J. Biol. Macromol., 14(1), 43-57 (2014)

ArticleRelationship between GAD/GABA and Sweet, Umami and Bitter Tastes,Those Received in Type II Taste Buds
Kimiko Sasaki,1,2,* Kaori Hamano1 and Hiroshi Ueno1
1

Lab. Appl. Microbiol. & Biochem., Nara Women's University, Nara 630-8506, JAPAN
2

Dept. Food, Mimasaka University, Tsuyama 708-8511, JAPANReceived February 25, 2014; Accepted March 3, 2014
Food extracts from spices and teas were examined for the taste altering ability on sweet,
umami and bitter tastes by using the taste sensation test. The idea arose based upon thefindings that 1) some food extracts from spices and teas act as salt taste enhancers and 2)
the enhancing effect is directly proportional to glutamate decarboxylase 67 (GAD67)
activity in vitro.

Our results indicated that those extracts significantly activated or

inhibited GAD67 enzyme activity were able to alter sweet, umami, and bitter tastes.
However, there were no relationships between the taste potency of each of the examined
extracts and the GAD67 relative activity ratio. While it is unlikely that the extracts havedirectly activated GAD67 activity and enhanced sweet, umami or bitter tastes, the results
do not exclude the idea that GABA may participate in the taste cell-to-cell communications.
Key words: GAD, GABA, taste signal transduction
In Japan, several decades ago people used toIntroduction

have struggled to obtain enough food to

Food supplies important nutrients andsatisfy their needs, but nowadays they canenergy to sustain life for humans. Humanchoose to eat from foods available in theireating behaviors vary depending on social

daily lives.

circumstances and cultural environments.

gradually has shifted from satisfying huger_________________________________*To whom correspondence should be
addressed: E-mail: sasaki@mimasaka.ac.jp
Abbreviations: GABA: γ-amino butyricacid; GAD, glutamate decarboxylase; PCA,
perchloric acid; PLP, pyridoxal 5'phosphate

The purpose of eating

to concerning nutritional aspects as well as
enjoyment of eating, and taste becomes a
critical factor in eating.
Five basic tastes, sweet, sour, bitter,salty, and umami, have been established (1).

44

Taste

Association of GAD/ GABA and tastes

substanceswithconnections (4). It has not been clear that

corresponding receptor proteins and the

how taste signals from type II cells

signals are transmitted to the taste nerve

transmitted to the nerves.

system. Those receptor proteins locate onrole of the chemical mediators that may act

the taste buds, which are classified into 4 cellon the taste signal transduction has not beentypes, I, II, III, and IV (2). Type II cells

clarified. Recently, Nakamura et al. haveexpress G-protein coupled receptors forshown that glutamate decarboxylase (GAD)

sweet, bitter and umami tastes, and type III

67, one of the isoforms to synthesize -cells express ion-channel receptors for sour

aminobutyric acid (GABA), is expressed inand salt tastes.

Although taste signals

the type III taste cells (5)(Fig.1). They alsotransmitted from receptors to taste nerves,

found that GABA-gated chloride iontype II cells appears to have no synaptic

channel, also known as GABAA receptor, isconnection with gustatory nerves (3).

expressed

Electron microscopic observation showed

Nakamura's

only

understanding of the role of type III cells,

type

III

first

cells

interact

havingsynaptic

in

the

study

Moreover, the

taste

buds

(5-7).

has

expanded

ourFig. 1 Schematic view for taste transduction route between
type II and type III cells
Sweet, umami and bitterness are received on type II cells.
Salty and sourness are received on type III cells that havesynaptic connections. GAD67 synthesizes GABA insideof type III cells.

Sasaki, Hamano and Ueno

45

where GABA may play an active role in thefluctuation of GABA level could occur

salt signal transduction.

within the type III cells, it should influence

GAD67 expressed in the type III cells issalty taste as well as other associatedan active enzyme and produces GABA (5).

activities. In fact, Hisaki and Ueno haveSince GABA is a known neurotransmitter inshown that extracts of spices and herbs

CNS, it is reasonable to assume that GABA

significantly altered GAD67 enzymatic

plays some biological role(s) in the type III

activity in vitro and this phenomenon

cells. In this extent, two types of GABA

coincided with the level of salty taste (6, 8).

actions involving the taste signaling areHence, it is in value to examine theproposed. One is an intracellular action offluctuation of GABA level and its influence

GABA in the sour taste signaling, and theon sour, umami and sweet tastes.

other an extracellular action in sweet, bitter,and umami taste signaling.

If anyTable 1 Characteristics of food extracts
A name and components of spices used in theextraction of food ingredients

The contrast effect has been known in
the food cookery field. A small amount of
added

salt

sweetnesssignificantly
and/orenhances

umami

of

the

foods.

Inhibition effect is also known that a small
added amount of salt weakens bitterness. It
is still a puzzle that how salt which acts on
type III cells could influence sweetness,
bitterness

and

umami

whose

taste

information is received on type II cells.
Since any signals traveling from type II totype III, or vice versa, have not been clearly
understood, the contrast and/or inhibition
effects on foods could not be explained atmolecular level.

If GABA could be

identified as a messenger acting between
type II and type III cells, the mechanism of
the contrast and/or inhibition effects could
be explained. In order to demonstrate the
GABA role as the messenger between thetwo cell types, we present preliminary
results of 1) how food extracts affect sweet,
bitter or umami tastes and 2) whether or not
those effects are related to GAD67 activity.

46

Association of GAD/ GABA and tastes

production. GAD reaction was initiated byadding the appropriate amount of enzyme

Materials and Methods
Extraction

andpreparation

of

food

solution into final 1.0 mL assay solution towhich 100 μL of the assay mixture

ingredients
Spices, herbs and tea leaves listed incontaining 0.2 M L-glutamate and 2 mM

Table 1 were obtained from Somatech

pyridoxal 5'-phosphate (PLP) in 0.5 M

Center, House Foods Co., and commercially

HEPES buffer, pH 7.0 was added.

available sources.

Most samples wereassay solution was incubated at 37 °C for 1

provided as a dry form and were processedh. At the end of incubation period, 50 μL of

into a small pieces. Ultrapure water was

60% perchloric acid (PCA) was added to

added to the samples and placed in aterminate the reaction. As a blank, PCA

refrigerator for overnight.

Then, water

solution was added to the assay solution

soluble components were filtered using 110

immediately after the enzyme addition.

mm diameter filter paper.

Crushed food

After the centrifugation, the supernatant was

samples were typically mixed with 5 times

collected, where an appropriate amount ofweight volume of water and powdered

the aliquots was placed into the vial andsamples were mixed with 10 times weight

injected to HPLC for GABA analysis as

volume of water.

described previously (8).

The supernatants werecollected and used as food extracts.

The

Area of the

GABA peak was compared to that of thestandard GABA and the amount of GABA
produced per incubation time was estimated.

Preparation of GAD67
Recombinant rat brain GAD67 wasTogether with protein assay data obtained byexpressed by growing E. coli strain

Bradford method (BioRad), specific activity

transformed with Rosetta-gamiB (DE3)of GAD67 was calculated as expressed

pLysS

GST-GAD67

mol/min/mg. Bovine serum albumin was

was cloned in thisused as a reference protein for the protein

vectorinencoding cDNA

which

laboratory. Purification of the recombinant

assays.

GAD67 protein from GST-GAD67 fusion
protein was carried out by glutathione

Evaluation of the effects of food extracts on

affinity chromatography.

GAD67 activity

GST-tag wasremoved with thrombin treatment to give an

Effects of food extracts on GAD67
activity was evaluated by carrying out theactive GAD67.

enzyme assay in the presence of an
appropriate amount of food extracts. The

Evaluating GABA production
by

GAD67 enzyme assay was carried out

amount of GABA produced was compared

quantitatively

with that obtained without the addition of thedetermining

GABA

Sasaki, Hamano and Ueno

47

food extracts. Any increase or decrease in

their effects on human taste sensation (9).

GAD67 activity was referred to relative

Basic tastes for sweet, umami and bitter

activity ratio (%) where no change inwere 3% sucrose, 0.08% MSG and 0.05%

activity was expressed as zero and any

caffeine, respectively (Table 2) (10-12).

inhibition was expressed as a minus %Reagents used were commercial grade

number.

purchased from NacalaiTesque, Ltd.

Eachof the extract solutions was diluted with
Taste sensory test

ultrapure

water

to

the

appropriate

Seventeen extracts obtained from theconcentrations and placed in a paper cup.

spices, herbs and teas were examined for

Typically, about 20 ml was supplied for theFig.2 Effects of food extracts on GAD67 Activity
GAD67 activity is plotted in the presence and absence of food extract.
Increased or decreased activity is indicated as positive or negative
number in %, respectively. Water was used in place of food extracts
as control that gave 100%.

48

Association of GAD/ GABA and tastes

Table 2 Reagents used in sensory test

tests. Detailed test procedure is described

2. Take the first tasting solution into

as follows:

mouth trying to spread over the entire(1)

surface of the tongue, and spit out or

Sensory test subjects were selectedfrom a group of students ranged in age fromswallow it.

19 to 21 at Mimasaka University where the3. Rinse mouth with ultrapure water,

tests were performed. They agreed withthen, take the second tasting solution

the purpose of this study.

into mouth.

They were

volunteers and did not receive any special

4. Complete the sensory evaluation sheet

training prior to the tests. The study wasin three step scale, “strong”, “same”

approved by the Mimasaka Universityand “weak” on any changes.

Ethics Review Committee.
(2)

The sensory test was proceeded

Results

according to the guideline "Fact of Food

Search of spices that affect the activity of

Sensory Test to Measure the Taste" (13).

GAD67

Subjects were allowed to repeat theWe have examined if spice, herb andtests if they were not fully confidence of thetea extracts influenced the activity of

results.

GAD67 by incubating with those ingredients(4)

Tests were limited to three or fourand measured any changes in GABA

times in order to avoid fatigue of the subjects.

production with and without the extracts(5)(Fig. 2).

(3)

The test site was at the cooking

Yuzu peel, anise, cumin, androom

celery raised GABA production whereas

temperature was kept at 23-24 °C. All tests

rose rugosa, chamomile, lemongrass, oolong,

were performed between 9:00 and 12:00 a.m.

and oregano showed strong inhibition.

(6) Test results were statistically processed

Among the examined, a small number of theaccording to the two-sample preference test

extracts activated GAD67 activity.

(14).

level of GABA production varied among the(7) The subjects proceeded the sensory test

extracts.

protocol as follows:

extracts was considered; however, it islaboratoryoncampus,where

1. Rinsed mouth with ultrapure water

The

GABA contamination in theSasaki, Hamano and Ueno

49

Table. 3 Change in the sweet taste sensitivity by food extracts （n＝30）
The subjects were evaluated three levels "strong", "same" and "weak" the strength ofbitter taste after tasting the food ingredients.
enhancing effect of sweet taste
Relationship between activity of GAD67 and enhancing effect of sweet by Food
Extracts.
Enhancing effect of taste by the food extract is subtracted a ratio of panelists who
evaluated that taste was weak from the rate of them who evaluated that taste was strong.
Enhancing effect of sweet taste points = strong - weak /n (number of subjects)

unlikely occurred since endogenous GABA

activity in vitro.

was subtracted from GAD assay results by
the control experiments.

Hence, it is likely

Sensory test

that there is a direct interaction between

We conducted a sensory test in which

GAD67 protein and food ingredient(s).

30 female college students answered the

Our results support the idea that spice or tea

strength of sweet, umami and bitter tastes,

components have an impact on GAD67

whereas the 17 extracts were tested due to50

Association of GAD/ GABA and tastes

their affects on GAD67 activity.

concentration for sucrose was shown to be
0.5%.

(1)Sweet taste

The amounts of the extract were

determined based upon their effects on

Sweet taste tests were carried out with

GAD67 activity. Results are summarized

3% sucrose solution in the presence andin Table 3. Those who tasted the extracts ofabsence of the extracts. This concentration

all 17 extracts answered that anise, poppyof sucrose was chosen according to theseed, celery, mace, lemongrass and oolong

tea enhanced sweet taste. On the other hand,reference(10).
The
threshold
Table. 4 Change in the umami taste sensitivity by food extracts （n＝29）
The subjects were evaluated three levels "strong", "same" and "weak" the strengthof bitter taste after tasting the food ingredients.
enhancing effect of umami taste
Relationship between activity of GAD67 and enhancing effect of umami by Food
Extracts .
Enhancing effect of taste by the food extract is subtracted a ratio of panelists who
evaluated that taste was weak from the rate of them who evaluated that taste wasstrong. Enhancing effect of umami taste points = strong– weak /n (number ofsubjects)

Sasaki, Hamano and Ueno

51

over 60% of the test subjects responded thatconcentration of 0.08% was used as anbasil, chamomile, ginger and rosa rugosa

umami compound. This concentration was

weakened sweet taste.

It is evident thatchosen as it was above the thresholdspices, herbs and teas could alter sweet taste.

(0.03%) (10, 11). Subjects were asked totaste the umami solution in the absence and(2)Umami taste
L-Monosodium glutamate solution atpresence of an appropriate amount of each of
the extracts. The subjects marked “strong”

Table. 5 Change in the bitter taste sensitivity by food extracts （n＝29）
The subjects were evaluated three levels "strong", "same" and "weak" the strength ofbitter taste after tasting the food ingredients.
enhancing effect of bitter taste
Relationship between activity of GAD67 and enhancing effect of bitter by Food
Extracts .
Enhancing effect of taste by the food extract is subtracted a ratio of panelists who
evaluated that taste was weak from the rate of them who evaluated that taste wasstrong.
Enhancing effect of bitter taste points = strong - weak /n (number of subjects)

52

Association of GAD/ GABA and tastes

and “weak” when they sensed umami taste

salty taste enhancement.

being

diminished,that the presence of food extracts could

Table 4 summarized the

reduce salt intake and the reduction of salt

results. Anise, cumin, lemongrass, oregano,intake could lead to the prevention ofcelery, mace and parsley significantly

hypertension.

enhanced umami taste. Perilla and ginger

mechanism has not been clarified, the study

significantly

taste.

suggests that the observed salt enhancement

Similar to sweet taste, the components ofis probably due to the effects on salt signal

spices and teas could have affected umami

transduction pathway locating on the type III

taste.

taste buds. In this study, we have focusedenhancedrespectively.

andinhibited

umami

They suggested

Althoughdetailed

on the effects of spice, herb, and tea extracts
on sweet, umami, and bitter tastes.

(3)Bitter taste
Caffeine concentration of 0.05% was

Spices, herbs, and teas may have someThis

unexplored activities besides on the taste

concentration of caffeine was typical for

sensations, possibly altering sweetness,regular coffee and tea (12).

umami and bitterness.

chosen for the bitter taste tests.

Chamomile,

It has not beenanise, paprika and celery enhanced the bitter

rigorously investigated on how spices, herbs

taste (Table 5). Interestingly, there were no

and teas affect the tastes except few cases

extracts that significantly reduced the bitter(16, 17).

taste. In addition, the dynamic range for thetransduction study has reported that GAD67

effects on bitter taste was relatively narrowis involved via producing GABA in type III

as compared with those for sweet and umami

cells, and some spice extracts exhibit salt

tastes. Similar to sweet and umami tastes,enhancing effects.

the examined extracts showed various

encouraged us to study the roles of spices in

effects on bitter taste.

relation with tastes on human subjects.

Recently, a salty taste signal

This finding has

We have measured the potency of taste
for each of the extracts in the sensory test.
The potency of taste was derived by

Discussion
and nutritional

subtracting the number of subjects who

sciences have shown that food not only

responded that the extract "weakened" theprovides energy, but also its components

taste from the number of subjects whoplay significant physiological roles on ourreported that the same extract "enhanced”.

health (15). Recently, Hisaki et al. reported

An index of the enhancing effect of taste bythat food extracts could be used as a salty

the food extract was thus calculated based

Modern biologicaltaste enhancer (6).

In their study, forexample, a small amount of anise exhibitedSasaki, Hamano and Ueno

53

upon the taste potency that was divided by

the extracts are responsible for the alteration

the total number of subjects (Tables 3 - 5).

of the tastes, and what the molecular basis or

The results indicate that almost all themechanism for such actions.

examined extracts did alter the sweet,

In addition to the roles of spice and tea

umami and bitter tastes. Some enhanced

extracts on sweet, umami and bitter tastes as

while some others reduced sensation of one

described above, the relationship between

or more of the three tastes. At the moment,

the effect on GAD67 activity (Fig. 2) and

it is not clear which chemical components ineach of the examined taste sensations werepoppy seed

Enhancing effect on sweet taste(points)

0.4○

lemongrass

○

○○

mace

oolongcelery

○

○

anise

○

paprika
0.2
perillaY = 0.3213x+ 13.956

0○yuzu peel

R2 = 0.1516

○
○

cumin

○

oregano-0.2○

parsley○

peppermint

rose rugosa○○

chamomile○

ginger

basil

○

-0.4
-80

-60

-40

-20

GAD67

Activity (%)

020

Fig. 3 Relationship for the effect of 17 food extracts on sweet taste
sensation and that on GAD67 activity
X axis indicates effects on GAD67 activity and Y axis indicates effect on
the sweet taste. Correlation between the effects is shown as a line (R2 =0.1516).

40

54

Association of GAD/ GABA and tastes

them.

analyzed (Fig. 3 - 5):

The results indicate that any

activation or inhibition of GAD67 activity
may not influence sweet taste. This may

Sweet Taste
We examined whether or not the effects

deny the possibility that any GABAof those food extracts on sweet taste haveproduced in the type III cells would

some of correlation with GAD67 activity.

influence the sweet taste signal transduction.

When the sweet taste potency vs relative

It has been reported that the degree of theactivity for GAD67 are plotted (Fig. 3), there

sweetness was unaffected by the GABA

seems to be no direct correlation between

presence in the food samples (18, 19). This

Enhancing effect on umami taste (points)

0.6○ cumin○lemongrass

○

0.5

○

oregano

anise
mace

0.4

○

parsley
0.3○

chamomile○

0.2

celerypoppy seedy = 0.06764x+ 28.52665
R2 = 0.01445

peppermint○

rose rugosa
0.1

○

○

paprika

○
○

○

○

basil

oolongyuzu peel

ginger0○○
perilla ○

-80

-60

-40

-20

0

20

GAD67 Activity (%)
Fig. 4 Relationship for the effect of 17 food extracts on umami taste
sensation and that on GAD67 activity
X axis indicates effects on GAD67 activity and Y axis indicates effect on
the umami taste. Correlation between the effects is shown as a line
(R2=0.01445).

40

Sasaki, Hamano and Ueno

55

suggests that GAD67 and GABA possibly

The effects of the extracts on GAD67

locating in the type III cells have no

relative activity ratio were plotted against

communication

taste

those on umami taste potency. As a result,transduction that is received by the type II

there was no correlation between thecells.

GAD67 relative activity ratio and the umami

with

the

sweet

taste potency (Fig. 4). It is of interests tonote that the degree of umami taste was not

Umami Taste
We examined if the umami taste wasaffected when GABA alone was added (18,affected by GAD67 activity and/or GABA.

19). This suggests that GAD67 activity and○chamomile

Enhancing effect on bitter taste(points)

0.4○

Y= 0.04409x+ 18.75393

aniseparsley ○R2 = 0.0169
0.3

paprika○○○ celeryrose rugosa

poppy seed
0.2○

oolong ○oregano○○○ gingerperilla○ cumin○ ○ mace

0.1

basil
○

○ peppermint

yuzu peellemongrass

0
-80

-60

○
-40

-20

0

20

GAD67 Activity (%)
Fig. 5 Relationship for the effect of 17 food extracts on bitter taste
sensation and that on GAD67 activity
X axis indicates effects on GAD67 activity and Y axis indicates effect on
the bitter taste. Correlation between the effects is shown as a line
(R2=0.0169).

40

56

Association of GAD/ GABA and tastes

GABA are unlikely to participate in the

involved in "contrast effect" known inumami taste transduction pathway, whichcooking field.

possibly is located in the type II cells.
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