beers was first
recognized as early as in 1875, however it was not until
the early sixties that the basic science underlying the
formation of LSF was established. Studies using model
systems showed that LSF was produced in a non-enzymic
light-induced reaction involving riboflavin (1) (as a
sensitizer), a suitable sulfur-containing compound, and
isohumulones, the main beer-bittering principles. The
typical skunky flavor was attributed to the formation of 3methylbut-2-ene-1-thiol (MBT), a pungent off-flavor in
beer with a flavor threshold of few ng per litre. Later it
was shown that also direct UV irradiation of isohumulones
affords radical precursors of MBT. Still, the exact reaction
mechanism remained elusive until recent detailed
investigations. Herein, the mechanistic details for the
formation of LSF in beer are comprehensively reviewed.
Cerevisia, 33(3) 2008
INTRODUCTION
Beer lightstruck flavor has been known for a long time.
Lintner (1875) was the first to report on the formation of
an offending taste and obnoxious odor in beer exposed to
light, which he called “lightstruck flavor” (LSF). In the
sixties, Kuroiwa et al. (1961, 1963) suggested that the
main constituent of the off-flavor was 3-methylbut-2-ene1-thiol (MBT) derived from photodecomposition of
isohumulones, the beer bittering principles, in the presence
of a photosensitizer, namely riboflavin (vitamin B 2). MBT,
one of the most powerful flavor substances known
(threshold of ca. 4 ng per liter beer) (Irwin et al., 1993), is
generally referred to as “skunky thiol”, because the odor
resembles that of secretions of the anal glands of skunks
(Mustela vison L.). Moreover, the Kuroiwa group
established that the blue part of the visible spectrum (350500 nm), in particular, is most efficient in generating LSF.
This key finding was described in a formal mechanism,
which involved light absorption by riboflavin, energy
transfer to the isohumulones, Norrish Type I
photocleavage within the excited isohumulones, release of
a carbonyl radical (4-methylpent-3-enoyl) followed by
decarbonylation to a stabilized dimethyl allyl radical (3methylbut-2-enyl), and, finally, trapping by a thiol radical
derived from „some‟ sulfur source (Fig. 1).
It is an inevitable fact: light leads to LSF and beer must be
protected from the light. Indeed, a pale yellow beer
absorbs some blue light, while a dark beer absorbs
throughout the whole visible spectrum. Brown glass cuts
off light around 500 nm, hence, beer in a brown bottle
seems protected. On the other hand, green glass has a cutoff around 400 nm indicating that the most energetic part
of the visible spectrum, namely blue light, may penetrate
through a green glass, hence, protection against LSF is
compromised.
O
O
O
R
HO
OH
O
O
h (350-500 nm)
R
HO
Sensitizer (riboflavin)
Norrish I
O
.
OH
.
ISOHUMULONES
4-METHYLPENT-3-ENOYL RADICAL
a: R = CH2CH(CH3)2
b: R = CH(CH3)2
c: R = CH(CH3)CH2CH3
-CO
.
HS
Sulfur Source
3-METHYLBUT-2-ENE-1-THIOL
3-METHYLBUT-2-ENYL RADICAL
(MBT)
Figure 1: Formal mechanism for formation of 3-methylbut-2-ene-1-thiol (MBT) (according to Kuroiwa et al. 1961, 1963).
PHOTOINDUCED OXIDATION OF ISOHUMULONES: KEY STEP IN THE FORMATION OF THE
LIGHTSTRUCK FLAVOR
The pivotal reaction in beer brewing is the isomerization
of humulones, present in all hop varieties, to
isohumulones, present in all beers (10-100 mg L-1) (Fig.
2). Humulones constitute a mixture of 3 analogs, socalled n-humulone (R = CH2CH(CH3)2), cohumulone (R =
CH(CH3)2), and adhumulone (R = CH(CH3)CH2CH3). Due
to the presence of two chiral centres in the isohumulones,
each individual humulone gives rise to the formation of
two isohumulones, called cis and trans, respectively.
These stereochemical denotations refer to the spatial
orientation of the tertiary alcohol group and the prenyl
side chain relative to the five-membered ring. Orientation
to the same face of the ring indicates a cis-configuration,
an opposite orientation indicates a trans-configuration.
The cis-compounds are more stable than the transcompounds due to the trans-configuration of the two long
side chains, hence, the cis-compounds predominate in the
mixture of cis and trans. In normal brewing conditions, the
ratio is 7:3 in favor of the cis-compounds, while the total
yield of isohumulones is only 20-30%.
During the last decades, „advanced‟ hop products have
come to the market. Modern hop technology has allowed
isolation of pure humulones from hop extracts that are
prepared using liquid or supercritical carbon dioxide.
Humulones can be very efficiently isomerized in alkaline
conditions to isohumulones(1a-c, 2a-c) which can further
be reduced to dihydroisohumulones (3a-c), commonly
known as rhoisohumulones or „rho‟ (reduction of carbonyl
using sodium borohydride), tetrahydroisohumulones (4ac), commonly known as „tetra‟ (hydrogenation of the
double bonds in the side chains using catalytic
hydrogenation), and hexahydroisohumulones (5a-c)
(combination of both reactions) (Verzele & De
Keukeleire, 1991) (Fig. 3). In current brewing practice,
dihydroisohumulones are used for brewing lightstable
beers, while tetrahydroisohumulones are used for
increased bitterness and for stabilization of the foam head.
Hexahydroisohumulones
find
only
occasional
applications.
Isohumulones and derivatives possess the same
chromophore being an enolized beta-tricarbonyl group
with UV absorption characteristics in the UV-B region
(280-320 nm). Consequently, photoreactions may be
induced by direct absorption of UV-B light. Since the
compounds are transparent in the visible spectrum,
photoreactivity can only occur via intervention of a
photosensitizer such as riboflavin (RF) or other flavins
which are yellow-colored and, thus, absorb blue light.
Flavins are present in beer at levels of a few hundreds of
micrograms. It is clear that photodecomposition of
isohumulones by direct irradiation (exposure to UV-B
light) follows a reaction pathway different from the
reaction course on indirect (photosensitized) irradiation.
The triplet-state energy of the isohumulones (ca. 300 kJ
mol-1) is significantly higher than the triplet-state energy
of RF (ca. 210 kJ mol -1), consequently, energy transfer
from RF to isohumulones must be an energetically
unfavorable and, therefore, highly unlikely process. It
occurred to us that the key reaction step towards formation
of LSF could be photooxidation of isohumulones by
triplet-excited RF (3RF*), since 3RF* is a strong electron
acceptor, which is able to oxidize various organic
substrates (Heelis et al., 1978; Heelis, 1982; Kino et al.,
1998). Furthermore, it should be realized that structural
similarities between isohumulones and derivatives can
lead to comparable photoreactivies. In this respect,
isohumulones and tetrahydroisohumulones possess the
same alpha-hydroxyketo group (acyloin group) comprised
of the tertiary alcohol on the ring and the carbonyl in the
acyl side chain, while dihydroisohumulones have a related
alpha-diol group. Our strategy emphasized the duality in
unraveling the mechanism of LSF and, therefore, our
studies were split into 2 separate parts, investigation of
OH
LSF under direct light exposure (UV-B) on the one hand,
and under indirect (sensitized) irradiation (visible light) on
the other hand. The aim was to identify excited states and
intermediate radicals using appropriate techniques
(electrochemical procedures, (time-resolved) electron
paramagnetic resonance spectrosocopy (EPR), laser flash
photolysis spectroscopy), thereby seeking confirmation by
analysis and identification of photoreaction products (GCMS, LC-MS, ESI-MS, NMR).
O
O
R
HO
O
O
O
R
HO
T
O
HO
+
OH
O
HO
R
OH
O
20-30%
ratio 3:7
10-100 mg per liter
Humulones (in hops)
Trans-isohumulones
Cis-isohumulones
Figure 2: Isomerization of humulones to isohumulones.
O
O
O
O
R
HO
R
HO
NaBH4
OH
O
OH
HO
Isohumulones
Dihydroisohumulones
-TRICARBONYL
H2
H2
n-: R = CH2CH(CH3)2
Pd/C
Pd/C
co-: R = CH(CH3)2
ad-: R = CH(CH3)CH2CH3
O
O
O
O
R
O
R
NaBH4
HO
OH
Tetrahydroisohumulones
HO
HO
OH
Hexahydroisohumulones
Figure 3: Formation of reduced isohumulones.
VISIBLE-LIGHT-INDUCED
PHOTODECOMPOSITION OF ISOHUMULONES
AND REDUCED ISOHUMULONES
Our first seminal paper on LSF was published in 2001
(Burns et al.) and, during the forthcoming years, a series
of 7 papers (Heyerick et al., 2003, 2005; Huvaere et al.,
2003, 2004a, 2004b, 2005, 2006) detailed all our studies
on LSF that ultimately culminated in establishing the full
mechanism of the formation of LSF. In daily practice, the
visible-light-induced formation of LSF is by far the most
important process, which, in this report, will be focused
upon.
Isohumulones and reduced isohumulones (dihydroisohumulones,
tetrahydroisohumulones)
in
their
undissociated forms were subjected to electrochemical
oxidation in acetonitrile, but, surprisingly, none of the
compounds was electroactive. In contrast, we found that
the corresponding anions (resulting from proton
abstraction
from
the
enolized
beta-tricarbonyl
chromophore, which is common to all substrates) were
readily oxidized and showed prominent oxidation waves
in the cyclic voltammograms (Fig. 4; Ep of ca. 1.4 V).
This feature has profound consequences, since, indeed,
isohumulones and reduced isohumulones with pKa values
around 3 occur predominantly as salts in lager beers (pH
usually between 4.2 and 4.4). Furthermore, the resulting
radical intermediates proved to be highly reactive, as the
oxidations were irreversible. The similar heights of the
anodic waves with respect to ferrocene oxidation indicated
that isohumulones and reduced isohumulones were
oxidized in a one-electron process suggesting oxidation of
a common species. The common species refers to the
enolized beta-tricarbonyl group, which is typical of all
compounds studied. It can be concluded that (reduced)
isohumulones are prone to undergo one-electron oxidation
in the presence of suitable electron acceptors.
0,00E+00
b
-1,00E-05
I/A
d
-2,00E-05
a
c
-3,00E-05
1600
1200
800
400
E/mV vs. SHE
Figure 4: Cyclic voltammograms of ferrocene (a), trans-isohumulones (b), dihydroisohumulones (c) and tetrahydroisohumulones (d) in their respective anionic forms (0.5 mM in MeCN).
Next, incipient radicals were investigated by EPR
spectroscopy, but low concentrations prevented direct
detection. Consequently, spin traps were applied, namely
2-methyl-2-nitrosopropane (MNP) and 5,5-dimethyl-1pyrroline N-oxide (DMPO). With DMPO, both oxygenand carbon-centered radicals were identified in the spintrapped adducts. A double triplet with hyperfine coupling
constants aN ~ 13.2 G and aH ~ 8.9 G (line width ~ 1.6 G)
3440
3460
3480
B/G
3500
3520
for isohumulones and aN ~ 13.1 G and aH ~ 9.5 G (line
width ~ 2.6 G) for dihydroisohumulones showed the
parallelism. Furthermore, MNP spin trapping of radicals
from isohumulones and dihydroisohumulones suggested
addition of a triacylmethyl radical as a result of electron
release from the beta-tricarbonyl group (aN ~ 13.6 G and
aH ~ 15.3 G, respectively.
3440
3460
3480
3500
3520
B/G
Figure 5: Experimental (upper trace) and simulated (lower trace) spin patterns of radicals derived from isohumulones (left)
and dihydroisohumulones (right) after electrolysis and trapping by DMPO under nitrogen (isohumulones) and oxygen
(dihydroisohumulones).
Then, RF was introduced, but solubility problems favored
the use of flavin mononucleotide (FMN) in subsequent
experiments. Both compounds exhibit very similar
spectroscopic properties as well as photoreactivities. Laser
flash photolysis spectroscopy gave insight into the
behavior of 3FMN* in the presence of (reduced)
isohumulones. The absorption maximum of FMN at 720
nm is attributed exclusively to 3RF* or 3FMN*, since its
decay is stimulated by addition of appropriate triplet
quenchers. Salts of isohumulones and reduced
isohumulones affected the decay of 3RF* considerably.
Bimolecular rate constants for the reaction of
isohumulones with 3RF* at pH 4.6 and pH 7.0 do not
differ, as expected (Fig. 6). Indeed, the molecules prevail
largely as salts at these pH values in view of the fact that
the pKa‟s are around 3. Strikingly, the reaction rate
constants for isohumulones and reduced isohumulones
only varied between 1.7 and 2.3 x 108 dm3 mol-1 s-1
suggesting comparable behavior. It should be noted that
these constants are very high and near difusion-controlled
thereby indicating that 3RF*-induced oxidation is
extremely efficient.
Next, radicals originating in the interaction between 3RF*
and (reduced) isohumulones were detected by EPR
spectroscopy following spin trapping by MNP under
nitrogen. A similar signal (aN ~ 14.5 G ± 0.5 G) was
observed for all compounds confirming the notion that
radicals arise from a reactive moiety common to
isohumulones and reduced isohumulones thereby
confirming the findings from electrochemical oxidation
(Fig. 7). It was apparent from kinetic analysis that the
reactivities of isohumulones and tetrahydroisohumulones
were quite similar, while dihydroisohumulones appeared
slightly less reactive (Fig. 8). Moreover, transisohumulones interacted slightly stronger with 3FMN*
than cis-isohumulones.
k obs /10 -5 dm 3 mol
-1
s -1
3
2
1
0
0
250
500
750
1000
Concentration/µM
Figure 6: Linear plot of the pseudo-first-order rate constant (kobs) for decay of 3RF*, observed at 720 nm, as a function of the
concentration of the potassium salts of isohumulones following a laser flash at 440 nm (pH 4.6 () and at pH 7 (▲)).
3440
3460
3480
B/G
3500
3520
3440
3460
3480
B/G
3500
3520
3440
3460
3480
3500
3520
B/G
Figure 7: Experimental (upper trace) and simulated (lower trace) spin patterns of radicals derived from isohumulones (left),
dihydroisohumulones (middle), and tetrahydroisohumulones (right) after photooxidation by triplet-excited flavin
mononucleotide and subsequent spin trapping by DMPO under nitrogen (isohumulones and tetrahydroisohumulones) and
oxygen (dihydroisohumulones).
20
Trans-isohumulones
Isohumulones
15
k obs /10 5 s
Tetrahydroisohumulones
Cis-tetrahydroisohumulones
10
Dihydroisohumulones
5
0
0
0,25
0,5
0,75
1
1,25
1,5
Concentration/mM
Figure 8: Plot of the rate constants for decay of triplet-excited flavin mononucleotide measured at 720 nm (kobs) as a function
of varying concentrations of trans-isohumulones, isohumulones, tetrahydroisohumulones, cis-tetrahydroisohumulones, and
dihydroisohumulones.
CONCLUSION
1. Radicals formed on photochemical oxidation by
triplet-excited riboflavin or flavin mononucleotide are
similar to radicals observed on electrochemical
oxidation of isohumulones and reduced isohumulones.
2. Formation of radicals derived from isohumulones is
the initial step in the decomposition to a 3-methylbut2-enyl radical, which is a key intermediate in the
pathway leading to the lightstruck flavor in beer.
3. Tetrahydroisohumulones are equally photoreactive as
isohumulones, but, remarkably, dihydroisohumulones
are also oxidized by triplet-excited riboflavin or
flavins. This result contradicts the commonly held
belief that these compounds are light-proof.
The mechanism of sulfur incorporation revealed
It is clear that sulfur-containing beer constituents such as
proteins, polypeptides or amino acids should be suitable
sulfur sources to deliver the sulfur that eventually is
incorporated in MBT. However, the mechanism of this
pivotal final reaction step in the photoinduced formation
of LSF, namely the intervention of a sulfhydryl radical,
remained elusive until recently. We used relevant sulfurcontaining amino acids and derivatives in model systems
involving flavin-mediated photooxidation. Highly
reactive, short-lived intermediates were analyzed using
laser flash photolysis spectroscopy and spin trapping with
subsequent
EPR
spectroscopy.
Furthermore,
photoreactions were the subject of a comprehensive
product analysis by mass spectroscopy in order to provide
support for the existence of the proposed reaction
intermediates (Huvaere et al., 2006).
In order to establish the feasibility of generating sulfhydryl
radicals by photooxidation, the kinetics of the interaction
of sulfur-containing amino acids and derivatives with
3
FMN* were investigated by laser flash photolysis
spectroscopy. Increasing concentrations of the substrates
proportionally affected the decay rate of 3FMN*, as shown
for S-methylcysteine in Fig. 9. As a result, the bimolecular
rate constants for the interaction could be determined from
the slope of the linear plot of the pseudo-first-order rate
constants (observed by transient absorption spectroscopy
at 720 nm) as a function of the concentration of sulfurcontaining compounds. The values (expressed as 106 dm3
mol-1 s-1) were 328.6 (pH 4.2) and 120.8 (pH 7.0) for Smethylcysteine and 1.4 (pH 4.2) and 0.5 (pH 7.0) for
cysteine. The much higher reactivity of S-methylcysteine
with respect to that of cysteine reflects the stability of the
respective radicals being formed (Cardoso et al., 2004).
Next, EPR was applied to model systems (FMN and
substrates) that were irradiated at 440 nm in the presence
of spin traps. Fig. 10 shows the experimental and
simulated patterns of spin adducts (DMPO) of cysteine
and S-methylcysteine. For cysteine, two adducts were
detected, a major adduct (80.5%) with coupling constants
of aN (G) ~ 15.2 and a H (G) ~ 17.2 (cysteinyl addict), and a
minor adduct (19.5%) with coupling constants of aN ~ 15.0
G and aH ~ 15.7 G (sulfhydryl adduct). S-methylcysteine,
on the other hand, gave only one adduct with coupling
constants of aN ~ 15.3 G and aH ~ 18.0 G (methylthio
adduct). These results were nicely corroborated by
headspace GC-MS analysis of the model systems
(including isohumulones as well) leading to the
identification of the major volatile analytes which proved
to be MBT for cysteine and S-methyl-MBT for Smethylcysteine (Fig. 11). The characterization of this
reaction step was the missing link in the unraveling of the
mechanism of visible-light-induced formation of LSF.
Hence, the overall mechanism has now been fully
established.
Figure 9: Transient absorption of triplet-excited flavin mononucleotide sodium salt (3FMN*) in water, observed at 720 nm, in
the presence of varying concentrations of S-methylcysteine (a: 0 mM, b: 1 mM, c: 2 mM, d: 3 mM, e: 4 mM) at pH 7.0. Inset:
Linear plot of the pseudo-first-order rate constants (kobs) for the decay of 3FMN* as a function of the concentration of Smethylcysteine.
Figure 10: Experimental (upper) and simulated (lower) EPR signals of spin adducts resulting from photooxidation of sulfurcontaining amino acids by triplet-excited flavin mononucleotide at pH 7.0 in water and subsequent trapping by DMPO. A:
cysteine, B: S-methylcysteine.
Figure 11: Upper panel: Mass spectrum of MBT, formed by visible-light irradiation (2 h) of a mixture containing flavin
mononucleotide (FMN), isohumulones, and cysteine at pH 4.2 in water (Inset: Monitoring of m/z 102 in the headspace. The
peak corresponding to MBT is indicated with an arrow). Lower panel: Mass spectrum of the methylthio ether of MBT (Smethyl-MBT), formed by visible-light irradiation (2 h) of a mixture containing FMN, isohumulones, and S-methylcysteine at
pH 4.2 in water (Inset: Monitoring of m/z 116 in the headspace).
CONCLUSION
1. Amino acids such as cysteine do not directly absorb
visible light, but, clearly, triplet-excited flavins may
interact with sulfur-containing amino acids via
electron abstraction, thus furnishing the corresponding
sulfur-centered radicals.
2. S-methylcysteine gave a DMPO spin adduct (a N ~
15.3 G and aH ~ 18.0 G) that was attributed to
trapping of a methylthio radical (SMe). The values of
the coupling constants for the major DMPO spin
adduct derived from cysteine (80.5%) were very
similar to the coupling constants reported for a
cysteinyl radical (aN ~ 15.2 G and a H ~ 17.2 G),
whereas the unknown adduct was considered to have
arisen from addition of SH to DMPO.
3. Sulfur-containing amino acids and, by extension, also
sulfur-containing polypeptides and proteins are prone
to undergo photooxidation by visible light in the
presence of riboflavin or other flavin derivatives.
4. Thiol-containing substrates such as cysteine and Smethylcysteine give rise to the formation of sulfhydryl
radicals and methylthio radicals, respectively.
5. It was demonstrated that formation of 3-methylbut-2ene-1-thiol (MBT) is the result of a recombination of
a sulfhydryl radical and a 3-methylbut-2-enyl radical,
derived from photodegradation of isohumulones.
Fully
detailed
mechanistic
sequences
of
photodecomposition of isohumulones and reduced
isohumulones
Photodecomposition of isohumulones on direct irradiation
(Fig. 12)
1. Formation of the excited singlet state of the
isohumulones
2. Intersystem crossing to the excited triplet state
3. Triplet energy transfer from the enolized tricarbonyl
to the acyloin
4. Norrish Type I cleavage of the acyloin
5. Decarbonylation of the carbonyl radical
6. Trapping of a sulfhydryl radical to form MBT
Figure 12: Mechanism for light-induced formation of 3-methylbut-2-ene-1-thiol (MBT) from isohumulones on direct
irradiation in the presence of a sulfur source.
Photodecomposition of isohumulones on sensitized
irradiation proceeds according to the following steps (Fig.
13)
1. Formation of the excited triplet state of riboflavin
2. Sequential electron and proton abstraction from
isohumulones by triplet-excited riboflavin
3. Alpha-cleavage of the oxy radical
4. Decarbonylation of the carbonyl radical
5. Formation of a sulfhydryl radical from cysteine by
triplet-excited riboflavin
6. Radical recombination to MBT
Note: The carbonyl radical derived from photodecomposition of tetrahydroisohumulones (4-methylpentanoyl) does not decarbonylate readily, as this would
lead to the very unstable 3-methylbutyl radical. Rather, the
radical could be trapped directly by a sulfhydryl radical or
by other S-centered radicals to form sulfury off-flavors.
However, S-containing reaction products have not been
identified until now. The misconception of tetrahydroisohumulones being light-stable is due to the fact
that MBT cannot be formed (the double bond is lacking).
Thus, it should be kept in mind that „a LSF‟, not „the LSF‟
associated to MBT, must undoubtedly result from
exposure of tetrahydroisohumulones to light. Similarly,
despite
clear
evidence
of
flavin-mediated
photodecomposition of dihydroisohumulones (Huvaere et
al., 2004b), these compounds continue to being marketed
as light-stable alternatives for „natural‟ isohumulones.
Photodecomposition
of
dihydroisohumulones
on
sensitized irradiation (Fig. 14)
1. Formation of the excited triplet state of riboflavin
2. Sequential electron and proton abstraction from
dihydroisohumulones by triplet-excited riboflavin
3. Alpha-cleavage of the oxy radical
4. Hydrogen abstraction from the ketyl radical
5. Formation of 4-methylpent-3-enal
Figure 13: Mechanism for light-induced formation of 3-methylbut-2-ene-1-thiol (MBT) from isohumulones on sensitized
irradiation (riboflavin) in the presence of cysteine as sulfur source.
Figure 14: Mechanism for light-induced formation of 4-methylpent-3-enal from dihydroisohumulones on sensitized irradiation
(riboflavin).
Inhibition of formation of the lightstruck flavor in beer
The formation of MBT, and therefore of LSF, can be
inhibited by addition of appropriate quenchers. Detection
of MBT at ppt-levels can be effected by GC-MS after
headspace sorptive extraction. As is shown in Fig. 15,
formation of MBT on visible-light illumination (12 h) is
clearly enhanced when isohumulones or FMN are added in
excess. Addition of various quenchers (A-C) led to
varying quenching efficiences, while quencher C is most
effective on irradiation with visible light and quencher A
exhibits the best performance on irradiation with UV-Blight. These results have been confirmed using commercial
lager beers, as it can unequivocally be demonstrated that
MBT production is significantly suppressed by the
quenching activity (Fig. 16). In accordance with the
proposed reaction mechanisms, inhibition of formation of
LSF should occur conjointly with increased resistance of
isohumulones to photodecomposition. Fig. 17 nicely
shows the efficacy of quencher C resulting in decreased
photodegradation of isohumulones and, thus, in increased
flavor stability.
Figure 15: Formation of 3-methylbut-2-ene-1-thiol (MBT) in
the presence of isohumulones or flavin mononucleotide
(FMN) (left) and in the presence of various quenchers (right)
under different illumination conditions.
Figure 16: Single-ion monitoring (ion 102.0) GC-MS chromatogram of a commercial lager beer upon irradiation at 300 nm
(upper panel) and upon irradiation at 300 nm in the presence of quencher A (lower panel). Arrow at t R = 7.5 min indicates peak
associated with 3-Methylbut-2-ene-1-thiol (MBT).
Figure 17: Photosensitized degradation of trans-isohumulones in the presence of riboflavin with or without addition of
quencher C (visible light).
GENERAL CONCLUSIONS
Beers are unstable to light, because of decomposition
of isohumulones and reduced isohumulones.
Photodecomposition by visible light occurs via oneelectron
oxidation
of
the
beta-tricarbonyl
chromophore, common to isohumulones and reduced
isohumulones.
Isohumulones and reduced isohumulones furnish
radicals on the route to the formation of the lightstruck
flavor.
Photooxidation of sulfur-containing amino acids by
triplet-excited riboflavin and other flavins delivers Scentered radicals.
Radicals derived from isohumulones and reduced
isohumulones recombine with S-centered radicals to
form off-flavors.
Riboflavin and derivatives function as photogenerated
oxidants that are sacrificed, not as „true‟
photosensitizers that are regenerated.
Reduced isohumulones are photoreactive, hence their
use to brew lightproof beers is questionable.
ACKNOWLEDGEMENTS
The authors are grateful for financial support by the
InBev-Baillet Latour Foundation (Leuven, Belgium),
InBev nv (Brussels, Belgium), the Institute for the
Promotion of Innovation by Science and Technology in
Flanders (IWT-Vlaanderen, Brussels, Belgium), the Fund
for Scientific Research – Flanders (FWO-Vlaanderen,
Brussels, Belgium), the Centre for Advanced Studies
(LMC, Copenhagen, Denmark). Scientific collaboration
with Prof. Dr. P. Sandra en Dr. F. David from the
Research Institute for Chromatography (Kortrijk,
Belgium) is most appreciated.
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