Effect of oxidized arachidonic acid and hexanal on the mouse taste perception of bitterness and umami

Article abstract of DOI:10.1271/bbb.100277

The oxidization of fatty acids generates many volatile compounds forming an aroma, but little is known whether mammals use gustatory sense to detect the oxidized products as a taste or only use olfactory sense to detect as an aroma. We examined in this study the effect of aqueous extracts of the compounds from autoxidized arachidonic acid (AA) ethyl ester or hexanal which is the predominant component generated from oxidized AA by the anosmic mouse licking performance to a tastant. The addition of the water extract from oxidized AA or hexanal to a quinine hydrochloride (QHC1) solution decreased the anosmic mice licking frequency at several concentrations of QHC1. Hexanal also reduced the licking frequency of anosmic mice conditioned to avoid MSG at several concentrations of monosodium glutamate (MSG). These results suggest that hexanal would affect mouse taste perception to QHC1 and MSG via the gustatory sensation.

Full text of DOI:10.1271/bbb.100277

Biosci. Biotechnol. Biochem., 74 (9), 1884–1890, 2010
Effect of Oxidized Arachidonic Acid and Hexanal
on the Mouse Taste Perception of Bitterness and Umami
Susumu YAMAGUCHI,1;2 Hidenori FUJIWARA,2;3 Ikukazu TASHIMA,2
y
1;3;
Daigo IWANAGA,1 and Hideki USHIO
1Laboratory of Food Function, Department of Food Science and Technology, Tokyo University
of Marine Science and Technology, 5-7 Konan, Minato-ku, Tokyo 108-8477, Japan
2Oil and Fats Laboratory, J-Oil Mills, Inc., 7-41 Daikokucho, Tsurumi, Yokohama, Kanagawa 230-0053, Japan
3Laboratory of Marine Biochemistry, Department of Aquatic Bioscience, Graduate School
of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received April 13, 2010; Accepted May 31, 2010; Online Publication, September 7, 2010
[doi:10.1271/bbb.100277]
The oxidization of fatty acids generates many volatile
compounds forming an aroma, but little is known
whether mammals use gustatory sense to detect the
oxidized products as a taste or only use olfactory sense
to detect as an aroma. We examined in this study the
effect of aqueous extracts of the compounds from
autoxidized arachidonic acid (AA) ethyl ester or hexanal
which is the predominant component generated from
oxidized AA by the anosmic mouse licking performance
to a tastant. The addition of the water extract from
oxidized AA or hexanal to a quinine hydrochloride
(QHCl) solution decreased the anosmic mice licking
frequency at several concentrations of QHCl. Hexanal
also reduced the licking frequency of anosmic mice
conditioned to avoid MSG at several concentrations of
monosodium glutamate (MSG). These results suggest
that hexanal would affect mouse taste perception to
QHCl and MSG via the gustatory sensation.
Rodents are generally extremely sensitive to odorants12)
and therefore the volatile compounds generated from
oxidized oil could affect their choice of oil. Both the
aroma and postingestively nutritive effects of oil could
reinforce the aroma preference, and the appetite for
dietary fats and oils would be a combined response to
the aroma and nutritive qualities of this class of
nutrient.13) The aroma qualities of several compounds
generated from oxidized fatty acids have been charac-
terized by many studies,14–16) although few studies have
reported the taste of oxidized oil. A human sensory test
to compare the effect on taste of diluted soy sauce
among several autoxidized fatty acids has indicated that
only oxidized arachidonic acid (AA) enhanced the
bitterness of the soy sauce.17) The results interestingly
suggest that autoxidized fatty acids should affect the
bitter taste perception, but it is not clear whether the
effect was induced by oxidized AA via the gustatory or
olfactory sensation of the panelists. We focused this
present study on the effect of oxidized AA on the bitter
taste sensation, and studied whether oxidized AA or the
compounds generated from oxidized AA affected the
bitter tastants via only the olfactory sensation or
gustatory sensation by using a behavioral study on
olfactory-blocked mice. We will show that hexanal, one
of the predominant components generated from oxidized
AA, affected the mouse gustatory sensation to quinine
hydrochloride (QHCl), a classic bitter taste,18) and that
monosodium glutamate (MSG) had an effect on the
classic umami taste.19–22)
Key words: arachidonic acid; hexanal; quinine hydro-
chloride; monosodium glutamate; taste
perception
Dietary fats and oils are composed of triacylglycerol
and create high biological energy. Mammals might
evolutionally develop a chemical sensing system for
such high-energy compounds. Recent studies using
rodent behavioral analyses and electrophysiological
analyses of the gustatory nerve have suggested that fats
and oils should be implicated in gustatory sense and
have defined some mechanisms behind the gustatory
sensation to the fats and oil.1–9) These studies have
indicated that free fatty acids hydrolyzed from triacyl-
glycerol in the oral cavity were involved in the detection
of oil and fat via the mammalian gustatory sensation.
Various changes occur in the chemical and physical
characteristics of fat and oil when it is oxidized,10)
including an increase in viscosity, development of color
and a decrease in its nutritional value. Many volatile
compounds derived from fatty acids are also generated
from the oxidized oils and express various aromas.11)
Materials and Methods
Animals. Eight to twelve weeks of age C57BL/6j male mice
(Nippon Clea, Tokyo, Japan) were used for all experimentation. These
mice were maintained under controlled conditions of a 12/12-h light-
dark cycle and a constant temperature of 23 ꢀ 3 ꢁC. All experiments
were conducted in accordance with the Guide for the Care and Use of
Laboratory Animals of Tokyo University of Marine Science and
Technology and were approved by the Committee for the Care and Use
of Laboratory Animals of Tokyo University of Marine Science and
Technology.
y
To whom correspondence should be addressed. Laboratory of Marine Biochemistry, Department of Aquatic Bioscience, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; Tel: +81-3-5841-5299; Fax: +81-3-
5841-8166; E-mail: aushio@mail.ecc.u-tokyo.ac.jp

Effect of AA and Hexanal on Mouse Taste Perception of QHCl and MSG
1885
Chemicals. Arachidonic acid ethyl ester (99% pure), hexanal
(>97% pure) and quinine hydrochloride (99% pure) were purchase
from Sigma Aldrich Japan (Tokyo, Japan). 1-Octen-3-ol (>97% pure),
2,4-decadienal (>90% pure) and lithium chloride (>99%) were from
Wako Chemicals (Osaka, Japan). 2-Pentylfuran (>98%), trans-2-
octenal (>95%) and trans-2-nonenal (>95%) was purchase from
Tokyo Chemical Industry (Tokyo, Japan). All other reagents used were
of analytical grade.
and 12 cm long) through a small opening (1:5 ꢂ 1:5 cm) in the cage
wall which could be automatically opened and closed at a set time by a
sliding door. The position of the bottle only enabled contact of the
spout with the mouse tongue during drinking. A direct-current
potential of 1.5 V was established between the copper grid on the
floor of the test chamber and a terminal fitted in the licking bottle. The
short-response latency of the detection circuit (<1 ms) provided a
precise measure of the start and end of licking. The licking signals
were captured via an NR2000 AD converter (Keyence, Osaka, Japan)
and input to a PC for analysis.
Autoxidation of AA ethyl. We used in this study ethyl ester of AA
for evaluating the AA-derived oxidized compounds in order to
eliminate the effects of fatty acids on the gustatory sense in mice as
reported by others.6–9) AA ethyl was autoxidized under the same
conditions as those in the previous report;17) briefly, 80 mg of AA ethyl
was pipetted into a glass bottle (ꢀ12 mm ꢂ 43 mm) and sealed with a
cap. The glass bottle was incubated in the dark for 24 h at 37 ꢁC.
Training the mice. The mice were trained to take the test fluid from
the spout of the licking bottle, after being deprived of water for 22 h
before training. On the first day of training, the sliding door was
opened and each mouse was placed in the test cage to give free access
to distilled water from a drinking tube for 1 h. On the second and third
days, each mouse was trained to drink distilled water on an interval
schedule providing a 10-s period during which distilled water was
available.
Analysis of oxidized AA ethyl. The peroxide value (PV) was
determined according to the AOCS method,23) and the carbonyl value
(CV) was determined by the standard method.24)
Olfactory blockage. The olfactory response was blocked 48 h before
the test session by infusing diethyl-ether anesthetized mice with 0.1 ml
of physiological saline containing 0.2 M ZnSO4 into each nasal cavity
with a blunted needle. Drainage to the pharynx was sucked into an
aspirator with a capillary pipette. The test session was conducted after
2 d of recovery with food and water provided ad libitum, and then
water was deprived for 22 h. It was confirmed that all mice used in the
licking test showed no aversion to NH3 gas prior to the test session.
Preparation of the water extract from oxidized AA ethyl. Oxidized
AA ethyl was suspended in a 1000-fold volume of distilled water. The
mixture of oxidized oil and water was agitated in a bath-type
ultrasonicator (W222, Honda, Tokyo) for 10 min. The mixture was
then centrifuged at 20;000 ꢂ g for 30 min, after which the water phase
was collected as a sample for the water-extracted compounds from
oxidized AA ethyl.
Fractionation of oxidized AA ethyl by column chromatography.
Silica gel column chromatography was used for fractionating the
oxidized AA ethyl. A chromatographic glass column with Wako gel
C-200 (ꢀ20 mm ꢂ 133 mm, 75–150 mm particle size; Wako, Tokyo,
Japan) was equilibrated with hexane. Two hundred milligrams of
oxidized AA ethyl was dissolved in 5 ml of hexane and loaded into
the glass column, and 150 ml of hexane was passed through the
column. The column was successively eluted with 150 ml each of
hexane:diethyl ether [70:30], hexane:diethyl ether [50:50], diethyl
ether, and ethanol. The eluting solvents were evaporated under vacuum
(R-114, Buchi, Switzerland) and dried with nitrogen gas to get four
fractions.
Licking frequency of the olfactory-blocked mice for QHCl with or
without the water extract from oxidized AA ethyl. In order to study the
effect of oxidized oil on the mouse detection of QHCl, the licking
frequency for a QHCl solution with or without the water extract from
oxidized AA ethyl was counted by the licking counter.
The licking frequency for distilled water and the water extract from
oxidized AA-ethyl by the olfactory-blocked mice was counted by the
licking counter. Each mouse was subjected to two test sessions. The
first was to count the licking frequency for distilled water and for the
0.05, 0.1, 0.3, 0.6 and 1.0 mM QHCl concentration series alone (n ¼ 8).
The other test session was to count the licking frequency for distilled
water and the concentration series of QHCl with the water extract from
oxidized AA ethyl (n ¼ 9). The number of licks of distilled water was
confirmed to be in the same range between the two test sessions. Each
concentration of the QHCl solution was randomly presented to the
mice. The licking ratio is the number of licks with the test stimulus
divided by the number of licks with distilled water for each mouse.
Each fractionated sample was suspended in 50 ml of distilled water.
The water phase was collected as the water-extracted sample from
fractionated oxidized AA ethyl after agitation and centrifugation as just
described.
GC-MS Analysis. The oxidized AA ethyl and column fractions were
analyzed with a 17A-GC chromatograph (Shimadzu, Kyoto, Japan)
equipped with a Supelco Wax10 capillary column (30 m ꢂ 0:25 mm
i.d.) and a QP-5000 (Shimadzu) mass spectrometer. Oxidized AA ethyl
was diluted 400 times with diethyl ether, and 0.5 ml was injected. A
5.0 mg amount of Fr. 1, 6.7 mg of Fr. 2, 9.5 mg of Fr. 3 and 2.2 mg of
Fr. 4 were each diluted by 2 ml of diethyl ether, and 0.5 ml was injected.
Helium was the carrier gas at flow rate of 1.8 ml/min, and the injector
port temperature was 230 ꢁC. The oven temperature was maintained at
40 ꢁC for 4 min, increased to 150 ꢁC at the rate of 30 ꢁC/min, then to
230 ꢁC at the rate of 5 ꢁC/min, and finally held for 10 min. The split
ratio was 1:50, the electron impact ionization voltage was 70 eV, and
the source temperature was 250 ꢁC.
Licking frequency of the olfactory-blocked mice for QHCl with or
without the water extract from fractionated oxidized AA ethyl. Each
fraction from oxidized AA ethyl was suspended in 50 ml of distilled
water, and a water extract from each fraction was prepared by the same
method as described above for the water extract from oxidized AA-
ethyl. The licking frequency of the olfactory blocked mice (n ¼ 6) to
QHCl with or without each water extract from the fraction of oxidized
AA ethyl was measured and the licking ratio calculated as described
above. Each mouse was tested in five sessions to give the licking
frequency for QHCl alone, and for QHCl with Fr. 1, Fr. 2, Fr. 3 and
Fr. 4. The licking frequency for distilled water was also tested in each
session. The number of licks for distilled water was confirmed to be in
the same range for each test session.
We identified hexanal, 1-octen-3-ol, 2,4-decadienal, 2-pentylfuran,
trans-2-octenal and trans-2-nonenal in each fractionated sample, these
substances having been reported as the main decomposition products
from oxidized AA,25) and the reagents for these compounds were
commercially available. These compounds were identified by compu-
terized matching of the acquired mass spectra with those stored in the
NIST107 mass spectral library of the GC-MS data system and by the
GC retention data for each reagent. These compounds and AA ethyl
contained in each fraction were quantitatively determined by using
linear calibration curves prepared from the peak area versus injected
amount of each reagent.
Licking frequency of the olfactory-blocked mice for QHCl with or
without hexanal. A 100 mM water solution of hexanal was prepared by
mixing 10 mg of hexanal with 1000 ml of distilled water and stirring by
a magnetic stirrer for 2 h at room temperature. The water solution was
centrifuged at 20;000 g for 30 min, and the water phase was collected
and used as a 100 mM hexanal solution. The resulting hexanal solution
was diluted with distilled water to obtain a 50 mM hexanal solution. The
licking frequency of the olfactory-blocked mice to distilled water and
to the 100 mM hexanal solution (n ¼ 8) was then counted. The licking
ratio was compared between QHCl alone and QHCl with the 50 mM
(n ¼ 6) and 100 mM hexanal solutions (n ¼ 6).
Licking counter. A glass spout was attached to an experimental fluid
bottle. The bottle was linked to a test chamber (7 cm wide, 12 cm high,

1886
S. YAMAGUCHI et al.
Table 1. Schedule for Conditioning of the MSG-Averse Mice
50
40
30
20
10
0
A
Day
Session
1
Olfactory blocking
Food and water ad libitum
2
3
4
Recovery: food and water ad libitum
Recovery: food and water ad libitum
Conditioned stimulus (CS): 100 mM MSG plus LiCl
Food and water ad libitum
5
6
7
8
Recovery: food and water ad libitum
22 h water deprivation
Conditioned stimulus (CS): 100 mM MSG plus LiCl
Food and water ad libitum
Recovery: food and water ad libitum
22 h water deprivation
Test session
Water
Oxidized AA
1.2
1.0
0.8
0.6
0.4
0.2
0
B
Licking frequency of the olfactory-blocked mice for MSG with or
without hexanal. In order to study the effect of hexanal on the mouse
taste perception to MSG, the licking frequency for the MSG solution
with and without hexanal was evaluated with the olfactory-blocked
mice conditioned for their aversion to the MSG taste. The olfactory-
blocked mice were conditioned to avoid MSG by an intraperitoneal
injection of 0.2 M LiCl as 2% of the mouse body weight after a 100 mM
MSG intake for 10 min. The schedule for treating the mice is indicated
in Table 1. The licking frequency for the mice (n ¼ 8) treated with
distilled water and with the water solution of 100 mM hexanal was then
counted with the licking counter. The licking frequency of the mice for
1, 5, 10, 30, 50 and 100 mM MSG with or without the 100 mM hexanal
solution (n ¼ 9) was also counted.
**
**
QHCl
QHCl + oxidized AA
**
0.01
0.1
QHCl concentration (mM)
1.0
Fig. 1. Effect of the Water Extract from Oxidized AA Ethyl on the
Mouse Licking Response.
Data analysis. The numbers of licks of distilled water and of the
water extract from oxidized AA or hexanal were each presented as the
group mean ꢀ SE. A comparison of the mean licking number between
the data for distilled water and each water extract was then analyzed by
one-way ANOVA.
A, Mean licking frequency during a 10-s period for distilled water
and the water extract from oxidized AA ethyl by anosmic mice.
Each value is expressed as the mean ꢀ SE (n ¼ 8). The filled
column and bar express the mean ꢀ SE of the number of licks for
distilled water (D.W.). The unfilled column and bar express the
mean ꢀ SE of the number of licks for the water extract from
oxidized AA ethyl. B, Licking ratio of anosmic mice for QHCl with
or without the water extract from oxidized AA ethyl in a 10-s period
after presenting the test taste stimulus. Symbols and bar express the
mean ꢀ SE (n ¼ 7) for the licking ratio to QHCl with (unfilled circle
and solid line) or without (filled circle and solid line) the water
extract from oxidized AA ethyl. Data were analyzed by two-way
ANOVA and then the statistical difference was determined by
Tukey’s multiple-comparison post hoc tests among the test solu-
tions. ꢃꢃ p < 0:01.
Licking ratios for QHCl with or without the water extract from
oxidized AA or hexanal were also each presented as the group
mean ꢀ SE. A comparison of the data between the mean licking ratio
for QHCl in the presence and absence of the water extract from
oxidized AA ethyl or hexanal was then analyzed by two-way ANOVA.
A post hoc analysis was performed with Tukey’s multiple-comparison
test when the F ratio for ANOVA was significant at p < 0:05.
The number of licks by the mice with blocked-olfactory response
and conditioned to avoid 100 mM MSG to distilled water and the water
extract from hexanal were then each presented as the group
mean ꢀ SE. The mean number of licks for distilled water and the
hexanal solutions were analyzed by one-way ANOVA.
Effect of oxidized AA ethyl on the mouse taste
perception of QHCl
The licking ratios for MSG with or without hexanal were each
presented as the group mean ꢀ SE. The mean licking ratios for MSG in
the presence and absence of the water extract from oxidized AA ethyl
or hexanal were then analyzed by two-way ANOVA with Tukey’s
multiple-comparison post hoc tests.
The licking frequency of each water extract from
oxidized AA ethyl was equal to that of distilled water
with the anosmic mice (Fig. 1A). There was no signifi-
cant difference among the licking frequency for water
and the water extract from oxidized AA ethyl for each
mouse [Fð1; 14Þ ¼ 0:539, p ¼ 0:475]. The water extract
from oxidized AA alone would not affect the licking
response of the anosmic mice in this examination.
The addition of the water extract from AA ethyl to
QHCl induced a decrease in the licking responses at
several concentrations (Fig. 1B). There were significant
effects of the QHCl concentration [Fð5; 96Þ ¼ 110:748,
p < 0:01] and of adding the oxidized AA extract to the
QHCl solution [Fð1; 96Þ ¼ 56:538, p < 0:01]. There
was also significant interaction between the QHCl
concentration and the presence of the oxidized AA
extract [Fð5; 96Þ ¼ 5:178, p < 0:01]. The post hoc
analysis identified that the licking response to QHCl
with the oxidized AA extract was significantly lower
The licking frequency of the mice to QHCl with or without
fractionated oxidized AA ethyl was examined by comparing the data
for the mean licking ratio of QHCl in the presence and absence of each
fractionated oxidized AA ethyl by two-way ANOVA with Tukey’s
multiple-comparison post hoc test between the QHCl solution alone
and each fraction with QHCl.
Results and Discussion
Analysis of autoxidized AA ethyl
We analyzed the peroxide value (PV) and carbonyl
value (CV) for oxidized AA ethyl. PV for the prepared
autoxidized AA ethyl was 928:8 ꢀ 23:8 meq/kg
(mean ꢀ SE, n ¼ 3), and CV was 140:4 ꢀ 7:8 mmol/g
(mean ꢀ SE, n ¼ 3).

Effect of AA and Hexanal on Mouse Taste Perception of QHCl and MSG
1887
Table 2. Amount of Decomposition Products in Fractionated Oxidized AA Ethyl
(mg/fraction)
Fr. 4
Oxidized AA-ethyl
Fr. 1
Fr. 2
Fr. 3
ꢃ
Hexanal
2:05 ꢀ 0:18
—
—
0:25 ꢀ 0:07
0:67 ꢀ 0:03
0:06 ꢀ 0:02
1-Octen-3-ol
2,4-Decadienal
2-Pentylfuran
trans-2-Octenal
trans-2-Nonenal
AA ethyl
0:18 ꢀ 0:01
0:02 ꢀ 0:00
0:03 ꢀ 0:00
0:01 ꢀ 0:00
1:51 ꢀ 0:07
—
tr
0:10 ꢀ 0:01
0:07 ꢀ 0:01
—
—
—
—
—
trꢃꢃ
tr
tr
tr
tr
tr
tr
tr
tr
tr
tr
176:16 ꢀ 1:56
174:57 ꢀ 0:11
0:13 ꢀ 0:02
0:60 ꢀ 0:00
ꢃnot detected; ꢃꢃtrace amount.
presence of Fr. 3 [Fð5; 60Þ ¼ 4:269, p < 0:01]. The post
hoc analysis identified a significantly lower licking
response to 0.3 mM QHCl with Fr. 3 than that to QHCl
alone at 0.3 mM (p < 0:01).
This result suggests that a compound included in Fr. 3
influenced the anosmic mice taste perception and
reduced the licking response to QHCl.
1.2
1.0
0.8
0.6
Determination of the water-extracted compounds
from oxidized AA ethyl
0.4
0.2
0
QHCl
**
QHCl + Fr. 1
QHCl + Fr. 2
QHCl + Fr. 3
QHCl + Fr. 4
The main compounds reported as predominant oxi-
dized decomposed products from AA ethyl by Arts
et al.25) were partly confirmed in the present study and
are generally consistent with their report (Table 2).
Hexanal in each fraction of this present study would
have been dissolved in the distilled water while prepar-
ing the water solution for the test stimulus, because the
concentration of hexanal was less than the literature
value for the solubility in water at 25 ꢁC.26) The
predominant effect of hexanal on the mice licking
response to QHCl was apparent among these fractions in
the test solution for Fr. 3. This test solution was
estimated to contain about 130 mM hexanal (0.67 mg of
hexanal dissolved in 50 ml of distilled water). We
subsequently focused our attention on the effect of
hexanal on the mice licking response to QHCl.
0.01
0.1
1.0
QHCl concentration (mM)
Fig. 2. Effect of the Water-Extracted Compounds from Oxidized AA
Ethyl on the Mouse Licking Response.
Presented are the licking ratios of anosmic mice for QHCl with or
without each fractionated water extract from oxidized AA ethyl in a
10-s period after presenting the test taste stimulus. Symbols and bar
express the mean ꢀ SE (n ¼ 6) of the licking ratio for QHCl with or
without the fractionated water extract from oxidized AA ethyl (filled
circle and solid line, QHCl alone; unfilled circleand solid line, QHCl
with Fr. 1; filled triangle and solid line, QHCl with Fr. 2; unfilled
triangle and solid line, QHCl with Fr. 3; filled square and solid line,
QHCl with Fr. 4). Data were analyzed by two-way ANOVA and
then the statistical difference was determined by Tukey’s multiple-
comparison post hoc tests between QHCl alone and QHCl with each
fraction. ꢃꢃ p < 0:01.
Effect of hexanal on the mouse licking response to
QHCl
than that to QHCl alone at 0.1, 0.3 and 0.6 mM (p < 0:01
at 0.1 mM,p < 0:01 at 0.3 mM, p < 0:01 at 0.6 mM,
Fig. 1B). This result suggests that the water extract from
oxidized AA ethyl affected the mouse taste perception
and induced a decrease in the licking response to QHCl.
We examined the licking response of the anosmic
mice to QHCl with and without 50 mM or 100 mM hexanal
(Fig. 3). There was no significant difference between the
licking frequency to distilled water and that to the
100 mM hexanal solution [n ¼ 8, Fð1; 14Þ ¼ 0:0853,
p ¼ 0:775] (Fig. 3A). There was also no significant
difference in the licking response of olfactory normal
mice to 100 mM hexanal and distilled water (data not
shown).
Effect of the fractionated water extracts from oxidized
AA ethyl on the mouse taste perception for QHCl
The examination of the mice licking response to
QHCl with the water extract from oxidized AA ethyl
indicates that oxidized AA ethyl contained a substance
which affected the mice licking response to QHCl via a
gustatory sensation. This substance was identified by
examining the fractions from oxidized AA ethyl for their
the effect on the licking response to QHCl by GC-MS.
The licking response of the anosmic mice to QHCl
with or without each fractionated water extract from
oxidized AA ethyl is indicated in Fig. 2A. Although
there was no significant effect of adding Fr. 1, Fr, 2 or
Fr. 4 to the QHCl solution, there was a significant effect
of the QHCl concentration [Fð4; 125Þ ¼ 104:643,
p < 0:01] and of adding Fr. 3 to the QHCl solution
[Fð1; 60Þ ¼ 14:060, p < 0:01]. There was significant
interaction between the QHCl concentration and the
A comparison of the licking ratio for anosmic mice to
QHCl with and without 50 mM hexanal (Fig. 3B) showed
a
significant effect of the QHCl concentration
[Fð5; 60Þ ¼ 24:280, p < 0:01], but none in the presence
and absence of 50 mM hexanal [Fð1; 60Þ ¼ 1:102,
p ¼ 0:298]. In the presence of 100 mM hexanal
(Fig. 3C), there was a significant effect on the anosmic
mice licking ratio of the QHCl concentration
[Fð5; 60Þ ¼ 112:258, p < 0:01] and of the addition of
hexanal to the QHCl solution [Fð1; 60Þ ¼ 41:030,
p < 0:01]. There was also significant interaction be-
tween the QHCl concentration and the presence of
hexanal [Fð5; 60Þ ¼ 2:565, p ¼ 0:036]. The post hoc
analysis showed a lower licking response to QHCl with

1888
S. YAMAGUCHI et al.
50
40
30
20
10
hexanal than that to QHCl alone at 0.1, 0.3 and 0.6 mM
(p ¼ 0:019 at 0.1 mM, p < 0:01 at 0.3 mM and p ¼
0:022 at 0.6 mM). This result suggests that 100 mM
hexanal influenced the anosmic mice taste perception
and reduced the licking response to QHCl. There was
thus dose dependency for hexanal.
The effect of fractionated oxidized AA ethyl on the
mice licking response to QHCl showed that the test
solution of Fr. 3 containing about 130 mM hexanal
markedly reduced the licking frequency to QHCl.
Hexanal is thus considered to be one active substance
in Fr. 3. Figure 2 shows that the water extract from Fr. 2
containing about 50 mM hexanal failed to sensitize the
mouse response, probably because of the dose depend-
ency of hexanal as shown in Fig. 3B and C.
The water extract from oxidized AA ethyl would have
contained about 0.01 g of hexanal in 1.0 g of oxidized
AA ethyl suspended in 1000 ml of water (Table 2).
About 100 mM hexanal would have been contained in the
water extract from the oxidized AA ethyl if all the
hexanal in the oxidized AA ethyl was dissolved in water
during the preparation. This result suggests that hexanal
was one of the compounds in the water extract from
oxidized AA ethyl affecting the anosmic mice licking
response to QHCl. We are now investigating the effects
of other compounds in the oxidized AA ethyl extract on
the QHCl taste perception.
Mice as well as humans dislike and avoid bitter,
astringent and stimulant tastes. A previous study has
reported that rats could distinguish oxidized oil from
fresh oil and preferred the fresh oil in a two-bottle
choice test.27) Other studies have also reported that
bitter, astringent and stimulant tastes were detected in
highly auto-oxidized soybean oil by human sensory
evaluations.28–31) Carbonyl compounds from oxidized
oil such as 2,4-decadienal and some other low-molecu-
lar-weight compounds have been found to have a
‘‘heated oil-like’’ taste.28) There has however been no
report on hexanal being involved in the mouse taste
perception to a bitter tastant. Our examination has
shown that the mixture of a water extract from oxidized
AA ethyl or hexanal with a moderately high concen-
tration of QHCl reduced the licking response of anosmic
mice, although these compounds did not elicit any
obvious decrease in the licking response to QHCl at
concentrations of these compounds lower than 0.05 mM.
This suggests that hexanal may have enhanced the
aversive taste stimulus of QHCl.
A
0
Water
Hexanal
1.2
B
1.0
0.8
0.6
0.4
0.2
QHCl
QHCl + 50 µM hexanal
0
1.2
C
1.0
0.8
0.6
0.4
0.2
0
*
**
QHCl
QHCl + 100 µM hexanal
*
0.01
0.1
1
QHCl concentration (mM)
Fig. 3. Effect of Hexanal on the Mouse Licking Response.
A, Mean mouse licking frequency during a 10-s period for
distilled water and 100 mM hexanal by anosmic mice. Each value is
expressed as the mean ꢀ SE (n ¼ 8). The filled column and bar
express the mean ꢀ SE of the licking number for distilled water
(D.W.). The unfilled column and bar express the mean ꢀ SE of the
licking number for 100 mM hexanal. B, Licking ratio by anosmic
mice for QHCl with or without hexanal in a 10-s period after
presenting the test taste stimulus. Symbols and bars express the
mean ꢀ SE (n ¼ 6) of the licking ratio for QHCl with (unfilled
circle and solid line) or without (filled circle and solid line) 50 mM
hexanal. Data were analyzed by two-way ANOVA and there was no
significant difference between adding 50 mM hexanal to QHCl and
only QHCl. C, Licking ratio by anosmic mice to QHCl with or
without hexanal during a 10-s period after presenting the test taste
stimulus. Symbols and bars express the mean ꢀ SE (n ¼ 6) of the
licking ratio for QHCl with (unfilled circle and solid line) or without
(filled circle and solid line) 100 mM hexanal. Data were analyzed by
two-way ANOVA and then the statistical difference among the test
solutions was determined by Tukey’s multiple-comparison post hoc
test. ꢃꢃ p < 0:01. ꢃ p < 0:05.
Effect of hexanal on the mouse licking response to
MSG
Our previous report has demonstrated that oxidized
AA ethyl strongly enhanced the umami taste of soy
sauce in human sensory tests.17) We then hypothesized
that hexanal in oxidized AA would also enhance the
umami taste perception and examined effect of hexanal
on the licking response of mice to MSG, a classic umami
taste,19–22) as a tastant other than QHCl. We used in this
present study anosmic mice that had been conditioned to
avoid the MSG taste in order to reflect the MSG taste
perception on licking behavior.
100 mM hexanal solution [n ¼ 8, Fð1; 14Þ ¼ 0:0029,
p ¼ 0:958]. The licking response of the mice to MSG
with or without hexanal is indicated in Fig. 4B. There
were significant effects from the MSG concentration
[Fð5; 96Þ ¼ 61:703, p < 0:01] and the addition of
hexanal to the MSG solution [Fð1; 96Þ ¼ 4:712,
As shown in Fig. 4A, there was no significant
difference between the licking frequency of the trained
and anosmic mice for distilled water and that for a

Effect of AA and Hexanal on Mouse Taste Perception of QHCl and MSG
1889
responsive to MSG and QHCl. We are now investigating
the mechanism for the effects of oxidized compounds on
taste perception.
50
40
30
20
10
A
Conclusion
Water extracts from oxidized AA ethyl and hexanal,
which is the predominant component generated from
autoxidized AA, sensitized the mouse taste perception of
QHCl and MSG. The decomposed products from
oxidized AA ethyl, including hexanal, might enhance
the mouse taste perception to QHCl and MSG via a
gustatory sensation.
0
Water
Hexanal
1.2
B
Acknowledgment
1.0
0.8
0.6
0.4
0.2
0
This study was supported in part by Grant-in-Aid
from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan.
*
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