GWAS of human bitter taste perception identifies new loci and reveals additional complexity of bitter taste genetics.

Human perception of bitterness displays pronounced interindividual variation. This phenotypic variation is mirrored by equally pronounced genetic variation in the family of bitter taste receptor genes. To better understand the effects of common genetic variations on human bitter taste perception, we conducted a genome-wide association study on a discovery panel of 504 subjects and a validation panel of 104 subjects from the general population of São Paulo in Brazil. Correction for general taste-sensitivity allowed us to identify a SNP in the cluster of bitter taste receptors on chr12 (10.88- 11.24 Mb, build 36.1) significantly associated (best SNP: rs2708377, P = 5.31 × 10(-13), r(2) = 8.9%, β = -0.12, s.e. = 0.016) with the perceived bitterness of caffeine. This association overlaps with-but is statistically distinct from-the previously identified SNP rs10772420 influencing the perception of quinine bitterness that falls in the same bitter taste cluster. We replicated this association to quinine perception (P = 4.97 × 10(-37), r(2) = 23.2%, β = 0.25, s.e. = 0.020) and additionally found the effect of this genetic locus to be concentration specific with a strong impact on the perception of low, but no impact on the perception of high concentrations of quinine. Our study, thus, furthers our understanding of the complex genetic architecture of bitter taste perception.

Identification of a bitter-taste receptor gene repertoire in different Lagomorphs species

The repertoires of bitter-taste receptor (T2R) gene have been described for several animal species, but these data are still scarce for Lagomorphs. The aim of the present work is to identify potential repertoires of T2R in several Lagomorph species, covering a wide geographical distribution. We studied these genes in Lepus timidus, L. europaeus, Oryctolagus cuniculus algirus, Romerolagus diazi, and Sylvilagus floridanus, using O. cuniculus cuniculus as control species for PCR and DNA sequencing. We studied the identities of the DNA sequences and built the corresponding phylogenetic tree. Sequencing was successful for both subspecies of O. cuniculus for all T2R genes studied, for five genes in Lepus, and for three genes in R. diazi and S. floridanus. We describe for the first time the partial repertoires of T2R genes for Lagomorphs species, other than the common rabbit. Our phylogenetic analyses indicate that sequence proximity levels follow the established taxonomic classification.

Taste receptors in the gut: a chemosensitive mechanism from fish to human

The ingestion of a meal evokes a series of digestive processes, which consist of the essential functions of the digestive system: food transport, secretory activity, absorption of nutrients and the expulsion of undigested residues do not absorbed. The gastrointestinal chemosensitivity is characterized by cellular elements of the endocrine gastrointestinal mucosa and nerve fibers, in particular of vagal nature. A wide range of mediators endocrine and/or paracrine can be released from various endocrine cells in response to nutrients in the diet. These hormones, in addition to their direct activity, act through specific receptors activating some of the most important functions in the control of energy intake and energy homeostasis in the body. For integration of this complex system of control of gastrointestinal chemosensitivity, recent evidence demonstrates the presence of taste receptors (TR) belonging to the family of G proteins coupled receptor expressed in the mucosa of the gastrointestinal tract of different mammals and human. This thesis is divided into several research projects that have been conceived in order to clarify the relationship between TR and nutrients. To define this relationship I have used various scientific approaches, which have gone on to evaluate changes in signal molecules of TR, in particular of the α-transducin in the fasting state and after refeeding with standard diet in the gastrointestinal tract of the pig, the mapping of the same molecule signal in the gastrointestinal tract of fish (Dicentrarchus labrax), the signaling pathway of bitter TR in the STC-1 endocrine cell line and finally the involvement of bitter TR in particular of T2R38 in patients with an excessive caloric intake. The results showed how there is a close correlation between nutrients, TR and hormonal release and how they are useful both in taste perception but also likely to be involved in chronic diseases such as obesity.

Sequence analysis of bitter taste receptor gene repertoires in different ruminant species

Bitter taste has been extensively studied in mammalian species and is associated with sensitivity to toxins and with food choices that avoid dangerous substances in the diet. At the molecular level, bitter compounds are sensed by bitter taste receptor proteins (T2R) present at the surface of taste receptor cells in the gustatory papillae. Our work aims at exploring the phylogenetic relationships of T2R gene sequences within different ruminant species. To accomplish this goal, we gathered a collection of ruminant species with different feeding behaviors and for which no genome data is available: American bison, chamois, elk, European bison, fallow deer, goat, moose, mouflon, muskox, red deer, reindeer and white tailed deer. The herbivores chosen for this study belong to different taxonomic families and habitats, and hence, exhibit distinct foraging behaviors and diet preferences. We describe the first partial repertoires of T2R gene sequences for these species obtained by direct sequencing. We then consider the homology and evolutionary history of these receptors within this ruminant group, and whether it relates to feeding type classification, using MEGA software. Our results suggest that phylogenetic proximity of T2R genes corresponds more to the traditional taxonomic groups of the species rather than reflecting a categorization by feeding strategy.

Astringent Food Compounds and Their Interactions with Taste Properties

Astringency is traditionally thought to be induced by plant tannins in foods. Because of this current research concerning the mechanism of astringency is focused on tannin‐protein interactions and thus on precipitation, which may be perceived by mechanoreceptors. However, astringency is elicited by a wide range of different phenolic compounds, as well as, some non‐phenolic compounds in various foods. Many ellagitannins or smaller compounds that contribute to astringent properties do not interact with salivary proteins and may be directly perceived through some receptors. Generally, the higher degree of polymerization of proanthocyanidins can be associated with more intense astringency. However, the astringent properties of smaller phenolic compounds may not be directly predicted from the structure of a compound, although glycosylation has a significant role. The astringency of organic acids may be directly linked to the perception of sourness, and this increases along with decreasing pH. Astringency can be divided into different sub‐qualities, including even other qualities than traditional mouth‐drying, puckering or roughing sensations. Astringency is often accompanied by bitter or sour or both taste properties. The different sub‐qualities can be influenced by different astringent compounds. In general, the glycolysation of the phenolic compound results in more velvety and smooth mouthdrying astringency. Flavonol glycosides and other flavonoid compounds and ellagitannins contribute to this velvety mouthdrying astringency. Additionally, they often lack the bitter properties. Proanthocyanidins and phenolic acids elicit more puckering and roughing astringency with some additional bitter properties. Quercetin 3‐O‐rutinoside, along with other quercetin glycosides, is among the key astringent compounds in black tea and red currants. In foods, there are always various other additional attributes that are perceived at the same with astringency. Astringent compounds themselves may have other sensory characteristics, such as bitter or sour properties, or they may enhance or suppress other sensory properties. Components contributing to these other properties, such as sugars, may also have similar effects on astringent sensations. Food components eliciting sweetness or fattiness or some polymeric polysaccharides can be used to mask astringent subqualities. Astringency can generally be referred to as a negative contributor to the liking of various foods. On the other hand, perceptions of astringent properties can vary among individuals. Many genetic factors that influence perceptions of taste properties, such as variations in perceiving a bitter taste or variations in saliva, may also effect the perception of astringency. Individuals who are more sensitive to different sensations may notice the differences between astringent properties more clearly. This may not have effects on the overall perception of astringency. However, in many cases, the liking of astringent foods may need to be learned by repetitive exposure. Astringency is often among the key sensory properties forming the unique overall flavour of certain foods, and therefore it also influences whether or not a food is liked. In many cases, astringency may be an important sub‐property suppressed by other more abundant sensory properties, but it may still have a significant contribution to the overall flavour and thus consumer preferences. The results of the practical work of this thesis show that the astringent phenolic compounds are mostly located in the skin fractions of black currants, crowberries and bilberries (publications I–III). The skin fractions themselves are rather tasteless. However, the astringent phenolic compounds can be efficiently removed from these skin fractions by consecutive ethanol extractions. Berries contain a wide range of different flavonol glycosides, hydroxycinnamic acid derivatives and anthocyanins and some of them strongly contribute to the different astringent and bitterness properties. Sweetness and sourness are located in the juice fractions along with the majority of sugars and fruit acids. The sweet and sour properties of the juice may be used to mask the astringent and bitterness properties of the extracts. Enzymatic treatments increase the astringent properties and fermented flavour of the black currant juice and decrease sweetness and freshness due to the effects on chemical compositions (IV). Sourness and sweetness are positive contributors to the liking of crowberry and bilberry fractions, whereas bitterness is more negative (V). Some astringent properties in berries are clearly negative factors, whereas some may be more positive. The liking of berries is strongly influenced by various consumer background factors, such as motives and health concerns. The liking of berries and berry fractions may also be affected by genetic factors, such as variations in the gene hTAS2R38, which codes bitter taste receptors (V).

Exploring the effects of genotypical and phenotypical variations in bitter taste sensitivity on perception, liking and intake of brassica vegetables in the UK

Brassicaceous vegetables (BV) have chemoprotective effects and yet consumption of BV in the UK is low. Previous studies suggest perception, liking and intake of BV are influenced by bitter taste sensitivity which this study further explores. Phenotypical taste sensitivity of 136 subjects was classified using propythiouracil (PROP) and sodium chloride and fungiform papillae density (FPD) was measured from tongue images. Polymorphisms of TAS2R38 and gustin (CA6) genes were analysed. Liking and bitterness of four raw vegetables (two BV (broccoli and white cabbage) and two non-BV (spinach and courgette)), as well as habitual consumption, were evaluated. There was a significant association between TAS2R38 genotype and PROP taster status (p<0.0001) and between FPD and PROP taster status (p=0.029). Individuals with greater sensitivity for PROP predominantly had TAS2R38 PAV/PAV genotype and greater FPD. BV were perceived as more bitter than non-BV (p<0.0001) with PAV/PAV subjects perceiving significantly stronger bitter intensity. There was a significant difference in liking for the four vegetables (p=0.002), and between consumers of different TAS2R38 genotype (p=0.0024). Individuals with TAS2R38 AVI/AVI genotype liked BV more. Regarding intake, both PAV/PAV and AVI/AVI individuals consumed more total vegetables and BV than PAV/AVI. Although PROP nontasters tended to consume more vegetables and BV than the other two phenotype groups, liking and vegetable intake were not significantly affected by taste phenotype. Although there was not a significant effect of CA6 genotype on bitterness ratings, there was a significant interaction between CA6 and TAS2R38, and in addition CA6 genotype was significantly associated with BV intake. However, these effects require validation as the proportions of the population with the CA6 G/G genotype was extremely small (7%). Our results confirmed that bitter taste perception in vegetables was influenced by both genotype and phenotype of bitter taste sensitivity. Moreover, our findings demonstrated that neither genotype nor phenotype of taste sensitivity alone accurately predict vegetable liking and intake as demographic factors were found to have a substantial influence.

The use of spray drying technology to reduce bitter taste of casein hydrolysate

The utilization of protein hydrolysates in food systems is frequently hindered due to their bitterness and hygroscopicity. Spray drying technology could be an alternative for reducing these problems. The aim of this work was to reduce or to mask the casein hydrolysate bitter taste using spray drying and mixtures of gelatin and soy protein isolate (SPI) as carriers. Six formulations were studied: three with 20% of hydrolysate and 80% of mixture (gelatine/SPI at proportions of 50/50, 40/60 and 60/40%) and three with 30% of hydrolysate and 70% of mixture (gelatine/SPI at proportions of 50/50, 40/60 and 60/40%). The spray-dried formulations were evaluated by SEM, hygroscopicity, thermal behavior (DSC), dissolution, and bitter taste, by a trained sensory panel using a paired-comparison test (free samples vs. spray-dried samples); all samples were presented in powder form. SEM analysis showed mostly spherically shaped particles, with many concavities and some particles with pores. All formulations were oil and water compatible and showed lower hygroscopicity values than free casein hydrolysate. At Aw 0.83, the free hydrolysate showed Tg about 25 degrees C lower than the formulations, indicating that the formulations may be more stable at Aw >= 0.65 since the glass transition should be prevented. The sensory panel found the formulations, tasted in the powder form, to be less bitter (P < 0.05) than the free casein hydrolysate. These results indicated that spray drying of casein hydrolysate with mixtures of gelatin and SPI was successful to attenuate the bitterness of casein hydrolysate. Thus, spray drying widens the possibilities of application of casein hydrolysates. (C) 2009 Elsevier Ltd. All rights reserved.

Differential Evolutionary Constraints in the Evolution of Chemoreceptors: A Murine and Human Case Study

Chemoreception is among the most important sensory modalities in animals. Organisms use the ability to perceive chemical compounds in all major ecological activities. Recent studies have allowed the characterization of chemoreceptor gene families. These genes present strikingly high variability in copy numbers and pseudogenization degrees among different species, but the mechanisms underlying their evolution are not fully understood. We have analyzed the functional networks of these genes, their orthologs distribution, and performed phylogenetic analyses in order to investigate their evolutionary dynamics. We have modeled the chemosensory networks and compared the evolutionary constraints of their genes in Mus musculus, Homo sapiens, and Rattus norvegicus. We have observed significant differences regarding the constraints on the orthologous groups and network topologies of chemoreceptors and signal transduction machinery. Our findings suggest that chemosensory receptor genes are less constrained than their signal transducing machinery, resulting in greater receptor diversity and conservation of information processing pathways. More importantly, we have observed significant differences among the receptors themselves, suggesting that olfactory and bitter taste receptors are more conserved than vomeronasal receptors.

Ric-8A, a G alpha protein guanine nucleotide exchange factor potentiates taste receptor signaling

Taste receptors for sweet, bitter and umami tastants are G-protein-coupled receptors (GPCRs). While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS), RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs) are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of G alpha subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with G alpha-gustducin and G alpha i2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

Genetic Analysis of Taste in Homo Sapiens

There are many known taste receptors specific to each taste attribute. This thesis examines the relationship between single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) in known taste and taste pathway receptors TAS2R38, Gustin, and TRPM5 and for PROP (6-n-propylthiouracil) taster status (PTS), thermal taster status (TTS), and orosensory sensation intensity ratings. PTS is a proxy for general taste responsiveness, and the ability to taste PROP classifies individuals into three phenotypes: super (PST), medium (PMT), and non-tasters (PNT). Another taste phenotype, also serving as a proxy for general taste responsiveness, is TTS, classifying individuals as thermal tasters (TTs) or thermal non-tasters (TnTs). DNA extractions from buccal cells obtained from 60 individuals were performed and analysis of TAS2R38, Gustin, and TRPM5 variations were conducted through Polymerase Chain Reaction (PCR), sequencing for SNPs, and upQMPSF for CNV analysis of TRPM5. Among the SNPs and CNVs studied, only TAS2R38 was found to be significantly associated with PTS and intensity ratings for sweet, bitter, and sour taste as well as astringency. However, not all PROP phenotypic differences can be explained by the variations at these three SNP sites in TAS2R38, suggesting the involvement of additional genes. No association was found between TTS and TAS2R38 or Gustin, confirming that PTS and TTS are not genetically associated. The examined TRPM5 SNPs and CNVs did not correlate with TTS. Therefore, further research is necessary into other factors contributing to PTS and TTS.

Dominant loss of responsiveness to sweet and bitter compounds caused by a single mutation in α-gustducin

Biochemical and genetic studies have implicated α-gustducin as a key component in the transduction of both bitter or sweet taste. Yet, α-gustducin-null mice are not completely unresponsive to bitter or sweet compounds. To gain insights into how gustducin mediates responses to bitter and sweet compounds, and to elicit the nature of the gustducin-independent pathways, we generated a dominant-negative form of α-gustducin and expressed it as a transgene from the α-gustducin promoter in both wild-type and α-gustducin-null mice. A single mutation, G352P, introduced into the C-terminal region of α-gustducin critical for receptor interaction rendered the mutant protein unresponsive to activation by taste receptor, but left its other functions intact. In control experiments, expression of wild-type α-gustducin as a transgene in α-gustducin-null mice fully restored responsiveness to bitter and sweet compounds, formally proving that the targeted deletion of the α-gustducin gene caused the taste deficits of the null mice. In contrast, transgenic expression of the G352P mutant did not restore responsiveness of the null mice to either bitter or sweet compounds. Furthermore, in the wild-type background, the mutant transgene inhibited endogenous α-gustducin's interactions with taste receptors, i.e., it acted as a dominant-negative. That the mutant transgene further diminished the residual bitter and sweet taste responsiveness of the α-gustducin-null mice suggests that other guanine nucleotide-binding regulatory proteins expressed in the α-gustducin lineage of taste cells mediate these responses.

Analytical investigation of chromium and zinc in sweet, sour and bitter tasting fruits, vegetables and medicinal plants

Sweet, sour and bitter tasting fruits, vegetables and medicinal plants are an important component of human diet. The role of chromium and zinc in carbohydrate metabolism for control of diabetes is highlighted in selected commodities. Average levels of chromium and zinc in sweet taste were 0.69 ± 0.48 mg kg-1 and 4.81 ± 4.31 mg kg-1 respectively with correlation of 0.545, while in sour taste the values were 22.5 ± 22.0 mg kg-1 and 24.5 ± 11.8 mg kg-1 respectively with the correlation of 0.239 and in bitter taste, 0.61 ± 0.33 mg kg-1 and 4.70 ± 3.54 mg kg-1 respectively with correlation of 0.343. Overall, sour tasting commodities were found higher in levels of chromium and zinc and are recommended as food supplement for diabeties. None of these species contain metals above the toxic level.

Comparison of a trained sensory panel and an electronic tongue in the assessment of bitter dairy protein hydrolsates

The bitter taste elicited by dairy protein hydrolysates (DPH) is a renowned issue for their acceptability by consumers and therefore incorporation into foods. The traditional method of assessment of taste in foods is by sensory analysis but this can be problematic due to the overall unpleasantness of the samples. Thus, there is a growing interest into the use of electronic tongues (e-tongues) as an alternative method to quantify the bitterness in such samples. In the present study the response of the e-tongue to the standard bitter agent caffeine and a range of both casein and whey based hydrolysates was compared to that of a trained sensory panel. Partial least square regression (PLS) was employed to compare the response of the e-tongue and the sensory panel. There was strong correlation shown between the two methods in the analysis of caffeine (R2 of 0.98) and DPH samples with R2 values ranging from 0.94-0.99. This study exhibits potential for the e-tongue to be used in bitterness screening in DPHs to reduce the reliance on expensive and time consuming sensory panels.

Reactivity of Beer Bitter Acids Toward the 1-Hydroxyethyl Radical as Probed by Spin-Trapping Electron Paramagnetic Resonance (EPR) and Electrospray Ionization-Tandem Mass Spectrometry (ESI-MS/MS)

The iso-alpha-acids or isohumulones are the major contributors to the bitter taste of beer, and it is well-recognized that they are degraded during beer aging. In particular, the trans-isohumulones seem to be less stable than the cis-isohumulones. The major radical identified in beer is the 1-hydroxyethyl radical; however, the reactivity between this radical and the isohumulones has not been reported until now. Therefore, we studied the reactivity of isohumulones toward the 1-hydroxyethyl radical through a competitive kinetic approach. It was observed that both cis- and trans-isohumulones and dihydroisohumulones are decomposed in the presence of 1-hydroxyethyl radicals, while the reactivities are comparable. On the other hand, the tetrahydroisohumulones did not react with 1-hydroxyethyl radicals. The apparent second-order rate constants for the reactions between the 1-hydroxyethyl radical and these compounds were determined by electron paramagnetic resonance (EPR) spectroscopy and electrospray ionization-tandem mass spectrometry [ESI(+)-MS/MS]. It follows that degradation of beer bitter acids is highly influenced by the presence of 1-hydroxyethyl radicals. The reaction products were detected by liquid chromatography electrospray ionization-ion trap-tandem mass spectrometry (LC-ESI-IT-MS/MS), and the formation of oxidized derivatives of the isohumulones was confirmed. These data help to understand the mechanism of beer degradation upon aging.