Sensors and Materials, Vol. 13, No. 3 (2001) 179-187
MYUTokyo
S &M0441

Development of Technology for Separating
and Identifying Bitter Substances
Ryuji Tak:amatsu, Kiyoshi Toko, Hiroshi Tak:eguchi 1 and Akira Kawabata2
Department of Electronics, Graduate School of Information Science and Electrical Engineering,
Kyushu University, Fukuoka 812-8581, Japan'Center for Analytical Chemistry and Science, Inc. Yokkaichi, Mie 510--8530, Japan
2Center for Analytical Chemistry and Science, Inc. Ibaraki 300--0332, Japan(Received December 15, 2000: accepted February 28, 2001)
Key words:
taste sensor

HPLC, lipid membranes, bitter substances, separation and identification, multichannel

A multichannel taste sensor is developed by imitating the mechanism in gustatory
systems. Various foods have been measured using a taste sensor, and identification andquantification of a taste have become possible. However, there is no method for detecting
and separating bitter substances in a solution. Development of such a method might
contribute greatly to the understanding of foods, chemicals and medicines. The purpose ofthis study is to examine a method for detecting and separating bitter substances using ataste sensor and high-performance liquid chromatography (HPLC) and to develop theequipment.

1.

Introduction

Humans have five senses: sight, hearing, touch, smell and taste. These senses are very
important because humans act after receiving information from the outside world. Sensors
have been produced to quantify the subjective and equivocal senses of humans by artificial
methods and materials. In the field of taste, a multichannel taste sensor has recently beendeveloped. This sensor imitates the mechanism in gustatory systems and produces as
output the taste itself as information_<l-3) Various foods have been measured using the taste
sensor, and identification and quantification of the taste have become possible.

179

180

Sensors and Materials, Yol. 13, No. 3 (2001)

There are five basic tastes: sweetness, saltiness, sourness, bitterness and umami taste.
Bitterness among these tastes can be considered to be the signal of poison. Many animals
mainly avoid bitter foods. Humans do not like bitterness either, aside from the bitterness ofbeverages like beer and coffee. When a sensory test is given to control the bitterness offoods or medicines, inspectors are under much stress.<4l Sometimes, it may also injure theirhealth; the inspectors may be in danger when they are checking drugs.
In addition, the mechanism of the taste reception systems of the taste sensor resembles
that of humans. The taste sensor imitates the mechanism in gustatory systems. Thus it
becomes possible to detect bitterness quantitatively, by means other than a sensory test.
However, it is difficult to separate bitterness and other taste qualities and to identify thebitter substance by the taste sensor. If such a detecting and separating method weredeveloped, it might contribute largely to the food, chemical and medical industries. Thus
we tried to examine a method for detecting and separating bitter substances using both thetaste sensor and high-performance liquid chromatography (HPLC) and to develop theequipment.

2.

Materials and Methods

2.1 Multichannel taste sensor

Generally, we used eight different membranes, each of which is an artificial lipid/
polymer membrane that imitates a lipid bilayer, as the transducer in the taste sensor. Thus
the taste sensor has a multichannel structure and eight outputs. There are two reasons weadopted these membranes as transducers. First, the biomembrane that receives taste
substances is composed of proteins and a lipid bilayer. Second, these membranes imitate alipid bilayer. < 1-3l
The taste sensor is superior to that of humans in sensitivity, durability and reproducibil­
ity of measurements. Thus it is expected that the transducer will be able to be applied tofood, medical and environment measurements.
The taste sensor shows large responses to bitter substances, since the bitter substances
are bound to lipid membranes, as clarified by the change of membrane potential caused byadsorption (CPA) method.<5l Taste qualities such as sourness, saltiness, sweetness andumami taste are barely detected using the CPA method. That implies that lipid membranes
have the potential to bind bitter substances selectively. This is reasonable because many
bitter substances as well as the hydrocarbon chains of lipids are hydrophobic. This is thereason we adopted lipid membranes for the taste sensor in this study as the material to bindwith bitter substances.
Table 1 shows the lipid membranes used in this study for each bitter substance. The
response to these bitter substances is large and stable_(5,6l For example, the response of thetaste sensor to caffeine is shown in Fig. 1.

2.2 HighTpe,formance liquid chromatography

Chromatography is a method used to separate a mixture into separate components.
Two phases, the stationary phase and the mobile phase, maintain an equilibrium. A sample
flows through a column. Each ingredient in the sample interacts with each phase and is181Sensors and Materials, Vol. 13, No. 3 (2001)
Table 1
Lipid membranes for each bitter substance.
Bitter substance

Lipid membrane

Quinine hydrochloride
Caffeine
Iso-a-acid

DOP (Dioctyl phosphate)
OA (Oleic acid)
OAm (Oleyl amine)

5Q)

0.

0Q)

0.
Q)

-5DA

OA DOP 9:1

5:5

3:7 TOMAOAm

ChannelFig. ;. Response of the taste sensor to caffeine. The horizontal axis is the channel of the taste
sensor. Measurements were made three times. Each abbreviation identifies the lipid: DA, decyl
alcohol; OA, oleic acid; DOP, dioctyl phosphate, TOMA, trioctyl methyl ammonium chloride;
OAm, oleyl amine; and 9:1 is the molar ratio C:T=9:l; 5:5, C:T=5:5; 3:7, C:T=3:7.

separated. This separated component is measured qualitatively and quantitatively. In thecase of using a liquid as the mobile phase, the chromatography is called liquid chromatog­
raphy (LC). Specially, it is called high-performance liquid chromatography (HPLC) if theseparation is carried out at high speed using a high-pressure pump and a high-performance
filler.
The basic equipment for HPLC consists of five parts: a pump for propelling a liquid(mobile phase), an injector for introducing a sample, a column, a detector and dataprocessing equipment. Figure 2 shows a schematic of typical HPLC equipment.

182

Sensors and Materials, Vol. 13, No. 3 (2001)

Homoiothermal water
Pump

Mobile phase tank

Waste fluid tankFig. 2. Schematic ofHPLC equipment. A solvent, the mobile phase, is propelled by the pump. The
mobile phase is sent to the column by passing through plumbing shown by the bold line. A solidpackage, the stationary phase, fills the column. A small amount of sample is injected on the column
from the sample injection port. The sample is separated in the column. The detection of each eluted
ingredient is transformed into an electronic signal in proportion to the concentration and is recordedby the data processing equipment.

The HPLC instrument in this study was a Shimadzu LC-IOAGVP equipped with aCTO- IOACVP colurnn oven, a Shimadzu SPD-IOAVP UV-VIS detector, a Shimadzu
DGU-12A degasser, a Shimadzu CBM-IOA communications bus module and a personalcomputer. The analytical column (0.46 x 15 cm) was a Shimpack ODS (Shimadzu Ltd.).
2.3 Measurement method
2.3. l Sample preparation and chromatography
Figure 3 shows the method of preparing samples for HPLC measurement. Bitter
substances adsorb to the lipid membrane in process (2), and the substances are extracted byethanol in process@. These steps successfully lead to the separation of bitter substances.
We used 1 mM quinine hydrochloride as a typical positively-charged substance to show
bitterness, 10 mM caffeine as a neutral substance and 20,000 ppm iso-a-acid as anegatively-charged substance. Some lipids can also be dissolved in ethanol in process@.
To take into consideration the effect of these lipids, we also prepared 1 mM KCl in place ofbitter substances in the above method. The washing time in@ was 5 s for iso-a-acid.
Table 2 shows chromatographic conditions for three kinds of bitter substances. Thesolution in process @ was measured by HPLC.

183

Sensors and Materials, Vol. 13, No. 3 (2001)

Lqll�md,,..--=-.J� -+ ,,..__...
�Samplesoak for 120 seconds

wash for 10 seconds

soak for 120 seconds

Fig. 3. Method for preparing samples for HPLC measurement.

Table 2Chromatographic conditions for each bitter substance. Solvent A is a molar ratio of 10 mM
phosphoric acid buffer: acetonitrile: methanol=2:1 :2. Solvent B is a molar ratio of 10 mM
phosphoric acid buffer: acetonitrile:methanol=9:1. Solvent C is methanol and water (72.5:27.5)
containing 85% phosphoric acid (1.7 g/100 ml) and 10% tetraethylammonium hydroxide (2.95 g/100ml).

Mobile phase
Flow rate (ml/min)
Temperature of column oven (°C)
Detection wavelength (nm)

Quinine hydrochloride

Caffeine

Iso-a-acid

Solvent A
0.130254

Solvent B
1.040237

Solvent C
1.550280

2.3.2 Calibration and calculation

The measurement of various concentrations of sample provided the calibration curves.
The concentration of each bitter substance extracted using HPLC was calculated, using thecalibration curves for quinine and caffeine in Fig. 4 and for iso-a-acid in Fig. 5.
Figure 5 shows three peaks: isocohumulon (co-), isohumulon (n-) and isoadhumulon(ad-). Five homologues exist in iso-a-acid because of differences in the acyl side chain,each homologue having two geometrical isomers: a cis form and a trans form. The ratio of
the homologues depends on the variety of hop. The major homologues included in beer are
co-, n- and ad-. Few differences in the bitter taste exist among the homologues.<7J184

Sensors and Materials,Vol. 13, No. 3 (2001)

(b)
4000

00.01
0.02
Concentration (mM)

0.03

010
Concentration (ppm)

20Fig. 4. Calibration curves of (a) quinine hydrochloride and (b) caffeine.

3.

Results and Discussion

Because the peak of the lipid overlapped the peak of quinine hydrochloride, wededuced the chromatogram of the lipid from the chromatogram of quinine hydrochloride.
The result is shown in Fig. 6(a). Figures 6(b) and 6(c) show the graphs of caffeine and iso­a-acid, respectively. These two chromatograms may be used as is because each analyte
gives a single peak. There are three peaks corresponding to the three homologues, i.e., co­' n-, and ad-, in Fig. 6(c). Table 3 is obtained from Fig. 6 using the calibration curves ofFigs. 4 and 5. It is clear from this result that the adsorption of these substances to therespective membranes brings about the response of the taste sensor to bitter substances, asis shown in the CPA measurement.C5l
We can thus obtain information about the adsorption of bitter substances to the lipid
membranes by measuring various concentrations of bitter substances using this method. In
addition, a response from the taste sensor implies a relationship between the strength ofbitter tastes and adsorption.
Quinine hydrochloride is positively charged in aqueous solution, while caffeine isneutral and the iso-a-acids are negatively charged. This result indicates the possibility ofdetecting any bitter substance using this method, if the chromatographic conditions areprepared for each bitter substance. However, the weak point in this method is its lowsensitivity. In general, substances whose adsorption power is weak seem to be difficult todetect. For example, 0.1 mM quinine hydrochloride could not be detected. The sensitivity
can be increased by improving the extraction conditions.
This method requires the chromatographic conditions to be changed for each substance.
When an unknown solution is measured, several chromatographic conditions must beprepared. We suggest the following process to achieve the separation and identification ofan unknown solution. First, the property of a bitter substance is understood using the taste
sensor; second, the chromatographic conditions are determined, and finally, samples are185Sensors and Materials, Vol. 13, No. 3 (2001)[x10·5J[x1o+a:i0(a)

jj
�

..

..

-"'-"'

rf.

00500
Concentration (ppm)

1000

0400
200
600
Concentration (ppm)

800[x10•5J(c)2a. 100100
Concentration (ppm)

200Fig. 5. Calibration curve of (a) iso-a-acid(co-), (b) iso-a-acid(n-) and (c) iso-a-acid(ad-).

measured. These improvements enable the practical use of this technology for separatingand identifying bitter substances.

186

Sensors and Materials, Vol. 13, No . 3 (2001)

(b(a)

�e

�
..:l'#"�

4000

10000g�01'--,-.10to0(c)Time(min)2030-�et..�
_j\c;'0to5Time (min)co-n-6000-e4000g�ad-20000s

Time (min)toFig. 6. The result of (a) 1mM quinine hydrochloride, (b) 10mM caffeine and (c) 20,000ppm iso­a-acid.

Table 3
Concentration of each chemical listed in Table 1extracted usingHPLC from the lipid membranes towhich each is bound.
1mM quinine hydrochloride
10mM caffeine
20,000ppm iso-a-acid (co-)
20,000ppm iso-a-acid (n-)
20,000ppm iso-a-acid (ad-)

l .OSµM
0.438µM8.99 ppm
7.52ppm. 2.60ppm

Sensors and Materials, Vol. 13, No. 3 (2001)

187

Acknowledgement
We sincerely thank Dr. H. Ikezaki of Anritsu Co., Ltd. for his support of this work.

References

2345678K. Toko: Biomimetic Sensor Technology (Cambridge University Press, Cambridge, 2000)
Chap. 6.
K. Toko: Sensors and Actuators B 64 (2000) 205.
K. Toko: Meas. Sci. Technol. 9 (1998) 1919.
M. Sato and N. Ogawa: Saishin Mikaku no Kagaku (Science of Taste, Asakura, Tokyo, 1997),
Chap. 2. [in Japanese]
H. Ikezaki, A. Taniguchi and K. Toko: IEE Japan 118-E (1998) 506. [in Japanese]
S. Takagi, K. Toko, K. Wada, H. Yamada and K. Toyoshima: J. Pharm. Sci. 87 (1998) 552.
N. Hashimoto: Biru no Hanashi Part 2 -Oisisa no Kagaku (Topics of Beer, Gihodo Publishing,Tokyo, 1998) Chap. 9. [in Japanese]
Y. Izumi, M. Ogawa, S. Kato, J. Shiokawa and T.Shiba: Kikibunseki no tebiki 2 (Kagakudojin,Kyoto, 1996) Chap. 2. [in Japanese]