Cells expressing bitter taste receptors and uses thereof

Abstract

The present disclosure generally relates to the expression of T2R taste receptors in eukaryotic cells, such as U2OS cells. The disclosure also relates to methods for expressing T2R taste receptors in such cells, and to methods for screening for substances that modulate the T2R receptors and, as a result, may be useful as modulators of bitter taste.

Description

[Technical field]

[0001] The present disclosure generally relates to the expression of T2R taste receptors in eukaryotic cells, such as U2OS cells. The disclosure also relates to methods for expressing T2R taste receptors in such cells, and to methods for screening for substances that modulate the T2R receptors and, as a result, may be useful as modulators of bitter taste.

[0002] 2. Description of Related Art The taste system provides sensory information about the chemical composition of the external world. In animals, taste transduction is one of the most sophisticated forms of chemically induced sensation. Taste signaling is found throughout the animal kingdom, from the simplest metazoans to the most complex vertebrates. There are thought to be five basic taste modalities in mammals: sweet, bitter, sour, salty, and umami.

[0003] The taste receptor can be functionally expressed in a cell, such as a human embryonic kidney 293 (HEK-293) cell. The taste receptor is coupled to one or more G proteins. When a test molecule binds to the taste receptor, the coupled G protein responds to the binding event by eliciting a response, such as a change in calcium cation concentration within the cell. The chemical change can be detected and measured, thereby providing a chemical signal indicative of the binding of the test molecule to the taste receptor. Multiple cells expressing the G protein-coupled taste receptor can be incorporated into a standard cellular assay. The assay can be used to screen various taste compounds to determine the extent and manner in which they modulate the taste receptor protein.

[0004] The cell type may affect the robustness of the assay. For example, most of the assays have a signal window within which the detectable signal is approximately well matched with the activity of the test molecule on the taste receptor. Outside the signal window, the correlation decreases and the assay does not provide useful information about the binding event. T2R bitter receptors can be expressed in HEK-293 cells, but for some of the approximately 25 bitter receptors, the signal window is significantly narrowed when the bitter receptor is expressed in HEK-293 cells. Therefore, there is a need to develop a method to express T2R bitter receptors in other cell types that can tolerate a wider signal window that can be used to evaluate the interaction of test compounds over a wider range of activity levels.

[0005] overview The present disclosure provides methods for expressing T2R bitter taste receptors in cell types other than HEK-293, and in some cases, assays incorporating such cells provide a broader signaling window than the same taste receptors expressed in comparable HEK-293 cells.

[0006] In a first aspect, the disclosure provides a U2OS cell comprising a T2R bitter taste receptor. In some embodiments, the cell further comprises a G protein coupled to the T2R bitter taste receptor. In some embodiments, the cell further comprises an exogenous nucleic acid sequence encoding a G protein. In some embodiments, the G protein is gustducin. In some other embodiments, the G protein is a G(i / o) protein. In some other embodiments, the G protein is a promiscuous G protein, such as G15 or G16gust25. In some embodiments, the U2OS cell is an isolated U2OS cell.

[0007] In a second aspect, the disclosure provides a U2OS cell comprising an exogenous nucleic acid sequence encoding a T2R bitter taste receptor. In some embodiments, the cell further comprises an exogenous nucleic acid sequence encoding a G protein. In some further embodiments, the G protein is gustducin. In some other embodiments, the G protein is a G(i / o) protein. In some other embodiments, the G protein is a promiscuous G protein, such as G15 or G16gust25. In some embodiments, the U2OS cell is an isolated U2OS cell.

[0008] In a third aspect, the present disclosure provides a method for identifying bitter taste modulators, comprising: (a) contacting one or more U2OS cells of the first aspect with a test compound; and (b) measuring T2R bitter taste receptor activity, wherein a change in T2R bitter taste activity indicates that the test compound is a bitter taste modulator. In some embodiments, U2OS cells are included in the assay. In some embodiments, U2OS cells are attached to a substrate.

[0009] In a fourth aspect, the present disclosure provides a method for identifying a compound that reduces bitterness, comprising: (a) contacting one or more U2OS cells of the first aspect with a test compound and a bitter tastant; and (b) measuring the response of the T2R bitter receptor by comparing the activity of the T2R bitter receptor to the bitter tastant in the presence and absence of the test compound, where a reduction in T2R bitter receptor activity indicates that the test compound is a bitter blocker. In some embodiments, the method comprises identifying the test compound as a bitter blocker based on a change in T2R bitter receptor activity. In some embodiments, the method comprises selecting the bitter blocker as a compound that reduces bitterness. In some embodiments, the U2OS cells are included in the assay. In some embodiments, the U2OS cells are attached to a substrate.

[0010] In a fifth aspect, the disclosure provides a use of an identified or selected active test compound of any of the specific embodiments of the third or fourth aspects for reducing the bitter taste of an ingestible composition.

[0011] In a sixth aspect, the disclosure provides a method of reducing the bitterness of an ingestible composition, the method comprising introducing an amount (such as a bitterness-reducing effective amount) of any identified or selected active test compound of any of the embodiments of any of the third or fourth aspects into the ingestible composition.

[0012] In a seventh aspect, the disclosure provides an ingestible composition comprising an identified or selected active test compound of any of the specific embodiments of the third or fourth aspects. In some embodiments, the ingestible composition comprises one or more bitter tastants.

[0013] In an eighth aspect, the present disclosure provides a flavor product comprising the ingestible composition of the seventh aspect. In some embodiments, the flavor product is a beverage, such as soda, flavored water, tea, and the like. In some other embodiments, the flavor product is a food product. In some other embodiments, the flavor product is a drug or over-the-counter drug.

[0014] Further aspects and embodiments thereof are set forth in the following detailed description, drawings, abstract, and claims.

[0015] The following drawings are provided for the purpose of illustrating various embodiments of the compositions and methods disclosed herein. The drawings are provided for illustrative purposes only and are not intended to describe any preferred compositions or preferred methods, or to serve as a basis for limiting the scope of the claimed invention. [Brief description of the drawings]

[0016] [Figure 1]1 shows the response of cells expressing five types of T2R bitter receptors at different MOI levels. Each of the five sets of bars in the bar graph represents cells expressing a different bitter receptor. The leftmost set represents cells expressing a polypeptide of SEQ ID NO: 1, where the bitter tastant is chloramphenicol. The set immediately to the right represents cells expressing a polypeptide of SEQ ID NO: 3, where the bitter tastant is strychnine. The set immediately to the right represents cells expressing a polypeptide of SEQ ID NO: 5, where the bitter tastant is aristolochic acid. The set immediately to the right represents cells expressing a polypeptide of SEQ ID NO: 7, where the bitter tastant is chloroquine. The set immediately to the right represents cells expressing a polypeptide of SEQ ID NO: 9, where the bitter tastant is strychnine. The rightmost bar represents a standard control.

[0017] Detailed Description The following detailed description illustrates various aspects and embodiments provided herein. This description is intended to be interpreted from the perspective of one of ordinary skill in the relevant art. As such, it may not necessarily contain information that is known to those of ordinary skill in the art.

[0018] definition The following terms and phrases have the meanings set forth below unless otherwise specified herein. Other terms and phrases may be used in this disclosure that are not expressly defined herein. Such other terms and phrases have the meanings that they would have to one of ordinary skill in the art within the context of this disclosure. In some cases, a term or phrase may be defined in the singular or plural. In such cases, it is understood that any term in the singular form may include its plural counterpart, and vice versa, any term in the plural form may include its singular counterpart, unless expressly indicated to the contrary.

[0019] As used herein, "solvate" refers to a compound formed by the interaction of one or more solvent molecules with one or more compounds described herein. In some embodiments, the solvate is a ingestible solvate, such as a hydrate.

[0020] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. For example, reference to "a substituent" includes a single substituent as well as two or more substituents, and the like.

[0021] As used herein, the words "for example," "for instance," "such as," or "including" are intended to provide examples that further clarify a more general subject matter. Unless expressly indicated otherwise, such examples are provided only as an aid in understanding the embodiments illustrated in this disclosure and are not intended to be limiting in any way. Additionally, these words and phrases do not imply any kind of selection of the disclosed embodiments.

[0022] As used herein, "comprise" or "comprises" or "comprising" or "comprised of" refers to an open-ended group, meaning that the group may contain additional elements in addition to those explicitly listed. For example, the phrase "comprising A" means that A must be present, but other elements may also be present. The terms "include", "have", and "composed of" and their grammatical variations have the same meaning. In contrast, "consist of" or "consists of" or "consisting of" refers to a group with limitations. For example, the phrase "consisting of A" means that A is present and only A is present.

[0023] As used herein, "optionally" means that the subsequently described event may or may not occur. In some embodiments, the optional event does not occur. In some other embodiments, the optional event occurs one or more times.

[0024] As used herein, "or" should be given its broadest reasonable interpretation and should not be limited to an either-or interpretation. Thus, the phrase "comprising A or B" means that A is present and B may not be present, or B is present and A is absent, or both A and B are present. Furthermore, for example, if A defines a set that may have multiple elements, e.g., A1 and A2, one or more elements of the set may be present at the same time.

[0025] "Bitter compound" refers to a compound that induces a detectable bitter flavor in a subject, e.g., a compound that activates a T2R receptor in vitro. "Bitter taste receptor blocker" or "bitter taste blocker" refers to a compound that antagonizes the activation of one or more T2R receptors in vitro by a bitter compound.

[0026] As used herein, the terms "sweetener," "sweet flavoring agent," "sweet flavoring substance," or "sweet compound" refer to a compound or an acceptable salt thereof that induces a detectable sweet flavor in a subject, e.g., a compound that activates the T1R2 / T1R3 receptor in vitro.

[0027] Other terms not included in this subsection are defined elsewhere in this description.

[0028] Cells expressing T2R receptors In certain embodiments, the present disclosure provides U2OS cells, which are cells that contain a T2R bitter receptor. In certain related embodiments, the present disclosure provides U2OS cells, which are cells that contain an exogenous nucleic acid sequence that encodes a T2R bitter receptor.

[0029] In humans, about 25 T2R receptors are known. In some embodiments, the T2R bitter receptor is a human T2R bitter receptor, or a functional fragment thereof.

[0030] In some embodiments, the T2R bitter taste receptor is a polypeptide set forth in SEQ ID NO: 1, or a functional fragment thereof. SEQ ID NO: 1 is [ka] It is.

[0031] In some embodiments, the polypeptide set forth in SEQ ID NO:1 corresponds to the polynucleotide set forth in SEQ ID NO:2. [ka] It is.

[0032] In some embodiments, the T2R bitter taste receptor is a polypeptide set forth in SEQ ID NO:3, or a functional fragment thereof. SEQ ID NO:3 is [ka] It is.

[0033] In some embodiments, the polypeptide set forth in SEQ ID NO:3 corresponds to the polynucleotide set forth in SEQ ID NO:4. [ka] It is.

[0034] In some embodiments, the T2R bitter taste receptor is a polypeptide set forth in SEQ ID NO:5, or a functional fragment thereof. SEQ ID NO:5 is [ka] It is.

[0035] In some embodiments, the polypeptide set forth in SEQ ID NO:5 corresponds to the polynucleotide set forth in SEQ ID NO:6. [ka] It is.

[0036] In some embodiments, the T2R bitter taste receptor is a polypeptide set forth in SEQ ID NO: 7, or a functional fragment thereof. SEQ ID NO: 7 is [ka] It is.

[0037] In some embodiments, the polypeptide set forth in SEQ ID NO:7 corresponds to the polynucleotide set forth in SEQ ID NO:8. [ka] It is.

[0038] In some embodiments, the T2R bitter taste receptor is a polypeptide set forth in SEQ ID NO: 9, or a functional fragment thereof. SEQ ID NO: 9 is [ka] It is.

[0039] In some embodiments, the polypeptide set forth in SEQ ID NO:9 corresponds to the polynucleotide set forth in SEQ ID NO:10. [ka] It is.

[0040] The sequences set forth above are described in detail in the Sequence Listing text file, which is submitted herewith and incorporated by reference as if set forth herein in its entirety.

[0041] In some embodiments, U2OS cells are engineered to overexpress one or more T2R bitter taste receptor proteins. U2OS cells can be engineered to overexpress any T2R bitter taste receptor, such as human bitter taste receptor. In some embodiments, U2OS cells are engineered to overexpress one or more polypeptides as set forth in SEQ ID NO: 1, 3, 5, 7, or 9.

[0042] In some embodiments, the cell further comprises an exogenous nucleic acid sequence encoding a G protein. In some further embodiments, the G protein is gustducin, such as α-gustducin. In some other embodiments, the G protein is a promiscuous G protein. Non-limiting examples of promiscuous G proteins include Gα16, Gα15, Gα16gust25, Gα15gust25, Gα16gust44, Gα15gust44, and Gα15-i / 3-5, as described in U.S. Pat. No. 10,107,794. In some other embodiments, the G protein is a G(i / o) protein.

[0043] In some embodiments, the cell further comprises a G protein coupled to a T2R bitter taste receptor. In some further embodiments, the G protein is a gustducin, such as α-gustducin. In some other embodiments, the G protein is a promiscuous G protein. Non-limiting examples of promiscuous G proteins include Gα16, Gα15, Gα16gust25, Gα15gust25, Gα16gust44, Gα15gust44, and Gα15-i / 3-5, as described in U.S. Pat. No. 10,107,794. In some other embodiments, the G protein is a G(i / o) protein.

[0044] Human U2OS cells are well known in the art and are generally commercially available, for example, from ATCC, catalog number HTB-96. In some embodiments, the U2OS cells are isolated U2OS cells. In some embodiments, the U2OS cells are contained in an assay. In some embodiments, the U2OS cells are attached to a substrate. Assays that measure changes in intracellular calcium cation concentration (e.g., Ca Fluorometric Imaging Plate Reader) 2+A variety of functional assays can be used, including a functional assay that measures pERK1 / 2 activation (e.g., high content imaging, HCI), and an assay that measures receptor internalization (e.g., TRANSFLUOR). A change in T2R bitter receptor activity by the test compound or by the bitter tastant in the presence of the test compound indicates modulation of the T2R bitter receptor by the test compound, thereby identifying the test compound as a T2R bitter taste modulator, i.e., a molecule that confers a bitter taste, blocks some or all of the bitter taste of a bitter tastant, or enhances the bitter taste of another molecule. An increase in T2R bitter receptor activity indicates that the test compound is a bitter taste modulator. Bitter taste modulators can be included in a variety of consumable products, including foods, beverages, and pharmaceuticals.

[0045] In some cases, cells other than human U2OS cells can be used. Non-limiting examples of such cells include the following cell types: 1A2, ARH-77, RWPE-1, WI-38, EJM, NCI-H1155, L-1236, NCI-H526, JM1, SHP-77, SNU-878, NCI-H2196, C3A, CA46, SNU-466, KS-1, SNU-738, MOLP-2, HDLM-2, Pfeiffer, HCC-15, Alexander cells, L-540, KMS-12-BM, JK-1, NCI-H1092, SW 1990, NCI-H1184, SU-DHL-1, Hep 3B2.1-7, P3HR-1, NCI-H2029, SU-DHL-5, SNU-1, MOLP-8, SUP-M2, MONO-MAC-1, SNU-1040, KYM-1, HEC-59, HCC1569, OCI-LY3, Hs 819.T, DU4475, CI-1, S-117, OVCAR-8, SNU-626, HL-60, SUIT-2, T3M-4, RKO, MOR / CPR, DK-MG, GA-1 0, OCUM-1, HCT-15, HT, MONO-MAC-6, G-402, Toledo, COV362, SU-DHL-8, Daoy, NCI-H1435, LS513, Hs 839.T, Hs 172.T, BT-483, KMS-21BM, AGS, NCI-H2172, LC-1 / sq-SF, SNU-201, NUGC-4, SK-HEP-1, SUP-B15, SNU-5, HT-1197, SUP-T1, AMO-1, KU812, AN3 CA, AML-193, VMRC-RCW, HLE, HuH28, Hs 751.<h2 style=";text-align:left;direction:ltr">T、NCI-H2110、MEG-01、MV-4-11、Hep G2、KYSE-30、KALS-1、BICR 6、RMUG-S、JHH-6、Ki-JK、IST-MES1、HCC-95、HPB-ALL、HSC-3、697、LOU-NH91、KARPAS-299、GI-1、COLO 792、SK-N-FI、D341 Med、HGC-27、SR-786、COLO-818、MHH-CALL-2、SF126、NCI-H322、A-253、NCI-H1623、MCF7、HCC-44、FU97、OCI-LY-19、Hs 766T、NCI-H522、RL、HCC1428、RPMI 6666、U-937、NCI-H460、SW 1088、NCI-H1792、NCI-H1693、UACC-257、JHUEM-2、HuT 78, UACC-893, NCI-H929, A-704, OV56, LN-229, OE19, SK-MEL-24, RD-ES, NCI-H211, KCI-MOH1, NCI-H1963, Hs 706.T, ChaGo-K-1, EPLC-272H, OPM-2, KHM-1B, A549, HuG1-N, NCI-H508, MHH-CALL-3, SNU-1076, A3 / KAW, MEL-HO, TO 175.T, Caki-1, Hs 936.T, SK-LU-1, WM-983B, K-562, EFE-184, SNU-520, NCI-H2291, HCC-1195, ABC-1, KE-39, NH-6, HCC2218, CMK, RS4;11, KYSE-450, OV7, K YSE-510, SK-UT-1, SNU-C1, OE33, P12-ICHIKAWA, DLD-1, COV434, HuNS1, SNU-899, SW480, COLO-678, LU99, KOPN-8, NCI-H2227, SW1463, Hs 675.T、JHH-4、NCI-H1703、HEC-1-A、BDCM、MIA PaCa-2, PC-3, TE-15, PK-45H, MKN-45, HCC-366, CAL-29, HEC-50B, CPC-N, KMRC-20, SW1116, EOL-1, COLO 205、EHEB、YD-38、MC116、SK-N-BE(2)、BV-173、NCI-H2347、LU65、RT4、U-87 MG、LK-2、KP-N-YN、HEC-251、NCI-H1651、GP2d、RERF-LC-MS、NB-4、NCI-H2286、SNU-61、T-47D、huH-1、SNU-18K-SW86 1353、M-07e、KASUMI-1、YH-13、NCI-H28、GAMG、JeKo-1、GOS-3、SNU -324、PA-TU-8902、MFE-280、SNU-245、NALM-1、RERF-LC-Sq1、BICR 22、ZR-75-1、COR-L23、SW579、COR-L88、KM12、Hs 611.T、OUMS-23、RERF-LC-Ad1、NCI-H1385、SK-LMS-1、COLO-320、BL-70、GRANTA-519、MCAS、Panc 08.13、AM-38、KMS-11、SIG-M5、SNU-407、JHOS-2、OVCAR-4、Set-2、OV-90、MeWo、HEL、HT-29、MDA -MB-231, TOV-21G, NCI-H1355, KMS-27, NALM-6, KMS-26, Caov-4, KASUMI-2, UACC-62, U266B1, Hs 695T, HT55, BICR 31, TCC-PAN2, KMS-20, Hs 578T, RI-1, Hs 606.T, NCI-H1341, THP-1, BCP-1, Hs 737.T、SW1417、MOLT-4、Raji、ESS-1、MEL-JUSO、SH-10-TC、Hs 683、ME-1、EB2、PLC / PRF / 5、NCI-H1339、A4 / Fuk、SEM、HEC-265、IST-MES2、KE-97、N CI-H1437, COLO-704, NCI-H1915, TE-5, NCI-H2023, NCI-H82, T1-73, SNU-840, HuT 102、NCI-H1944、KYSE-520、Kasumi-6、1321N1、Hs 742.T, IM95, PL45, CL-40, WM1799, KMM-1, SNU-449, JHUEM-1, KARPAS-620, Loucy, SNU-1079, Da are HCC-56, HSC-2, COR-L47, PA-TU-89885, OAW28, COR-L311, L-363, Malme-3M, NOMO-1, Hs 870.T、SU-DHL-10、Hs 229.T、NCI-H810、KYSE-410、RPMI-8402、SNU-175、EBC-1、RVH-421、K029AX、PA-TU-8988T、LXF-289、HOVSA-H8 940.T、MM1-S、SUP-HD1、LNCaP、、ーンFGC、HSC-4、NU-DHL-1、NCI-H2228、BEN、CAL-78、Sq-1、NCI-H1993、NU-S C2A, MDA-MB-134-VI, COV318, KE-37, TYK, MOTN-1, T98G, SW837, EB1, Becker, PE / CA-PJ34, C 616.T, NCI-H446, WM-88, CHP-126, Calu-1, SNU-283, NCI-H1573, SW 1271, SNU-16, JHOS-4, ACHN, Calu-3, MRC-1 1783, TE-11, TE-9, HuH-6, P31 / FUJ, HT-1376, NCI-H520, 786-O, KNS-60, Caki-2, OVK18, PL-21, NCI-21 H2452, JURL-MK1, TEN, JHH-7, MDA-MB-157, Calu-6, RKN, NUGC-2, ONS-76, J82, OUMS-27, SNU-1196, Hs 739.T、RPMI-7951、NCI-H854、JHH-5、JVM-2、Hey-A8、5637、KYSE-140、Capan-2、KYSE-150、HEC-1-B16、HEC-1-B16、H.7、MHH-NB-11、SNU-387、SK-OV-3、SK-MEL-28、IGROV1、ML-1、HLF-a、C HL-1, YKG1, A-204, OCI-M1, 8505C, JVM-3, NCI-H647, DB, COLO-800, P K-59, FaDu, HLF, OVMANA, EFO-27, PF-382, NCI-H747, LS123, SU-DHL- 6、SJRH30、PANC-1、NCI-H2342、KM-H2、DND-41、HH、HuCCT1、F-36P、DMS 454、Hs 274.T、AU565、NCI-H1666、EN、RH-41、NCI-H1373、NCI-H838、SK-MEL-30、MOLM-6、DEL、NCI-H226、NC I-H1648, NCI-H661, 143B, Mino, C32, KMS-34, NCI-H1694, SK-ES-1, UACC-812, GDM-1, NCI-H23, Panc 02.03、CCF-STTG1、LOX IMVI、SJSA-1、MDST8、PK-1、NCI-H716、SU-DHL-4、MPP 89、MJ、COLO 829、PE / CA-PJ15、HD-MY-Z、BxPC-3、WM-793、COLO 668、T84、JHOM-1、PEER、LS411N、GMS-10、KMBC-2、RMG-I、KELLY、SNU-761、NALM-19、HEC-151、G-361、OV-AKOW488 900、LCLC-103H、FTC-133、QGP-1、Reh、CMK-11-5、NU-DUL-1、BT-20、Hs 600.T、Hs 604.T、KATO III、SNU-410、NCI-H2126、SK-MEL-5、MDA-MB-468、AsPC-1、HUP-T3、KP-N-S I9s、L-428、SNU-1105、HUP-T4、769-P、LMSU、NCI-H1869、NCO2、MOLM-16、CAL 27、HCC70、NCI-H1930、COV644、Hs 863.T、HCC-2279、D283 Med、Hs 944.<h2 style=";text-align:left;direction:ltr">T、HCC1599、MDA-MB-415、HCC2157、NCI-H1618、SNU-308、HCC1954、DMS 153、HPAF-II、T24、CJM、VM-CUB1、UM-UC-3、LAMA-84、NCI-H1734、JHH-2、VMRC-RCZ、MFE-319、MDA-MB-453、SNU-503、TOV-1 12D、B-CPAP、GSU、HCC-78、NCI-H2171、CAMA-1、HEC-108、HCC4006、CAL-85-1、NCI-H2122、COLO-699、NCI-H196、LUDLU-1、SW 780、RPMI 8226、LP-1、PC-14、HuTu 80、TT、SW948、22Rv1、HARA、NCI-H596、IPC-298、SCaBER、NCI-H1838、NB-1、Hs 934.T、Hs 895.T、DMS 114、KYSE-70、KP-3、KP4、DAN-G、NCI-H2009、OC 316、SCC-25、U-138 MG、RCC10RGB、MFE-296、NCI-H1755、RERF-LC-KJ、8305C、WSU-DLCL2、ES-2、MSTO-211H、SCC- 15、ZR-75-30、PSN1、SNU-423、NCI-H2106、TE-1、UT-7、KMS-28BM、NCI-H2081、SK-MM-2、COLO 741、OC 314、HCC1395、MOLT-13、LN-18、Panc 10.05、PE / CA-PJ41(クローンD2)、Hs 746T、CW-2、SKM-1、NUGC-3、TE-10、NCI-H358、NCI-H69、BFTC-909、HOS、BICR 18、NCI-H1395、OVKATE、Hs 698.T、EFM-19、COLO-783、MHH-CALL-4、ACC-MESO-1、NCI-H1436、KP-N-RT-BM- 1、SK-MEL-31、NCI-H1105、CAL-51、YD-15、NCI-H2085、NCI-H2444、HCC1187、Hs 939.T、CAL-120、SCC-9、TUHR14TKB、KMRC-2、KG-1-C、ECC10、CG.<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">TH-W-1, NCI-H841, C2BBe1, SUP-T11, RCH-ACV, CADO-ES1, JURKAT, 647-V, SK-MEL-2, MDA-MB-175-VII, MKN74, SNU-C4, LCLC-97TM1, SCC-4, BHY, IGR-37, KYO-1, Hs 281.T, TT, TUHR4TKB, HT-1080, NCI-H660, TE 441.T, LS1034, KNS-42, Panc 04.03、HCC1419、AZ-521、SNG-M、NCI-N87、G-292、クローンA141 B1、KPL-1、MDA-MB-361、CL-14、NCI-H2170、HuH-7、RD、NCI-H 2066、IGR-1、TE-14、VCaP、BL-41、SNU-620、SK-MES-1、MEC-2、NCI-H1299、IGR-39、RT112 / 84、SF-295、DV-90、A2780、BICR 56、NCI-H510、NCI-H2141、YD-8、NCI-H2405、TF-1、MEC-1、CCK-81、NCI-H1048、Hs 822.T、NCI-H2052、K052、CAL-54、Hs 840.T、SW620、SK-CO-1、BT-474、CL-11、KNS-62、NCI-H1650、G-401、MOLT-16、SNU-398、COLO-680N、EM-2、Hs 294T、CAL-62、KMRC-3、A101D、KG-1、BT-549、HT115、A-375、SW-1710、WM-115、KLE、JHUEM-3、MKN7、CHP-212、HC C202、BC-3C、NCI-H1568、KMS-18、PE / CA-PJ49、COLO-849、SIMA、OCI-AML3、GSS、EC-GI-10、EFO-21、RCM-1、DMS 273、KU-19-19、RERF-GC-1B、SH-4、SK-MEL-3、RERF-LC-Ad2、M059K、JHOM-2B、MDA PCa 2b、Hs 852.T、RL95-2、Panc 03.27、SNU-216、Panc 02.13、CFPAC-1、SK-N-SH、OCI-AML2、LoVo、SBC-5、NCI-H1876、NCI-H44 1、SK-N-AS、COR-L24、HCC38、NCI-H1781、DOHH-2、NCI-H1563、U-251 MG、HPAC、JIMT-1、U-2 OS、A-673、TC-71、NCI-H650、NIH:OVCAR-3、CAS-1、JL-1、SK-MEL-1、MDA-MB-4355、Ii Sikawa (TEER) 617.T、SU.86.86、RERF-LC-AI、TT2609-C02、LS 180、YAPC、HDQ-P1、KNS-81、FU-OV-1、KP-2、DMS 53、SNU-1722Detroit 562、42-MG-BA、L3.3、COLO-679、NCI-H2087、NCI-H2030、GCT、NCI-H889、Caov -3, MDA-MB-436, NCI-H524, MKN1, KCL-22, Capan-1, CML-T1, H4, NCI-H727, Hs 343.T、MHH-ES-1、NMC-G1、HCC-1171、REC-1、Hs 618.T、A172、YD-10B、SW48、MUTZ-5、TE-6、JHH-1、HCT 116、TE-4、IA-LM、MG-63、NCI-H1975、TALL-1、HCC1806、HMCB、SCLC-21H、HCC1500、CL-34、Panc 05.04、SW403、TM-31、HCC1937、JMSU-1、DMS 79、SNB-19、NCI-H1836、Li-7、HCC827、639-V、MOLM-13、SK-BR-3、IMR-32、TUHR10TKB、 OAW42、SK-N-MC、TGBC11TKB、NCI-H1581、EFM-192A、YMB-1、HCC2935、ECC12、HCC-33、DU 145、NCI-H146、SNU-1214、SNU-1077、23132 / 87、HT-144、SNU-182、Hs 888.T、SNU-475、GCIY、Hs 729、JHOC-5SW 1573、HEC-6、OCI-AMLS、Hs 688(A).T、Hs 821.T, PCM6, RT-112, SK-N-DZ, SNU-478, SNU-119, HCC1143, NCI-H209, 8-MG-BA, COR-L105, COR-L95, SNU-46, CO V504, CAL-148, SNU-05, DBTRG-05MG, BHT-101, WM-266-4, BFTC-905, KYSE-270, TE-8, SNU-213, and SH-SYSY. .

[0046] Screening Methods In certain aspects, the present disclosure provides a method for identifying bitter taste modulators, comprising: (a) contacting one or more U2OS cells expressing bitter taste receptors (according to any of the embodiments shown above) with a test compound; and (b) measuring T2R bitter taste receptor activity, wherein a change in T2R bitter taste activity indicates that the test compound is a bitter taste modulator. In some embodiments, the method comprises identifying an active test compound that modulates bitter taste based on the measured response. In some embodiments, the method comprises selecting an active test compound as a compound that modulates bitter taste.

[0047] In certain aspects, the present disclosure provides a method for identifying a compound that reduces bitterness, comprising: (a) contacting one or more U2OS cells expressing the bitter receptor of the first aspect with a test compound and a bitter tastant; and (b) measuring the response of the T2R bitter receptor by comparing the activity of the T2R bitter receptor to the bitter tastant in the presence and absence of the test compound, wherein a reduction in T2R bitter receptor activity indicates that the test compound is a bitter blocker. In some embodiments, the method comprises identifying the test compound as a bitter blocker based on a change in T2R bitter receptor activity. In some embodiments, the method comprises selecting the bitter blocker as a compound that reduces bitterness.

[0048] The contacting can be carried out in any suitable manner.For example, in some non-limiting examples, the introduction can be carried out in a cell-based assay, where the test compound (and in certain embodiments, the bitter compound) is contacted with a cell containing a T2R bitter taste receptor.Such cell-based assays are well known in the art.

[0049] The foregoing embodiments involve measuring the response of the taste receptor protein to the test compound. This measurement can be performed by any suitable means. For example, in some embodiments, each taste receptor protein is expressed on the surface of a cell, and compositions comprising the test compound (and in certain aspects the bitter compound) are screened against cells expressing the taste receptor protein in a standard cell assay. Measurement of binding can be performed by any suitable means typically used in determining protein binding in cell assays. Suitable methods include, but are not limited to, the use of calcium fluorescent dyes, calcium labeled proteins, fluorescent cAMP labels, and the like. Activity of the test compound is determined by the ability of the test compound to antagonize the binding of the bitter compound to one or more taste receptor proteins. In some embodiments, the U2OS cells contain a detectable label or an exogenous nucleic acid sequence encoding a detectable label. In some embodiments, the detectable label is β-arrestin GFP.

[0050] In some embodiments of any of the above embodiments, the method further comprises identifying an active test compound that reduces or modulates bitterness based on the measured response.In some embodiments, the identified active test compound antagonizes the binding of the bitter compound to taste receptor protein.For example, in some embodiments, the identified active test compound is a compound that antagonizes the binding of the bitter compound to one of the above taste receptor proteins.

[0051] In some embodiments of any of the foregoing embodiments, the test compound is determined to be an active compound because it antagonizes at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the binding activity of the bitter compound.

[0052] In some further embodiments, the method further comprises selecting an active test compound as a compound that reduces bitterness. After identifying an active test compound, the selection can be performed by any suitable means.

[0053] The test compound may be any suitable compound suitable for use in a cell screening assay. For example, in some embodiments, the test compound is an organic compound. In some embodiments, the test compound is a naturally occurring compound or a glycosylated derivative thereof. In some other embodiments, the test compound is a non-naturally occurring compound.

[0054] In aspects and embodiments using bitter tastants, the bitter tastant can be any suitable compound that functions as a bitter tastant, i.e., any compound that agonizes one or more T2R bitter taste receptors. Non-limiting examples of bitter tastants suitable for use in the assay include picric acid, chloroquine, picoline, andrographolide, ofloxacin, strychnine, oxyphenonium, aristolochic acid, salicin, denatonium benzoate, N-propylthiouracil, cinchonine, caffeine, oleuropein, steviol glycosides (such as rebaudioside A or rebaudioside M), mogrosides (such as mogroside V, siamenoside I, isomogroside V, alpha isomer of mogroside V), bitter plant extracts (e.g., citrus extracts containing liminin, nomellin, naringin, etc.), and active pharmaceutical ingredients.

[0055] Uses, Methods, and Ingestible Compositions In other aspects, the disclosure provides for the use of an identified or selected active compound of any of the preceding aspects, including any embodiment or combination of embodiments set forth above.

[0056] In certain aspects, the present disclosure provides for the use of an active test compound identified or selected by any of the screening methods disclosed herein to reduce the bitter taste of an ingestible composition.

[0057] In a related aspect, the disclosure provides a method for reducing the bitterness of an ingestible composition, the method comprising introducing into the ingestible composition an amount (such as a bitterness-reducing effective amount) of an active test compound identified or selected by any of the screening methods disclosed herein.

[0058] The aforementioned uses and methods generally involve the use of the identified or selected active compound in a composition containing one or more additional ingredients. For example, in at least one aspect, the present disclosure provides a composition comprising the identified or selected active compound of any of the aforementioned aspects, including any embodiment or combination of those embodiments shown above, wherein the identified or selected active compound comprises at least 50% by weight of the composition on a dry weight basis (e.g., the total weight of the composition excluding the weight of any liquid carrier). In a related aspect, the present disclosure provides a solid composition comprising the identified or selected active compound of any of the aforementioned aspects, including any embodiment or combination of those embodiments shown above, wherein the identified or selected active compound comprises at least 50% by weight of the solid composition on a total weight basis of the composition. In another related aspect, the present disclosure provides an ingestible composition comprising the identified or selected active compound of any of the aforementioned aspects, including any embodiment or combination of those embodiments shown above, wherein the concentration of the identified or selected active compound in the ingestible composition is 200 ppm or less. In another related aspect, the disclosure provides an ingestible composition comprising the identified or selected active compound of any of the preceding aspects, including any embodiment set forth above or combinations of those embodiments, wherein the ingestible composition comprises 1000 ppm or less, or 900 ppm or less, or 800 ppm or less, or 700 ppm or less, or 600 ppm or less, or 500 ppm or less, or 400 ppm or less, or 300 ppm or less, or 200 ppm or less, or 100 ppm or less, or 50 ppm or less, etc.

[0059] Of any of the aspects and embodiments presented herein that refer to an ingestible composition, in certain embodiments, the ingestible composition is a non-naturally occurring product, such as a composition that is specially manufactured for the production of a flavored product, such as a food or beverage item.

[0060] Thus, in some embodiments, the composition according to any of the above aspects (including any of the uses or methods) comprises an identified or selected active compound and a sweetener. In some embodiments, the composition further comprises a vehicle. In some embodiments, the vehicle is water.

[0061] In some other embodiments, the sweetener is present in an amount between 10 ppm and 1000 ppm. In some embodiments, the sweetener is present in an amount between 20 ppm and 800 ppm. In some embodiments, the sweetener is present in an amount between 30 ppm and 600 ppm. In some embodiments, the sweetener is present in an amount between 40 ppm and 500 ppm. In some embodiments, the sweetener is present in an amount between 50 ppm and 400 ppm. In some embodiments, the sweetener is present in an amount between 50 ppm and 300 ppm. In some embodiments, the sweetener is present in an amount between 50 ppm and 200 ppm. In some embodiments, the sweetener is present in an amount between 50 ppm and 150 ppm. In some further embodiments of the embodiments set forth in this paragraph, the sweetener is a derivative of any of the foregoing, such as a steviol glycoside, a mogroside, a glycoside derivative (e.g., a glucosylate), or any combination thereof. In some embodiments, the sweetener is a steviol glycoside, such as rebaudioside A, rebaudioside M, rebaudioside D, rebaudioside B, or any combination thereof. In some embodiments, the sweetener is mogroside V, isomogroside V, isomogroside IV, E , mogroside IIIE, siamenoside I, the alpha isomer of siamenoside I, or any combination thereof. In some embodiments, the sweetener is sucrose, fructose, glucose, allulose, erythritol, or any combination thereof.

[0062] The sweeteners may be natural or synthetic, including, but not limited to, agave inulin, agave nectar, agave syrup, amazake, brazzein, brown rice syrup, coconut crystals, coconut sugar, coconut syrup, date sugar, fructans (also called inulin fiber, fructooligosaccharides, or oligofructose), green stevia powder, stevia rebaudiana), rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside N, rebaudioside O, rebaudioside M, and other sweet stevia glycosides, stevioside, stevioside extract, honey, Jerusalem artichoke syrup, licorice root, Monk fruit (fruit, powder, or extract), lucuma (fruit, powder, or extract), maple sap (e.g., sugar maple (Acer saccharum), black maple (Acer nigrum), red maple (Acer rubrum, silver maple (Acer saccharinum), Norway maple (Acer platanoides), ash maple (Acer negundo), bigleaf maple (Acer macrophyllum), bigleaf maple (Acer grandidentatum), Rocky Mountain maple (Acer glabrum), and silver maple (Acer mono), maple syrup, maple sugar, walnut sap (including sap extracted from, for example, butternut (Juglans cinerea), black walnut (Juglans nigra), Japanese walnut (Juglans ailatifolia), and Persian walnut (Juglans regia), birch sap (including sap extracted from, for example, silver birch (Betula papyrifera), yellow birch (Betula alleghaniensis), sweet beach (Betula lenta), river birch (Betula nigra), gray birch (Betula populifolia),including sap extracted from Betula pendula), sycamore sap (such as sap extracted from Platanus occidentalis), ironwood sap (such as sap extracted from Ostrya virginiana), muscovado, molasses (such as blackstrap molasses), molasses sugar, monatin, monellin, cane sugar (also called natural sugar, unrefined cane sugar, or sucrose), palm sugar, panocha, piloncillo, rapadura, raw sugar, rice syrup, sorghum, sorghum syrup, cassava syrup (also called tapioca syrup), thaumatin, yacon root, malt syrup, barley malt syrup, barley malt flour Powder, beet sugar, cane sugar, crystalline fruit juice crystals, caramel, carbitol, carob syrup, white sugar, hydrogenated starch hydrolysates, hydrolyzed sugar cane juice, hydrolyzed starch, invert sugar, anethole, arabinogalactan, arop, syrup, P-4000, acesulfame potassium (also called acesulfame K or ace-K), alitame (also called aclame), advantame, aspartame, bayunoside, neotame, benzamide derivatives, vernadame ( bernadame), canderel, carrelam and other guanidine sweeteners, vegetable fiber, corn sugar, coupling sugar, curculin, cyclamate, cyclocaryoside I, demerara, dextran, dextrin, malt sugar, dulcin, sucrose, vulgin, dulcoside A, dulcoside B, emulin, enoxolone, maltodextrin, saccharin, estragole, ethyl maltol, glycine, gluconic acid, gluconolactone, glucosamine, glucuronic acid ... Ronic acid, glycerol, glycine, glycyphylline, glycyrrhizin, glycyrrhetinic acid monoglucuronide, golden sugar, yellow sugar, golden syrup, granulated sugar, Jiaogulan, Hernandursin, isomerized liquid sugar, Jalab, chicory root dietary fiber, kynurenine derivatives (including N'-formyl-kynurenine, N'-acetyl-kynurenine, 6-chloro-kynurenine), galactitol, ritesse, ligicane, lycasin, ragduname,Guanidine, Falernum, Mabinlin I, Mabinlin II, Maltol, Multisorb, Maltodextrin, Maltotriol, Mannosamine, Miraculin, Starch Syrup, Mogrosides (including, for example, Mogroside IV, Mogroside V, and Neomogroside), Muclodiosides, Nanosugar, Naringin Dihydrochalcone, Neohesperidin Dihydrochalcone, Nib Sugar, Nigerooligosaccharides, Norbu, Orgate Syrup, Osladin, Pekmez, Pentasin, Perian Dorin I, Perillaldehyde, Perillartine, Petphyllum, Phenylalanine, Phlomisoside I, Phlorodizin, Phyllodulcin, Polyglycitol Syrup, Polypodoside A, Pterocaryoside A, Pterocaryoside B, Rebiana, Refiner Syrup, Rub Syrup, Rubusoside, Serigeain A, Shugr, Siamenoside I, Siraitia grosvenorii, soy oligosaccharides, Splenda, SRI oxime V, steviol glycosides, steviolbioside, stevioside, strogin 1, 2, and 4, sucronate, sucrononate, sugar, suosan, phlorizin, super aspartame, tetrasaccharide, threitol, molasses, trilobtain, tryptophan and derivatives (6-trifluoromethyl-tryptophan, 6-chloro-D-tryptophan), vanilla sugar, volemitol, birch syrup, aspartame-acesulfame, asguline, and combinations or blends of any two or more thereof.

[0063] The identified or selected active compound can be present in the ingestible composition in any suitable amount. In some embodiments, the identified or selected active compound is present in an amount sufficient to enhance the taste of the composition (reduce bitterness). Thus, in some embodiments, the ingestible composition contains the identified or selected active compound at a concentration of 200 ppm or less, or 150 ppm or less, or 100 ppm or less, or 50 ppm or less, or 40 ppm or less, or 30 ppm or less, or 20 ppm or less. In some embodiments, the identified or selected active compound is present in a minimum amount, such as 1 ppm or 5 ppm. Thus, in some embodiments, the ingestible composition comprises the identified or selected active compound at a concentration in the range of 1 ppm to 200 ppm, or 1 ppm to 150 ppm, or 1 ppm to 100 ppm, or 1 ppm to 50 ppm, or 1 ppm to 40 ppm, or 1 ppm to 30 ppm, or 1 ppm to 20 ppm, or 5 ppm to 200 ppm, or 5 ppm to 150 ppm, or 5 ppm to 100 ppm, or 5 ppm to 50 ppm, or 5 ppm to 40 ppm, or 5 ppm to 30 ppm, or 5 ppm to 20 ppm. In embodiments in which a sweetener, such as sucrose or fructose, is present, the weight to weight ratio of sweetener to the identified or selected active compound in the ingestible composition ranges from 1000:1 to 50000:1, or from 1000:1 to 10000:1, or from 2000:1 to 8000:1.

[0064] In certain embodiments, the ingestible composition can include any additional ingredient or combination of ingredients commonly used in food and beverage products, including, but not limited to, Acids including, for example, citric acid, phosphoric acid, ascorbic acid, sodium sulfate, lactic acid, or tartaric acid; bitter ingredients, including, for example, caffeine, quinine, green tea, catechins, polyphenols, green robusta coffee extract, green coffee extract, potassium chloride, menthol, or proteins (such as proteins and protein isolates derived from plants, algae, or fungi); Colorants containing, for example, caramel color, Red No. 40, Yellow No. 5, Yellow No. 6, Blue No. 1, Red No. 3, purple carrot, black carrot juice, purple sweet potato, vegetable juice, fruit juice, beta carotene, turmeric curcumin, or titanium dioxide; Preservatives including, for example, sodium benzoate, potassium benzoate, potassium sorbate, sodium metabisulfite, sorbic acid, or benzoic acid; Antioxidants including, for example, ascorbic acid, calcium disodium EDTA, alpha tocopherol, mixed tocopherols, rosemary extract, grape seed extract, resveratrol, or sodium hexametaphosphate; For example, resveratrol, Co-Q10, omega-3 fatty acids, theanine, choline chloride (citocoline), Fibersol, inulin (chicory root), taurine, ginseng extract, guanine extract, ginger extract, L-phenylalanine, L-carnitine, L-tartrate, D-glucoronolactone, inositol, bioflavonoids, echinacea, ginkgo biloba, yerba mate, flaxseed oil, garcinia cambogia husk extract, white tea extract, ribose, milk thistle extract, grape seed extract, pyrodixine HCl (vitamin B6), cyanocobalamin. Vitamins or functional ingredients including (vitamin B12), niacinamide (vitamin B3), biotin, calcium lactate, calcium pantothenate (pantothenic acid), calcium phosphate, calcium carbonate, chromium chloride, chromium polynicotinate, copper sulfate, folic acid, ferric pyrophosphate, iron, magnesium lactate, magnesium carbonate, magnesium sulfate, monopotassium phosphate, monosodium phosphate, phosphorus, potassium iodide, potassium phosphate, riboflavin, sodium sulfate, sodium gluconate, sodium polyphosphate, sodium bicarbonate, thiamine mononitrate, vitamin D3, vitamin A palmitate, zinc gluconate, zinc lactate, or zinc sulfate; clouding agents, such as ester gum, brominated vegetable oil (BVO), or sucrose acetate isobutyrate (SAIB); For example, sodium citrate, potassium citrate, or salt-containing buffers; For example, propylene glycol, ethyl alcohol, glycerin, gum arabic (gum acacia), maltodextrin, modified corn starch, dextrose, natural flavors, natural flavors containing other natural flavors (natural flavors WONF), natural and artificial flavors, artificial flavors, flavors containing silicon dioxide, magnesium carbonate, or tricalcium phosphate; or Examples include starches and stabilizers, including pectin, xanthan gum, carboxymethylcellulose (CMC), polysorbate 60, polysorbate 80, medium chain triglycerides, cellulose gel, cellulose gum, sodium caseinate, modified food starch, gum arabic (gum acacia), inulin, or carrageenan.

[0065] The ingestible composition can have any suitable pH. In some embodiments, the identified or selected active compound enhances the sweetness of a sweetener over a wide range of pH, for example, from low to neutral pH. Low and neutral pH include, but are not limited to, pHs of about 2.5 to about 8.5; about 3.0 to about 8.0; about 3.5 to about 7.5; and about 4.0 to about 7. In certain embodiments, the compounds disclosed and described herein, individually or in combination, can enhance the perceived sweetness of a fixed concentration of a sweetener in a taste test at a compound concentration of about 50 μM, 40 μM, 30 μM, 20 μM, or 10 μM at both low and neutral pH values. In certain embodiments, the enhancement factor at low pH of the compounds disclosed and described herein, individually or in combination, is substantially similar to the enhancement factor of the compounds at neutral pH. Such consistent sweetness enhancing properties over a wide range of pH allow the compounds disclosed and described herein to be widely used, individually or in combination, in a wide variety of foods and beverages.

[0066] Flavor Products and Concentrates In certain aspects, the present disclosure provides a flavor product comprising the composition of any of the preceding aspects. In some embodiments, the flavor product is a beverage, such as soda, flavored water, tea, and the like. In some other embodiments, the flavor product is a food product, such as yogurt.

[0067] In embodiments where the flavored product is a beverage, the beverage may be selected from the group consisting of enhanced sparkling beverages, cola, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grape flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger ale, root beer, fruit juice, fruit flavored juice, fruit juice drink, nectar, vegetable juice, vegetable flavored juice, sports drink, energy drink, enhanced water drink, vitamin enhanced water, near water drink, coconut water, tea type beverage, coffee, cocoa beverage, beverage containing dairy ingredients, beverage containing grain extract, and smoothie. In some embodiments, the beverage may be a soft drink.

[0068] Of any of the aspects and embodiments presented herein that refer to a flavor product, in certain embodiments, the flavor product is a non-naturally occurring product, such as a packaged food or beverage item.

[0069] Further non-limiting examples of edible and drinkable products or formulations include sweet coatings, frostings, or glazes for such products or any elements included in the soup category, the dried prepared foods category, the beverage category, the ready meals category, the canned or preserved foods category, the frozen prepared foods category, the refrigerated prepared foods category, the snack foods category, the baked goods category, the confectionery category, the dairy category, the ice cream category, the meal replacements category, the pasta and noodle category, and the sauces, dressings, condiments category, the baby food category, and / or the spreads category.

[0070] In general, the soup category refers to canned / preserved, dried, instant, refrigerated, UHT, and frozen soups. For the purposes of this definition, soup means food prepared from meat, poultry, fish, vegetables, grains, fruits, and other ingredients and cooked in a liquid that may contain visible particles of some or all of these ingredients. Soups may be clear (broth-like) or thick (chowder-like), smooth, pureed, or filled, ready-to-drink, semi-concentrated, or concentrated, and may be served hot or cold as a meal or main course, or as a snack (sipped like a beverage). Soups may be used as ingredients to prepare other meal components and may vary from broths (consomme) to sauces (cream or cheese-based soups).

[0071] The dry and culinary food category generally refers to: (i) cooking aid products such as powders, granules, pastes, concentrated liquid products, e.g., pressed cubes, tablets, or concentrated bouillons, bouillons, and bouillon-like products in powder or granular form, sold separately as finished products or as ingredients in products, sauces, and recipe mixes (regardless of technology); (ii) meal solution products such as dried soup mixes, dried instant soups, dried semi-cooked soups, dried or ambient prepared ready-made dishes, dried soups and freeze-dried soups, and meals and a la carte dishes, including pasta, potato, and rice dishes; and (iii) meal garnish products such as condiments, marinades, salad dressings, salad toppings, dips, bread crumbs, batter mixes, shelf-stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, including recipe mixes for salads, whether dried, liquid, or frozen, sold as finished products or as ingredients in products.

[0072] The beverage category generally refers to beverages, beverage mixes, and concentrates, including but not limited to carbonated and non-carbonated beverages, alcoholic and non-alcoholic beverages, ready-to-drink beverages, liquid concentrated formulations for preparing beverages such as sodas, and dry powdered beverage precursor mixes. The beverage category also includes alcoholic beverages, soft drinks, sports drinks, isotonic drinks, and hot drinks. Alcoholic beverages include but are not limited to beer, cider / perry, FAB, wine, and spirits. Soft drinks include but are not limited to carbonated beverages, such as cola and non-cola carbonated drinks; fruit juices, such as fruit juices, nectars, juice drinks, and fruit-flavored drinks; bottled waters, including sparkling water, spring water, and purified water / table water; functional beverages, such as sports drinks, energy drinks, or elixirs, which may be carbonated or non-carbonated; and concentrates, such as ready-to-drink liquid concentrates and powder concentrates. Hot or cold beverages include, but are not limited to, coffee or iced coffee, such as fresh coffee, instant coffee, and blended coffee; tea or iced tea, such as black tea, green tea, white tea, oolong tea, and flavored tea; and other beverages, including flavored, malted, or plant-based powders, granules, blocks, or tablets mixed with milk or water.

[0073] The snack food category generally refers to any food that can be an informal snack, including but not limited to sweet and savory snacks and snack bars. Examples of snack foods include, but are not limited to, fruit snacks, chips / crisps, formed snacks, tortilla / corn chips, popcorn, pretzels, nuts, and other sweet and savory snacks. Examples of snack bars include, but are not limited to, granola / muesli bars, breakfast bars, nutrition bars, fruit bars, and other snack bars.

[0074] The baked goods category generally refers to any edible product whose cooking process involves exposure to heat or excessive sunlight. Examples of baked goods include, but are not limited to, bread, buns, cookies, muffins, cereals, toaster pastries, pastries, waffles, tortillas, biscuits, pies, bagels, tarts, quiches, cakes, any baked food product, and any combination thereof.

[0075] The ice cream category generally refers to frozen desserts that contain cream, sugar, and flavors. Examples of ice cream include, but are not limited to, ice cream for store consumption; ice cream for takeaway; frozen yogurt and specialty ice cream; soy, oat, bean (e.g., red and mung bean), and rice-based ice cream.

[0076] The confectionery category generally refers to sweet edible products. Examples of confectionery include, but are not limited to, candy, gelatin, chocolate confectionery, sugar confectionery, gum, and the like, and any combination products.

[0077] The meal replacement category generally refers to any food product intended to replace a regular meal, especially for health or fitness conscious people. Examples of meal replacements include, but are not limited to, weight loss products and illness products.

[0078] The ready meal category generally refers to any food that can be served as a meal without extensive cooking or processing. Ready meals include products where the manufacturer adds recipe "skill" to enhance speed, completeness, and convenience. Examples of ready meals include, but are not limited to, canned / preserved, frozen, dehydrated, and refrigerated ready meals; dinner mixes; frozen pizza; refrigerated pizza; and pre-prepared salads.

[0079] The pasta and noodles category includes all pastas and / or noodles, including, but not limited to, canned, dried, and refrigerated / fresh pastas; and plain, instant, refrigerated, frozen, and snack noodles.

[0080] Canned / preserved food categories include, but are not limited to, canned / preserved meat and meat products, fish / seafood, vegetables, tomatoes, beans, fruit, ready meals, soups, pasta, and other canned / preserved foods.

[0081] Frozen processed food categories include, but are not limited to, frozen processed red meat, processed poultry, processed fish / seafood, processed vegetables, meat substitutes, processed potatoes, bakery products, desserts, ready meals, pizza, soups, noodles, and other frozen foods.

[0082] Dry packaged food categories include, but are not limited to, cooked rice, dessert mixes, dry ready meals, dry soups, instant soups, dry pastas, plain noodles, and instant noodles. Refrigerated packaged food categories include, but are not limited to, refrigerated processed meats, processed fish / seafood products, lunch kits, fresh cut fruit, ready meals, pizza, prepared salads, soups, fresh pastas, and noodles.

[0083] The sauces, dressings, and condiments category includes, but is not limited to, tomato paste and puree, bouillon / stock cubes, herbs and spices, monosodium glutamate (MSG), table sauces, soy-based sauces, pasta sauces, wet / cooking sauces, dry sauces / powder mixes, ketchup, mayonnaise, mustard, salad dressings, vinaigrettes, dips, pickle products, and other sauces, dressings, and condiments.

[0084] Baby food categories include, but are not limited to, milk-based or soy-based formulas; as well as cooked, dry, and other baby foods.

[0085] Spreads categories include, but are not limited to, jams and preserves, honey, chocolate spreads, nut-based spreads, and yeast-based spreads.

[0086] The dairy category generally refers to edible products made from mammalian milk. Examples of dairy products include, but are not limited to, drinking dairy products, cheese, yogurt, and sour milk beverages, as well as other dairy products.

[0087] Further examples of flavor products, particularly edible and drinkable products or formulations, are given below. Exemplary ingestible compositions include one or more of confectionery, chocolate confectionery, tablets, count lines, self-lined / soft lines in bags, boxed assortments, standard boxed assortments, twist-wrapped miniatures, seasonal chocolates, chocolates with toys, alfajores, other chocolate confectionery, mints, standard mints, strong mints, hard candies, lozenges, gums, jellies and chewing confectionery, toffees, caramels and nougat, medicated confectionery, lollipops, liquorice, other sugar confectionery, bread, packaged / commercial bread, unpackaged / specialty bread, pastries, cakes, packaged / commercial cakes, unpackaged / specialty cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, rte cereals, home breakfast cereals, flakes, muesli, other cereals, children's breakfast cereals, hot cereals, ice cream, ice cream for store consumption, individually packaged dairy products, ice cream, individually wrapped ices, multipack dairy ice cream, multipack ices, takeaway ice cream, takeaway dairy ice cream, ice cream desserts, bulk ice cream, takeaway ice cream, frozen yogurt, specialty ice cream, dairy products, milk, raw / pasteurized milk, raw / pasteurized full fat milk, raw / pasteurized semi-skimmed milk, long life / uht milk, long life / uht full fat milk, long life / uht semi-skimmed milk, long life / uht non-fat milk, goat's milk, condensed / evaporated milk , Plain Condensed Milk / Evaporated Milk, Flavoured, Functional and Other Condensed Milk, Flavoured Milk Drinks, Dairy Only Flavoured Milk Drinks, Flavoured Milk Drinks with Fruit Juice, Soy Milk, Sour Milk Drinks, Fermented Dairy Drinks, Coffee Whitener, Milk Powder, Flavoured Powdered Milk Drinks, Cream, Cheese, Processed Cheese, Spreadable Processed Cheese, Non-Spreadable Processed Cheese, Non-Processed Cheese, Spreadable Non-Processed Cheese, Hard Cheese, Packaged Hard Cheese, Unpackaged Hard Cheese, Yogurt,Plain / Natural Yogurt, Flavoured Yogurt, Yogurt with Fruit, Probiotic Yogurt, Drinkable Yogurt, Drinkable Yogurt, Probiotic Drinkable Yogurt, Refrigerated & Shelf-Stable Desserts, Dairy-Based Desserts, Soy-Based Desserts, Chilled Snacks, Fromagee Frais & Quark, Plain Fromagee Frais & Quark, Flavoured Fromagee Frais & Quark, Flavoured Fromagee Frais & Quark, Sweet & Savoury Snacks, Fruit Snacks, Chips / Crisps, Formed Snacks, Tortilla / Corn Chips, Popcorn, Pretzels, Nuts, Other Sweet & Savoury Snacks, Snack Bars, Granola Bars, Breakfast Bars, Nutrition Bars, Fruit Bars, Other Snack Bars, Meal Replacement Products, Weight Loss Products, Recovery Drinks, Ready Meals, Canned Ready Meals, Frozen Ready Meals, Dried Ready Meals, Refrigerated Ready Meals, Dinner Mixes, Frozen Pizza, Refrigerated Pizza, Soup, Canned Soups, Dried Soups, Instant Soups, Refrigerated Soups, Hot Soups, Frozen Soups, Pasta, Canned Pasta, Dried Pasta, Refrigerated / Raw Pasta, Noodles, Plain noodles, Instant noodles, Cup / bowl instant noodles, Pouch instant noodles, Refrigerated noodles, Snack noodles, Canned foods, Canned meat and meat products, Canned fish / seafood, Canned vegetables, Canned tomatoes, Canned beans, Canned fruit, Canned ready meals, Canned soups, Canned pasta, Other canned foods, Frozen foods, Frozen processed red meat, Frozen processed chicken, Frozen processed fish / seafood, Frozen processed vegetables, Frozen meat substitutes, Frozen potatoes, Oven baked potato chips, Other oven baked potato products, Non oven baked frozen potatoes, Frozen bakery products, Frozen Desserts, Frozen ready meals, Frozen pizza, Frozen soups, Frozen noodles, Other frozen foods, Dry foods, Dessert mixes, Dry ready meals, Dry soups, Instant soups, Dry pasta, Plain noodles, Instant noodles, Cup / bowl instant noodles, Pouch instant noodles, Refrigerated foods, Refrigerated processed meat, Refrigerated fish / seafood products, Refrigerated processed fish, Refrigerated battered fish, Refrigerated smoked fish, Refrigerated lunch kits, Refrigerated ready meals, Refrigerated pizza, Refrigerated soups, Refrigerated / fresh pasta, Refrigerated noodles, Oils and fats, Olive oil, Vegetable oils and seed oils, Cooking fats, Butter, Margarine,spreadable fats and oils, functional spreadable fats and oils, sauces, dressings and condiments, tomato paste and puree, bouillon / stock cubes, stock cubes, gravy granules, liquid stocks and fonds, herbs and spices, fermented sauces, soy based sauces, pasta sauces, wet sauces, dry sauces / powder mixes, ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings, regular salad dressings, low fat salad dressings, vinaigrettes, dips, pickle products, other sauces, dressings and condiments, baby food, formula, standard formula, follow-up formula, toddler formula, hypoallergen formula, prepared baby food, dry baby food, other baby food, spreads, jams and preserves, honey, chocolate spreads, nut based spreads, and yeast based spreads. Exemplary ingestible compositions also include confectionery, bakery products, ice cream, dairy products, sweet and savory snacks, snack bars, meal replacement products, ready meals, soups, pastas, noodles, canned foods, frozen foods, dry foods, refrigerated foods, fats and oils, baby foods, or spreads, or mixtures thereof. Exemplary ingestible compositions also include breakfast cereals, sweetened drinks, or solid or liquid concentrate compositions for preparing beverages, ideally allowing for reduced concentrations of known sugar or artificial sweeteners.

[0088] Some embodiments provide chewable compositions that may or may not be intended to be swallowed. In some embodiments, the chewable compositions may be gums, chewing gums, sweetened gums, sugarless gums, functional gums, bubble gums, containing the compounds disclosed and described herein, individually or in combination.

[0089] In some embodiments, the compounds disclosed and described herein, individually or in combination, modulate the bitterness or other taste characteristics of other natural or synthetic sweet tastants and ingestible compositions made therefrom. In one embodiment, the compounds disclosed and described herein, individually or in combination, may be used or provided at their bitter tastant-inhibiting concentrations. For example, the compounds disclosed and described herein, individually or in combination, may be present in amounts ranging from 0.001 ppm to 100 ppm, or narrower ranges of options ranging from 0.1 ppm to 50 ppm, 0.01 ppm to 40 ppm, 0.05 ppm to 30 ppm, 0.01 ppm to 25 ppm, or 0.1 ppm to 30 ppm, or 0.1 ppm to 25 ppm, or 1 ppm to 30 ppm, or 1 ppm to 25 ppm.

[0090] In some embodiments, the identified or selected active compounds disclosed and described herein can be provided individually or in combination, for example, in a flavored concentrated formulation suitable for further processing to produce a ready-to-use product (i.e., a product that can be served as is). By "flavored concentrated formulation" is meant a formulation that needs to be reconstituted with one or more diluent media to be suitable for a ready-to-use composition. The term "ready-to-use composition" is used synonymously herein with "ingestible composition" and means any substance that can be orally ingested, alone or together with other substances, whether or not it is intended to be consumed. In one embodiment, a ready-to-use composition includes a composition that can be directly consumed by humans or animals. A flavored concentrated formulation is typically used by mixing with or diluting with one or more diluent media, such as any edible or ingestible ingredient or product, to impart or modify one or more flavors to the diluent media. Such a use process is often referred to as reconstitution. Reconstitution can be carried out in a domestic or industrial environment. For example, a frozen fruit juice concentrate can be reconstituted with water or other aqueous medium by a consumer in their kitchen to obtain a ready-to-drink fruit juice beverage. In another example, soft drink syrup concentrates can be reconstituted by manufacturers on an industrial scale with water or other aqueous media to produce ready-to-drink soft drinks.Flavored concentrated formulations have higher concentrations of flavoring agents or flavor modifiers than ready-to-use compositions, so flavored concentrated formulations are typically not suitable for direct consumption without reconstitution.There are many advantages to using and producing flavored concentrated formulations.For example, one advantage is that flavored concentrated formulations can be reconstituted at the time of use by adding a suitable solvent, solid, or liquid, thus reducing weight and volume during shipping.

[0091] The flavor products provided according to any of the foregoing embodiments also, in certain embodiments, comprise one or more additional flavor modifying compounds, such as a sweetness enhancing compound (e.g., hesperetin, naringenin, glucosylated steviol glycosides, etc.), a bitterness blocking compound, an umami enhancing compound, a sourness reducing compound, a saltiness enhancing compound, a cooling effect enhancing compound, or any combination of the foregoing.

[0092] Of any of the aspects and embodiments presented herein that refer to a sweetened or flavored concentrate, in certain embodiments, the sweetened or flavored concentrate is a non-naturally occurring product, such as a composition that is specially manufactured for the production of a flavored product, such as a food or beverage item.

[0093] In one embodiment, the flavored concentrated formulation comprises i) the compounds disclosed and described herein, either individually or in combination; ii) a carrier; and iii) optionally at least one auxiliary. The term "carrier" refers to a normally inert accessory substance, such as a solvent, binder, or other inert medium, that is used in combination with the compound and one or more optional auxiliary to form a formulation. For example, water or starch can be a carrier for the flavored concentrated formulation. In some embodiments, the carrier is the same as the dilution medium for reconstituting the flavored concentrated formulation, and in other embodiments, the carrier is different from the dilution medium. The term "carrier" as used herein includes, but is not limited to, ingestible carriers.

[0094] The term "auxiliary agent" refers to an additive that complements, stabilizes, maintains, or enhances the intended function or effectiveness of an active ingredient, such as a compound of the present invention. In one embodiment, the at least one auxiliary agent comprises one or more flavoring agents. The flavoring agent may be any flavor known to a person skilled in the art or a consumer, such as chocolate, coffee, tea, mocha, French vanilla, peanut butter, chai, or a combination thereof. In another embodiment, the at least one auxiliary agent comprises one or more bitterness blockers identified as above. In another embodiment, the at least one auxiliary agent comprises one or more ingredients selected from the group consisting of emulsifiers, stabilizers, antimicrobial preservatives, antioxidants, vitamins, minerals, fats, starches, protein concentrates and isolates, salts, and combinations thereof. Examples of emulsifiers, stabilizers, antimicrobial preservatives, antioxidants, vitamins, minerals, fats, starches, protein concentrates and isolates, and salts are described in U.S. Pat. No. 6,468,576, the contents of which are incorporated herein by reference in their entirety for any purpose.

[0095] In one embodiment, the flavored concentrated formulation may be in a form selected from the group consisting of liquids, including solutions and suspensions, solids, foams, pastes, gels, creams, and combinations thereof, such as liquids containing a certain amount of solids. In one embodiment, the flavored concentrated formulation is in the form of a liquid, including aqueous and non-aqueous bases. In some embodiments, the flavored concentrated formulation may be carbonated or non-carbonated.

[0096] The flavored concentrated formulation may further include a freezing point depressant, a nucleating agent, or both as at least one auxiliary. A freezing point depressant is an ingestible compound or agent that can lower the freezing point of the liquid or solvent to which the compound or agent is added. That is, a liquid or solution containing the freezing point depressant has a lower freezing point than a liquid or solvent without the freezing point depressant. In addition to lowering the starting freezing point, the freezing point depressant can also lower the water activity of the flavored concentrated formulation. Examples of freezing point depressants include, but are not limited to, carbohydrates, oils, ethyl alcohol, polyols such as glycerol, and combinations thereof. A nucleating agent refers to an ingestible compound or agent that can promote nucleation. The presence of a nucleating agent in a flavored concentrated formulation can improve the texture of the frozen blushes of the frozen slush by increasing the number of desirable ice crystallization centers, and aid in maintaining the physical properties and performance of the slush at freezing temperatures. Examples of nucleating agents include, but are not limited to, calcium silicate, calcium carbonate, titanium dioxide, and combinations thereof.

[0097] In one embodiment, the flavored concentrated formulation is formulated to have a low water activity to extend shelf life. Water activity is the ratio of the vapor pressure of water in the formulation to the vapor pressure of pure water at the same temperature. In one embodiment, the flavored concentrated formulation has a water activity of less than about 0.85. In another embodiment, the flavored concentrated formulation has a water activity of less than about 0.80. In another embodiment, the flavored concentrated formulation has a water activity of less than about 0.75.

[0098] In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 2 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 5 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 10 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 15 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 20 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 30 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 40 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration of at least 50 times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration at least 60 times that of the compound in the ready-to-use composition. In one embodiment, the flavored concentrated formulation has the compound at a concentration up to 100 times that of the compound in the ready-to-use composition.

[0099] Blocking the bitterness of pharmaceutical APIs Many drug compounds impart a bitter taste, limiting the ways in which they can be formulated and administered. Thus, in certain aspects, the present disclosure provides pharmaceutical compositions comprising a bitter tasting active pharmaceutical ingredient and a test compound identified or selected by the screening method (as provided herein). The pharmaceutical composition can be in any form suitable for oral administration, such as tablets, lozenges, capsules, powders, solutions, suspensions, etc. The pharmaceutical composition can include any suitable pharmaceutical excipients, binders, etc., as provided in Remington's Pharmaceutical Sciences. In some embodiments, the bitter tasting active pharmaceutical ingredient is an ion channel inhibitor, such as a proton channel inhibitor. Other examples of bitter tasting APIs whose bitterness is reduced by the selected compounds include, but are not limited to, acetaminophen, atropine, brinzolamide, chloramphenicol, chloroquine, clindamycin, dexamethasone, dextromethorphan, digoxin, diltiazem, diphenhydramine, docusate, dorzolamide, doxepin, doxylamine, enalapril, erythromycin, esomeprazole, famotidine, gabapentin, ginkgolide A, guaifenesin, L-histidine, ibuprofen, lomefloxacin, methylprednisolone, ofloxacin, oleuropein, oxyphenonium, phenylephrine, pirenzepine, prednisone, pseudoephedrine, ranitidine, trapidil, trimethoprim, and cetirizine.

[0100] Use in Oral Care Products Oral care products often contain ingredients that impart an astringent or bitter taste. Thus, in certain aspects, the present disclosure provides oral care products that include a bitter-tasting active ingredient and a test compound identified or selected by the screening method (presented herein). Such ingredients include menthol, menthol analogs, mint extracts, sodium bicarbonate, alkali metal peroxymonosulfate (potassium peroxymonosulfate), cetylpyridinium chloride, lauramidopropyl betaine, cocamidopropyl betaine, arginine, hydrogen peroxide, chlorhexidine gluconate, zinc phosphate, zinc chloride, zinc citrate, potassium nitrate, pentasodium triphosphate, tetrasodium pyrophosphate, stannous fluoride, thymol, methyl salicylate, eucalyptol, or any combination thereof. Suitable oral care products include toothpastes, mouthwashes, whitening agents, dentifrices, and the like.

[0101] Working Example U2OS cells stably expressing G16gust25 were transduced with Bacmam recombinant viruses encoding the indicated human bitter receptors and multiplicities of infection (MOI). After 24 hours, the response of the cells to known bitter receptor agonists was evaluated in a FLIPR assay. Figure 1 shows the response of cells expressing five T2R bitter receptors at different MOI levels. Each of the five sets of bars in the bar graph represents cells expressing a different bitter receptor. The leftmost set represents cells expressing the polypeptide of SEQ ID NO: 1, where the bitter tastant is chloramphenicol. The set immediately to the right represents cells expressing the polypeptide of SEQ ID NO: 3, where the bitter tastant is strychnine. The set immediately to the right represents cells expressing the polypeptide of SEQ ID NO: 5, where the bitter tastant is aristolochic acid. The set immediately to the right represents cells expressing the polypeptide of SEQ ID NO: 7, where the bitter tastant is chloroquine. The next set to the right represents cells expressing the polypeptide of SEQ ID NO: 9, in which the bitter tastant is strychnine. The rightmost bar represents the standard control.

Claims

1. A U2OS cell comprising a T2R bitter taste receptor and a G protein, the G protein being functionally coupled to the T2R bitter taste receptor.

2. The U2OS cell of claim 1, wherein the T2R bitter receptor is a polypeptide sequence of SEQ ID NO: 1, or a polypeptide sequence of a functional fragment thereof; a polypeptide sequence of SEQ ID NO: 3, or a polypeptide sequence of a functional fragment thereof; a polypeptide sequence of SEQ ID NO: 5, or a polypeptide sequence of a functional fragment thereof; a polypeptide sequence of SEQ ID NO: 7, or a polypeptide sequence of a functional fragment thereof; or a polypeptide sequence of SEQ ID NO: 9, or a polypeptide sequence of a functional fragment thereof.

3. The U2OS cell of claim 1 or 2, wherein the G protein is gustducin or a G(i / o) polypeptide, or a promiscuous G protein such as G15 or G16gust25.

4. The U2OS cell of claim 1 or 2, which has been modified to overexpress the T2R bitter taste receptor.

5. The U2OS cell of claim 1 or 2, which has been modified to overexpress the G protein.

6. The U2OS cell of claim 1 or 2, which is an isolated cell.

7. The U2OS cell of claim 1 or 2, which is included in a cell-based assay or disposed on a solid support.

8. 1. A method for identifying a bitter taste modulator, comprising: (a) contacting the U2OS cell of claim 1 or 2 with a test compound; (b) measuring T2R bitter taste receptor activity, where a change in T2R bitter taste activity indicates that the test compound is a bitter taste modulator. A method comprising:

9. The method of claim 8, further comprising (c) identifying the bitter taste modulator as a compound that modulates bitterness.

10. 1. A method for identifying a compound that reduces bitterness, comprising the steps of: (a) contacting the U2OS cell of claim 1 with a test compound and a bitter substance; (b) measuring the response of the T2R bitter receptor by comparing the activity of the T2R bitter receptor to the bitter tastant in the presence and absence of the test compound, wherein a change in T2R bitter receptor activity indicates that the test compound is a bitter blocker. A method comprising:

11. The method of claim 10, further comprising: (c) identifying the test compound as a bitter taste blocker based on a change in the T2R bitter taste receptor activity.

12. 12. The method of claim 11, further comprising: (d) selecting the bitter taste blocker as a compound that reduces bitterness.

13. 13. Use of a compound identified in the method of claim 11 or a compound selected in the method of claim 12 to reduce the bitter taste of an ingestible composition.

14. 13. An ingestible composition comprising one or more bitter tastants and a compound identified in the method of claim 11 or selected in the method of claim 12.

15. 15. A flavored product comprising the ingestible composition of claim 14, the flavored product being a food, beverage, pharmaceutical, or oral care product.

Images