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May 19, 2017 - Of the bitter-modulating compounds tested, eriodictyol, matairesinol, enterolacton, lariciresinol, and homoeriodictyol reduced the effe...
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Letter

Characterization of Bitter Compounds via Modulation of Proton Secretion in Human Gastric Parietal Cells in Culture Kathrin I. Liszt, Joachim Hans, Jakob P. Ley, Elke Köck, and Veronika Somoza J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 19 May 2017 Downloaded from http://pubs.acs.org on May 21, 2017

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Journal of Agricultural and Food Chemistry

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Characterization of Bitter Compounds via Modulation of Proton Secretion in

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Human Gastric Parietal Cells in Culture

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Kathrin I. Liszt1,2,#, Joachim Hans3, Jakob P. Ley3, Elke Köck1, Veronika Somoza1,2$

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1

Department of Nutritional and Physiological Chemistry, Faculty of Chemistry, University of Vienna, Althanstraße 14, 1090 Vienna, Austria

2

Christian Doppler Laboratory for Bioactive Aroma Compounds, University of Vienna, Althanstraße 14, 1090 Vienna, Austria 3

Symrise AG, Ingredient Research Flavor & Nutrition, Mühlenfeldstraße, 37603 Holzminden, Germany

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#

Author to whom correspondence should be addressed for characterization of HGT-1

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cells: Email: [email protected]; Phone: +43-4277-70610; Fax: +43-4277-

15

970610

16 17

$

Author to whom correspondence should be addressed for all other requests: E-mail

[email protected]; Phone: +43-4277-70610; Fax: +43-4277-970610.

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Short Title:

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Identification of bitter modulators

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ABSTRACT

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Humans perceive bitterness via around 25 different bitter receptors. Therefore, the

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identification of antagonists remains a complex challenge. We previously

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demonstrated several bitter tasting compounds such as caffeine to induce acid

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secretion in the stomach and in a human gastric tumor cell line (HGT-1). Here, the

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results of a fluorescent-based in vitro assay using HGT-1 cells and a human sensory

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panel testing nine selected potential bitter modulators with or without the bitter

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compounds caffeine or theobromine, were compared. Of the bitter modulating

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compounds

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homoeriodictyol reduced the effect of caffeine on proton secretion by -163 ± 14.0 %,

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-152 ± 12.4 %, -74 ± 16.4 %, -58 ± 7.2 %, and -44.6 ± 16.5 %, respectively, and

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reduced the bitter intensity of caffeine in the human sensory panel. In contrast,

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naringenin and 5,7-dihydroxy-4(4-hydroxyphenyl)chroman-2-one neither reduced the

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caffeine-induced proton secretion in HGT-1 cells nor showed an effect on bitter

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intensity perceived by the sensory panel. Results for theobromine were not as

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pronounced as those for caffeine, but followed a similar trend. The results

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demonstrate that the HGT-1 in vitro assay is a useful tool to identify potential bitter

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masking compounds. Nevertheless, a sensory human panel is necessary to quantify

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the bitter-masking potency.

tested,

eriodictyol,

matairesinol,

enterolacton,

lariciresinol,

and

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Key words:

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bitter masking, TAS2Rs, HGT-1 cells, bitter modulators, bitter tastants

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INTRODUCTION

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The taste and smell of food is an indicator for its quality. Especially bitter or sour

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taste helps to identify potentially harmful foods and food constituents. Bitterness is

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perceived by activation of at least one or more of 25 different bitter taste receptors

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(TAS2Rs) located in the taste buds of the tongue1, 2, although TAS2Rs are not only

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present in the oral cavity. Their expression has also been reported for extra-oral

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tissues as the upper and lower respiratory tract3, 4, the urinary tract5, the heart6, the

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brain7, and the gastrointestinal tract2, including the stomach8-10 in mammals. In

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previous studies, we have demonstrated that bitter compounds like procyanidin B2

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and catechin as wine constituents11, hop-derived beer bitter acids12, and caffeine in

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coffee13 can stimulate proton secretion in HGT-1 cells, a cell line that represents the

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characteristics of an acid producing parietal cell14. Parietal cells are located in the

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gastric fundus and corpus region of the human stomach and produce gastric acid,

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which is necessary to maintain a low pH for the activation of gastric enzyme to digest

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proteins and absorb calcium and vitamin B1215. Regulation of digestion by bitter

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compounds derived from the human diet is suggested16. Furthermore, we

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demonstrated that mRNA of several TAS2Rs is expressed in HGT-1 cells and in

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human gastric biopsies by RT-qPCR. As demonstrated in HGT-1 cells, the proposed

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physiological role of TAS2Rs in parietal cells is the regulation of proton secretion. In

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previous experiments by means of a CRISPR-Cas9 knockout approach, caffeine- as

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well as aristocholic acid-induced proton secretion in HGT-1 cells was demonstrated

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to depend on TAS2R4317. Moreover, the caffeine effect on proton secretion in HGT-1

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cells was reduced by addition of the bitter masking compound homoeriodictyol

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(HED)17. HED is an antagonist of TAS2R43, 31, 20, and 50, and an agonist of

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TAS2R14, as identified by measuring the percent reduction of Calcium efflux in

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stimulated transfected HEK cells after addition of HED17. Homoeriodictyol can be

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extracted from the leaves of Herba Santa (Eriodictyon californicum), a plant which

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has been used to reduce the bitterness for some pharmaceutical applications for

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many years18. In 2005, Ley and colleagues18 identified homoeriodictyol and

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eriodictyol to be the active bitter masking compounds of this plant. Since then,

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several structurally similar compounds have been identified to reduce the bitter

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intensity of caffeine tested by human sensory panels. Although sensory evaluations

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by human panels are most valuable for the identification of bitter masking

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compounds, this approach is time consuming, requires a certain quantity of test

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material, and is limited to compounds for which toxic effects can be reliably excluded

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for concentrations being tested. Another experimental approach for the identification

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of bitter modulators are heterologous cell assays19, 20 or assays based on native21 or

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immortalized taste cells22. In heterologous cell assays, usually only one of 25 bitter

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taste receptors is expressed. Although this approach offers the identification of

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TAS2R agonists and antagonists, correlations with sensory data are often weak,

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since bitter compounds or bitter modulators may well target several TAS2Rs19, with

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the overall interaction of all targeted receptors presumably generating the

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physiological response of bitter reception. Also, some TAS2Rs are broadly tuned,

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whereas others show a high ligand specificity23. An optimized cellular screening

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approach for bitter modulating compounds for which neither large quantities nor

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toxicity data exist would need to include an orchestrated functionality of all TAS2Rs

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with good correlation to human sensory data.

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In previous studies, we have demonstrated that HGT-1 cells are sensitive to bitter

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compounds. Here, we demonstrate that the HGT-1 cell line is a suitable model for

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the identification of various bitter-modulating compounds, e.g. by measuring the 4 ACS Paragon Plus Environment

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inhibitory effect on caffeine- or theobromine-induced proton secretion. The effect of

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the bitter compound naringin, present in grapefruits, was also tested. Since the

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treatment with naringin in the cell model did not showed a significant effect nine

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potentially bitter masking compounds in combination with or without caffeine or

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theobromine were tested in HGT-1 cells and in a human sensory panel. The

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structures were selected to their general similarity to homoeriodictyol and

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eriodictyol18 according to a structure activity-based approach as described for the

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lignans in Ley et al.24 and neoflavonoids in Backes et al.25.

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MATERIALS AND METHODS

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Chemicals. Cell culture materials and histamine were purchased from Sigma-Aldrich,

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salts for buffer preparation from Carl Roth (Karlsruhe, Germany). Bitter compounds

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caffeine, naringin and theobromine were obtained from Sigma Aldrich (Munich,

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Germany) in a quality suitable for food and flavor use (min. 95 % according to GC or

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HPLC); bitter masking compounds eriodictyol (Carl Roth GmbH, Karlsruhe,

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Germany), phloretin, naringenin (Sigma Aldrich, Munich, Germany), (-)-matairesinol,

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and (+)-lariciresinol, enterodiol, enterolacton (all from Phytolab, Verstenbergsgreuth,

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Germany). Homoeriodictyol mono sodium salt, racemic, was prepared according to

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Ley et al.18 and 5,7-dihydroxy-4(4-hydroxyphenyl)chroman-2-one, racemic, prepared

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according to Backes et al.26, see supporting information) were all >95 % (according

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to HPLC) and suitable for food or flavor use. For the in vitro tests, stock solution of

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caffeine, theobromine, homoeriodictyol sodium salt were prepared in water, while

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naringin, eriodictyol,

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hydroxyphenyl)chroman-2-one (DH) were dissolved in ethanol and phloretin,

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enterodiol, enterolacton in DMSO. Test solutions for the cell culture study were

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prepared in phenol red free Dulbecco's Modified Eagle's Medium (DMEM) containing

matairesinol, lariciresinol,

naringenin, 5,7-dihydroxy-4(4-

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10% fetal bovine serum with a maximum solvent concentration of 1 % ethanol or

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0.1 % dimethylsulfoxide (DMSO). Test solutions for the sensory study were prepared

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as 1% (w/v) solutions in ethanol. Final solutions for sensory evaluation were adjusted

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for the required compound concentrations in water, not exceeding a final ethanol

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concentration of 1% (v/v).

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Cell culture. The human gastric tumor cell line HGT-1, obtained from Dr. C.

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Laboisse (Laboratory of Pathological Anatomy, Nantes, Frances), was used in cell

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culture experiments. Cells were cultured in DMEM with 4 g/L glucose, supplemented

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with 10 % fetal bovine serum, 3 % L-glutamine, and 1 % penicillin/streptomycin

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under standard conditions at 37 °C, and 5 % CO2. In Table S1 we provide results of

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the genotype analysis of 86 SNPs at the 25 TAS2R genes, described by Pirsatu et

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al.27 in HGT-1 cells. The data was obtained from a whole genome sequencing,

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including a variant calling, as described in Liszt et al.17.

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Cell Cytotoxicity. Cytotoxic effects of test compounds were excluded by staining

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the cells with the ViaCount reagent (Merck Millipore, Darmstadt, Germany) and

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measuring the number of dead and living cells according to the manufacturer’s

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protocol. A total number of 500 000 cells were seeded in a 24 well plate and

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incubated at 37 °C and 5 % CO2. After around 24 hours, cells were treated with test

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compounds diluted in DMEM for 1h at 37 °C and 5 % CO2. Cells were washed once

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with 500 µL Krebs-Hepes-buffer and harvested with 50 µL Trypsin/EDTA solution.

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The reaction was stopped with 250 µL DMEM. Then, 20 µL of this cell suspension

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were diluted in 180 µL ViaCount reagent and incubated at room temperature for 5

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min. Viability was measured at a guava easyCyte™ flow cytometer (Merck Millipore,

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Darmstadt,

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theobromine, matairesinol and lariciresinol were tested in concentrations of 0.003

Germany).

Enterolacton,

enterodiol,

DH,

naringin,

naringenin,

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mM, 0.03 mM and 0.3 mM while eriodictyol, homoeriodictyol and phloretin were

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additionally tested in the concentration of 0.3 µM and caffeine in the concentrations

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0.03 mM, 0.3 mM and 3 mM. Concentrations were chosen based on the publication

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of Ley et. al.18 in which a 0.3 mM homoeriodictyol reduced the bitterness of 2.6 mM

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caffeine in a human sensory panel. The cell concentrations of dead and living cells

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after treatment with test compounds were compared to untreated cells as a control

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as well as to a solvent control.

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Intracellular pH measurement in HGT-1 cells. The intracellular pH was measured

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as a marker of proton secretion in HGT-1 cells by means of the pH-sensitive

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fluorescence dye SNARF-1-AM® (Invitrogen) as previously described11,

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100 000 HGT-1 cells were seeded in a black 96 well plate. After 24 hours, cells were

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stained with 3 µM SNARF-1-AM® for 30 min at standard conditions, and treated with

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the bitter compounds with or without the bitter masking compounds for 10 min.

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Histamine 1 mM was used as positive control. As control, cells were exposed to

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DMEM only. In addition, a solvent control, DMEM containing either 1 % ethanol or

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0.1 % ethanol or 0.1 % DMSO, and a positive control, 1 mM histamine, were used.

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Fluorescence was measured at 580 nm and 640 nm emission after excitation at 488

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nm using an Infinite 200 Pro Plate Reader. Using a calibration curve the intracellular

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pH and following the intracellular H+ concentration was calculated. The ratio between

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treated and non-treated cells (medium only) was calculated and log2 transformed to

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determine the intracellular proton index (IPX)11, 28-30. The lower the IPX the stronger

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the proton secretion.

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SENSORY EVALUATION

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. Briefly,

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Sensory evaluation of the potential bitter masking effects for the tested compounds

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was performed by a trained panel (n ≥ 9) in multiple sessions. For each session, a

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pair of test solutions (bitter agent vs bitter agent + modulator) was offered in a

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randomized, blinded design and scored by panelists on a non-calibrated visual

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analogue scale ranging from 0 (not bitter) to 10 (extremely bitter). Panelists were

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asked to neutralize with water and keep a waiting time of 2 min between samples.

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STATISTICS

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Statistical analysis was performed with Excel 2007 (Microsoft) and SigmaPlot

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software 11.0 (Systat Software). Significant differences for data derived from cell

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culture experiments were calculated applying a one-way ANOVA on Ranks vs.

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control. Outliers were excluded by Nalimov outlier analysis. At least three biological

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replicates and 2 technical replicates were analyzed for each cell culture experiment.

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Data is presented as mean ± SEM.

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Sensory data were analyzed by calculating the average of responses to the bitter

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compound alone and of the responses to the combination of the bitter compound

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with the respective masking substance. Significance of the differences in response

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were determined by Student’s t-test, with a p-value of lower than 0.05 being

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considered as significant.

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RESULTS AND DISCUSSION

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We aimed at demonstrating the HGT-1 cell line as a suitable model for the

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identification of bitter modulating compounds, providing data with good correlations

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to evaluations by human sensory panels.

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First, nine potentially bitter masking compounds in combination with or without

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caffeine or theobromine as well as caffeine, theobromine and naringin itself

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(structures shown in Figure 1A) were tested for their cytotoxic effects on HGT-1 cells

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by counting living and dead cells after staining with the ViaCount reagent. The

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ViaCount reagent includes two dyes, one solely stains cells with an intact cell

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nucleus, and therefore only living cells, while the other one only stains dead cells.

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Neither the bitter masking compounds nor any of the bitter masking compounds in

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the concentration range of 3 mM and 0.003 mM showed a reduction beneath 90 %

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cell viability (data not shown). Therefore, these concentrations were tested in further

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cell culture experiments.

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The methylxanthines caffeine and theobromine, as well as naringin are commonly

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consumed bitter compounds present in the human diet. Caffeine and theobromine

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are consumed via e.g. coffee, tea or chocolate, whereas naringin is the most

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relevant bitter compound of grapefruit. Caffeine and theobromine induced proton

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secretion in HGT-1 cells, while naringin showed no stimulation compared to its

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solvent control (1% ethanol) when used in a concentration range between 0.03 – 300

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µM (Figure 1 B,C,D). Therefore, no further tests with naringin were carried out,

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although we cannot exclude one of the TAS2Rs being targeted by higher naringin

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concentrations. The effect for caffeine has been previously demonstrated17,

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Interestingly, no TAS2R has yet been identified as target of naringin23, even though it

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tastes very bitter. In contrast, 5 TAS2Rs, TAS2R7, 10, 14, 43, 4623 have been

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reported to be targeted by caffeine. For theobromine, no data regarding its role as

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TAS2R agonist has been available. The effect of the selected nine potential bitter

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masking compounds on proton secretion were tested in two concentrations, as

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demonstrated in Figure 1D. Concentrations selected for testing reflected the

30

.

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concentrations of the compounds typically utilized in their respective applications in

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aromas or food. For two of the nine compounds, enterodiol and phloretin, a

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stimulation of proton secretion in HGT-1 cells with IPX values of IPX -0.48 ± 0.031

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and IPX -0.21 ± 0.042, respectively, in concentrations of 300 µM or 30 µM was

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demonstrated. While only for one compound, matairesinol (300 µM), an inhibition of

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the proton secretion with an IPX of 0.35 ± 0.039 was demonstrated. To evaluate

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whether these compounds can reduce the stimulatory effect of a bitter compound on

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proton secretion, target compounds were tested in combination with either 3 mM

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caffeine or 300 µM theobromine. The percent reduction of the potential bitter

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masking compound on the effect of either caffeine or theobromine is presented in

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Figure 2. Eriodictyol, matairesinol, enterolacton, lariciresinol, and homoeriodictyol

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reduced the effect of caffeine on proton secretion by -163 ± 14.0 %, -152 ± 12.4 %, -

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74 ± 16.4 %, -58 ± 7.2 %, and -44.6 ± 16.5 %, respectively (Figure 2A). The results

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for the percent reduction of the theobromine effect are similar: Eriodictyol,

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matairesinol and homoeriodictyol reduced the effect of theobromine on proton

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secretion in HGT-1 cells by -190 ± 20.4 %, -153 ± 9.94 %, and -181 ± 15.6 %,

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respectively (Figure 2B). A reduction of the methylxanthine-mediated effect lower

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than -100 % indicates that the inhibition is even stronger compared to the stimulation

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of the methylxanthine as individual compound. At least for matairesinol, it is shown

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that it inhibits proton secretion independently of a concomitant treatment with a

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stimulating compound. These results indicate that the here tested bitter masking

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compounds might not act only as an antagonist on a specific receptor rather than, at

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least for matairesinol, act as an agonist that activate a signaling pathway to inhibit

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proton secretion.

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To investigate the hypothesis that the data obtained from the cellular HGT-1

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screening of bitter modulators correlates with human sensory data, the bitter

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masking compounds were also tested in a human sensory panel in food-relevant

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concentrations (25 to 100 ppm) as stated in the FEMA-GRAS report 22 on flavoring

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substances. Similar to the results in HGT-1 cells, eriodictyol (-46% p=0.0014),

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homoeriodictyol (-43%, p=0.0003), lariciresinol (-38%, p=0.0005), matairesinol (-35%,

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p=0.0078) and phloretin (-28%, p=0.0235) showed strong and significant reduction of

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bitter intensity perceived from caffeine; enterodiol (-30%, p =0.08) also exhibited a

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strong, albeit not significant reduction of caffeine induced bitterness (Figure 3). The

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bitter intensity of theobromine was significantly reduced by eriodictyol (-36%,

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p=0.0098) and matairesinol (-24%, p=0.0626) showed a strong but not significant

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reduction of theobromine bitterness. Interestingly, homoeriodyctiol, showing a strong

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and clear reduction of caffeine-induced bitterness, has been demonstrated to be

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much less effective in reducing theobromine bitterness (-12%, p=0.3526). In Table 1

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the effects of the bitter modulators on caffeine or theobromine in the sensory panel

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and in the in vitro study are summarized. While naringenin and DH showed no

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significant effect in the sensory panel and the in vitro study, homoeriodictyol,

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lariciresinol, matairesinol and eriodictyol reduced the bitter intensity of caffeine in the

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human sensory data and also reduced the caffeine induced proton secretion in HGT-

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1 cells. In the in vitro measurements enterolacton significantly reduced the caffeine

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induced proton secretion, while in the human sensory study the reduction of caffeine

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bitterness was not significant. Homoeriodictyol, lariciresinol, matairesinol and

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eriodictyol reduced the effect of theobromine on proton secretion in HGT-1 cells, but

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only eriodictyol and matairesinol showed a reduction of bitter intensity in the human

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sensory test. For enterodiol and phloretin, the results from the HGT-1 cells and the

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sensory evaluation were not as conclusive. We, therefore, propose testing a wider

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concentration range, exceeding those of food applications, to validate the effects of

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enterodiol and phloretin on bitter-mediated proton secretion in HGT-1 cells

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associated with bitterness-perception in sensory studies.

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In the in vitro model, different concentrations of compounds can be tested easily.

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Therefore, the effect of modulators can measured more efficiently in different

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concentrations.

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experiments might elucidate the detailed mechanism of action, e.g. clarify whether (i)

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a bitter modulator either blocks the access of a bitter compound to the TAS2R or (ii)

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directly interacts with a bitter compound, thereby changing its structure and

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preventing TAS2R binding.

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These results demonstrate that the HGT-1 in vitro assay is suitable for the

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identification of potential bitter masking compounds, however quantification of the

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bitter masking effect requires a human sensory panel.

Future

mechanistic

studies

involving

TAS2R

knock

down

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ASSOCIATED CONTENT

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Results of HGT-1 DNA sequence analysis for genotyping of TAS2R genes and

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chemical characterization of 5,7-Dihydroxy-4(4-hydroxyphenyl)chroman-2-one.

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ABBREVIATIONS USED

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HGT-1 - human gastric tumor cell line -1, TAS2Rs - bitter taste receptors, HED -

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homoeriodictyol, DH - 5,7-dihydroxy-4(4-hydroxyphenyl)chroman-2-one, DMSO –

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dimethylsulfoxide, DMEM - Dulbecco's Modified Eagle's Medium, IPX – intracellular

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proton index,

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AUTHOR INFORMATION

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The authors Dr. Joachim Hans and Dr. Jakob P. Ley are employees at Symrise AG.

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Holzminden/Germany.

299

300

FUNDING

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This work was financially supported by the Austrian Federal Ministry of Economy,

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Family and Youth and the Austrian National Foundation for Research, Technology

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and Development and by Symrise AG, Holzminden/Germany.

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REFERENCES

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1. Behrens, M.; Meyerhof, W., Oral and extraoral bitter taste receptors. Results Probl Cell Differ 2010, 52, 87-99. 2. Calvo, S. S.; Egan, J. M., The endocrinology of taste receptors. Nat Rev Endocrinol 2015, 11, 213-227. 3. Deshpande, D. A.; Wang, W. C.; McIlmoyle, E. L.; Robinett, K. S.; Schillinger, R. M.; An, S. S.; Sham, J. S.; Liggett, S. B., Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction. Nat Med 2010, 16, 1299-304. 4. Lee, R. J.; Cohen, N. A., Sinonasal solitary chemosensory cells "taste" the upper respiratory environment to regulate innate immunity. Am J Rhinol Allergy 2014, 28, 366-73. 5. Deckmann, K.; Filipski, K.; Krasteva-Christ, G.; Fronius, M.; Althaus, M.; Rafiq, A.; Papadakis, T.; Renno, L.; Jurastow, I.; Wessels, L.; Wolff, M.; Schutz, B.; Weihe, E.; Chubanov, V.; Gudermann, T.; Klein, J.; Bschleipfer, T.; Kummer, W., Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes. Proc Natl Acad Sci U S A 2014, 111, 8287-92. 6. Foster, S. R.; Porrello, E. R.; Stefani, M.; Smith, N. J.; Molenaar, P.; dos Remedios, C. G.; Thomas, W. G.; Ramialison, M., Cardiac gene expression data and in silico analysis provide novel insights into human and mouse taste receptor gene regulation. Naunyn Schmiedebergs Arch Pharmacol 2015, 388, 1009-27. 7. Singh, N.; Vrontakis, M.; Parkinson, F.; Chelikani, P., Functional bitter taste receptors are expressed in brain cells. Biochem Biophys Res Commun 2011, 406, 146-51. 8. Wu, S. V.; Rozengurt, N.; Yang, M.; Young, S. H.; Sinnett-Smith, J.; Rozengurt, E., Expression of bitter taste receptors of the T2R family in the gastrointestinal tract and enteroendocrine STC-1 cells. Proc Natl Acad Sci U S A 2002, 99, 2392-7. 9. Prandi, S.; Bromke, M.; Hübner, S.; Voigt, A.; Boehm, U.; Meyerhof, W.; Behrens, M., A subset of mouse colonic goblet cells expresses the bitter taste receptor tas2r131. PLoS One 2013, 8, e82820. 10. Avau, B.; Rotondo, A.; Thijs, T.; Andrews, C. N.; Janssen, P.; Tack, J.; Depoortere, I., Targeting extra-oral bitter taste receptors modulates gastrointestinal motility with effects on satiation. Sci Rep 2015, 15985. 11. Liszt, K. I.; Eder, R.; Wendelin, S.; Somoza, V., Identification of Catechin, Syringic Acid, and Procyanidin B2 in Wine as Stimulants of Gastric Acid Secretion. J Agric Food Chem 2015, 63, 7775-83. 12. Walker, J.; Hell, J.; Liszt, K. I.; Dresel, M.; Pignitter, M.; Hofmann, T.; Somoza, V., Identification of beer bitter acids regulating mechanisms of gastric acid secretion. J Agric Food Chem 2012, 60, 1405-12. 13. Rubach, M:, Lang, R.; Seebach, E.; Somoza, M.M.; Hofmann, T.; Somoza, V., Multi-parametric approach to identify coffee components that regulate mechanisms of gastric acid secretion. Mol Nutr Food Res 2012, 56, 325-35. 14. Carmosino, M.; Procino, G.; Casavola, V.; Svelto, M.; Valenti, G., The cultured human gastric cells HGT-1 express the principal transporters involved in acid secretion. Pflugers Arch 2000, 440, 871-80. 15. Schubert, M. L., Gastric secretion. Curr Opin Gastroenterol 2010, 26 (6), 598-603. 16. Janssen, S.; Laermans, J.; Verhulst, P. J.; Thijs, T.; Tack, J.; Depoortere, I., Bitter taste receptors and α-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying. Proc Natl Acad Sci U S A 2011, 108, 2094-9. 17. Liszt, K. I.; Ley, J. P.; Lieder, B.; Behrens, M.; Reiner, A.; Stöger, V.; Hochkogler, C. M.; Köck, E.; Marchiori, A.; Hans, J.; Widder, S.; Krammer, G.; Sanger, G. J. S., Mark Manuel; Meyerhof, W.; Somoza , V., Caffeine induces gastric acid secretion via bitter taste signaling in gastric parietal cells. submitted to PNAS 2017. 14 ACS Paragon Plus Environment

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Journal of Agricultural and Food Chemistry

354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377

18. Ley, J. P.; Krammer, G.; Reinders, G.; Gatfield, I. L.; Bertram, H. J., Evaluation of bitter masking flavanones from Herba Santa (Eriodictyon californicum (H. and A.) Torr., Hydrophyllaceae). J Agric Food Chem 2005, 53), 6061-6. 19. Slack, J. P.; Brockhoff, A.; Batram, C.; Menzel, S.; Sonnabend, C.; Born, S.; Galindo, M. M.; Kohl, S.; Thalmann, S.; Ostopovici-Halip, L.; Simons, C. T.; Ungureanu, I.; Duineveld, K.; Bologa, C. G.; Behrens, M.; Furrer, S.; Oprea, T. I.; Meyerhof, W., Modulation of bitter taste perception by a small molecule hTAS2R antagonist. Curr Biol 2010, 20, 1104-9. 20. Roland, W. S.; Gouka, R. J.; Gruppen, H.; Driesse, M.; van Buren, L.; Smit, G.; Vincken, J. P., 6methoxyflavanones as bitter taste receptor blockers for hTAS2R39. PLoS One 2014, 9 (4), e94451. 21. Ozdener, M. H.; Brand, J. G.; Spielman, A. I.; Lischka, F. W.; Teeter, J. H.; Breslin, P. A.; Rawson, N. E., Characterization of human fungiform papillae cells in culture. Chem Senses 2011, 36, 601-12. 22. Hochheimer, A.; Krohn, M. Human taste cells capable of continuous proliferation. 2013. 23. Meyerhof, W.; Batram, C.; Kuhn, C.; Brockhoff, A.; Chudoba, E.; Bufe, B.; Appendino, G.; Behrens, M., The molecular receptive ranges of human TAS2R bitter taste receptors. Chem Senses 2010, 35, 157-70. 24. Ley, J. P.; Dessoy, M.; Paetz, S.; Blings, M.; Hoffmann-Lücke, P.; Reichelt, K. V.; Krammer, G. E.; Pienkny, S.; Brandt, W.; Wessjohann, L., Identification of enterodiol as a masker for caffeine bitterness by using a pharmacophore model based on structural analogues of homoeriodictyol. J Agric Food Chem 2012, 60, 6303-11. 25. Backes; M.; Vössing, T.; Aust, S.; Pienkny, S.; Brandt, W.; Wessjohann, L.; Ley, J. P., Identification of nitrogen containing flavanoids as a potent bitter masker supported by combined gustophore modeling and docking studies. Deutsche Forschungsanstalt fuer Lebensmittelchemie: Garching 2014, 29-34.

378 379

26. Backes, M.; Vössing, T.; Ley, J. P.; Paetz, S. Use of neoflavanoids for modifying taste. 2013 EP 2570035 A1

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27. Pirastu, N.; Kooyman, M.; Traglia, M.; Robino, A.; Willems, S. M.; Pistis, G.; d'Adamo, P.; Amin, N.; d'Eustacchio, A.; Navarini, L.; Sala, C.; Karssen, L. C.; van Duijn, C.; Toniolo, D.; Gasparini, P., Association analysis of bitter receptor genes in five isolated populations identifies a significant correlation between TAS2R43 variants and coffee liking. PLoS One 2014, 9, e92065. 28. Liszt, K. I.; Walker, J.; Somoza, V., Identification of Organic Acids in Wine That Stimulate Mechanisms of Gastric Acid Secretion. J Agric Food Chem 2012. 29. Weiss, C.; Rubach, M.; Lang, R.; Seebach, E.; Blumberg, S.; Frank, O.; Hofmann, T.; Somoza, V., Measurement of the intracellular ph in human stomach cells: a novel approach to evaluate the gastric acid secretory potential of coffee beverages. J Agric Food Chem 2010, 58, 1976-85. 30. Rubach, M.; Lang, R.; Seebach, E.; Somoza, M. M.; Hofmann, T.; Somoza, V., Multiparametric approach to identify coffee components that regulate mechanisms of gastric acid secretion. Mol Nutr Food Res 2012, 56, 325-35.

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Figure captions

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Figure 1. Chemical structures of test compounds (A) and intracellular proton index

398

(IPX) of HGT-1 cells treated for 10 min with (B) theobromine or (C) naringin or (D)

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different bitter masking compounds in different concentrations and 3000 µM caffeine

400

and 300 µM theobromine. Treated cells were normalized to their solvent control.

401

Data: mean±SEM, n= 3-4, tr=6, except for (D) caffeine n= 16, theobromine n=9, tr=6,

402

statistics: one-way ANOVA on Ranks with Dunn’s post hoc test vs. control (control =

403

basal 0) and (C) vs 1% EtOH (solvent control). Significant differences are indicated

404

by **; p < 0.01, *; p < 0.05, n.s., not significant

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Figure 2. Percent reduction of the effect of the methylxanthines (A) 3 mM caffeine or

407

(B) 300 µM theobromine on HGT-1 cells after concomitant treatment with different

408

bitter masking compounds either in a concentration of 3, 30 or 300 µM for 10 min.

409

The percent reduction is calculated by IPX of the methylxanthine divided by IPX

410

methylxanthine + bitter masker. Data: mean±SEM, (A) n= 3-13, (B) n= 3, tr=6,

411

statistics: one-way ANOVA on Ranks with Dunn’s post hoc test vs. control (control =

412

basal 0). Significant differences are indicated by **; p < 0.01, *; p < 0.05.

413 414

Figure 3. Results of the sensory evaluation by 9-20 panelists. Perceived bitter

415

intensity, rated on a scale from 0 to 10, induced by either (A) 2.6 mM caffeine or (B)

416

1.7 mM theobromine in comparison to addition of bitter modulators in a sensory

417

panel. Concentration of bitter modulators: Naringenin 0.37 mM, Enterodiol 0.08 mM,

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Phloretin 0.18 mM, DH 0.09 mM, Homoeriodictyol 0.31 mM, Lariciresinol 0.07 mM,

419

Enterolacton 0.08 mM, Matairesinol 0.07 mM, Erioditctyol 0.35 mM. Data: mean±

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SEM statistics: Student’s t-test; Significant differences are indicated by *; p < 0.05. 16 ACS Paragon Plus Environment

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Table 1. Summary of the effect of potential bitter masking compounds on bitter intensity in a human sensory panel and proton secretion in HGT-1 cells Sensory evaluation

IPX

Caffeine Theobromine Caffeine

Theobromine

Naringenin

0

0

Enterodiol

0

Phloretin

+

DH

0

0

0

Homoeriodictyol

+

0

+

+

Lariciresinol

+

0

+

0

Enterolacton

0

Matairesinol

+

+*

+

+

Eriodictyol

+

+

+

+

0

0

+

0; no effect, +; reduction of bitter taste/proton secretion, -; enhancing proton secretion *; Matairesinol reduced the bitter intensity of theobromine with a p value of 0.06.

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Figure 1.

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Figure 2

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Figure 3

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In vitro

+ bitter Bitter masking compound

+ Homoeriodictyol In vitro

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bitter