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Masking the bitter taste of foods is one of the key strategies to improve their taste and palatability, particularly in the context of clean labeling, where natural compounds are preferred. Despite the demand, the availability of natural bitter-masking compounds remains limited. Here, we identified the bitter-masking compound 4'-demethyl-3,9-dihydroeucomin ( DMDHE ) isolated from the resin of Daemonorops draco by means of an activity-guided in vivo (sensory bitterness rating of quinine) and in vitro (cell-based bitter response assays) approach. First, a mean bitter-masking effect of -29.6 ± 6.30% on the bitterness perceived from quinine [10 ppm] was demonstrated for an organic solvent extract of the resin of D. draco (= DD [500 ppm]) in a sensory trial. The results were verified in a cell-based bitter assay in which the bitter taste receptor (TAS2R)-dependent proton secretion serves as an outcome measure of the cellular bitter response in parietal HGT-1 cells. By means of preparative RP-18 high-performance liquid chromatography (HPLC) analysis combined with activity-guided sensory evaluations, the most potent bitter-masking fractions were identified. Subsequent quantitative liquid chromatography/high-resolution mass spectrometry/charged aerosol detection/ultraviolet (LC-HRMS/CAD/UV), NMR analysis, followed by gram-scale synthesis, led to the characterization of DMDHE as bitter-masking homoisoflavanone. DMDHE decreased the sensory bitterness of quinine by 14.8 ± 5.00%. Functional involvement of TAS2R14 was demonstrated by means of a CRISPR-Cas9 approach, which revealed a reduction of the DMDHE -evoked bitter-masking effect by 40.4 ± 9.32% in HGT-1 TAS2R14ko versus HGT-1 wt cells.

A microfluidic tongue-on-a-chip platform has been evaluated relative to the known sensory properties of various sweeteners. Analogous metrics of typical sensory features reported by human panels such as sweet taste thresholds, onset, and lingering, as well as bitter off-flavor and blocking interactions were deduced from the taste receptor activation curves and then compared. To this end, a flow cell containing a receptor cell array bearing the sweet and six bitter taste receptors was transiently exposed to pure and mixed sweetener samples. The sample concentration gradient across time was separately characterized by the injection of fluorescein dye. Subsequently, cellular calcium responses to different doses of advantame, aspartame, saccharine, and sucrose were overlaid with the concentration gradient. Parameters describing the response kinetics compared to the gradient were quantified. Advantame at 15 μM recorded a significantly faster sweetness onset of 5 ± 2 s and a longer lingering time of 39 s relative to sucrose at 100 mM with an onset of 13 ± 2 s and a lingering time of 6 s. Saccharine was shown to activate the bitter receptors TAS2R8, TAS2R31, and TAS2R43, confirming its known off-flavor, whereas addition of cyclamate reduced or blocked this saccharine bitter response. The potential of using this tongue-on-a-chip to bridge the gap with in vitro assays and taste panels is discussed.

Beyond the key bitter compound kaempferol 3- O -(2‴- O -sinapoyl-β-d-sophoroside) previously described in the literature ( 1 ), eight further bitter and astringent-tasting kaempferol glucosides ( 2 - 9 ) have been identified in rapeseed protein isolates ( Brassica napus L.). The bitterness and astringency of these taste-active substances have been described with taste threshold concentrations ranging from 3.3 to 531.7 and 0.3 to 66.4 μmol/L, respectively, as determined by human sensory experiments. In this study, the impact of 1 and kaempferol 3- O -β-d-glucopyranoside ( 8 ) on TAS2R-linked proton secretion by HGT-1 cells was analyzed by quantification of the intracellular proton index. mRNA levels of bitter receptors TAS2R3, 4, 5, 13, 30, 31, 39, 40, 43, 45, 46, 50 and TAS2R8 were increased after treatment with compounds 1 and 8 . Using quantitative UHPLC-MS/MS MRM measurements, the concentrations of 1 - 9 were determined in rapeseed/canola seeds and their corresponding protein isolates. Depending on the sample material, compounds 1 , 3 , and 5 - 9 exceeded dose over threshold (DoT) factors above one for both bitterness and astringency in selected protein isolates. In addition, an increase in the key bitter compound 1 during industrial protein production (apart from enrichment) was observed, allowing the identification of the potential precursor of 1 to be kaempferol 3- O -(2‴- O -sinapoyl-β-d-sophoroside)-7- O -β-d-glucopyranoside ( 3 ). These results may contribute to the production of less bitter and astringent rapeseed protein isolates through the optimization of breeding and postharvest downstream processing.

In the past, taste interactions between sodium chloride (NaCl) and bitter tastants were investigated in human sensory studies, and the suppression of bitterness by sodium was observed. It is currently not clear if this phenomenon occurs predominantly peripherally or centrally and if the effect is general or only particular bitter compounds are blocked. Therefore, the influence of NaCl at the receptor level was tested by functional expression assays using four out of ∼25 human bitter taste receptors together with prototypical agonists. It was observed that NaCl affected only the responses of particular bitter taste receptor-compound pairs, whereas other bitter responses remained unchanged upon variations of the sodium concentrations. Among the tested receptors, TAS2R16 showed a reduction in signaling in the presence of NaCl. This demonstrates that for some receptor-agonist pairs, NaCl reduces the activation at the receptor level, whereas central effects may dominate the NaCl-induced bitter taste inhibition for other substances.

Recent data have shown anti-inflammatory effects for trans -resveratrol (RSV) and rosmarinic acid (RA) in various immune-competent cell models through reduction of lipopolysaccharide (LPS)-induced interleukin 6 (IL-6) release. Because both compounds have been reported to taste bitter, we hypothesized an involvement of human bitter taste sensing receptors (TAS2Rs) on IL-6 release in LPS-treated human gingival fibroblasts (HGF-1). First, the bitter taste intensity of RSV and RA was compared in a sensory trial with 10 untrained panelists, of whom 90% rated a 50 ppm of RSV in water solution more bitter than 50 ppm of RA. A mean 19 ± 6% reduction of the RSV-induced bitter taste intensity was achieved by co-administration of 50 ppm of the bitter-masking, TAS2R43 antagonist homoeriodictyol (HED). Mechanistic experiments in a stably CRISPR-Cas9-edited TAS2R43ko gastric cell model revealed involvement of TAS2R43 in the HED-evoked effect on RSV-induced proton secretion, whereas the cellular response to RSV did not depend upon TAS2R43 . Next, the IL-6 modulatory effect of 100 μM RSV was studied in LPS-treated immune-competent HGF-1 cells. After 6 h of treatment, RSV reduced the LPS-induced IL-6 gene expression and protein release by -46.2 ± 12.7 and -73.9 ± 2.99%, respectively. This RSV-evoked effect was abolished by co-administration of HED. Because real-time quantitative polymerase chain reaction analyses revealed a regulation of TAS2R50 in RSV with or without HED-treated HGF-1 cells, an siRNA knockdown approach of TAS2R50 was applied to verify TAS2R50 involvement in the RSV-induced reduction of the LPS-evoked IL-6 release in HGT-1 cells.

The typical bitter taste of beer is caused by adding hops ( Humulus lupulus L.) during the wort boiling process. The bitter taste of hop-derived compounds was found to be mediated by three bitter taste receptors: TAS2R1, TAS2R14, and TAS2R40. In this work, structural bioinformatics analyses were used to characterize the binding modes of trans -isocohumulone, trans -isohumulone, trans -isoadhumulone, cis -isocohumulone, cis -isohumulone, cis -isoadhumulone, cohumulone, humulone, adhumulone, and 8-prenylnaringenin into the orthosteric binding site of their cognate receptors. A conserved asparagine in transmembrane 3 was found to be essential for the recognition of hop-derived compounds, whereas the surrounding residues in the binding site of the three receptors encode the ligand specificity. Hop-derived compounds are renowned bioactive molecules and are considered as potential hit molecules for drug discovery to treat metabolic diseases. A chemoinformatics analysis revealed that hop-derived compounds cluster in a different region of the chemical space compared to known bitter food-derived compounds, pinpointing hop-derived compounds as a very peculiar class of bitter compounds.

TAS2R14 is of great potential as a therapeutic target against asthma, and the discovery of TAS2R14 agonists can be very valuable for treating this disease. Herein, we developed a strategy using virtual screening and affinity screening based on a fabricated biosensor combined with UPLC-MS analysis to screen TAS2R14 agonists from Platycodon grandiflorum. By ligand-based virtual screening, 16 best-fit candidates were yielded. A novel TAS2R14-functionalized high-electron-mobility transistor (HEMT) sensor was applied to detect and fish out the potential TAS2R14 agonists from P. grandiflorum extracts. Those components captured by the immobilized TAS2R14 were eluted and characterized on UPLC-QTOF MS. As a result, six potential TAS2R14 agonists were screened out and identified. Among them, platycodin L was confirmed to be a special agonist of TAS2R14 for the first time and had an EC 50 of 15.03 ± 1.15 μM via intracellular calcium mobilization assay ( n = 6). The results indicated that the proposed strategy was efficient to discover TAS2R14 agonists from the herb directly.

Polyphenols may contribute directly to plant-based foodstuffs flavor, in particular to astringency and bitterness. In this work, the bitterness of a small library of polyphenols from different classes [procyanidin dimers type B, ellagitannins (punicalagin, castalagin, and vescalagin) and phenolic acid ethyl esters (protocatechuic, ferulic, and vanillic acid ethyl esters] was studied by a cell-based assay. The bitter taste receptors (TAS2Rs) activated by these polyphenols and the half-maximum effective concentrations (EC 50 ) of each agonist-TAS2Rs pair were determined. Computational methodologies were used to understand the polyphenol molecular region responsible for receptor activation and to get insights into the type of bonds established in the agonist-TAS2Rs pairs. The results show the combinatorial pattern of TAS2Rs activation. TAS2R5 seems to be the only receptor exhibiting a bias toward the activation by condensed tannins, while TAS2R7 seems more tuned for hydrolyzable (ellagi)tannins. Additionally, at the concentrations usually found for these compounds in foodstuffs, they can actively contribute to bitter taste, especially ellagitannins.

The aim of this work was to determine the T2R4 bitter taste receptor-blocking ability of enzymatic beef protein hydrolysates and identified peptide sequences. Beef protein was hydrolyzed with each of six commercial enzymes (alcalase, chymotrypsin, trypsin, pepsin, flavourzyme, and thermoase). Electronic tongue measurements showed that the hydrolysates had significantly ( p < 0.05) lower bitter scores than quinine. Addition of the hydrolysates to quinine led to reduced bitterness intensity of quinine with trypsin and pepsin hydrolysates being the most effective. Addition of the hydrolysates to HEK293T cells that heterologously express one of the bitter taste receptors (T2R4) showed alcalase, thermoase, pepsin, and trypsin hydrolysates as the most effective in reducing calcium mobilization. Eight peptides that were identified from the alcalase and chymotrypsin hydrolysates also suppressed quinine-dependent calcium release from T2R4 with AGDDAPRAVF and ETSARHL being the most effective. We conclude that short peptide lengths or the presence of multiple serine residues may not be desirable structural requirements for blocking quinine-dependent T2R4 activation.

Sensory studies showed the volatile fraction of lemon grass and its main constituent, the odor-active citronellal, to significantly decrease the perceived bitterness of a black tea infusion as well as caffeine solutions. Seven citronellal-related derivatives were synthesized and shown to inhibit the perceived bitterness of caffeine in a structure-dependent manner. The aldehyde function at carbon 1, the ( R)-configuration of the methyl-branched carbon 3, and a hydrophobic carbon chain were found to favor the bitter inhibitory activity of citronellal; for example, even low concentrations of 25 ppm were observed to reduce bitterness perception of caffeine solution (6 mmol/L) by 32%, whereas ( R)-citronellic acid (100 pm) showed a reduction of only 21% and ( R)-citronellol (100 pm) was completely inactive. Cell-based functional experiments, conducted with the human bitter taste receptors TAS2R7, TAS2R10, TAS2R14, TAS2R43, and TAS2R46 reported to be sensitive to caffeine, revealed ( R)-citronellal to completely block caffeine-induced calcium signals in TAS2R43-expressing cells, and, to a lesser extent, in TAS2R46-expressing cells. Stimulation of TAS2R43-expressing cells with structurally different bitter agonists identified ( R)-citronellal as a general allosteric inhibitor of TAS2R43. Further structure/activity studies indicated 3-methyl-branched aliphatic aldehydes with a carbon chain of ≥4 C atoms as best TAS2R43 antagonists. Whereas odor-taste interactions have been mainly interpreted in the literature to be caused by a central neuronal integration of odors and tastes, rather than by peripheral events at the level of reception, the findings of this study open up a new dimension regarding the interaction of the two chemical senses.

Stevia is one of the sweeteners with the greatest consumer demand because of its natural origin and minimal calorie content. Steviol glycosides (SG) are the main active compounds present in the leaves of Stevia rebaudiana and are responsible for its sweetness. However, recent in vitro studies in HEK 293 cells revealed that SG specifically activate the hT2R4 and hT2R14 bitter taste receptors, triggering this mouth feel. The objective of this study was to characterize the interaction of SG with these two receptors at the molecular level. The results showed that SG have only one site for orthosteric binding to these receptors. The binding free energy (ΔG binding ) between the receptor and SG was negatively correlated with SG bitterness intensity, for both hT2R4 (r = -0.95) and hT2R14 (r = -0.89). We also determined, by steered molecular dynamics simulations, that the force required to extract stevioside from the receptors was greater than that required for rebaudioside A, in accordance with the ΔG values obtained by molecular docking. Finally, we identified the loop responsible for the activation by SG of both receptors. As a whole, these results contribute to a better understanding of the resulting off-flavor perception of these natural sweeteners in foods and beverages, allowing for better prediction, and control, of the resulting bitterness.

Bitter taste receptors (Tas2rs) and downstream effectors are responsible for mediating bitterness perception and regulation of food choice in mammals. Using RT-PCR, we demonstrated the expression of three Tas2rs and taste signal transduction molecules, α-gustducin, PLCβ2, and TRPM5, in the palate, tongue, and gastrointestinal tract sections in chicken. The bitter tastant quinine activates all three chicken Tas2rs in vitro as shown using calcium-imaging assays of transfected cells. Administration of quinine postnatally or perinatally (both pre- and posthatch) to chickens increased the expression of Tas2r genes in the palate by 6.45-fold (ggTas2r1 postnatal treatment), 4.86-fold (ggTas2r1 perinatal treatment), and 4.48-fold (ggTas2r7 postnatal treatment) compared to the genes' expression in the naı̈ve group respectively, and affected taste related gene expression in the duodenum. Whereas no-choice intake of quinine solution was not significantly lower than that of water in naı̈ve chicks, the treatment groups postnatal, prenatal, and perinatal showed significantly lower intake of quinine by 56.1, 47.7, and 50.2%, respectively, suggesting a possible trend toward sensitization. These results open new venues toward unraveling the formative stages shaping food intake and nutrition in chicken.

Many flavonoids and isoflavonoids have an undesirable bitter taste, which hampers their use as food bioactives. The aim of this study was to investigate the effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 and to predict their structural requirements to activate these receptors. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, among which 58 ligands were overlapping. Their activation threshold values varied over a range of 3 log units between 0.12 and 500 μM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure-activity relationships. The 2D models demonstrated excellent predictive power in identifying bitter (iso)flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 (or hTAS2R39) were determined by 3D-pharmacophore models to be composed of two (or three) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. The additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behaviors on the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)flavonoids and provided a powerful tool in the prediction of their potential bitterness. By understanding the "bitter motif", introduction of bitter taste in functional foods enriched in (iso)flavonoid bioactives might be avoided.

Amino acids and peptides represent important flavor molecules eliciting various taste sensations. Here, we present a comprehensive assessment of the interaction of various peptides and all proteinogenic amino acids with the 25 human TAS2Rs expressed in cell lines. L-Phenylalanine and L-tryptophan activate TAS2R1 and TAS2R4, respectively, whereas TAS2R4 and TAS2R39 responded to D-tryptophan. Structure-function analysis uncovered the basis for the lack of stereoselectivity of TAS2R4. The same three TAS2Rs or subsets thereof were also sensitive to various dipeptides containing L-tryptophan, L-phenylalanine, or L-leucine and to Trp-Trp-Trp, whereas Leu-Leu-Leu specifically activated TAS2R4. Trp-Trp-Trp also activated TAS2R46 and TAS2R14. Two key bitter peptides from Gouda cheese, namely, Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser and Leu-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn, both activated TAS2R1 and TAS2R39. Thus, the data demonstrate that the bitterness of amino acids and peptides is not mediated by specifically tuned TAS2Rs but rather is brought about by an unexpectedly complex pattern of sensitive TAS2Rs.

Steviol glycosides, the sweet principle of Stevia Rebaudiana (Bertoni) Bertoni, have recently been approved as a food additive in the EU. The herbal non-nutritive high-potency sweeteners perfectly meet the rising consumer demand for natural food ingredients in Europe. We have characterized the organoleptic properties of the most common steviol glycosides by an experimental approach combining human sensory studies and cell-based functional taste receptor expression assays. On the basis of their potency to elicit sweet and bitter taste sensations, we identified glycone chain length, pyranose substitution, and the C16 double bond as the structural features giving distinction to the gustatory profile of steviol glycosides. A comprehensive screening of 25 human bitter taste receptors revealed that two receptors, hTAS2R4 and hTAS2R14, mediate the bitter off-taste of steviol glycosides. For some test substances, e.g., stevioside, we observed a decline in sweet intensity at supra-maximum concentrations. This effect did not arise from allosteric modulation of the hTAS1R2/R3 sweet taste receptor but might be explained by intramolecular cross-modal suppression between the sweet and bitter taste component of steviol glycosides. These results might contribute to the production of preferentially sweet and least bitter tasting Stevia extracts by an optimization of breeding and postharvest downstream processing.

The aim of this study was to identify the bitter receptor(s) that recognize the bitter taste of the soy isoflavone genistein. Screening of all 25 human bitter receptors revealed genistein as agonist of hTAS2R14 and hTAS2R39. Genistein displayed threshold values of 4 and 8 μM on hTAS2R14 and hTAS2R39 and EC(50) values of 29 and 49 μM, respectively. In addition, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar toward most isoflavonoid aglycones. By trend, threshold values were slightly lower on hTAS2R14. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely, glycitin, genistin, acetylgenistin, and malonylgenistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than fewer hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the determined bitter receptor threshold values, whereas those of other soy isoflavones are around or below their respective threshold value. Despite its low concentration, genistein might be one of the main contributors to the bitterness of soy products. Furthermore, the bioactive isoflavonoids equol and coumestrol activated both receptors, indicating that their sensory impact should be considered when used as food ingredients.

Bitterness perception in mammals is mediated through activation of dedicated bitter taste receptors located in the oral cavity. Genomic analyses revealed the existence of orthologous mammalian bitter taste receptor genes, which presumably recognize the same compounds in different species, as well as species-specific receptor gene expansions believed to fulfill a critical role during evolution. In man, 8 of the 25 bitter taste receptors (hTAS2Rs) are closely related members of such an expanded subfamily of receptor genes. This study identified two natural bitter terpenoids, andrographolide and amarogentin, that are agonists for the orphan receptor hTAS2R50, the most distant member of the subfamily. This paper presents the pharmacological characterization of this receptor and analyzes its functional relationship with the previously deorphaned hTAS2R43, hTAS2R44, hTAS2R46, and hTAS2R47. Insights into the general breadth of tuning, functional redundancies, and relationships between pharmacological activation patterns and amino acid homologies for this receptor subfamily are presented.

Some acidic peptides are known to reduce bitterness, but the detailed mechanism underlying this effect remains to be elucidated. In this study, we analyzed the effects of acidic dipeptides on the inhibition of the human bitter taste receptor hTAS2R16. Calcium imaging analysis of HEK293T cells expressing hTAS2R16 revealed that their response to the bitter tastant salicin was reduced in the presence of acidic dipeptides. A similar inhibitory effect was observed in a variety of other acids. The inhibition depended on the pH values resulting from the addition of acids but not on their concentrations. Our results suggest that the inhibition of the bitter taste receptors can be attributed to the bitterness-masking effect of the acidic dipeptides and that acidic pH may be one of the critical factors responsible for this sensory event.

Sesquiterpene lactones are a major class of natural bitter compounds occurring in vegetables and culinary herbs as well as in aromatic and medicinal plants, where they often represent the main gustatory and pharmacologically active component. Investigations on sesquiterpene lactones have mainly focused on their bioactive potential rather than on their sensory properties. In the present study, we report about the stimulation of heterologously expressed human bitter taste receptors, hTAS2Rs, by the bitter sesquiterpene lactone herbolide D. A specific response to herbolide D was observed i.a. for hTAS2R46, a so far orphan bitter taste receptor without any known ligand. By further investigation of its agonist pattern, we characterized hTAS2R46 as a bitter receptor broadly tuned to sesquiterpene lactones and to clerodane and labdane diterpenoids as well as to the unrelated bitter substances strychnine and denatonium.