Comprehensive metabolome characterization of leaves, internodes, and aerial roots of Vanilla planifolia by untargeted LC-MS and GC × GC-MS.

INTRODUCTION: Untargeted metabolomics is a powerful tool that provides strategies for gaining a systematic understanding of quantitative changes in the levels of metabolites, especially when combining different metabolomic platforms. Vanilla is one of the world's most popular flavors originating from cured pods of the orchid Vanilla planifolia. However, only a few studies have investigated the metabolome of V. planifolia, and no LC-MS or GC-MS metabolomics studies with respect to leaves have been performed. OBJECTIVE: The aim of the study was to comprehensively characterize the metabolome of different organs (leaves, internodes, and aerial roots) of V. planifolia. MATERIAL AND METHODS: Characterization of the metabolome was achieved using two complementary platforms (GC × GC-MS, LC-QToF-MS), and metabolite identification was based on a comparison with in-house databases or curated external spectral libraries. RESULTS: In total, 127 metabolites could be identified with high certainty (confidence level 1 or 2) including sugars, amino acids, fatty acids, organic acids, and amines/amides but also secondary metabolites such as vanillin-related metabolites, flavonoids, and terpenoids. Ninty-eight metabolites showed significantly different intensities between the plant organs. Most strikingly, aglycons of flavonoids and vanillin-related metabolites were elevated in aerial roots, whereas its O-glycoside forms tended to be higher in leaves and/or internodes. This suggests that the more bioactive aglycones may accumulate where preferably needed, e.g. for defense against pathogens. CONCLUSION: The results derived from the study substantially expand the knowledge regarding the vanilla metabolome forming a valuable basis for more targeted investigations in future studies, e.g. towards an optimization of vanilla plant cultivation.

An enzymatic tandem reaction to produce odor-active fatty aldehydes.

Aldehydes represent a versatile and favored class of flavoring substances. A biocatalytic access to odor-active aldehydes was developed by conversion of fatty acids with two enzymes of the α-dioxygenase pathway. The recombinant enzymes α-dioxygenase (α-DOX) originating from Crocosphaera subtropica and fatty aldehyde dehydrogenase (FALDH) from Vibrio harveyi were heterologously expressed in E. coli, purified, and applied in a coupled (tandem) repetitive reaction. The concept was optimized in terms of number of reaction cycles and production yields. Up to five cycles and aldehyde yields of up to 26% were achieved. Afterward, the approach was applied to sea buckthorn pulp oil as raw material for the enzyme catalyzed production of flavoring/fragrance ingredients based on complex aldehyde mixtures. The most abundant fatty acids in sea buckthorn pulp oil, namely palmitic, palmitoleic, oleic, and linoleic acid, were used as substrates for further biotransformation experiments. Various aldehydes were identified, semi-quantified, and sensorially characterized by means of headspace-solid phase microextraction-gas chromatography-mass spectrometry-olfactometry (HS-SPME-GC-MS-O). Structural validation of unsaturated aldehydes in terms of double-bond positions was performed by multidimensional high-resolution mass spectrometry experiments of their Paternò-Büchi (PB) photoproducts. Retention indices and odor impressions of inter alia (Z,Z)-5,8-tetradecadienal (Z,Z)-6,9-pentadecadienal, (Z)-8-pentadecenal, (Z)-4-tridecenal, (Z)-6-pentadecenal, and (Z)-8-heptadecenal were determined for the first time. KEY POINTS: • Coupled reaction of Csα-DOX and VhFALDH yields chain-shortened fatty aldehydes. • Odors of several Z-unsaturated fatty aldehydes are described for the first time. • Potential for industrial production of aldehyde-based odorants from natural sources.

Separation of Dihydro-Isocoumarins and Dihydro-Stilbenoids from Hydrangea macrophylla ssp. serrata by Use of Counter-Current Chromatography.

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar 'Yae-no-amacha' using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data.

Discovery of Novel Bacterial Chalcone Isomerases by a Sequence-Structure-Function-Evolution Strategy for Enzymatic Synthesis of (S)-Flavanones.

Chalcone isomerase (CHI) is a key enzyme in the biosynthesis of flavonoids in plants. The first bacterial CHI (CHIera ) was identified from Eubacterium ramulus, but its distribution, evolutionary source, substrate scope, and stereoselectivity are still unclear. Here, we describe the identification of 66 novel bacterial CHIs from Genbank using a novel Sequence-Structure-Function-Evolution (SSFE) strategy. These novel bacterial CHIs show diversity in substrate specificity towards various hydroxylated and methoxylated chalcones. The mutagenesis of CHIera according to the substrate binding models of these novel bacterial CHIs resulted in several variants with greatly improved activity towards these chalcones. Furthermore, the preparative scale conversion catalyzed by bacterial CHIs has been performed for five chalcones and revealed (S)-selectivity with up to 96 % ee, which provides an alternative biocatalytic route for the synthesis of (S)-flavanones in high yields.

Caffeine induces gastric acid secretion via bitter taste signaling in gastric parietal cells.

Caffeine, generally known as a stimulant of gastric acid secretion (GAS), is a bitter-tasting compound that activates several taste type 2 bitter receptors (TAS2Rs). TAS2Rs are expressed in the mouth and in several extraoral sites, e.g., in the gastrointestinal tract, in which their functional role still needs to be clarified. We hypothesized that caffeine evokes effects on GAS by activation of oral and gastric TAS2Rs and demonstrate that caffeine, when administered encapsulated, stimulates GAS, whereas oral administration of a caffeine solution delays GAS in healthy human subjects. Correlation analysis of data obtained from ingestion of the caffeine solution revealed an association between the magnitude of the GAS response and the perceived bitterness, suggesting a functional role of oral TAS2Rs in GAS. Expression of TAS2Rs, including cognate TAS2Rs for caffeine, was shown in human gastric epithelial cells of the corpus/fundus and in HGT-1 cells, a model for the study of GAS. In HGT-1 cells, various bitter compounds as well as caffeine stimulated proton secretion, whereby the caffeine-evoked effect was (i) shown to depend on one of its cognate receptor, TAS2R43, and adenylyl cyclase; and (ii) reduced by homoeriodictyol (HED), a known inhibitor of caffeine's bitter taste. This inhibitory effect of HED on caffeine-induced GAS was verified in healthy human subjects. These findings (i) demonstrate that bitter taste receptors in the stomach and the oral cavity are involved in the regulation of GAS and (ii) suggest that bitter tastants and bitter-masking compounds could be potentially useful therapeutics to regulate gastric pH.

Identification of Interleukin-8-Reducing Lead Compounds Based on SAR Studies on Dihydrochalcone-Related Compounds in Human Gingival Fibroblasts (HGF-1 cells) In Vitro.

Background: In order to identify potential activities against periodontal diseases, eighteen dihydrochalcones and structurally related compounds were tested in an established biological in vitro cell model of periodontal inflammation using human gingival fibroblasts (HGF-1 cells). Methods: Subsequently to co-incubation of HGF-1 cells with a bacterial endotoxin (Porphyromonas gingivalis lipopolysaccharide, pgLPS) and each individual dihydrochalcone in a concentration range of 1 µM to 100 µM, gene expression of interleukin-8 (IL-8) was determined by qPCR and cellular interleukin-8 (IL-8) release by ELISA. Results: Structure-activity analysis based on the dihydrochalcone backbone and various substitution patterns at its aromatic ring revealed moieties 2',4,4',6'-tetrahydroxy 3-methoxydihydrochalcone (7) to be the most effective anti-inflammatory compound, reducing the pgLPS-induced IL-8 release concentration between 1 µM and 100 µM up to 94%. In general, a 2,4,6-trihydroxy substitution at the A-ring and concomitant vanilloyl (4-hydroxy-3-methoxy) pattern at the B-ring revealed to be preferable for IL-8 release inhibition. Furthermore, the introduction of an electronegative atom in the A,B-linker chain led to an increased anti-inflammatory activity, shown by the potency of 4-hydroxybenzoic acid N-vanillylamide (13). Conclusions: Our data may be feasible to be used for further lead structure designs for the development of potent anti-inflammatory additives in oral care products.

Biological Evaluation of Natural and Synthesized Homovanillic Acid Esters as Inhibitors of Intestinal Fatty Acid Uptake in Differentiated Caco-2 Cells.

With raising prevalence of obesity, the regulation of human body fat is increasingly relevant. The modulation of fatty acid uptake by enterocytes represents a promising target for body weight maintenance. Recent results demonstrated that the trigeminal active compounds capsaicin, nonivamide, and trans-pellitorine dose-dependently reduce fatty acid uptake in differentiated Caco-2 cells as a model for the intestinal barrier. However, non-pungent alternatives have not been investigated and structural determinants for the modulation of intestinal fatty acid uptake have not been identified so far. Thus, based on the previous results, we synthesized 23 homovanillic acid esters in addition to the naturally occurring capsiate and screened them for their potential to reduce intestinal fatty acid uptake using the fluorescent fatty acid analog Bodipy-C12 in differentiated Caco­2 cells as an enterocyte model. Whereas pre-incubation with 100 µM capsiate did not change fatty acid uptake by Caco-2 enterocytes, a maximum inhibition of -47% was reached using 100 µM 1­methylpentyl-2-(4-hydroxy-3-methoxy-phenyl)acetate. Structural analysis of the 24 structural analogues tested in the present study revealed that a branched fatty acid side chain, independent of the chain length, is one of the most important structural motifs associated with inhibition of fatty acid uptake in Caco-2 enterocytes. The results of the present study may serve as an important basis for designing potent dietary inhibitors of fatty acid uptake.

The advanced glycation end product Nϵ -carboxymethyllysine and its precursor glyoxal increase serotonin release from Caco-2 cells.

Advanced glycation end products (AGEs), comprising a highly diverse class of Maillard reaction compounds formed in vivo and during heating processes of foods, have been described in the progression of several degenerative conditions such as Alzheimer's disease and diabetes mellitus. Nϵ -Carboxymethyllysine (CML) represents a well-characterized AGE, which is frequently encountered in a Western diet and is known to mediate its cellular effects through binding to the receptor for AGEs (RAGE). As very little is known about the impact of exogenous CML and its precursor, glyoxal, on intestinal cells, a genome-wide screening using a customized microarray was conducted in fully differentiated Caco-2 cells. After verification of gene regulation by qPCR, functional assays on fatty acid uptake, glucose uptake, and serotonin release were performed. While only treatment with glyoxal showed a slight impact on fatty acid uptake (P < 0.05), both compounds reduced glucose uptake significantly, leading to values of 81.3% ± 22.8% (500 µM CML, control set to 100%) and 68.3% ± 20.9% (0.3 µM glyoxal). Treatment with 500 µM CML or 0.3 µM glyoxal increased serotonin release (P < 0.05) to 236% ± 111% and 264% ± 66%, respectively. Co-incubation with the RAGE antagonist FPS-ZM1 reduced CML-induced serotonin release by 34%, suggesting a RAGE-mediated mechanism. Similarly, co-incubation with the SGLT-1 inhibitor phloridzin attenuated serotonin release after CML treatment by 32%, hinting at a connection between CML-stimulated serotonin release and glucose uptake. Future studies need to elucidate whether the CML/glyoxal-induced serotonin release in enterocytes might stimulate serotonin-mediated intestinal motility.

Nonivamide enhances miRNA let-7d expression and decreases adipogenesis PPARγ expression in 3T3-L1 cells.

Red pepper and its major pungent principle, capsaicin (CAP), have been shown to be effective anti-obesity agents by reducing energy intake, enhancing energy metabolism, decreasing serum triacylglycerol content, and inhibiting adipogenesis via activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the binding of CAP to the TRPV1 receptor is also responsible for its pungent sensation, strongly limiting its dietary intake. Here, the effects of a less pungent structural CAP-analog, nonivamide, on adipogenesis and underlying mechanisms in 3T3-L1 cells were studied. Nonivamide was found to reduce mean lipid accumulation, a marker of adipogenesis, to a similar extent as CAP, up to 10.4% (P < 0.001). Blockage of the TRPV1 receptor with the specific inhibitor trans-tert-butylcyclohexanol revealed that the anti-adipogenic activity of nonivamide depends, as with CAP, on TRPV1 receptor activation. In addition, in cells treated with nonivamide during adipogenesis, protein levels of the pro-adipogenic transcription factor peroxisome-proliferator activated receptor γ (PPARγ) decreased. Results from miRNA microarrays and digital droplet PCR analysis demonstrated an increase in the expression of the miRNA mmu-let-7d-5p, which has been associated with decreased PPARγ levels.

Astringency is a trigeminal sensation that involves the activation of G protein-coupled signaling by phenolic compounds.

Astringency is an everyday sensory experience best described as a dry mouthfeel typically elicited by phenol-rich alimentary products like tea and wine. The neural correlates and cellular mechanisms of astringency perception are still not well understood. We explored taste and astringency perception in human subjects to study the contribution of the taste as well as of the trigeminal sensory system to astringency perception. Subjects with either a lesion or lidocaine anesthesia of the Chorda tympani taste nerve showed no impairment of astringency perception. Only anesthesia of both the lingual taste and trigeminal innervation by inferior alveolar nerve block led to a loss of astringency perception. In an in vitro model of trigeminal ganglion neurons of mice, we studied the cellular mechanisms of astringency perception. Primary mouse trigeminal ganglion neurons showed robust responses to 8 out of 19 monomeric phenolic astringent compounds and 8 polymeric red wine polyphenols in Ca(2+) imaging experiments. The activating substances shared one or several galloyl moieties, whereas substances lacking the moiety did not or only weakly stimulate responses. The responses depended on Ca(2+) influx and voltage-gated Ca(2+) channels, but not on transient receptor potential channels. Responses to the phenolic compound epigallocatechin gallate as well as to a polymeric red wine polyphenol were inhibited by the Gαs inactivator suramin, the adenylate cyclase inhibitor SQ, and the cyclic nucleotide-gated channel inhibitor l-cis-diltiazem and displayed sensitivity to blockers of Ca(2+)-activated Cl(-) channels.

Synthesis and sensory studies of umami-active scaffolds.

The class of 2-isopropyl-5-methylbicyclo[4.1.0]heptane-7-carboxamides, 1-4, has been identified as potent umami-tasting molecules. A scalable synthesis of this challenging scaffold and new sensory insights will be presented. Interestingly, the umami characteristics differ remarkably, depending on constitutional and stereochemical features of the parent scaffold. During our studies, we could identify the carboxamide moiety as a crucial factor to influence the umami intensity of these scaffolds. In addition, the configuration of the cyclopropyl moiety exerts some influence, whereas the absolute configuration of the menthyl scaffold, at least the tested D- and L-configuration, is less important.

Enzymatic conversion of flavonoids using bacterial chalcone isomerase and enoate reductase.

Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction.

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

Identification of 4'-Demethyl-3,9-dihydroeucomin as a Bitter-Masking Compound from the Resin of Daemonorops draco.

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.

Human Gingival Fibroblasts as a Novel Cell Model Describing the Association between Bitter Taste Thresholds and Interleukin-6 Release.

Human gingival fibroblast cells (HGF-1 cells) present an important cell model to investigate the gingiva's response to inflammatory stimuli such as lipopolysaccharides from Porphyromonas gingivalis (Pg-LPS). Recently, we demonstrated trans-resveratrol to repress the Pg-LPS evoked release of the pro-inflammatory cytokine interleukin-6 (IL-6) via involvement of bitter taste sensing receptor TAS2R50 in HGF-1 cells. Since HGF-1 cells express most of the known 25 TAS2Rs, we hypothesized an association between a compound's bitter taste threshold and its repressing effect on the Pg-LPS evoked IL-6 release by HGF-1 cells. To verify our hypothesis, 11 compounds were selected from the chemical bitter space and subjected to the HGF-1 cell assay, spanning a concentration range between 0.1 µM and 50 mM. In the first set of experiments, the specific role of TAS2R50 was excluded by results from structurally diverse TAS2R agonists and antagonists and by means of a molecular docking approach. In the second set of experiments, the HGF-1 cell response was used to establish a linear association between a compound's effective concentration to repress the Pg-LPS evoked IL-6 release by 25% and its bitter taste threshold concentration published in the literature. The Pearson correlation coefficient revealed for this linear association was R2 = 0.60 (p < 0.01), exceeding respective data for the test compounds from a well-established native cell model, the HGT-1 cells, with R2 = 0.153 (p = 0.263). In conclusion, we provide a predictive model for bitter tasting compounds with a potential to act as anti-inflammatory substances.

Comprehensive Metabolite Profiling of Hydrangea macrophylla ssp. serrata Extracts Using Liquid Chromatography Coupled with Electrospray Ionization Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry.

A wide range of secondary metabolites has been described for various Hydrangea species, including the sweet-tasting phenyldihydroisocoumarin phyllodulcin, which is found in the leaves of Hydrangea macrophylla ssp. serrata. This work aims at the development and validation of an analytical workflow for comprehensive semi-polar metabolite profiling using liquid chromatography coupled with electrospray ionization ion mobility quadrupole time-of-flight mass spectrometry (UPLC-ESI-IMS-QToF-MS) to complement existing analytical studies. The unsupervised analysis of this data set demonstrates the capability of this analytical workflow to distinguish different H. macrophylla ssp. serrata cultivars. In combination with supervised analysis, a list of metabolites responsible for the differentiation of the cultivars studied has been obtained. Suspect screening of phenyldihydroisocoumarins provides comprehensive information, which could help in the search for key enzymes related to the biosynthesis of phyllodulcin.

Impact of lactisole on the time-intensity profile of selected sweeteners in dependence of the binding site.

Currently, there is limited insight into the influence of the different binding sites of agonists and antagonists of the sweet taste receptor TAS1R2/TAS1R3 on temporal sensory properties of sweet tasting compounds. We investigated whether the binding site and a competitive or allosteric inhibition of TAS1R2/TAS1R3 influence the time-dependent sensory perception and in vitro TAS1R2/TAS1R3-activation profiles. We compared time-intensity ratings of cyclamate, NHDC, acesulfame K, and aspartame with and without lactisole with the corresponding TAS1R2/TAS1R3-activation in transfected HEK293 cells. In combination with lactisole, cyclamate and NHDC demonstrated a shift of the dose-response curve corresponding to a competitive inhibition by lactisole in the sensory and the cell experiments. Allosteric inhibition by lactisole for aspartame and acesulfame K was seen in the cell experiments, but not the sensory ratings. In conclusion, the data do not support a major impact of the binding site on the time-intensity profile of the tested sweeteners.

Reducing the Bitter Taste of Pharmaceuticals Using Cell-Based Identification of Bitter-Masking Compounds.

The palatability of a pharmaceutical preparation is a significant obstacle in developing a patient-friendly dosage form. Bitter taste is an important factor for patients in (i) selecting a certain drug from generic products available in the market and (ii) adhering to a therapeutic regimen. The various methods developed for identification of bitter tasting and bitter-taste modulating compounds present a number of limitations, ranging from limited sensitivity to lack of close correlations with sensory data. In this study, we demonstrate a fluorescence-based assay, analyzing the bitter receptor TAS2R-linked intracellular pH (pHi) of human gastric parietal (HGT-1) cells as a suitable tool for the identification of bitter tasting and bitter-taste modulating pharmaceutical compounds and preparations, which resembles bitter taste perception. Among the fluorometric protocols established to analyze pHi changes, one of the most commonly employed assays is based on the use of the pH-sensitive dye SNARF-1 AM. This methodology presents some limitations; over time, the assay shows a relatively low signal amplitude and sensitivity. Here, the SNARF-1 AM methodology was optimized. The identified bicarbonate extrusion mechanisms were partially inhibited, and measurements were carried out in a medium with lower intrinsic fluorescence, with no need for controlling external CO2 levels. We applied the assay for the screening of flavonoids as potential bitter-masking compounds for guaifenesin, a bitter-tasting antitussive drug. Our findings revealed that eriodictyol, hesperitin and phyllodulcin were the most potent suitable candidates for bitter-masking activity, verified in a human sensory trial.

Individual Sweet Taste Perception Influences Salivary Characteristics After Orosensory Stimulation With Sucrose and Noncaloric Sweeteners.

Emerging evidence points to a major role of salivary flow and viscoelastic properties in taste perception and mouthfeel. It has been proposed that sweet-tasting compounds influence salivary characteristics. However, whether perceived differences in the sensory properties of structurally diverse sweet-tasting compounds contribute to salivary flow and saliva viscoelasticity as part of mouthfeel and overall sweet taste perception remains to be clarified. In this study, we hypothesized that the sensory diversity of sweeteners would differentially change salivary characteristics in response to oral sweet taste stimulation. Therefore, we investigated salivary flow and saliva viscoelasticity from 21 healthy test subjects after orosensory stimulation with sucrose, rebaudioside M (RebM), sucralose, and neohesperidin dihydrochalcone (NHDC) in a crossover design and considered the basal level of selected influencing factors, including the basal oral microbiome. All test compounds enhanced the salivary flow rate by up to 1.51 ± 0.12 g/min for RebM compared to 1.10 ± 0.09 g/min for water within the 1st min after stimulation. The increase in flow rate was moderately correlated with the individually perceived sweet taste (r = 0.3, p < 0.01) but did not differ between the test compounds. The complex viscosity of saliva was not affected by the test compounds, but the analysis of covariance showed that it was associated (p < 0.05) with mucin 5B (Muc5B) concentration. The oral microbiome was of typical composition and diversity but was strongly individual-dependent (permutational analysis of variance (PERMANOVA): R 2 = 0.76, p < 0.001) and was not associated with changes in salivary characteristics. In conclusion, this study indicates an impact of individual sweet taste impressions on the flow rate without measurable changes in the complex viscosity of saliva, which may contribute to the overall taste perception and mouthfeel of sweet-tasting compounds.

Bitter Sensing TAS2R50 Mediates the trans-Resveratrol-Induced Anti-inflammatory Effect on Interleukin 6 Release in HGF-1 Cells in Culture.

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.