Chem. Percept.

(2008) 1:58–77
DOI 10.1007/s12078-008-9008-2

Masking Bitter Taste by Molecules

Jakob P. Ley

Received: 21 November 2007 / Accepted: 24 January 2008 / Published online: 13 February 2008
# 2008 Springer Science + Business Media, LLC

Abstract Combating bitter taste in food, pharmaceuticals, dATP 2-deoxyadenosintriphosphate

and beverages remains a huge challenge. In the past, FLIPR fluorescence-induced plate reader
bitterness reduction was focused on pharmaceuticals and GRK G protein-coupled receptor kinases
drugs; however, more recently, the most intense research is HEK293 human embryonic kidney cells type 293
performed on the reduction of bitter or astringent taste in IP3 inositoltriphosphate
functional food or beverage applications. These foods and L-DOPA L-3,4-dihydroxyphenylalanine
beverages possess inherent off-tastes due to fortification Leu-Trp L-leucinyl-L-tryptophan
with healthy but poor-tasting actives. During the last γ-PGA poly-γ-glutamic acid
10 years, tremendous progress in the elucidation of bitter PDE phosphodiesterase
taste reception and transduction on the cellular level was PLCβ2 phospholipase C subtype β2
made and many new molecules and compounds to reduce TRC taste receptor cells
bitter off-tastes were reported. The following review will be TRPM5 transient receptor potential channel, type M5
focused on the advances, in the area of bitter-masking T2R taste receptor type 2
molecules, during the last 10 years. It will not cover other
debittering strategies such as process optimization or
biotransformations to reduce the amount of bitter ingre-
Introduction
dients, encapsulation, and other physical formulation
technologies. The review will close with a short compar-
Bitter taste is a major problem in the food and pharmaceu-
ative study of various bitter maskers and some suggestions
tical industries due to its negative hedonic impact on
for flavor development of poor-tasting ingredients.
ingestion (Drewnoswki 2001; Drewnoswki and Gomez-
Carneros 2000). Only in rare cases, consumers prefer a
Keywords Off-taste . Bitter Taste . Masking Technologies .
strong bitter taste for food and beverages, e.g., in black
Taste Masking
coffee, black or green tea, beer, red wine, grapefruit
products, or bitter lemon. In most other cases, the bitter
Abbreviations
taste is not desirable and has to be eliminated from or
AMP adenosine monophosphate
masked in the product. As an example, most legumes,
ATP adenosine triphosphate
fruits, and staple foods were extensively optimized using
CMP cytosine monophosphate
breeding and cultivation technology to become less bitter,
cTDA comparative taste dilution analysis
astringent, or sour variants over the course of time. Another
example is in the juice industry, whereby raw orange juices
are processed to be debittered by cleaving the bitter
J. P. Ley (*) naringin to the less bitter naringenin or naringin-7-O-
Flavor & Nutrition Research & Innovation, Flavor Research,
glucoside. Most cloudy raw apple juices are treated to
Symrise GmbH & Co. KG,
P.O. Box 1253, 37601 Holzminden, Germany remove most of the polyphenols, which can taste bitter or
e-mail: jakob.ley@symrise.com astringent to yield clear beverages (Oszmianski et al. 2007).
Chem. Percept. (2008) 1:58–77
In the pharmaceutical area, there is also a large demand
for bitter reduction techniques due to the low compliance
of patients taking bitter drugs for longer times. In young
children, the problem is more serious, due to their higher
taste sensitivity and because in many cases, it is not
possible to supply large enough capsules containing the
active pharmaceutical ingredient. Currently, the old rule
“only bitter medicine is good medicine,” is no longer
valid.
In recent years, the problem of bitter- or bad-tasting food
products is surfacing again, due to the demand for healthier
food or beverages. Reduced sugar, fat, and sodium for
healthy benefits can also accentuate sourness, bitterness,
and astringency in the base matrices. Many artificial
sweeteners exhibit astringent, metallic, or bitter aftertaste.
In salt or sodium replacers, potassium chloride is common-
ly used in many applications, which leaves a very metallic
bitter taste that most people find undesirable. Compounds
such as certain polyphenols (e.g., tea catechins), soy
products, phytosterols, vitamins, minerals, fish oil, etc.
used for fortification of functional food can cause serious
taste deficiencies and reduced consumer demand for such
products (Eckert and Riker 2007).
One of the major problems of masking of off-taste is the
complex mixture of sensations. The ingestible is not only
perceived as bitter, but is also astringent and/or sour. Each
modality is transduced by different molecular sensing
systems in the mouth, and the sensation consciously
recognized is again a difficult mixture to separate into
individual taste qualities.
Many techniques to reduce bitterness or off-taste have
evolved through the years:
& Removal of bad tasting components, where possible
& Physical barriers (e.g., [micro, nano] encapsulation,
coatings, emulsions, suspensions)
& Scavengers, complexing agents
& Strong flavors or tastants (e.g., salt, sweeteners, acid,
strong fruit flavors)
& Congruent flavors (e.g., chocolate, grapefruit, coffee)
& Masking flavors (e.g., against rancid or fishy flavor of
polyunsaturated fats)
& Bitter taste reducing compounds on a molecular level
The use of physical barriers is a common approach for
pharmaceutical actives, and there are several comprehen-
sive reviews (Sohi et al. 2004; Stier 2004). In most cases,
these technologies cannot or in limited use be adapted for
food or beverage applications because the latter applica-
tions contain much more water and often use raw materials
which are not permissible for food use. The use of classical
flavors and tastants was reviewed by Pszczola (2004).
Therefore, the following review will focus mainly on the
last topic, the masking of bitter taste on a molecular level.
59
The last general review covering such molecules was
compiled by Roy in his book (1997). Since then, the
knowledge of taste, especially bitter taste detection, and
transduction on a cellular level, has heavily evolved, and as
a result, several new approaches for detecting and devel-
oping bitter-masking molecules were reported.
Detection of Bitter Taste
Since the identification of the receptor proteins responsible
for bitter taste reception by Chandreshekar et al. (2000;
Adler et al. 2000), the mechanism of bitter reception by
taste receptor cells seems to be generally known nowadays
and was thoroughly reviewed in recent time (Margolskee
2002; Montmayeur and Matsunami 2002; Meyerhof 2005;
Chandrashekar et al. 2006; Behrens and Meyerhof 2006).
Below, the mechanism is briefly summarized (for a
schematic summary see Fig. 1).
Bitter molecules bind to a G protein-coupled receptor-
type T2R on the apical membrane of the taste receptor cells
(TRC) located in the taste buds. In humans, roughly 25
different T2R are described. Additionally, several alleles are
known and about 100 different bitter phenotypes exist in
man. TRC are specialized to a certain taste quality. For
sweet taste, this was demonstrated by genetic experiments
on mice in a labeled-line model. Most probably, sweet cells
are linked directly to positive hedonic centers of the brain.
The authors constructed a mouse expressing T2Rs on sweet
cells and they preferred a bitter and toxic solution and not
the sweet one (Zhang et al. 2003). For the bitter modality,
one TRC expresses more than one T2R type but not in
all variants. On the other hand, it is now known that
one particular bitter compound can bind to several T2R
subtypes with distinct affinity and that at least some of
the bitter receptor proteins, e.g., the hT2R47, are
broadly tuned for several structural classes of bitter
molecules (Meyerhof et al. 2007). As a result, a bitter
taste pattern (Fig. 2) for the cells occurs in a similar way
to the olfaction process; however, the final signal to the
brain is mainly “negative” or “bitter”. Discussions con-
tinue that bitter qualities may be distinguishable but not
yet proven by combined sensory and molecular biological
experiments.
Following the binding of agonists to the T2R, phospho-
lipase C is activated via a β-subunit of a G protein of the
TRC which activates the IP3 (inositoltriphosphate) pathway
in the cell. Calcium will be released from internal stores
and at least the co-expressed ion channel TRPM5 will be
activated and the cell depolarizes. In addition, the α-unit of
the TRC-specific G protein gustducin may activate the PDE
pathway of transduction (Ming et al. 1999), but there is no
final proof of concept at this time.
60
Fig. 1 Actual bitter taste transduction mechanisms (combined and
adapted according to: Gilbertson et al. 2000; Perez et al. 2003; Bufe et
al. 2002) exemplified for the human salicin receptor hT2R16.
Probably only (but not all of the 25) bitter receptors are expressed
on a single “bitter” taste receptor cell. The generally accepted
In contrast to the intracellular mechanisms, the intercel-
lular transduction of the signal generated by agonist
receptor interaction is not yet elucidated in detail. Most
TRC which can be stimulated by bitter tastants (or other
taste qualities) are not directly linked to synapses but they
release neurotransmitters (Clapp et al. 2006). The picture is
still not fully clear, and serotonin, ATP, and some neuro-
peptides are under discussion (Herness et al. 2005;
Romanov et al. 2007; Huang et al. 2007). This does not
preclude there being other transmitters that have not yet
been identified. The neurotransmitters subsequently activate
the so-called output cells in the taste bud, which are
connected to synapses of afferent gustatory nerves (Roper
2007).
In addition to this generally accepted pathway, there
were some studies which suggest possible further mecha-
nisms of bitter reception but are still under discussion.
Naim et al. claimed that tastants can rapidly enter taste cells
and act on intracellular proteins (Peri et al. 2000). One
example may be the general quenching mechanisms of G
protein-coupled receptors by inhibition of signal termina-
tion-related kinases which may cause the lingering bitter
aftertaste of sweeteners (Zubare-Samuelov et al. 2005).
Another protein discussed by the group was activation of
Chem. Percept. (2008) 1:58–77
mechanism follows the IP3 pathway, the PDE branch is not fully
proven. The ATP (or alternative neurotransmitter) release mechanism
is not yet fully known (Romanov et al. 2007; Huang et al. 2007). PLC
Phospholipase, IP3 inositoltriphosphate, PDE phosphodiesterase,
NMP nucleosidemonophosphate
adenylyl cyclase by several sweeteners and bitter tastants
(Zubare-Samuelov et al. 2003). There were some hypoth-
eses that the prominent bitter caffeine may be detected by
activation of PDE (phosphodiesterase) due to its known
activity and the difficulties to identify the responsible T2R
(Yan et al. 2001). Until now, no mammalian bitter receptor
for caffeine is known but it was described recently for fruit
flies (Moon et al. 2006).
From a molecular standpoint, there are several potential
targets to suppress bitter taste transduction:
& Molecules, which can complex or scavenge the bitter
tastants (molecular encapsulation) or can disrupt the
transport to the receptor
& Antagonists of T2r binding sites
& Modulators of T2r binding sites
& Modulators of other proteins involved in taste trans-
duction, e.g., gustducin, PLCβ2 (phospholipase C β-2),
PDE pathway
& Modulators of TRPM5 function
& Compounds which can influence the neurotransmitter
release, binding, or reuptake
& Modulators of signal quenching, e.g., reactivation of G
proteins or receptors
Chem. Percept. (2008) 1:58–77 61

agonists receptor hTAS2R values: threshold conc. in log M max. response

1 3 4 5 7 8 9 10 13 14 16 38 39 41 43 44 45 46 47 48 49 50 55 60 61 76 on receptor
arbutin low
amygdalin
PROP
Diphenylthiourea
aristolochic acid
saccharin
absinthin
picrotoxinin strong
chloramphenicol
humolone + not
alpha-thujon quantified
herbolid A
phenylisothiocyanate orphan
denatoium benzoate receptor
PTC
strychnin
brucin
1-naphthoic acid
piperonylic acid
sodium benzoate
salicine
nitrosaccharine
acesulfam K
sucroseoctaacetat
sesquiterpene lactones
papaverine
quinacrine
chloroquine

Fig. 2 Bitter receptor matrix (compiled from data from: Meyerhof et Known agonists are presented in column 1, the human T2R are given
al. 2007; Bufe et al. 2002; Xu and Li 2006; Pronin et al. 2004; Kuhn in line 1. The gray areas are not yet characterized. Only a few
et al. 2004; Prodi et al. 2004; Behrens et al. 2004). The picture is only interactions were not only qualified but additionally quantified by
an actual spotlight of the whole matrix in respect to potential agonists. Meyerhof et al. (2007) and Bufe et al. (2002)

Whereas scavenging is an established and a well-known
mechanism, it is not clear whether the transport can be
selectively influenced. Saliva flow and its constituents
certainly play an important role for the complex transport
of tastants to the taste cells (Matsuo 2000), but early
discussions regarding the role of lipocalins secreted by von
Ebner glands as “tastant-binding proteins” for bitter
compounds could not be verified (Creuzenet and Mangroo
1998). Unfortunately, in contrast to the agonist/T2R studies
(Fig. 2), similar data regarding agonist/antagonist/T2R
interactions were not published yet. These data would be
of very high value for development of selective bitter
inhibitors. The influence of neurotransmitter release on
real tasting experiments was very rarely reported until
now. In a recent paper of Heath et al. (2006), the
modulation of human taste thresholds by changes of the
serotonin and noradrenalin levels induced by certain drugs
was determined. Significantly enhancing the serotonin level
caused a reduction of the sucrose taste threshold by 27% and
the quinine taste threshold by 53%. An increased noradrenalin
titer significantly reduced bitter taste threshold by 39% and
sour threshold by 22%. As a conclusion, influencing
neurotransmitter levels, e.g., by drugs can dramatically change
the taste response and may be a possible target for further
developments of taste modulation compounds. But it is too
early to decide which neurotransmitter or its receptor may be
the most important target for bitter masking.
Potential modulators/antagonists of receptors and pro-
teins will be discussed later in greater detail. In nearly all
presented cases, the exact mechanism of masking activity is
not yet known.
Bitter-Tasting Molecules: Structure–Activity
Relationships
In contrast to the other taste qualities of sweet, umami, sour,
and salty, there is a large number of molecules which are
described as bitter. Generally speaking, the bitter modality
is an aversive taste which protects animals against
potentially toxic or harmful substances in nature. In
parallel, in bitter (and sometimes toxic) plants, molecules
have evolved to deter herbivores (Simmonds 2001). It will
not be the intention of this review to list all relevant bitter
tastants; therefore, in the following paragraphs only some
examples will be discussed.
Bitterness is widely distributed in nature and principally
each chemical class can contain bitter molecules. Simple
salts such as sodium sulfate or magnesium sulfate show a
strong bitterness. Some higher peptides, terpenoids, alka-
loids, polyphenols, heterocycles, and macrolides can also
exhibit bitterness. A review of the most important bitter
classes found in plants was given by Belitz and Wieser
(1985). Bitter molecules occur in many variations; however,
62 Chem. Percept. (2008) 1:58–77

the strongest and most important representatives are from
certain alkaloids (e.g., nicotine, quinine, caffeine, strych-
nine), terpenoids (e.g., isoalpha acid, amarogentine, limo-
noids), and flavonoids (e.g., neohesperedin, epigallocatechin
gallate, Fig. 3).
Besides this extreme wide structural range of bitterness,
it is a surprising effect that the bad taste is very specific to
isomers of similar molecular structure. Small structural
variations can change the taste profile or strongly influence
the threshold. As examples (Fig. 4), the amino acid L-
tryptophan is bitter but the D-enantiomer shows a distinct
sweet taste (Belitz et al. 2001); the hesperetin rutinoside
(hesperidin) is tasteless but the positional isomer hesperetin
neohesperidoside (neohesperedin) is strongly bitter (Steglich
et al. 1997); quercetin is only weakly astringent but the 3-
hydroxyderivative taxifoline exhibits a strong bitterness at
the same concentration (own trials, each tested at 100 ppm in
water). Sometimes the bitterness of a molecule depends on
the molecular environment. Neat linoleic acid is more or less
tasteless; however, the same molecule shows a distinct
bitterness in emulsions (Stephan and Steinhart 2000).
Due to the wide variations of the structural basis of bitter
tasting molecules, it is difficult to generalize the molecular
requirements. Nevertheless in the past, there were several
attempts to correlate structural elements with bitter taste to
get a clue of how taste perception works. According to
Belitz and Wieser (1985), a bitter molecule needs a polar
group and a hydrophobic moiety (monopolar-hydrophobic
concept). But as mentioned above, the spatial distribution
of the two structural features seems to be of much more

Fig. 3 Important bitter tastants H

of different structural classes. O OH
Denatonium benzoate is the OH
N HO
most bitter compound known N N
H H O
O N N OH OH
CH3O
O
Caffeine
N Quinine OH
HO O
OH
O OH
Epigallocatechin gallate
Acetaminophen/ OH
N
H Paracetamol

OH
O O O
+
N HO
N O
H
HO O
Denatonium benzoate OH
Salicine OH
O
OH
OH O
O
HO
O O
O
OH
HO O O
O
O H O O O
O
O
H
HO OH Neohesperidin
Limonin
OH
O O O
OH
O O

H NH
O
NH2
H O
O OH O HO OH N
H
O O
HO
O
cis-Isohumolone H-L-Leu-L-Trp-OH
HO OH
OH
Amarogentin
Chem. Percept. (2008) 1:58–77 63

Fig. 4 Small structural varia- O

OH O
tions cause dramatic changes of OH
taste quality as a exemplified for
enantiomers, regioisomers, and NH2
structure changes by simple re- NH2
duction of a skeleton
N N
H H
L-Tryptophan: bitter D-Tryptophan: sweet

OH
HO OH
OH O
O HO
HO O
O OH
OH O HO O O
HO O O
O O
OH
HO O O HO OH
OH OH
O
Hesperidin: tasteless Neohesperidin: bitter

OH O
OH O
OH
OH
OH
OH HO O
HO O
OH
OH
Quercetin: weakly astringent Taxifolin: strongly bitter

importance, and even small structural changes cause
dramatic differences in taste attributes (as examples,
Fig. 4). Recently, a more detailed structure–activity model
regarding necessary molecular features for bitterness was
reported (Rodgers et al. 2006). Beginning with nearly 650
known bitter compounds (excluding bitter peptides) and
13,500 randomly selected non-bitter molecules a model
using MOLPRINT 2D circular fingerprints was developed.
By using this model, it was possible to predict 72% of the
bitter molecules. Unfortunately, only a small subset (33
compounds) selected from the original structures was
published due to confidentiality reasons. Just recently, a
study regarding the bitterness of the important structural
class of sesquiterpene lactones was published. Starting with
cynaropicrin and grosheimin from artichocke extracts, a
QSAR model was developed and could be established for
the prediction of bitterness of several analogues (Scotti et
al. 2007). Bitterness prediction was much more successful
in the more focused structural class of peptides (Asao et al.
1987; Opris and Diudea 2001; Ramos de Armas et al.
2004). Generally speaking, the higher the hydrophobicity of
terminal amino acids of the peptide chain, the higher the
bitterness of the peptide. Peptides with more than three to
four amino acid residues are in most cases more or less
tasteless (exception: sweet tasting proteins such as thaumatin,
brazzein, lysozyme). As a short summary, although it would
be of great value for food engineers, it is actually very difficult
to predict the bitterness properties of molecules that were
never tasted and that will probably be the same in the near
future.
Identification of Bitter-Masking Molecules
Unfortunately, until now, there was no description of a three-
dimensional structure of one of the T2r proteins or that of a
complex of a bitter agonist or antagonists and the T2r.
Because there are no reports regarding three-dimensional
structures of proteins with a tight relationship to T2r,
molecular modeling or computational docking experiments
are actually very difficult and flawed. Therefore, it is
generally by trial and error that new bitter-masking mole-
cules are discovered. Several methods for identification of
such compounds were reported in the literature; in the
following paragraphs, the sensory and molecular biological
methods will be described.
64
The promising studies based on taste sensors made from
modified polymer electrodes (e.g., Toko 2000; Takagi et al.
2001; Miyanag et al. 2003) will be not reviewed due to
their very different nature. However, some studies cited
later are based on such results. Another method is based on
affinity chromatography using molecular-imprinted poly-
mers: a polymer constructed using quinine as a template
was used for identification of L-arginine as suppressant
(Ogawa et al. 2005). In sensory tests, these results have
been validated.
Test systems based on receptors or cell constructs are
most promising to detect selective antagonists using high-
throughput screening assays of known or new molecules.
The classical sensory methods are limited to molecules
principally safe for human consumption. The main advan-
tage of sensory screening is that the findings are not limited
to a single-masking mechanism. Therefore, they can be
used directly for more realistic food models. The physico-
chemical methods (taste sensors) perform well in simple
test systems (e.g., solutions of pharmaceuticals) and for
known bitter molecules, especially in scavenging or
complexing systems. For detection of new bitter or bitter-
masking compounds, the methods are in many cases only
of limited value because they are based on very different
physicochemical mechanisms compared to taste cells.
Sensory Methods
Until now, the oldest but most successful method is to
detect bitter-masking molecules by simply tasting. The
classical masking systems of sodium chloride, sugar, or
sugar and salt in combination with acids were found by trial
and error. Many spices and flavors derived from plants,
especially aromatic herbs, were introduced most likely for
masking purposes in ancient times. For pharmaceuticals,
more sophisticated techniques were developed. In the
nineteenth century, the first sensory studies regarding
bitter-masking extracts, e.g., from Herba Santa, Miracle
Fruit, or Gymnema spp. were published by Lewin (1894).
Jellinek (1966) first reported standardized recommenda-
tions for sensorial tests for masking compounds. These
methods are still state of the art and are used for screening
of taste-influencing substances. An example is the simple
duo difference test using caffeine as bitter standard for
screening bitter-masking compounds (Ley et al. 2006a).
Most important for reliable results is a trained panel with
sufficient participants (10–20), a randomized and blinded
sampling and preferably only one tasting session per day,
best performed in the morning. The panelists have to
quote the bitterness impression on a hedonic scale (e.g., 1
[weak]–9 [strong]); a quantitative descriptive panel can be
combined with the ranking exercise for further direction.
Quantification can be improved by using reference
Chem. Percept. (2008) 1:58–77
samples of known concentration and comparing test
solutions against these references to determine the bitter
equivalents (Ley et al. 2005a). Some other working
groups have developed the half-site tongue test which
may be preferred for very small sample volumes (Shikata
et al. 2000).
Soldo and Hofmann (2005) suggest not using the
absolute bitterness ratings but the change in threshold of
bitterness perception for the detection of bitterness inhib-
itors. To improve the speed of screening, the comparative
taste dilution analysis (cTDA) was developed. This quan-
titative screening method is a combined tasting using the
well-established taste dilution analysis (Scharbert et al.
2004) and the tastant whose taste quality should be modified.
Unfortunately, the cTDA is a very time-consuming method
and cannot be used for a quick sampling. To improve the
speed, further improvements of LC analysis with directly
consumable solvents (water, ethanol) to yield an online
tasting result (LC Taste®) were recently reported (Krammer
et al. 2006).
Regardless which sensory method is considered, they are
of high value for development of bitter-masking compounds
due to the holistic approach: in contrast to the more focused
biochemical assays the whole flavor and taste attributes,
especially the common off-taste characteristics of candi-
dates, can be determined in a small set of tasting sessions.
Biological Test Systems
During the last decade, several assays to determine agonist
or antagonist activities on bitter receptors were developed
(McGregor 2004). Recently, some promising successes in
cultivating primary taste receptor cells were reported (Kishi
et al. 2005; Ozdener et al. 2006). Generally, the assays are
not based on primary taste receptor cells due to their limited
life span. In most cases, easy-to-handle transfected immor-
tal cell lines such as HEK293 cell systems (Bufe et al.
2002; Ruiz-Avila et al. 2000; Margolskee and Ming 2000;
Bufe et al. 2004, 2003; Pronin et al. 2003; Gravina et al.
2003) are used. Frequently, constructs of T2r genes together
with expression and transporting parts and/or with other
segments of the gustatory signaling system are used for
transfection. In many cases, existing G-protein signaling
systems of the HEK293 cells are used, and the change of
Ca2+ levels of the cell most often determined, e.g., using
fluorescence methods (FLIPR, etc.; Fig. 5).
The cell-based test systems can be used to identify the
agonists as well as the antagonists or modulators of the
bitter taste receptors. Whereas, some handful of agonists
and their receptors are described in between, only rare data
regarding antagonists on a T2r level exists (only nucleo-
tides such as adenosine monophosphate (AMP) were
characterized thus far; McGregor and Gravina 2002).
Chem. Percept. (2008) 1:58–77 65

Fig. 5 Schematic and simpli- HEK293

fied HEK293 transient cell cul-
2+
ture-based bitter antagonist Ca
2+
Ca Ca
2+

assay using calcium signaling

exemplified for salicin as ago- virus containing
nist transfection can also be transfection taste receptor gene
induced by chemicals. Read out
is performed as fluorescence 2+ inactive Ca2+
Ca
2+
Ca Ca
2+
sensitive
detection. Control cells are pre- fluorescence dye
pared using the same protocol
without transfection step incubation HO
HO
O
OH
HO OH HO OH
O HO O HO O
HO HO
O
HO
O

OH OH OH

taste receptor
Ca
2+ incubation read out 2+ 2+
2+ 2+
Ca
2+ Ca Ca
Ca Ca 2+ 2+
Ca Ca 2+
Ca

cell nucleus

inhibitor

The newest methodology is the screening of TRPM5
influencing compounds (Servant et al. 2007) and recently,
some new taste inhibitors based on TRPM5 antagonistic
mechanisms were identified (Bryant et al. 2007). Another
assay is focused on modulation of activity of the GRK
(G protein-coupled receptor kinases; Passe 2007) which are
responsible for signal deletion of activated G protein-
coupled receptors, but the value of both methods was not
yet proven by sensory methods.
Bitter-Masking Compounds
In the following paragraphs, most of the molecule-based
masking technologies will be reviewed. The majority of the
studies were not published in peer-reviewed journals, but as
patent applications because masking is of much more
importance to the pharmaceutical and food industries than
to scientific working groups. It is not always possible to
quantify or validate the reported results in patent applica-
tions. Quantitative sensory or other physical data of such
sources were not covered, unless the results are of high
importance and seemed to be reasonable. In nearly all
cases, no data regarding the possible mechanisms of
masking were published. Due to these limitations, hypoth-
eses regarding mechanisms will be excluded unless there
are supporting data.
One important requirement for applicability of potential
bitter-masking compounds is absence of side effects,
especially taste or flavor side effects. There is less value
in using a general taste inhibitor than a selective bitterness
inhibitor. Therefore, as an example, modulators of PLCβ2
or TRPM5 can impart bitter, umami, and sweet taste at the
same time and may be therefore of lower value for masking
purposes.
Masking by Strong and/or Congruent Flavors and Tastants
It is known to most food technologists that bitter taste can
be masked by strong flavors, especially by using so-called
congruent flavors. These flavors cause a certain acceptance
of bitterness due to their inherent occurrence. Examples are
cocoa or chocolate flavor preparations which mask the
bitterness of quinine (Reid and Becker 1956) or grapefruit
flavors which are widely used to mask pharmaceutical
actives. In cola-type beverages, most consumers cannot
detect the bitterness of caffeine due to the high dosage of
sucrose, sweetener and acid. Another classical system is the
suppression of bitterness by sodium salts. Sodium salts
which are low in saltiness such as gluconate or acetate are
the most successful maskers (Keast et al. 2001, 2004). As a
side effect, the preferred flavors and taste qualities are
enhanced (Breslin and Beauchamp 1997). A combination
of sodium salts and L-arginine was used for the reduction of
bitterness of certain peptides (Ogawa et al. 2004). In a more
detailed study, the bitterness reduction of quinine hydro-
chloride by using sucrose, sodium chloride, and tannic acid,
a strong astringent, was evaluated (Nakamura et al. 2002).
For 80% suppression of the taste of a 0.1-mM quinine
solution, 800 mM of sucrose, 300 mM NaCl, or 8 mM
aspartame, respectively, was necessary. Bitter taste of
caffeine in a tablet was reduced by using a umami/sweet
mixture of erythritol–CaHPO4, L-glutamic acid, inosinic
acid, and 5-ribonucleotides (Kitamura and Uokyu 2001).
In the latter method, the addition of high potency sweet-
eners is a commonly used method to reduce the off-taste of
66
other ingredients. Sometimes, the amount of sweetener is not
sufficient to elicit a sweet taste, this is important for non-
sweet applications. Examples are thaumatin for reduction of
protein off-tastes (Hamisch and Valentin 2001), thaumatin for
KCl bitter reduction (Takahiro 1988) a combination of zinc
and sodium cyclamate for pharmaceuticals (Keast and
Breslin 2005), neohesperidin dihydrochalcone for general
bitter reduction (Cano et al. 2000), and stevioside or
rebaudiosides for proanthocyanidine-rich tea beverages
(Uchida et al. 2007).
The mechanisms of the aforementioned bitter-masking
technologies are not known. Probably, the masking activ-
ities are mostly caused by psychophysical effects due to the
suppression of the off-taste by camouflage. Unfortunately,
the use of strong flavors or tastants is not acceptable in a lot
of applications. For example, it is not possible to use higher
amounts of sodium salts in sweet beverages or sweeteners
in savory applications. Therefore, the applicability of such
compounds is only limited.
Polymers and Complexing Agents
The use of ion exchangers to catch poor-tasting pharma-
ceutical actives is very well established for pharmaceuticals
and reviewed elsewhere (Sohi et al. 2004; Stier 2004). In
the following paragraphs, the focus will be more set on
food applications, natural structures, or molecules derived
from nature as scavengers. All of the previously mentioned
sequestering and complexing agents need to be used in
relatively high concentrations to be effective, and it seems
to be unlikely that they act on receptor or even cellular
level. Biopolymers such as alginates and other charged
polysaccharides may cause severe problems in applications
due to their gelling properties and influence on texture,
flavor release, and other sensorial qualities.
Scavenging molecules have been described several times
such as cyclodextrins (Binello et al. 2004) or cyclofructans
(Nishioka et al. 2004) or combinations thereof (Mori et al.
2006) which can complex bitter molecules. Such com-
plexes, e.g., combined with isohumolone, can be used to
encapsulate the product and to improve taste and stability
(Tatewaki et al. 2007). β-Cyclodextrin (Fig. 7) at 0.4% is
able to reduce the bitterness of a 0.05% caffeine solution by
about 90%. The α- and the γ-cyclodextrins are much less
active and higher concentrations of β-cyclodextrin taste
sweet. In the same study, the authors demonstrated that the
bitterness of various plant extracts such as artichoke or
gentian can be selectively reduced by β-cyclodextrin
(Binello et al., 2004). A polymer-supported cyclodextrin
using chitin as base was also successfully tested as a bitter-
masking agent (Binello et al. 2004).
Complexes of phospholipids with proteins were sug-
gested to mask bitterness of pharmaceuticals (Katsugari
Chem. Percept. (2008) 1:58–77
et al. 1995). In a 0.5-mM solution of quinine, 1% of a
phosphatidic acid/β-lactoglobulin complex was able to
reduce the bitterness by 90%, and 0.1% of the complex
reduced bitterness by 50%. Other bitter tastants such as
pharmaceutical actives propanolol and promethazine were
masked to a similar extent whereas, the bitter taste of
caffeine and naringin were less effectively reduced. The
effect is caused mainly by sequestering the frequently basic
and hydrophobic bitter molecules, as determined by
binding studies. It was found that these lipoproteins
reversibly suppressed the responses of the frog glossophar-
yngeal nerve to the bitter substance. The results suggested
that binding of lipoproteins to the hydrophobic region of
the receptor membranes leads to suppression of the
responses to the bitter substances (Katsugari et al. 1995).
However, another study dealing with these complexes
showed that there might be a individual component of the
reported effects. Some people were not able to perceive any
masking effect using these lipoproteins (Ishimaru et al.
2001).
Poly-γ-glutamic acid (γ-PGA) was described to relieve
poor taste, especially the bitterness of amino acids and
peptides (Sonoda et al. 2000). Bitterness of a 2% solution
of a mixture of L-leucine, L-isoleucine, and L-valine was
reduced by 70% using 1% γ-PGA. Bitterness of a 0.1%
caffeine solution was reduced in a dose-dependent manner
down to 30% using 1% of γ-PGA.
Partially phosphorylated oligosaccharides derived from
potatoes or their salts were used to reduce bitterness of
certain beverages (Kamsaka et al. 2002). In the same
application, sodium alginates (average molecular weight
50,000± 10,000 Da) were suggested for reduction of
unpleasant off-tastes caused by tea catechins (Shirata et al.
2003). The chitin derivative, chitosan, (Fig. 6) at a
concentration of 0.4 to 1.2% in water, is also able to
reduce bitterness of caffeine and various plant extracts but
also exhibits a strong astringency.
In a study using taste sensors rather than sensory panels,
the astringency of various tea catechins at 100 ppm was
reduced using pectin at concentrations <0.1% (Hayashi
et al. 2005). Only the catechins containing a gallate group
were affected. Unfortunately, there is no correlation to
human sensory data provided. Sulfated polysaccharides,
such as carrageenan, were used to reduce the undesirable
taste of amino acids mixtures (e.g., L-histidine, L-isoleucine,
L-leucine, L-methionine, L-phenylalanine, L-tryptophan, and
L-valine, each at 10%). In a ratio of 9:1 carrageenan/amino
acid cocktail, e.g., in beverages, the bitterness of an aqueous
solution of such a mixture was reduced to 1 (weak bitterness)
compared to 9 (strong bitterness) for the neat amino acid
cocktail (Calton and Wood 2002).
Egg white proteins treated with enzymes such as papain,
ficin, bromelain, and Aspergillus oryzae protease could
Chem. Percept. (2008) 1:58–77 67

Fig. 6 Oligo- and polymeric OH

bitter maskers or scavengers:
OH O NH3
+
cyclodextrins, polyamino acids,
OH
and charged carbohydrates
O OH NH2
O + O OH
O OH H3N n
OH O OH OHOH
O OH O
OH O O O
HO
OH n
OH HO OH
O O OH NH
OH HO O
HO O O
O OH
HO OH O NH3
+
OH HO
O HO
O O
O O
HO HO HO OH
HO
β-cyclodextrin poly-γ-glutamic acid chitosan

suppress bitterness or unpleasant taste of foods based on
soybean milk and green vegetable juice (Kittaka et al.
2005). A defatted (<10%) egg yolk was used in a mixture
with green tea extract (70% polyphenols; Sugiura et al.
2001). The complex product was less astringent and bitter
when compared to the free catechins.
Low-Molecular-Weight Substances
Until the third quarter of the last century, there were only
rare examples about small bitter-masking molecules in the
literature. The bitter-masking activity of a 0.5% gymnemic
acid solution was described early on (Lewin 1894); how-
ever, the same molecule inhibits sweet taste as well. In
1979, the flavanoid neodiosmin (Fig. 7) was described as a
bitter-masking agent using the threshold method (Guadagni
et al. 1979). Neodiosmin (10 ppm) increased the averaged
threshold of 40 panelists for caffeine from 128 up to
230 ppm by nearly 80%. The bitterness of a 100-ppm
caffeine and 8-ppm quinine solution, respectively, each
containing 10 ppm of the flavonoid, was rated as “not bitter”
in contrast to the solutions without flavonoid (“extremely
bitter”). Unfortunately, neodiosmin is not readily available.
One of the smallest “molecules” which can inhibit bitter
taste is the Zn2+ ion. It was shown that that ZnSO4 (25 mM)
can reduce bitterness of a quinine–HCl solution (0.04–
0.4 mM) by at least 50% to 70% (Keast 2003). On the other
hand, sweetness of a glucose solution (0.4 to 2.4 M) was
reduced to 20% whereas salty, umami, and sour taste were
not affected. In addition, zinc salt solutions show a
Fig. 7 Bitter-masking com-
pounds first described
O H
HO
O H
HO
HO O
H OH
OH
OH
prominent astringency and therefore the use in food or
beverage applications is very limited. Interestingly, magne-
sium salts (25 mM) can also reduce bitterness of quinine–
HCl to a somewhat lesser extent without significantly
affecting sweet, salty, sour, and umami taste.
Ornithine derivatives such as L-ornithyl-β-alanine or
L-ornithinyltaurine (for structures, Fig. 8), showed a bitter-
masking effect against potassium salts (Fuller and Kurtz
1997b). A solution of 1.8 g KCl and 0.2 g NaCl per liter
showed no bitterness after addition of the actives. Neat
taurine and its salts showed the same effect according to the
reference. Similar effects towards bitterness of potassium
containing test solutions were described for different
imidazole derivatives, one example shown in Fig. 8 (Fuller
and Kurtz 1997c). L-Ornithine itself was described as an
off-taste suppressant in combination with the bitter-branched
chain amino acids L-isoleucine, L-leucine, and L-valine as
determined by taste sensor measurements (Kawabe et al.
2004; Tokuyama et al. 2006). γ-Amino butyric acid in low
concentrations (<100 ppm) demonstrates masking activity
against caffeine and quinine (Ley et al. 2005b) but can also
suppress off-tastes of catechin-rich applications such as
cocoa, chocolate (Fujita et al. 2007), or of potassium
containing low salt products (Yamakoshi et al. 2006). The
effect of γ-amino butyric acid seems to be of high interest
because this well-known neurotransmitter and its receptor
were suggested as potential members of signal transduction
or modulation between taste receptor cells and output cells
(Herness et al. 2005). According to a Japanese patent, the
bitter aftertaste of high potency sweeteners can be reduced
OH O
O
HO
O HO O OH
OH HO O O
O O
OH OH O
O
O OH
neodiosmin
gymnemic acid I
68 Chem. Percept. (2008) 1:58–77

COOH -
SO3
Ph
-
N Ph - O O
O NH SO3
O NH N N
H
+
NH3
NH2 NH2 NH2
H2N
H2N H2N -
SO3

H-L-Orn-β-Ala-OH H-L-Orn-Tau-OH imidazole derivative taurine L-ornithine

O
O
HO O
HO O
O OH
OH N
OH NH
O NH
O NH N
NH2 HO O
O
NH2
O HO NH2
N-(1-methyl-4-oxo-2-
γ-amino butyric acid diglutamate H-L-Asp-L-Phe-OH imidazolin-2-yl)alanin

O -
O

NH NC HO
O +
O O N
-
N N SO3Na O
N OH
H H H
NH2 O

COOH N-(4-cyanophenyl)-N-
benzoyl-ε-amino caproic acid (sodiumsulfomethyl)- L-theanine pyridinium glycinyl betain
urea
Fig. 8 Amino acid and peptide derivatives

using the amino acids L-asparagine, L-methionine, L-
tyrosine, L-serine, L-aspartic acid, L-glutamine, L-alanine,
L-leucine, or L-proline (Takahishi and Kawai 2000). The
bitter taste of peptide hydrolysates as well as for brucin and
caffeine was eliminated by the dipeptide L-Glu-L-Glu
(Belikov and Gololobov 1986). Unfortunately, the authors
did not report any quantitative data.
Several dipeptides containing asparaginic acid were
disclosed as bitter maskers (Fuller and Kurtz 1997a). For
example, L-aspartyl-L-phenylalanine potassium salt (0.6 g/L)
suppressed the bitterness of potassium chloride (20 g/L) and
resulted in a taste reminiscent of sodium chloride. N-(1-
methyl-4-oxo-2-imidazolin-2-yl)alanine at about 500 ppm
concentration is able to reduce bitter and astringent taste
impressions of sweeteners and can suppress the lingering
sweet aftertaste of artificial sweeteners such as saccharin,
steviosides, or acesulfame K (Harada and Kamada 2000).
Further bitter-masking molecules based on amino acids were
described. The sour-tasting N-benzoyl-ɛ-aminocapronic acid
(Nakamura et al. 1997) suppresses sweet and bitter taste as
well as certain sulfomethylaryl ureas (Roy et al. 1990)
shown in Fig. 8. L-Theanine, a unique amino acid of green
tea, exhibits a masking effect besides its effects on brain
waves (Juneja et al. 1999).
A reaction mixture of amino acids such as L-arginine,
L-lysine, and L-ornithine with citric acid was used to
improve the taste of foodstuffs (Okai 2003). Not only was
bitter taste reduced but sweetness and saltiness, respective-
ly. Umami impressions were also increased in a beverage or
soy sauce containing 1–5% of the mixture. Some pyridinium
betain derivatives based on amino acids, isolated from
Maillard reaction mixtures, demonstrate bitter-masking
effects (Soldo and Hofmann 2005). The pyridinium glycinyl
betaine was able to reduce the bitterness ratings of various
concentrations of caffeine (250–2,500 ppm) by about 3 U
using a scale of 0 (no bitterness) to 5 (very strong).
According to Tomotake et al. (1998), simple-sodium-
saturated fatty acid salts such as sodium stearate, palmitate,
and laurate in relatively high concentrations of about 1%
were able to reduce the bitter taste of a 100-ppm quinine
solution significantly (Fujita and Kuroki 2004). In a more
extensive study, the influence of fatty acids such as linoleic
acid on the five basic taste qualities was investigated. Fatty
acids can increase the threshold (i.e., lower sensitivity) for
sodium chloride, citric acid, and caffeine and the bitter
rating for caffeine was reduced (Mattes 2007). In further
sensory experiments, it was demonstrated that edible oils
(tuna, soybean, high oleic acid corn oil) in 10% oil-in-water
Chem. Percept. (2008) 1:58–77
emulsions can reduce sour, bitter, and umami taste. In
general, however, in sweet and salty taste (Koriyama et al.
2002) and the time intensity profile of tastants will be
extended for all taste qualities. In the same study, the effect
of free fatty acids was tested. The highly unsaturated fatty
acids linoleic, eicosapentaenoic, and hexadocosaenoic acid
showed a highly significant masking effect against quinine
sulfate and L-leucine. Further lipids which are able to
reduce bitterness were described. Plant stanol esters (8% in
water using guar gum as emulsifier) can reduce the
bitterness of 600 ppm caffeine solution by 15% as
compared to a simple rapeseed oil at 8% (Pouru et al.
2004). Some hydrogenated ethoxylated glycerol esters
show masking effects at concentrations of about 1–2%
against several pharmaceutical actives such as dextro-
methorphan, chlorhexidine, guaifenesin, caffeine, aspirin,
or acetaminophen in combination with sweeteners such as
sucralose and mono-ammonium glycyrrhizinate (Roger
2006). Phospholipid mixtures containing phosphatidylino-
sitol and phosphatidic acid were described as useful for
masking bitter taste of certain drugs (Tadokoro and Goto
2007). The effect of some of the lipids is not very strong,
and the amount of masking agents is very high in the
majority of applications. Therefore, it seems that they do
not interact with receptors or taste receptor cells but may
work by scavenging bitter molecules or by acting as
surfactants.
The possible inhibition of intracellular phosphatases as a
mechanism of bitter taste suppression was claimed (but not
proven) for several organic phosphates, phosphonates,
vanadates, thiophosphates, and biphosphates such as
eugenylmonophosphate, thymylmonophosphate, menthyl-
monophosphate, phosphotyrosine, or phosphoserine (Nelson
1998). As example, vanillylmonophosphate (Fig. 9) at 0.3%
was able to significantly increase the threshold and to
decrease the bitterness of pharmaceutical-active ingredients
such as dextromorphan, acetaminophen, or denatonium
benzoate. However, the effect was not the same for caffeine
(Nelson et al. 1998).
O O
O OH
O
P
O
N impact sweeteners is of limited value for general applica-
HO H
O bility. Further gluconates such as Cu-(I)-gluconate can
HO
capsaicin
vanillylmonophosphate
O O
O N
H L-tryptophan, L-arginine, or L-lysine (each 0.2%) was
OH
L-menthyl lactate spilanthol
Fig. 9 Phosphatase inhibitors and trigeminals
69
Some trigeminal active molecules such as capsaicin
(Fig. 9) may reduce bitter response of the gustatory nerves
according to experiments on rats (Simons et al. 2003). The
mechanism is not limited to bitterness but affects sweet and
umami taste as well. From a traditional point of view, this
may be the reason why hot spices are so commonly used to
suppress off-tastes in staple food. Capsaicin typically
produces hotness, and the sensation of pain and must be
dosed very carefully and its applicability may be therefore
limited to spicy food or beverages. In addition, higher
dosages of trigeminal stimulants such as capsaicin and
menthol can cause intrinsic bitterness (Green and Schullery
2003). Some physiological cooling compounds such as
L-menthyl lactate, L-menthon glycerol acetal, N-ethyl-L-
menthancarboxamide, or L-menthyl propylenglycolcarbonate
in combination with high intensity sweeteners, demonstrate
masking effects against bitter-tasting antitussives or expec-
torants such as dextromorphan (Yano 2000). The tingling
compound spilanthol was used as a masking compound for
bitter or astringent aftertaste of artificial or high intensity
sweeteners (Miyazawa et al. 2006). The interaction of
trigeminal and gustatory sense is of high interest due to the
co-expression of trigeminal fibres in fungiform taste papilla
and the known phenomena of thermally induced taste effects
(Talavera et al. 2007). But recently it was shown that the
intensities of gustatory and trigeminal sensations are not
directly correlated (Green et al. 2005). Therefore, the de-
scribed taste modulation effects may be due to direct inter-
actions with taste transduction mechanisms. Interestingly, the
TPRM5 channel involved in taste transduction is a close
relative of the capsaicin (TRPV1) and menthol (TRPM8)
receptors (Talavera et al. 2007).
A combination of a high-impact sweetener and ginger
oleoresin was suggested to mask bitter pharmaceuticals
such as acetaminophen (Lindley 2003). A mixture of ginger
oil (20 ppm) with thaumatin (12.5 ppm), magnesium
gluconate (4%), and starch is able to decrease the bitterness
of a 1.6% acetaminophen solution down to the comparable
bitterness of a 0.96% acetaminophen solution without
masking ingredients; most probably the thaumatin/Mg
gluconate combination was the effective masking part of
the formulation. But as mentioned earlier, the use of high-
improve the taste of food or beverages (Fujii and Yasuda
2006). The addition of 50 ppm of the salt remarkably
reduces bitterness of a coffee beverage.
The bitterness of amino acids such as L-valine, L-leucine,
L-isoleucine (each 1% in aqueous solution), L-phenylalanine,
masked by addition of 1 wt.% of the non-reducing and
sweet carbohydrate α,α-trehalose (Uchida et al. 2003;
Fig. 10). Isomaltulose was suggested for reduction of the
70 Chem. Percept. (2008) 1:58–77

Fig. 10 Bitter-masking OH
carbohydrates OH
HO OH OH

OH O OH
O O O O OH
OH OH HO OH
O OH
HO OH OH OH
HO
OH OH
OH

isomaltulose xylitol
α,α-trehalose
OH
HO OH OH
HO OH HO OH HO
OH
HO O O OH O OH
HO O
O OH O O
n O
HO O
O HO HO HO
OH
OH
HO
pannitose (GlcP-α-1-6-GlcP-α-1-4-GlcP) cellooligosaccharides (n = 0 - 2)

bitter taste of tea beverages containing polyphenols (Doerr Two flavanones, eriodictyol and homoeriodictyol (each at
et al. 2007). For such carbohydrates, the masking effect may 100 ppm, Fig. 11), exhibit a 40–60% reduction effect on a
be due to their intrinsic sweetness. Bitterness inhibitors based 500-ppm caffeine solution. In addition, effects were found
on the trisaccharides pannitose (Fig. 11) or the reduced also against bitterness of quinine, amarogentine, para-
derivative pannitol were described to be able to reduce the off- cetamol, denatonium benzoate, and salicin, whereas the
tastes and flavors of soy bean products and other problematic bitterness of potassium salts, linoleic acid emulsions, and of
plant materials such as whole grain biscuits, carrot juice, and a bitter peptide, L-leucyl-L-tryptophan was not eliminated.
others (Nakanishi et al. 2005). Cellooligosaccharide such as Various structural relatives were screened for their bitter-
cellotetraose in an amount of 1% can suppress bitterness, masking activity but only few active molecules were found
e.g., of caffeine (Saski et al. 2002); these oligosaccharides (Fig. 11): hydroxylated benzoic acid N-vanillylamides (Ley
are more or less tasteless and therefore would be useful for a et al. 2006a), some hydroxylated deoxybenzoins (Ley et al.
lot of applications. 2006b), and short chain gingerdiones such as [2]-gingerdione
The active principle of the long-known bitter-reducing (Ley et al. 2007). All of these compounds are somewhat
activity of liquid extracts of Herba Santa (Yerba Santa, tasteless, and the masking activity and pattern is very similar.
Eriodictyon ssp.; Lewin 1894) was found by sensory- This seems to be specific but the underlying molecular
guided fractionation of the plant extract (Ley et al. 2005a). mechanism has not yet elucidated. The compounds can be
used in a lot of applications to combat bitterness but are most
active in beverage-type applications.
OH O Lactisol (Fig. 12), a known sweet taste inhibitor also on
OH O
the receptor level (Xu et al. 2004), can suppress the off-
NH
tastes of potassium chloride and artificial sweeteners (Kurtz
O
HO O
O
R
HO and Fuller 1997a; Kurtz and Fuller 1993). A solution
OH
containing 20 g/L of a mixture of 95% KCl and 5% NaCl
OH and 500 ppm lactisol sodium salt tastes like neat sodium
R = H: eriodictyol 2,4-dihydroxybenzoic
R = CH3: homoeriodictyol acid N-vanillylamide chloride solution with virtually none of the KCl bitterness.
OH O Unfortunately, such high concentrations of lactisol block
OH the sweetness impression, and therefore, broad applicability
OH O
is questionable. The activity of lactisol may be limited to
O
O certain elicitors of bitter taste due to a extensive sensory
HO
study on various bitter tastants which show no broad-
OH
reducing effect for lactisol (Johnson et al. 1994). For the
2,4,4'-trihydroxy-3'-methoxy-
deoxybenzoine [2]-gingerdione same application area, some other molecules such as orotic
Fig. 11 Bitter-masking molecules related to homoeriodictyol or dihydroorotic acid (Fuller and Kurtz 1997d), aspartame
Chem. Percept. (2008) 1:58–77 71

O
O O
OH O
NH OH
O NH CN OH
HOOC N O O O
O COOH H
HO
HO N O
H
N-(p-cyanophenylcarbamoyl)- 2,4-dihydroxybenzoic acid
lactisol orotic acid
L-aspartyl-L-phenylalanine

O O
O
HO O
OH OH
NH2 HO
HO
O
O

L-DOPA sinapic acid flavone

OH HO OH

HO
O O N O OH
O HO O H2N O
OH O N P O
HO O
OH OH
HO HO

ferulic acid chlorogenic acid CMP

OH
N HO OH H
N N N O
N N
H2N N O O
O H2N N O OH O
N O P OH
n N O
P O
OH OH
dATP (n=2) AMP acylated arylhydrazones
Fig. 12 Various molecules reducing bitter or metallic aftertaste

derivatives such as N-(p-cyanophenylcarbamoyl)-L-aspartyl-
L-phenylalanine (Fuller and Kurtz 1997e), and phenolic
acids such as 2,4-dihydroxybenzoic acid, sinapic acid, or
L-DOPA (L-3,4-dihydroxyphenylalanine) (Fuller and Kurtz
1997f) as well as some flavonoids such as flavone itself
(Kurtz and Fuller 1997b) were reported.
Ferulic acid (and other hydroxycinnamic acids, Fig. 12)
in concentrations of 0.001–0.2% was suggested to combat
the bitter aftertaste of artificial sweeteners as well the
bitterness of caffeine or quinine (Riemer 1994). The bitter
aftertaste of a 500-ppm solution of Na-saccharin was
significantly reduced by using 550 ppm ferulic acid. The
bitterness and off-taste of artificial sweeteners such as
aspartame, sodium saccharin, and acesulfame K can be
reduced using chlorogenic acid or other cinnamic acid
esters of quinic acid (Lee et al. 1975; Chien et al. 2002;
Takagaki 2006). In an acidic beverage containing aspar-
tame, acesulfame K, sodium benzoate, phosphoric acid, and
citric acid, an amount of 30 ppm of chlorogenic acid in the
form of an extract prepared from green coffee beans the
metallic and bitter off-taste was markedly reduced (Chien
et al. 2002). A similar effect was obtained using only the
parent compound, quinic acid (Togawa et al. 2001). The
bitter taste of high dosages of L-menthol, which may be
experienced, e.g., in chewing gums, can be reduced also by
chlorogenic acid and its analogues (Matsumoto et al. 2006).
The advantage of the hydroxycinnamic acid derivatives is
their low intrinsic taste and their occurrence in a lot of
natural extracts, e.g., chlorogenic acid can be extracted
from green coffee beans (Matsumoto et al. 2006).
As mentioned earlier, the first bitter inhibitors found by
screening with receptor assays were reported in 2002
(McGregor and Gravina 2002; McGregor and Homan
2003). The simple nucleotides CMP (cytosine monophos-
phate) and dATP (2-deoxyadenosine triphosphate) cause at
10 mM a 40% and 60% decrease in bitterness of a quinine
solution, respectively. In combination with taurine, AMP
shows a well-accepted masking effect against KCl bitter-
ness in sodium-reduced formulations (Salemme and Barndt
2006).
Very uncommon compounds to combat poor taste were
developed just recently (Bryant et al. 2007): some acylated
arylhydrazones (Fig. 12) reduce the TRPM5 activity in a
HEK293 cell system to 40% residual activity at 10 μM.
72 Chem. Percept. (2008) 1:58–77

Unfortunately, no sensory data has been provided thus far,
so this might be of high interest to see whether the
compounds can selectively block only one taste quality or
block taste in general.
Comparability of Sensory Results and Consequences
for Flavor Development
One of the major problems for a selection and assessment
of potential bitter-masking molecules is the compatibility of
data generated with different methods, standards, and
panels. Therefore, the duo screening using caffeine as a
model bitter compound was described in Ley et al. (2006a).
As an example, we evaluated some of the most promising
molecules from literature and compared it to our own
developments (Table 1). The compounds tested were
selected from flavonoids, polymeric and monomeric amino
acids, carbohydrates, and polyphenols to cover different
possible bitter-masking mechanisms according to the list of
possible targets mentioned in the “Introduction”. For poly-
γ-glutamic acid, a scavenging mechanism may be assumed
as well as for cellotrioside and α,α-trehalose, whereas γ-
amino butyric acid may influence the intercellular commu-
nication. The flavonoids can possibly act as modulators of
T2R or other proteins of the signal transduction cascade.
But for all these molecules, no proof of the basic
mechanism does yet exist. The test concentration was
selected as recommended in the relevant publication and, in
some cases, it was limited by solubility.
In our trials, none of the molecules was able to decrease
the bitterness of caffeine totally. Some of the tested
compounds could not inhibit caffeine bitterness at all. In
the original papers, these compounds were not tested on
caffeine and generally, the activity against caffeine is not
limited to a set of structural classes.
For applicability, it is not only important to determine
the activity for one concentration, but also to compile a
dose activity plot. In most of the cited studies, these data
are only rarely found. In several cases, there is a strong
dose activity response of the masking effect and the activity
is mostly limited to a certain level. For some of the
compounds, we determined the dose response against a
500-ppm caffeine solution. In all investigated cases, there
was a ceiling effect of activity as shown in Fig. 13. γ-
Amino butyric acid and [2]-gingerdione showed decreased
activity at the highest concentration (100 and 500 ppm,
respectively) which may in the case of γ-amino butyric acid
be caused by intrinsic astringent or sour taste. The typical
saturation effect is not limited to caffeine, as shown for
activity of homoeriodicytol against the bitter principle of
gentian, amarogentin (Fig. 14). Such effects were also
described for other compounds such as phospholipoproteins
(Katsugari et al. 1995). In biological screening systems,
similar effects can be seen, as has been reported for activity
of nucleotides against denatonium benzoate bitterness
(McGregor and Gravina 2002).
For some of the masking molecules listed in Fig. 13, we
have tested their activity against different bitter molecules,
e.g., salicin, quinine, KCl and the bitter dipeptide L-
leucinyl-L-tryptophan (Leu-Trp; Fig. 15). The activity
against caffeine and quinine and the peptide is comparable
for all tested masking molecules. However, for salicine,
there are remarkable differences. For salicin, the main bitter
receptor was described earlier, and in this case, it may be a
selective blocking effect which needs to be demonstrated
on a biological level. It would be of great value to
determine the pattern of antagonistic activity (according to

Table 1 Relative reduction of bitterness in randomized, blinded duo tests using 500 ppm caffeine solution caused by some known bitter maskers
from literature

Compound Reference Concentration Masking effect (%) Significance

Neodiosmine Guadagni et al. (1979) 100 ppm 28

Poly-γ-glutamic acid Sonoda et al. (2000) 1% 31 p<0.05
Cellotrioside Saski et al. (2002) 500 ppm 29
Homoeriodictyol sodium salt Ley et al. (2005a) 100 ppm 43 p<0.05
Homoeriodictyol Ley et al. (2005a) 100 ppm 28 p<0.05
Eriodictyol Ley et al. (2005a) 100 ppm 47 p<0.05
γ-Amino butyric acid Ley et al. (2005b) 50 ppm 33 p<0.05
α,α-trehalose Uchida et al. (2003) 1% 10
Taurine Fuller and Kurtz (1997b) 50 ppm 0
L-Theanine Juneja et al. (1999) 500 ppm −6
2,4-Dihydroxybenzoic acid N-vanillyl amide Ley et al. (2006a) 100 ppm 33 p<0.05
2,4-Dihydroxybenzoic acid Fuller and Kurtz (1997f) 100 ppm 2
[2]-Gingerdione Ley et al. (2007) 100 ppm 34 p<0.05

Ratings were determined by a trained panel (n=12–16) on a scale from 1 (no bitterness) to 10 (strong bitterness) and the relative inhibiting effect
was recalculated from these data.
Chem. Percept. (2008) 1:58–77
relative masking effect 100%
80%
60%
40%
20%
0%
0 200 400
concentration inhibitor (ppm)
Fig. 13 Dose response of selected masking molecules from Table 1
against bitter taste of a 500-ppm caffeine solution. For each
concentration, a randomized, blinded duo test according to the method
described in Ley et al. (2006a) was used. Ratings were determined by
a trained panel (n=12–16) on a scale from 1 (no bitterness) to 10
(strong bitterness), and the relative inhibiting effect was recalculated
from these data
Fig. 2) of the masking compounds mentioned in Table 1
and to compare it with the sensory results. Probably the
molecular targets of the compounds differ as suggested by
the results with various bitter molecules shown in Fig. 15.
As a consequence for flavor development using masking
molecules, it is important to know which bitter principle
has to be blocked. After choosing the best performing
blocking agent, it must be validated for activity in the final
matrix. One of the most challenging issues is the concen-
tration of the bitter tastant. Although there might be a
masking molecule that can reduce the bitterness relatively,
e.g., by 40%, the residual bitterness might be too high for
acceptance by the end consumer. To our knowledge there is
no complete or total masking technology available, and
therefore it is most important to reduce the bitter principle
as much as possible by additional methods such as
debittering, complexation, or encapsulation to yield a
successful end product. In some cases, new problems arise
caused by selective bitter masking. As an example, green
tea exhibits a bitter and a strong astringent taste. When a
typical bitter-masking agent is used for improving taste,
100%
relative masking effect
80%
60%
40%
20%
0%
0 100
concentration inhibitor (ppm)
Fig. 14 Dose response curves of homoeriodictyol sodium salt against
30 ppb amarogentin (as described in Ley et al. 2005a). Test conditions
as described for Fig. 13
73
2,4-Dihydroxybenzoic
acid N-vanillylamide
[2]-Gingerdione
GABA
Eriodictyol
homoeriodictyol
sodium salt
600 800 1000
Fig. 15 Activity of five bitter-masking compounds against caffeine,
salicin, quinine, the dipeptide Leu-Trp, and KCl determined using the
randomized and blinded duo testing (described in Ley et al. 2005a).
Test conditions as described for Fig. 13
sometimes an increased astringency can be perceived
because bitter taste can mask astringency to a certain
degree. It is absolutely necessary to begin with base
optimization, different masking technologies, and finally a
good flavor which will mask distinct off-tastes for ready-to-
use food or beverages for acceptance by the consumer.
Conclusions and Outlook
Combating bitter taste in food, pharmaceuticals, and
beverages remains a large challenge. Most existing masking
technologies and more specifically, the bitter-masking
molecules, were found by trial and error methods. In the
past, bitterness reduction was generally focused on phar-
maceuticals and drug actives. Today, the most intensive
research is performed to reduce bitter or astringent taste of
functional food or beverage applications which show off-
tastes due to enrichment with healthy, poor-tasting actives.
During the last 10 years, tremendous progress in the
elucidation of bitter taste reception and transduction on
the cellular level was made. This was fueled by the human
genome project and the Nobel Prize for Medicine and
Physiology issued to Buck and Axel in 2004 for their basic
findings regarding the olfactory mechanisms. Unfortunate-
ly, bitter taste seems to be the most complex quality of all
basic taste modalities due to the large number and diversity
of T2r bitter receptors. It seems feasible to develop effective
bitter-masking molecules of high potency which are strong
modulators of cellular signal transduction pathways as they
200 300 400 500
may not be acceptable due to their side effects on other
taste qualities and perhaps even other non-taste cell
mechanisms. Especially the use of screening methods based
on agonist/antagonist/T2R interactions may be very useful
74
to identify lead compounds for development of new
selective bitter-masking compounds.
Actually there is no principal all in one solution for
masking issues. However, in most cases, combinations of
different technologies such as encapsulation/formulation,
and selective removal or biotransformation of bitter
molecules, using strong or congruent tastants or flavors
and/or masking molecules have to be used to produce a
superior tasting and healthy food or beverage. Nevertheless,
in the future selective inhibitors for special bitter com-
pounds (e.g., bitter plant polyphenols such as catechins or
high intensity sweeteners such as saccharin) may be found
and developed.
Acknowledgements I would like to thank Gerhard Krammer,
Gerald Reinders, Ian Gatfield, Gabi Vielhaber, and Kathrin Freiherr
for all the helpful discussions and valuable suggestions related to the
topic and especially Heinz-Jürgen Bertram who initiated and
supported the topic. Special thanks to Debbie Kennison for proof-
reading the paper.
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