Bitter taste receptor blockers and methods for identifying same

Abstract

The present disclosure generally provides methods for identifying compounds that block bitter taste receptors, particularly those activated by certain compounds commonly present in citrus extracts. The present disclosure also provides compositions containing such compounds and methods of their use.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application Publication No. 62 / 897,844, filed September 9, 2019, and U.S. Provisional Patent Application Publication No. 63 / 010,655, filed April 15, 2020, and European Patent Application No. 19216401.0, filed December 16, 2019, the entire contents of each of which are incorporated herein by reference.

[0002] Reference to the electronic sequence listing This application submits an Electronic Sequence Listing entitled SNMX054WOSEQLIST.txt, which is 13,621 bytes in size and was created on September 4, 2020. The information in the Electronic Sequence Listing is incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure generally provides methods for identifying compounds that block bitter taste receptors, particularly those activated by certain compounds commonly present in citrus extracts. The present disclosure also provides compositions containing such compounds and methods of their use.

[0004] 2. Description of Related Art The gustatory system provides sensory information about the chemical composition of the outside world. Gustatory transduction is one of the more sophisticated forms of chemically induced sensation in animals. Taste signaling is found throughout the animal kingdom, from simple metazoans to the most complex vertebrates. Mammals are thought to have five basic taste modalities: sweet, bitter, sour, salty, and umami.

[0005] Bitterness is one of the most sensitive of these five taste modalities, and humans generally perceive it as unpleasant, sharp, and unpleasant. Many bitter compounds are known to be toxic. Therefore, the ability to detect bitter compounds at low concentrations provided certain evolutionary advantages. Nevertheless, many non-toxic food and beverage products, such as coffee, tea, green leafy vegetables, and citrus fruits, contain bitter compounds. For many humans, the concentrations of bitter compounds in such foods and beverages are below the level that elicits an unpleasant response. However, others may have a lower threshold for perceiving bitterness, which may reduce the enjoyment of consuming these products. The addition of sweeteners, such as sucrose, to such products can help offset the bitterness. However, the addition of sweeteners may increase the calorie content of the product and significantly alter the taste for others who can easily tolerate the mild, natural bitterness of these products without sweetening.

[0006] Humans perceive bitter taste when bitter compounds activate one or more of the 25 different bitter taste receptors typically found on the tongue. These bitter taste receptors are members of the TAS2R (taste receptor, type 2) receptor family. Each of the 25 different bitter taste receptors contains a protein, the sequence of which varies from one bitter taste receptor to another. These 25 different bitter taste receptors are commonly referred to by number, such as TAS2R1, TAS2R3, TAS2R4, etc. Different bitter compounds may activate different groups of bitter taste receptors. Therefore, a compound that blocks the bitterness of a particular bitter compound may not effectively block the bitterness of others.

[0007] Citrus fruits contain various compounds that humans generally perceive as bitter. Many of these compounds are found in large amounts in the peel or rind of citrus fruits, but may also be present in the flesh or pulp of the fruit. Some bitter taste receptor blockers effectively block the bitterness of certain foods or beverages, such as the bitter compounds present in coffee. However, these compounds are not as effective at blocking the bitterness of citrus products. Therefore, there is a continuing need to discover compounds that can block the bitter taste receptors responsible for humans' tendency to perceive certain citrus products as bitter.

[0008] summary The present disclosure relates to the discovery of a collection of TAS2R taste receptors primarily responsible for the human perception of the bitter taste of certain compounds naturally occurring in citrus fruits.

[0009] In a first aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 1, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0010] In a second aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0011] In a third aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 3, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0012] In a fourth aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 4, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0013] In a fifth aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 5, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0014] In a sixth aspect, the present disclosure provides a method for identifying a compound that reduces bitterness, the method comprising: introducing a test compound and a bitter compound into one or more taste receptor proteins, wherein the one or more taste receptor proteins are polypeptides comprising the polypeptide sequence of SEQ ID NO: 1 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 2 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 3 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 4 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 5 or a functional fragment thereof, or a polypeptide sequence at least 90% identical to any of the sequences; and measuring the response of each of the one or more taste receptor proteins to the test compound by comparing the activity of the one or more taste receptor proteins to the bitter compound in the presence and absence of the test compound. In some embodiments, the method further comprises identifying an active test compound that reduces bitterness based on the measured response, and selecting the active test compound as a compound that reduces bitterness.

[0015] In a seventh aspect, the present disclosure provides a use of any of the identified or selected active test compounds of certain embodiments of the sixth aspect for reducing the bitter taste of an ingestible composition. In some embodiments thereof, the ingestible composition is a composition derived from citrus fruits. In some embodiments, the ingestible composition is a naturally occurring composition. In some other embodiments, the ingestible composition is a non-naturally occurring composition. In some embodiments, the identified or selected active compound is a polymethoxyflavone (PMF), such as a PMF derived from citrus fruits. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof.

[0016] In an eighth aspect, the present disclosure provides the use of polymethoxyflavones (PMFs), e.g., citrus-derived PMFs, to reduce the bitterness of one or more bitter compounds. In some embodiments thereof, the one or more bitter compounds are comprised by an ingestible composition. In some embodiments thereof, the ingestible composition is a composition derived from citrus fruits. In some embodiments, the ingestible composition is a naturally occurring composition. In some other embodiments, the ingestible composition is a non-naturally occurring composition. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof. In some embodiments, the bitter compound is a compound that modulates (activates) a polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, at least 95% identical, or at least 97% identical to any of the foregoing. In some embodiments, the bitter compound is a limonoid, e.g., limonin, nomilinic acid, or any combination thereof.

[0017] In a ninth aspect, the present disclosure provides methods for reducing the bitterness of an ingestible composition, comprising introducing into the ingestible composition an amount (e.g., a bitterness-reducing effective amount) of any of the identified or selected active test compounds of certain embodiments of the sixth aspect. In some embodiments thereof, the ingestible composition comprises one or more compounds derived from citrus fruits. In some embodiments, the ingestible composition is a non-naturally occurring composition. In some embodiments, the identified or selected active compound is a polymethoxyflavone (PMF), such as a PMF derived from citrus fruits. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof.

[0018] In a tenth aspect, the present disclosure provides a method for reducing the bitterness of an ingestible composition, comprising introducing an amount (e.g., a bitterness-reducing amount) of a polymethoxyflavone (PMF) into the ingestible composition. In some embodiments thereof, the ingestible composition comprises one or more bitter compounds. In some embodiments thereof, the ingestible composition is a composition derived from citrus fruits. In some embodiments, the ingestible composition is a naturally occurring composition. In some other embodiments, the ingestible composition is a non-naturally occurring composition. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof. In some embodiments, the bitter compound is a compound that modulates (activates) the polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some embodiments, the bitter compound is a limonoid, such as limonin, nomilin, nomilic acid, or any combination thereof.

[0019] In an eleventh aspect, the present disclosure provides ingestible compositions comprising any of the identified or selected active test compounds of certain embodiments of the sixth aspect. In some embodiments thereof, the ingestible composition comprises one or more compounds derived from citrus fruits. In some embodiments, the ingestible composition is a non-naturally occurring composition.

[0020] In a twelfth aspect, the present disclosure provides an ingestible composition comprising one or more polymethoxyflavones (PMFs). In some embodiments, the one or more PMFs are present in the ingestible composition in an amount effective to reduce bitterness. In some embodiments thereof, the ingestible composition comprises one or more bitter compounds. In some embodiments thereof, the ingestible composition is a composition derived from citrus fruits. In some embodiments, the ingestible composition is a naturally occurring composition. In some other embodiments, the ingestible composition is a non-naturally occurring composition. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof. In some embodiments, the bitter compound is a compound that modulates (activates) the polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some embodiments, the bitter compound is a limonoid, such as limonin, nomilin, nomilic acid, or any combination thereof.

[0021] In a thirteenth aspect, the present disclosure provides a flavored product comprising the ingestible composition of the eleventh aspect. In some embodiments, the flavored product is a beverage product, such as soda, flavored water, and tea. In some embodiments, the flavored product is a food product.

[0022] In a fourteenth aspect, the present disclosure provides a flavored product comprising the ingestible composition of the twelfth aspect. In some embodiments, the flavored product is a beverage product, such as soda, flavored water, and tea. In some embodiments, the flavored product is a food product.

[0023] In a fifteenth aspect, the present disclosure provides a method for reducing the bitterness of a citrus composition derived from citrus fruits having huanglongbing disease, the method comprising: (a) obtaining a citrus composition from the citrus fruits, wherein at least some of the citrus fruits have huanglongbing disease; and (b) introducing a bitterness-reducing composition into the citrus composition, the bitterness-reducing composition comprising a polymethoxyflavones (PMFs), e.g., an amount (e.g., a bitterness-reducing effective amount) of polymethoxyflavones (PMFs). In some embodiments, the citrus composition comprises an abnormally high concentration of one or more bitter compounds. In some such embodiments, the PMFs are selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof. In some embodiments, the bitter compound is a compound that modulates (activates) the polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, at least 95% identical, or at least 97% identical to any of the foregoing. In some embodiments, the bitter compound is a limonoid, such as limonin, nomilin, nomilic acid, or any combination thereof. In some embodiments, the bitterness-reducing composition is derived from citrus waste.

[0024] Further aspects and embodiments thereof are described in the following detailed description, drawings, abstract, and claims.

[0025] 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 illustrate preferred compositions or preferred methods or to serve as a source of any limitation on the scope of the claimed invention. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows six polymethoxyflavones (PMFs) that were discovered to have antagonistic activity to the activity of bitter compounds in citrus fruits.

[0027] Detailed Description of the Invention The following detailed description describes various aspects and embodiments provided herein. The description should be read from the perspective of one skilled in the relevant art and, as such, may not necessarily include information that is known to such skilled artisans.

[0028] definition The following terms and phrases have the meanings indicated below unless otherwise defined herein. This disclosure may use other terms and phrases not expressly defined herein. Such other terms and phrases have the meaning they would have within the written context of this disclosure to one of ordinary skill in the art. In some cases, terms or phrases may be defined in the singular or plural. In such cases, unless specifically indicated to the contrary, it is understood that any term in the singular may include its plural counterpart, and vice versa.

[0029] A "bitter compound" refers to a compound that induces a detectable bitter flavor in a subject, e.g., a compound that activates a TAS2R receptor in vitro. A "bitter taste receptor blocker" or "bitter taste blocker" refers to a compound that antagonizes the activation of one or more TAS2R receptors in vitro by a bitter compound.

[0030] "Polymethoxyflavone" refers to a flavone derivative having three or more, or four or more, or five or more methoxy substituents at the 3, 5, 6, 7, 8, 2', 3', 4', 5', and 6' positions, where the numbering follows the conventional flavone numbering, as shown below: [ka]

[0031] Non-limiting examples include the following compounds, where the left column lists common names and the right column lists alternative names: [ka]

[0032] "Functional fragment" refers to a portion of a polypeptide sequence to which a bitter compound binds. Polypeptide sequences often contain specific amino acids that are not actively involved in binding but may serve other purposes. In some cases, these non-functional portions of the polypeptide sequence can be removed or partially replaced while leaving the functional portion of the sequence intact. These modified proteins are said to contain functional fragments of the original polypeptide sequence.

[0033] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "substituent" includes a single substituent as well as two or more substituents, etc. As used herein, "for example," "for instance," "such as," or "including" is meant to introduce an example that further clarifies a more general subject matter. Unless otherwise specified, such examples are provided solely as an aid in understanding the embodiments set forth in this disclosure and are not meant to be limiting in any way. Nor do these words or phrases imply any preference of any kind for the disclosed embodiments.

[0034] As used herein, "comprise" or "comprises" or "comprising" or "comprised of" refers to an open group, meaning that the group can include additional components in addition to those explicitly listed. For example, the phrase "comprises A" means that A must be present, but other components can 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 closed group. For example, the phrase "consisting of A" means that A and only A are present.

[0035] 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 other embodiments, the optional event occurs one or more times.

[0036] As used herein, "or" should be given its broadest reasonable interpretation and should not be limited to an either / or construction. Thus, the phrase "comprising A or B" means that A is present and B is not present, or B is present and A is not present, or both A and B are present. Furthermore, for example, if A defines a class that can have multiple members, e.g., A1 and A2, one or more members of the class can be present at the same time.

[0037] Other terms not included in this subsection are defined elsewhere in this description.

[0038] Polypeptide sequences activated by bitter compounds The present disclosure provides specific polypeptide sequences that are useful for simulating in vitro the response that specific human bitter taste receptors would exhibit to such compounds when ingested orally.

[0039] In a first aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 1, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. The polypeptide sequence corresponding to SEQ ID NO: 1 is set forth in the Sequence Listing, which is incorporated herein by reference. For purposes of clarity and to avoid any confusion, the polypeptide sequence corresponding to SEQ ID NO: 1, starting at its N-terminus, using the standard one-letter amino acid code, is: MFSPADNIFIILITGEFILGILGNGYIALVNWIDWIKKKKISTVDYILTNLVIARICLISVMVVNGIVIVLNPDVYTKNKQQIVIFTFWTFANYLNMWITTCLNVFYFLKIASSSHPLFLWLKWKIDMVVHWILLGCFAISLLVSLIAAIVLSCDYRFHAIAKHKRNITEMFHVSKIPYFEPLTLFNLFAIVPFIVSLISFFLLVRSLWRHTKQIKLYATGSRDPSTEVHVRAIKTMTSFIFFFFLYYISSILMTFSYLMTKYKLAVEFGEIAAILYPLGHSLILIVLNNKLRQTFVRMLTCRKIACMI

[0040] In a second aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. The polypeptide sequence corresponding to SEQ ID NO: 2 is set forth in the Sequence Listing, which is incorporated herein by reference. For purposes of clarity and to avoid any confusion, the polypeptide sequence corresponding to SEQ ID NO: 2, starting at its N-terminus and using the standard one-letter amino acid code, is: MITFLPIIFSILIVVIFVIGNFANGFIALVNSIEWVKRQKISFVDQILTALAVSRVGLLWVLLLHWYATQLNPAFYSVEVRITAYNVWAVTNHFSSWLATSLSMFYLLRIANFSNLIFLRIKRRVKSVVLVILLGPLLFLVCHLFVINMDETVWTKEYEGNVTWKIKLRSAMYHSNMTLTMLANFVPLTLTLISFLLLICSLCKHLKKMQLHGKGSQDPSTKVHIKALQTVTSFLLLCAIYFLSMIISVCNLGRLEKQPVFMFCQAIIFSYPSTHPFILILGNKKLKQIFLSVLRHVRYWVKDRSLRLHRFTRGALCVF.

[0041] In a third aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 3, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. The polypeptide sequence corresponding to SEQ ID NO: 3 is set forth in the Sequence Listing, which is incorporated herein by reference. For purposes of clarity and to avoid any confusion, the polypeptide sequence corresponding to SEQ ID NO: 3, starting at its N-terminus, using the standard one-letter amino acid code, is: MLTLTRIRTVSYEVRSTFLFISVLEFAVGFLTNAFVFLVNFWDVVKRQPLSNSDCVLLCLSISRLFLHGLLFLSAIQLTHFQKLSEPLNHSYQAIIMLWMIANQANLWLAACLSLLYCSKLIRFSHTFLICLASWVSRKISQMLLGIILCSCICTVLCVWCFFSRPHFTVTTVLFMNNNTRLNWQIKDLNLFYSFLFCYLWSVPPFLLFLVSSGMLTVSLGRHMRTMKVYTRNSRDPSLEAHIKALKSLVSFFCFFVISSCAAFISVPLLILWRDGVMVCVGIMAACPSGHAAVLISGNAKLRRAVMTILLWAQSSLKVRADHKADSRTLC

[0042] In a fourth aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 4, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. The polypeptide sequence corresponding to SEQ ID NO: 4 is set forth in the Sequence Listing, which is incorporated herein by reference. For purposes of clarity and to avoid any confusion, the polypeptide sequence corresponding to SEQ ID NO:4, starting from its N-terminus, using the standard one-letter amino acid code, is: MMCFLLIISSILVVFAFVLGNVANGFIALVNIIDWVNTRKISSAEQILTALVVSRIGLLWVMLFLWYATVFNSALYGLEVRIVASNAWAVTNHFSMWLAASLSIFCLLKIANFSNLISLHLKKRIKSVVLVILLGPLVFLICNLAVITMDERVWTKEYEGNVTWKIKLRNAIHLSSLTVTTLANLIPFTLSLICFLLLICSLCKHLKKMRLHSKGSQDPSTKVHIKALQTVTSFLMLFAIYFLCIITSTWNLRTQQSKLVLLLCQTVAIMYPSFHSFILIMGSRKLKQTFLSVLWQMTR

[0043] In a fifth aspect, the present disclosure provides a polypeptide sequence of SEQ ID NO: 5, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. The polypeptide sequence corresponding to SEQ ID NO: 5 is set forth in the Sequence Listing, which is incorporated herein by reference. For purposes of clarity and to avoid any confusion, the polypeptide sequence corresponding to SEQ ID NO:5, starting at its N-terminus, using the standard one-letter amino acid code, is: MITFLPIIFSILIVVTFVIGNFANGFIALVNSIEWFKRQKISFADQILTALAVSRVGLLWVLVLNWYATELNPAFNSIEVRITAYNVWAVINHFSNWLATSLSIFYLLKIANFSNLIFLHLKRRVKSVVLVILLGPLLFLVCHLFVINMNQIIWTKEYEGNMTWKIKLRSAMYLSNTTVTILANLVPFTLTLISFLLLICSLCKHLKKMQLHGKGSQDPSMKVHIKALQTVTSFLLLCAIYFLSIIMSVWSFESLENKPVFMFCEAIAFSYPSTHPFILIWGNKKLKQTFLSVLWHVRYWVKGEKPSSS.

[0044] The polypeptide sequences of the above aspects and embodiments may be present in any suitable composition. In some embodiments, one or more polypeptide sequences of the above aspects and embodiments are present in a non-naturally occurring composition, such as in an in vitro assay. In some further such embodiments, the polypeptide sequences of the above aspects and embodiments are expressed on the surface of a cell, for example, in a cell of a eukaryotic cell line.

[0045] Screening Methods In a sixth aspect, the present disclosure provides a method for identifying a compound that reduces bitterness, the method comprising: introducing a test compound and a bitter compound into one or more taste receptor proteins, wherein the one or more taste receptor proteins are polypeptides comprising the polypeptide sequence of SEQ ID NO: 1 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 2 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 3 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 4 or a functional fragment thereof, or the polypeptide sequence of SEQ ID NO: 5 or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical to any of the foregoing sequences; and measuring the response of each of the one or more taste receptor proteins to the test compound by comparing the activity of the one or more taste receptor proteins to the bitter compound in the presence and absence of the test compound.

[0046] In some such embodiments, the introducing step comprises introducing the test compound and the bitter compound to a taste receptor protein, wherein the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some further embodiments, the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical thereto. In some embodiments, the polypeptide sequence of SEQ ID NO: 1 may be referred to as hT2R80.

[0047] In some further embodiments of any of the foregoing embodiments, the introducing step further comprises introducing the test compound and the bitter compound to a taste receptor protein, wherein the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some further embodiments, the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical thereto. In some embodiments, the polypeptide sequence of SEQ ID NO:2 may be referred to as hT2R44.

[0048] In some further embodiments of any of the foregoing embodiments, the introducing step further comprises introducing the test compound and the bitter compound to a taste receptor protein, wherein the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some further embodiments, the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical thereto. In some embodiments, the polypeptide sequence of SEQ ID NO: 3 may be referred to as hT2R51.

[0049] In some further embodiments of any of the foregoing embodiments, the introducing step further comprises introducing the test compound and the bitter compound to a taste receptor protein, wherein the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some further embodiments, the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical thereto. In some embodiments, the polypeptide sequence of SEQ ID NO: 4 may be referred to as hT2R65.

[0050] In some further embodiments of any of the foregoing embodiments, the introducing step further comprises introducing the test compound and the bitter compound to a taste receptor protein, wherein the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some further embodiments, the taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical thereto. In some embodiments, the polypeptide sequence of SEQ ID NO: 5 is hT2R75.

[0051] It should be noted that the introduction step can include introducing the test compound and bitter compound into any combination of the peptide sequences described in the preceding five paragraphs. This can be performed in any suitable manner. For example, in some non-limiting examples, when the test compound and bitter compound are introduced into two or more different taste receptor proteins, the introduction can be performed in a single cell-based assay. However, in some other non-limiting examples, when the test compound and bitter compound are introduced into two or more different taste receptor proteins, the introduction can be performed in two or more cell-based assays, for example, one separate assay for each taste receptor protein.

[0052] The present disclosure contemplates screening test compounds against any combination of two or more, or three or more, or four or more, or five different taste receptor proteins associated with each of the five different polypeptide sequences described herein.

[0053] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:1, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0054] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:1, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:3, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0055] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:1, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:4, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0056] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:1, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:5, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0057] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:3, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0058] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:4, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0059] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:5, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0060] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:3, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:4, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0061] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:3, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:5, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0062] Thus, in some embodiments, introducing comprises introducing a test compound and a bitter compound to each of a first taste receptor protein and a second taste receptor protein (e.g., in the same assay or different assays), wherein the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and measuring comprises measuring the response of each of the first receptor protein and the second receptor protein to the test compound by comparing the activity of each of the first receptor protein and the second receptor protein to the bitter compound in the presence and absence of the test compound. In some further embodiments, the first taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:4, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto, and the second taste receptor protein is a polypeptide comprising the polypeptide sequence of SEQ ID NO:5, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical thereto.

[0063] Similarly, introduction and measurement can include any combination of three different taste receptor proteins, such as: a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO:3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO:4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO:3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO:5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO:2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO:4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO:5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or A polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0064] Similarly, introduction and measurement can include any combination of four different taste receptor proteins, such as: a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; or A polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0065] Similarly, introduction and measurement can include five different taste receptor proteins, such as: a polypeptide comprising the polypeptide sequence of SEQ ID NO: 1, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 2, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; a polypeptide comprising the polypeptide sequence of SEQ ID NO: 3, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 4, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing; and a polypeptide comprising the polypeptide sequence of SEQ ID NO: 5, a functional fragment thereof, or a polypeptide whose sequence is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0066] The embodiment includes measuring the response of each taste receptor protein to a test compound by comparing the activity of the taste receptor protein to the bitter compound in the presence and absence of 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 a composition containing the bitter compound (both in the presence and absence of the test compound) is screened against cells expressing the taste receptor protein in a standard cell-based assay. Binding measurements can be performed by any suitable means typically used to determine protein binding in cell-based assays. Suitable methods include, but are not limited to, the use of fluorescent dyes, calcium indicator proteins, fluorescent cAMP indicators, and the like. The activity of a test compound is determined by its ability to antagonize the binding of the bitter compound to one or more taste receptor proteins.

[0067] In some further embodiments of any of the above embodiments, the method further includes identifying an active test compound that reduces bitterness based on the measured response. According to the above embodiments, the identified active test compound antagonizes the binding of the bitter compound to one, two, three, four, or five taste receptor proteins. 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 taste receptor proteins. In some other embodiments, the identified active test compound is a compound that antagonizes the binding of the bitter compound to two of the above different combinations of taste receptor proteins. In some other embodiments, the identified active test compound is a compound that antagonizes the binding of the bitter compound to three of the above different combinations of taste receptor proteins.

[0068] 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.

[0069] 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, selection can be performed by any suitable means.

[0070] The test compound may be any suitable compound suitable for use in a cellular 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.

[0071] Any suitable composition may be used to provide the bitter compound. In some embodiments, the bitter compound is an extract of a plant from the Meliaceae family or a plant from the Rutacaea family. In some further embodiments, the bitter compound is an extract of a plant from the Rutaceae family and the Citrus genus, such as an extract from orange, lemon, grapefruit, lime, kumquat, pomelo, tangelo, agri, tangerine, or yuzu trees or fruits. In some further embodiments, the bitter compound is a triterpene. In some embodiments, the bitter compound is a tetranortriterpenoid. In some embodiments, the bitter compound is a limonoid. In some embodiments, the bitter compound is limonin, nomilin, nomilic acid, azadirachtin, or any combination thereof. In some further embodiments, the bitter compound is limonin.

[0072] Use and Method In other aspects, the present disclosure provides the use of any identified or selected active compound of the above aspects, including any embodiment or combination of embodiments described above. In certain related aspects, the present disclosure provides the use of any identified or selected active compound of the above aspects, including any embodiment or combination of embodiments described above, to enhance the sweetness of an ingestible composition. In some embodiments thereof, the ingestible composition includes a sweetener, e.g., a caloric sweetener. In certain other related aspects, the present disclosure provides the use of any identified or selected active compound of the above aspects, including any embodiment or combination of embodiments described above, to reduce the sourness of an ingestible composition. In some embodiments, the identified or selected active compound is a polymethoxyflavone (PMF), such as a PMF derived from citrus fruits. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof.

[0073] In certain related aspects, the present disclosure provides the use of polymethoxyflavones (PMFs), such as citrus-derived PMFs, to reduce the bitterness of one or more bitter compounds. In some embodiments thereof, the one or more bitter compounds are comprised by an ingestible composition. In some embodiments thereof, the ingestible composition is a citrus-derived composition. In some embodiments, the ingestible composition is a naturally occurring composition. In some other embodiments, the ingestible composition is a non-naturally occurring composition. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof.

[0074] The bitter compound may be any suitable bitter compound commonly found in natural foods. In some embodiments, the bitter compound is a compound that modulates (activates) the polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0075] In some embodiments, the bitter compound is a compound extracted from a plant from the Meliaceae family or a plant from the Rutaceae family. In some further embodiments, the bitter compound is an extract of a plant from the Rutaceae family and the genus Citrus, such as an extract from orange, lemon, grapefruit, lime, kumquat, pomelo, tangelo, agri, tangerine, or yuzu trees or fruits. In some embodiments, the bitter compound is a triterpene. In some further embodiments, the bitter compound is a tetranortriterpenoid. In some further embodiments, the bitter compound is a limonoid. In some such embodiments, the bitter compound is limonin, nomilin, nomilic acid, azadirachtin, or any combination thereof. In some embodiments, the bitter compound is limonin. In some embodiments, the bitter compound is nomilin.

[0076] The present disclosure also provides methods corresponding to each of the above uses. Accordingly, in certain related aspects, the present disclosure provides a method for enhancing the sweetness of an ingestible composition, comprising introducing into the ingestible composition an amount (e.g., a sweetness-enhancing amount) of an identified or selected active compound of any of the above aspects, including any of the above embodiments or combinations thereof. In some other related aspects, the present disclosure provides a method for reducing the bitterness of an ingestible composition, comprising introducing into the ingestible composition an amount (e.g., a bitterness-reducing amount) of an identified or selected active compound of any of the above aspects, including any of the above embodiments or combinations thereof. In some embodiments, the identified or selected active compound is a polymethoxyflavone (PMF), such as a PMF derived from citrus fruits. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof.

[0077] In certain related aspects, the present disclosure provides methods for reducing the bitterness of an ingestible composition, comprising introducing an amount (e.g., a bitterness-reducing amount) of a polymethoxyflavone (PMF) into the ingestible composition. In some embodiments thereof, the ingestible composition comprises one or more bitter compounds. In some embodiments thereof, the ingestible composition is a composition derived from citrus fruits. In some embodiments, the ingestible composition is a naturally occurring composition. In some other embodiments, the ingestible composition is a non-naturally occurring composition. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof.

[0078] The bitter compound may be any suitable bitter compound commonly found in natural foods. In some embodiments, the bitter compound is a compound that modulates (activates) the polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0079] In some embodiments, the bitter compound is a compound extracted from a plant from the Meliaceae family or a plant from the Rutaceae family. In some further embodiments, the bitter compound is an extract of a plant from the Rutaceae family and the genus Citrus, such as an extract from orange, lemon, grapefruit, lime, kumquat, pomelo, tangelo, agri, tangerine, or yuzu trees or fruits. In some embodiments, the bitter compound is a triterpene. In some further embodiments, the bitter compound is a tetranortriterpenoid. In some further embodiments, the bitter compound is a limonoid. In some such embodiments, the bitter compound is limonin, nomilin, nomilic acid, azadirachtin, or any combination thereof. In some embodiments, the bitter compound is limonin. In some embodiments, the bitter compound is nomilin.

[0080] The uses and methods generally involve the use of the identified or selected active compound or PMF in a composition that includes one or more additional ingredients. For example, in at least one aspect, the present disclosure provides a composition comprising any of the identified or selected active compounds of the above aspects, including any embodiment or combination of embodiments described above, wherein the identified or selected active compound constitutes at least 50% of the composition by dry weight (e.g., by total weight of the composition excluding the weight of any liquid carrier). In a related aspect, the present disclosure provides a solid composition comprising any of the identified or selected active compounds of the above aspects, including any embodiment or combination of embodiments described above, wherein the identified or selected active compound constitutes at least 50% of the solid composition by total weight of the composition. In another related aspect, the present disclosure provides an ingestible composition comprising any of the identified or selected active compounds of the above aspects, including any embodiment or combination of embodiments described above, wherein the concentration of the identified or selected active compound in the ingestible composition is 200 ppm or less. In other related aspects, the disclosure provides ingestible compositions comprising any of the identified or selected active compounds of the above aspects, including any embodiment or combination of embodiments described above, wherein the ingestible composition comprises no more than 1000 ppm, or no more than 900 ppm, or no more than 800 ppm, or no more than 700 ppm, or no more than 600 ppm, or no more than 500 ppm, or no more than 400 ppm, or no more than 300 ppm, or no more than 200 ppm, or no more than 100 ppm, or no more than 50 ppm of steviol glycosides (including rebaudioside A). In other related aspects, the disclosure provides ingestible compositions comprising any of the identified or selected active compounds of the above aspects, including any embodiment or combination of embodiments described above, wherein the ingestible composition comprises a caloric sweetener, such as sucrose, fructose, xylitol, erythritol, or a combination thereof.In another related aspect, the present disclosure provides a concentrated sweetening composition comprising any identified or selected active compound of the above aspects and a sweetener, including any embodiment or combination of embodiments described above.

[0081] In certain embodiments of any of the aspects and embodiments described herein that refer to an ingestible composition, the ingestible composition is a non-naturally occurring product, e.g., a flavored product, such as a composition specially manufactured for the production of a food or beverage product.

[0082] In certain aspects, the present disclosure provides ingestible compositions comprising one or more polymethoxyflavones (PMFs). In some embodiments, the one or more PMFs are present in the ingestible composition in an amount effective to reduce bitterness. In some embodiments thereof, the ingestible composition comprises one or more bitter compounds. In some embodiments thereof, the ingestible composition is a composition derived from citrus fruits. In some embodiments, the ingestible composition is a naturally occurring composition. In some other embodiments, the ingestible composition is a non-naturally occurring composition. In some such embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof.

[0083] The bitter compound may be any suitable bitter compound commonly found in natural foods. In some embodiments, the bitter compound is a compound that modulates (activates) the polypeptide sequence of SEQ ID NO: 2, or a functional fragment thereof, or a polypeptide sequence that is at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing.

[0084] In some embodiments, the bitter compound is a compound extracted from a plant from the Meliaceae family or a plant from the Rutaceae family. In some further embodiments, the bitter compound is an extract of a plant from the Rutaceae family and the genus Citrus, such as an extract from orange, lemon, grapefruit, lime, kumquat, pomelo, tangelo, agri, tangerine, or yuzu trees or fruits. In some embodiments, the bitter compound is a triterpene. In some further embodiments, the bitter compound is a tetranortriterpenoid. In some further embodiments, the bitter compound is a limonoid. In some such embodiments, the bitter compound is limonin, nomilin, nomilic acid, azadirachtin, or any combination thereof. In some embodiments, the bitter compound is limonin. In some embodiments, the bitter compound is nomilin.

[0085] In the above aspects, or any other aspect referring to an ingestible composition, the PMF may be present in the composition at any suitable concentration. For example, in some embodiments, the PMF is present in the composition at a concentration of 1 ppm to 1000 ppm, or 1 ppm to 900 ppm, or 1 ppm to 800 ppm, or 1 ppm to 700 ppm, or 1 ppm to 600 ppm, or 1 ppm to 500 ppm, or 1 ppm to 400 ppm, or 1 ppm to 300 ppm, or 1 ppm to 250 ppm, or 1 ppm to 200 ppm, or 1 ppm to 150 ppm, or 1 ppm to 100 ppm, or 1 ppm to 80 ppm, or 1 ppm to 70 ppm, or 1 ppm to 60 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 is present in the ingestible composition at a concentration ranging from 5 ppm to 1000 ppm, or from 5 ppm to 900 ppm, or from 5 ppm to 800 ppm, or from 5 ppm to 700 ppm, or from 5 ppm to 600 ppm, or from 5 ppm to 500 ppm, or from 5 ppm to 400 ppm, or from 5 ppm to 300 ppm, or from 5 ppm to 250 ppm, or from 5 ppm to 200 ppm, or from 5 ppm to 150 ppm, or from 5 ppm to 100 ppm, or from 5 ppm to 80 ppm, or from 5 ppm to 70 ppm, or from 5 ppm to 60 ppm, or from 5 ppm to 50 ppm, or from 5 ppm to 40 ppm, or from 5 ppm to 30 ppm, or from 5 ppm to 20 ppm. In some embodiments, at least the protein of the PMF (e.g., at least 10% by weight, or at least 20% by weight, or at least 30% by weight) is incorporated into the ingestible composition, e.g., the concentration of the PMF is higher than that which may be present in a natural product extract.

[0086] Generally, the compounds disclosed and described herein can be provided individually or in combination in a composition, e.g., an ingestible composition. In one embodiment, the compounds disclosed and described herein can be provided individually or in combination to impart a more sugar-like temporal or flavor profile to a sweetener composition by combining one or more compounds disclosed and described herein with one or more sweeteners in a sweetening composition. In another embodiment, the compounds disclosed and described herein can be provided individually or in combination to increase or enhance the sweetness of a composition by contacting the composition with a compound disclosed and described herein to form an altered composition.

[0087] Thus, in some embodiments, the composition of any of the above aspects (including any use or method) comprises the identified or selected active compound and a sweetener. In some embodiments, the composition further comprises an excipient. In some embodiments, the excipient is water. In some embodiments, the identified or selected active compound is present at a concentration below its sweetness perception threshold.

[0088] For example, in some embodiments, the sweetener is present in an amount of about 0.1% to about 12% by weight. In some embodiments, the sweetener is present in an amount of about 0.2% to about 10% by weight. In some embodiments, the sweetener is present in an amount of about 0.3% to about 8% by weight. In some embodiments, the sweetener is present in an amount of about 0.4% to about 6% by weight. In some embodiments, the sweetener is present in an amount of about 0.5% to about 5% by weight. In some embodiments, the sweetener is present in an amount of about 1% to about 2% by weight. In some embodiments, the sweetener is present in an amount of about 0.1% to about 5% by weight. In some embodiments, the sweetener is present in an amount of about 0.1% to about 4% by weight. In some embodiments, the sweetener is present in an amount of about 0.1% to about 3% by weight. In some embodiments, the sweetener is present in an amount of about 0.1% to about 2% by weight. In some embodiments, the sweetener is present in an amount of about 0.1% to about 1% by weight. In some embodiments, the sweetener is present in an amount of about 0.1% to about 0.5% by weight. In some embodiments, the sweetener is present in an amount of about 0.5% to about 10% by weight. In some embodiments, the sweetener is present in an amount of about 2% to about 8% by weight. In some further embodiments of the embodiments described in this paragraph, the sweetener is sucrose, fructose, glucose, xylitol, erythritol, or a combination thereof.

[0089] 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 described in this paragraph, the sweetener is a steviol glycoside, a mogroside, a derivative of any of the foregoing, e.g., a glycoside derivative (e.g., glucosylates), 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 a mogroside, such as mogroside V, isomogroside V, isomogroside IV, or any combination thereof. 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.

[0090] The composition may include any suitable sweetener or combination of sweeteners. In some embodiments, the sweetener is a conventional sugar sweetener, such as sucrose, fructose, glucose, and a sweetener composition containing natural sugars, such as corn syrup (including high-fructose corn syrup) or other syrups, or a sweetener concentrate derived from natural fruit and vegetable sources. In some embodiments, the sweetener is sucrose, fructose, or a combination thereof. In some embodiments, the sweetener is sucrose. In some other embodiments, the sweetener is selected from rare natural sugars, including D-allose, D-psicose, L-ribose, D-tagatose, L-glucose, L-fucose, L-albinose, D-turanose, and D-leucrose. In some embodiments, the sweetener is selected from semi-synthetic "sugar alcohol" sweeteners, such as erythritol, isomalt, lactitol, mannitol, sorbitol, xylitol, maltodextrin, etc. In some embodiments, the sweetener is selected from artificial sweeteners such as aspartame, saccharin, acesulfame K, cyclamate, sucralose, and alitame. In some embodiments, the sweetener is selected from the group consisting of cyclamic acid, mogrosides, tagatose, galactose, mannose, sucrose, fructose, lactose, neotame and other aspartame derivatives, aspartame glucose, glucose, D-tryptophan, glycine, maltitol, lactitol, isomalt, hydrogenated glucose syrup (HGS), hydrogenated starch hydrolysates (HSH), stevioside, rebaudioside A, other sweet stevia-based glycosides, chemically modified steviol glycosides (e.g., glucosylated steviol glycosides), mogrosides, chemically modified mogrosides (e.g., glucosylated mogrosides), karerame, and other guanidine-based sweeteners. In some embodiments, the sweetener is a combination of two or more of the sweeteners described in this paragraph. In some embodiments, the sweetener may be a combination of two, three, four, or five of the sweeteners disclosed herein. In some embodiments, the sweetener may be a sugar.In some embodiments, the sweetener may be a combination of one or more sugars and other natural and artificial sweeteners. In some embodiments, the sweetener is a sugar. In some embodiments, the sugar is sucrose. In some embodiments, the sugar is sugar beet sugar. In some embodiments, the sugar may be sucrose, fructose, glucose, or a combination thereof. In some embodiments, the sugar may be sucrose. In some embodiments, the sugar may be a combination of fructose and glucose.

[0091] Sweeteners may also include, for example, sweetener compositions comprising one or more natural or synthetic carbohydrates, such as corn syrup, high fructose corn syrup, high maltose corn syrup, glucose syrup, sucralose syrup, hydrogenated glucose syrup (HGS), hydrogenated starch hydrolysates (HSH), or other syrups or sweetener concentrates derived from natural fruit and vegetable sources, or semi-synthetic "sugar alcohol" sweeteners, such as polyols. Non-limiting examples of polyols in some embodiments include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomaltooligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, isomaltulose, maltodextrin, and the like, as well as sugar alcohols or other carbohydrates, or combinations thereof that can be reduced without adversely affecting taste.

[0092] Sweeteners may be natural or synthetic sweeteners, 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 known as inulin fiber, fructooligosaccharides, or oligo-fructose), green stevia powder, stevia rebausia. 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-based glycosides, stevioside, stevioside extract, honey, Jerusalem artichoke Artichoke syrup, licorice root, Monk fruit (fruit, powder, or extract), Lucuma (fruit, powder, or extract), maple sap (including sap extracted from, for example, 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 (for example, white walnut (Juglans cinerea), black walnut (Juglans nigra), and Japanese walnut (Juglans ailatifolia, including sap extracted from Japanese walnut (Juglans regia), birch sap (e.g., American white birch (Betula papyrifera), yellow birch (Betula alleghaniensis), American beetle (Betula lenta), river birch (Betula nigra), gray birch (Betula populifolia), and silver birch (Betulapendula), sycamore maple sap (e.g., sap extracted from American sycamore maple (Platanus occidentalis), ironwood sap (e.g., sap extracted from American asada (Ostrya virginiana)), muscovado, molasses (e.g., blackstrap molasses), molasses sugar, monatin, monellin, sucrose (also known as natural sugar, unrefined sucrose, or sucrose), palm sugar, raw sugar, piloncillo, rapadura, raw sugar, rice syrup, sorghum, sorghum syrup, cassava syrup (also known as tapioca syrup), thaumatin, yacon root, malt syrup, barley malt syrup, barley malt powder, sugar beet sugar, sucrose, crystalline juicecrystals), caramel, carbitol, carob syrup, castor sugar, hydrogenated starch hydrolysate, hydrolyzed canned juice, hydrolyzed starch, invert sugar, anethole, arabinogalactan, arop, syrup, P-4000, acesulfame potassium (also known as acesulfame K or ace-K), alitame (also known as acrame), advantame, aspartame, bayunoside, neotame, benzamide derivatives, bernadam, canderel, karerame and other guanidine-based sweeteners, vegetable fiber, corn sugar, cups Ring sugar, curculin, cyclamate, cyclocaryoside I, demerara, dextran, dextrin, saccharified malt, dulcin, sucrose, valgin, dulcoside A, dulcoside B, emulin, enoxolone, maltodextrin, saccharin, estragole, ethyl maltol, glucine, gluconic acid, gluconolactone, glucosamine, glucoronic acid, glycerol, glycine, glycyphilin, glycyrrhizin, glycyrrhetinic acid monoglucuronide, golden sugar, yellow sugar, golden syrup, granulated sugar, almond oil Machagulan, hernandurcin, isomerized liquid sugar, jalab, chicory root dietary fiber, kynurenine derivatives (including N'-formyl-kynurenine, N'-acetyl-kynurenine, 6-chloro-kynurenine), galactitol, ritesse, lydican, 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), mukuroziosides, nanosh Gar, Naringin Dihydrochalcone, Neohesperidin Dihydrochalcone, Nib Sugar, Nigerooligosaccharide, Norbu, Orje Syrup, Osladin, Pekmez, Pentajin, Periandrin I, Perillaldehyde, Perillartine, Petophyllum, Phenylalanine, Phlomisoside I, Phlologizin, Phyllodulcin, Polyglycitol Syrup, Polypodoside A, Pterocaryoside A, Pterocaryoside B, Rebiana, Refiner Syrup, Rub Syrup, Rubusoside, Serigeain A, Sugrue, Shamenoside I, Siraitiagrosvenorii), soy oligosaccharides, Splenda, SRI oxime V, steviol glycosides, steviolbioside, stevioside, strogin 1, 2 and 4, sucronate, sucrose, suosan, phloridzin, super aspartame, tetrasaccharide, threitol, treacle, trilobutene, 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.

[0093] In yet other embodiments, the sweetener may be a chemically or enzymatically modified natural high-potency sweetener. Modified natural high-potency sweeteners include glycosylated natural high-potency sweeteners, such as glucosyl, galactosyl, or fructosyl derivatives containing 1 to 50 glycoside residues. Glycosylated natural high-potency sweeteners can be prepared by enzymatic transglycosylation catalyzed by various enzymes exhibiting transglycosylation activity. In some embodiments, the modified sweetener may be substituted or unsubstituted.

[0094] Additional sweeteners also include any combination of two or more of the foregoing sweeteners. In some embodiments, the sweetener may include a combination of two, three, four, or five of the sweeteners disclosed herein. In some embodiments, the sweetener may be a sugar. In some embodiments, the sweetener may be a combination of one or more sugars with other natural and artificial sweeteners. In some embodiments, the sweetener is a caloric sweetener, such as sucrose, fructose, xylitol, erythritol, or a combination thereof. In some embodiments, the ingestible composition is free of, or (in some embodiments) substantially free of, stevia-derived sweeteners, such as steviol glycosides, glucosylated steviol glycosides, or rebaudiosides. For example, in some embodiments, the ingestible composition either does not contain stevia-derived sweeteners, or contains stevia-derived sweeteners at a concentration of 1000 ppm or less, or 500 ppm or less, or 200 ppm or less, or 100 ppm or less, or 50 ppm or less, or 20 ppm or less, or 10 ppm or less, or 5 ppm or less, or 3 ppm or less, or 1 ppm or less.

[0095] 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 (e.g., enhance sweetness, reduce sourness, or 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 minimal amount, for example, 1 ppm or 5 ppm. Thus, in some embodiments, the ingestible composition comprises the identified or selected active compound at a concentration ranging from 1 ppm to 200 ppm, or from 1 ppm to 150 ppm, or from 1 ppm to 100 ppm, or from 1 ppm to 50 ppm, or from 1 ppm to 40 ppm, or from 1 ppm to 30 ppm, or from 1 ppm to 20 ppm, or from 5 ppm to 200 ppm, or from 5 ppm to 150 ppm, or from 5 ppm to 100 ppm, or from 5 ppm to 50 ppm, or from 5 ppm to 40 ppm, or from 5 ppm to 30 ppm, or from 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 5000:1, or from 1000:1 to 10000:1, or from 2000:1 to 8000:1.

[0096] The ingestible composition or sweetener concentrate may, in certain embodiments, include any additional ingredient or combination of ingredients as conventionally used in food and beverage products, including, but not limited to: Acids including, for example, citric acid, phosphoric acid, ascorbic acid, sodium acid sulfate, lactic acid, or tartaric acid; bitter components, including, for example, caffeine, quinine, green tea, catechins, polyphenols, green robusta coffee extract, green coffee extract, potassium chloride, menthol, or proteins (e.g., proteins and protein isolates derived from plants, algae, or fungi); Colorants, such as caramel color, red #40, yellow #5, yellow #6, blue #1, red #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 metabisulfate, 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; Vitamins or functional ingredients, such as resveratrol, Co-Q10, omega-3 fatty acids, theanine, choline chloride (cytocholine), Fibersol, inulin (chicory root), taurine, ginseng extract, guanana extract, ginger extract, L-phenylalanine, L-carnitine, L-tartrate, D-glucoronolactone, inositol, bioflavonoids, echinacea, ginkgo biloba, yerba mate, flaxseed oil, garcinia cambogia rind extract, white tea extract, ribose, milk thistle thistle) extract, grape seed extract, pyrodixine HCl (vitamin B6), cyanobalamin (vitamin B12), niacinamide (vitamin B3), biotin, calcium lactate, calcium pantothenate (pantothenic acid), calcium phosphate, calcium carbonate, chromium chloride, chromium polynicotinate, ferric sulfate, folic acid, ferric pyrophosphate, iron, magnesium lactate, magnesium carbonate, magnesium sulfate, monopotassium phosphate, monosodium phosphate, phosphorus, potassium iodide, potassium phosphate, riboflavin, sodium sulfate, Vitamins or functional ingredients, including sodium gluconate, sodium polyphosphate, sodium bicarbonate, thiamine mononitrate, vitamin D3, vitamin A palmitate, zinc gluconate, zinc lactate, or zinc sulfate; Clouding agents, including, for example, ester guns, brominated vegetable oil (BVO), or sucrose acetate isobutyrate (SAIB); For example, sodium citrate, potassium citrate, or buffers containing salts; For example, propylene glycol, ethyl alcohol, glycerin, gum arabic (gum acacia), maltodextrin, modified corn starch, dextrose, natural flavors, natural flavors with other natural flavors (natural flavors WONF), natural and artificial flavors, flavors containing artificial flavors, silicon dioxide, magnesium carbonate, or tricalcium phosphate; or For example, starches and stabilizers including pectin, xanthan gum, carboxymethyl cellulose (CMC), polysorbate 60, polysorbate 80, medium chain triglycerides, cellulose gel, cellulose gum, sodium caseinate, modified food starch, gum arabic (gum arabic), inulin, or carrageenan.

[0097] The ingestible composition or sweetener concentrate 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 lower to neutral pH. Lower and neutral pH includes, but is not limited to, pHs from about 2.5 to about 8.5, from about 3.0 to about 8.0, from about 3.5 to about 7.5, and from 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 sweetener in taste tests at compound concentrations 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 of the compounds disclosed and described herein, individually or in combination, at lower pH 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 for broad use of the compounds disclosed and described herein, individually or in combination, in a wide variety of foods and beverages.

[0098] The ingestible compositions provided according to any of the above embodiments, in certain embodiments, include one or more additional identified or selected active compounds, such as a compound that enhances sweetness (e.g., hesperetin, naringenin, glucosylated steviol glycosides, etc.), a compound that blocks bitterness, a compound that enhances umami, a compound that reduces sourness or licorice taste, a compound that enhances saltiness, a compound that enhances a cooling effect, or any combination of the foregoing.

[0099] Flavored products and concentrates In certain aspects, the present disclosure provides a flavored product comprising the composition of any of the preceding aspects. In some embodiments, the flavored product is a beverage product, such as soda, flavored water, and tea. In some embodiments, the flavored product is a food product, such as yogurt.

[0100] In embodiments in which the flavored product is a beverage, the beverage may be selected from the group consisting of fortified 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, juice drink, nectar, vegetable juice, vegetable flavored juice, sports drink, energy drink, fortified water drink, vitamin fortified water, near water drink, coconut water, tea-type drink, coffee, cocoa drink, dairy beverage, cereal extract beverage, and smoothie. In some embodiments, the beverage may be a soft drink.

[0101] In certain embodiments of any of the aspects and embodiments described herein that refer to a flavored product, the flavored product is a non-naturally occurring product, such as a packaged food or beverage product.

[0102] Further non-limiting examples of food and beverage products or formulations include sweet coatings, frostings or glazes for such products, or any entity included in the soup category, the dried prepared foods category, the beverage category, the prepared foods 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 replacement category, the pasta and noodle category, and the sauces, dressings, condiments category, the baby food category, and / or the spreads category.

[0103] Further non-limiting examples of food and beverage products or formulations include sweet coatings, frostings or glazes for such products, or any entity included in the soup category, the dried prepared foods category, the beverage category, the ready meal 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 replacement category, the pasta and noodle category, and the sauces, dressings, condiments category, the baby food category, and / or the spreads category.

[0104] Generally, the soup category refers to canned / preserved, dehydrated, instant, refrigerated, UHT, and frozen soups. For purposes of this definition, soup means a food prepared from meat, poultry, fish, vegetables, grains, fruit, and other ingredients and cooked in a liquid that may contain some or all visible parts of these ingredients. It may be clear (like bouillon) or thick (like chowder), smooth, pureed, or chunky, ready-to-use, semi-condensed, or condensed, and may be served hot or cold, as a first or main course of a meal, or as a snack (sipped like a drink) between meals. Soup may be used as an ingredient for preparing other meal ingredients and may vary from bouillons (consommes) to sauces (cream- or cheese-based soups).

[0105] The dehydrated and cooking food category generally refers to: (i) cooking aid products, such as powders, granules, pastes, concentrated liquid products (including concentrated bouillons, bouillons, and bouillon-like products in compressed cube, tablet, powder, or granule form, sold separately as finished products or as an ingredient in products), sauces, and recipe mixes (regardless of technology); (ii) meal solution products, such as dehydrated and freeze-dried soups (including dehydrated soup mixes, dehydrated instant soups, dehydrated cooked soups, and dehydrated or circumstantial cooking of ready-to-eat dishes), meals, and single-serve entrees (including pasta, potato, and rice dishes); and (iii) meal garnish products, such as seasonings, marinades, salad dressings, salad toppings, dips, breads, batter mixes, shelf-stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces, or sauce mixes (including recipe mixes for salads, whether dehydrated, liquid, or frozen, sold as finished products or as an ingredient in products).

[0106] 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 concentrate formulations for preparing beverages, such as soda, and dry powdered beverage precursor mixes. The beverage category also includes alcoholic beverages, soft drinks, sports drinks, isotonic beverages, and hot drinks. Alcoholic beverages include, but are not limited to, beer, cider / perry, FAB, wine, and spirits. Soft drinks include carbonated, e.g., cola and non-cola carbonated; fruit juices, e.g., juices, nectars, juice drinks, and fruit-flavored beverages; bottled waters, including sparkling water, spring water, and purified / table water; functional beverages, which may be carbonated or distilled, including sports drinks, energy drinks, or elixir drinks; and concentrates, e.g., ready-to-drink liquid and powder concentrates. Drinks, either hot or cold, include, but are not limited to, coffee or iced coffee, such as fresh coffee, instant coffee, and coffee combinations; tea or iced tea, such as black tea, green tea, white tea, oolong tea, flavored tea; and other drinks, including flavored, malt, or plant-based powders, granules, blocks, or tablets mixed with milk or water.

[0107] The snack food category generally refers to any food that can be a light, casual meal, 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, extruded 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, energy bars, fruit bars, and other snack bars.

[0108] The category of baked goods generally refers to any edible product whose preparation involves a process of 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 goods, and any combination thereof.

[0109] The ice cream category generally refers to frozen desserts containing cream, sugar, and flavorings. Examples of ice cream include, but are not limited to, impulse ice cream; takeaway ice cream; frozen yogurt and artisanal ice cream; soy, oat, pea (e.g., adzuki bean, mung bean), and rice-based ice cream.

[0110] The confectionery category generally refers to edible products that are sweet in taste. Examples of confectionery include, but are not limited to, candy, gelatin, chocolate confectionery, sugar confectionery, gum, and the like, and any combination thereof.

[0111] 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, slimming products and recovery products.

[0112] The ready meal category generally refers to any food that can be served as a meal without extensive preparation or processing. Ready meals include products with the "skill" of a recipe attached by the manufacturer, providing a high degree of preparation, perfection, and convenience. Examples of ready meals include, but are not limited to, canned / preserved, frozen, dried, and refrigerated ready meals; dinner mixes; frozen pizza; refrigerated pizza; and pre-prepared salads.

[0113] The pasta and noodles category includes pasta and / or noodles, including, but not limited to, canned, dried, refrigerated / fresh pasta; and plain, instant, frozen, refrigerated, and snack noodles.

[0114] The canned / preserved food category includes, but is not limited to, canned / preserved meat and meat products, fish / seafood, vegetables, tomatoes, beans, fruit, ready meals, soups, pasta, and other canned / preserved foods.

[0115] The frozen processed food category includes, but is 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.

[0116] The dry processed food category includes, but is not limited to, rice, dessert mixes, dried ready meals, dehydrated soups, instant soups, dried pasta, plain noodles, and instant noodles. The refrigerated processed food category includes, but is not limited to, refrigerated processed meats, processed fish / seafood products, lunch kits, fresh cut fruit, ready meals, pizza, pre-prepared salads, soups, and fresh pasta and noodles.

[0117] 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, pickles, and other sauces, dressings and condiments.

[0118] The baby food category includes, but is not limited to, milk-based or soy-based formulas; and cooked, dried, and other baby foods.

[0119] The spreads category includes, but is not limited to, jams and preserves, honey, chocolate spreads, nut-based spreads, and yeast-based spreads.

[0120] The dairy category generally refers to edible products made from mammalian milk. Examples of dairy products include, but are not limited to, milk products, cheese, yogurt and sour milk drinks, and other dairy products.

[0121] Further examples of flavored products, particularly food and beverage products or formulations, are given below. Exemplary ingestible compositions include one or more of confectionery, chocolate confectionery, tablets, count lines, bagged self-lined / soft lines, boxed assortments, standard boxed assortments, twist wrapped miniatures, seasonal chocolates, toy chocolates, alfajores, other chocolate confectionery, mints, standard mints, power mints, boiled confectionery, pastilles, gum, jellies and chews, toffee, caramel and nougat, medicated confectionery, lollipops, licorice, other sugar confectionery, bread, bagged / factory-made bread, bagged / factory-made bread, pastries, cakes, bagged / factory-made cakes, bagged / factory-made cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, flavored biscuits and crackers, bread substitutes, breakfast cereal, rte cereal, family breakfast cereal, flakes, muesli, other cereals, children's breakfast cereal, hot cereal, ice cream, impulse ice cream, single portion dairy ice cream, single portion aqueous ice cream. Ice cream, multipack dairy ice cream, multipack water-based ice cream, take away ice cream, take away dairy ice cream, ice cream desserts, bulk ice cream, take away water-based ice cream, frozen yogurt, artisanal ice cream, dairy products, milk, raw / pasteurized milk, full fat raw / pasteurized milk, semi-skimmed fresh / pasteurized milk, long shelf life / uht milk, full fat long shelf life / uht milk, semi-skimmed long shelf life / uht milk, non-fat long shelf life / uht milk, goat milk, concentrated / evaporated milk, plain concentrated / evaporated milk, flavoured, functional and other concentrated milks, flavoured milk drinks, dairy only flavoured milk drinks, flavoured milk drinks with fruit juice, soy milk, sour milk drinks, fermented milk drinks, coffee whitener, powdered milk, flavoured powdered milk drinks, cream, cheese, processed cheese, spreadable processed cheese, non-spreadable processed cheese, raw cheese,Spreadable raw cheese, hard cheese, packaged hard cheese, unpackaged hard cheese, yogurt, plain / natural yogurt, flavored yogurt, fruit yogurt, probiotic yogurt, drinking yogurt, regular drinking yogurt, probiotic drinking yogurt, refrigerated and shelf stable desserts, dairy based desserts, soy based desserts, refrigerated snacks, fromage fries and quark, plain fromage fries and quark, flavored fromage fries and quark, flavored fromage fries and quark, sweet and savory snacks, fruit snacks, chips / crisps, extruded snacks, tortillas / corn chips, popcorn, pretzels, nuts, sweet and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal replacements, slimming products, recovery drinks, ready meals, canned ready meals, frozen ready meals, dried ready meals, refrigerated ready meals, dinner mixes, frozen pizza, refrigerated pizza, soups, canned soups, dehydrated snacks soup, instant soup, refrigerated soup, hot soup, frozen soup, pasta, canned pasta, dried pasta, refrigerated / fresh pasta, noodles, plain noodles, instant noodles, cup / bowl instant noodles, pouch instant noodles, chilled noodles, snack noodles, canned food, canned meat and meat products, canned fish / seafood, canned vegetables, canned tomatoes, canned beans, canned fruit, canned ready meals, canned soup, canned pasta, other canned food, frozen food, frozen processed red meat, frozen processed poultry, frozen processed fish / fish, 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 soup, Frozen noodles, Other frozen foods, Dried foods, Dessert mixes, Dried ready meals, Dehydrated soups, Instant soups, Dried 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 coated fish, Refrigerated smoked fish, Refrigerated lunch kits, Refrigerated ready meals, Refrigerated pizza, Refrigerated soup,Includes refrigerated / fresh pasta, refrigerated noodles, oils and fats, olive oil, vegetable and seed oils, cooking fats, butter, margarine, spreadable oils and fats, functional spreadable oils and fats, 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, pickles, other sauces, dressings and condiments, milk powder, formula, standard formula, follow-on formula, toddler formula, hypoallergenic formula, prepared baby food, dry baby food, other baby food, spreads, jams and preserves, honey, chocolate spreads, nut-based spreads, yeast-based spreads. Exemplary ingestible compositions also include confectionery, bakery products, ice cream, dairy products, sweetened snacks, snack bars, meal replacements, ready meals, soups, pasta, noodles, canned foods, frozen foods, dried 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 to allow for reduced concentrations of known sugar sweeteners or artificial sweeteners.

[0122] 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, sugared gums, sugarless gums, functional gums, bubble gums, or the like, comprising the compounds disclosed and described herein, individually or in combination.

[0123] Typically, at least a sweet taste receptor-modulating amount, a sweet taste receptor ligand-modulating amount, a sweet flavor-modulating amount, a sweet flavor-imparting amount, a sweet flavor-enhancing amount, or a therapeutically effective amount of one or more compounds of the present invention is added to an ingestible composition, optionally in the presence of a sweetener, such that the sweetness-altered ingestible composition has an increased sweetness, as judged by humans or animals generally through procedures generally known in the art, or, in the case of formulation testing, by a majority of a panel of at least eight human taste testers, compared to an ingestible composition prepared without the compounds of the present invention.

[0124] In some embodiments, the compounds disclosed and described herein, individually or in combination, modulate the sweetness 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, can be used or provided at their ligand-enhanced concentrations. For example, the compounds disclosed and described herein, individually or in combination, can be present in amounts of 0.001 ppm to 100 ppm, or in narrower alternative ranges of 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.

[0125] In some embodiments, the identified or selected active compounds disclosed and described herein may be provided, individually or in combination, in a flavor concentrate formulation, suitable for subsequent processing, for example, to produce a ready-to-use (i.e., readily available) product. By "flavor concentrate formulation" is meant a formulation that must be reconstituted with one or more dilution media to become a ready-to-use composition. The term "ready-to-use composition" is used interchangeably herein with "ingestible composition," which refers to any substance that can be orally ingested, alone or with other substances, whether or not intended for consumption. In one embodiment, a ready-to-use composition includes a composition that may be directly consumed by humans or animals. A flavor concentrate formulation is typically used by mixing or diluting it with one or more dilution media, e.g., any consumable or ingestible ingredient or product, to impart or modify one or more flavors to the dilution media. Such a use process is often referred to as reconstitution. Reconstitution can be performed in a home or industrial setting. For example, a frozen fruit juice concentrate can be reconstituted by a consumer in their kitchen with water or another aqueous medium to obtain a ready-to-use fruit juice beverage. In another example, soft drink syrup concentrates can be reconstituted with water or other aqueous media by large-scale industrial manufacturers to produce ready-to-use soft drinks. Because flavor concentrate formulations contain flavoring agents or flavor modifiers at higher concentrations than ready-to-use compositions, flavor concentrate formulations are typically not suitable for direct consumption without reconstitution. There are many advantages to using and producing flavor concentrate formulations. For example, one advantage is a reduction in weight and volume for transportation, since the flavor concentrate formulation can be reconstituted at the time of use by adding a suitable solvent, solid, or liquid.

[0126] The flavored products described according to any of the above embodiments, 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.

[0127] In certain embodiments of any of the aspects and embodiments described herein that refer to a sweetening or flavoring concentrate, the sweetening or flavoring concentrate is a composition that is specially prepared for the production of a non-naturally occurring product, e.g., a flavored product, e.g., a food or beverage product.

[0128] In one embodiment, a flavor concentrate formulation comprises i) a compound disclosed and described herein, either individually or in combination, ii) a carrier, and iii) optionally at least one adjuvant. The term "carrier" refers to a typically inert auxiliary substance, such as a solvent, binder, or other inert medium, used in combination with a compound of the present invention and one or more optional adjuvants to form a formulation. For example, water or starch may be a carrier for a flavor concentrate formulation. In some embodiments, the carrier is the same as the dilution medium for reconstituting the flavor concentrate formulation, and in other embodiments, the carrier is different from the dilution medium. As used herein, the term "carrier" includes, but is not limited to, ingestible and acceptable carriers.

[0129] The term "adjuvant" refers to an additive that supplements, 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 adjuvant comprises one or more flavoring agents. The flavoring agents may be any flavor known to those skilled in the art or consumers, such as chocolate, coffee, tea, mocha, French vanilla, peanut butter, chai, or combinations thereof. In another embodiment, the at least one adjuvant comprises one or more sweeteners. The one or more sweeteners may be any of the sweeteners described herein. In another embodiment, the at least one adjuvant 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 by reference in their entirety for all purposes.

[0130] In one embodiment, the flavor concentrate formulations of the present invention may be in a form selected from the group consisting of liquids, including solutions and suspensions, solids, foamable materials, pastes, gels, creams, and combinations thereof, e.g., liquids containing a certain amount of solids. In one embodiment, the flavor concentrate formulation has a water activity of less than about 0.85. In some embodiments, the flavor concentrate formulations of the present invention may be carbonated or non-carbonated.

[0131] The flavor concentrate formulation may further include at least one adjuvant: a freezing point depressant, a nucleating agent, or both. A freezing point depressant is an ingestible compound or agent capable of depressing the freezing point of a 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 depressing the initial freezing point, the freezing point depressant may also reduce the water activity of the flavor concentrate 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 capable of promoting nucleation. The presence of a nucleating agent in a flavor concentrate formulation can improve the mouthfeel of frozen blushes in frozen slushes and help maintain the physical properties and performance of the slush at freezing temperatures by increasing the number of desired ice crystal centers. Examples of nucleating agents include, but are not limited to, calcium silicate, calcium carbonate, titanium dioxide, and combinations thereof.

[0132] In one embodiment, the flavor concentrate formulation is formulated with 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 flavor concentrate formulation has a water activity of less than about 0.85. In another embodiment, the flavor concentrate formulation has a water activity of less than about 0.80. In another embodiment, the flavor concentrate formulation has a water activity of less than about 0.75.

[0133] In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least twice the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least five times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least ten times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least fifteen times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least twenty times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least thirty times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least forty times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least fifty times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the present invention at a concentration that is at least sixty times the concentration of the compound in the ready-to-use composition. In one embodiment, the flavor concentrate formulation has a compound of the invention at a concentration that is up to 100 times the concentration of the compound in the ready-to-use composition.

[0134] The sweetness or flavor concentrate provided according to any of the above embodiments may, in certain embodiments, comprise one or more additional flavor-modifying compounds, such as a compound that enhances sweetness (e.g., hesperetin, naringenin, glucosylated steviol glycosides, etc.), a compound that masks bitterness (e.g., eriodictyol, homoeriodictyol, sterubin, and salts or glycoside derivatives thereof, and vanillyl lignans such as matairesinol and other compounds described in WO 2012 / 146584), a compound that enhances umami (e.g., rubemamine, rubesenamine, (E)-3-(3,4-dimethoxyphenyl)-N-(4-methoxyphenethyl)acrylamide, etc.), a compound that reduces sourness and / or licorice taste, a compound that enhances saltiness, a compound that enhances cooling effect, or any combination of the foregoing.

[0135] Treatment of diseased citrus fruits compositions In a fifteenth aspect, the present disclosure provides a method for reducing the bitterness of a citrus composition derived from citrus fruits having huanglongbing disease, the method comprising: (a) obtaining a citrus composition from citrus fruits, wherein at least some of the citrus fruits have huanglongbing disease; and (b) introducing a bitterness-reducing composition into the citrus composition, the bitterness-reducing composition comprising polymethoxyflavones (PMFs), for example, an amount (e.g., a bitterness-reducing effective amount) of polymethoxyflavones (PMFs).

[0136] In some embodiments, the citrus composition contains an abnormally high concentration of one or more bitter compounds, such as abnormally high levels of limonin or nomilin. In some embodiments, the bitter compound is a compound that modulates (activates) the polypeptide sequence of SEQ ID NO:2, or a functional fragment thereof, or a polypeptide sequence at least 90% identical, or at least 95% identical, or at least 97% identical to any of the foregoing. In some embodiments, the bitter compound is a limonoid, such as limonin, nomilin, nomilic acid, or any combination thereof.

[0137] Any suitable PMF can be used. In some embodiments, the PMF is selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof. In some embodiments, the bitterness-reducing composition comprising a PMF is derived from citrus waste.

[0138] Bitterness Blocking in Pharmaceutical APIs Many pharmaceutical compounds impart a bitter taste, thus limiting the ways in which they can be formulated and administered. Accordingly, in certain aspects, the present disclosure provides pharmaceutical compositions comprising a bitter-tasting active pharmaceutical ingredient and one or more polymethoxyflavones (PMFs). In some embodiments, the one or more PMFs are selected from the group consisting of sinensetin, 4'-hydroxy-sinensetin, quercetogetin, nobiletin, tangeretin, heptamethoxyflavone, and any combination thereof. Such pharmaceutical compositions may be in any form suitable for oral administration, such as tablets, lozenges, capsules, powders, liquid solutions, liquid suspensions, etc. Such pharmaceutical compositions may include any suitable pharmaceutical excipients, binders, etc., such as those listed in Remington's Pharmaceutical Sciences. In some embodiments, the bitter-tasting active pharmaceutical ingredient is an ion channel inhibitor, e.g., a proton channel inhibitor. Other examples of bitter-tasting APIs whose bitterness is reduced by one or more PMFs include, but are not limited to, atropine, brinzolamide, chloramphenicol, chloroquine, clindamycin, dexamethasone, digoxin, diltiazem, diphenhydramine, docusate, dorzolamide, doxepin, doxylamine, enalapril, erythromycin, esomeprazole, famotidine, gabapentin, ginkgolide A, guaifenesin, L-histidine, lomefloxacin, methylprednisolone, ofloxacin, oleuropein, oxyphenonium, pirenzepine, prednisone, ranitidine, trapidil, trimethoprim, and cetirizine.

[0139] Example The following examples are included to further illustrate the present invention. Of course, these examples should not be construed as specifically limiting the invention. Variations of these examples within the scope of the claims are within the purview of one of ordinary skill in the art and are considered to be within the scope of the invention as described and claimed herein. The reader will recognize that one of ordinary skill in the art armed with this disclosure will be able to make and use the invention without the need for exhaustive examples.

[0140] Example 1 – Identification of taste receptors Cells expressing the promiscuous G protein (G16g44) were transfected with an expression vector encoding 22 functional bitter receptor cDNAs, one receptor per transfection, using TransIT-293 (Mirus). Cells were directly transfected into high-density plates in a 384-well format and incubated for 28 hours. The cells were then loaded with the Ca-specific dye Fluo4-AM. After a 60-minute incubation, excess dye was washed away, and the cells were stimulated with each bitter agonist while simultaneously recording changes in intracellular fluorescence using a FLIPR instrument. The bitter agonist and the responding receptor were identified, and a full dose-response analysis was performed with each bitter agonist using each stable bitter receptor cell line in the same assay.

[0141] The protocol was performed using limonin as a non-limiting example of a bitter agonist. With limonin, positive dose responses were recorded for five different bitter receptor proteins expressed in cells over a concentration range of 12.5 μM to 50 μM. The five expressed receptor proteins correspond to the amino acid sequences identified herein as SEQ ID NOS: 1, 2, 3, 4, and 5.

[0142] Example 2 - Bitterness antagonism Certain PMFs were tested for their ability to antagonize the binding of limonin to cells expressing the polypeptide identified herein as SEQ ID NO: 2. Table 1 shows the percent activity of limonin against the polypeptide at a concentration of 25 μM both in the absence of test antagonist and in the presence of test antagonist at concentrations ranging from 50 μM to 200 μM.

[0143] [Table 1-1] [Table 1-2]

Claims

1. 1. A method for identifying a compound that reduces bitterness, comprising: (i) introducing a test compound and a bitter compound into one or more taste receptor proteins; wherein the one or more taste receptor proteins are polypeptides comprising the polypeptide sequence of SEQ ID NO: 1 or a functional fragment thereof, the polypeptide sequence of SEQ ID NO: 2 or a functional fragment thereof, the polypeptide sequence of SEQ ID NO: 4 or a functional fragment thereof, the polypeptide sequence of SEQ ID NO: 5 or a functional fragment thereof, or a polypeptide sequence at least 90% identical to any of the foregoing sequences; (ii) measuring the response of each of the one or more taste receptor proteins to the test compound by comparing the activity of the one or more taste receptor proteins to the bitter compound in the presence and absence of the test compound, wherein the one or more taste receptor proteins are each expressed on the surface of a cell, and the activity of the test compound is determined by its ability to antagonize the binding of the bitter compound to the one or more taste receptor proteins in a cellular assay; and (iii) identifying active test compounds as compounds that reduce bitter taste based on the measured response. Including, A method for identifying a compound that reduces bitterness, wherein the bitter compound is a limonoid.

2. 10. The method of claim 1, the introducing step comprises introducing the test compound and the bitter compound into two or more taste receptor proteins, and the two or more taste receptor proteins are independently polypeptides comprising a polypeptide sequence of SEQ ID NO: 2 or a functional fragment thereof, a polypeptide sequence of SEQ ID NO: 4 or a functional fragment thereof, a polypeptide sequence of SEQ ID NO: 5 or a functional fragment thereof, or a polypeptide sequence at least 90% identical to any of the foregoing sequences; The method, wherein the measuring step includes measuring the response of each of two or more taste receptor proteins to the test compound by comparing the activity of one or more taste receptor proteins to the bitter compound in the presence and absence of the test compound.

3. 3. The method of claim 1 or 2, wherein the bitter compound is limonin, nomilin, nomilic acid, azadirachtin, or any combination thereof.

4. 4. The method of claim 3, wherein the bitter compound is limonin.

5. 5. The method of any one of claims 1 to 4, wherein the cell is a eukaryotic cell.

6. 6. The method of any one of claims 1 to 5, wherein the one or more taste receptor proteins comprise the polypeptide sequence of SEQ ID NO: 4 or a functional fragment thereof.

7. A method described in any one of claims 1 to 6, wherein the compound that reduces bitterness is a polymethoxyflavone.

8. Polymethoxyflavones include 5,6,7,3',4'-pentamethoxyflavone, 3,5,6,7,3',4'-hexamethoxyflavone, 5,6,7,8,3',4'-hexamethoxyflavone, 5,6,7,4'-tetramethoxyflavone, 3,5,6,7,8,3',4'-heptamethoxyflavone, 5,6,7,8,4'-pentamethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, and 5-hydroxy-3,6,7,3',4'-pentamethoxyflavone.

8. The method of claim 7, wherein the compound is selected from the group consisting of lavone, 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone, 5-hydroxy-6,7,4'-trimethoxyflavone, 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone, 5-hydroxy-6,7,8,4'-tetramethoxyflavone, 4'-hydroxy-5,6,7,3'-tetramethoxyflavone and 4'-hydroxy-3,5,6,7,8,3'-hexamethoxyflavone.

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