Chem. Senses 27: 739-745,
2002
© Oxford University Press 2002
Taste Enhancements Between Various Amino Acids and IMP
Food Research and Development Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan
Correspondence to be sent to: Misako Kawai, Institute of Life Sciences, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. e-mail: misako_kawai{at}ajinomoto.com
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Abstract |
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 Top Abstract IntroductionExperiments and resultsDiscussionReferences |
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It is well known that a strong synergistic interaction of umami occurs between L-
-amino acids with an acidic side chain, such as L-Glu or
L-Asp, and 5'-mononucleotides, such as inosine 5'-monophosphate
(IMP). We tested taste interactions between various L--amino acids and
IMP by the psychophysical method and found that taste enhancement occurred
when IMP was added to several sweet amino acids, such as L-Ala, L-Ser and Gly.
The enhanced quality of taste was recognized as umami, and was not blocked by
the sweetness inhibitor ±2-(p-methoxyphenoxy)propanoic acid.
The total taste intensities of various concentrations of the amino acid and
IMP mixtures were measured using magnitude estimation. The results showed that
the potentiation ratios were larger than 1 in the cases of L-Ala, L-Ser and
Gly. However, the ratio was 
1 in the case of D-Ala, which had an
enhanced taste of sweetness. Thus the umami taste enhancement of several sweet
L--amino acids by IMP was synergistic rather than additive as that of
acidic amino acids. |
Introduction |
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TopAbstractIntroductionExperiments and resultsDiscussionReferences |
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Each amino acid contributes, to differing degrees, to the taste of foods. Although individual amino acids do not have a strong taste in themselves, their taste is intensified in the presence of other compounds, such as nucleotides (Kuninaka, 1960
) or
inorganic salts (Fuke and Konosu,
1991; Ugawa et al.,
1992). Among various taste interactions, the most prominent
phenomenon is a large umami enhancement by mixing of the monosodium salt of
L-Glu (MSG) and inosine 5'-monophosphate (IMP) or guanosine
5'-monophosphate (GMP), which has been well investigated. This quality
of taste sensation, umami, is the characteristic taste of MSG, IMP or GMP and
their mixtures, and constitutes one of the five basic tastes, along with
sweetness, saltiness, sourness and bitterness. Yamaguchi measured umami
interaction between MSG and IMP using the MSG point of subjective equality
method and demonstrated that the enhancement occurs synergistically
(Yamaguchi, 1967). Rifkin and
Bartoshuk showed that MSG—GMP interaction is a synergistic umami
enhancement by the method of magnitude estimation
(Rifkin and Bartoshuk, 1980).
Only acidic analogues of L-Glu, such as L-Asp, L-homocysteinic acid and
ibotenic acid, have been reported to show synergistic umami enhancement by the
addition of purine 5'-mononucleotides
(Yamaguchi et al.,
1971; Furukawa,
1991; Kurihara,
1998). Some kinds of acidic peptides, such as L-Glu-Gly-L-Ser,
which express weak umami by themselves, have been shown to express enhanced
umami by the addition of IMP (Arai et
al., 1973; Maehashi
et al., 1999). Furukawa concluded that among protein
amino acids, only L-Glu and L-Asp show umami enhancement by the addition of
IMP (Furukawa, 1991). However,
this result was obtained at a very low concentration, and a relatively small
enhancement might have been missed. In addition, several L--amino acids
with neutral side chains elicit umami at high concentrations
(Ninomiya et al.,
1966; Yoshida and Saito,
1969), therefore they show potential umami enhancement by adding a
5'-mononucleotide. In this human psychophysical study, taste interactions between various amino acids and IMP were re-examined at various concentrations to investigate whether other non-acidic amino acids have umami synergistic potential.
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Experiments and results |
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TopAbstractIntroductionExperiments and resultsDiscussionReferences |
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General procedure
Twenty- to 40-year-old male or female researchers from the Food Research and Development Laboratories of Ajinomoto Co., Inc., Japan, participated as subjects. All psychophysical tests followed the regulations for sensory evaluation of the Ajinomoto Co., Inc.
Stimulus compounds were as follows. Amino acids and succinic acid of
guaranteed reagent grade were from Nacalai Tesque Inc. (Kyoto, Japan);
peptides were from Sigma-Aldrich (Tokyo, Japan); food-additive grade
IMP·2Na (IMP) was from Ajinomoto Co., Inc. (Tokyo, Japan). In
Experiment 3, the sweetness inhibitor ±2-(p-methoxy-phenoxy)
propanoic acid (PMP) (Schiffman et
al., 1999) was purchased from Sigma-Aldrich. Deionized water
purified using a Milli-Q water purification system (Millipore, Bedford, MA)
was used both for stimuli and as mouth rinse.
Stimulus solutions were poured into plastic cups and served at room temperature. Subjects were asked to rinse their mouths thoroughly, then sip the stimulus solution, swirl it around in the mouth for several seconds to taste it, and spit it out. In Experiments 2 and 3, subjects were asked to sip the entire 10 ml aliquot in the cup at once.
Experiment 1
This experiment was conducted to examine how the taste quality changed when IMP at near-threshold concentration was added to each amino acid solution.
Test procedure
In the first test, each amino acid solution was adjusted to a concentration
that had an almost `moderate' total-taste intensity to allow the perception of
taste quality (Table 1). These
concentrations showed agreement with previous measurements of taste intensity
of amino acid solutions by a NaCl standard scale
(Ninomiya et al.,
1966), and with those of taste intensity of NaCl solutions by
labelled magnitude scale (Kawai and
Okiyama, 1998). Several non-amino acid compounds were also tested
under the following concentrations: succinic acid, 2 mM; peptides:
Gly—Gly, 60.6 mM and L-Glu—L-Glu, 3.62 mM. Pairs of stimulus
solutions with and without 0.5 mM IMP were presented randomly to 10 subjects.
The subjects were asked to taste at will in volume of sipping and way of
repetition and to describe the difference in intensity between solutions with
and without IMP for each quality of taste (sweetness, saltiness, sourness,
bitterness, umami, and other tastes) and for total taste using the symbols
>, 
or =. Based on these responses, numerical values were applied to
the solutions with IMP as follows: `with IMP > without IMP': +2; `with
without': +1, `with = without': 0; `with 
without': -1, and `with <
without': -2. Values for the solution without IMP were assumed to be zero. The
average value for each taste quality was calculated and a Wilcoxon's signed
rank test was performed.
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We then recruited 50 subjects for the second sensory test to identify the enhanced quality of taste by adding IMP to each amino acid solution of Gly, L-Ser, L-Ala and D-Ala. Pairs of 50 mM solutions of each amino acid with and without 0.5 mM IMP were presented to subjects. The pair of water and 0.5 mM IMP solution was also presented. The order of presentation of these pairs was randomized and the order within pairs was counter-balanced. The subjects were asked to taste at will as in the previous evaluations and to select the stronger one, and to choose the most different quality of taste in intensity between the two among sweetness, saltiness, sourness, bitterness, umami, and other tastes.
Results
The results from the first test of 26 compounds are presented in
Table 1. The taste of many
amino acids, except for bitter amino acids with aliphatic side chains, was
modified by adding IMP at 0.5 mM, which was its approximate threshold
concentration. Several sweet amino acids that do not have acidic side chains
showed significant umami enhancement. The dipeptide, L-Glu—L-Glu, also
showed umami enhancement.
The results from the latter test of Gly, L-Ser, L-Ala, D-Ala, and water are summarized in Table 2. In the cases of Gly, L-Ser, L-Ala and D-Ala, solutions containing IMP were chosen as the stronger-tasting solutions with significantly greater frequency, while in the case of water the difference in choice was not significant. In the cases of Gly, L-Ser and L-Ala, umami was perceived as the most enhanced quality of taste by addition of IMP. In the case of D-Ala, on the other hand, sweetness was perceived as the most enhanced quality of taste.
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Experiment 2
The taste intensities of solution pairs for which the solution with IMP showed significant taste enhancement (Experiment 1) were further examined by modulus-free magnitude estimation.
Test procedure
Eight subjects who had previously participated in magnitude estimation
sessions participated in two sessions for each stimulus set. The stimulus set
consisted of 19 concentrations of solutions of IMP (0, 0.5, 1.0 and 2.0 mM)
and the amino acids Gly, L-Ser, L-Ala or D-Ala (0, 25, 50, 100, 200 mM) except
for pure water. Gly, L-Ser and L-Ala were shown to enhance umami, and D-Ala,
which is an enantiomer of L-Ala, was shown to enhance sweetness in Experiment
1. Ten millilitre samples of the 19 solutions were served in random order,
with the exception that a solution of approximately middle taste intensity was
presented as the first sample so that the subjects could make the subsequent
ratings easily. Subjects were asked to rate the total-taste intensity of the
first sample using any number and to give single magnitude estimates in the
form of a ratio to the taste previously sampled. For each of the ratings,
subjects were instructed to rinse their mouths more than twice to eliminate
the effect of IMP, and sip all of the aliquot in a cup at once to rate
total-taste intensity sensed during sample solution in their mouths. Collected
data were taken as 16 independent estimates.
Data analysis
Prior to analysis, the stimulus was assigned a value of zero in a session,
and then this value was replaced with a value equal to 1% of the second
smallest number of the session. To eliminate arbitrary numbers between
sessions, standardization was performed by multiplication as if the sum of the
numbers of each session was 100. The geometric mean of standardized values for
each stimulus, i.e. the taste intensity, was calculated by taking the
arithmetic mean of the logarithmic values. Then, each geometric mean value was
converted to a relative value by division with the value for the 2.0 mM IMP
unmixed solution. The potentiation ratio was calculated by dividing the taste
intensity of the mixture by the sum of taste intensities of the individual
components in the mixture.
Results
In all tested amino acids at low concentrations, the potentiation ratio
exceeded 1. At higher concentrations, the potentiation ratios of Gly, L-Ser
and L-Ala exceeded 1, but that of D-Ala was 1 (Figures
1 and
2).
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Experiment 3
The results from Experiment 1 show that umami was the perceived quality of the enhanced taste when several sweet L-amino acids were mixed with IMP. In this experiment, we attempted to qualify the quality of the enhanced taste, whether umami or sweetness.
Test procedure
Four male subjects performed 80 sessions of triangle tests. Two cups of 100
mM Gly and one cup of 100 mM Gly + 0.5 mM IMP or 200 mM Gly were presented in
the presence of 0.5 mM PMP. The latter two stimuli showed almost the same
total taste intensity as they did in Experiment 2
(Figure 1). Sets of three 10 ml
solutions were presented randomly, and for each set the subjects were asked to
choose the odd one, and then to label the discriminability of this choice as
`Very easy', `Easy' or `Difficult'. Based on signal detection measures,
d' was estimated according to the correct ratio
(Ennis, 1993;
Bi et al., 1997).
Results
The ratio of each response is listed in
Table 3. In the presence of
PMP, although 100 mM Gly + 0.5 mM IMP was easily discriminated from 100 mM
Gly, the discrimination of 200 mM Gly from 100 mM Gly was not easy. The
difference between the two d' values was highly significant
(Z = 5.74) (Bi et al.,
1997). Though dispersion was observed in the correct ratio and
discriminability among the answers of the four subjects, the above tendency
showed consistency among subjects.
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Discussion |
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TopAbstractIntroductionExperiments and resultsDiscussionReferences |
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Umami was the enhanced quality of taste when IMP was added to some sweet L-
-amino acids
In prior human sensory evaluation studies, only acidic amino acids have
been reported to have enhanced umami by addition of purine
5'-monophosphates (Yamaguchi et
al., 1971; Furukawa,
1991). In Experiment 1 of the present study, however, addition of
IMP changed the taste of various amino acids as follows: (1) umami was
enhanced for L-Ser, L-Gln, L-Asp, L-Asn, L-Thr, L-Glu, Gly, L-Ala, L-Met,
L-Cys and peptide L-Glu—L-Glu; (2) sweetness was enhanced for L-Ala,
D-Ala, 4-hydroxy-L-Pro, L-Arg, L-His and L-Cys; and (3) Sourness was reduced
for L-Glu, L-Asn, L-Thr, L-Gln, and succinic acid. With regards to (1),
although many of the amino acids had a dominant sweet taste in themselves,
umami was perceived as the basic quality of the enhanced taste. Next, we used
50 subjects to investigate the perceived quality of enhanced taste by adding
IMP at near-threshold concentration to L-Ala, D-Ala, Gly and L-Ser, the
total-taste intensities of which were found to be significantly enhanced by
IMP addition in the first test. Subjects did not agree as to quality of
enhanced taste by addition of 0.5 mM IMP to pure water, while umami was
clearly perceived as the quality of the enhancement for L-Ala, Gly and L-Ser,
and sweetness was perceived as that for D-Ala.
Although umami and sweetness are independent basic tastes, people sometimes
integrate the two and cannot accurately discriminate between them in
mixed-taste solutions, such as in mixtures of sweet amino acids and IMP. For
this reason, the quality of the enhanced taste should be identified not only
by a subjective test but also by a more objective method. In Experiment 3, we
further investigated whether the quality of the enhanced taste was umami or
sweetness using Gly—IMP solutions with a sweetness inhibitor, PMP. This
compound is an analogue of a sweetener, dulcin, and reduces sweet taste as a
competitive antagonist having a broad inhibitory spectrum
(Schiffman et al.,
1999). One hundred mM Gly + 0.5 mM IMP was determined to be
equivalent to 200 mM Gly in total-taste intensity in Experiment 2, and both
solutions were easily distinguished from 100 mM Gly. In the presence of PMP,
only the discrimination between 200 mM Gly and 100 mM Gly became difficult.
PMP inhibited the sweetness that was perceived as the intrinsic dominant taste
of Gly, and did not inhibit the taste enhanced by addition of IMP. Similar
phenomena were observed in the cases of L-Ser and L-Ala (data not shown).
Thus, the quality of the enhanced taste was perceived as umami and was not
inhibited by the sweetness inhibitor, PMP.
Umami enhancement is synergistic
We measured the taste intensity of amino acid and amino acid—IMP solutions by magnitude estimation. We designed our study to give estimates not of the individual basic taste qualities, but of the total-taste intensity of amino acid and amino acid—IMP solutions in order to eliminate idiosyncrasies in taste discrimination of sweetness—umami mixed solutions. The slopes of the psychometric amino acid concentration—taste intensity curve got steeper as the concentration of IMP was higher in the cases of Gly, L-Ser and L-Ala, while in the case of D-Ala the slope did not change (Figure 1).
Based on the fact that magnitude estimates have shown a high correlation
with neural responses (Borg et
al., 1967), we applied the function of potentiation ratio
(PR) to our results, as has been done in nerve response animal studies
(Yamamoto et al.,
1991). The PR value patterns of Experiment 2
(Figure 2) can be classified
into two cases: Case 1: L-Ala, Gly and L-Ser; PR > 1; and Case 2: D-Ala; PR
= 1 (PR > 1 at 25 mM, approximate threshold concentration). Thus, the umami
enhancement in Case 1 was synergistic rather than additive, while the
sweetness enhancement in Case 2 was additive. The larger PRs at low stimulus
concentrations might have been due to quasi-zero ratings for both amino acids
and IMP unmixed solutions.
We further tested whether our result met Rifkin and Bartoshuk's criteria of
synergism (Rifkin and Bartoshuk,
1980). Figure 3
compares three values: the observed taste intensity of mixture solutions, the
predicted value made by the sum of perceived intensities of single-ingredient
solutions, and the value by stimulus addition analysis calculated in molar
concentration. In the cases of Gly, L-Ala and L-Ser, observed intensity of
mixtures exceeded not only the sum of perceived intensities but also the value
by stimulus addition analysis. Therefore, the enhancement patterns agreed with
Rifkin and Bartoshuk's criteria of synergism. On the other hand, in the case
of D-Ala, these values were almost the same.
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The essential structure for synergistic umami is L--amino
acid
Amino acids that showed umami enhancement by IMP in our study have umami in
addition to their dominant sweet taste at high concentrations
(Yoshida and Saito, 1969).
Noguchi et al. reported that some peptides, such as
L-Glu—L-Glu, also have umami at neutral pH; L-Glu—L-Glu also
showed umami enhancement in our study
(Noguchi et al.,
1975). These findings indicate that many amino acids other than
L-Glu and L-Asp and some peptides might have weak interactions with umami
receptor(s) in a mode similar to that of MSG. Our findings that umami
enhancement occurs synergistically verify the action of this mechanism. On the
other hand, the sweetness enhancement of D-Ala, an enantiomer of L-Ala, by IMP
occurred only in an additive manner. Furthermore, succinic acid, which was
isolated as an umami compound from clams, did not show umami enhancement by
the addition of IMP. Our results suggest that the basic structure necessary
for synergistic umami might be L--amino acid. Umami has been said to be
the guide taste for protein, and our result that among protein amino acids not
only acidic but also various neutral amino acids showed umami enhancement
might support this idea.
Chaudhari et al. cloned cDNA of a truncated analogue of the brain
metabotropic glutamate receptor, taste mGluR4, from rat taste cells
(Chaudhari et al.,
2000). More recently, the hetero-dimeric G-protein-coupled
receptors, T1R1—T1R3, from human taste cells was found to respond to
L-Glu synergistically with IMP (Li et
al., 2002; Nelson et
al., 2002). It is shown that human T1R1 influences
sensitivity to L-Ala and L-Ser as well as L-Glu. In other words, L-Ala and
L-Ser activate robustly receptors containing human T1R1, and IMP potentiates
the response synergistically. These latest reports support our psychophysical
findings in a molecular manner. Though the subunit T1R3 is shared with the
sweet taste receptor T1R2—T1R3
(Nelson et al.,
2001), the umami elicited by mixing various sweet L--amino
acids and IMP was barely reduced by PMP in Experiment 3, and the umami of MSG
and MSG—IMP mixture was also barely inhibited (data not shown). If we
can find and use an umami-specific taste inhibitor, our findings from the
above psychophysical experiments to divide total taste into each quality would
be strongly supported.
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Acknowledgments |
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We are grateful to Dr G.K. Beauchump and Dr P.A.S. Breslin for practical suggestions and helpful advice for taste measurement with ratio scale.
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Accepted July 26, 2002
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