A psychophysical investigation of binary bitter-compound interactions

The aim of this study was to determine if taste interactions occur when bitter stimuli are mixed. Eight bitter stimuli were employed: denatonium benzoate (DB), quinine-HCl (QHCl), sucrose octaacetate (SOA), urea, L-tryptophan (L-trp), L-phenylalanine (L-phe), ranitidine-HCl, and Tetralone. The first experiment constructed individual psychophysical curves for each subject (n = 19) for each compound to account for individual differences in sensitivities when presenting bitter compounds in experiment 2. Correlation analysis revealed two groupings of bitter compounds at low intensity (1, L-trp, L-phe, and ranitidine; 2, SOA and QHCl), but the correlations within each group decreased as the perceived intensity increased. In experiment 2, intensity ratings and two-alternative forced-choice discrimination tasks showed that bitter compounds generally combine additively in mixture and do not show interactions with a few specific exceptions. The methods employed detected synergy among sweeteners, but could not detect synergy among these eight bitter compounds. In general, the perceived bitterness of these binary bitter-compound mixtures was an additive function of the total bitter-inducing stimuli in the mouth.     

Enzymes of auxin biosynthesis and their regulation I. Tryptophan and phenylalanine aminotransferase in pea plants

The transaminations of L-tryptophan (L-trp) and of L-phenylalanine (L-phe) are catalysedin vitro by the same non-specific aminotransferase. The transaminations procceed at the same pH (pH 8.5) and temperature (45 °C) optima, have parallel increases in activity with addition of the coenzyme pyridoxal phosphate (PRP) and have identical elution characteristics in gel chromatography. The enzyme from pea seedlings has a relatively weak affinity for both amino acids (K_m L-trp = 4.16 × 10⁻¹ mmol 1⁻¹; K_m L-phe = 2.10 × 10⁻¹ mmol 1⁻¹). Differences in affinity for a series of keto acids in the pea enzyme were observed, with pyruvate having the strongest and glyoxylate the weakest affinity. Transamination of L-trp and L-phe was demonstrated by enzyme extracts from pea, maize and tomato, but was not detected in kohlrabi. The amino acids L-asparagine (L-asn), L-phe, L-lysine (L-lys), L-methionine (L-met) have distinct inhibitory effects on the transamination of L-trp. Indolylacetylaspartate and tryptophol were shown to be competitive inhibitors. The regulation at the molecular level of L-trp transaminase activity is discussed.     

The influence of sodium salts on binary mixtures of bitter-tasting compounds

In order to study potential mixture interactions among bitter compounds, selected sodium salts were added to five compounds presented either alone or as binary bitter-compound mixtures. Each compound was tested at a concentration that elicited 'weak' perceived bitterness. The bitter compounds were mixed at these concentrations to form a subset of possible binary mixtures. For comparison, the concentration of each solitary compound was doubled to measure bitterness inhibition at the higher intensity level elicited by the mixtures. The following sodium salts were tested for bitterness inhibition: 100 mM sodium chloride (salty), 100 mM sodium gluconate (salty), 100 and 20 mM monosodium glutamate (umami), and 50 mM adenosine monophosphate disodium salt (umami). Sucrose (sweet) was also employed as a bitterness suppressor. The sodium salts differentially suppressed the bitterness of compounds and their binary combinations. Although most bitter compounds were suppressed, the bitterness of tetralone was not suppressed, nor was the bitterness of the binary mixtures that contained it. In general, the percent suppression of binary mixtures of compounds was predicted by the average percent suppression of its two components. Within the constraints of the present study, the bitterness of mixtures was suppressed by sodium salts and sucrose independently, with few bitter interactions. This is consistent with observations that the bitter taste system integrates the bitterness of multi-compound solutions linearly.     

Individual differences in sensitivity to bitter-tasting substances

Perception of several bitter-tasting compounds was tested in52 subjects. Stable individual differences in the perceivedintensity of the bitterness of suprathreshold concentrationsof quinine sulfate (QSO₄) and urea were found. Whereas 18 subjectsjudged selected concentrations of these compounds to be equallybitter, 17 found QSO₄ to be more bitter than urea, and 17 foundurea to be more bitter than QSO₄. These reliable individualdifferences were significantly related to threshold sensitivityto QSO₄; that is, individuals who perceived QSO₄ to be moreintense than urea at suprathreshold concentrations also hadlower QSO₄ thresholds than did those who perceived urea to bemore intense than QSO₄. There appeared to be no relationshipbetween the relative perceived intensities of these compoundsand rating of the bitterness of PROP (6-n-propylthiouracil).However, QSO₄-sensitive individuals tended to find the bitternessof suprathreshold caffeine and sucrose octaacetate to be greaterthan that of suprathreshold magnesium sulfate, whereas the reversewas true for urea-sensitive individuals. This pattern parallelsthe pattern of cross-adaptation among these compounds reportedby other investigators. These results are consistent with theexistence of multiple bitter transduction sequences and suggestthat individual differences in response to various bitter compoundsmay reflect differences in teh relative availability of specifictransduction sequences.     

Relationship between Bitterness of Peptides and their Chemical Structures

Various peptides and derivatives of peptides and amino acids were synthesized and tasted, systematically, to elucidate the relationship between bitterness and chemical structures of peptides.

We have found that: 1. Peptides become more bitter than the original amino acids when their amino and carboxyl groups are blocked and when peptide bond is formed. 2. A peptide molecule with a high content of amino acids with hydrophobic side chains will develop bitter taste. 3. The amino acids in a peptide chain independently contribute to bitterness regardless of amino acid sequences and configuration.     

Formation of Dendrolasin,Sesquirosefuran, Perillene and Rosefuran by Biomimetic Autooxidation

To estimate the steric distance between the bitter taste determinant sites in peptides, some cyclic dipeptides, amino acid anilides, amino acid cyclohexylamides, and benzoyl amino acids were synthesized and their tastes were evaluated. The diketopiperazine ring of cyclic dipeptides acted as a bitter taste determinant site due to its hydrophobicity. The steric distance between 2 sites was estimated as 4.1 Å from the molecule models of cyclic dipeptides composed of typical amino acids in the bitter peptides. Due to the hypothesis of two bitter taste determinant sites, which bind with the bitter taste receptor via a “binding unit” and a “stimulating unit,” a mechanism for the bitterness in peptides was postulated.     

Reduction of saltiness and bitterness after a chlorhexidine rinse

Chronic rinsing with chlorhexidine, an oral-antiseptic, has been shown to decrease the saltiness of NaCl and the bitterness of quinine. The effect of acute chlorhexidine on taste has not been investigated. The purpose of the present study was to examine the effect of acute chlorhexidine rinses on taste intensity and quality of 11 stimuli representing sweet, salt, sour, bitter and savory. All stimuli were first matched for overall intensity so the effects of chlorhexidine would be directly comparable across compounds. As a control treatment, the bitter taste of chlorhexidine digluconate (0.12%) was matched in intensity to quinine HCl, which was found to cross-adapt the bitterness of chlorhexidine. Subjects participated in four experimental conditions: a pre-test, a quinine treatment, a chlorhexidine treatment, and a post-test condition, while rating total taste intensity and taste qualities in separate test sessions. Relative to the quinine treatment, chlorhexidine was found to decrease the salty taste of NaCl, KCl and NH4Cl, and not to significantly affect the tastes of sucrose, monosodium glutamate (MSG), citric acid, HCl and the taste of water. The bitter taste of urea, sucrose octa-acetate and quinine were suppressed after chlorhexidine rinses relative to water rinses, but were only marginally suppressed relative to quinine rinses. Potential mechanisms are discussed.     

Individual differences in perception of bitterness from capsaicin, piperine and zingerone

It was recently shown that in some subjects capsaicin can evoke bitterness as well as burning and stinging, particularly in the circumvallate (CV) region of the tongue. Because perception of bitterness from capsaicin is characterized by large individual differences, the main goal of the present study was to learn whether people who taste capsaicin as bitter also report bitterness from structurally similar sensory irritants that are known to stimulate capsaicin-sensitive neurons. The irritancy and taste of capsaicin and two of its most commonly studied congeners, piperine and zingerone, were measured in individuals who had been screened for visibility of, and reliable access to, the CV papillae. Approximately half of these individuals reported tasting bitterness from all three irritants when the stimuli were swabbed directly onto the CV papillae. Concentrations that produced similar levels of burning sensation across subjects also produced similar (though lower) levels of bitter taste. These results are consistent with the hypothesis that capsaicin and its congeners stimulate bitterness via a common sensory receptor that is distributed differentially among individuals. Additionally, bitter tasters rated gustatory qualities (but not burning and stinging) slightly but significantly higher than did bitter non-tasters, which suggests that perception of capsaicin bitterness is associated with a higher overall taste responsiveness (but not chemesthetic responsiveness) in the CV region.     

Suppression of Bitterness by Sodium: Variation Among Bitter Taste Stimuli

Taste interactions between salts (NaCl, LiCl, KCl, L-arginine:L-asparticacid, Na-acetate and Na-gluconate) and bittertasting compounds(urea, quinine HCI, magnesium sulphate, KCI, amiloride HCI andcaffeine) were investigated. In each study binary combinationsof three or four concentrations of one bitter compound withfour concentrations (0, 0.1, 0.3 and 0.5 M) of one salt wererated for bitterness and saltiness using the method of magnitudeestimation. In most cases, perceived bitterness was suppressedby salts, although the degree of suppression varied. In general,bitterness suppression was not accompanied by an equivalentreciprocal suppression of saltiness. Only MgSO₄ and amiloridehad suppressing effects on the saltiness of NaCl at the intermediateconcentrations and no bitter compound affected the saltinessat the high concentrations of NaCl. Since salt suppressed thebitterness of urea effectively, a detailed analysis of suppressionof the bitterness of urea by different salts was conducted.Those studies indicated that the key component in this effectwas the sodium or lithium ion for two reasons: first, all threesodium salts and the lithium salt had a suppressive effect onbitterness, whereas KCl did not; secondly, the effect of a salton suppression of the bitterness of urea was independent ofits perceived saltiness; that is, NaCl, Na-acetate (which isperceived as less salty than NaCl), and Na-gluconate (whichis perceived as less salty than Na-acetate) reduced bitternesscomparably. These results suggest that there is a major peripheralcomponent to the suppression of the bitterness of urea, andperhaps other bitter tasting compounds, by sodium. Chem. Senses20: 609–623, 1995.     

Tolerance of bitter stimuli and attenuation/accumulation of their bitterness in humans

Some components of bitterness make key flavor contributions to promote the palatability of foods, whereas other components are recognized as aversive signals to avoid consuming harmful substances. These contradictory behaviors suggest that humans tolerate tastes of bitterants based on certain criteria. Here, we investigated human taste tolerance and sensory cues leading to diverse taste tolerance of bitter compounds. Tolerance of eight bitter compounds, which are typically contained in foods, was evaluated by measuring detection and rejection thresholds. The results revealed that the level of tolerance of each compound was variable, and some compounds showed an acceptable concentration regarding the suprathreshold intensity. Tolerance did not depend on the nutritive value or attenuation and accumulation characteristics of bitterness and bitter taste receptors. These results suggest that the criteria controlling tolerance of bitter compounds may be derived from a complex relationship between the taste quality and cognitive process.     

Riboflavin-binding protein is a novel bitter inhibitor

Riboflavin-binding protein (RBP) from chicken egg, which was recently reported to be a selective sweet inhibitor for protein sweeteners, was also found to be a bitter inhibitor. RBP elicited broadly tuned inhibition of various bitter substances including quinine-HCl, naringin, theobromine, caffeine, glycyl-L-phenylalanine (Gly-Phe), and denatonium benzoate, whereas several other proteins, such as ovalbumin (OVA) and beta-lactoglobulin, were ineffective in reducing bitterness of these same compounds. Both the bitter tastes of quinine and caffeine were reduced following an oral prerinse with RBP. It was found that RBP binds to quinine but not to caffeine, theobromine, naringin, and Gly-Phe. However, the binding of RBP to quinine was probably not responsible for the bitter inhibition because OVA bound to quinine as well as RBP. Based on these results, it is suggested that the bitter inhibitory effect of RBP is the consequence of its ability to interact with taste receptors rather than because it interacts with the bitter tastants themselves. RBP may have practical uses in reducing bitterness of foods and pharmaceuticals. It may also prove a useful tool in studies of mechanisms of bitter taste.     

Clinical bitterness masking test for phantogeusia

It is difficult to determine the reason why a patient complains of a bitter taste when their mouth is empty. We examined a new diagnostic test using a bitterness masking substance. The bitterness masking substance, 'Benecoat BMI-60' (hereafter BMI-60), is a masking substance specific to the taste cells' bitterness receptors. After patients gargled with BMI-60 solutions, the phantom sensation of bitterness was masked in some patients, but was not masked in others. Bitter substances in saliva seemed to be masked by BMI-60, but bitterness did not seem to be masked when the locus of the phantom sensation was within the peripheral nerve and/or the brain. The bitterness masking test is useful for diagnosis of the phantom sensation of bitter taste.     

Marked Increase in PROP Taste Responsiveness Following Oral Supplementation with Selected Salivary Proteins or Their Related Free Amino Acids

The genetic predisposition to taste 6-n-propylthiouracil (PROP) varies among individuals and is associated with salivary levels of Ps-1 and II-2 peptides, belonging to the basic proline-rich protein family (bPRP). We evaluated the role of these proteins and free amino acids that selectively interact with the PROP molecule, in modulating bitter taste responsiveness. Subjects were classified by their PROP taster status based on ratings of perceived taste intensity for PROP and NaCl solutions. Quantitative and qualitative determinations of Ps-1 and II-2 proteins in unstimulated saliva were performed by HPLC-ESI-MS analysis. Subjects rated PROP bitterness after supplementation with Ps-1 and II-2, and two amino acids (L-Arg and L-Lys) whose interaction with PROP was demonstrated by ¹H-NMR spectroscopy. ANOVA showed that salivary levels of II-2 and Ps-1 proteins were higher in unstimulated saliva of PROP super-tasters and medium tasters than in non-tasters. Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter taste responsiveness, and this effect was specific to the non-taster group.¹H-NMR results showed that the interaction between PROP and L-Arg is stronger than that involving L-Lys, and taste experiments confirmed that oral supplementation with these two amino acids increased PROP bitterness intensity, more for L-Arg than for L-Lys. These data suggest that Ps-1 protein facilitates PROP bitter taste perception and identifies a role for free L-Arg and L-Lys in PROP tasting.     

Studies on a Model of Bitter Peptides Including Arginine,Proline and Phenylalanine Residues. II. Bitterness Behavior of a Tetrapeptide (Arg-Pro-Phe-Phe) and Its Derivatives

In order to investigate the production of a strong bitter taste of the tetrapeptide, Arg-Pro-Phe-Phe (1), we synthesized 16 kinds of analogs and tasted them. From the results, it was clarified that all the constituent amino acid residues in Arg-Pro-Phe-Phe (1) were necessary for its strong bitter taste. For a further increase in bitterness potency, it was found that the bitterness production units necessary should be concentrated together. In addition, Arg-Pro-Gly-Gly (6) and Gly-Gly-Arg-Pro (7) were found to have no bitterness. This will be very useful not only for studies on debittering of food but also for basic studies on the taste production mechanism.     

In Vitro Stimulation by Parathion of Porcine Pancreatic Carboxylesterase Activity toward Triacetin

In order to elucidate the relationship between bitter taste and chemical structure in peptides, various kinds of model bitter peptides containing arginine, proline and phenylalanine were synthesized, and the contribution of the individual amino acids to the bitter taste was made clear. It was confirmed that, in order to strengthen the bitterness in di- and tripeptides, the hydrophobic amino acid needs to be located at the C-terminal and, conversely, the basic amino acid should be located at the N-terminal Furthermore, a strong bitter taste was observed when arginine was contiguous to proline such as Arg-Pro, Gly-Arg-Pro and Arg-Pro-Gly. A synergistic effect for bitter taste was observed in the peptides whose structure is (Arg)_l-(Pro)_m-(Phe)_n (l=1, 2; m, n = 1 ~ 3) by increasing the number of amino acids. Among them, the octapeptide (Arg-Arg-Pro-Pro-Pro-Phe-Phe-Phe) possessed an extremely bitter taste with its threshold value of 0.002 mm and was found to be the most bitter among the peptides.     

Peripheral coding of bitter taste in Drosophila

Taste receptors play a crucial role in detecting the presence of bitter compounds such as alkaloids, and help to prevent the ingestion of toxic food. In Drosophila, we show for the first time that several taste sensilla on the prothoracic legs detect bitter compounds both through the activation of specific taste neurons but also through inhibition of taste neurons activated by sugars and water. Each sensillum usually houses a cluster of four taste neurons classified according to their best stimulus (S for sugar, W for Water, L1 and L2 for salts). Using a new statistical approach based on the analysis of interspike intervals, we show that bitter compounds activate the L2 cell. Bitter-activated L2 cells were excited with a latency of at least 50 ms. Their sensitivity to bitter compounds was different between sensilla, suggesting that specific receptors to bitter compounds are differentially expressed among L2 cells. When presented in mixtures, bitter compounds inhibited the responses of S and W, but not the L1 cell. The inhibition was effective even in sensilla where bitter compounds did not activate the L2 cell, indicating that bitter compounds directly interact with the S and W cells. Interestingly, this inhibition occurred with latencies similar to the excitation of bitter-activated L2 cells. It suggests that the inhibition in the W and S cells shares similar transduction pathways with the excitation in the L2 cells. Combined with molecular approaches, the results presented here should provide a physiological basis to understand how bitter compounds are detected and discriminated.     

Receptor sites for amino acids in the facial taste system of the channel catfish

1. Receptor sites for different amino acids in the facial taste system of the channel catfish, Ictalurus punctatus, were determined from in vivo electrophysiological cross-adaptation experiments. 2. Relatively independent receptor sites were indicated for L-proline, D-proline, D-arginine, L-histidine and L-lysine, as well as those previously reported for L-alanine, L-arginine and D-alanine. 3. The functional isolation of two nerve twigs that were more responsive to D-alanine than to L-alanine or to other test stimuli provided further evidence for the existence of D-alanine sites that are independent from those to L-alanine. 4. Under all cross-adaptation regimes, the taste responses to the majority of test stimuli were reduced. Various possible mechanisms accounting for this generalized reduction in action potential activity during adaptation are discussed.     

Bitter Peptide Isolated from Milk Cultures of Streptococcus cremoris

Certain cultures of Streptococcus cremoris produced a bitter taste that occurred in the whey portion of milk cultures. Whey from a culture which produced bitterness was fractionated on Sephadex. The fraction in which the bitter taste was concentrated was chromatographed successively on paper with butanol-acetic acid-water (5:1:4), and then butanol-2-butanone-water (2:2:1). In each instance, the bitter component was in the most rapidly moving band that gave a positive ninhydrin test. The bitterness was observed to be caused by a peptide containing the following numbers of each amino acid: arginine, 1; glutamic acid, 2; glycine, 2; isoleucine, 2; leucine, 2; phenylalanine, 1; proline, 5; and valine, 4. N-terminal amino acids could be detected by coupling with 2,4-dinitrofluorobenzene or phenylisothiocyanate, or by hydrolysis with leucine aminopeptidase. When treated with carboxypeptidase, only leucine and valine appeared at the C-terminal end, and these were detected simultaneously.     

Debittering effect of Monascus carboxypeptidase during the hydrolysis of soybean protein

The actions of pepsin and the admixture of pepsin and Monascus pilosus carboxypeptidase 1 (MpiCP-1) on the hydrolysis of soybean protein were studied. The results showed that the pepsin hydrolyzate of soybean protein was much more bitter and contained relatively smaller amounts of total free amino acids than the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. In addition, hydrophilic and hydrophobic amino acids were present in almost equal proportions in the pepsin hydrolyzate, while mainly hydrophobic amino acids made up the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. These results suggest that MpiCP-1 suppresses and reverses the development of the bitterness taste that results from the pepsin hydrolysis of soybean protein by releasing mainly hydrophobic amino acids from the C-termini of the bitter components.     

The molecular basis of individual differences in phenylthiocarbamide and propylthiouracil bitterness perception

Individual differences in perception are ubiquitous within the chemical senses: taste, smell, and chemical somesthesis . A hypothesis of this fact states that polymorphisms in human sensory receptor genes could alter perception by coding for functionally distinct receptor types . We have previously reported evidence that sequence variants in a presumptive bitter receptor gene (hTAS2R38) correlate with differences in bitterness recognition of phenylthiocarbamide (PTC) . Here, we map individual psychogenomic pathways for bitter taste by testing people with a variety of psychophysical tasks and linking their individual perceptions of the compounds PTC and propylthiouracil (PROP) to the in vitro responses of their TAS2R38 receptor variants. Functional expression studies demonstrate that five different haplotypes from the hTAS2R38 gene code for operatively distinct receptors. The responses of the three haplotypes we also tested in vivo correlate strongly with individuals' psychophysical bitter sensitivities to a family of compounds. These data provide a direct molecular link between heritable variability in bitter taste perception to functional variations of a single G protein coupled receptor that responds to compounds such as PTC and PROP that contain the N-C=S moiety. The molecular mechanisms of perceived bitterness variability have therapeutic implications, such as helping patients to consume beneficial bitter-tasting compounds-for example, pharmaceuticals and selected phytochemicals.