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A Review of Cyclic and Oxidative Bitter Derivatives of Alpha, Iso-Alpha and

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The Science of Beer

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A Review of Cyclic and Oxidative Bitter Derivatives

of Alpha, Iso-Alpha and Beta-Hop Acids

Junguang Hao, R.A. Speers, Heliang Fan, Yang Deng & Ziru Dai

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JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS
https://doi.org/10.1080/03610470.2020.1712641

REVIEW ARTICLE

A Review of Cyclic and Oxidative Bitter Derivatives of Alpha, Iso-Alpha and Beta-
Hop Acids
Junguang Haoa , R.A. Speersb,c,d , Heliang Fana , Yang Denge, and Ziru Daia
a
Key Laboratory of Characteristic Fruits and Vegetables Fermentation, College of Food Science and Engineering, Beibu Gulf University,
Qinzhou, Guangxi, P.R. China; bCanadian Institute of Fermentation Technology, Dalhousie University, Halifax, Nova Scotia Canada;
c
International Centre of Brewing and Distilling, Heriot Watt University, Riccarton, Edinburgh, Scotland; dDepartment of Brewing Science, Qilu
University, Jinan, Shandong, P.R. China; eCollege of Food Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong,
P.R. China

ABSTRACT KEYWORDS
In the last decade, with the combined application of high efficiency preparation techniques, high a-acids; b-acids; cyclic
resolution HPLC MS/MS and sophisticated NMR technologies, novel bitter derivatives of a-, iso-a-, derivative; iso-a-acids;
b-acids have been identified in beer and model solutions, which has updated our knowledge of oxidative derivative
hop chemistry. In this review, newly identified cyclic and oxidative products of a-, iso-a-, b-acids in
hops and beer, have been comprehensively described to give brewers a more in depth under-
standing of hop and beer bitterness chemistry. A total of 39 unique substances: 15 derived from
a-acids, 15 from iso-a-acids, and 9 derived from b-acids are elucidated, including their structures,
suggested formation mechanism, distribution profiles in model solutions or beer, reactions during
fermentation, thresholds of some of these compounds, and their evolution during storage.

Introduction role in beer bitterness.[1,12–16] Nevertheless, it should be kept

Beer is a fermented alcoholic beverage with centuries of
brewing tradition, consisting at a minimum of malt, yeast
and hops (Humulus lupulus). The refreshing character, allur-
ing aroma, and desirable bitter taste profile of a fresh beer
are its main attractions to consumers. Hops in the form of
cone, pellet, extract or derivate extracts, are essential ingre-
dients and play key roles in beer, defining its unique aroma
and desirable bitterness.[1–3] Of course, hops also contribute
in multiple roles in beer foam stability and quality, micro-
bial stability and colloidal stability.[1–3] Among the factors
that hops provide, bitterness is a primary characteristic that
distinguishes beer from other beverages.
With the development of hop research over the last cen-
tury, hop chemistry, utilization and varietal development
have evolved into distinct and separate sub-fields of the dis-
cipline.[1,3–8] Of all of the bitter sources of beer, the most
important are the a-acids in the hop’s soft resin followed by
the b-acids,[1,3–8] also derived from the soft resin, and finally
the bitter compounds such as xanthohumol and desmethyl-
xanthohumol, which exist in the hard resin.[1,3,9–13]
In the beer industry, the complex taste profile of beer is
reflected by the molecular blueprint of its sensometabolites
defined by molecular sensory science. However, iso-a-acids,
namely the isomerization products of a-acid that occur dur-
ing wort boiling, are the preeminent contributors of bitter
taste of beer.[1,3] It is well known that bitter substances,
which originate from the soft and the hard resins, play a key
in mind that bitter intensity and quality can also be affected
by the levels of non-hop-resin compounds such as polyphe-
nols and hop aroma volatiles.[17–20] Nowadays, hop research-
ers focus on the effect of new varieties and new hopping
technologies (especially dry hopping) on beer aroma and
bitterness.[21–27]
The bitterness of fresh beer is refreshing, but decreases in
intensity and changes from a desirable bitterness into linger-
ing and undesirable harsh bitter flavors with time.[16,28–32]
However, aged hops are sometime used exclusively in trad-
itional recipes such as lambic beer and also in novel recipes
developed recently by some craft brewers.[13,33] For this rea-
son, a comprehensive and clear knowledge of the aging reac-
tions of hop during storage including the oxidation a- and
b-acids into the hard resin would be useful to brewers.
Until recently, the identifications of potential derivatives
of bitter substances present in trace quantities were ham-
pered due to the complexity of the beer and hop matrix in
addition to their low detection limits.[34–39] Many hop oxi-
dation products were suggested in the 1950s via studies in
model solutions, while the existence of some of them were
not confirmed in real beer samples, with the exception of
humulinones and hulupones.[34,36,37,40,41]
In the last decade, with the combined application of high
resolution HPLC MS/MS using the ECHO technique,
sophisticated NMR with B1-field-compensated preparation
technologies, new transformation products of a-, iso-a-,
b-acids have been identified in beers and/or model

CONTACT Ziru Dai 3239212883@qq.com

ß 2020 American Society of Brewing Chemists, Inc.
2 J. HAO ET AL.
solutions.[42–46] To compensate for the coextracted matrix
effect in the routine HPLC MS/MS analysis, the ECHO tech-
nique was achieved by Intelmann et al. using a nonlabelled
target compound as an internal standard, which is injected
into the HPLC-MS system shortly after the injection of the
analyte.[43,44] This methodology has opened new avenues for
a deeper understanding of hop and beer bitterness
chemistry.[42–46]
Additionally, several excellent reviews related to hop bitter-
ness have been published in the last two decades.[1,3,16,47–51]
However, unfortunately, the newly found cyclic and oxidative
products of a-, iso-a- and b-acids in hop and beer, their reac-
tion routes, as well as their evolution during brewing and
storage are not discussed in these reviews.[1,3,16,47–51] To
address this gap, this short review will attempt to elucidate
them, which will help brewers obtain some answers to intri-
guing questions such as why only trans-iso-a-acids in bottled
beer degrade exclusively to cyclic products. Nevertheless, his-
toric oxidation products such as humulinones and hulupones
will be briefly introduced as background to make the ‘picture’
of cyclic and oxidative bitter derivatives unabridged. The a-,
iso-a- and b-acids, are all composed of six congeners varying
in the carbon skeleton of the alkanoyl side chain, namely 2-
methylpropanoyl, 2-methybutanoyl, 3-methylbutanoyl, propa-
noyl, methylpentanoyl, hexanoyl, corresponding to co-, n-,
ad-, post-, pre- and adpre-congeners, respectively.[43] As the
last three of these hop congeners exist in trace amounts, they
are difficult to quantify.[43] There are no related reports on
derivative products of these congeners and hence they are not
discussed in this review.[42–46]
Transformation products from a-acids (I)
It is well known that a-acids contribute negligible bitterness
to lager beer, in contrast to their isomerized products: cis-
iso-a-acids (II) and trans-iso-a-acids (III), which are the
most important bitter contributors of beer.[1–3,8,17,52] The
documented iso-a-acids and other products that originate
from a-acids will be discussed here, especially newly
reported derivates. Besides the isomerization of a-acids,
there are two other main reactions related to the transform-
ation products of a-acids. One is the oxidation reaction of
humulinones to 40 -hydroxyallohumulinones and to its fur-
ther derivative products, the other is a cyclization reaction.
The oxidized products, compound IV to XV, listed in Figure
1 are due mainly to the research of the Taniguchi team
within last ten years.[13,45,55,56]
Humulinones (V)
Alpha and b-acids undergo a rapid oxidation during hop
storage, and the hard resin fractions are considered to be
mainly composed of the oxidation products derived from a-
and b-acids.[11,36,37,57,58] Humulinones were first identified
as oxidized a-acids in 1950, and their bitterness was found
to be 35% as bitter as iso-a-acids.[34,59] This value was
recently adjusted upwards to be 66% as bitter as iso-a-acids
by Algazzali and Shellhammer via dosing purified
humulinones into unhopped beer and evaluating with a
trained panel.[60]
Taniguchi et al. developed a high resolution HPLC
method to identify trace oxidative products of a-acids in
hops.[56] In the chromatograms produced by this method,
cohumulinone and n-humulinone were eluted as a single
peak, and adhumulinone, existing as two diastereomers, was
eluted as two equal intensity peaks.[56] The oxidation mech-
anism of a-acids leading to humulinones in hops was pro-
posed in analogy to the reaction of isolated a-acids with
peroxide reagent.[61]
Hops are susceptible to oxygen degradation during hop
storage, and humulinones have long been thought to be the
main compound in oxidized hops. Mikyska and Krofta
found that changes of a-acids during storage were tempera-
ture and air dependent.[62] In the absence of air, a-acids in
T90 pellets of four hop varieties at 2  C remained relatively
constant during storage.[62] Under that condition, the con-
centrations of a-acids exhibited a loss of 20–25% after one
year.[62] In contrast, with storage at 20  C with air access,
the concentrations of a-acids dropped substantially, and
showed an acceleration of their decrease after six months,
with a final loss of 64–88% at the end of twelve
months storage.[62]
Abeo-isohumulone with molecular weight 394 （IV）
Abeo-isohumulone, an oxidation product of a-acid with a
molecular weight 394 and a five-membered structure, was
found in forced oxidized hop by Garc
ıa-Villalba et al.[54]
Its formation was suggested to be directly from humuli-
nones via the oxidation of their 3-methyl-2-butenyl side
chain followed by cyclization via either intramolecular
dehydration or nucleophilic cleavage of the intermediate
oxirane ring.[54] Beside humulinones, this oxidized product
was reported to show up in naturally aged Saaz and
Nugget hops over a longer period of storage.[54] This oxi-
dation product belongs to the abeo-isohumulone group,
which is not derived from isohumulones but obtained dir-
ectly through the oxidation of humulone.[54] Although no
detection of this compound in beer was reported, it still
might possibly affect the bitterness quality of the beers
brewed with rich amounts of aged hops.
40 -Hydroxy-allohumulinones (VI)
Taniguchi et al. identified 40 -hydroxy-allohumulinones as
oxidative products of humulinones,[56] and proposed that
the oxidation pathway resembled iso-a-acids to hydroxy-
alloisohumulones,[56] which was illustrated by Intelmann
and Hofmann[63] and Almeida et al.[64]
It has been asserted that humulinones are the major
products of oxidized hops.[56] In a verification experiment,
hop pellets were analyzed during storage at 20, 40, 60  C in
the dark for 40, 40, and 8 weeks, respectively.[56] During
40 weeks of storage at 20  C, humulinones and 40 -hydroxy-
allohumulinones increased gradually to 117% and 114% of
the initial a-acids amount, at expense of 81.2% of the
a-acids.[56] When stored at 40  C, a rapid decrease in a-acids
 JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 3

Figure 1. Structures of a-acids (I) [40, 43] and respective transformation products in beer and hop, namely, cis-iso-a-acids (II),[43, 53] trans-iso-a-acids (III),[43, 53] abeo-
isohumulone (IV),[54] humulinones (V),[36] 40 -hydroxy-allohumulinones (VI),[45] 40 -hydroxyallo-cis-humulinones (VII),[45] cis-oxyhumulinic acids (VIII),[45] scorpiohumuli-
nols A/B (IX),[13] dicyclohumulinols A/B (X),[13] hemiacetal of tricycloperoxyhumulone A (XI),[55] tricycloperoxyisohumulone A (XII),[55] deisopropyltricycloisohumulone
(XIII),[55] tricyclooxyisohumulones A and B (XIV/XV).[55]

to less than 5% of the initial amount was found after eight
weeks.[56] Under these conditions, humulinone levels also
decreased after reaching their maximum concentration at
three weeks, while the amount of 40 -hydroxy-allohumuli-
nones increased rapidly for 2–5 weeks and slowly for
6–11 weeks, reaching their highest level at 11 weeks, followed
by slow degradation with time.[56] The compositional change
at 60  C resembled those at 40  C, although the reaction
speed was 10 times faster.[56] The results suggested that the
higher storage temperature accelerated oxidative reaction
speed, however, did not change the end product compos-
ition.[56] This speculation was confirmed by the detection of
the final generated hydrophilic oxidation products after
40 weeks at 20  C, four weeks at 40  C, and 48 h at 60  C,
which were almost identical, including unidentified
minor peaks.[56]
4 J. HAO ET AL.
Using their optimized HPLC method to analyze a metha-
nol extract of the stored hops and its soft and hard resin
fraction, it was the first time that 40 -hydroxyallohumuli-
nones were identified as principal constituents of the hard
resin, in addition to humulinones and hulupones in the soft
resin fraction.[45] The concentrations of 40 -hydroxy-allohu-
mulinones by LC MS/MS in hops are thought to be poten-
tially useful prediction indices of the degree of hop
oxidation, as these compounds are more stable than their
precursor humulinones. They might have some effect on the
bitterness quality of the beers brewed with rich amounts of
aged hops.
40 -Hydroxyallo-cis-humulinones (VII) and cis-oxyhumulinic
acids (VIII)
To further investigate the possible transformation of 40 -
hydroxyallohumulinones during the wort boiling process,
changes in the chemical composition of purified 40 -hydroxy-
allohumulinones were determined using a model boiling
experiment conducted in an aqueous buffer (pH 5.5). Based
on this model boiling experiment, 40 -hydroxy-allohumulinones
were shown to be isomerized into 40 -hydroxyallo-cis-humuli-
nones and further decomposed into cis-oxyhumulinic acids.[45]
To further mimic the conversion of 40 -hydroxyallo-humuli-
nones in the wort boiling process, purified 40 -hydroxy-allohu-
mulinones were boiled in an aqueous buffer (pH 5.5) for
120 min and the concentrations of reactants and products were
traced sequentially.[45] It was found that 40 -hydroxyallo-cis-
humulinones increased for about 60 min and then slowly
decreased thereafter, along with the amount of cis-oxyhumu-
linic acids, which increased correspondingly.[45] The total
amount of generated 40 -hydroxyallo-cis-humulinones and cis-
oxyhumulinic acids accounted for roughly 40–50% of the
decomposed 40 -hydroxy-allohumulinones.[45] These results sug-
gested that during boiling, 40 -hydroxy-allohumulinones pri-
marily isomerized into 40 -hydroxyallo-cis-humulinones, which
further decomposed into cis-oxyhumulinic acids. This finding
is helpful in understanding of the effect of the hard resin com-
position on beer bitterness quality.
Scorpiohumulinols a/B (IX) and dicyclohumulinols a/B(X)
Scorpiohumulinols A/B and dicyclohumulinols A/B were iden-
tified by Taniguchi et al. as cyclic products of 40 -hydroxy-allo-
humulinones during model solution storage.[13] To verify the
composition change of the hop hard resin during storage,
composition of the hard resin, dissolved in a pH 4.0 acidic
buffer and storage at 40  C, was traced.[13] During the process,
a decrease of 40 -hydroxy-allohumulinones and a subsequent
appearance of an unknown compound were clearly seen.[13]
To understand this reaction, further incubation experiments of
different reference compounds, namely 40 -hydroxy-allohumuli-
nones, 40 -hydroxyallo-cis-humulinones, cis-oxyhumulinic acids,
tricyclooxyisohumulones A/B, were conducted.[13] It was veri-
fied that these unknown compounds originated from 40 -
hydroxy-allohumulinones, which were identified as scorpiohu-
mulinols A/B and dicyclohumulinols A/B. Scorpiohumulinols
A/B and dicyclohumulinols A/B were proposed to be produced
via proton-catalyzed reactions of 40 hydroxy-allohumulinones,
which resemble that from hydroxyl-trans-alloisohumulones to
scorpiohumols suggested by Intelmann et al.[13,65] To form
scorpiohumulinols A/B, the reaction starts via protonation of
the carbonyl group of the hydroxyisohexenoyl side chain at
C(4), followed by cyclization onto the prenyl group at C(5),
then addition of H2O occurs to give rise to a bicyclic inter-
mediate. Upon protonation of the hydroxyl group at C(40 ) in
this intermediate, followed by the loss of H2O, the second cyc-
lization reaction gives rise to scorpiohumulinols A/B. In the
case of the formation of dicyclohumulinols, an initial proton-
ation reaction occurs at the hydroxyl group of the hydroxyiso-
hexenoyl side chain at C(4).[13] Upon elimination of this
group as H2O, the resonating hybrid intermediate would
undergo the cyclization onto the prenyl group at C(5), and
then addition of H2O to give rise to dicyclohumulinols
A/B.[13] To get an intuitive view, please refer to the reaction
cascade reported by Tanighuchi et al.[13]
Furthermore, prolonged (12 weeks) time-dependent ex-
periments of 40 -hydroxyallo-humulinones model solutions at
40  C were carried out and proved that scorpiohumulinols
A/B and dicyclohumulinols A/B were found to be more sta-
ble than their precursors.[13] The generated transformation
products accounted for approximately 70% of the decom-
position of 40 -hydroxy-allohumulinones. The finding of
time-dependent changes of 40 -hydroxy-allohumulinones into
scorpiohumulinols A/B and dicyclohumulinols A/B during
mimicked beer storage is valuable both for positively utiliz-
ing and controlling the hard resin in hops during brewing
and for evaluating the influence of beer aging on beer
brewed with hard-resin-enriched hops or extracts.[13]
Cyclization products of humulone（XI-XV）
To confirm the occurrence of the oxidized a-acids, an inves-
tigation of the autoxidation of humulone in model dissolved
humulone hexane solutions held at 20  C for 144 h was
undertaken by Taniguchi et al.[55] As a result, the hemiacetal
of tricycloperoxyhumulone A (XI), tricycloperoxyisohumu-
lone A (XII), deisopropyltricycloisohumulone (XIII) and tri-
cyclooxyisohumulones A (XIV) and B (XV) were isolated
and their structures were elucidated for the first time.[55]
An autoxidation cascade from humulone to these com-
pounds was similar to the b-acids oxidation pathway.[44,55,66]
The reaction cascade was initiated by a single electron oxi-
dation at C(4) in humulone, followed by a twice stereo-
selective cyclization and one addition of triplet oxygen,
leading to intermediate epimers tricycloperoxyhumulone A
and B.[55] Only tricycloperoxyhumulone A could isomerize
to a stable hemiacetal of tricycloperoxyhumulone, while
both tricycloperoxyhumulone A and B could rapidly isomer-
ize into their corresponding epimers of tricycloperoxyisohu-
mulone A/B via acyloin rearrangement.[55] The epimers
tricycloperoxyisohumulone A/B would then transform into
corresponding alkoxy radical, which could be saturated via
either hydrogen abstraction to lead to alcohols tricyclooxyi-
sohumulones A and B, or the loss of one molecule of acet-
one to give rise to deisopropyltricycloisohumulone.[55]
 JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 5

Figure 2. Structures of iso-a-acids (II) (III) [43, 53] and respective transformation products in beer and hop, namely, hydroperoxy-cis-alloisohumulones (XVI),[63]
hydroxyl-cis-alloisohumulone (XVII)),[63] cis-alloisohumulones (XVIII),[53] trans-humulinic acids (XIX),[66] cis-humulinic acids (XX),[67] trans-alloisohumulones (XXI),[53]
hydroperoxy-trans-alloisohumulones (XXII),[63] hydroxyl-trans-alloisohumulone (XXIII),[63] scorpiohumols (XXIV),[65] tricyclohumulactols (XXV),[65] tricyclohumols
(XXVI),[32, 42] tricylohumenes (XXVII),[32, 42] isotricyclohumenes (XXVIII),[32, 42] tetracyclohumol (XXIX),[32, 42] epiteracyclohumol (XXX).[32, 42]
6 J. HAO ET AL.
The occurrence of these autoxidation products in hops
stored at 40  C up to forty weeks was verified with the
exception of hemiacetal of tricycloperoxyhumulone.[55]
Humulone concentration rapidly decreased during the first
five weeks to less than 10.7% of the initial amount.[55] The
concentration of tricycloperoxyisohumulone A reached its
maximum after four weeks and then decreased rapidly.[55]
In contrast, the amounts of tricyclooxyisohumulones A/B
and deisopropyltricycloisohumulone increased rapidly dur-
ing the first five weeks and were then stable until week 40.
The total amount of oxidation products generated after five
weeks accounted for 9.1% of the amount of humulone that
decomposed.[55]
Using their optimized HPLC method, Taniguchi et al.
analyzed a methanol extract of the stored hops, and tricy-
cloisohumulones A and B were proven to be the hard resin
components.[45] In addition to humulinones, 40 -hydroxyallo-
humulinones, tricyclooxyisohumulones A and B, deisopro-
pyltricycloisohumulone, and tricycloperoxyisohumulone A
were all suggested to serve as aging indicators of hop
oxidation.[55,56]
Transformation products from iso-a-acids (II, III)
During the brewing process, a-acids are extracted and iso-
merized into iso-a-acids via an acyloin-type ring contrac-
tion, a key step in the bittering reaction.[67] Besides being
the principal bitter components in beer, iso-a-acids contrib-
ute to foam stability and gram-positive bacteria inhib-
ition.[68,69] Iso-a-acids are made up of two diastereomeric
isomers, namely trans- and cis-iso-a-acids, depending on the
spatial orientation of the tertiary alcohol function at C(4)
and the prenyl side chain at C(5). Trans-isomers are in the
S-configuration, while cis-isomers are in the R-configur-
ation.[67] Iso-a-acids contribute the primary bitterness to
beer, while multiple studies have revealed that different cis/
trans congeners of iso-a-acid showed varying effects, includ-
ing differing sensory time intensity behavior of the iso-
a-acids.[1,70–72] It was proposed by Rigby that the high pro-
portion of the isocohumulone in beer triggers a rough and
strong bitter intensity,[73] while Wackerbauer and Balzer
held the opposite position.[70] Nevertheless, in the current
brewing industry, some hop breeders still tend to breed low
cohumulone hop varieties for brewers who insist that con-
trolling the proportion of the different humulones in the
beer can improve bitter taste quality.[74]
During the aging of beer, a significant decrease in the
intensity of bitterness and a deterioration of a pleasant bitter
taste (e.g., toward a long lasting, lingering, harsh bitterness)
was reported to be common for bottled beers, even when
held under quasi-oxygen free conditions.[75,76] This has been
regarded as a shelf life limiting factor of beer by the indus-
try, in particular for lager beer producers.[75,76]
Multiple studies have revealed that trans-isomers decrease
substantially in the beer aging process, whereas the concen-
trations of cis-counterparts seem to be relatively con-
stant.[6,8,30,76–78] The Trans to Cis (T/C) ratio was verified
by Intelmann et al. to be around 0.4 for the fresh beer
samples and decreased to a mean value of 0.16 after storage
of eight months at 28  C.[43] As the ratio of the individual
congeners did not vary, it was suggested that the degrad-
ation of the trans-iso-a-acids is independent of the side
chain structure.[43,76] Total iso-a-acids decreased by about
27%, whereas the loss of the trans-isomers accounted for
66% of the degradation of iso-a-acids. As the T/C ratios
between the isomers did not differ substantially, it was
implied that a similar degradation mechanism might be
applicable to all of the beers under investigation.[43] The
molecular basis of the trans-specific degradation and minor
decrease of cis-a-acid during storage had remained unknown
until discovered by Intelmann et al.[32,42–44,63,65]
Figure 2 depicts the recent discovered cyclized and oxi-
dized products of iso-a-acids as well as other transformation
products, namely component XVI to XXX, which can be
detected by modern analytical tools in model solutions as
well as in hop and/or beer samples.[32,42,53,63,67]
Alloisohumulones（XVIII, XXI）and humulinic acids
(XIX, XX)
Allo-iso-n-humulones form via the isomerization of the dou-
ble bond of the iso-3-hexenoyl side chain of iso-n-humulone
and the consecutive hydration of the enone. The conforma-
tions of cis-alloisohumulone (XVIII）and trans-alloisohumu-
lone (XXI） correspond to their precursors.[51,53,65] Cis-
alloisohumulones were found in trace levels in pilsner-type
beers of different ages, while trans-alloisoadhumulones could
not be unequivocally detected.[65]
As non-bitter compounds, with a pKa value 2.7, trans-
humulinic acid (XIX) and cis-humulinic acid (XX) form by
proton catalyzing reaction independent of the iso-a-acid or
alkali catalyzed formation.[65,67,79] Cis/trans-humulinic acids
were detected in minor amounts in wort and were relatively
stable in the brewing process of pilsner-type beer, although
the concentrations of trans-humulinics acids were less than
those of cis-counterparts.[80] The concentration of humulinic
acids was lower in glass bottled beer, compared to oxygen
permeable PET packaged beer.[65] During a prolonged stor-
age of glass bottled beer (of up to 582 days), the trans- and
cis-humulinic acids showed a strong increase, while trans-
iso-a-acids were almost fully depleted.[65] This observation
agrees with the fact that humulinic acids could form from
both trans- and cis-iso-a-acids.[65] The concentrations of dif-
ferent congers of cis-humulinic acids and trans-humulinic
acids in pilsner-type beer before (A) and after (B) storage
for 8 months at 28  C, pilsner-type beer stored for 4 years at
20  C in a glass bottle (C), pilsner-type beer stored for
10 years at room temperature in a can (D), pilsner-type beer
stored for 4 years at room temperature in PET-bottle (E)
were all relatively high. For example, the concentration of
cis-cohumulinic acid in beer A-E was 0.25, 0.54, 1.14, 2.36,
5.15 lmol/L, while that of the trans-counterpart was 0.10,
0.14, 0.24, 0.51, 0.60 lmol/L, respectively. It was suggested
that humulinic acids markedly affect the bitterness evolution
of beers packaged in either low-oxygen or oxygen-permeable
bottles during storage.

Hydroperoxy-alloisohumulones (XVI, XXII) and hydroxyl-
alloisohumulones （XVII, XXIII）
Studies of autoxidation products of iso-a-acids were performed
by Intelmann et al.,[63,65] with suitable model solutions and
modern analysis techniques such as LC MS/MS and NMR.
The slight decline of cis-iso-a-acids concentrations in aging
beer was clarified[62,63,76,77] and minor unknown peaks were
encountered in purified cis-iso-a-acids model solutions.[42] As
a result, hydroperoxy-cis-alloiso-a-acids (XVI) and hydroper-
oxy-trans-alloiso-a-acids (XXII) were found derived from
respective cis- and trans-iso-a-acids, which further turned into
hydroxyl-cis-alloiso-a-acids (XVII) and hydroxyl-trans-alloiso-a-
acids (XXIII).[63,65] Hydroperoxy-cis-alloiso-a-acids and hydroxyl-
cis-alloiso-a-acids are in the R-configuration, while their trans-
counterparts are in the S-configuration.[63]
A reaction cascade capable of forming hydroperoxides
was suggested by Intelmann et al. similar to the lipid autoxi-
dation of unsaturated fatty acids.[63] Initially, a hydrogen
atom is abstracted by a starter radical leading to a resonance
stabilized radical in the isohexenoyl side chain of the iso-
a-acids. After addition of triplet oxygen, a peroxy radical is
formed, which can abstract a hydrogen atom from another
donor molecule to induce of another reaction cascade leading
to the formation of hydroperoxy-cis-alloisohumulone and
hydroperoxy-trans-alloisohumulone, respectively.[63,65] Via
cleavage of the hydroperoxy function by means of transition
metal ions such as iron ion or by light, an intermediate
alkoxyl radical is generated, which either gives rise to the gen-
eration of hydroxyl-cis-alloisohumulones and hydroxyl-trans-
alloisohumulones by abstraction of a hydrogen atom from
another donor molecule,[63,65] or cyclizes into an epoxy
intermediate.[65]
After treatment of the beer in the presence of oxygen for
only two hours at room temperature, substantially higher
amounts of hydroperoxides were found, thus demonstrating
that both the cis- and trans-iso-a-acids do undergo oxida-
tion in the presence of oxygen.[63] Meanwhile, chromato-
grams obtained after incubating the beer spiked by cis- and
trans-isohumulone, respectively, for two weeks under an
atmosphere of nitrogen at 37  C clearly indicated that the
addition of cis-isohumulone favored the oxidative generation
of hydroperoxy-cis-alloisohumulones, whereas the addition
of trans-isohumulone accelerated the oxidative production
of hydroperoxy-trans-alloiso humulones along with the acid
catalyzed formation of tricyclohumol.[63]
In freshly prepared wort and resultant beers, hydroper-
oxy-alloisohumulones and hydroxyl-alloisohumulones were
clearly detected, which implied these kinds of oxidations
occurred during wort boiling.[63,65,80] Among them, cis-alloi-
sohumulone hydroxide, trans-alloisohumulone hydroxide
and cis-alloisohumulone hydroperoxide were found in rela-
tively high concentrations of 0.164, 0.090, 0.088 mg/L.[80]
Meanwhile, the concentrations of oxidation products
decreased to less than 50% during the brewing process in
the final beer.[80]
The total amount of hydroperoxy-alloisohumulones in
fresh bottled beer decreased from 0.52 lmol/L after storage
to either eight months at 28  C, four years at 20  C, or for
JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 7
ten years at 20  C to 0.51, 0.52, 0.32 lmol/L, respectively.[63]
During these prolonged storage periods, absolute concentra-
tions of hydroperoxides were decreased, while the ratios to
their iso-acids ‘parents’ clearly increased. Furthermore,
hydroperoxy-trans-alloisocohumulone accumulated during
storage, while the cis-isomer decreased drastically.[63] In the
beer stored for four years in the gas permeable PET bottle,
the sum of hydroperoxy-alloisohumulones concentrations
dropped to near zero.[63] Nevertheless, when incubated for
24 h at 20  C accessible to air, the hydroperoxides were
detected at twice the elevated amount in beer, compared to
fresh beer.[63] This implied that the oxidation speed of iso-
a-acids to the hydroperoxides was rapid initially under aer-
obic conditions, while hydroperoxides tended to degrade in
prolonged storage under an air atmosphere.[63]
The sum of hydroxyl-alloisohumulones in fresh beer was
0.39 lmol/L and after storage for eight months at 28  C,
four years at 20  C, and for 10 years at 20  C was 1.29, 1.33,
2.11 lmol/L, respectively.[63] Similar to their hydroperoxide
precursors, hydroxyl-trans-alloisocohumulone accumulated
during storage, while the cis-isomer decreased drastically.[63]
Conversely, in the beer stored for four years in the gas per-
meable PET bottle, the sum of hydroxyl-alloisohumulones
concentration dropped to 0.08 lmol/L.[63] Nevertheless,
when incubated for 24 h at 20  C accessible to air, the
hydroxyl-alloisohumulones in beer were detected in an
11-fold elevated amount, compared to the fresh control.[63]
This implied that the transformation of hydroperoxy-
alloisohumulones to hydroxyl-alloisohumulones was also
very rapid initially (aerobic condition stage) and tended to
degrade with prolonged storage under air.[63] As the primary
autoxidation products of both cis- and trans-iso-a-acids,
these are helpful for brewers to understand the detailed pro-
cess and its influential factors of iso-a-acids degradation in
beers, in particular, beer in oxygen-permeable PET bottles.
Because of their pronounced instability, these compounds
cannot be recommended as suitable indicator molecules of
the oxidation degree of beer.
Scorpiohumols (XXIV) and tricyclohumulactols (XXV)
Subsequently, tricyclohumulactols and scorpiohumols were iden-
tified as oxidative products of hydroperoxy-alloiso-a-acids by
Intelman et al.[65] As mentioned above, an intermediate alkoxyl
radical generated from hydroperoxy-alloiso-a-acids could
either lead to hydroxyl-cis-alloisohumulones and hydroxyl-
trans-alloisohumulones or form an epoxy intermediate.[65]
This epoxy intermediate then undergoes rearrangement, fol-
lowed by an additional peroxidation, to convert into a hemi-
ketal. Then, one molecule of acetone from this hemiketal is
released, followed sequentially by enolization, a proton-cata-
lyzed cyclization, a molecule of H2O addition as well as intra-
molecular ring closure, and finally gives rise to a
tricyclohumulactol.[65]
For the formation of scorpiohumol, a hydroxyl-trans-
alloisohumulone undergoes a proton-catalyzed carbonylene
reaction, followed by addition of one molecule of H2O to
the tertiary alcohol intermediate.[65] Proton-catalyzed elimin-
ation of one molecule of H2O from the allylic alcohol,
8 J. HAO ET AL.
followed by the nucleophilic attack of the tertiary hydroxyl
group, gives rise to scorpiohumols.[65]
In contrast to their precursors, tricyclohumulactols and
scorpiohumols were only clearly detected in a model solu-
tion, and not in either fresh or aged beers due to their low
concentration and/or coelution with isobaric molecules.[65]
The lack of detection in the stored beer sample emphasized
the complexity of the matrix effect of beer on the formation
of specific bitter sensometabolites.
Trans-specific cyclization reaction products (XXVI-XXX)
Five cyclization products, i.e., tricyclohumols (XXVI), tricy-
clohumenes (XXVII), isotricyclohumenes (XXVIII), tetracy-
clohumols (XXIX), epitetracyclohumols (XXX) were identified
as previously unknown trans-specific degradation products
via HPLC MS/MS and NMR.[32,42] In these identifications,
suitable model solutions were examined. That is, spiked pure
trans- and cis-a-acids in an aqueous solution (pH 4.0) were
incubated for 6 h at 60  C in closed glass vessels, instead of
the previous rather artificial conditions such as high alkaline
pH or with the participation of hydrogen peroxide.[32,42]
Furthermore, 18O stable isotope labeling along with
model quantitative studies were adopted to explore the
transformation routes from trans-iso-a-acids to their specific
degradation products, and a proton-catalyzed intramolecular
nucleophilic cyclization of trans conversion was revealed by
Intelmann et al.[32] An intermediary carbocation is formed
via protonation of the carbonyl group of the isohexenoyl
side chain, which converts into another carbocation via cyc-
lization triggered by an intramolecular attack of the p-elec-
trons of the methane carbon of the prenyl chain.[32] The
second carbonation might undergo a second intramolecular
attack of the olefinic carbon of the isoprenyl side chain to
form the third carbonation, which can further turn into dif-
ferent stable end products via three alternative routes.
Nucleophilic addition of a molecule of H2O gives rise to
tricyclic alcohols such as tricyclohumols, whereas elimin-
ation of a proton leads to the formation of tricyclic olefince,
e.g., tricyclohumenes, isotricyclohumenes.[32] Alternatively,
the second carbocation might add one molecule of H2O to
give the intermediary alcohol, followed by protonation at
the carbonyl function of the alkanoyl side chain. The follow-
ing intramolecular cyclization between the double isoprenyl
side chain and vinylogous carbonyl moiety in the tricarbonyl
system gives rise to either tetracyclic degradation products
such as tetracyclohumulone or epitetracyclic compounds
such as epitetracyclohumulone, depending on the initial
configuration of the second intermediary carbocation.[32]
After fourteen days at 37  C, the addition of cis-isohumu-
lone to beer did not lead to any increase of the aforemen-
tioned cyclic products. In contrast, spiking the beer with
trans-isohumulone induced a significant increase of those
cyclic products.[32] To verify whether their formation was
oxygen and temperature dependent, their concentrations
were traced in canned beers with very low oxygen content
(less than 0.1 mg/L) and maintained at either 6, 27 or 37  C
for up to eighteen weeks.[32] Storing beer at 6  C marginally
influenced cyclic product formations, while maintaining beer
at 37  C induced a strong degradation of the parent trans-
iso-a-acids and 1–5 lmol/L target degradation products
formed after eighteen weeks.[32] Among the cyclic product
formations, tricyclocohumol was found to be the most pre-
dominant and with epitetracyclocohumol as the minor reac-
tion product. The total concentration of these products
accounted for about 91% of the initial trans-iso-a-acids deg-
radation.[32] Without doubt, this study clearly demonstrated
that oxidative and/or photooxidative reactions do not play
any major role in the transformation of trans-iso-a-acids
specific degradation compounds.[32] In further studies, aque-
ous solutions of trans-isocohumulene adjusted to pH 1, 3, 4
and 6 respectively, were incubated at 60  C for up to 50 h
under an atmosphere of nitrogen to find out whether these
reactions were pH dependent.[32] The most rapid degrad-
ation of trans-isocohumulone was found at pH 1 (i.e., less
than 5% of this reactant remained after 4 h).[32] Increasing
the pH value to 3.0 induced a substantial decrease of the
reaction speed.[32] None of these compounds were formed at
pH 6 (32). Thus, it was concluded that the degradation of
trans-isocohumulene was strongly pH dependent, which was
in line with the accelerated degradation of iso-a-acids with
decreasing pH.[32,81]
To quantitatively monitor the evolution of tricyclic trans-
iso-a-acid degradation products, samples from the wort, six
intermediate beer sample (beers I-VI) taken at different stages
of brewing process and the final beer (sample VII) were col-
lected from an industrial, full-scale pilsner-type beer manufac-
turing process by Haseleu et al.[80] The results showed that
tricyclic trans-iso-a-acid degradation products were not detect-
able in the wort, but were rapidly formed in increasing con-
centrations in the fermentation process.[80] For example, the
amount of tricyclocohumol in beers I-VlI was 0.624, 0.828,
0.852, 1.019, 0.959, 1.017, 0.972 mg/L, respectively.[80] It was
suggested that decreasing the pH accelerates the formation of
a degradation products of trans-iso-a-acids, however the con-
centrations of the trans-iso-a-acids did not show a clearly
decreasing trend during the brewing process.[80]
To clarify the evolution of cyclic trans-iso-a-acids degrad-
ation products during storage, their concentrations in
brown-glass bottled fresh beer, samples stored for eight
months at 28  C, four years at 20  C, and ten years at 20  C,
respectively as well as beer sample stored for four years at
0  C in a PET bottle were compared.[63,65] Noticeable
amounts of trans-specific iso-a-acid degradation products
were detected in fresh beer, e.g., the sum amount of cyclic
degradation products was 5.75 lmol/L. Tricyclohumol and
tricyclocohumol were proven to be the most abundant
molecular species with concentrations of 1.6 and 1.0 lmol/
L.[65] After storage for eight months at 28  C, the sum of
cyclic products increased rapidly to 25.29 lmol/L, about 4-
fold of that in fresh beer.[65] Moreover, tricyclohumol was
found to be most abundant among cyclic degradation prod-
ucts with concentration of 7.6, 9.2, 7.4 lmol/L, in glass bot-
tled beer stored for eight months at 28  C, four years at
20  C and ten years at 20  C, respectively.[65] Furthermore,
the stability of tricyclohumol to oxidation was demonstrated
by the fact that its concentration in the oxygen-permeable

PET bottle was at same level (i.e., 8.3 lmol/L) as in glass
bottled beers.[63,65] Interestingly, the total amounts of cohu-
mulone, trans-isocohumulone, cis-isocohumulone, cyclic deg-
radation of trans-isocohumulone, trans- and cis-cohumulinic
remained rather constant during the 582 days of the pilsner
beer storage at 20  C.[65]
To further clarify the influence of different pH values on
beer, beers with pH values of 4.2, 4.3 and 4.4 stored for up to
360 days at 20  C were compared.[65] Although the pH differ-
ence between beer samples was only 0.1, the tricyclocohumol
concentrations showed a distinct difference.[65] In addition, the
difference of tricyclocohumol concentration of beer between
pH 4.2 and pH 4.4, increased to 0.8 lmol/L after 360 days.[65]
Therefore, it confirmed again that a more acidic pH value trig-
gered the degradation of trans-iso-a-acids more easily during
beer storage.[65] The time, pH, and temperature dependent
cyclic degradation of trans-iso-a-acids was clearly demon-
strated to be the predominant reaction leading to lingering,
harsh bitter tasting tricyclic and tetracyclic compounds during
beer aging.[65] Trans-specific iso-a-acids degradation products
were the most abundant aging transformation products in bot-
tled beer, along with minor occurrences of oxidized products
such as humulinic acids.[63,65] It is noteworthy that no degrad-
ation products of the isohumulones in substantial amounts
were detected in hop during long term storage, which implies
that the molecular transformation of bitter components
reported for beer might not be applicable to hops.[82] The
thresholds of cyclic degradation products of trans-isocohumu-
lone, determined in water (pH 4.5) using a triangle test by
Intelmann et al., are listed in Table 1.[42,83] Depending on their
chemical structure, the bitter thresholds varied from 5 to
70 lmol/L.[42,83] The thresholds of tricyclocohumene and iso-
tricyclocohumene were 5 and 10 lmol/L, respectively, which
was somewhat lower than that of trans-isocohumulone.[42,83]
In contrast, the thresholds of tricyclocohumol, tetracyclocohu-
mol, and epitetracyclocohumol were 1.5, 3.5, 3.5 fold to that of
trans-isocohumulone, respectively.[42,83]
These studies on trans-specific cyclization reaction prod-
ucts offer the scientific basis for a knowledge-based exten-
sion of the shelf life of desirable beer’s bitter taste and delay
of the onset of the less preferred harsh bitter aftertaste by
controlling the initial pH value of the beer via technological
means and by keeping the temperature as low as possible
during storage of the final product.[65,80]
Transformation products from b-acids (XXXI)
Along with a-acid, b-acids are also important compounds of
the soft hop resin. The b-acids occur as six congeners,
namely co-, n-, ad-, post-, pre- and adpre-congeners, corre-
sponding to their different acyl side chains, sharing similar
nomenclature with a-acids.[43] In the brewing industry, their
low solubility in aqueous solutions and the ability to oxidize
into more soluble products are defining characteristics
of b-acids.
For some time, the main oxidation products of b-acids
were regarded to be hulupones, occurring in cohulupone,
hulupone and adhulupone, which were proposed to degrade
JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 9
Table 1. Bitter recognition threshold concentrations of trans-iso-cohumulone
and its cyclic degradation products.[42,83]
Compound Threshold concentration (lmol/L)
trans-isocolumulone 20
tricyclocohumol 30
tricyclocohumene 5
isotricyclocohumene 10
tetracyclocohumol 70
eptitetracyclocohumol 70
into hulupinic acids.[40,41,58] Hulupones have been found to
contribute to beer bitterness, with an obvious distribution in
aged hops, as well as in beer at levels of 2–10 mg/L.[40,41,58]
Some other oxidative degradation products of b-acids, such
as lupoxes, lupdoxes, lupdeps, lupodols, as well as tricycle-
oxycolupulones, were also reported very early, while their
bitterness characteristics remained unclear and their exist-
ence has not been further determined in real beer sam-
ples.[84–87] Mikyska and Krofta found that the changes of
b-acids during storage were temperature and air depend-
ent.[62] Without air access, b-acids in T90 pellets of four
hop varieties storage at 2  C remained constant after one
year at 20  C.[62] In contrast, when stored at 20  C with air
access, the concentrations of b-acids dropped significantly,
with a final loss of 51–83% at the end of twelve months
of storage.[62]
Figure 3 depicts the oxidation and cyclization products,
component XXXII to XXXIX, verified by LC MS/MS within
the last decade. Briefly, there are three main reaction possi-
bilities related to transformation of b-acids. One is hulupone
transformation into hulupinic acids. The second one is a
cyclization reaction found by Haseleu et al.[44,88] While the
third is the formation of epoxyhulupone from hulupone
found by Dusek et al.[46]
Hulupones (XXXII) and hulupinic acid （XXXIII）
Recently, hulupones, together with humulinones, were pro-
ven to be abundant oxidized hop acids in aged
hops[46,54,90,91] and could be rich in beer in substantial
quantities.[44,65,88] Beers hopped by pre-oxidized b-acids
have a strong sensory bitterness.[60,92] The bitterness inten-
sity of hulupones were reported to be 50%,[58] 200%[89] and
84%[60] as bitterness as iso-a-acids by different researchers,
respectively. Algazzali and Shellhammer were confident with
their established bitter intensity value, as they had adopted
refined sensory methodology and modern purifying techni-
ques.[60] In contrast to its precursor, hulupinic acid was
regarded to contribute marginal bitterness to beer.[41,93]
Duseket al. analyzed colupulone, lupulone, cohulupone,
hulupone, and hulupinic acid in two stored Sladek hop pel-
lets, with storage periods of 24 and 60 months, and also in
beer brewed with these pellets.[46] It was found that the
amount of starting materials (lupulone and colupulone) in
hops stored for 24 months, was higher than in hops stored
for 60 months. Meanwhile, lower levels of cohulupone, hulu-
pone, and hulupinic acid were found in hops stored for
24 months and in the corresponding beers in comparison to
those in hops stored for 60 months and their resultant
beers.[46]
10 J. HAO ET AL.

Figure 3. Structures of b-acids (XXXI)[43, 58] and respective transformation products in beer and hop, namely, hulupones (XXXII),[44, 80, 88] hulupinic acid (XXXIII),[89]
tricyclolupones epimers (XXXIV),[44, 88] dehydrotricyclolupones epimers (XXXV),[44, 88] hydroperoxytricyclolupones epimers (XXXVI),[44, 88] hydroxytricyclolupones epi-
mers (XXXVII),[44, 88] nortricyclolupones (XXXVIII),[44, 88] epoxyhulupones (XXXIX).[46]

Taniguchi et al. also traced the changes of hulupones in
hop pellets stored at different temperatures and found that
the formation of hulupones in hops was temperature and
time dependent.[56] At 20  C, hulupones increased to 17% of
the initial amount of b-acids after forty weeks storage. In
the case of 40  C, hulupones reached their maximum of 30%
of the initial amount of b-acids after four weeks storage,
and existed stably until week 40. At 60  C, hulupones
reached their maximum at 72 h and were stable for eight
weeks.[56] The initial concentrations of these hulupones in
wort decreased to a lower level, corresponding to a utiliza-
tion rate of 4.8–10.4%. A comparatively low concentration
was found for hulupinic acid.[80] Interestingly, the decreas-
ing concentration of hulupones during beer processing was
not accompanied by an accelerated formation of hulupinic
acid, which had been implied that there might be unknown
pathways contributing to the decrease of hulupones.[80]
Cyclization products of b-acids (XXXIV-XXXVIII)
Thermal treatment of colupulone in a pH 5.8 aqueous solu-
tion with MgCl2 at 110  C in a closed vial, to mimic the wort
boiling condition, followed by an analysis using LC TOF MS
and 1 D/2D NMR spectroscopy, led to revealing that cohulu-
pone, hulupinic acid, two tricyclocolupone epimers (XXXIV),
two dehydrotricyclocolupones (XXXV), two hydroperoxy-tri-
cyclocolupone epimers (XXXVI), two hydroxytricyclolupone
epimers (XXXVII), and nortricyclocolupone (XXXVIII) were
the major bitter-tasting transformation products.[44,88]
The formation routes are depicted in the name of colupu-
lone as follows: the reaction cascade starts by a hydrogen loss
from the hydroxyl function of colupulone to give its corre-
sponding alkoxyl radical, which, upon mesomerization and
radical cyclisation, generates the novel C(6)-C(12) linkage in
the intermediate.[44,88] The transient alkyl radical takes
another cyclization by the attack of the alkyl radical at the
double bond of the isoprenyl moiety at C(6), thus leads to an
intermediate radical with novel C(4)-C(14) linkage.[44,88] This
radical is the common intermediate to generate the nine tri-
cyclic compounds via three alternative reactive routes. Firstly,
the radical may lose a proton at C(2) of the isopropenyl moi-
ety leading to the formation of dehydrotricyclocolupone epimer
A/B.[44,88] Secondly, the radical may abstract a proton from a
hydrogen donor molecule to yield tricyclocolupone A/B,

respectively.[44,88] Thirdly, the radical may convert to a hydro-
peroxy radical by addition of triplet oxygen, then to hydro-
peroxy-tricyclocolupone A/B by abstracting one hydrogen
atom from a donor molecule.[44,88] Furthermore, these hydro-
peroxides are prone to liberate the corresponding alkoxy moi-
ety to form an intermediate radical in the presence of
transition metals by means of a Fenton-type reaction.[44,88]
This intermediary alkoxy radical can be saturated by hydro-
gen abstraction from a donor molecule to give hydroxytricy-
clocolupone epimer A/B or can be split off of 2-propanone to
form nortricyclocolupone.[44,88] The generation of these com-
pounds seems to be independent of the variable alkanoyl side
chain of the b-acids, all of the degradation products from
colupulone show the isopropyl moiety in their chemical
structure.[44,88]
The thresholds of these oxidation transformation prod-
ucts of colupulones, determined by means of a triangle test
in 5% ethanol solution with pH 4.4, are listed in Table
2.[44,88] Depending on their chemical structure, these com-
pounds show rather low recognition thresholds, ranging
from 7.9 to 90.3 lmol/L, with a clear bitter taste. The lowest
threshold of 7.9 lmol/L was found for cohulupone, giving a
short-lasting a-acid-like bitterness. In contrast, all tricyclic
compounds exhibited a long-lasting lingering and harsh bit-
terness perceived on the posterior tongue and throat.[44,88]
Among them, the thresholds of the hydroxytricyclic and
hydroperoxytricyclic compounds were near the threshold of
19 lmol/L of the trans-isocohumulone, implying a strong
potential of bitterness.[44,83,88]
Large differences were found in the distribution of the indi-
vidual colupulone transformation products in the different-
type beers under investigation, namely, dark, lager, pilsner,
bitter, and wheat beers, which might be due to the variations
in the composition of hop products, the hop dosage rate, as
well as other related specific parameters affecting b-acid trans-
formations during brewing.[44] In all beer samples of the
different types, the concentrations of the hydroperoxytricyclo-
colupone epimer A/B were all less than their detection limita-
tions, which were in line with the observations of their
instability and tendency to convert into their corresponding
alcohols.[44] In the pilsner beer brewing process, among the
cyclization products of b-acids, hydroxytricyclo-colupone epi-
mer A/B and hydroxytricyclolupone epimer A/B could be
clearly detected in the wort. Degradation products of b-acids,
namely hulupones, hydroxyltricyclolupone epimer A/B, and
hulupinic acid accounted for 20% of the starting amount of
b-acids added with the hops in the final wort.[80] However,
only tricyclocolupone epimer A and epimer B could be
detected in the final beer, of which concentrations decreased
to less than 50% of those in wort.[80]
Using HR/AM-LC-MS/MS, Dusek et al. analyzed b-acids
and their cyclic transformation products in two stored Sladek
hop pellets, with storage periods of 24 and 60 months, and in
beers brewed with them.[46] It was found that the amounts of
starting materials (lupulone and colupulone in hops stored
for 24 months) were higher than those in hops stored for
60 months. It was clearly shown that cyclic degradation
products, namely nortricyclo-, dehydrotricyclo-, tricyclo-,
JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 11
Table 2. Bitter recognition threshold concentrations of colupulone and its
transformation products.[44,88]
Compound Threshold concentration (lmol/L)
colupulone 39.3
cohulupone 7.9
hulupinic acid 68.5
nortricyclocolupone 90.3
dehydrotricyclocolupone epimer A 40.5
dehydrotricyclocolupone epimer B 40.8
tricyclocolupone epimer A 54.4
tricyclocolupone epimer B 37.9
hydroxytricyclocolupone epimer A 17.1
hydroxytricyclocolupone epimer B 14.7
hydroperoxytricyclocolupone epimer A 20.5
hydroperoxytricyclocolupone epimer B 20.9
hydroperoxytricyclo-, hydroxytricyclo- transformation prod-
ucts of colupulone and lupulone were all found in these two
stored hops. Lower concentrations of different cyclic products
were seen in hops stored for 24 months, which were about
45–77% of the corresponding concentrations in hops stored
for 60 months, respectively.[46] In contrast to hops, no meas-
urable dehydrotricyclic, hydroxytricyclic, and hydroperoxytri-
cyclic products were found in these beers.[46] As for beers
produced from hops stored for 24 months, nortricyclocolu-
pone and nortricyclo-lupone were detectable, while the con-
centrations of them in beer brewed with hops stored for
24 months was 67% and 77% of that in beer brewed with
hops stored for 60 months.[46] It appears certain that long-
term storage of hops leads to an appreciable formation of all
cyclization products of b-acids. However an understanding of
the impact of these individual cyclic compounds on beer bit-
terness remains unsatisfactory, as most of these are difficult
to detect in beer.
Epoxyhulupones (XXXIX)
In addition to the above mentioned cyclization products of
b-acids in hops, two new cyclic compounds were identified
by Dusek et al.[46] The tentative structures were proposed to
be epoxycohulupone and epoxyhulupone for these newly
identified compounds in model solution, based on the high
resolution MS/MS spectra. Up to now, only trace amounts
of epoxyhulupone were found in real hop samples.[46] The
structures of these two compounds were not confirmed by
NMR and their formation routes were not yet proposed.[46]
The ongoing discovery of new cyclic compounds of b-acids
illustrates the complexity of hop bitterness chemistry and
more work is still needed for a thorough understanding of
the chemistry of hop acid derivations and effects on hop
and beer bitterness in the future.
Conclusions
This review discusses the cyclic and oxidation products of
a-, iso-a-, and b-acids in hops and beer, with a focus on the
majority of these products identified in last decade. Thirty-
nine substances, namely 15 related to a-acids, 15 to iso-
a-acids, and 9 to b-acids are elucidated, including their pro-
posed formation mechanisms, distribution profiles in model
solutions or in hop or beer samples, thresholds of some, their
12 J. HAO ET AL.
alterations during fermentation and their evolution during
storage. These 39 substances should not be strictly referred to
as 39 compounds, as some of them are different congeners.
For example cis-iso-co-, n-, ad-humulones are described as
cis-iso-a-acids or different epimers for example hydroperoxy-
tricyclic co-, n-, ad-lupones epimer A and epimer B are
described as hydroperoxytricyclolupones epimers.
The literature surveyed by this review attempts to give a
complete picture of a-, b-, iso-a-acids and their related
transformations during brewing and to provide some
answers to long-time confusing questions encountered by
brewers, such as “which compounds are responsible for the
lingering and harsh bitterness of aged low-oxygen beer?”,
“which reaction is responsible for the degradation of cis-iso-
acids in PET bottled beer?” etc.
The detection of the concentrations of indicator com-
pounds of hop aging such as humulinones, 40 -hydroxy-allo-
humulinones, deisopropyltricycloisohumulone, tricyclo-oxyiso
humulones A and B, and tricycloperoxyisohumulone A is
suggested as a potential means for quality assurance testing,
although the popularization of these expensive and compli-
cated instruments in the brewing industry will take time.
Nevertheless, with more research focused on these cyclic
and oxidative bitter derivatives in the future, the science
behind brewing bitterness will become more clarified and
help the brewer to better control beer bitter quality.
Funding
This work was supported by the Science foundation of Beibu Gulf
University under grant no.17kjjh04 and no.18KYQD05, National
Natural Science Foundation of China under grant no. 31801517, and
Shandong Provincial Natural Science Foundation under grant no.
ZR2019BC010.
ORCID
Junguang Hao http://orcid.org/0000-0003-1519-9019
R.A. Speers http://orcid.org/0000-0002-9820-1657
Heliang Fan http://orcid.org/0000-0001-7889-8963
Literature cited
[1] Hughes, P. S. The Significance of Iso-Alpha Acid for Beer
Quality. J. Inst. Brew. 2000, 106, 271–276. DOI: 10.1002/j.2050-
0416.2000.tb00066.x
[2] Fritsh, A.; Shellhammer, T. H. Relative Bitterness of Reduced
and Nonreduced Iso-a-Acids in Lager Beer. J. Am. Soc. Brew.
Chem. 2008, 66, 88–93. DOI: 10.1094/ASBCJ-2008-0313-01.
[3] Ting, P. L.; Ryder, D. S. The Bitter, Twisted Truth of the Hop:
50 Years of Hop Chemistry. J. Am. Soc. Brew. Chem. 2017, 75,
161–180. DOI: 10.1094/ASBCJ-2017-3638-01.
[4] Irwin, A. J.; Murray, C. R.; Thompson, D. J. An Investigation
of the Relationship between Hopping Rating, Time of Boil and
Individual Alpha Acid Utilization. J. Am. Soc. Brew. Chem.
1985, 43, 145–152. DOI: 10.1094/ASBCJ-43-0145.
[5] Hughes, P. S.; Marinova, G. Variations in Iso-Alpha-Acids
Distribution and Behavior of Different Products. Brew.
Guardian 1997, 126, 26–29.
[6] Jaskula, B.; Syryn, E.; Goiris, K.; Rouck, G. D.; Opstaele, F. V.;
Clippeleer, J. D.; Aerts, G.; Cooman, L. D. Hopping Technology
in Relation to Beer Bitterness Consistency and Flavor Stability.
J. Am. Soc. Brew. Chem. 2007, 65, 38–46. DOI: 10.1094/ASBCJ-
2007-0112-03.
[7] Jaskula, B.; Kafarski, P.; Aerts, G.; Cooman, L. D. A Kinetic
Study on the Isomerization of Hop a-Acids. J. Agric. Food
Chem. 2008, 56, 6408–6415. DOI: 10.1021/jf8004965.
[8] Jaskula, B.; Goiris, K.; Opstaele, F. V.; Rouck, G. D.; Aerts, G.;
Cooman, L. D. Hopping Technology in Relation to a-Acids
Isomerization Yield, Final Utilization, and Stability of Beer
Bitterness. J. Am. Soc. Brew. Chem. 2009, 67, 44–57. DOI: 10.
1094/ASBCJ-2009-0106-01.
[9] Stevens, J. F.; Taylor, A. W.; Clawson, J. E.; Deinzer, M. L. Fate
of Xanthohumol and Related Prenylflavonoids from Hops to
Beer. J. Agric. Food Chem. 1999, 47, 2421–2428. DOI: 10.1021/
jf990101k.
[10] Ceslova, L.; Holcapek, M.; Fidler, M.; Drstickova, J.; Lisa, M.
Characterization of Prenylflavonoids and Hop Bitter Acids in
Various Classes of Czech Beers and Hop Extracts Using High-
Performance Liquid Chromatography-Mass Spectrometry. J.
Chromatogr. A 2009, 216, 7249–7257. DOI: 10.1016/j.chroma.
2009.09.022.
[11] Almaguere, C.; Gastl, M.; Arendt, E. K.; Becker, T.
Contributions of Hop Hard Resins to Beer Quality. Brewing Sci.
2012, 65, 118–129.
[12] Dresel, M.; Dunkel, A.; Hofmann, T. Sensomics Analysis of Key
Bitter Compounds in the Hard Resin of Hops (Humulus lupu-
lus L.) and Their Contribution to the Bitter Profile of Pilsner-
Type Beer. J. Agric. Food Chem. 2015, 63, 3402–3418. DOI: 10.
1021/acs.jafc.5b00239.
[13] Taniguchi, Y.; Yamada, M.; Taniguchi, H.; Matsukura, Y.;
Shindo, K. Chemical Characterization of Beer Aging Products
Derived from Hard Resin Components in Hops (Humulus
lupulus L.). J. Agric. Food Chem. 2015, 63, 10181–10191. DOI:
10.1021/acs.jafc.5b04138.
[14] Araki, S.; Takashio, M.; Shinotsuka, K. A New Parameter for
Determination of the Extent of Staling in Beer. J. Am. Soc.
Brew. Chem. 2002, 60, 26–30. DOI: 10.1094/ASBCJ-60-0026.
[15] Kappler, S.; Krahl, M.; Geissinger, C.; Becker, T.; Krottenthaler,
M. Degradation of Iso-a-Acids during Wort Boiling. J. Inst.
Brew. 2010, 116, 332–338. DOI: 10.1002/j.2050-0416.2010.
tb00783.x.
[16] Caballero, I.; Blanco, C. A.; Porras, M. Iso-a-Acids, Bitterness
and Loss of Beer Quality during Storage. Trends Food Sci.
Technol. 2012, 26, 21–30. DOI: 10.1016/j.tifs.2012.01.001.
[17] Oladokun, O.; Tarrega, A.; James, S.; Smart, K.; Hort, J.; Cook,
D. The Impact of Hop Bitter Acid and Polyphenol Profiles on
the Perceived Bitterness of Beer. Food Chem. 2016, 205,
212–220. DOI: 10.1016/j.foodchem.2016.03.023.
[18] Oladokun, O.; Tarrega, A.; James, S.; Cowley, T.; Dehrmann, F.;
Smart, K.; Cook, D.; Hort, J. Modification of Perceived Beer
Bitterness Intensity, Character and Temporal Profile by Hop
Aroma Extract. Food Res. Int. 2016, 86, 104–111. DOI: 10.1016/
j.foodres.2016.05.018.
[19] Oladokun, O.; James, S.; Cowley, T.; Dehrmann, F.; Smart, K.;
Hort, J.; Cook, D. Perceived Bitterness Character of Beer in
Relation to Hop Variety and the Impact of Hop Aroma. Food
Chem. 2017, 230, 215–224. DOI: 10.1016/j.foodchem.2017.03.
031.
[20] Hahn, C. D.; Lafontaine, S. R.; Pereira, C. B.; Shellhammer,
T. H. Evaluation of Nonvolatile Chemistry Affecting Sensory
Bitterness Intensity of Highly Hopped Beers. J. Agric. Food
Chem. 2018, 66, 3505–3513. DOI: 10.1021/acs.jafc.7b05784.
[21] Wietstock, P.; Kunz, T.; Methner, F. J. Influence of Hopping
Technology on Oxidative Stability and Staling-Related
Carbonyls in Pale Lager Beer. Brew. Sci. 2016, 69, 73–83.
[22] Parkin, E.; Shellhammer, T. H. Toward Understanding the
Bitterness of Dry-Hopped Beer. J. Am. Soc. Brew. Chem. 2017,
75, 363–368. DOI: 10.1094/ASBCJ-2017-4311-01.
[23] Lafontaine, S. R.; Shellhammer, T. H. Impact of Static Dry-
Hopping Rate on the Sensory and Analytical Profiles of Beer. J.
Inst. Brew. 2018, 124, 434–442. DOI: 10.1002/jib.517.

[24] Mikyska, A.; Olsovsk
a, J.; Slab
y, M.; Sterba, K.; Cerenak,
 A.;
Kosir, I. J.; Pavlovic, M.; Kolenc, Z.; Krofta, K. Analytical and
Sensory Profiles of Slovenian and Czech Hop Genotypes in
Single Hopped Beers. J. Inst. Brew. 2018, 124, 209–221. DOI:
10.1002/jib.494.
[25] Lafontaine, S. R.; Shellhammer, T. H. Sensory Directed Mixture
Study of Beers Dry-Hopped with Cascade, Centennial, and
Chinook. J. Am. Soc. Brew. Chem. 2018, 76, 199–208. DOI: 10.
1080/03610470.2018.1487747.
[26] Lafontaine, S. R.; Shellhammer, T. H. Investigating the Factors
Impacting Aroma, Flavor, and Stability in Dry-Hopped Beers.
Tech. Quart. Master Brew. Assoc. Am. 2019, 56, 13–23.
[27] Junior, J. C. M.; Faria, M. A.; Melo, A.; Martins, Z. E.; Ferreira,
I. Modeling of a-Acids and Xanthohumol Extraction in Dry-
Hopped Beers. Food Chem. 2019, 278, 216–222. DOI: 10.1016/j.
foodchem.2018.11.050.
[28] Schieberle, P. Primary Odorants of Pale Lager Beer. Differences
to Other Beers and Changes during Storage. Z. Lebensm.
Unters. Forch. 1991, 193, 558–565.
[29] Vanderhaegen, B.; Neven, H.; Coghe, S.; Verstrepen, K. J.;
Verachtert, H.; Derdelinckx, G. Evolution of Chemical and
Sensory Properties during Aging of Top-Fermented Beer. J.
Agric. Food Chem. 2003, 51, 6782–6790. DOI: 10.1021/
jf034631z.
[30] Vanderhaegen, B.; Neven, H.; Verachtert, H.; Derdelinckx, G.
The Chemistry of Beer Aging - a Critical Review. Food Chem.
2006, 95, 357–381. DOI: 10.1016/j.foodchem.2005.01.006.
[31] Vanderhaegen, B.; Delvaux, F.; Daenen, L.; Verachtert, H.;
Delvaux, F. R. Aging Characteristics of Different Beer Types.
Food Chem. 2007, 103, 404–412. DOI: 10.1016/j.foodchem.2006.
07.062.
[32] Intelmann, D.; Demmer, O.; Desmer, N.; Hofmann, T. 18O
Stable Isotope Labeling, Quantitative Model Experiments, and
Molecular Dynamics Simulation Studies on the Trans-Specific
Degradation of the Bitter Tasting Iso-a-Acids of Beer. J. Agric.
Food Chem. 2009, 57, 11014–11023. DOI: 10.1021/jf903000c.
[33] Maye, J. P.; Smith, R. Dry Hopping and Its Effects on
International Bitterness Unit Test and Beer Bitterness. Tech.
Quart. Master Brew. Assoc. Am. 2016, 53, 134–136.
[34] Cook, A. H.; Harris, G. The Chemistry of Hop Constituents.
Part I. Humulinone, a New Constituent of Hops. J. Chem. Soc.
1950, 1950, 1873–1876. DOI: 10.1039/jr9500001873.
[35] Meilgaard, M. Hop Analysis, Cohumulone Factor and the
Bitterness of Beer: Review and Critical Evaluation. J. Inst. Brew.
1960, 66, 35–50. DOI: 10.1002/j.2050-0416.1960.tb01696.x
[36] Burton, J. S.; Stevens, R. Evaluation of Hops. XI. The Hard
Resin and Presence of Hulupinic Acid. J. Inst. Brew. 1965, 71,
51–56. DOI: 10.1002/j.2050-0416.1965.tb02024.x.
[37] Laws, D. R. J. Hop Resins and Beer Flavor. V. The Significance
of Oxidized Hop Resins in Brewing. J. Inst. Brew. 1968, 74,
178–182. DOI: 10.1002/j.2050-0416.1968.tb03112.x
[38] Verzele, M.; Vewaele, C.; Kerrebroeck, M. V. Analysis of Beer
Iso-a-Acids by High-Performance Liquid Chromatography
without Sample Pre-Treatment. J. Chromatogr. A 1982, 244,
321–326. DOI: 10.1016/S0021-9673(00)85696-4.
[39] Ono, M.; Kakudo, Y.; Yamamoto, Y.; Nagami, K.; Kumada, J.
Quantitative Analysis of Hop Bittering Components and Its
Application to Hop Evaluation. J. Am. Soc. Brew. Chem. 1984,
42, 167–172. DOI: 10.1094/ASBCJ-42-0167.
[40] Stevens, R. The Chemistry of Hop Constituents. Chem. Rev.
1967, 67, 19–71.
[41] Aitken, R. A.; Bruce, A.; Harris, J. O.; Seaton, J. C. The
Bitterness of Hop-Derived Materials in Beer. J. Inst. Brew.
1970, 76, 29–37. DOI: 10.1002/j.2050-0416.1970.tb03255.x.
[42] Intelmann, D.; Kummerl€ owe, G.; Haseleu, G.; Desmer, N.;
Schulze, K.; Fr€ ohlich, R.; Frank, O.; Luy, B.; Hofmann, T.
Structures of Storage-Induced Transformation Products of the
Beer’s Bitter Principles, Revealed by Sophisticated NMR
Spectroscopic and LC-MS Techniques. Chem. Eur. J. 2009, 15,
13047–13058. DOI: 10.1002/chem.200902058.
JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 13
[43] Intelmann, D.; Haseleu, G.; Hofmann, T. LC-MS/MS
Quantitation of Hop-Derived Bitter Compounds in Beer Using
the ECHO Technique. J. Agric. Food Chem. 2009, 57,
1172–1182. DOI: 10.1021/jf803040g.
[44] Haseleu, G.; Intelmann, D.; Hofmann, T. Identification and RP-
HPLC-ESI-MS/MS Quantitation of Bitter-Tasting b-Acid
Transformation Products in Beer. J. Agric. Food Chem. 2009,
57, 7480–7489. DOI: 10.1021/jf901759y.
[45] Taniguchi, Y.; Taniguchi, H.; Yamada, M.; Matsukura, Y.;
Koizumi, H.; Furihata, K.; Shindo, K. Analysis of the
Components of Hard Resin in Hops (Humulus lupulus L.) and
Structural Elucidation of Their Transformation Products
Formed during the Brewing Process. J. Agric. Food Chem. 2014,
62, 11602–11612. DOI: 10.1021/jf504394h.
[46] Dusek, M.; Olsovsk
a, J.; Krofta, K.; Jurkov
a, M.; Mikyska, A.
Qualitative Determination of b-Acids and Their Transformation
Products in Beer and Hop Using HR/AM-LC-MS/MS. J. Agric.
Food Chem. 2014, 62, 7690–7697. DOI: 10.1021/jf501852r.
[47] Jaskula-Goiris, B.; Aerts, G.; Cooman, L. D. Hop a-Acids
Isomerisation and Utilisation: An Experimental Review.
Cerevisia 2010, 35, 57–70. DOI: 10.1016/j.cervis.2010.09.004.
[48] Sch€onberger, C.; Kostelecky, T. 125th Anniversary Review: The
Role of Hops in Brewing. J. Inst. Brew. 2011, 117, 259–267.
DOI: 10.1002/j.2050-0416.2011.tb00471.x.
[49] Clippeleer, J. D.; Cooman, L. D.; Aerts, G. Beer’s Bitter
Compounds-A Detailed Review on Iso-a-Acids: current
Knowledge of the Mechanisms for Their Formation and
Degradation. Brewing Sci. 2014, 67, 167–182.
[50] Almaguer, C.; Sch€ onberger, C.; Gastl, M.; Arendt, E. K.; Becker,
T. Humulus lupulus - a Story That Begs to Be Told. A Review.
J. Inst. Brew. 2014, 120, 289–314. DOI: 10.1002/jib.160.
[51] Steenackers, B.; Cooman, L. D.; Vos, D. D. Chemical
Transformations of Characteristic Hop Secondary Metabolites
in Relation to Beer Properties and the Brewing Process: A
Review. Food Chem. 2015, 172, 742–756.
[52] Kostrzewa, D.; Dobrzy
nska–Inger, A.; R
oj, E.; GrzeR da, K.;
Kozłowski, K. Isomerization of Hop Extract a-Acids. J. Inst.
Brew. 2016, 122, 493–499. DOI: 10.1002/jib.349.
[53] Clarke, B. J.; Hildebrand, R. P. The Isomerization of
Humulone. III. The Degradation of Isohumulone. J. Inst. Brew.
1967, 73, 282–293. DOI: 10.1002/j.2050-0416.1967.tb03045.x.
[54] Garc
ıa-Villalba, R.; Cortacero-Ram
ırez, S.; Segura-Carretero, A.;
Contreras, J. A. M.-L.; Fern
andez-Guti
errez, A. Analysis of Hop
Acids and Their Oxidized Derivatives and Iso-a-Acids in Beer
by Capillary-Electrophoresis-Electrospray Ionization Mass
Spectrometry. J. Agric. Food Chem. 2006, 54, 5400–5409. DOI:
10.1021/jf060207x.
[55] Taniguchi, Y.; Taniguchi, H.; Matsukura, Y.; Kawachi, Y.;
Shindo, K. Structural Elucidation of Humulone Autoxidation
Products and Analysis of Their Occurrence in Stored Hops. J.
Nat. Prod. 2014, 776, 1252–1261. DOI: 10.1021/np4008427.
[56] Taniguchi, Y.; Matsukura, Y.; Ozaki, H.; Nishimura, K.; Shindo,
K. Identification and Quantification of the Oxidation Products
Derived from a- Acids and b- Acids during Storage of Hops
(Humulus lupulus L.). J. Agric. Food Chem. 2013, 61,
3121–3130. DOI: 10.1021/jf3047187.
[57] Keukeleire, S. D. Oxidation Products of Hop a-Acids and Iso-
a-Acids. Cerevisia 1981, 2, 73–80.
[58] Palamand, S. R.; Aldenhoff, J. M. Bitter Tasting Compounds of
Beer. Chemistry and Taste Properties of Some Hop Resin
Compounds. J. Agric. Food Chem. 1973, 21, 535–543. DOI: 10.
1021/jf60188a005.
[59] Whitear, A. L.; Hudson, J. R. Hop Resins and Beer Flavor III:
Hop Resins in Beers. J. Inst. Brew. 1964, 70, 24–30. DOI: 10.
1002/j.2050-0416.1964.tb01962.x.
[60] Algazzali, V.; Shellhammer, T. Bitterness Intensity of Oxidized
Hop Acids: Humulinones and Hulupones. J. Am. Soc. Brew.
Chem. 2016, 74, 36–43. DOI: 10.1094/ASBCJ-2016-1130-01.
[61] Cook, A. H.; Howard, G. A.; Slater, C. A. Chemistry of Hop
Constituents. VIII. Oxidation of Humulone and Cohumulone.
14 J. HAO ET AL.
J. Inst. Brew. 1955, 61, 321–325. DOI: 10.1002/j.2050-0416.1955.
tb02803.x.
[62] Mikyska, A.; Krofta, K. Assessment of Changes in Hop Resins
and Polyphenols during Long-Term Storage. J. Inst. Brew. 2012,
118, 269–279. DOI: 10.1002/jib.40.
[63] Intelmann, D.; Hofmann, T. On the Autoxidation of Bitter-
Tasting Iso-a-Acids in Beer. J. Agric. Food Chem. 2010, 58,
5059–5067. DOI: 10.1021/jf100083e.
[64] de Almeida, N. E. C.; Homem-de-Mello, P.; De Keukeleire, D.;
Cardoso, D. R. Reactivity of Beer Bitter Acids Toward the 1-
Hydroxyethyl Radical as Probed by Spin-Trapping Electron
Paramagnetic Resonance (EPR) and Electrospray Ionization-
Tandem Mass Spectrometry (ESI-MS/MS). J. Agric. Food Chem.
2011, 59, 4183–4191. DOI: 10.1021/jf104797d.
[65] Intelmann, D.; Haseleu, G.; Dunkel, A.; Lagemann, A.; Stephan,
A.; Hofmann, T. Comprehensive Sensomics Analysis of Hop-
Derived Bitter Compounds during Storage of Beer. J. Agric.
Food Chem. 2011, 59, 1939–1953. DOI: 10.1021/jf104392y.
[66] Potter, M. D.; Keukeleire, D. D.; Bruyn, A. D.; Verzele, M.
Tricyclo-Oxy and Tricycle-Hydro-Peroxycolupulones. Bull. Soc.
Chum, Belg. 1978, 23, 5407–5418.
[67] Keukeleire, D. D.; Verzele, M. The Absolute Configuration of
the Isohumulones and the Humulinic Acids. Tetrahedron 1971,
27, 4939–4945. DOI: 10.1016/S0040-4020(01)98199-2.
[68] Simpson, W. J.; Smith, A. R. W. Factors Affecting Antibacterial
Activity of Hop Compounds and Their Derivatives. J. Appl.
Bacteriol. 1992, 72, 327–334. DOI: 10.1111/j.1365-2672.1992.
tb01843.x.
[69] Kunimune, T.; Shellhammer, T. H. Foam-Stabilizing Effects and
Cling Formation and Cling Formation Patterns of Iso-Alpha-
Acids in Lager Beer. J. Agric. Food Chem. 2008, 56, 8629–8634.
DOI: 10.1021/jf8011079.
[70] Wackerbauer, K.; Balzer, U. Hop Bitter Compounds in Beer.
Part II: The Influence of Cohumulone on Beer Quality. Brew.
Int. 1993, 11, 116–118.
[71] Hughes, P. S.; Simpson, W. J. Bitterness of Congeners and
Stereoisomers of Hop-Derived Bitter Acids Found in Beer. J.
Am. Soc. Brew. Chem. 1996, 54, 234–237. DOI: 10.1094/ASBCJ-
54-0234.
[72] Sch€onberger, C. Why Cohumulone is Better than Its
Reputation. Brauwelt Int. 2009, 3, 159–160.
[73] Rigby, F. L. A Theory on the Hop Flavor of Beer. Proc. Am.
Soc. Brew. Chem. 1972, 30, 46–50.
[74] Kusche, M.; Stettner, G.; Stephan, A. Influence of the New
High Alpha Hop Variety Herkules on Beer Quality. Proc. Eur.
Brew. Conv. Congr 2007, Fachverlag Hans Carl: N€ urnberg,
Germany, CD ROM 2007, Contribution 24.
[75] King, B. M.; Duineveld, C. A. A. Changes in Bitterness as Beer
Ages Naturally. Food Qual. Prefer. 1999, 10, 315–324. DOI: 10.
1016/S0950-3293(98)00040-8.
[76] Cooman, L.; Aerts, G.; Overmeire, H.; Keukeleire, D. D.
Alterations of the Profiles of Iso-Alpha-Acids during Beer
Ageing, Marked Instability of Trans-Iso-Alpha Acids and
Implications for Beer Bitterness Consistency in Relation to
Tetrahydroiso-a-Acids. J. Inst. Brew. 2000, 106, 169–178. DOI:
10.1002/j.2050-0416.2000.tb00054.x.
[77] Malfliet, S.; Opstaele, F. V.; Clippeleer, J. D.; Syryn, E.; Goiris,
K.; Cooman, L. C.; Aerts, G. Flavour Instability of Pale Lager
Beers: Determination of Analytical Markers in Relation to
View publication stats
Sensory Ageing. J. Inst. Brew. 2008, 114, 180–192. DOI: 10.
1002/j.2050-0416.2008.tb00324.x.
[78] Nimubona, D.; Blanco, C. A.; Caballero, I.; Rojas, A.; Andr
es-
Iglesias, C. An Approximate Shelf Life Prediction of Elaborated
Lager Beer in Terms of Degradation of Its Iso-a-Acids. J. Food
Eng. 2013, 116, 138–143. DOI: 10.1016/j.jfoodeng.2012.11.019.
[79] Schulze, W. G.; Ting, P. L.; Henckel, L. A.; Goldstein, H.
Separation of Humulinic Acids by Reverse-Phase High
Performance Liquid Chromatography. J. Am. Soc. Brew. Chem.
1981, 39, 12–15. DOI: 10.1094/ASBCJ-39-0012.
[80] Haseleu, G.; Lagemann, A.; Stephan, A.; Intelmann, D.; Dunkel,
A.; Hofmann, T. Quantitative Sensomics Profiling of Hop-
Derived Bitter Compounds throughout a Full-Scale Beer
Manufacturing Process. J. Agric. Food Chem. 2010, 58,
7930–7939. DOI: 10.1021/jf101326v.
[81] Kaneda, H.; Takashio, M.; Tamaki, T.; Osawa, T. Influence of
pH on Flavor Staling during Beer Storage. J. Inst. Brew. 1997,
103, 21–23. DOI: 10.1002/j.2050-0416.1997.tb00932.x.
[82] Dresel, M.; Vogt, C.; Dunkel, A.; Hofmann, T. The Bitter
Chemodiversity of Hops (Humulus lupulus L.). J. Agric. Food
Chem. 2016, 64, 7789–7799. DOI: 10.1021/acs.jafc.6b03933.
[83] Intelmann, D.; Batram, C.; Kuhn, C.; Haseleu, G.; Meyerhof,
M.; Hofmann, T. Three TAS2R Bitter Taste Receptors Mediate
the Psychophysical Responses to Bitter Compounds of Hops
(Humulus lupulus L.) and Beer. Chem. Percept. 2009, 2,
118–132. DOI: 10.1007/s12078-009-9049-1.
[84] Kokubo, E.; Kowaka, M.; Kuroiwa, Y. New Bitter Substances:
Lupoxes a, Lupoxes b and Lupdoxes a. Proc. Am. Soc. Brew.
Chem. 1971, 29, 265–279. DOI: 10.1080/00960845.1971.
12007025.
[85] Kowaka, M.; Kokubo, E.; Kuroiwa, Y. New Bitter Substances of
Beer: Lupoxes c and Lupdoxes b. Proc. Am. Soc. Brew. Chem.
1972, 30, 42–46. DOI: 10.1080/00960845.1972.12005958.
[86] Kowaka, M.; Kokubo, E.; Kuroiwa, Y. New Bitter Substances of
Beer: Lupdeps and Lupdols. Proc. Am. Soc. Brew. Chem. 1973,
31, 66–71. DOI: 10.1080/00960845.1973.12006004.
[87] Verzele, M.; de Velde, N. V.; Dewaele, C. High Performance
Liquid Chromatography Analysis of Colupone Oxidation
Products in Hops and in Beer. J. Am. Soc. Brew. Chem. 1983,
41, 57–60. DOI: 10.1094/ASBCJ-41-0057.
[88] Haseleu, G.; Intelmann, D.; Hofmann, T. Structure
Determination and Sensory Evaluation of Novel Bitter
Compounds Formed from b-Acids of Hop (Humulus lupulus
L.) upon Wort Boiling. Food Chem. 2009, 116, 71–81. DOI: 10.
1016/j.foodchem.2009.02.008.
[89] Briggs, D. E.; Boulton, C. A.; Brookes, P. A.; Stevens, R. The
Chemistry of Hop Constituents. Chapter 8. In: Brewing Science
and Practice; Woodhead: Cambridge, U.K, . 2004; pp. 255–305.
[90] Krofta, K.; Vrabcova, S.; Mikyska, A.; Jurkova, M. The Effect of
Hop Beta-Acids Oxidation Products on Beer Bitterness. Kvasn
y
Průmysl. 2013, 59, 306–312.
[91] Krofta, K.; Mikyska, A. Hop Beta-Acids: Properties, Significance
and Utilization. Kvasn
y Průmysl. 2014, 60, 96–105.
[92] Maye, J. P.; Smith, R.; Leker, R. Humulinone Formation in
Hops and Hop Pellets and Its Implications for Dry Hopped
Beers. Tech. Quart. Master Brew. Assoc. Am. 2016, 53, 23–27.
[93] Burton, J. S.; Stevens, R.; Elvidge, J. A. Chemistry of Hop
Constituents. Part XVIII. Hulupinic Acid. J. Chem. Soc. 1964,
1964, 952–955. DOI: 10.1039/jr9640000952.