Bitter peptides activate hTAS2Rs, the human bitter receptors

Fermented food contains numerous peptides derived from material proteins. Bitter peptides formed during the fermentation process are responsible for the bitter taste of fermented food. We investigated whether human bitter receptors (hTAS2Rs) recognize bitterness of peptides with a heterologous expression system. HEK293 cells expressing hTAS2R1, hTAS2R4, hTAS2R14, and hTAS2R16 responded to bitter casein digests. Among those cells, the hTAS2R1-expressing cell was most strongly activated by the synthesized bitter peptides Gly-Phe and Gly-Leu, and none of the cells was activated by the non-bitter dipeptide Gly-Gly. The results showed that these bitter peptides, as well as many other bitter compounds, activate hTAS2Rs, suggesting that humans utilize these hTAS2Rs to recognize and perceive the structure and bitterness of peptides.     

Inducibility of Aryl Hydrocarbon Hydroxylase Activity in Cultured Cell as Food-screening for Safety Assessment

In order to investigate the effect of leucine residues on the taste of peptides, some oligo peptides containing leucine residues were synthesized and their taste was evaluated. The hydrophobicity of leucine residues markedly caused the bitterness of peptides and stronger bitterness was always found when a leucine residue was located at the C-terminus of peptides. The possibility of 2 binding sites between the bitter peptides and the bitter taste receptors of the gustation cells was postulated.     

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.     

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.     

Bitterness of Phenylalanine- and Tyrosine-containing Peptides

In order to investigate the role of phenylalanine and tyrosine residues in the bitter taste of peptides, some oligopeptides containing phenylalanine or tyrosine were synthesized and their taste was evaluated. The hydrophobicity of the phenylalanine or tyrosine molecule markedly caused the bitter taste in peptide. The bitterness was more intense when phenylalanine was located at the C- terminus and when the content of phenylalanine or tyrosine was increased in peptides. The hydrophobic residue in peptides functioned as a bitter taste determinant site. The experimental results suggest the existence of an additional site for the bitter taste of peptides.     

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.     

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.     

灵长类苦味受体基因研究进展

在鲜味、甜味、苦味、咸味和酸味5种味觉形式中,苦味能避免动物摄入有毒有害物质,在动物的生存中发挥着特别重要的作用。苦味味觉的产生依赖于苦味物质与苦味受体的相互作用。苦味受体由苦味受体基因Tas2rs编码,此类基因在不同物种中数量变化较大以适应不同的需求。目前的研究在灵长类中鉴别出了若干苦味受体的配体,并发现有的苦味受体基因所经受的选择压在类群之间、基因之间甚至同一基因不同功能区之间都存在着变化。本文从苦味受体作用的多样性特点,受体与配体的对应关系、受体基因进化模式与食性之间的关系、苦味受体基因的适应性进化方面对灵长类苦味受体基因进行了综述,以期为苦味受体基因在灵长类中的深入研究提供参考。     

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.     

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.     

Cross-adaptation and bitterness inhibition of L-tryptophan, L-phenylalanine and urea: further support for shared peripheral physiology

A previous study investigating individuals' bitterness sensitivities found a close association among three compounds: L-tryptophan (L-trp), L-phenylalanine (L-phe) and urea (Delwiche et al., 2001, Percept. Psychophys. 63, 761-776). In the present experiment, psychophysical cross-adaptation and bitterness inhibition experiments were performed on these three compounds to determine whether the bitterness could be differentially affected by either technique. If the two experimental approaches failed to differentiate L-trp, L-phe and urea's bitterness, then we may infer they share peripheral physiological mechanisms involved in bitter taste. All compounds were intensity matched in each of 13 subjects, so the judgments of adaptation or bitterness inhibition would be based on equal initial magnitudes and, therefore, directly comparable. In the first experiment, cross-adaptation of bitterness between the amino acids was high (>80%) and reciprocal. Urea and quinine-HCl (control) did not cross-adapt with the amino acids symmetrically. In a second experiment, the sodium salts, NaCl and Na gluconate, did not differentially inhibit the bitterness of L-trp, L-phe and urea, but the control compound, MgSO(4), was differentially affected. The bitter inhibition experiment supports the hypothesis that L-trp, L-phe and urea share peripheral bitter taste mechanisms, while the adaptation experiment revealed subtle differences between urea and the amino acids indicating that urea and the amino acids activate only partially overlapping bitter taste mechanisms.     

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.     

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.     

Activation of human bitter taste receptors by polymethoxylated flavonoids

Tangeretin and nobiletin are polymethoxylated flavonoids in citrus peel. Both tangeretin and nobiletin are bitter; however, their bitterness has not been evaluated using human bitter taste receptors (hTAS2Rs). We screened 25 kinds of hTAS2Rs and found that hTAS2R14 and hTAS2R46 received both compounds.     

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.     

TASTE INTENSITIES OF OIL-IN-WATER EMULSIONS WITH VARYING FAT CONTENT

The objective of this study was to determine the effect fat has on the intensity of sweet, salty, sour, bitter and umami tastes in oil-in-water emulsions. The first experiment used two levels of fat (9% and 17% in oil-in-water emulsions) and two intensities of each taste (high and low). We compared the taste intensities of these emulsions to the intensities of oil-free samples with equal total volume, and to oil free samples of the same aqueous taste compound concentrations. Because of potential confusion between taste intensity and viscosity, we repeated the experiment, having panelists rate both thickness and taste intensity. Diluting with oil, compared to diluting with water, decreased bitterness, but increased the intensity of salty, sweet, sour and umami tastes. When compared to samples with equal aqueous taste compound concentrations, fat suppressed bitterness, but had no effect on the other tastes.     

Stimulation of bitterness by capsaicin and menthol: differences between lingual areas innervated by the glossopharyngeal and chorda tympani nerves

Capsaicin is viewed as a purely chemesthetic stimulus that selectively stimulates the somatosensory system. Here we show that when applied to small areas of the tongue, capsaicin can produce a bitter taste as well as sensory irritation. In experiment 1, individuals were screened for the ability to perceive bitterness from capsaicin on the circumvallate papillae. Fifteen of 25 subjects who reported at least weak bitterness rated the intensity of taste, irritation and coolness produced by 100-320 microM capsaicin and 100-320 mM menthol applied via cotton swabs to the tip (fungiform region), the posterior edge (foliate region), and the dorsal posterior surface (circumvallate region) of the tongue. Sucrose, citric acid, sodium chloride and quinine hydrochloride were applied to the same areas to assess tastes responsiveness. On average, capsaicin and menthol produced "moderate" bitterness (and no other significant taste qualities) in the circumvallate region, and weaker bitterness on the side and tip of the tongue. Sensory irritation from capsaicin was rated significantly higher at the tongue tip, whereas menthol coolness was rated higher in the circumvallate region. In experiment 2 we applied sucrose and quinine hydrochloride together with capsaicin to investigate the effects other taste stimuli might have on capsaicin's reported bitterness. As expected, adding quinine produced stronger bitterness in the circumvallate and fungiform regions, and adding sucrose significantly reduced the bitterness of capsaicin in the circumvallate region. Overall, the results suggest that capsaicin and menthol are capable of stimulating a subset of taste neurons that respond to bitter substances, perhaps via receptor-gated ion channels like those recently found in capsaicin- and menthol-sensitive trigeminal ganglion neurons, and that the glossopharyngeal nerve may contain more such neurons than the chorda tympani nerve. That some people fail to perceive bitterness from capsaicin further implies that the incidence of capsaicin-sensitive taste neurons varies across people as well as between gustatory nerves.     

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.     

Specific alleles of bitter receptor genes influence human sensitivity to the bitterness of aloin and saccharin

Variation in human taste is a well-known phenomenon. However, little is known about the molecular basis for it. Bitter taste in humans is believed to be mediated by a family of 25 G protein-coupled receptors (hT2Rs, or TAS2Rs). Despite recent progress in the functional expression of hT2Rs in vitro, up until now, hT2R38, a receptor for phenylthiocarbamide (PTC), was the only gene directly linked to variations in human bitter taste. Here we report that polymorphism in two hT2R genes results in different receptor activities and different taste sensitivities to three bitter molecules. The hT2R43 gene allele, which encodes a protein with tryptophan in position 35, makes people very sensitive to the bitterness of the natural plant compounds aloin and aristolochic acid. People who do not possess this allele do not taste these compounds at low concentrations. The same hT2R43 gene allele makes people more sensitive to the bitterness of an artificial sweetener, saccharin. In addition, a closely related gene's (hT2R44's) allele also makes people more sensitive to the bitterness of saccharin. We also demonstrated that some people do not possess certain hT2R genes, contributing to taste variation between individuals. Our findings thus reveal new examples of variations in human taste and provide a molecular basis for them.     

Supertasting and PROP bitterness depends on more than the TAS2R38 gene

Polymorphisms in the TAS2R38 gene provide insight to phenotypes long associated 6-n-propylthiouracil (PROP) and phenylthiocarbamide bitterness. We tested relationships between TAS2R38 genotype, taste phenotype, and fungiform papillae (FP) number in 139 females and 59 males (age range 21-60 years), primarily of European ancestry. DNA was analyzed for 3 polymorphic sites, identifying common (alanine-valine-isoleucine [AVI/AVI], heterozygotes, proline-alanine-valine [PAV/PAV]) and rare (proline-valine-isoleucine, alanine-alanine-valine, AAI) forms. Individuals with PROP threshold >0.15 mM were almost exclusively AVI/AVI; those with threshold <0.1 mM could have any genotype. PAV/PAVs were more difficult to identify with PROP taste measures, although perceived bitterness of moderate PROP concentrations (0.32, 1 mM) had better correspondence with genotype than did threshold. For AVI/AVIs, increases in bitterness from 1 to 3.2 mM PROP nearly paralleled those of TAS2R38 heterozygotes and PAV/PAVs. Some bitterness gains were related to FP number sampled from a standard area on the tongue tip, yet the PROP bitterness-FP relationship differed across genotype. Among homozygotes, FP was a significant determinant of PROP bitterness; heterozygotes showed a flat relationship. Those tasting concentrated PROP as more bitter also tasted concentrated sucrose, citric acid, sodium chloride, and quinine as more intense, even after statistically controlling for TAS2R38 genotype, FP, and intensity of tones (nonoral standard). To summarize, although PROP threshold generally exhibited single-gene complete dominance, PROP bitterness may involve additional bitter receptors as evidenced by misclassification of some nontaster homozygotes and the bitterness functions for concentrated PROP. Variability in receptor expression may explain attenuated bitterness-FP relationships. PROP bitterness does associate with heightened taste sensations (i.e., supertasting), but this is not due to TAS2R38 polymorphisms.