Journal of the American Society of Brewing Chemists

The Science of Beer

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ujbc20

Evaluation of Components Contributing to the

International Bitterness Unit of Wort and Beer

Toru Kishimoto, Satoko Teramoto, Akiko Fujita & Osamu Yamada

To cite this article: Toru Kishimoto, Satoko Teramoto, Akiko Fujita & Osamu Yamada (2021):
Evaluation of Components Contributing to the International Bitterness Unit of Wort and Beer,
Journal of the American Society of Brewing Chemists, DOI: 10.1080/03610470.2021.1878684

To link to this article: https://doi.org/10.1080/03610470.2021.1878684

Published online: 01 Mar 2021.

Submit your article to this journal

Article views: 122

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=ujbc20
JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS
https://doi.org/10.1080/03610470.2021.1878684

Evaluation of Components Contributing to the International Bitterness Unit

of Wort and Beer
Toru Kishimoto , Satoko Teramoto, Akiko Fujita, and Osamu Yamada
National Research Institute of Brewing, Hiroshima, Japan

ABSTRACT KEYWORDS
This study evaluated the components that contribute to the International Bitterness Unit (IBU) of International bitterness unit;
wort and beer. A range of samples were analyzed in this study, including beer with 47.5 IBU con- isomerization temperature;
taining 39.9 ppm iso-a-acid, and one with 49.9 IBU containing only 2.2 ppm iso-alpha acid. The IBU oxidative polar compounds;
aged hop
value of wort and beer is mainly contributed by iso-a-acid, non-isomerized a-acid, oxidative polar
compounds, and malt-derived polar components. The isomerization of a-acid to iso-a-acid is initi-
ated between 60
C and 70
C and the isomerization ratios accelerate rapidly at higher tempera-
ture. In the wort boiled below 60
C, over 50% of the IBU value was contributed by non-
isomerized a-acid. Most of the non-isomerized a-acid disappeared during fermentation, whereas
the concentration of iso-a-acid was stable. The disappearance of a-acid resulted in a large
decrease in the IBU value during fermentation, and the isomerization ratio influenced the degree
of the decrease. Over 80% of the IBU value was contributed by the oxidative polar compounds in
the beer hopped with the pellet stored at 40
C for 90 days. Bitterness intensity was less in the
beers with deteriorated hops than in those with non-deteriorated hops, despite both having the
same IBU values.

Introduction The IBU value is used to express bitterness intensity in

beers and is measured by the method defined by the
Hops (Humulus lupulus L.) are used in the brewing process
American Society of Brewing Chemists (ASBC)[19] and
to add a characteristic bitterness and distinctive aroma to European Brewery Convention (EBC).[20] In the measurement
beer. The bitterness of beer is ascribed to the iso a-acid of the IBU value, components of worts and beers are extracted
(IAA) produced by the isomerization of a-acid (AA) during using 2,2,4-trimethylpentane, and the IBU values are calcu-
wort boiling and the whirlpool process of brewing. AA is lated from the absorbance values of the 2,2,4-trimethylpentane
composed of homologues, normal-humulone, co-humulone, layer at 275 nm, as measured by a spectrophotometer. The
and ad-humulone, and IAA is also composed of its corre- double bond structures of the compounds in the 2,2,4-trime-
sponding homologues, iso-normal-humulone, iso-co-humu- thylpentane layer absorb ultraviolet (UV) light in the 275 nm
lone, and iso-ad-humulone. region.[21] Although the IBU value is believed to reflect the
Hops are susceptible to oxygen degradation during stor- concentration of IAA, this method measures IAA, AA, b-acid,
age, and oxidative polar compounds (OPCs) are present in and OPCs, including polyphenols in beer[9,11–14,22] and the
freshly baled, aged, or improperly stored hops in varying sum of these compounds is then incorporated in the IBU cal-
levels.[1,2] It had been reported that OPCs are composed of culation. Hahn et al.[11] examined the components that influ-
many kinds of hop-derived compounds, such as oxidative ence sensory bitterness perception in highly hopped beers and
degradation products of bitter acids, polyphenols, fatty acids concluded that the IBU cannot predict bitterness in a linear
and aroma components.[1,3–9] Hao et al. reviewed the struc- response to sensory perception. They proposed an alternative
model for predicting bitterness based on isohumulones,
tures and suggested the formation mechanism and distribu-
humulinones, and ethanol concentrations.
tion profiles of a total of 39 oxidative substances, including
In this study, worts with different compositions, contain-
15 derived from AA, 15 from IAA, and 9 from b-acid.[10] ing normal and highly oxidized hops, and different degrees
These components have impacts on the sensory bitterness of of isomerization ratio, were prepared and fermented. Then,
beer,[9,11–14] though their contributions vary. It was reported the impact of the components contributing to the IBU value
that the bitterness intensity of OPCs were lower,[9,15–18] and of the wort and beer were examined.
Algazzali and Shellhammer reported that oxidized hop acids, In the sensory evaluation, the variations among interindi-
hulupones, and humulinones are 84% and 66% as bitter as vidual panelists are the large issue. Humans perceive at least
isohumulones, respectively.[9] five taste modalities, as sweet, salty, sour, bitter, and umami.

CONTACT Toru Kishimoto t.kishimoto@nrib.go.jp

ß 2021 American Society of Brewing Chemists, Inc.
2 T. KISHIMOTO ET AL.

Individual differences in taste appear to be caused by alcohol content and apparent extracts were analyzed using
physiological differences in the gustatory system, cognitive Alcolyzer Beer ME equipped with DMA4500M density
processing of taste signals in the brain, genetics, or environ- meter (Anton PaarVR GmbH, Graz, Austria).

mental influences, and large variations occur in the percep-

tion of bitterness and umami.[23–26] To elucidate the issue, a
Measurements of IBU values
taste sensing system with less variability was adopted in this
study. This particular sensor does not simply respond to a The IBU values of wort and beers were analyzed according
concentration of a substance but to the intensity of the bitter- to the method defined by the American Society of Brewing
ness, and the higher correlation with organoleptic evaluation Chemists (Method Beer-23A)[19] and the European Brewery
and the taste sensing system has been previously studied in Convention (Method 9.6).[20] To 10 mL of wort or beer,
detail.[27] The bitterness intensity of the finished beers were 0.5 mL of 6 mol/L hydrochloric acid and 20 mL of 2,2,4-tri-
compared using this instrumental sensory method. methylpentane were added. The mixture was shaken vigor-
ously for 15 min on a wrist action shaker, and then
centrifuged at 1580  g for 5 min at room temperature. The
Experimental 2,2,4-trimethylpentane layer was subjected to an IBU meas-
Reagents and materials for brewing urement, followed by HPLC analysis.
The absorbance of the 2,2,4-trimethylpentane layer at
HPLC analysis standards of IAA (DCHA - ISO Standard) 275 nm was measured by a spectrophotometer, using
and AA (ICE-4 Hops Extract Standard) were purchased pure 2,2,4-trimethylpentane as a control, and then the
from the American Society of Brewing Chemists IBU values were calculated by multiplying the absorb-
(Minnesota, U.S.A.). The chemicals 2,2,4-trimethylpentane, ance by 50.
toluene, 6 mol/L hydrochloric acid, and methanol for
HPLC analysis, phosphoric acid, and 10% tetraethylammo-
nium hydroxide solution were purchased from FUJIFILM HPLC analysis of bitter components in beer
Wako Pure Chemical Corporation (Osaka, Japan). The The 2,2,4-trimethylpentane layer, filtered with a 0.5 lm
malt extract was obtained from Coopers Brewery PTFE membrane filter, was subjected to HPLC analysis.
(Australia). Bottom-fermenting sludgy yeast was purchased The HPLC (Shimadzu, Kyoto, Japan) was connected to a
from the Chuetsu Yeast Co., Ltd. (Niigata, Japan). Hop pel- UV detector set at 275 nm using a previously described
lets of Hallertau Magnum, containing 14% a-acid, was pur- method with slight modifications.[7] The mobile phase was
chased from MoreFlavor, Inc. (Pittsburg, CA, U.S.A.). prepared as follows: 2800 mL of methanol was added to
Forcibly deteriorated hops were prepared by storing the 1200 mL of pure distilled water containing 150 mL of 10%
Hallertau Magnum hop pellets at 40
C in opened alumi- tetraethylammonium hydroxide solution and 150 g of phos-
num bags and sampled at the 3rd, 5th, 7th, 10th, 14th, 21st, phoric acid. Twenty microliters of sample volume was
30th, and 90th day from the start of storage. injected, and the separation was performed on a Sim-pack
CLC-ODS/H column (250 mm length x 4.6 mm i.d.;
Small scale brewing processes Shimadzu) set at 40
C with a flow rate of 1.0 mL/min. The
analytical HPLC column was washed using pure distilled
Malt extract (250 g) was dissolved in 1.5 L of distilled water and 0.05% phosphoric acid for every analysis. The
water. Boiling processes were conducted on a two-liter concentrations of AA and IAA were calculated with the
scale after the adjustment of the pH to 5.1 and the con- absolute calibration curve method using the areas of each
centration of extract to 11.6%. At the beginning of the analytical peak and those of the authentic standards,
wort boiling processes, 2.1 g of hop pellet was added to DCHA-ISO Standard and ICE-4 Hops Extract Standard.
1.5 L of wort. The wort was boiled for 60 min with an
evaporation rate of 8.0% per hour. Apart from the above
boiling experiment, the wort temperatures were kept at HPLC analysis of bitter components in hop pellets
20, 30, 40, 50, 60, 70, 80, and 90
C for 60 min to examine The bitter components were extracted from 5 g of milled
the isomerization ratio. The worts were then cooled hop pellet using 100 mL of toluene for 2 h at room tempera-
immediately to 4
C to prevent further isomerization, and ture. Thereafter, 50 lL of each toluene extract was evapo-
the water to replenish the evaporated amount was added rated and dried under the flow of N2 gas in a tube. Then
after the boiling process. 2500 lL of methanol was added to the tube to dissolve the
The wort (850 mL) was fermented on a one-liter scale. bitter compounds.
The sludgy yeast was washed two times by suspending it in The methanol layer, filtered with a 0.5 lm PTFE mem-
1 L of unhopped wort and centrifuging at 4700  g. After brane filter, was analyzed using an HPLC system
centrifugation, 4 g of the precipitated yeast was added to (Shimadzu, Kyoto, Japan) connected to a UV detector set
850 mL of hopped wort and fermented for 7 days at 10
C at 302 nm. The mobile phase was prepared as follows:
with stirring at a speed of 1000 rpm. After 7 days of fermen- 800 mL of methanol was added to 200 mL of pure distilled
tation, the beer was centrifuged at 10,000  g for 30 min. water, containing 29.5 mL of 10% tetraethylammonium
The supernatants were subjected to further analysis. The hydroxide solution and 17 g of phosphoric acid. Ten lL of
 JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 3

Figure 1. HPLC chromatograms of the beer extracts with (A) normally stored hop pellet at 4
C, (B) hop pellets stored at 40
C for 10 days, (C) 21 days, (D) 90 days,
and (E) unhopped beer.

Measurement of bitterness intensity using a taste

sample was injected and separation was performed on a
sensing system
Sim-pack CLC-ODS/H column (250 mm length x 4.6 mm
i.d.; Shimadzu) set at 40
C with a flow rate of 1.0 mL/min. Prior to analysis by the taste sensing system, the IBU values of
The analytical HPLC column was washed using pure dis- all the sample beers were adjusted to 40 IBU using unhopped
tilled water and 0.05% of phosphoric acid for every ana- beer. Bitterness intensity values were measured by the SA402
lysis. The concentrations of AA and IAA were calculated taste sensing system (Intelligent Sensor Technology, Inc.,
by the absolute calibration curve method using the areas of Kanagawa, Japan), fitted with a sensor probe for bitter taste
each analytical peak and those of the authentic standards, (SB2C00) and a reference probe.[28] The taste sensor probe
ICE-4 Hops Extract Standard. consisted of a polymer membrane, an Ag/AgCl electrode, and
4 T. KISHIMOTO ET AL.

Figure 2. HPLC chromatograms of the wort extracts (F) boiled at 100
C for 60 min, (G) kept at 80
C, (H) 50
C, (I) 20
C for 60 min after the addition of normally
stored hop pellet, and (J) unhopped wort.

an internal cavity filled with 3.3 M KCl aqueous solution satu- were dipped into sample solutions or standard substance
rated with AgCl. The polymer membrane of the bitter taste solutions for 30 s to detect the change in membrane
sensor was composed of tetradodecylammonium bromide, 2- potential. The bitter taste intensity of the sample solution
nitrophenyl octyl ether, and poly vinyl chloride. The reference was defined as the difference between the change in mem-
probe consisted of a liquid junction made with ceramics, an brane potential in the sample solution and that in the ref-
Ag/AgCl electrode, and an internal cavity filled with 3.3 M KCl erence solution, generated by taste substances adsorbed
aq. solution, saturated with AgCl. on the polymer membrane of the sensor probe. The value
The sensor measurement was automatically carried out of each sample was obtained from the average of three
at 25
C. The taste sensor probe and the reference probe measurements.
 JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 5

4.2
4.2
4.2
4.2
4.2
4.2
4.2
4.2
4.2
4.1
pH
extract (%)
Apparent

2.4
2.3
2.3
2.3
2.4
2.4
2.4
2.3
2.4
2.4
Alcohol (%)
4.6
4.6
4.6
4.6
4.6
4.5
4.6
4.5
4.7
4.8
of AA to IBU value
Contribution (%)

14.6
14.6
14.3
14.2
15.6
14.0
10.0
4.8
3.1
Figure 3. Correlations between the total peak areas of HPLC chromatograms

-　
and IBU values of wort and beer extracts.

Beer
Results and discussion
Components contributing to the IBU value

(ppm)
3.3
3.2
3.2
3.3
3.4
4.2
4.1
2.8
2.5
AA

0
Figure 1 and Figure 2 show the HPLC chromatograms of
the components in 2,2,4-trimethylpentane layers that con-
tribute to the IBU value of beer. No chromatographic peaks

(ppm)
7.2
6.9
7.1
7.4
7.3
11.1
17.2
28.5
39.9
IAA
were detected outside the chromatographic time. Figure 3

0
shows the correlation between the total peak areas of the
HPLC chromatograms and the IBU values of wort and beer.
The total peak areas were proportional to the IBU value of
IBU decrease during
fermentation (%)
wort and beer as shown in Figure 3, indicating that the IBU
values were attributed to the components included in the
43.5
42.7
44.2
43.5
44.2
36.7
30.2
23.3
14.9

Wort was kept at each temperature for 60 min, after the adjustment of pH to 5.1 and apparent extract to 11.6.
Table 1. Concentrations of iso-a-acid and a-acid in wort and beer produced at different boiling temperatures.

　
chromatogram in Figure 1 and 2.
The peaks of IAA (iso-co-humulone, iso-normal-humu-
lone, and iso-ad-humulone) and AA (co-humulone, normal

The contribution (%) of AA to IBU value was calculated from the peak area of HPLC chromatograms.
and ad-humulone) were identified by comparing those of
authentic compounds. As shown in Figure 1(C and D), the
19.1
19.1
18.7
19.5
20.0
22.6
27.8
38.4
47.5
3.7
IBU

fractions of polar components that appeared before the peak

of iso-co-humulone increased as hop deterioration pro-
gressed and were identified as OPCs in previous reports.[7,8]
of AA to IBU value
Contribution (%)

As shown in Table 1, unhopped beer and wort have IBU

values of 3.7 and 5.6, respectively, and slight amounts of
56.5
56.2
56.8
55.1
51.6
44.0
35.8
21.2
11.9
-　

malt-derived polar components contributed to the IBU

value, as depicted in Figure 1(E) and 2(J). Thus, the IBU
values of wort and beer were mainly attributed to IAA, AA,
OPCs, and malt-derived polar components. Almost all the
AA disappeared during fermentation (Figures 1 and 2).
Wort

(ppm)
27.3
27.5
26.9
26.8
26.2
23.6
23.2
18.1
10.3
AA

The effect of non-isomerized a-acid during fermentation

(ppm)
6.9
7.0
6.6
7.3
8.7
11.8
18.7
34.4
40.1
IAA

The contribution of non-isomerized AA to the IBU value of

0

wort and beers has been described above. Worts with differ-
ent isomerization ratios were prepared by changing the iso-
merization temperature. The hopped wort was kept at 20,
33.7
33.4
33.4
34.5
35.8
35.7
39.9
50.1
55.8
5.6
IBU

30, 40, 50, 60, 70, 80, 90, and 100
C for 60 min.
Table 1 shows the concentrations of IAA and AA in wort
and beer produced at different boiling temperatures. Figure
temperature (
C)

4 shows the correlation between the boiling temperature and

unhopped

isomerization ratio of AA in worts. The isomerization ratio

was calculated by the formula; IAA (mg)/(AA (mg) þ IAA
Boiling

100
20
30
40
50
60
70
80
90

(mg)), as the ratio of the homologues (normal, co, ad-form)

6 T. KISHIMOTO ET AL.

and the molecular weight of the AA does not change after during fermentation as in Table 1, although those of IAA
the isomerization. As shown in Figure 4 and Table 1, the were stable before and after fermentation. Thus, the decrease
isomerization ratio and IAA concentration of beer increased in the IBU value during fermentation was mainly caused by
from the boiling temperature of 60
C to 70
C and the the disappearance of AA, and the isomerization ratio influ-
increase in ratio is accelerated at higher temperatures. The enced the decrease in the IBU value during fermentation.
wort kept at 20
C already contained 6.9 ppm of IAA, which The reasons for the disappearance of hydrophobic compo-
was assumed to have been derived from the drying process nents are discussed in the literature.[29–32] As the pH
or pelletizing process of hop cones as no IAA was detected decreases during fermentation, hydrophobic components
from the undried fresh hop cone (data not shown). such as AA become insoluble in the beer. These hydropho-
As shown in Table 1, for the wort boiled at 20
C to bic components also adhere to yeast cell walls; moreover,

the degree of adsorption to the cell surface depends on the
60 C, over 50% of the IBU value was contributed by the
AA content. Larger decreases in IBU values were observed degree of hydrophobicity of the cell surface.[31]
during fermentation in the wort with the lower isomeriza- Figure 4 shows that isomerization started at a tempera-
tion ratio, as depicted in Figure 1(A, F), and Table 1. The ture between 60
C and 70
C. This indicates that isomeriza-
concentration of AA in each wort dramatically decreased tion progresses not only in the wort boiling process, but
also in the subsequent whirlpool process, wherein the wort
is exposed to higher temperatures. In order to control the
bitterness value of the final beer product, the time spent in
the whirlpool rest process should also be considered.

Content of OPCs in beer depends on the degradation of

used hops
The contribution of OPCs to the IBU values of wort and beers
are as mentioned earlier. Worts with different ratios of OPCs
were prepared using hops with different degrees of oxidization.
Hop pellets were stored at 40
C in order to prepare forcibly
deteriorated hops, and they were sampled at the 3rd, 5th, 7th,
10th, 14th, 21st, 30th, and 90th day from the start of storage.
Table 2 shows the concentration of the bitter compo-
Figure 4. Correlations between boiling temperatures and isomerization ratio of nents in beer hopped with forcibly deteriorated pellet. As
a-acid. The wort was kept at each temperature for 60 min after the addition of shown in Table 2 and Table 3, as the storage period of the
hops. Isomerization ratios were calculated by the formula; iso-a-acid (ppm)/(iso-
a-acid (ppm) þ a-acid (ppm)).
hopped pellet became longer, the AA content in the hop
pellet, and the corresponding IAA in beer, decreased, and

Table 2. Content of bitter components of beer hopped with forcibly deteriorated hop pellet.
Storage periods a-acid (%) IBU of iso-co- iso-normal- iso-ad- normal,
of hop pellet content in finished humulone humulone humulone co-humulone ad-humulone
at 40
C hopped pellet beer (ppm) (ppm) (ppm) (ppm) (ppm)
0 day 14.0 47.5 12.9 21.6 5.3 1.3 1.2
3 days 13.4 48.2 11.4 19.1 4.6 0.8 0.8
5 days 12.8 48.8 11.8 19.3 4.6 1.0 0.9
7 days 12.1 47.1 12.4 20.4 4.9 0.9 0.8
10 days 10.9 49.9 11.4 18.6 4.6 0.9 0.9
14 days 9.8 48.9 11.0 17.9 4.4 0.8 0.7
21 days 4.4 54.7 6.6 10.5 2.6 0.4 0.4
30 days 1.2 51.1 3.4 5.1 1.3 0.1 0.1
90 days 0.0 49.9 1.0 1.0 0.2 0.0 0.0
The wort, including 2.1 g of each stored hop pellet, was boiled for 60 min and fermented for 7 days.

Table 3. Contributions (%) to IBU value of beer calculated from peak area of HPLC chromatograms.
Storage periods of hop pellet at 40
C Malt-derived compounds (%) Oxidative polar components (%) iso-a-acid (%) a-acid (%)
0 day 7.7 8.4 80.7 3.3
3 days 7.6 9.3 80.7 2.4
5 days 7.5 9.5 80.3 2.8
7 days 7.8 10.7 79.2 2.3
10 days 7.3 12.2 78.0 2.5
14 days 7.5 13.6 76.7 2.3
21 days 6.7 36.9 55.0 1.4
30 days 7.1 59.1 33.5 0.3
90 days 7.3 83.4 9.3 0.0
The contribution was calculated from the ratio of the area of each peak to the total peaks.
 JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 7

Figure 5. The bitterness intensity evaluated by taste sensing system and total iso-a-acid content. The IBU values of all beers were adjusted to the same 40 IBU
using unhopped beer and were subjected to taste sensing analysis. The bar graph shows the bitterness intensity by the taste sensing system, and the line graph
shows the iso-alpha-acid content (ppm) after adjusting the bitterness value to 40 IBU.

the ratio of the OPC (%) in the total peak area of the chro- for 21, 30, and 90 days, where the total IAA also decreased
matogram of beer, increased. However, the IBU values of markedly. It was found that the bitterness intensities of the
the beer did not vary, as it depended on the storage peri- beer composed of larger ratios of OPCs were lower. Our
ods of the hopped pellet, and the contents of IAA and observations are consistent with previous reports that beers
OPCs did not decrease during fermentation to contribute made with deteriorated hops have less bitterness than that
to the IBU values of beer. It should be noted that the beer expected from the IBU values.[9,22] This is because the bit-
derived from the hop, which was forcibly deteriorated at terness intensities derived from OPCs are lower as the
40
C for 90 days, contained only 2.2 ppm of IAA and threshold levels of OPCs are generally higher.[9,15–18]
an IBU value of 49.9. The beer with normally stored hop
had almost the same IBU value and contained 39.9 ppm
of IAA.
Conclusions
The IBU value had been believed to reflect the concentration
of iso-a-acid, however, in the current study, samples with
Evaluation of bitterness intensity of beers with forcibly
disparate iso-a-acid content were analyzed: one was a beer
deteriorated hop pellets using a taste sensing system
sample with 47.5 IBU containing 39.9 ppm iso-a-acid, and
The intensity of bitterness is expected to vary, even in beers the other was a beer sample with 49.9 IBU containing only
with the same IBU values: while some beers have IBU values 2.2 ppm iso-alpha acid. The IBU value of the wort and beer
mainly composed of IAA, the others have IBU values mainly is mainly contributed by iso-a-acid, non-isomerized a-acid,
composed of OPCs. The bitterness intensity of the beers OPCs, and malt-derived polar components. It was observed
hopped with oxidized hops was evaluated using an elec- that the isomerization of a-acid to iso-a-acid was initiated
tronic tongue, a taste sensing system, after the adjustment of between 60
C to 70
C and the isomerization ratios acceler-
their IBU value to 40 using unhopped beer. ated rapidly at higher temperatures. In the wort boiled at
The taste sensing system is an instrumental method for temperatures below 60
C, over 50% of the IBU value was
evaluating the taste attributes of food and beverages, such as contributed by non-isomerized a-acid, and most of the non-
beer.[27,28,33–37] The electronic tongue of the taste sensing isomerized a-acid disappeared during fermentation.
system consists of sensor electrodes with lipid/polymer However, the concentration of iso-a-acid was stable. Thus,
membranes prepared for each primary taste.[28] The taste the disappearance of the a-acid resulted in the larger
intensities are recorded as membrane potential changes decrease in IBU value during fermentation, and the isomer-
caused by the interaction between the lipid/polymer mem- ization ratio influenced the degree of the decrease.
branes and the taste substances. A difference of 0.5 in the The OPCs that increase with the deterioration of hop
response value between samples, from the taste sensing sys- contribute to the IBU value of beer. When non-deteriorated
tem, can be recognized by a panelist who is accustomed to hop with small amounts of OPCs were used in brewing, the
drinking beer. If there is a difference of 1.0 in the response IBU was mainly contributed by IAA and only 8.4% was con-
value between samples, ordinary people can recognize the tributed by the OPCs. However, in the beer hopped with the
difference in bitterness intensity.[27] pellet stored at 40
C for 90 days, over 80% of the IBU value
Figure 5 shows the bitterness intensity evaluated by the was contributed by the OPCs. The bitterness intensity of
taste sensing system and the total IAA content. The bitter- beers hopped with deteriorated hops were examined using
ness intensity largely decreased in the beer with hops stored the taste sensing system. The beers with the deteriorated
8 T. KISHIMOTO ET AL.

hop had lesser bitterness intensity, despite having the same [14] Shellhammer, T. H.; McLaughlin, I. R.; Lederer, C. Bitterness
IBU values as the beer with non-deteriorated hop. Modifying Properties of Hop Polyphenols Extracted from Spent
Hop Material. J. Am. Soc. Brew. Chem. 2008, 66, 174–183. DOI:
10.1094/ASBCJ-2008-0619-01.
Disclosure statement [15] Intelmann, D.; Kummerlowe, G.; Haseleu, G.; Desmer, N.;
Schulze, K.; Frohlich, R.; Frank, O.; Luy, B.; Hofmann, T.
No potential competing interest was reported by the authors. Structures of Storage-Induced Transformation Products of the
Beer’s Bitter Principles, Revealed by Sophisticated NMR
Spectroscopic and LC-MS Techniques. Chemistry. 2009, 15,
ORCID 13047–13058. DOI: 10.1002/chem.200902058.
[16] Haseleu, G.; Intelmann, D.; Hofmann, T. Identification and RP-
Toru Kishimoto http://orcid.org/0000-0003-4556-0709 HPLC-ESI-MS/MS Quantitation of Bitter-Tasting Beta-Acid
Transformation Products in Beer. J. Agric. Food Chem. 2009,
57, 7480–7489. DOI: 10.1021/jf901759y.
Literature cited [17] Intelmann, D.; Batram, C.; Kuhn, C.; Haseleu, G.; Meyerhof,
M.; Hofmann, T. Three TAS2R Bitter Taste Receptors Mediate
[1] Maye, J. P.; Smith, R.; Leker, J. Humulinone Formation in
the Psychophysical Responses to Bitter Compounds of Hops
Hops and Hop Pellets and Its Implications for Dry Hopped
(Humulus lupulus L.) and Beer. Chem. Percept. 2009, 2,
Beers. Tech. Q. Master Brew. Assoc. Am. 2016, 53, 23–27.
118–132. DOI: 10.1007/s12078-009-9049-1.
[2] Oladokun, O.; Cowley, S. J.; Smart, K.; Hort, J.; Cook, D. Dry-
[18] Haseleu, G.; Intelmann, D.; Hofmann, T. Structure
Hopping: The Effects of Temperature and Hop Variety on the
Determination and Sensory Evaluation of Novel Bitter
Bittering Profiles and Properties of Resultant Beers.
Compounds Formed from b-Acid of Hop (Humulus lupulus L.)
BrewingScience. 2017, 70, 187–196.
upon Wort Boiling. Food Chem. 2009, 116, 71–81. DOI: 10.
[3] Haseleu, G.; Lagemann, A.; Stephan, A.; Intelmann, D.; Dunkel,
1016/j.foodchem.2009.02.008.
A.; Hofmann, T. Quantitative Sensomics Profiling of Hop-
[19] American Society of Brewing Chemists. Method Beer-23A,
Derived Bitter Compounds throughout a Full-Scale Beer
Methods of Analysis of the ASBC, 8th ed.; Minnesota: American
Manufacturing Process. J Agric Food Chem. 2010, 58,
Society of Brewing Chemists, 1992.
7930–7939. DOI: 10.1021/jf101326v. [20] European Brewery Convention. Analytica-EBC Method 9.6, 4th
[4] Dresel, M.; Dunkel, A.; Hofmann, T. Sensomics Analysis of Key ed.; Brauerei- und Getr€anke-Rundschau: Zurich, Switzerland.
Bitter Compounds in the Hard Resin of Hops (Humulus lupu- 1987; E155.
lus L.) and Their Contribution to the Bitter Profile of Pilsner- [21] University of Toronto Scarborough. Interpreting UV-Vis
Type Beer. J Agric Food Chem. 2015, 63, 3402–3418. DOI: 10. Spectra, In Chemistry Online, https://www.utsc.utoronto.ca/
1021/acs.jafc.5b00239. webapps/chemistryonline/production/uv/uv_spectra.php.
[5] Kishimoto, T.; Wanikawa, A.; Kono, K.; Aoki, K. Odorants [22] Peacock, V. The International Bitterness Unit, Its Creation and
Comprising Hop Aroma of Beer: hop-Derived Odorants What It Measures. In Hop Flavor and Aroma, Proceedings of
Increased in the Beer Hopped with Aged Hops. In Proc. Eur. the 1st International Brewers Symposium, Master Brewers
Brew. Conv. Congr.Venice, Italy. CD Fachverlag Hans Carl: Association of the Americas: Minnesota, 2009; pp 157–166.
N€urenberg, 2007; pp 226–235. [23] Puputti, S.; Aisala, H.; Hoppu, U.; Sandell, M. Factors
[6] Taniguchi, Y.; Matsukura, Y.; Ozaki, H.; Nishimura, K.; Shindo, Explaining Individual Differences in Taste Sensitivity and Taste
K. Identification and Quantification of the Oxidation Products Modality Recognition among Finnish Adults. J. Sens. Stud.
Derived from a-Acids and b-Acids during Storage of Hops 2019, 34, e12506. DOI: 10.1111/joss.12506.
(Humulus lupulus L.). J Agric Food Chem. 2013, 61, 3121–3130. [24] Knaapila, A.; Hwang, L. D.; Lysenko, A.; Duke, F. F.; Fesi, B.;
DOI: 10.1021/jf3047187. Khoshnevisan, A.; James, R. S.; Wysocki, C. J.; Rhyu, M.;
[7] Ono, M.; Kakudo, Y.; Nagami, K.; Kumada, J.; Ueda, S. Tordoff, M. G.; et al. Genetic Analysis of Chemosensory Traits
Simultaneous Analysis of Hop Bittering Components by High- in Human Twins. Chem. Senses. 2012, 37, 869–881. DOI: 10.
Performance Liquid Chromatography. III. Development of on- 1093/chemse/bjs070.
Line Sample Enrichment by Precolumn Switching. J. Am. Soc. [25] Lugaz, O.; Pillias, A.-M.; Faurion, A. A. A New Specific
Brew. Chem. 1987, 45, 70–76. DOI: 10.1094/ASBCJ-45-0070. Ageusia: Some Humans Cannot Taste L-Glutamate. Chem.
[8] Kowaka, M.; Fukuoka, Y.; Kawasaki, H.; Asano, K. The True Senses. 2002, 27, 105–115. DOI: 10.1093/chemse/27.2.105.
Value of Aroma Hops in Brewing. In Proc. Eur. Brew. Conv. [26] Puputti, S.; Aisala, H.; Hoppu, U.; Sandell, M. Multidimensional
Congr., London UK. IRL Press: Oxford, 1983; pp 71–78. Measurement of Individual Differences in Taste Perception.
[9] Algazzali, V. A.; Shellhammer, T. H. Bitterness Intensity of Food Qual. Pref. 2018, 65, 10–17. DOI: 10.1016/j.foodqual.2017.
Oxidized Hop Acid: Humulinones and Hulupones. J. Am. Soc. 12.006.
Brew. Chem. 2016, 74, 36–43. [27] Kobayashi, Y.; Habara, M.; Ikezazki, H.; Chen, R.; Naito, Y.;
[10] Hao, J.; Speers, R. A.; Fan, H.; Deng, Y.; Dai, Z. A Review of Toko, K. Advanced Taste Sensors Based on Artificial Lipids
Cyclic and Oxidative Bitter Derivatives of Alpha, Iso-Alpha and with Global Selectivity to Basic Taste Qualities and High
Beta-Hop Acid. J. Am. Soc. Brew. Chem. 2020, 78, 89–102. Correlation to Sensory Scores. Sensors (Basel). 2010, 10,
DOI: 10.1080/03610470.2020.1712641. 3411–3443. DOI: 10.3390/s100403411.
[11] Hahn, C. D.; Lafontaine, S. R.; Pereira, C. B.; Shellhammer, [28] Toko, K. Taste Sensors. In Biomimetic Sensor Technology;
T. H. Evaluation of Nonvolatile Chemistry Affecting Sensory Cambridge University Press: Cambridge, UK, 2000; pp
Bitterness Intensity of Highly Hopped Beers. J. Agric Food 113–180.
Chem. 2018, 66, 3505–3513. DOI: 10.1021/acs.jafc.7b05784. [29] Popescu, V.; Soceanu, A.; Dobrinas, S.; Stanciu, G. A Study of
[12] Malowicki, M. G.; Shellhammer, T. H. Isomerization and Beer Bitterness Loss during the Various Stages of the Romanian
Degradation Kinetics of Hop (Humulus lupulus) acids in a Beer Production Process. J. Inst. Brew. 2013, 119, 111–115.
model wort-boiling system. J. Agric Food Chem. 2005, 53, DOI: 10.1002/jib.82.
4434–4439. DOI: 10.1021/jf0481296. [30] Kishimoto, T.; Wanikawa, A.; Kono, K.; Shibata, K.
[13] Maye, J. P.; Smith, R. Dry Hopping and Its Effects on the Comparison of the Odor-Active Compounds in Unhopped Beer
International Bitter Unit Test and Beer Bitterness. Tech. Q. and Beers Hopped with Different Hop Varieties. J Agric Food
Master Brew. Assoc. Am. 2016, 53, 134–136. Chem. 2006, 54, 8855–8861.
 JOURNAL OF THE AMERICAN SOCIETY OF BREWING CHEMISTS 9

[31] Kishimoto, T.; Uemura, K.; Aizawa, M. A New Insight for with High Selectivity and Sensitivity to the Bitterness of Drugs
Controlling the Hop Aroma Using Hydrophobicity of Yeast and with High Correlation with Sensory Score. IEEJ Trans.
Cell Surface. In Proc. Eur. Brew. Conv. Congr., Luxembourg, CD Elec. Electron. Eng. 2009, 4, 710–719. DOI: 10.1002/tee.20469.
Fachverlag Hans Carl: N€ urenberg, 2013. DOI: 10.13140/RG.2.2. [35] Takagi, S.; Toko, K.; Wada, K.; Ohki, T. Quantification of
34844.64643. Suppression of Bitterness Using an Electronic Tongue. J.
[32] Haslbeck, K.; Bub, S.; Sch€onberger, C.; Zarnkow, M.; Jacob, F.; Pharm. Sci. 2001, 90, 2042–2048. DOI: 10.1002/jps.1155.abs.
Coelhan, M. On the Fate of b-Myrcene during Fermentation – [36] Hayashi, N.; Ujihara, T.; Chen, R.; Irie, K.; Ikezaki, H.
The Role of Stripping and Uptake of Hop Oil Components by Objective Evaluation Methods for the Bitter and Astringent
Brewer’s Yeast in Dry-Hopped Wort and Beer. Brew. Sci. 2017, Taste Intensities of Black and Oolong Teas by a Taste Sensor.
70, 159–169. Food Res. Int 2013, 53, 816–821. DOI: 10.1016/j.foodres.2013.
[33] Ezaki, S.; Yuki, T.; Toko, K.; Tsuda, Y.; Nakatani, K. Analysis 01.017.
of Taste Qualities and Ingredients of Beer by Taste Sensing [37] Hayashi, N.; Chen, R.; Hiraoka, M.; Ujihara, T.; Ikezaki, H.
System. IEEJ Transsm. 1997, 117, 449–455. DOI: 10.1541/ieejs- Beta-Cyclodextrin/Surface Plasmon Resonance Detection
mas.117.449. System for Sensing Bitter-Astringent Taste Intensity of Green
[34] Kobayashi, Y.; Hamada, H.; Yamaguchi, Y.; Ikezaki, H.; Toko, Tea Catechins. J Agric Food Chem. 2010, 58, 8351–8356. DOI:
K. Development of an Artificial Lipid-Based Membrane Sensor 10.1021/jf1012693.