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,and beverages remains a huge challenge. In the past,bitterness reduction was focused on pharmaceuticals anddrugs; however, more recently, the most intense research isperformed on the reduction of bitter or astringent taste infunctional food or beverage applications. These foods andbeverages possess inherent off-tastes due to fortificationwith healthy but poor-tasting actives. During the last10 years, tremendous progress in the elucidation of bittertaste reception and transduction on the cellular level wasmade and many new molecules and compounds to reducebitter off-tastes were reported. The following review will befocused on the advances, in the area of bitter-maskingmolecules, during the last 10 years. It will not cover otherdebittering strategies such as process optimization orbiotransformations to reduce the amount of bitter ingre-dients, encapsulation, and other physical formulationtechnologies. The review will close with a short compar-ative study of various bitter maskers and some suggestionsfor flavor development of poor-tasting ingredients.

Keywords Off-taste . Bitter Taste . Masking Technologies .

Taste Masking

AbbreviationsAMP adenosine monophosphateATP adenosine triphosphateCMP cytosine monophosphatecTDA comparative taste dilution analysis

dATP 2-deoxyadenosintriphosphateFLIPR fluorescence-induced plate readerGRK G protein-coupled receptor kinasesHEK293 human embryonic kidney cells type 293IP3 inositoltriphosphateL-DOPA L-3,4-dihydroxyphenylalanineLeu-Trp L-leucinyl-L-tryptophanγ-PGA poly-γ-glutamic acidPDE phosphodiesterasePLCβ2 phospholipase C subtype β2TRC taste receptor cellsTRPM5 transient receptor potential channel, type M5T2R taste receptor type 2

Introduction

Bitter taste is a major problem in the food and pharmaceu-tical industries due to its negative hedonic impact oningestion (Drewnoswki 2001; Drewnoswki and Gomez-Carneros 2000). Only in rare cases, consumers prefer astrong bitter taste for food and beverages, e.g., in blackcoffee, black or green tea, beer, red wine, grapefruitproducts, or bitter lemon. In most other cases, the bittertaste is not desirable and has to be eliminated from ormasked in the product. As an example, most legumes,fruits, and staple foods were extensively optimized usingbreeding and cultivation technology to become less bitter,astringent, or sour variants over the course of time. Anotherexample is in the juice industry, whereby raw orange juicesare processed to be debittered by cleaving the bitternaringin to the less bitter naringenin or naringin-7-O-glucoside. Most cloudy raw apple juices are treated toremove most of the polyphenols, which can taste bitter orastringent to yield clear beverages (Oszmianski et al. 2007).

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

J. P. Ley (*)Flavor & Nutrition Research & Innovation, Flavor Research,Symrise GmbH & Co. KG,P.O. Box 1253, 37601 Holzminden, Germanye-mail: jakob.ley@symrise.com

In the pharmaceutical area, there is also a large demandfor bitter reduction techniques due to the low complianceof patients taking bitter drugs for longer times. In youngchildren, the problem is more serious, due to their highertaste sensitivity and because in many cases, it is notpossible to supply large enough capsules containing theactive pharmaceutical ingredient. Currently, the old rule“only bitter medicine is good medicine,” is no longervalid.

In recent years, the problem of bitter- or bad-tasting foodproducts is surfacing again, due to the demand for healthierfood or beverages. Reduced sugar, fat, and sodium forhealthy benefits can also accentuate sourness, bitterness,and astringency in the base matrices. Many artificialsweeteners exhibit astringent, metallic, or bitter aftertaste.In salt or sodium replacers, potassium chloride is common-ly used in many applications, which leaves a very metallicbitter taste that most people find undesirable. Compoundssuch as certain polyphenols (e.g., tea catechins), soyproducts, phytosterols, vitamins, minerals, fish oil, etc.used for fortification of functional food can cause serioustaste deficiencies and reduced consumer demand for suchproducts (Eckert and Riker 2007).

One of the major problems of masking of off-taste is thecomplex mixture of sensations. The ingestible is not onlyperceived as bitter, but is also astringent and/or sour. Eachmodality is transduced by different molecular sensingsystems in the mouth, and the sensation consciouslyrecognized is again a difficult mixture to separate intoindividual taste qualities.

Many techniques to reduce bitterness or off-taste haveevolved 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 forpharmaceutical 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 forfood or beverage applications because the latter applica-tions contain much more water and often use raw materialswhich are not permissible for food use. The use of classicalflavors and tastants was reviewed by Pszczola (2004).Therefore, the following review will focus mainly on thelast topic, the masking of bitter taste on a molecular level.

The last general review covering such molecules wascompiled by Roy in his book (1997). Since then, theknowledge of taste, especially bitter taste detection, andtransduction on a cellular level, has heavily evolved, and asa 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 responsiblefor bitter taste reception by Chandreshekar et al. (2000;Adler et al. 2000), the mechanism of bitter reception bytaste receptor cells seems to be generally known nowadaysand was thoroughly reviewed in recent time (Margolskee2002; Montmayeur and Matsunami 2002; Meyerhof 2005;Chandrashekar et al. 2006; Behrens and Meyerhof 2006).Below, the mechanism is briefly summarized (for aschematic 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 25different T2R are described. Additionally, several alleles areknown and about 100 different bitter phenotypes exist inman. TRC are specialized to a certain taste quality. Forsweet taste, this was demonstrated by genetic experimentson mice in a labeled-line model. Most probably, sweet cellsare linked directly to positive hedonic centers of the brain.The authors constructed a mouse expressing T2Rs on sweetcells and they preferred a bitter and toxic solution and notthe sweet one (Zhang et al. 2003). For the bitter modality,one TRC expresses more than one T2R type but not inall variants. On the other hand, it is now known thatone particular bitter compound can bind to several T2Rsubtypes with distinct affinity and that at least some ofthe bitter receptor proteins, e.g., the hT2R47, arebroadly tuned for several structural classes of bittermolecules (Meyerhof et al. 2007). As a result, a bittertaste pattern (Fig. 2) for the cells occurs in a similar wayto the olfaction process; however, the final signal to thebrain is mainly “negative” or “bitter”. Discussions con-tinue that bitter qualities may be distinguishable but notyet proven by combined sensory and molecular biologicalexperiments.

Following the binding of agonists to the T2R, phospho-lipase C is activated via a β-subunit of a G protein of theTRC which activates the IP3 (inositoltriphosphate) pathwayin the cell. Calcium will be released from internal storesand at least the co-expressed ion channel TRPM5 will beactivated and the cell depolarizes. In addition, the α-unit ofthe TRC-specific G protein gustducin may activate the PDEpathway of transduction (Ming et al. 1999), but there is nofinal proof of concept at this time.

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In contrast to the intracellular mechanisms, the intercel-lular transduction of the signal generated by agonistreceptor interaction is not yet elucidated in detail. MostTRC which can be stimulated by bitter tastants (or othertaste qualities) are not directly linked to synapses but theyrelease neurotransmitters (Clapp et al. 2006). The picture isstill 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 notpreclude there being other transmitters that have not yetbeen identified. The neurotransmitters subsequently activatethe so-called output cells in the taste bud, which areconnected to synapses of afferent gustatory nerves (Roper2007).

In addition to this generally accepted pathway, therewere 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 cellsand act on intracellular proteins (Peri et al. 2000). Oneexample may be the general quenching mechanisms of Gprotein-coupled receptors by inhibition of signal termina-tion-related kinases which may cause the lingering bitteraftertaste of sweeteners (Zubare-Samuelov et al. 2005).Another protein discussed by the group was activation of

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 byactivation of PDE (phosphodiesterase) due to its knownactivity and the difficulties to identify the responsible T2R(Yan et al. 2001). Until now, no mammalian bitter receptorfor caffeine is known but it was described recently for fruitflies (Moon et al. 2006).

From a molecular standpoint, there are several potentialtargets to suppress bitter taste transduction:

& Molecules, which can complex or scavenge the bittertastants (molecular encapsulation) or can disrupt thetransport 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

Fig. 1 Actual bitter taste transduction mechanisms (combined andadapted according to: Gilbertson et al. 2000; Perez et al. 2003; Bufe etal. 2002) exemplified for the human salicin receptor hT2R16.Probably only (but not all of the 25) bitter receptors are expressedon a single “bitter” taste receptor cell. The generally accepted

mechanism follows the IP3 pathway, the PDE branch is not fullyproven. The ATP (or alternative neurotransmitter) release mechanismis not yet fully known (Romanov et al. 2007; Huang et al. 2007). PLCPhospholipase, IP3 inositoltriphosphate, PDE phosphodiesterase,NMP nucleosidemonophosphate

60 Chem. Percept. (2008) 1:58–77

Whereas scavenging is an established and a well-knownmechanism, it is not clear whether the transport can beselectively influenced. Saliva flow and its constituentscertainly play an important role for the complex transportof tastants to the taste cells (Matsuo 2000), but earlydiscussions regarding the role of lipocalins secreted by vonEbner glands as “tastant-binding proteins” for bittercompounds could not be verified (Creuzenet and Mangroo1998). Unfortunately, in contrast to the agonist/T2R studies(Fig. 2), similar data regarding agonist/antagonist/T2Rinteractions were not published yet. These data would beof very high value for development of selective bitterinhibitors. The influence of neurotransmitter release onreal tasting experiments was very rarely reported untilnow. In a recent paper of Heath et al. (2006), themodulation of human taste thresholds by changes of theserotonin and noradrenalin levels induced by certain drugswas determined. Significantly enhancing the serotonin levelcaused a reduction of the sucrose taste threshold by 27% andthe quinine taste threshold by 53%. An increased noradrenalintiter significantly reduced bitter taste threshold by 39% andsour threshold by 22%. As a conclusion, influencingneurotransmitter levels, e.g., by drugs can dramatically changethe taste response and may be a possible target for furtherdevelopments of taste modulation compounds. But it is tooearly to decide which neurotransmitter or its receptor may bethe most important target for bitter masking.

Potential modulators/antagonists of receptors and pro-teins will be discussed later in greater detail. In nearly allpresented cases, the exact mechanism of masking activity isnot yet known.

Bitter-Tasting Molecules: Structure–ActivityRelationships

In contrast to the other taste qualities of sweet, umami, sour,and salty, there is a large number of molecules which aredescribed as bitter. Generally speaking, the bitter modalityis an aversive taste which protects animals againstpotentially toxic or harmful substances in nature. Inparallel, in bitter (and sometimes toxic) plants, moleculeshave evolved to deter herbivores (Simmonds 2001). It willnot be the intention of this review to list all relevant bittertastants; therefore, in the following paragraphs only someexamples will be discussed.

Bitterness is widely distributed in nature and principallyeach chemical class can contain bitter molecules. Simplesalts such as sodium sulfate or magnesium sulfate show astrong bitterness. Some higher peptides, terpenoids, alka-loids, polyphenols, heterocycles, and macrolides can alsoexhibit bitterness. A review of the most important bitterclasses found in plants was given by Belitz and Wieser(1985). Bitter molecules occur in many variations; however,

agonists receptor hTAS2R values: threshold conc. in log M1 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

arbutin lowamygdalinPROPDiphenylthioureaaristolochic acidsaccharinabsinthinpicrotoxinin strongchloramphenicolhumolone + not alpha-thujon quantifiedherbolid Aphenylisothiocyanate orphandenatoium benzoate receptorPTCstrychninbrucin1-naphthoic acidpiperonylic acidsodium benzoatesalicinenitrosaccharineacesulfam Ksucroseoctaacetatsesquiterpene lactonespapaverinequinacrinechloroquine

max. responseon receptor

Fig. 2 Bitter receptor matrix (compiled from data from: Meyerhof etal. 2007; Bufe et al. 2002; Xu and Li 2006; Pronin et al. 2004; Kuhnet al. 2004; Prodi et al. 2004; Behrens et al. 2004). The picture is onlyan actual spotlight of the whole matrix in respect to potential agonists.

Known agonists are presented in column 1, the human T2R are givenin line 1. The gray areas are not yet characterized. Only a fewinteractions were not only qualified but additionally quantified byMeyerhof et al. (2007) and Bufe et al. (2002)

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the strongest and most important representatives are fromcertain alkaloids (e.g., nicotine, quinine, caffeine, strych-nine), terpenoids (e.g., isoalpha acid, amarogentine, limo-noids), and flavonoids (e.g., neohesperedin, epigallocatechingallate, Fig. 3).

Besides this extreme wide structural range of bitterness,it is a surprising effect that the bad taste is very specific toisomers of similar molecular structure. Small structuralvariations can change the taste profile or strongly influencethe threshold. As examples (Fig. 4), the amino acid L-tryptophan is bitter but the D-enantiomer shows a distinctsweet taste (Belitz et al. 2001); the hesperetin rutinoside(hesperidin) is tasteless but the positional isomer hesperetinneohesperidoside (neohesperedin) is strongly bitter (Steglichet al. 1997); quercetin is only weakly astringent but the 3-

hydroxyderivative taxifoline exhibits a strong bitterness atthe same concentration (own trials, each tested at 100 ppm inwater). Sometimes the bitterness of a molecule depends onthe molecular environment. Neat linoleic acid is more or lesstasteless; however, the same molecule shows a distinctbitterness in emulsions (Stephan and Steinhart 2000).

Due to the wide variations of the structural basis of bittertasting molecules, it is difficult to generalize the molecularrequirements. Nevertheless in the past, there were severalattempts to correlate structural elements with bitter taste toget a clue of how taste perception works. According toBelitz and Wieser (1985), a bitter molecule needs a polargroup and a hydrophobic moiety (monopolar-hydrophobicconcept). But as mentioned above, the spatial distributionof the two structural features seems to be of much more

N

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CaffeineQuinine

Epigallocatechin gallateAcetaminophen/Paracetamol

cis-Isohumolone

Salicine

Amarogentin

Denatonium benzoate

Limonin Neohesperidin

H-L-Leu-L-Trp-OH

Fig. 3 Important bitter tastantsof different structural classes.Denatonium benzoate is themost bitter compound known

62 Chem. Percept. (2008) 1:58–77

importance, and even small structural changes causedramatic differences in taste attributes (as examples,Fig. 4). Recently, a more detailed structure–activity modelregarding necessary molecular features for bitterness wasreported (Rodgers et al. 2006). Beginning with nearly 650known bitter compounds (excluding bitter peptides) and13,500 randomly selected non-bitter molecules a modelusing MOLPRINT 2D circular fingerprints was developed.By using this model, it was possible to predict 72% of thebitter molecules. Unfortunately, only a small subset (33compounds) selected from the original structures waspublished due to confidentiality reasons. Just recently, astudy regarding the bitterness of the important structuralclass of sesquiterpene lactones was published. Starting withcynaropicrin and grosheimin from artichocke extracts, aQSAR model was developed and could be established forthe prediction of bitterness of several analogues (Scotti etal. 2007). Bitterness prediction was much more successfulin 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 ofterminal amino acids of the peptide chain, the higher thebitterness of the peptide. Peptides with more than three to

four amino acid residues are in most cases more or lesstasteless (exception: sweet tasting proteins such as thaumatin,brazzein, lysozyme). As a short summary, although it wouldbe of great value for food engineers, it is actually very difficultto predict the bitterness properties of molecules that werenever tasted and that will probably be the same in the nearfuture.

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 acomplex of a bitter agonist or antagonists and the T2r.Because there are no reports regarding three-dimensionalstructures of proteins with a tight relationship to T2r,molecular modeling or computational docking experimentsare actually very difficult and flawed. Therefore, it isgenerally by trial and error that new bitter-masking mole-cules are discovered. Several methods for identification ofsuch compounds were reported in the literature; in thefollowing paragraphs, the sensory and molecular biologicalmethods will be described.

O

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Neohesperidin: bitter

L-Tryptophan: bitter

Hesperidin: tasteless

D-Tryptophan: sweet

Taxifolin: strongly bitterQuercetin: weakly astringent

Fig. 4 Small structural varia-tions cause dramatic changes oftaste quality as a exemplified forenantiomers, regioisomers, andstructure changes by simple re-duction of a skeleton

Chem. Percept. (2008) 1:58–77 6363

The promising studies based on taste sensors made frommodified polymer electrodes (e.g., Toko 2000; Takagi et al.2001; Miyanag et al. 2003) will be not reviewed due totheir very different nature. However, some studies citedlater are based on such results. Another method is based onaffinity chromatography using molecular-imprinted poly-mers: a polymer constructed using quinine as a templatewas used for identification of L-arginine as suppressant(Ogawa et al. 2005). In sensory tests, these results havebeen validated.

Test systems based on receptors or cell constructs aremost promising to detect selective antagonists using high-throughput screening assays of known or new molecules.The classical sensory methods are limited to moleculesprincipally safe for human consumption. The main advan-tage of sensory screening is that the findings are not limitedto a single-masking mechanism. Therefore, they can beused directly for more realistic food models. The physico-chemical methods (taste sensors) perform well in simpletest systems (e.g., solutions of pharmaceuticals) and forknown bitter molecules, especially in scavenging orcomplexing systems. For detection of new bitter or bitter-masking compounds, the methods are in many cases onlyof limited value because they are based on very differentphysicochemical mechanisms compared to taste cells.

Sensory Methods

Until now, the oldest but most successful method is todetect bitter-masking molecules by simply tasting. Theclassical masking systems of sodium chloride, sugar, orsugar and salt in combination with acids were found by trialand error. Many spices and flavors derived from plants,especially aromatic herbs, were introduced most likely formasking purposes in ancient times. For pharmaceuticals,more sophisticated techniques were developed. In thenineteenth century, the first sensory studies regardingbitter-masking extracts, e.g., from Herba Santa, MiracleFruit, or Gymnema spp. were published by Lewin (1894).Jellinek (1966) first reported standardized recommenda-tions for sensorial tests for masking compounds. Thesemethods are still state of the art and are used for screeningof taste-influencing substances. An example is the simpleduo difference test using caffeine as bitter standard forscreening bitter-masking compounds (Ley et al. 2006a).Most important for reliable results is a trained panel withsufficient participants (10–20), a randomized and blindedsampling and preferably only one tasting session per day,best performed in the morning. The panelists have toquote the bitterness impression on a hedonic scale (e.g., 1[weak]–9 [strong]); a quantitative descriptive panel can becombined with the ranking exercise for further direction.Quantification can be improved by using reference

samples of known concentration and comparing testsolutions against these references to determine the bitterequivalents (Ley et al. 2005a). Some other workinggroups have developed the half-site tongue test whichmay be preferred for very small sample volumes (Shikataet al. 2000).

Soldo and Hofmann (2005) suggest not using theabsolute bitterness ratings but the change in threshold ofbitterness perception for the detection of bitterness inhib-itors. To improve the speed of screening, the comparativetaste dilution analysis (cTDA) was developed. This quan-titative screening method is a combined tasting using thewell-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 methodand cannot be used for a quick sampling. To improve thespeed, further improvements of LC analysis with directlyconsumable solvents (water, ethanol) to yield an onlinetasting result (LC Taste®) were recently reported (Krammeret al. 2006).

Regardless which sensory method is considered, they areof high value for development of bitter-masking compoundsdue to the holistic approach: in contrast to the more focusedbiochemical 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 agonistor antagonist activities on bitter receptors were developed(McGregor 2004). Recently, some promising successes incultivating primary taste receptor cells were reported (Kishiet al. 2005; Ozdener et al. 2006). Generally, the assays arenot based on primary taste receptor cells due to their limitedlife 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 togetherwith expression and transporting parts and/or with othersegments of the gustatory signaling system are used fortransfection. In many cases, existing G-protein signalingsystems of the HEK293 cells are used, and the change ofCa2+ levels of the cell most often determined, e.g., usingfluorescence methods (FLIPR, etc.; Fig. 5).

The cell-based test systems can be used to identify theagonists as well as the antagonists or modulators of thebitter taste receptors. Whereas, some handful of agonistsand their receptors are described in between, only rare dataregarding antagonists on a T2r level exists (only nucleo-tides such as adenosine monophosphate (AMP) werecharacterized thus far; McGregor and Gravina 2002).

64 Chem. Percept. (2008) 1:58–77

The newest methodology is the screening of TRPM5influencing compounds (Servant et al. 2007) and recently,some new taste inhibitors based on TRPM5 antagonisticmechanisms were identified (Bryant et al. 2007). Anotherassay is focused on modulation of activity of the GRK(G protein-coupled receptor kinases; Passe 2007) which areresponsible for signal deletion of activated G protein-coupled receptors, but the value of both methods was notyet proven by sensory methods.

Bitter-Masking Compounds

In the following paragraphs, most of the molecule-basedmasking technologies will be reviewed. The majority of thestudies were not published in peer-reviewed journals, but aspatent applications because masking is of much moreimportance to the pharmaceutical and food industries thanto scientific working groups. It is not always possible toquantify or validate the reported results in patent applica-tions. Quantitative sensory or other physical data of suchsources were not covered, unless the results are of highimportance and seemed to be reasonable. In nearly allcases, no data regarding the possible mechanisms ofmasking were published. Due to these limitations, hypoth-eses regarding mechanisms will be excluded unless thereare supporting data.

One important requirement for applicability of potentialbitter-masking compounds is absence of side effects,especially taste or flavor side effects. There is less valuein using a general taste inhibitor than a selective bitternessinhibitor. Therefore, as an example, modulators of PLCβ2or TRPM5 can impart bitter, umami, and sweet taste at the

same time and may be therefore of lower value for maskingpurposes.

Masking by Strong and/or Congruent Flavors and Tastants

It is known to most food technologists that bitter taste canbe masked by strong flavors, especially by using so-calledcongruent flavors. These flavors cause a certain acceptanceof bitterness due to their inherent occurrence. Examples arecocoa or chocolate flavor preparations which mask thebitterness of quinine (Reid and Becker 1956) or grapefruitflavors which are widely used to mask pharmaceuticalactives. In cola-type beverages, most consumers cannotdetect the bitterness of caffeine due to the high dosage ofsucrose, sweetener and acid. Another classical system is thesuppression of bitterness by sodium salts. Sodium saltswhich are low in saltiness such as gluconate or acetate arethe most successful maskers (Keast et al. 2001, 2004). As aside effect, the preferred flavors and taste qualities areenhanced (Breslin and Beauchamp 1997). A combinationof sodium salts and L-arginine was used for the reduction ofbitterness of certain peptides (Ogawa et al. 2004). In a moredetailed 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 quininesolution, 800 mM of sucrose, 300 mM NaCl, or 8 mMaspartame, respectively, was necessary. Bitter taste ofcaffeine in a tablet was reduced by using a umami/sweetmixture of erythritol–CaHPO4, L-glutamic acid, inosinicacid, 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

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HEK293Fig. 5 Schematic and simpli-fied HEK293 transient cell cul-ture-based bitter antagonistassay using calcium signalingexemplified for salicin as ago-nist transfection can also beinduced by chemicals. Read outis performed as fluorescencedetection. Control cells are pre-pared using the same protocolwithout transfection step

Chem. Percept. (2008) 1:58–77 6565

other ingredients. Sometimes, the amount of sweetener is notsufficient to elicit a sweet taste, this is important for non-sweet applications. Examples are thaumatin for reduction ofprotein off-tastes (Hamisch and Valentin 2001), thaumatin forKCl bitter reduction (Takahiro 1988) a combination of zincand sodium cyclamate for pharmaceuticals (Keast andBreslin 2005), neohesperidin dihydrochalcone for generalbitter reduction (Cano et al. 2000), and stevioside orrebaudiosides for proanthocyanidine-rich tea beverages(Uchida et al. 2007).

The mechanisms of the aforementioned bitter-maskingtechnologies are not known. Probably, the masking activ-ities are mostly caused by psychophysical effects due to thesuppression of the off-taste by camouflage. Unfortunately,the use of strong flavors or tastants is not acceptable in a lotof applications. For example, it is not possible to use higheramounts of sodium salts in sweet beverages or sweetenersin savory applications. Therefore, the applicability of suchcompounds is only limited.

Polymers and Complexing Agents

The use of ion exchangers to catch poor-tasting pharma-ceutical actives is very well established for pharmaceuticalsand reviewed elsewhere (Sohi et al. 2004; Stier 2004). Inthe following paragraphs, the focus will be more set onfood applications, natural structures, or molecules derivedfrom nature as scavengers. All of the previously mentionedsequestering and complexing agents need to be used inrelatively high concentrations to be effective, and it seemsto be unlikely that they act on receptor or even cellularlevel. Biopolymers such as alginates and other chargedpolysaccharides may cause severe problems in applicationsdue to their gelling properties and influence on texture,flavor release, and other sensorial qualities.

Scavenging molecules have been described several timessuch 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 toencapsulate the product and to improve taste and stability(Tatewaki et al. 2007). β-Cyclodextrin (Fig. 7) at 0.4% isable to reduce the bitterness of a 0.05% caffeine solution byabout 90%. The α- and the γ-cyclodextrins are much lessactive and higher concentrations of β-cyclodextrin tastesweet. In the same study, the authors demonstrated that thebitterness of various plant extracts such as artichoke orgentian can be selectively reduced by β-cyclodextrin(Binello et al., 2004). A polymer-supported cyclodextrinusing 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

et al. 1995). In a 0.5-mM solution of quinine, 1% of aphosphatidic acid/β-lactoglobulin complex was able toreduce the bitterness by 90%, and 0.1% of the complexreduced bitterness by 50%. Other bitter tastants such aspharmaceutical actives propanolol and promethazine weremasked to a similar extent whereas, the bitter taste ofcaffeine and naringin were less effectively reduced. Theeffect is caused mainly by sequestering the frequently basicand hydrophobic bitter molecules, as determined bybinding studies. It was found that these lipoproteinsreversibly suppressed the responses of the frog glossophar-yngeal nerve to the bitter substance. The results suggestedthat binding of lipoproteins to the hydrophobic region ofthe receptor membranes leads to suppression of theresponses to the bitter substances (Katsugari et al. 1995).However, another study dealing with these complexesshowed that there might be a individual component of thereported effects. Some people were not able to perceive anymasking effect using these lipoproteins (Ishimaru et al.2001).

Poly-γ-glutamic acid (γ-PGA) was described to relievepoor taste, especially the bitterness of amino acids andpeptides (Sonoda et al. 2000). Bitterness of a 2% solutionof a mixture of L-leucine, L-isoleucine, and L-valine wasreduced by 70% using 1% γ-PGA. Bitterness of a 0.1%caffeine solution was reduced in a dose-dependent mannerdown to 30% using 1% of γ-PGA.

Partially phosphorylated oligosaccharides derived frompotatoes or their salts were used to reduce bitterness ofcertain beverages (Kamsaka et al. 2002). In the sameapplication, sodium alginates (average molecular weight50,000±10,000 Da) were suggested for reduction ofunpleasant off-tastes caused by tea catechins (Shirata et al.2003). The chitin derivative, chitosan, (Fig. 6) at aconcentration of 0.4 to 1.2% in water, is also able toreduce bitterness of caffeine and various plant extracts butalso exhibits a strong astringency.

In a study using taste sensors rather than sensory panels,the astringency of various tea catechins at 100 ppm wasreduced using pectin at concentrations <0.1% (Hayashiet al. 2005). Only the catechins containing a gallate groupwere affected. Unfortunately, there is no correlation tohuman sensory data provided. Sulfated polysaccharides,such as carrageenan, were used to reduce the undesirabletaste of amino acids mixtures (e.g., L-histidine, L-isoleucine,L-leucine, L-methionine, L-phenylalanine, L-tryptophan, andL-valine, each at 10%). In a ratio of 9:1 carrageenan/aminoacid cocktail, e.g., in beverages, the bitterness of an aqueoussolution of such a mixture was reduced to 1 (weak bitterness)compared to 9 (strong bitterness) for the neat amino acidcocktail (Calton and Wood 2002).

Egg white proteins treated with enzymes such as papain,ficin, bromelain, and Aspergillus oryzae protease could

66 Chem. Percept. (2008) 1:58–77

suppress bitterness or unpleasant taste of foods based onsoybean milk and green vegetable juice (Kittaka et al.2005). A defatted (<10%) egg yolk was used in a mixturewith green tea extract (70% polyphenols; Sugiura et al.2001). The complex product was less astringent and bitterwhen compared to the free catechins.

Low-Molecular-Weight Substances

Until the third quarter of the last century, there were onlyrare examples about small bitter-masking molecules in theliterature. The bitter-masking activity of a 0.5% gymnemicacid solution was described early on (Lewin 1894); how-ever, the same molecule inhibits sweet taste as well. In1979, the flavanoid neodiosmin (Fig. 7) was described as abitter-masking agent using the threshold method (Guadagniet al. 1979). Neodiosmin (10 ppm) increased the averagedthreshold of 40 panelists for caffeine from 128 up to230 ppm by nearly 80%. The bitterness of a 100-ppmcaffeine and 8-ppm quinine solution, respectively, eachcontaining 10 ppm of the flavonoid, was rated as “not bitter”in contrast to the solutions without flavonoid (“extremelybitter”). Unfortunately, neodiosmin is not readily available.

One of the smallest “molecules” which can inhibit bittertaste 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 otherhand, sweetness of a glucose solution (0.4 to 2.4 M) wasreduced to 20% whereas salty, umami, and sour taste werenot affected. In addition, zinc salt solutions show a

prominent astringency and therefore the use in food orbeverage applications is very limited. Interestingly, magne-sium salts (25 mM) can also reduce bitterness of quinine–HCl to a somewhat lesser extent without significantlyaffecting sweet, salty, sour, and umami taste.

Ornithine derivatives such as L-ornithyl-β-alanine orL-ornithinyltaurine (for structures, Fig. 8), showed a bitter-masking effect against potassium salts (Fuller and Kurtz1997b). A solution of 1.8 g KCl and 0.2 g NaCl per litershowed no bitterness after addition of the actives. Neattaurine and its salts showed the same effect according to thereference. Similar effects towards bitterness of potassiumcontaining test solutions were described for differentimidazole derivatives, one example shown in Fig. 8 (Fullerand Kurtz 1997c). L-Ornithine itself was described as anoff-taste suppressant in combination with the bitter-branchedchain amino acids L-isoleucine, L-leucine, and L-valine asdetermined by taste sensor measurements (Kawabe et al.2004; Tokuyama et al. 2006). γ-Amino butyric acid in lowconcentrations (<100 ppm) demonstrates masking activityagainst caffeine and quinine (Ley et al. 2005b) but can alsosuppress off-tastes of catechin-rich applications such ascocoa, chocolate (Fujita et al. 2007), or of potassiumcontaining low salt products (Yamakoshi et al. 2006). Theeffect of γ-amino butyric acid seems to be of high interestbecause this well-known neurotransmitter and its receptorwere suggested as potential members of signal transductionor modulation between taste receptor cells and output cells(Herness et al. 2005). According to a Japanese patent, thebitter aftertaste of high potency sweeteners can be reduced

OH

O

OH O

NH

OH

OO

NH2

n

O

O

OH

OH

OH

O

O

OH

OH

OOH

O

OH

OH

OH

O

O

OHOH

O

OH

OOH

OH

OH

O

O

OH

OH O

OH

O

OH

OHOH

O

OOH NH3

+

OH

OH

OH

OOH NH3

+

O

OH

O

OH

NH3

+

OH

n

poly-γ -glutamic acid β-cyclodextrin chitosan

Fig. 6 Oligo- and polymericbitter maskers or scavengers:cyclodextrins, polyamino acids,and charged carbohydrates

O

O

O

OH

OH

O

O

O

OH

OHOH

OOH

OHOH

OOH

OOH

OHOH

O

OH

H

H

H

OHO

O

O

OH

O

neodiosmingymnemic acid I

Fig. 7 Bitter-masking com-pounds first described

Chem. Percept. (2008) 1:58–77 6767

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). Thebitter taste of peptide hydrolysates as well as for brucin andcaffeine was eliminated by the dipeptide L-Glu-L-Glu(Belikov and Gololobov 1986). Unfortunately, the authorsdid not report any quantitative data.

Several dipeptides containing asparaginic acid weredisclosed as bitter maskers (Fuller and Kurtz 1997a). Forexample, L-aspartyl-L-phenylalanine potassium salt (0.6 g/L)suppressed the bitterness of potassium chloride (20 g/L) andresulted in a taste reminiscent of sodium chloride. N-(1-methyl-4-oxo-2-imidazolin-2-yl)alanine at about 500 ppmconcentration is able to reduce bitter and astringent tasteimpressions of sweeteners and can suppress the lingeringsweet aftertaste of artificial sweeteners such as saccharin,steviosides, or acesulfame K (Harada and Kamada 2000).Further bitter-masking molecules based on amino acids weredescribed. The sour-tasting N-benzoyl-ɛ-aminocapronic acid(Nakamura et al. 1997) suppresses sweet and bitter taste aswell as certain sulfomethylaryl ureas (Roy et al. 1990)shown in Fig. 8. L-Theanine, a unique amino acid of greentea, exhibits a masking effect besides its effects on brainwaves (Juneja et al. 1999).

A reaction mixture of amino acids such as L-arginine,L-lysine, and L-ornithine with citric acid was used toimprove the taste of foodstuffs (Okai 2003). Not only wasbitter taste reduced but sweetness and saltiness, respective-ly. Umami impressions were also increased in a beverage orsoy sauce containing 1–5% of the mixture. Some pyridiniumbetain derivatives based on amino acids, isolated fromMaillard reaction mixtures, demonstrate bitter-maskingeffects (Soldo and Hofmann 2005). The pyridinium glycinylbetaine was able to reduce the bitterness ratings of variousconcentrations of caffeine (250–2,500 ppm) by about 3 Uusing 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 quininesolution significantly (Fujita and Kuroki 2004). In a moreextensive study, the influence of fatty acids such as linoleicacid on the five basic taste qualities was investigated. Fattyacids can increase the threshold (i.e., lower sensitivity) forsodium chloride, citric acid, and caffeine and the bitterrating for caffeine was reduced (Mattes 2007). In furthersensory experiments, it was demonstrated that edible oils(tuna, soybean, high oleic acid corn oil) in 10% oil-in-water

N

NO

NH

OH

O

NHO

NH2

NH2

COOH

NHO

NH2

NH2

SO3-

NN

Ph

NH

Ph

SO3-

OH

O

NHO

OH

O

NH2

OH

O

NHO

NH2

O

OH

OH

O

O

NH3

+

NH2

O

NH2

SO3-

-

-

-

OOH

NH2

NH

O

COOH

NH

NH

O

SO3Na

NC

OH

O

NH2

O

NH

N+

O

O

O

OH

N-(1-methyl-4-oxo-2-imidazolin-2-yl)alanin

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

H-L-Asp-L-Phe-OHdiglutamate

L-ornithinetaurine

γ -amino butyric acid

benzoyl-ε -amino caproic acidN-(4-cyanophenyl)-N-(sodiumsulfomethyl)-urea

L-theanine pyridinium glycinyl betain

Fig. 8 Amino acid and peptide derivatives

68 Chem. Percept. (2008) 1:58–77

emulsions can reduce sour, bitter, and umami taste. Ingeneral, however, in sweet and salty taste (Koriyama et al.2002) and the time intensity profile of tastants will beextended for all taste qualities. In the same study, the effectof free fatty acids was tested. The highly unsaturated fattyacids linoleic, eicosapentaenoic, and hexadocosaenoic acidshowed a highly significant masking effect against quininesulfate and L-leucine. Further lipids which are able toreduce bitterness were described. Plant stanol esters (8% inwater using guar gum as emulsifier) can reduce thebitterness of 600 ppm caffeine solution by 15% ascompared to a simple rapeseed oil at 8% (Pouru et al.2004). Some hydrogenated ethoxylated glycerol estersshow 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 assucralose and mono-ammonium glycyrrhizinate (Roger2006). Phospholipid mixtures containing phosphatidylino-sitol and phosphatidic acid were described as useful formasking bitter taste of certain drugs (Tadokoro and Goto2007). The effect of some of the lipids is not very strong,and the amount of masking agents is very high in themajority of applications. Therefore, it seems that they donot interact with receptors or taste receptor cells but maywork by scavenging bitter molecules or by acting assurfactants.

The possible inhibition of intracellular phosphatases as amechanism of bitter taste suppression was claimed (but notproven) for several organic phosphates, phosphonates,vanadates, thiophosphates, and biphosphates such aseugenylmonophosphate, thymylmonophosphate, menthyl-monophosphate, phosphotyrosine, or phosphoserine (Nelson1998). As example, vanillylmonophosphate (Fig. 9) at 0.3%was able to significantly increase the threshold and todecrease the bitterness of pharmaceutical-active ingredientssuch as dextromorphan, acetaminophen, or denatoniumbenzoate. However, the effect was not the same for caffeine(Nelson et al. 1998).

Some trigeminal active molecules such as capsaicin(Fig. 9) may reduce bitter response of the gustatory nervesaccording to experiments on rats (Simons et al. 2003). Themechanism is not limited to bitterness but affects sweet andumami taste as well. From a traditional point of view, thismay be the reason why hot spices are so commonly used tosuppress off-tastes in staple food. Capsaicin typicallyproduces hotness, and the sensation of pain and must bedosed very carefully and its applicability may be thereforelimited to spicy food or beverages. In addition, higherdosages of trigeminal stimulants such as capsaicin andmenthol can cause intrinsic bitterness (Green and Schullery2003). Some physiological cooling compounds such asL-menthyl lactate, L-menthon glycerol acetal, N-ethyl-L-menthancarboxamide, or L-menthyl propylenglycolcarbonatein combination with high intensity sweeteners, demonstratemasking effects against bitter-tasting antitussives or expec-torants such as dextromorphan (Yano 2000). The tinglingcompound spilanthol was used as a masking compound forbitter or astringent aftertaste of artificial or high intensitysweeteners (Miyazawa et al. 2006). The interaction oftrigeminal and gustatory sense is of high interest due to theco-expression of trigeminal fibres in fungiform taste papillaand the known phenomena of thermally induced taste effects(Talavera et al. 2007). But recently it was shown that theintensities of gustatory and trigeminal sensations are notdirectly correlated (Green et al. 2005). Therefore, the de-scribed taste modulation effects may be due to direct inter-actions with taste transduction mechanisms. Interestingly, theTPRM5 channel involved in taste transduction is a closerelative of the capsaicin (TRPV1) and menthol (TRPM8)receptors (Talavera et al. 2007).

A combination of a high-impact sweetener and gingeroleoresin was suggested to mask bitter pharmaceuticalssuch as acetaminophen (Lindley 2003). A mixture of gingeroil (20 ppm) with thaumatin (12.5 ppm), magnesiumgluconate (4%), and starch is able to decrease the bitternessof a 1.6% acetaminophen solution down to the comparablebitterness of a 0.96% acetaminophen solution withoutmasking ingredients; most probably the thaumatin/Mggluconate combination was the effective masking part ofthe formulation. But as mentioned earlier, the use of high-impact sweeteners is of limited value for general applica-bility. Further gluconates such as Cu-(I)-gluconate canimprove the taste of food or beverages (Fujii and Yasuda2006). The addition of 50 ppm of the salt remarkablyreduces 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,L-tryptophan, L-arginine, or L-lysine (each 0.2%) wasmasked by addition of 1 wt.% of the non-reducing andsweet carbohydrate α,α-trehalose (Uchida et al. 2003;Fig. 10). Isomaltulose was suggested for reduction of the

OP

O

OH

OOH

O

OH

ONH

O

O

O

OH

NH

O

vanillylmonophosphate capsaicin

L-menthyl lactate spilanthol

Fig. 9 Phosphatase inhibitors and trigeminals

Chem. Percept. (2008) 1:58–77 6969

bitter taste of tea beverages containing polyphenols (Doerret al. 2007). For such carbohydrates, the masking effect maybe due to their intrinsic sweetness. Bitterness inhibitors basedon the trisaccharides pannitose (Fig. 11) or the reducedderivative pannitol were described to be able to reduce the off-tastes and flavors of soy bean products and other problematicplant materials such as whole grain biscuits, carrot juice, andothers (Nakanishi et al. 2005). Cellooligosaccharide such ascellotetraose in an amount of 1% can suppress bitterness,e.g., of caffeine (Saski et al. 2002); these oligosaccharidesare more or less tasteless and therefore would be useful for alot of applications.

The active principle of the long-known bitter-reducingactivity of liquid extracts of Herba Santa (Yerba Santa,Eriodictyon ssp.; Lewin 1894) was found by sensory-guided fractionation of the plant extract (Ley et al. 2005a).

Two flavanones, eriodictyol and homoeriodictyol (each at100 ppm, Fig. 11), exhibit a 40–60% reduction effect on a500-ppm caffeine solution. In addition, effects were foundalso against bitterness of quinine, amarogentine, para-cetamol, denatonium benzoate, and salicin, whereas thebitterness of potassium salts, linoleic acid emulsions, and ofa bitter peptide, L-leucyl-L-tryptophan was not eliminated.Various structural relatives were screened for their bitter-masking activity but only few active molecules were found(Fig. 11): hydroxylated benzoic acid N-vanillylamides (Leyet al. 2006a), some hydroxylated deoxybenzoins (Ley et al.2006b), and short chain gingerdiones such as [2]-gingerdione(Ley et al. 2007). All of these compounds are somewhattasteless, and the masking activity and pattern is very similar.This seems to be specific but the underlying molecularmechanism has not yet elucidated. The compounds can beused in a lot of applications to combat bitterness but are mostactive in beverage-type applications.

Lactisol (Fig. 12), a known sweet taste inhibitor also onthe receptor level (Xu et al. 2004), can suppress the off-tastes of potassium chloride and artificial sweeteners (Kurtzand Fuller 1997a; Kurtz and Fuller 1993). A solutioncontaining 20 g/L of a mixture of 95% KCl and 5% NaCland 500 ppm lactisol sodium salt tastes like neat sodiumchloride solution with virtually none of the KCl bitterness.Unfortunately, such high concentrations of lactisol blockthe sweetness impression, and therefore, broad applicabilityis questionable. The activity of lactisol may be limited tocertain elicitors of bitter taste due to a extensive sensorystudy on various bitter tastants which show no broad-reducing effect for lactisol (Johnson et al. 1994). For thesame application area, some other molecules such as oroticor dihydroorotic acid (Fuller and Kurtz 1997d), aspartame

O

O

OH

OH

O

OH

O

OH OH

OH

OH

O

OH

OH

OH

OHn

O

OH

OH

O

OH

OHO

OH

OH

OH

OH

OHO

OH

OH

OH

O

OH

O

OH

OH

O

O

OH

OH

OH

OH

O O

OHOH

OH

O

OH

OH

OH

OHOH

OH

OH OH

OH OH

cellooligosaccharides (n = 0 - 2)

α,α-trehalose

pannitose (GlcP-α -1-6-GlcP-α -1-4-GlcP)

isomaltulose xylitol

Fig. 10 Bitter-maskingcarbohydrates

OH

OH

O

O

OH

OR

OH

OH

NH

O

OH

O

OH O

OH

O

OH

O

OH

OH O

R = H: eriodictyolR = CH3: homoeriodictyol

2,4-dihydroxybenzoicacid N-vanillylamide

[2]-gingerdione2,4,4'-trihydroxy-3'-methoxy-deoxybenzoine

Fig. 11 Bitter-masking molecules related to homoeriodictyol

70 Chem. Percept. (2008) 1:58–77

derivatives such as N-(p-cyanophenylcarbamoyl)-L-aspartyl-L-phenylalanine (Fuller and Kurtz 1997e), and phenolicacids such as 2,4-dihydroxybenzoic acid, sinapic acid, orL-DOPA (L-3,4-dihydroxyphenylalanine) (Fuller and Kurtz1997f) 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 combatthe bitter aftertaste of artificial sweeteners as well thebitterness of caffeine or quinine (Riemer 1994). The bitteraftertaste of a 500-ppm solution of Na-saccharin wassignificantly reduced by using 550 ppm ferulic acid. Thebitterness and off-taste of artificial sweeteners such asaspartame, sodium saccharin, and acesulfame K can bereduced using chlorogenic acid or other cinnamic acidesters 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, andcitric acid, an amount of 30 ppm of chlorogenic acid in theform of an extract prepared from green coffee beans themetallic and bitter off-taste was markedly reduced (Chienet al. 2002). A similar effect was obtained using only the

parent compound, quinic acid (Togawa et al. 2001). Thebitter taste of high dosages of L-menthol, which may beexperienced, e.g., in chewing gums, can be reduced also bychlorogenic acid and its analogues (Matsumoto et al. 2006).The advantage of the hydroxycinnamic acid derivatives istheir low intrinsic taste and their occurrence in a lot ofnatural extracts, e.g., chlorogenic acid can be extractedfrom green coffee beans (Matsumoto et al. 2006).

As mentioned earlier, the first bitter inhibitors found byscreening with receptor assays were reported in 2002(McGregor and Gravina 2002; McGregor and Homan2003). The simple nucleotides CMP (cytosine monophos-phate) and dATP (2-deoxyadenosine triphosphate) cause at10 mM a 40% and 60% decrease in bitterness of a quininesolution, respectively. In combination with taurine, AMPshows a well-accepted masking effect against KCl bitter-ness in sodium-reduced formulations (Salemme and Barndt2006).

Very uncommon compounds to combat poor taste weredeveloped just recently (Bryant et al. 2007): some acylatedarylhydrazones (Fig. 12) reduce the TRPM5 activity in aHEK293 cell system to 40% residual activity at 10 μM.

OH

OH

O

O

OH

OH

OH

OH

O

O

NH

N

O

O

O

OH

OH

O

O

O COOH

OH

O

NHO

OH

O

NH

O

OCNN

H

NH

O

OHOOC

O

O

OH

O

NH2

OH

OH

OH

O

O

OH

O

OH

O

OH

OH

N

N

ON

OHN

NH2

OH

O P

OH

OHO

N

N

ON

N

NH2

OH

O P

O

OH

OHn

N O

OH OH

O P

OH

OHON O

NH2

chlorogenic acid

acylated arylhydrazones

ferulic acid

lactisol N-(p-cyanophenylcarbamoyl)-L-aspartyl-L-phenylalanine

orotic acid

flavoneL-DOPA sinapic acid

2,4-dihydroxybenzoic acid

dATP (n=2)

CMP

AMP

Fig. 12 Various molecules reducing bitter or metallic aftertaste

Chem. Percept. (2008) 1:58–77 7171

Unfortunately, no sensory data has been provided thus far,so this might be of high interest to see whether thecompounds can selectively block only one taste quality orblock taste in general.

Comparability of Sensory Results and Consequencesfor Flavor Development

One of the major problems for a selection and assessmentof potential bitter-masking molecules is the compatibility ofdata generated with different methods, standards, andpanels. Therefore, the duo screening using caffeine as amodel bitter compound was described in Ley et al. (2006a).As an example, we evaluated some of the most promisingmolecules from literature and compared it to our owndevelopments (Table 1). The compounds tested wereselected from flavonoids, polymeric and monomeric aminoacids, carbohydrates, and polyphenols to cover differentpossible bitter-masking mechanisms according to the list ofpossible targets mentioned in the “Introduction”. For poly-γ-glutamic acid, a scavenging mechanism may be assumedas 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 ofT2R or other proteins of the signal transduction cascade.But for all these molecules, no proof of the basicmechanism does yet exist. The test concentration wasselected as recommended in the relevant publication and, insome cases, it was limited by solubility.

In our trials, none of the molecules was able to decreasethe bitterness of caffeine totally. Some of the testedcompounds could not inhibit caffeine bitterness at all. Inthe original papers, these compounds were not tested on

caffeine and generally, the activity against caffeine is notlimited to a set of structural classes.

For applicability, it is not only important to determinethe activity for one concentration, but also to compile adose activity plot. In most of the cited studies, these dataare only rarely found. In several cases, there is a strongdose activity response of the masking effect and the activityis mostly limited to a certain level. For some of thecompounds, we determined the dose response against a500-ppm caffeine solution. In all investigated cases, therewas a ceiling effect of activity as shown in Fig. 13. γ-Amino butyric acid and [2]-gingerdione showed decreasedactivity at the highest concentration (100 and 500 ppm,respectively) which may in the case of γ-amino butyric acidbe caused by intrinsic astringent or sour taste. The typicalsaturation effect is not limited to caffeine, as shown foractivity of homoeriodicytol against the bitter principle ofgentian, amarogentin (Fig. 14). Such effects were alsodescribed for other compounds such as phospholipoproteins(Katsugari et al. 1995). In biological screening systems,similar effects can be seen, as has been reported for activityof nucleotides against denatonium benzoate bitterness(McGregor and Gravina 2002).

For some of the masking molecules listed in Fig. 13, wehave 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 activityagainst caffeine and quinine and the peptide is comparablefor all tested masking molecules. However, for salicine,there are remarkable differences. For salicin, the main bitterreceptor was described earlier, and in this case, it may be aselective blocking effect which needs to be demonstratedon a biological level. It would be of great value todetermine 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 maskersfrom literature

Compound Reference Concentration Masking effect (%) Significance

Neodiosmine Guadagni et al. (1979) 100 ppm 28Poly-γ-glutamic acid Sonoda et al. (2000) 1% 31 p<0.05Cellotrioside Saski et al. (2002) 500 ppm 29Homoeriodictyol sodium salt Ley et al. (2005a) 100 ppm 43 p<0.05Homoeriodictyol Ley et al. (2005a) 100 ppm 28 p<0.05Eriodictyol Ley et al. (2005a) 100 ppm 47 p<0.05γ-Amino butyric acid Ley et al. (2005b) 50 ppm 33 p<0.05α,α-trehalose Uchida et al. (2003) 1% 10Taurine Fuller and Kurtz (1997b) 50 ppm 0L-Theanine Juneja et al. (1999) 500 ppm −62,4-Dihydroxybenzoic acid N-vanillyl amide Ley et al. (2006a) 100 ppm 33 p<0.052,4-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 effectwas recalculated from these data.

72 Chem. Percept. (2008) 1:58–77

Fig. 2) of the masking compounds mentioned in Table 1and to compare it with the sensory results. Probably themolecular targets of the compounds differ as suggested bythe results with various bitter molecules shown in Fig. 15.

As a consequence for flavor development using maskingmolecules, it is important to know which bitter principlehas to be blocked. After choosing the best performingblocking agent, it must be validated for activity in the finalmatrix. One of the most challenging issues is the concen-tration of the bitter tastant. Although there might be amasking molecule that can reduce the bitterness relatively,e.g., by 40%, the residual bitterness might be too high foracceptance by the end consumer. To our knowledge there isno complete or total masking technology available, andtherefore it is most important to reduce the bitter principleas much as possible by additional methods such asdebittering, complexation, or encapsulation to yield asuccessful end product. In some cases, new problems arisecaused by selective bitter masking. As an example, greentea exhibits a bitter and a strong astringent taste. When atypical bitter-masking agent is used for improving taste,

sometimes an increased astringency can be perceivedbecause bitter taste can mask astringency to a certaindegree. It is absolutely necessary to begin with baseoptimization, different masking technologies, and finally agood 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, andbeverages remains a large challenge. Most existing maskingtechnologies and more specifically, the bitter-maskingmolecules, were found by trial and error methods. In thepast, bitterness reduction was generally focused on phar-maceuticals and drug actives. Today, the most intensiveresearch is performed to reduce bitter or astringent taste offunctional food or beverage applications which show off-tastes due to enrichment with healthy, poor-tasting actives.During the last 10 years, tremendous progress in theelucidation of bitter taste reception and transduction onthe cellular level was made. This was fueled by the humangenome project and the Nobel Prize for Medicine andPhysiology issued to Buck and Axel in 2004 for their basicfindings regarding the olfactory mechanisms. Unfortunate-ly, bitter taste seems to be the most complex quality of allbasic taste modalities due to the large number and diversityof T2r bitter receptors. It seems feasible to develop effectivebitter-masking molecules of high potency which are strongmodulators of cellular signal transduction pathways as theymay not be acceptable due to their side effects on othertaste qualities and perhaps even other non-taste cellmechanisms. Especially the use of screening methods basedon agonist/antagonist/T2R interactions may be very useful

0%

20%

40%

60%

80%

100%

0 200 400 600 800 1000

concentration inhibitor (ppm)

rela

tive

mas

kin

g e

ffec

t

2,4-Dihydroxybenzoicacid N-vanillylamide[2]-Gingerdione

GABA

Eriodictyol

homoeriodictyolsodium salt

Fig. 13 Dose response of selected masking molecules from Table 1against bitter taste of a 500-ppm caffeine solution. For eachconcentration, a randomized, blinded duo test according to the methoddescribed in Ley et al. (2006a) was used. Ratings were determined bya trained panel (n=12–16) on a scale from 1 (no bitterness) to 10(strong bitterness), and the relative inhibiting effect was recalculatedfrom these data

0%

20%

40%

60%

80%

100%

0 100 200 300 400 500

concentration inhibitor (ppm)

rela

tive

mas

kin

g e

ffec

t

Fig. 14 Dose response curves of homoeriodictyol sodium salt against30 ppb amarogentin (as described in Ley et al. 2005a). Test conditionsas described for Fig. 13

Fig. 15 Activity of five bitter-masking compounds against caffeine,salicin, quinine, the dipeptide Leu-Trp, and KCl determined using therandomized and blinded duo testing (described in Ley et al. 2005a).Test conditions as described for Fig. 13

Chem. Percept. (2008) 1:58–77 7373

to identify lead compounds for development of newselective bitter-masking compounds.

Actually there is no principal all in one solution formasking issues. However, in most cases, combinations ofdifferent technologies such as encapsulation/formulation,and selective removal or biotransformation of bittermolecules, using strong or congruent tastants or flavorsand/or masking molecules have to be used to produce asuperior 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 orhigh intensity sweeteners such as saccharin) may be foundand developed.

Acknowledgements I would like to thank Gerhard Krammer,Gerald Reinders, Ian Gatfield, Gabi Vielhaber, and Kathrin Freiherrfor all the helpful discussions and valuable suggestions related to thetopic and especially Heinz-Jürgen Bertram who initiated andsupported the topic. Special thanks to Debbie Kennison for proof-reading the paper.

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