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: [email protected] astringent to yield clear beverages (Oszmianski et al. 2007). Chem. Percept. (2008) 1:58–77 59

In the pharmaceutical area, there is also a large demand The last general review covering such molecules was for bitter reduction techniques due to the low compliance compiled by Roy in his book (1997). Since then, the of patients taking bitter drugs for longer times. In young knowledge of taste, especially bitter taste detection, and children, the problem is more serious, due to their higher transduction on a cellular level, has heavily evolved, and as taste sensitivity and because in many cases, it is not a result, several new approaches for detecting and devel- possible to supply large enough capsules containing the oping bitter-masking molecules were reported. active pharmaceutical ingredient. Currently, the old rule “only bitter medicine is good medicine,” is no longer valid. Detection of Bitter Taste In recent years, the problem of bitter- or bad-tasting food products is surfacing again, due to the demand for healthier Since the identification of the receptor proteins responsible food or beverages. Reduced sugar, fat, and sodium for for bitter taste reception by Chandreshekar et al. (2000; healthy benefits can also accentuate sourness, bitterness, Adler et al. 2000), the mechanism of bitter reception by and astringency in the base matrices. Many artificial taste receptor cells seems to be generally known nowadays sweeteners exhibit astringent, metallic, or bitter aftertaste. and was thoroughly reviewed in recent time (Margolskee In salt or sodium replacers, potassium chloride is common- 2002; Montmayeur and Matsunami 2002; Meyerhof 2005; ly used in many applications, which leaves a very metallic Chandrashekar et al. 2006; Behrens and Meyerhof 2006). bitter taste that most people find undesirable. Compounds Below, the mechanism is briefly summarized (for a such as certain polyphenols (e.g., tea catechins), soy schematic summary see Fig. 1). products, phytosterols, vitamins, minerals, fish oil, etc. Bitter molecules bind to a G protein-coupled receptor- used for fortification of functional food can cause serious type T2R on the apical membrane of the taste receptor cells taste deficiencies and reduced consumer demand for such (TRC) located in the taste buds. In humans, roughly 25 products (Eckert and Riker 2007). different T2R are described. Additionally, several alleles are One of the major problems of masking of off-taste is the known and about 100 different bitter phenotypes exist in complex mixture of sensations. The ingestible is not only man. TRC are specialized to a certain taste quality. For perceived as bitter, but is also astringent and/or sour. Each sweet taste, this was demonstrated by genetic experiments modality is transduced by different molecular sensing on mice in a labeled-line model. Most probably, sweet cells systems in the mouth, and the sensation consciously are linked directly to positive hedonic centers of the brain. recognized is again a difficult mixture to separate into The authors constructed a mouse expressing T2Rs on sweet individual taste qualities. cells and they preferred a bitter and toxic solution and not Many techniques to reduce bitterness or off-taste have the sweet one (Zhang et al. 2003). For the bitter modality, evolved through the years: one TRC expresses more than one T2R type but not in all variants. On the other hand, it is now known that & Removal of bad tasting components, where possible one particular bitter compound can bind to several T2R & Physical barriers (e.g., [micro, nano] encapsulation, subtypes with distinct affinity and that at least some of coatings, emulsions, suspensions) the bitter receptor proteins, e.g., the hT2R47, are & Scavengers, complexing agents broadly tuned for several structural classes of bitter & Strong flavors or tastants (e.g., salt, sweeteners, acid, molecules (Meyerhof et al. 2007).Asaresult,abitter strong fruit flavors) taste pattern (Fig. 2) for the cells occurs in a similar way & Congruent flavors (e.g., chocolate, grapefruit, coffee) to the olfaction process; however, the final signal to the & Masking flavors (e.g., against rancid or fishy flavor of brain is mainly “negative” or “bitter”. Discussions con- polyunsaturated fats) tinue that bitter qualities may be distinguishable but not & Bitter taste reducing compounds on a molecular level yet proven by combined sensory and molecular biological The use of physical barriers is a common approach for experiments. pharmaceutical actives, and there are several comprehen- Following the binding of agonists to the T2R, phospho- sive reviews (Sohi et al. 2004; Stier 2004). In most cases, lipase C is activated via a β-subunit of a G protein of the these technologies cannot or in limited use be adapted for TRC which activates the IP3 (inositoltriphosphate) pathway food or beverage applications because the latter applica- in the cell. Calcium will be released from internal stores tions contain much more water and often use raw materials and at least the co-expressed ion channel TRPM5 will be which are not permissible for food use. The use of classical activated and the cell depolarizes. In addition, the α-unit of flavors and tastants was reviewed by Pszczola (2004). the TRC-specific G protein gustducin may activate the PDE Therefore, the following review will focus mainly on the pathway of transduction (Ming et al. 1999), but there is no last topic, the masking of bitter taste on a molecular level. final proof of concept at this time. 60 Chem. Percept. (2008) 1:58–77

Fig. 1 Actual bitter taste transduction mechanisms (combined and mechanism follows the IP3 pathway, the PDE branch is not fully adapted according to: Gilbertson et al. 2000; Perez et al. 2003; Bufe et proven. The ATP (or alternative neurotransmitter) release mechanism al. 2002) exemplified for the human salicin receptor hT2R16. is not yet fully known (Romanov et al. 2007; Huang et al. 2007). PLC Probably only (but not all of the 25) bitter receptors are expressed Phospholipase, IP3 inositoltriphosphate, PDE phosphodiesterase, on a single “bitter” taste receptor cell. The generally accepted NMP nucleosidemonophosphate

In contrast to the intracellular mechanisms, the intercel- adenylyl cyclase by several sweeteners and bitter tastants lular transduction of the signal generated by agonist (Zubare-Samuelov et al. 2003). There were some hypoth- receptor interaction is not yet elucidated in detail. Most eses that the prominent bitter caffeine may be detected by TRC which can be stimulated by bitter tastants (or other activation of PDE (phosphodiesterase) due to its known taste qualities) are not directly linked to synapses but they activity and the difficulties to identify the responsible T2R release neurotransmitters (Clapp et al. 2006). The picture is (Yan et al. 2001). Until now, no mammalian bitter receptor still not fully clear, and serotonin, ATP, and some neuro- for caffeine is known but it was described recently for fruit peptides are under discussion (Herness et al. 2005; flies (Moon et al. 2006). Romanov et al. 2007; Huang et al. 2007). This does not From a molecular standpoint, there are several potential preclude there being other transmitters that have not yet targets to suppress bitter taste transduction: been identified. The neurotransmitters subsequently activate the so-called output cells in the taste bud, which are & Molecules, which can complex or scavenge the bitter connected to synapses of afferent gustatory nerves (Roper tastants (molecular encapsulation) or can disrupt the 2007). transport to the receptor In addition to this generally accepted pathway, there & Antagonists of T2r binding sites were some studies which suggest possible further mecha- & Modulators of T2r binding sites nisms of bitter reception but are still under discussion. & Modulators of other proteins involved in taste trans- Naim et al. claimed that tastants can rapidly enter taste cells duction, e.g., gustducin, PLCβ2 (phospholipase C β-2), and act on intracellular proteins (Peri et al. 2000). One PDE pathway example may be the general quenching mechanisms of G & Modulators of TRPM5 function protein-coupled receptors by inhibition of signal termina- & Compounds which can influence the neurotransmitter tion-related kinases which may cause the lingering bitter release, binding, or reuptake aftertaste of sweeteners (Zubare-Samuelov et al. 2005). & Modulators of signal quenching, e.g., reactivation of G Another protein discussed by the group was activation of 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 Potential modulators/antagonists of receptors and pro- mechanism, it is not clear whether the transport can be teins will be discussed later in greater detail. In nearly all selectively influenced. Saliva flow and its constituents presented cases, the exact mechanism of masking activity is certainly play an important role for the complex transport not yet known. 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 Bitter-Tasting Molecules: Structure–Activity compounds could not be verified (Creuzenet and Mangroo Relationships 1998). Unfortunately, in contrast to the agonist/T2R studies (Fig. 2), similar data regarding agonist/antagonist/T2R In contrast to the other taste qualities of sweet, umami, sour, interactions were not published yet. These data would be and salty, there is a large number of molecules which are of very high value for development of selective bitter described as bitter. Generally speaking, the bitter modality inhibitors. The influence of neurotransmitter release on is an aversive taste which protects animals against real tasting experiments was very rarely reported until potentially toxic or harmful substances in nature. In now. In a recent paper of Heath et al. (2006), the parallel, in bitter (and sometimes toxic) plants, molecules modulation of human taste thresholds by changes of the have evolved to deter herbivores (Simmonds 2001). It will serotonin and noradrenalin levels induced by certain drugs not be the intention of this review to list all relevant bitter was determined. Significantly enhancing the serotonin level tastants; therefore, in the following paragraphs only some caused a reduction of the sucrose taste threshold by 27% and examples will be discussed. the quinine taste threshold by 53%. An increased noradrenalin Bitterness is widely distributed in nature and principally titer significantly reduced bitter taste threshold by 39% and each chemical class can contain bitter molecules. Simple sour threshold by 22%. As a conclusion, influencing salts such as sodium sulfate or magnesium sulfate show a neurotransmitter levels, e.g., by drugs can dramatically change strong bitterness. Some higher peptides, terpenoids, alka- the taste response and may be a possible target for further loids, polyphenols, heterocycles, and macrolides can also developments of taste modulation compounds. But it is too exhibit bitterness. A review of the most important bitter early to decide which neurotransmitter or its receptor may be classes found in plants was given by Belitz and Wieser the most important target for bitter masking. (1985). Bitter molecules occur in many variations; however, 62 Chem. Percept. (2008) 1:58–77 the strongest and most important representatives are from hydroxyderivative taxifoline exhibits a strong bitterness at certain alkaloids (e.g., nicotine, quinine, caffeine, strych- the same concentration (own trials, each tested at 100 ppm in nine), terpenoids (e.g., isoalpha acid, amarogentine, limo- water). Sometimes the bitterness of a molecule depends on noids), and flavonoids (e.g., neohesperedin, epigallocatechin the molecular environment. Neat linoleic acid is more or less gallate, Fig. 3). tasteless; however, the same molecule shows a distinct Besides this extreme wide structural range of bitterness, bitterness in emulsions (Stephan and Steinhart 2000). it is a surprising effect that the bad taste is very specific to Due to the wide variations of the structural basis of bitter isomers of similar molecular structure. Small structural tasting molecules, it is difficult to generalize the molecular variations can change the taste profile or strongly influence requirements. Nevertheless in the past, there were several the threshold. As examples (Fig. 4), the amino acid L- attempts to correlate structural elements with bitter taste to tryptophan is bitter but the D-enantiomer shows a distinct get a clue of how taste perception works. According to sweet taste (Belitz et al. 2001); the hesperetin rutinoside Belitz and Wieser (1985), a bitter molecule needs a polar (hesperidin) is tasteless but the positional isomer hesperetin group and a hydrophobic moiety (monopolar-hydrophobic neohesperidoside (neohesperedin) is strongly bitter (Steglich concept). But as mentioned above, the spatial distribution et al. 1997); quercetin is only weakly astringent but the 3- 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 most bitter compound known N HO N H H O O N N OH OH CH O 3 O Caffeine N Quinine OH HO O OH O OH Epigallocatechin gallate OH N Acetaminophen/ 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 NH H O 2 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 O tions cause dramatic changes of OH OH taste quality as a exemplified for enantiomers, regioisomers, and NH 2 NH structure changes by simple re- 2 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 four amino acid residues are in most cases more or less dramatic differences in taste attributes (as examples, tasteless (exception: sweet tasting proteins such as thaumatin, Fig. 4). Recently, a more detailed structure–activity model brazzein, lysozyme). As a short summary, although it would regarding necessary molecular features for bitterness was be of great value for food engineers, it is actually very difficult reported (Rodgers et al. 2006). Beginning with nearly 650 to predict the bitterness properties of molecules that were known bitter compounds (excluding bitter peptides) and never tasted and that will probably be the same in the near 13,500 randomly selected non-bitter molecules a model future. 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 Identification of Bitter-Masking Molecules compounds) selected from the original structures was published due to confidentiality reasons. Just recently, a Unfortunately, until now, there was no description of a three- study regarding the bitterness of the important structural dimensional structure of one of the T2r proteins or that of a class of sesquiterpene lactones was published. Starting with complex of a bitter agonist or antagonists and the T2r. cynaropicrin and grosheimin from artichocke extracts, a Because there are no reports regarding three-dimensional QSAR model was developed and could be established for structures of proteins with a tight relationship to T2r, the prediction of bitterness of several analogues (Scotti et molecular modeling or computational docking experiments al. 2007). Bitterness prediction was much more successful are actually very difficult and flawed. Therefore, it is in the more focused structural class of peptides (Asao et al. generally by trial and error that new bitter-masking mole- 1987; Opris and Diudea 2001; Ramos de Armas et al. cules are discovered. Several methods for identification of 2004). Generally speaking, the higher the hydrophobicity of such compounds were reported in the literature; in the terminal amino acids of the peptide chain, the higher the following paragraphs, the sensory and molecular biological bitterness of the peptide. Peptides with more than three to methods will be described. 64 Chem. Percept. (2008) 1:58–77

The promising studies based on taste sensors made from samples of known concentration and comparing test modified polymer electrodes (e.g., Toko 2000; Takagi et al. solutions against these references to determine the bitter 2001; Miyanag et al. 2003) will be not reviewed due to equivalents (Ley et al. 2005a). Some other working their very different nature. However, some studies cited groups have developed the half-site tongue test which later are based on such results. Another method is based on may be preferred for very small sample volumes (Shikata affinity chromatography using molecular-imprinted poly- et al. 2000). mers: a polymer constructed using quinine as a template SoldoandHofmann(2005) suggest not using the was used for identification of L-arginine as suppressant absolute bitterness ratings but the change in threshold of (Ogawa et al. 2005). In sensory tests, these results have bitterness perception for the detection of bitterness inhib- been validated. itors. To improve the speed of screening, the comparative Test systems based on receptors or cell constructs are taste dilution analysis (cTDA) was developed. This quan- most promising to detect selective antagonists using high- titative screening method is a combined tasting using the throughput screening assays of known or new molecules. well-established taste dilution analysis (Scharbert et al. The classical sensory methods are limited to molecules 2004) and the tastant whose taste quality should be modified. principally safe for human consumption. The main advan- Unfortunately, the cTDA is a very time-consuming method tage of sensory screening is that the findings are not limited and cannot be used for a quick sampling. To improve the to a single-masking mechanism. Therefore, they can be speed, further improvements of LC analysis with directly used directly for more realistic food models. The physico- consumable solvents (water, ethanol) to yield an online chemical methods (taste sensors) perform well in simple tasting result (LC Taste®) were recently reported (Krammer test systems (e.g., solutions of pharmaceuticals) and for et al. 2006). known bitter molecules, especially in scavenging or Regardless which sensory method is considered, they are complexing systems. For detection of new bitter or bitter- of high value for development of bitter-masking compounds masking compounds, the methods are in many cases only due to the holistic approach: in contrast to the more focused of limited value because they are based on very different biochemical assays the whole flavor and taste attributes, physicochemical mechanisms compared to taste cells. especially the common off-taste characteristics of candi- dates, can be determined in a small set of tasting sessions. Sensory Methods Biological Test Systems Until now, the oldest but most successful method is to detect bitter-masking molecules by simply tasting. The During the last decade, several assays to determine agonist classical masking systems of sodium chloride, sugar, or or antagonist activities on bitter receptors were developed sugar and salt in combination with acids were found by trial (McGregor 2004). Recently, some promising successes in and error. Many spices and flavors derived from plants, cultivating primary taste receptor cells were reported (Kishi especially aromatic herbs, were introduced most likely for et al. 2005; Ozdener et al. 2006). Generally, the assays are masking purposes in ancient times. For pharmaceuticals, not based on primary taste receptor cells due to their limited more sophisticated techniques were developed. In the life span. In most cases, easy-to-handle transfected immor- nineteenth century, the first sensory studies regarding tal cell lines such as HEK293 cell systems (Bufe et al. bitter-masking extracts, e.g., from Herba Santa, Miracle 2002; Ruiz-Avila et al. 2000; Margolskee and Ming 2000; Fruit, or Gymnema spp. were published by Lewin (1894). Bufe et al. 2004, 2003; Pronin et al. 2003; Gravina et al. Jellinek (1966) first reported standardized recommenda- 2003) are used. Frequently, constructs of T2r genes together tions for sensorial tests for masking compounds. These with expression and transporting parts and/or with other methods are still state of the art and are used for screening segments of the gustatory signaling system are used for of taste-influencing substances. An example is the simple transfection. In many cases, existing G-protein signaling duo difference test using caffeine as bitter standard for systems of the HEK293 cells are used, and the change of screening bitter-masking compounds (Ley et al. 2006a). Ca2+ levels of the cell most often determined, e.g., using Most important for reliable results is a trained panel with fluorescence methods (FLIPR, etc.; Fig. 5). sufficient participants (10–20), a randomized and blinded The cell-based test systems can be used to identify the sampling and preferably only one tasting session per day, agonists as well as the antagonists or modulators of the best performed in the morning. The panelists have to bitter taste receptors. Whereas, some handful of agonists quote the bitterness impression on a hedonic scale (e.g., 1 and their receptors are described in between, only rare data [weak]–9 [strong]); a quantitative descriptive panel can be regarding antagonists on a T2r level exists (only nucleo- combined with the ranking exercise for further direction. tides such as adenosine monophosphate (AMP) were Quantification can be improved by using reference 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+ Ca ture-based bitter antagonist 2+ 2+ Ca Ca 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 2+ 2+ inactive Ca is performed as fluorescence Ca 2+ 2+ Ca Ca sensitive detection. Control cells are pre- fluorescence dye pared using the same protocol without transfection step HO OH incubation HO O HO OH HO OH O HO O HO O HO O O HO HO OH OH OH taste receptor 2+ incubation read out Ca 2+ 2+ 2+ 2+ 2+ Ca Ca Ca Ca Ca 2+ 2+ 2+ Ca Ca Ca

cell nucleus

inhibitor

The newest methodology is the screening of TRPM5 same time and may be therefore of lower value for masking influencing compounds (Servant et al. 2007) and recently, purposes. some new taste inhibitors based on TRPM5 antagonistic mechanisms were identified (Bryant et al. 2007). Another Masking by Strong and/or Congruent Flavors and Tastants assay is focused on modulation of activity of the GRK (G protein-coupled receptor kinases; Passe 2007) which are It is known to most food technologists that bitter taste can responsible for signal deletion of activated G protein- be masked by strong flavors, especially by using so-called coupled receptors, but the value of both methods was not congruent flavors. These flavors cause a certain acceptance yet proven by sensory methods. 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 Bitter-Masking Compounds flavors which are widely used to mask pharmaceutical actives. In cola-type beverages, most consumers cannot In the following paragraphs, most of the molecule-based detect the bitterness of caffeine due to the high dosage of masking technologies will be reviewed. The majority of the sucrose, sweetener and acid. Another classical system is the studies were not published in peer-reviewed journals, but as suppression of bitterness by sodium salts. Sodium salts patent applications because masking is of much more which are low in saltiness such as gluconate or acetate are importance to the pharmaceutical and food industries than the most successful maskers (Keast et al. 2001, 2004). As a to scientific working groups. It is not always possible to side effect, the preferred flavors and taste qualities are quantify or validate the reported results in patent applica- enhanced (Breslin and Beauchamp 1997). A combination tions. Quantitative sensory or other physical data of such of sodium salts and L-arginine was used for the reduction of sources were not covered, unless the results are of high bitterness of certain peptides (Ogawa et al. 2004). In a more importance and seemed to be reasonable. In nearly all detailed study, the bitterness reduction of quinine hydro- cases, no data regarding the possible mechanisms of chloride by using sucrose, sodium chloride, and tannic acid, masking were published. Due to these limitations, hypoth- a strong astringent, was evaluated (Nakamura et al. 2002). eses regarding mechanisms will be excluded unless there For 80% suppression of the taste of a 0.1-mM quinine are supporting data. solution, 800 mM of sucrose, 300 mM NaCl, or 8 mM One important requirement for applicability of potential aspartame, respectively, was necessary. Bitter taste of bitter-masking compounds is absence of side effects, caffeine in a tablet was reduced by using a umami/sweet especially taste or flavor side effects. There is less value mixture of erythritol–CaHPO4, L-glutamic acid, inosinic in using a general taste inhibitor than a selective bitterness acid, and 5-ribonucleotides (Kitamura and Uokyu 2001). inhibitor. Therefore, as an example, modulators of PLCβ2 In the latter method, the addition of high potency sweet- or TRPM5 can impart bitter, umami, and sweet taste at the eners is a commonly used method to reduce the off-taste of 66 Chem. Percept. (2008) 1:58–77 other ingredients. Sometimes, the amount of sweetener is not et al. 1995). In a 0.5-mM solution of quinine, 1% of a sufficient to elicit a sweet taste, this is important for non- phosphatidic acid/β-lactoglobulin complex was able to sweet applications. Examples are thaumatin for reduction of reduce the bitterness by 90%, and 0.1% of the complex protein off-tastes (Hamisch and Valentin 2001), thaumatin for reduced bitterness by 50%. Other bitter tastants such as KCl bitter reduction (Takahiro 1988) a combination of zinc pharmaceutical actives propanolol and promethazine were and sodium cyclamate for pharmaceuticals (Keast and masked to a similar extent whereas, the bitter taste of Breslin 2005), neohesperidin dihydrochalcone for general caffeine and naringin were less effectively reduced. The bitter reduction (Cano et al. 2000), and stevioside or effect is caused mainly by sequestering the frequently basic rebaudiosides for proanthocyanidine-rich tea beverages and hydrophobic bitter molecules, as determined by (Uchida et al. 2007). binding studies. It was found that these lipoproteins The mechanisms of the aforementioned bitter-masking reversibly suppressed the responses of the frog glossophar- technologies are not known. Probably, the masking activ- yngeal nerve to the bitter substance. The results suggested ities are mostly caused by psychophysical effects due to the that binding of lipoproteins to the hydrophobic region of suppression of the off-taste by camouflage. Unfortunately, the receptor membranes leads to suppression of the the use of strong flavors or tastants is not acceptable in a lot responses to the bitter substances (Katsugari et al. 1995). of applications. For example, it is not possible to use higher However, another study dealing with these complexes amounts of sodium salts in sweet beverages or sweeteners showed that there might be a individual component of the in savory applications. Therefore, the applicability of such reported effects. Some people were not able to perceive any compounds is only limited. masking effect using these lipoproteins (Ishimaru et al. 2001). Polymers and Complexing Agents Poly-γ-glutamic acid (γ-PGA) was described to relieve poor taste, especially the bitterness of amino acids and The use of ion exchangers to catch poor-tasting pharma- peptides (Sonoda et al. 2000). Bitterness of a 2% solution ceutical actives is very well established for pharmaceuticals of a mixture of L-leucine, L-isoleucine, and L-valine was and reviewed elsewhere (Sohi et al. 2004; Stier 2004). In reduced by 70% using 1% γ-PGA. Bitterness of a 0.1% the following paragraphs, the focus will be more set on caffeine solution was reduced in a dose-dependent manner food applications, natural structures, or molecules derived down to 30% using 1% of γ-PGA. from nature as scavengers. All of the previously mentioned Partially phosphorylated oligosaccharides derived from sequestering and complexing agents need to be used in potatoes or their salts were used to reduce bitterness of relatively high concentrations to be effective, and it seems certain beverages (Kamsaka et al. 2002). In the same to be unlikely that they act on receptor or even cellular application, sodium alginates (average molecular weight level. Biopolymers such as alginates and other charged 50,000±10,000 Da) were suggested for reduction of polysaccharides may cause severe problems in applications unpleasant off-tastes caused by tea catechins (Shirata et al. due to their gelling properties and influence on texture, 2003). The chitin derivative, chitosan, (Fig. 6)ata flavor release, and other sensorial qualities. concentration of 0.4 to 1.2% in water, is also able to Scavenging molecules have been described several times reduce bitterness of caffeine and various plant extracts but such as cyclodextrins (Binello et al. 2004) or cyclofructans also exhibits a strong astringency. (Nishioka et al. 2004) or combinations thereof (Mori et al. In a study using taste sensors rather than sensory panels, 2006) which can complex bitter molecules. Such com- the astringency of various tea catechins at 100 ppm was plexes, e.g., combined with isohumolone, can be used to reduced using pectin at concentrations <0.1% (Hayashi encapsulate the product and to improve taste and stability et al. 2005). Only the catechins containing a gallate group (Tatewaki et al. 2007). β-Cyclodextrin (Fig. 7)at0.4%is were affected. Unfortunately, there is no correlation to able to reduce the bitterness of a 0.05% caffeine solution by human sensory data provided. Sulfated polysaccharides, about 90%. The α- and the γ-cyclodextrins are much less such as carrageenan, were used to reduce the undesirable active and higher concentrations of β-cyclodextrin taste taste of amino acids mixtures (e.g., L-histidine, L-isoleucine, sweet. In the same study, the authors demonstrated that the L-leucine, L-methionine, L-phenylalanine, L-tryptophan, and bitterness of various plant extracts such as artichoke or L-valine, each at 10%). In a ratio of 9:1 carrageenan/amino gentian can be selectively reduced by β-cyclodextrin acid cocktail, e.g., in beverages, the bitterness of an aqueous (Binello et al., 2004). A polymer-supported cyclodextrin solution of such a mixture was reduced to 1 (weak bitterness) using chitin as base was also successfully tested as a bitter- compared to 9 (strong bitterness) for the neat amino acid masking agent (Binello et al. 2004). cocktail (Calton and Wood 2002). Complexes of phospholipids with proteins were sug- Egg white proteins treated with enzymes such as papain, gested to mask bitterness of pharmaceuticals (Katsugari 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 + cyclodextrins, polyamino acids, NH OH 3 and charged carbohydrates O OH NH2 + O OH O O OH H N OH 3 n O OH OHOH O OH OHO O O O HO OH n OH HO OH O O OH OH HO NH O HO O O O HO OH OH O + OH HO NH3 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 prominent astringency and therefore the use in food or soybean milk and green vegetable juice (Kittaka et al. beverage applications is very limited. Interestingly, magne- 2005). A defatted (<10%) egg yolk was used in a mixture sium salts (25 mM) can also reduce bitterness of quinine– with green tea extract (70% polyphenols; Sugiura et al. HCl to a somewhat lesser extent without significantly 2001). The complex product was less astringent and bitter affecting sweet, salty, sour, and umami taste. when compared to the free catechins. Ornithine derivatives such as L-ornithyl-β-alanine or L-ornithinyltaurine (for structures, Fig. 8), showed a bitter- Low-Molecular-Weight Substances masking effect against potassium salts (Fuller and Kurtz 1997b). A solution of 1.8 g KCl and 0.2 g NaCl per liter Until the third quarter of the last century, there were only showed no bitterness after addition of the actives. Neat rare examples about small bitter-masking molecules in the taurine and its salts showed the same effect according to the literature. The bitter-masking activity of a 0.5% gymnemic reference. Similar effects towards bitterness of potassium acid solution was described early on (Lewin 1894); how- containing test solutions were described for different ever, the same molecule inhibits sweet taste as well. In imidazole derivatives, one example shown in Fig. 8 (Fuller 1979, the flavanoid neodiosmin (Fig. 7) was described as a and Kurtz 1997c). L-Ornithine itself was described as an bitter-masking agent using the threshold method (Guadagni off-taste suppressant in combination with the bitter-branched et al. 1979). Neodiosmin (10 ppm) increased the averaged chain amino acids L-isoleucine, L-leucine, and L-valine as threshold of 40 panelists for caffeine from 128 up to determined by taste sensor measurements (Kawabe et al. 230 ppm by nearly 80%. The bitterness of a 100-ppm 2004; Tokuyama et al. 2006). γ-Amino butyric acid in low caffeine and 8-ppm quinine solution, respectively, each concentrations (<100 ppm) demonstrates masking activity containing 10 ppm of the flavonoid, was rated as “not bitter” against caffeine and quinine (Ley et al. 2005b) but can also in contrast to the solutions without flavonoid (“extremely suppress off-tastes of catechin-rich applications such as bitter”). Unfortunately, neodiosmin is not readily available. cocoa, chocolate (Fujita et al. 2007), or of potassium One of the smallest “molecules” which can inhibit bitter containing low salt products (Yamakoshi et al. 2006). The 2+ taste is the Zn ion. It was shown that that ZnSO4 (25 mM) effect of γ-amino butyric acid seems to be of high interest can reduce bitterness of a quinine–HCl solution (0.04– because this well-known neurotransmitter and its receptor 0.4 mM) by at least 50% to 70% (Keast 2003). On the other were suggested as potential members of signal transduction hand, sweetness of a glucose solution (0.4 to 2.4 M) was or modulation between taste receptor cells and output cells reduced to 20% whereas salty, umami, and sour taste were (Herness et al. 2005). According to a Japanese patent, the not affected. In addition, zinc salt solutions show a bitter aftertaste of high potency sweeteners can be reduced

Fig. 7 Bitter-masking com- OH O pounds first described O HO O HO O OH OH HO O O H O O O HO OH OH O O H O HO O OH HO O H OH OH neodiosmin OH 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 + NH NH 3 2 NH NH 2 2 H N - 2 H2N H N 2 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 NH O NH OH O NH N O NH2 HO 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 - O N N SO3Na 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- A reaction mixture of amino acids such as L-arginine, tyrosine, L-serine, L-aspartic acid, L-glutamine, L-alanine, L-lysine, and L-ornithine with citric acid was used to L-leucine, or L-proline (Takahishi and Kawai 2000). The improve the taste of foodstuffs (Okai 2003). Not only was bitter taste of peptide hydrolysates as well as for brucin and bitter taste reduced but sweetness and saltiness, respective- caffeine was eliminated by the dipeptide L-Glu-L-Glu ly. Umami impressions were also increased in a beverage or (Belikov and Gololobov 1986). Unfortunately, the authors soy sauce containing 1–5% of the mixture. Some pyridinium did not report any quantitative data. betain derivatives based on amino acids, isolated from Several dipeptides containing asparaginic acid were Maillard reaction mixtures, demonstrate bitter-masking disclosed as bitter maskers (Fuller and Kurtz 1997a). For effects (Soldo and Hofmann 2005). The pyridinium glycinyl example, L-aspartyl-L-phenylalanine potassium salt (0.6 g/L) betaine was able to reduce the bitterness ratings of various suppressed the bitterness of potassium chloride (20 g/L) and concentrations of caffeine (250–2,500 ppm) by about 3 U resulted in a taste reminiscent of sodium chloride. N-(1- using a scale of 0 (no bitterness) to 5 (very strong). methyl-4-oxo-2-imidazolin-2-yl)alanine at about 500 ppm According to Tomotake et al. (1998), simple-sodium- concentration is able to reduce bitter and astringent taste saturated fatty acid salts such as sodium stearate, palmitate, impressions of sweeteners and can suppress the lingering and laurate in relatively high concentrations of about 1% sweet aftertaste of artificial sweeteners such as saccharin, were able to reduce the bitter taste of a 100-ppm quinine steviosides, or acesulfame K (Harada and Kamada 2000). solution significantly (Fujita and Kuroki 2004). In a more Further bitter-masking molecules based on amino acids were extensive study, the influence of fatty acids such as linoleic described. The sour-tasting N-benzoyl-ɛ-aminocapronic acid acid on the five basic taste qualities was investigated. Fatty (Nakamura et al. 1997) suppresses sweet and bitter taste as acids can increase the threshold (i.e., lower sensitivity) for well as certain sulfomethylaryl ureas (Roy et al. 1990) sodium chloride, citric acid, and caffeine and the bitter shown in Fig. 8. L-Theanine, a unique amino acid of green rating for caffeine was reduced (Mattes 2007). In further tea, exhibits a masking effect besides its effects on brain sensory experiments, it was demonstrated that edible oils waves (Juneja et al. 1999). (tuna, soybean, high oleic acid corn oil) in 10% oil-in-water Chem. Percept. (2008) 1:58–77 69 emulsions can reduce sour, bitter, and umami taste. In Some trigeminal active molecules such as capsaicin general, however, in sweet and salty taste (Koriyama et al. (Fig. 9) may reduce bitter response of the gustatory nerves 2002) and the time intensity profile of tastants will be according to experiments on rats (Simons et al. 2003). The extended for all taste qualities. In the same study, the effect mechanism is not limited to bitterness but affects sweet and of free fatty acids was tested. The highly unsaturated fatty umami taste as well. From a traditional point of view, this acids linoleic, eicosapentaenoic, and hexadocosaenoic acid may be the reason why hot spices are so commonly used to showed a highly significant masking effect against quinine suppress off-tastes in staple food. Capsaicin typically sulfate and L-leucine. Further lipids which are able to produces hotness, and the sensation of pain and must be reduce bitterness were described. Plant stanol esters (8% in dosed very carefully and its applicability may be therefore water using guar gum as emulsifier) can reduce the limited to spicy food or beverages. In addition, higher bitterness of 600 ppm caffeine solution by 15% as dosages of trigeminal stimulants such as capsaicin and compared to a simple rapeseed oil at 8% (Pouru et al. menthol can cause intrinsic bitterness (Green and Schullery 2004). Some hydrogenated ethoxylated glycerol esters 2003). Some physiological cooling compounds such as show masking effects at concentrations of about 1–2% L-menthyl lactate, L-menthon glycerol acetal, N-ethyl-L- against several pharmaceutical actives such as dextro- menthancarboxamide, or L-menthyl propylenglycolcarbonate methorphan, chlorhexidine, guaifenesin, caffeine, aspirin, in combination with high intensity sweeteners, demonstrate or acetaminophen in combination with sweeteners such as masking effects against bitter-tasting antitussives or expec- sucralose and mono-ammonium glycyrrhizinate (Roger torants such as dextromorphan (Yano 2000). The tingling 2006). Phospholipid mixtures containing phosphatidylino- compound spilanthol was used as a masking compound for sitol and phosphatidic acid were described as useful for bitter or astringent aftertaste of artificial or high intensity masking bitter taste of certain drugs (Tadokoro and Goto sweeteners (Miyazawa et al. 2006). The interaction of 2007). The effect of some of the lipids is not very strong, trigeminal and gustatory sense is of high interest due to the and the amount of masking agents is very high in the co-expression of trigeminal fibres in fungiform taste papilla majority of applications. Therefore, it seems that they do and the known phenomena of thermally induced taste effects not interact with receptors or taste receptor cells but may (Talavera et al. 2007). But recently it was shown that the work by scavenging bitter molecules or by acting as intensities of gustatory and trigeminal sensations are not surfactants. directly correlated (Green et al. 2005). Therefore, the de- The possible inhibition of intracellular phosphatases as a scribed taste modulation effects may be due to direct inter- mechanism of bitter taste suppression was claimed (but not actions with taste transduction mechanisms. Interestingly, the proven) for several organic phosphates, phosphonates, TPRM5 channel involved in taste transduction is a close vanadates, thiophosphates, and biphosphates such as relative of the capsaicin (TRPV1) and menthol (TRPM8) eugenylmonophosphate, thymylmonophosphate, menthyl- receptors (Talavera et al. 2007). monophosphate, phosphotyrosine, or phosphoserine (Nelson A combination of a high-impact sweetener and ginger 1998). As example, vanillylmonophosphate (Fig. 9) at 0.3% oleoresin was suggested to mask bitter pharmaceuticals was able to significantly increase the threshold and to such as acetaminophen (Lindley 2003). A mixture of ginger decrease the bitterness of pharmaceutical-active ingredients oil (20 ppm) with thaumatin (12.5 ppm), magnesium such as dextromorphan, acetaminophen, or denatonium gluconate (4%), and starch is able to decrease the bitterness benzoate. However, the effect was not the same for caffeine of a 1.6% acetaminophen solution down to the comparable (Nelson et al. 1998). bitterness of a 0.96% acetaminophen solution without masking ingredients; most probably the thaumatin/Mg gluconate combination was the effective masking part of O O OH the formulation. But as mentioned earlier, the use of high- O O P N impact sweeteners is of limited value for general applica- O H O HO bility. Further gluconates such as Cu-(I)-gluconate can HO improve the taste of food or beverages (Fujii and Yasuda vanillylmonophosphate capsaicin 2006). The addition of 50 ppm of the salt remarkably reduces bitterness of a coffee beverage. O O The bitterness of amino acids such as L-valine, L-leucine, L-isoleucine (each 1% in aqueous solution), L-phenylalanine, O N H L-tryptophan, L-arginine, or L-lysine (each 0.2%) was OH masked by addition of 1 wt.% of the non-reducing and L-menthyl lactate spilanthol sweet carbohydrate α,α-trehalose (Uchida et al. 2003; Fig. 9 Phosphatase inhibitors and trigeminals Fig. 10). Isomaltulose was suggested for reduction of the 70 Chem. Percept. (2008) 1:58–77

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

OH O OH O O O O OH OH OH HOHO O OH OH OH HO HO OH OH OH OH xylitol α,α-trehalose isomaltulose OH HO OH OH HO OH HO OH HO OH HO O OH OH O HO O O O OH O O n O HO O O HO OH HO HO 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 O HO and Fuller 1997a; Kurtz and Fuller 1993). A solution HO O R containing 20 g/L of a mixture of 95% KCl and 5% NaCl OH 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 study on various bitter tastants which show no broad- HO 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 CN OH HOOC N O O NH H O O O COOH HO HO N O H N-(p-cyanophenylcarbamoyl)- lactisol orotic acid 2,4-dihydroxybenzoic acid L-aspartyl-L-phenylalanine

O O O HO O OH OH NH HO 2 HO O O

L-DOPA sinapic acid flavone OH HO OH HO O O N O OH O HO O H N O OH O 2 N P HO O O OH OH HO HO

ferulic acid chlorogenic acid CMP

OH N HO OH H N N N O N N H N N O O 2 O H N N O OH O N OH 2 O P N O n 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- parent compound, quinic acid (Togawa et al. 2001). The L-phenylalanine (Fuller and Kurtz 1997e), and phenolic bitter taste of high dosages of L-menthol, which may be acids such as 2,4-dihydroxybenzoic acid, sinapic acid, or experienced, e.g., in chewing gums, can be reduced also by L-DOPA (L-3,4-dihydroxyphenylalanine) (Fuller and Kurtz chlorogenic acid and its analogues (Matsumoto et al. 2006). 1997f) as well as some flavonoids such as flavone itself The advantage of the hydroxycinnamic acid derivatives is (Kurtz and Fuller 1997b) were reported. their low intrinsic taste and their occurrence in a lot of Ferulic acid (and other hydroxycinnamic acids, Fig. 12) natural extracts, e.g., chlorogenic acid can be extracted in concentrations of 0.001–0.2% was suggested to combat from green coffee beans (Matsumoto et al. 2006). the bitter aftertaste of artificial sweeteners as well the As mentioned earlier, the first bitter inhibitors found by bitterness of caffeine or quinine (Riemer 1994). The bitter screening with receptor assays were reported in 2002 aftertaste of a 500-ppm solution of Na-saccharin was (McGregor and Gravina 2002; McGregor and Homan significantly reduced by using 550 ppm ferulic acid. The 2003). The simple nucleotides CMP (cytosine monophos- bitterness and off-taste of artificial sweeteners such as phate) and dATP (2-deoxyadenosine triphosphate) cause at aspartame, sodium saccharin, and acesulfame K can be 10 mM a 40% and 60% decrease in bitterness of a quinine reduced using chlorogenic acid or other cinnamic acid solution, respectively. In combination with taurine, AMP esters of quinic acid (Lee et al. 1975; Chien et al. 2002; shows a well-accepted masking effect against KCl bitter- Takagaki 2006). In an acidic beverage containing aspar- ness in sodium-reduced formulations (Salemme and Barndt tame, acesulfame K, sodium benzoate, phosphoric acid, and 2006). citric acid, an amount of 30 ppm of chlorogenic acid in the Very uncommon compounds to combat poor taste were form of an extract prepared from green coffee beans the developed just recently (Bryant et al. 2007): some acylated metallic and bitter off-taste was markedly reduced (Chien arylhydrazones (Fig. 12) reduce the TRPM5 activity in a et al. 2002). A similar effect was obtained using only the 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, caffeine and generally, the activity against caffeine is not so this might be of high interest to see whether the limited to a set of structural classes. compounds can selectively block only one taste quality or For applicability, it is not only important to determine block taste in general. 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 Comparability of Sensory Results and Consequences dose activity response of the masking effect and the activity for Flavor Development is mostly limited to a certain level. For some of the compounds, we determined the dose response against a One of the major problems for a selection and assessment 500-ppm caffeine solution. In all investigated cases, there of potential bitter-masking molecules is the compatibility of was a ceiling effect of activity as shown in Fig. 13. γ- data generated with different methods, standards, and Amino butyric acid and [2]-gingerdione showed decreased panels. Therefore, the duo screening using caffeine as a activity at the highest concentration (100 and 500 ppm, model bitter compound was described in Ley et al. (2006a). respectively) which may in the case of γ-amino butyric acid As an example, we evaluated some of the most promising be caused by intrinsic astringent or sour taste. The typical molecules from literature and compared it to our own saturation effect is not limited to caffeine, as shown for developments (Table 1). The compounds tested were activity of homoeriodicytol against the bitter principle of selected from flavonoids, polymeric and monomeric amino gentian, amarogentin (Fig. 14). Such effects were also acids, carbohydrates, and polyphenols to cover different described for other compounds such as phospholipoproteins possible bitter-masking mechanisms according to the list of (Katsugari et al. 1995). In biological screening systems, possible targets mentioned in the “Introduction”. For poly- similar effects can be seen, as has been reported for activity γ-glutamic acid, a scavenging mechanism may be assumed of nucleotides against denatonium benzoate bitterness as well as for cellotrioside and α,α-trehalose, whereas γ- (McGregor and Gravina 2002). amino butyric acid may influence the intercellular commu- For some of the masking molecules listed in Fig. 13,we nication. The flavonoids can possibly act as modulators of have tested their activity against different bitter molecules, T2R or other proteins of the signal transduction cascade. e.g., salicin, quinine, KCl and the bitter dipeptide L- But for all these molecules, no proof of the basic leucinyl-L-tryptophan (Leu-Trp; Fig. 15). The activity mechanism does yet exist. The test concentration was against caffeine and quinine and the peptide is comparable selected as recommended in the relevant publication and, in for all tested masking molecules. However, for salicine, some cases, it was limited by solubility. there are remarkable differences. For salicin, the main bitter In our trials, none of the molecules was able to decrease receptor was described earlier, and in this case, it may be a the bitterness of caffeine totally. Some of the tested selective blocking effect which needs to be demonstrated compounds could not inhibit caffeine bitterness at all. In on a biological level. It would be of great value to the original papers, these compounds were not tested on 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 73

100%

80% 2,4-Dihydroxybenzoic acid N-vanillylamide [2]-Gingerdione 60% GABA

40% Eriodictyol homoeriodictyol 20% sodium salt relative masking effect

0% 0 200 400 600 800 1000 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 Fig. 15 Activity of five bitter-masking compounds against caffeine, from these data 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 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 sometimes an increased astringency can be perceived the results with various bitter molecules shown in Fig. 15. because bitter taste can mask astringency to a certain As a consequence for flavor development using masking degree. It is absolutely necessary to begin with base molecules, it is important to know which bitter principle optimization, different masking technologies, and finally a has to be blocked. After choosing the best performing good flavor which will mask distinct off-tastes for ready-to- blocking agent, it must be validated for activity in the final use food or beverages for acceptance by the consumer. 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, Conclusions and Outlook e.g., by 40%, the residual bitterness might be too high for acceptance by the end consumer. To our knowledge there is Combating bitter taste in food, pharmaceuticals, and no complete or total masking technology available, and beverages remains a large challenge. Most existing masking therefore it is most important to reduce the bitter principle technologies and more specifically, the bitter-masking as much as possible by additional methods such as molecules, were found by trial and error methods. In the debittering, complexation, or encapsulation to yield a past, bitterness reduction was generally focused on phar- successful end product. In some cases, new problems arise maceuticals and drug actives. Today, the most intensive caused by selective bitter masking. As an example, green research is performed to reduce bitter or astringent taste of tea exhibits a bitter and a strong astringent taste. When a functional food or beverage applications which show off- typical bitter-masking agent is used for improving taste, 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 100% the cellular level was made. This was fueled by the human

80% genome project and the Nobel Prize for Medicine and Physiology issued to Buck and Axel in 2004 for their basic 60% findings regarding the olfactory mechanisms. Unfortunate- ly, bitter taste seems to be the most complex quality of all 40% basic taste modalities due to the large number and diversity

20% of T2r bitter receptors. It seems feasible to develop effective relative masking effect bitter-masking molecules of high potency which are strong 0% modulators of cellular signal transduction pathways as they 0 100 200 300 400 500 may not be acceptable due to their side effects on other concentration inhibitor (ppm) taste qualities and perhaps even other non-taste cell Fig. 14 Dose response curves of homoeriodictyol sodium salt against 30 ppb amarogentin (as described in Ley et al. 2005a). Test conditions mechanisms. Especially the use of screening methods based as described for Fig. 13 on agonist/antagonist/T2R interactions may be very useful 74 Chem. Percept. (2008) 1:58–77 to identify lead compounds for development of new Cano J, Mintijano H, Lopez-Cremades F, Borrego F (2000) Masking selective bitter-masking compounds. the bitter taste of pharmaceuticals. Manufacturing Chemist (July), – Actually there is no principal all in one solution for 16 17 Chandreshekar J, Mueller K, Hoon MA, Adler E, Feng L, Guo W, masking issues. However, in most cases, combinations of Zuker CS, Ryba NJP (2000) T2Rs function as bitter taste different technologies such as encapsulation/formulation, receptor. Cell (Cambridge, Mass.) 100(6):703–711 and selective removal or biotransformation of bitter Chandrashekar J, Hoon MA, Ryba NJP, Zuker CS (2006) The molecules, using strong or congruent tastants or flavors receptors and cells for mammalian taste. Nature 444(7117):288 Chien M, Haeusler A, Van Leersum H (2002) Taste modifiers and/or masking molecules have to be used to produce a comprising a chlorogenic acid. WO 2002 100,192 superior tasting and healthy food or beverage. Nevertheless, Clapp TR, Medler KF, Damak S, Margolskee RF, Kinnamon SC in the future selective inhibitors for special bitter com- (2006) Mouse taste cells with G protein-coupled taste receptors pounds (e.g., bitter plant polyphenols such as catechins or lack voltage-gated calcium channels and SNAP-25. BMC Biol 4 (7), nmo pages (http://www.biomedcentral.com/1741-7007/4/7) high intensity sweeteners such as saccharin) may be found Creuzenet C, Mangroo D (1998) Physico-chemical characterization of and developed. human von Ebner gland protein expressed in Escherichia coli: implications for its physiological role. Protein Expr Purif 14 Acknowledgements I would like to thank Gerhard Krammer, (2):254–260 Gerald Reinders, Ian Gatfield, Gabi Vielhaber, and Kathrin Freiherr Doerr T, Hausmanns S, Kowalczyk J, Becker M, Killeit U (2007) for all the helpful discussions and valuable suggestions related to the Formulation for masking bitterness, especially in tea extracts, topic and especially Heinz-Jürgen Bertram who initiated and comprising a polyphenol-containing composition and isomaltu- supported the topic. Special thanks to Debbie Kennison for proof- lose. WO 2007 59,953 (in German) reading the paper. Drewnoswki A (2001) The science and complexity of bitter taste. Nutr Rev 59(6):163–169 Drewnowski A, Gomez-Carneros C (2000) Bitter taste, phytonutrients, – References and the consumer: a review. Am J Clin Nutr 72(6):1424 1435 Eckert M, Riker P (2007) Overcoming challenges in functional beverages. 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