J. Biol. Macromol., 14(1), 3-14 (2014)

ArticleScreening and Identification of Food Extracts that Are Useful
For Reducing Salt Intake- An Approach from a GABA-synthesizing Enzyme Kumiko Hisaki*§, Kaori Hamano* and Hiroshi Ueno**Laboratory of Applied Microbiology and Biochemistry,
Nara Women's University, Nara 630-8506, JAPAN§
Osaka International College, Moriguchi, Osaka 570-8555, JAPANReceived January 22, 2014; Accepted February 20, 2014
Potential use of plant extracts for reducing salt intake was explored. Recent
finding of GAD67 and GABA-gated chloride ion channel GABAA receptors being
expressed in type III taste buds supported an idea that GABA could be a signal forsalty taste mechanism. In this study, we have shown that food extracts effected on
both GAD67 activity in vitro and taste sensations in vivo. Furthermore, those foodextracts tended to enhance GAD67 activity showed direct correlation with salty tasteenhancement. It is concluded that food extracts can be a useful salt enhancing
materials.
Key words:

GABA, GAD67, spices, teas, salt intake

1. Introduction
Comprehensive Survey of Living
Conditions 2004 1) reported that strokes
were the main cause for nursing care andbedridden patients, as Japan becomes anaging society.
Prevention of strokes
would facilitate longevity by allowing
people to maintain a healthy life. Fewerstrokes could help reduce healthcare costs
significantly.
The main strategy forpreventing strokes is to manage
hypertension, a major risk factor forstrokes 2-5).
It is widely accepted that reduced
Abbreviations: GABA: -aminobutyrate, GAD67: an isoform ofglutamate decarboxylase, GST:
glutathione S transferase, PCA:
perchloric acid§
To whom correspondence should be
addressed: hisaki@oiu.jp

dietary salt intake can lower the
possibilities of developing hypertension.
On the other hand, there are difficulties in
reducing the salt intake, since saltenhances the flavor of foods.
It isdesirable to seek alternative approaches
that provide us with flavor in a low-salt
diet without sacrificing the taste. In thiscontext, understanding how the salty taste
signal transduction mechanism in taste
buds works is important.
Humans perceive five basic tastes,
including umami, bitter, sweet, sour, andsalty, and commonly recognized taste
substances are monosodium glutamate(MSG), quinine sulfate, sucrose, citricacid, and sodium chloride (NaCl),
respectively.
The sense of taste is
mediated by taste buds, approximately
5,000 located on the tongue. A single
taste bud is constituted of approximately
50-100 cells, and these cells are4

Screening food extracts for reducing salt intake

morphologically divided into four groups,
namely type I, II, III and IV 6, 7). Type II
cells are shown to contain receptors forumami, bitter and sweet substances andtype III cells for sour and salt substances.
Recently, a number of thecandidates for taste receptors have beenclarified. Umami, bitter and sweet tastes
are mediated by G protein-coupled
receptors (GPCRs) located on type II cells
7, 8). Sour tastes are transmitted by ion
channel-linked receptors, such as
PKD2L1 and PKD1L3, both of which
belong to the transient receptor potential(TRP) family, expressed on type III cells
9). Salty taste is similarly transmitted by
the sodium ion channel since intracellular
Na+ influx induces depolarization to givethe salty taste sensation.
Currently,
amiloride-sensitive epithelial sodium
channels (ENaCs) that are located on taste
cells, are regarded as one of the major
contributors to Na+ transduction. Details
of ENaCs’ molecular expression including
the types of the taste cells are not fully
understood. Several salty taste receptive
mechanisms other than ENaCs are also
proposed 10, 11).
NaCl, a chemical component forcommon table salt, is the prototypic salty
tastant. Frequent use of table salt has
been suspected as a cause of hypertension;
however, NaCl intake itself is not directly
associated with hypertension 5, 12). The
association is clear for Na+ 5, 13); hence,
KCl is often used as a dietary substitute for
NaCl. KCl exhibits saltiness together
with bitter taste 14). Since KCl has salty
taste, Na+ is not the only element that
contributes to the salty sensation. Salty
taste may be also attributed to chloride ion,
Cl-, because the substitution of Cl- to
iodine ion, I-, gives completely different
taste from NaCl 15). It is important tonote that KCl may not be a universal
substitute for NaCl, since high blood K+
level may cause heart failure and people
with kidney and heart problems should

avoid KCl intake. Hence, other suitable
substitutes to NaCl are needed.
In taste sensations, sometimes crosstalks between two different tastes takeplace, which are called hidden tastes
known in the field of cooking. One of
the hidden tastes is a contrast effect inwhich a small amount of salt enhances
both sweetness and umami taste 16, 17).
There is an inhibitory effect in which asmall amount of salt can weaken bitter
taste 18). Although detailed molecularmechanisms of the hidden tastes are still
under investigation, it is of interest to notethat the phenomenon can be interpreted ascell-to-cell communication which occurs
between type II and III cells. In order toexamine the hidden tastes in molecularlevel, one has to consider how the taste
signal be transmitted from the taste bud tothe gustatory nerves. It has been shownthat gustatory nerves are connected to thetype III cells via synaptic connection but
there is no such interaction with type II
cells 7, 8). It is not fully established how
the signal from type II cells is transmitted
to the gustatory nerves 19-21) .
Nakamura et al. found thatglutamate decarboxylase 67 (GAD67),
one of the isoforms of GAD, is expressedin the type III taste cells 22). GAD is an
enzyme to catalyze the synthetic reactionof γ-aminobutyrate (GABA).
In
mammals, there are two distinct genes
which encode GAD proteins, called
isoforms, and those exhibit almost
identical catalytic activity 23). Nakamura
et al. also found that GAD67 in the type
III taste cells is enzymatically active and
GABA is produced in the type III taste
cells 24). Since GABA is an amino acidthat acts as a major inhibitory
neurotransmitter in the central nervous
system, its existence in the type III taste
cells raises some questions: What is therole of GABA in the taste cells? Is
GABA involved in taste? Since GABA
is a neurotransmitter, it would be highlyHisaki, Hamano and Ueno

likely to act as a part of taste signaling
although it has yet to be ascertained.
There are several kinds of evidence tosupport GABA being involved in taste: 1)
By performing taste sensation tests withhuman subjects, GABA was shown to bea tasteless compound. Furthermore, italso gave an enhancement effect for sourand bitter when taking together with sourand bitter substances, respectively 25, 26).
2) GABA is a ligand for GABA receptors
in which GABAA and GABAC receptors
are GABA-gated chloride ion channels.
Since chloride ion participates as a salty
substance, it is highly likely that GABA is
involved in salt signal transduction.
Salty taste could be enhanced whena small amount of GABA is present 16).
It raises the possibilities that any
substances that affect GABA levels and/or
GABA production in the type III taste
cells would alter the salty taste signaling.
In the present study, we aim to identify
food extracts that affect GAD67 activity.
In order to find such food extracts, various
food extracts from spices, herbs, teas, andcommon food materials are examined.
The food extracts that influence GAD67
activity should be further examined for theeffects on taste sensation by taste

5

sensation tests with human subjects.
2. Materials and Methods
1. Spices, herbs and teas used for thescreening
Fifteen types of spices, herbs andteas used in this study are listed in Table 1
They are classified into six categories of1) leaves (oolong tea, lemongrass, celery,basil, oregano, peppermint, perilla, andparsley), 2) seeds (anise, cumin, andpoppy seed), 3) aril (mace), 4) fruit(paprika), 5) root (ginger), and 6) peel
(yuzu peel). These materials were gifts
from Somatech Center of House Foods
Corporationorpurchasedfromcommercially available sources.
2. Preparation of food extracts
Extraction of food was carried out
by incubating the crushed food materials
in 5 times the weight of water andpowdered foods in 10 times the weight ofwater at 4-6 °C for overnight.
Thesupernatant of the incubated samples was
individually collected and they were used
as food extracts. The pH and dry weightof the extracts are summarized in Table 1.

Table 1 Characteristics of Food Extracts
No.

Samples

Part of plant used

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

anise
basil
celerycumin
gingerlemongrassmaceoolongoreganopaprikaparsley
peppermint
perillapoppy seedyuzu peel

seed
leaf
setm and leaf
seedroot and rhizome
leaf
aril
leaf
leaf
fruit
leaf
leaf
leaf
seed
peel

pH
5.3
6.1
7.5
6.0
6.0
5.6
4.0
5.8
6.5
4.6
5.3
6.0
6.0
7.0
3.7

Food extract
Dry weight(mg/ml)
32
33～48
66
25～32
25～33
27～28
13
30
30
22～28
40
50
20～21
13
71～76

GABA
(μg/ml)
0.29
0.14
1.70
0.14
0.96
0.21
0.10
0.30
0.16
0.30
0.24
1.50
0.45
0.03
0.21

FamilyApiaceae
Lamiaceae
Lamiaceae
Apiaceae
Zingiberaceae
Poaceae
Myristicaceae
Theaceae
Lamiaceae
Solanaceae
Apiaceae
Lamiaceae
Lamiaceae
Papaveraceae
Rutaceae

6

Screening food extracts for reducing salt intake

3. Preparation of GAD67
A recombinant rat brain GAD67
was prepared by growing E. coli strain
Rosetta-gamiB (DE3) pLysS that wastransformed by GST-GAD67 containing
vector.
Since amino acid sequencehomology between the rat brain andhuman brain GAD67s is high enough, thecommercial anti-GAD67 antibody from
Sigma (St. Louis, MO, USA) which
recognizes both species was used for thedetection of rat brain GAD67 in thepresent study.
Purification of therecombinant GAD67 was performing byglutathione affinity chromatography.
The protein concentration was estimatedby using a Bio-Rad protein assay dye
reagent
purchased from Bio-Rad
Laboratories (Hercules, CA, USA) withbovine serum albumin as a standard.
GAD enzyme activity was determined byincubating the enzyme solution with Lglutamate in the presence of PLP and pH
7.4 HEPES buffer at 37 °C. Amounts ofGABA produced were determined by
HPLC after derivatization with NBD-F
and by using Agilent ZORBAX-C18
Table 2 GAD67 Activity in the
Presence of Food Extracts
No.

Samples

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

anise
basil
celerycumin
gingerlemongrassmaceoolongoreganopaprikaparsley
peppermint
perillapoppy seedyuzu peel

GAD67 activity
%
28.0
-2.2
15.2
15.8
-30.6
-66.7
3.9
-63.6
-62.5
-16.7
-8.2
-43.3
-30.9
-11.8
36.0

column (3.0 x 50 mm, 1.8 m particle
diameter). A typical purified GAD67
exhibited a specific activity of 0.4
mol/min/mg.
4. Effects of food extracts on GAD67
activity
The amounts of GABA produced in
GAD67 assay with and without the foodextracts were evaluated. The effect ofeach of the food extracts was indexed as aratio of relative activity (%) by comparing
amounts of GABA produced in thepresence and the absence of the foodextracts.
Positive and negative %numbers indicate that food extracts
activate or inhibit GAD67 enzyme activity,
respectively.
5. Quantitative analysis of GABAGAD67 enzyme solution was mixed
with 100 μL of assay mixture containing
0.5 M HEPES buffer (pH 7.0), 0.2 M
sodium L-glutamate, and 2 mM PLP,
where water and the food extracts wereadded to make up the total assay volumeof 1 mL.
The assay mixture wasincubated at 37 C for 1 h. At the end of
the incubation period, 50 μL of 60%
perchloric acid (PCA) was added to stop
the reaction. As a blank, PCA solution
was added at time zero.
Afterterminating the reaction, the mixture wascentrifuged and the supernatant wastransferred to a vial and used for GABA
analysis on an HPLC system as described
previously 27, 28). The obtained GABA
peak area was compared with the standard
GABA to estimate the GABA contents inthe assay mixture. The effect of each of
the food extracts on GAD activity wasevaluated by comparing GABA contents
in the reaction mixture in the presence andabsence of the examined food extract.

Hisaki, Hamano and Ueno

6. Taste sensation tests
Forty one healthy women between
the ages of 18 and 25 were chosen for thetaste sensation test subjects. The tests
were carried out in the cooking practiceroom at Osaka International College.
Prior to the tests, we explained the purposeof the sensory taste testes to the subjects.
Given the possible regulatory effect of theextracted food components from spices,
herbs and teas on GAD67 activity, theeffects of the prepared food extracts were
evaluated on the intensity of the salty
sensation. First, the subjects rinsed theirmouth with water, then, standard 0.8%
NaCl solution of 5 mL was put into theirmouth and they were allow to taste for awhile. After spitting out the solution,
they rinsed their mouth again with water.
Then, 0.5 - 1.0 mL of the sample solution
containing the appropriately diluted foodextract was tasted. They were allow totaste the solution for a while and could
swallow it, if they wanted it. Finally, thesubjects were given standard 0.8% NaClsolution again to test any changes in thesalty taste.
They evaluated the last
solution for the saltiness on the basis ofthree step scale from strong, same, to weak.
The scores of the taste sensation test were
derived as follows.
The number ofsubjects who answered weak was
subtracted from those who answered
strong.
The subtracted number was
divided by the total number of the subjects
and expressed as salty taste enhancement
points.
3. Results
1. Screening of food extracts that affect
GAD67 activity
Common spices, herbs and teas
were chosen and extracts prepared were
examined for their effects on GAD67
activity. Table 2 indicates that foodextracts were able to alter GAD67 activity
and the degree of inhibition/activation is7varied widely. Lemongrass, oolong tea,
and oregano extracts showed inhibition,
while, yuzu peel, anise, cumin and celery
extracts activated GAD67 extensively.
Mace, poppy seed, and basil extracts
hardly affected GAD67 activity. The
results suggest that those common foodextracts affect GAD67 activity in vitro andmay also affect in vivo.
2. Effects of food extracts on the salty taste
The extracts from the chosen spices,
herbs and teas exhibited various effects on
GAD67 activity by in vitro assay system.
All the food extracts listed in Table 1 wereused for the taste sensation test. The
results of the taste sensation test weresummarized and shown in Table 3A.
More than 50% of the subjects answeredthat peppermint, oregano and lemongrass
extracts weakened salty taste. On theother hand, more than 50% of the subjects
answered that celery, anise and cumin
extracts strengthened salty taste.
3. Effects of GABA on sweet taste
The effect of GABA on salty taste
sensation was evident in the taste test.
As type III cells utilize GABA for the salty
taste sensation, it is of interests to know if
GABA might influence to another taste
sensations, such as the sweet sensation
located on type II cells. The effect onsweet taste was chosen since it has been
known that sweet taste can be enhanced byadding a small amount of salt eliciting acontrast effect. Three-percent sucrose
solution,
which
is
a
commonconcentration for sweet solutions incooking, was used in the taste sensation
test.
We have previously demonstrated
that additional 0.0025% of GABA, about
threshold level, did not enhance sweet
taste for 3% sucrose with human subjects
26). We have examined further whether
or not higher concentration of GABA may
enhance sweet taste using commercial soy

8

Screening food extracts for reducing salt intake

Hisaki, Hamano and Ueno

sauce containing GABA.
The salt
content in the GABA soy sauce is a half ofthat in regular soy sauces 29).
Considering both GABA and salt contents
in the GABA containing soy sauce, wehave employed the same GABA level
used in the soy sauce for our experiment.
The amount of GABA in the soy sauce
was determined as follows: According
to the label of the GABA soy sauce, 1.3 g
GABA is added per 100 g, and salt content
is 7.8%. The amount of GABA per g of
NaCl is converted to be 0.17 g. The
contrast effect observed for sweetness

9with salt was demonstrated by adding
1.7% salt to the sweet material (Hisaki K,
personal unpublished results).
Basedupon this concept, 0.01% GABA wasestimated to give the contrast effecttoward 3% sucrose solution.
Theestimated amount of GABA wasequivalent to 0.05% salt.
When the test solution containing
0.01% GABA in 3% sucrose was
examined, it did not show any
enhancement of sweetness as shown in Fig.
1. For the comparison, our previous datausing 0.0025% GABA, almost at the10

Screening food extracts for reducing salt intake

threshold value, was presented in Fig. 1 26).
Significantly higher GABA showed nocontrast effect, rather it gave some sour
taste 25, 26). We did not carry out any
further experiment by raising GABA
concentration because of the sour taste.
Our results suggest that GABA itself may
not replace the action of salty taste toward
sweetness.
4. Relationship between GAD67 activity
and taste alteration with food extracts
We examined the effects of foodextracts on the salty taste alteration. The
salty taste enhancement in points werecompared with the effects on GAD67
activity (%) (Fig. 2A).
The results represent a strong
positive correlation between GAD67
relative activity and the enhancement ofsalty taste (R2=0.6707), and suggest that a
promoted GAD67 enzyme activity by thepresence of food extracts enhances salty
taste. It appears likely that the salty taste
is enhanced as a result of GABA actionthat could be synthesized inside the type
III cells.
5. Effects of food extracts on GAD67
activity and sweet taste
As described in the previous section,
the food extracts, that activated GAD67,
enhanced salty taste strongly.
Thissuggests a possibility that food extracts
acts on GAD67 located in type III taste
cells; hence, GABA production level
might be elevated. It is likely that GABA
may act as a taste effector or messenger.
Based on this assumption, we examined
the possible effect of GABA on theintensity of the sweet taste. For theevaluation method used in sweet taste, 3%
sucrose solution was used for the taste
sensation test. The 15 types of foodextracts examined for the salty taste tests
were used. The methodology of the taste
sensation test is the same as described forthe salty taste tests.

As shown in Table 3B, more than
50% of the subjects replied that mace,
poppy seed, anise, celery, oolong tea andlemongrass enhanced the sweet taste,
where mace, poppy seed and celery
showed significant differences of p<0.001,
anise and lemongrass showed p<0.01, andoolong tea showed p<0.05. On the other
hand, it was only oregano in which morethan 60% of the subjects expressed thesweet taste was weakened, p<0.01. The
obtained relative sweet taste enhancement
point was compared with GAD67 relative
activity (%). The results of the sweet
taste enhancement point were shown in
Table 3B. When the effect of foodextracts on GAD67 activity was plotted in
X axis and that on sweet tasteenhancement was in Y axis (Fig. 2B),
almost no correlation between the effectsof the GAD67 activity and the degree ofsweet taste enhancing effect of foodextracts was observed (R2=0.0846) with agreater variability of the data compared tothe salty taste enhancement shown in Fig.
2A. These results suggest that GABA
synthesized in taste bud cells would be
unlikely to influence the sweet taste.
The results do not conflict with the findingthat addition of free GABA in sucrose
solution did not enhance the sweetness as
described in the previous section.
4. Discussion
A number of questions have beenraised since the discovery of GAD67
protein expression in type III taste cells 22,
24). One major question was about therole of GABA in taste signalingmechanisms:
Whether or not it isinvolved in the salty taste signaltransduction, and if it is involved, how
GABA acts as a messenger to other cell
types. We have attempted to answer tothe questions by using the taste sensation
tests.
Since experiments utilizing
animals cannot indicate clearly theperception of tastes, it is necessary toHisaki, Hamano and Ueno

include human as a subject.
Our preliminary data indicate that
GAD67 and possibly its product GABA
are active in type III taste buds 30). When
GABA alone was subjected to the smell
and taste tests, it was found that the
majority of subjects expressed GABA
exhibiting sour and bitter tastes but very
little smell 25). Additional studies werecarried out in order to examine the effectsof GABA with the pre-existing basic 5
taste substances. The concentrations for11

5 taste substances are 3% sucrose, 0.5%
citric acid, 0.6% sodium chloride (NaCl),
0.00125% quinine sulfate, 0.06%
monosodium glutamate (MSG), and
0.0025% GABA (GABA). The presenceof GABA had no effect on sweet taste(p<0.01), bitter taste (p<0.001), and sour
taste. On the contrary, the presence of
GABA significantly enhanced umami andsalty tastes. In particular, the addition of
GABA enhanced salty taste without any
changes in taste quality 25, 26).

12

Screening food extracts for reducing salt intake

Based upon our preliminary results,
it was clear that GABA plays a role insalty taste transduction, possibly via
GABA-gated chloride ion channel or any
other unknown pathways that may havesome contacts with the taste nerves.
Designing an experiment directly proof
the involvement of GABA in the taste
signal pathway is difficult; thus, we havechosen indirect ways to examine therole(s) of GABA in taste by utilizing foodextracts. Effects of these food extracts
on both GAD67 activity and the taste
sensation tests were compared.
Fifteen food extracts, most of them
are classified as spices and herbs, werechosen for GAD67 activity tests (Table 1).
Our results indicate that the effect of foodextracts on GAD67 activity varied widely;some activated and others inhibited. It
was surprising that such food extracts as
yuzu peel, anise, celery, and cumin,
activated the enzyme activity since, in
general, activation of enzyme by natural
products is quite rare. To make sure of
the elimination of possible contaminationof GABA included in the food extracts, wehave carried out the assays by using proper
controls. At the time zero, PCA wasadded to the assay mixture that contained
all the components including GAD67 andthe food extract. The obtained GABA
level was subtracted from the analyzed
data where the assay reaction was
terminated by the addition of PCA at theend of the reaction time. Therefore, any
residual GABA presented in the foodsamples was not counted in the assay
results. This means that if there is any
increased GABA production in the assay
conditions, it would be due to the resultsof enzymatic reactions. Those in vitro
results encouraged us to advance to thenext stage, an examination of food extracts
with human subjects.
We have considered that the effects
of food extracts could be due to theendogenous GABA present in theextracted samples. Since any residual
GABA was not counted in the GAD67
activity assay system, residual GABA
possibly present in the sample wasdetermined (Table 1). Those samples
exhibiting significantly high levels of
GABA, such as celery, peppermint, andginger, do not necessarily show the saltenhancing effects. Poppy seed, mace,
basil, cumin, and oregano showed medium
level salty taste enhancing effects, butthese extracts do not contain significant
amount of GABA.
Hence, we haveconcluded that endogenous GABA in theextracted samples do not affect our taste
sensation tests.
The presence of GABA in NaClsolution showed the enhancement of thesalty taste 25, 26).
When those foodcomponents that activated GAD67 were
present in the salty solution, our subjects
expressed changes in saltiness. It isnoted that those food extracts tend to
inhibit GAD67 gave less salt enhancing
effects and those to enhance GAD67
activity showed the enhancement of thesaltiness. Our present results indicatethat food extracts have a direct effect on
the salty taste signal transduction.
Results of the 15 food extracts
examined for their activities on GAD67
and the human taste test are shown in Fig.
2A. X-axis represents GAD67 activity
in % scale and Y-axis represents effects onsalty taste in points. There is a positive
correlation between the elevated GAD67
activity by food extracts and theenhancement of the salty taste. The
results imply that GABA synthesized in
type III taste buds by GAD67 is likely toactivate the salty taste signal transduction.
The taste contrast effect is known
for sweet taste enhanced by the presenceof a small amount of salt. If GABA is
involved in the salt signaling mechanism,
it might be possible to influence sweet
taste according to the principle of the taste
contrast effect. Hence, we have carriedHisaki, Hamano and Ueno

out the experiment how GABA iseffective on sweet taste.
Fig. 1
represents the effect of two different
concentrations of GABA on sweet taste as
judged by the human taste tests.
Itsuggests that the addition of GABA tosugar does not affect the sweetness.
Additionally, 15 types of food extracts
examined were plotted in order to examine
the correlation between GAD67 activity
and sweet taste (Fig. 2B). The results
suggest there seem to be no correlation
between GAD67 activity and sweet taste
when various food extracts were added tosugar. It is reasonable to exclude theidea that GABA might be involved in thesweet taste signal pathway via the taste
contrast effect as expected for the actionof salt.
We have demonstrated that yuzu
peel, anise, cumin and celery are capableof enhancing salty taste. The detailed
mechanism of the salty enhancement is
not clear at the moment, but it is most
likely that those food extracts raise GABA
level by acting on GAD67. We shouldbe able to develop a dietary menu by using
yuzu peel, anise, cumin or celery as salty
taste enhancers for those who need low
salt diet.
In the present study, a possible
regulation of GAD67 activity in type III
taste buds by the food extracts from
common foods, such as spices, herbs andteas. The food extracts which increase
GAD67 activity can enhance salty taste, inother words, it can be effective forprevention of hypertension and useful toprevent lifestyle-related diseases such as
strokes. These food extracts can also be
used in the diets of patients with dialysis.
Among 15 types of foods that are analyzed
in this study, the extracts from anise,celery, cumin, and yuzu peel may bepromising additions to our diets toincrease GAD67 activity and to raise theintensity of the salty taste; therefore, they
may be useful for reducing salt intake.

13

Acknowledgements
The authors are indebted to The
Society for Research on Umami Taste,
JSPS Grants-in-Aid for Scientific
Research (B) Grant Number 22300258,
Nara Women’s University International
Exchange Center for the financial support,
and Somatech Center of House Foods
Corporation for providing food samples.
We also thank to Kathya Cho and Yumiko
Ueno for reading the manuscript.
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Communicated by Ashitaka Emiko