Peptide for adjusting calcium sensing receptor activity to adjust rich taste, and pet food product containing the same

Abstract

To provide a flavor composition that can be used for improving rich taste and / or palatability of a pet food product, and includes at least one peptide which activates calcium sensing receptor or increases the activity, and a method for identifying the peptide.SOLUTION: Palatability, taste, and / or flavor of a pet food product can be enhanced or modified by using a flavor composition. The flavor composition can include a combination of a plurality of compounds, and can be added to a pet food product by various delivery systems, or can be created in a production process of a pet food product.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 814,082, filed March 5, 2019, which is incorporated by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated by reference in its entirety into this application. The ASCII copy, created on February 27, 2020, is named 069269_0391_SL.txt and is 41,076 bytes in size. [Technical field]

[0003] The subject matter of the present disclosure relates to a flavor composition comprising at least one peptide that interacts with calcium sensing receptor (CaSR) to modulate kokumi. The flavor composition can be used to enhance or modify the palatability, taste, and / or flavor of a pet food product. The flavor composition can include a combination of compounds and can be added to the pet food product in various delivery systems or can be generated during the manufacturing process of the pet food product. [Background technology]

[0004] The taste profile of an edible composition includes the basic tastes of sweet, salty, bitter, sour, umami, and kokumi. Compounds that elicit these tastes are sometimes called tastants. Without being bound by theory, it is hypothesized that tastants are sensed by taste receptors in the mouth and throat, which send signals to the brain where these tastants and the resulting taste profiles are recorded. Taste receptors include the calcium-sensing receptor (CaSR), which is a G-protein coupled receptor (GPCR) that detects extracellular calcium levels and is close to the T1R1, T1R2, and T1R3 receptors, i.e., sweet and umami receptors. The calcium-sensing receptor has been shown to function as a receptor for kokumi.

[0005] It has long been a desire of pet food manufacturers to provide pet food products with high nutritional value. Moreover, especially with regard to cat and dog foods, pet food manufacturers desire a high degree of palatability so that pets can fully enjoy the nutritional benefits from the food. Domestic animals, especially cats, are known to be fussy about their food preferences and may refuse to eat a pet food product that they have accepted for a long time or refuse to eat more than a minimal amount of the pet food product. This phenomenon may be due in part to slight differences in the sensory profile of the ingredients, which domestic animals can perceive through their taste and olfactory systems. As a result, pet owners frequently change the type and brand of pet food to keep their pets healthy and satisfied. Summary of the Invention [Problem to be solved by the invention]

[0006] Despite recent advances in taste and flavor technology, there continues to be a need for compounds that can enhance or modify the taste, texture, and / or flavor profile of a pet food product, thereby enhancing or modifying the palatability of the pet food product. The enhancement or modification may be an increase in the intensity of a desired attribute, a replacement of a desired attribute that is not present in the pet food product or that is lost for some reason, or a decrease in the intensity of an undesirable attribute. In particular, it is desirable to increase the intensity of a desired tastant in a pet food product. Thus, there continues to be a need in the art for compositions for enhancing the palatability of a pet food product and / or for adjusting the body taste. [Means for solving the problem]

[0007] The subject matter of the present disclosure is directed to flavor compositions and methods for producing and modifying said compositions across a variety of pet food products. Specifically, the present disclosure is directed to compositions comprising one or more peptides that modulate kokumi flavor by enhancing, enhancing, reducing, and / or modulating the activity of calcium sensing receptor (CaSR).

[0008] In certain embodiments, the flavor composition comprises an oligopeptide. In certain embodiments, the oligopeptide comprises a tripeptide motif. In certain embodiments, the tripeptide motif is: (a) a first amino acid residue at the N-terminus which is a negatively charged amino acid residue or a polar uncharged amino acid residue; (b) a second amino acid residue having a molecular mass of 150 daltons or less; and (c) a C-terminal third amino acid residue that is a negatively charged amino acid residue or a polar uncharged amino acid residue, wherein said tripeptide binds to the calcium sensing receptor (CaSR) and confers a kokumi (good taste) to companion animals.

[0009] In certain embodiments, the first amino acid residue is a negatively charged amino acid residue. In certain embodiments, the third amino acid residue is a negatively charged amino acid residue. In certain embodiments, the negatively charged amino acid residue is selected from the group consisting of aspartic acid (Asp), glutamic acid (Glu), and any phosphorylated amino acid residue. In certain embodiments, the negatively charged amino acid residue is phosphorylated serine (pSer), phosphorylated tyrosine (pTyr), or phosphorylated threonine (pThr).

[0010] In certain embodiments, the first amino acid residue is a polar, uncharged amino acid residue. In certain embodiments, the third amino acid residue is a polar, uncharged amino acid residue. In certain embodiments, the polar, uncharged amino acid residue is selected from the group consisting of cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asp), serine (Ser), tyrosine (Tyr), and threonine (Thr).

[0011] In certain embodiments, the second amino acid residue is selected from the group consisting of lysine (Lys), isoleucine (Ile), leucine (Leu), alanine (Ala), methionine (Met), proline (Pro), valine (Val), aspartic acid (Asp), glutamic acid (Glu), cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asn), serine (Ser), and threonine (Thr). In certain embodiments, the second amino acid residue is alanine (Ala), valine (Val), or glutamic acid (Glu).

[0012] In certain embodiments, the oligopeptide is a tripeptide selected from the group consisting of Asp-Val-Glu, Glu-Val-Asp, Asp-Glu-Glu, pSer-Glu-pSer, pSer-Val-pSer, pSer-Val-Glu, Ser-Glu-Ser, Cys-Val-Cys, pTyr-Glu-pTyr, pThr-Glu-pThr, Asp-Ala-Glu, Glu-Val-Glu, Asp-Val-Asp, and any combination thereof.

[0013] In certain embodiments, the oligopeptide is selected from the group consisting of Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln, Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln-Ala, Glu-Ile-Val-Pro-Asn-pSer-Ala-Glu-Glu, Asp-Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln-Ala, and any combination thereof.

[0014] In certain embodiments, the companion animal is a cat or a dog. In certain embodiments, the companion animal is a cat.

[0015] In certain embodiments, the oligopeptide is produced during the manufacturing process of a food product.

[0016] The subject matter of the present disclosure provides a food product comprising any of the flavor compositions disclosed herein, wherein the flavor composition is present in an amount effective to enhance the fullness of the food product as determined by a panel of taste testers. The subject matter of the present disclosure provides a food product comprising any of the flavor compositions disclosed herein, wherein the flavor composition is present in an amount effective to enhance the palatability of the food product as determined by a panel of taste testers. In certain embodiments, the flavor composition is present in the food product at a concentration of about 1 nM to about 1 M, about 1 μM to about 1 M, about 0.0001% to about 10% w / w, about 0.001% to about 5% w / w, or about 0.01% to about 1% w / w. In certain embodiments, the food product comprises a pet food product. In certain embodiments, the pet food product is a pet food product for cats or a pet food product for dogs. In certain embodiments, the pet food product is a wet pet food product. In certain embodiments, the pet food product is a dry pet food product.

[0017] The presently disclosed subject matter provides a method for enhancing kokumi intensity of a food product, the method comprising blending the food product with any of the flavor compositions disclosed herein, the flavor composition being present in an amount effective to enhance kokumi intensity of the food product as determined by a panel of taste testers. In certain embodiments, the flavor composition is present in the blend at a concentration of about 1 nM to about 1 M, about 1 μM to about 1 M, about 0.0001% to about 10% w / w, about 0.001% to about 5% w / w, or about 0.01% to about 1% w / w.

[0018] In certain embodiments, the flavor composition is produced during the manufacturing process of the food product.

[0019] The presently disclosed subject matter provides a method of modulating activity of a calcium sensing receptor (CaSR), the method comprising contacting the CaSR with any of the flavor compositions disclosed herein.

[0020] The presently disclosed subject matter provides methods for identifying compositions that modulate the activity of CaSR. In certain embodiments, the method comprises: (a) contacting a test agent with the CaSR; (b) detecting an in silico interaction between the test agent and one or more amino acids selected from the group consisting of Pro39, Arg66, Gly67, Arg69, Trp70, Gly146, Ser147, Gly148, Tyr167, Ala168, Ser171, Ile187, Tyr218, Ser271, Glu297, Ser301, Ile416, and any combination thereof at the Venus Flytrap (VFT) domain interaction site of the CaSR; and (c) selecting a test agent that interacts with one or more of the amino acids as the composition. Includes.

[0021] In certain embodiments, step (b) further comprises detecting an interaction between the test agent and one or more amino acids selected from the group consisting of Asn64, Asn102, Thr145, Ser169, Ser170, Ser272, Ala298, Trp299, Ala300, Ser302, and any combination thereof, at the Venus Flytrap (VFT) domain interaction site of the CaSR.

[0022] In certain embodiments, the method further comprises determining the activity of the CaSR after step (a), and in certain embodiments, step (c) further comprises selecting a test agent for the composition that enhances the activity of the CaSR. In certain embodiments, the CaSR is expressed by a cell and the test agent is contacted with the cell.In certain embodiments, the cell expresses a calcium-binding photoprotein.

[0023] The foregoing has outlined rather broadly the features and technical advantages of the present application in order that the detailed description that follows may be better understood. Additional features and advantages of the present application will be described hereinafter and form the subject of the claims of the present application. Those skilled in the art will appreciate that the conception and specific embodiments disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present application. Those skilled in the art will also appreciate that such equivalent constructions do not depart from the spirit and scope of the present application as set forth in the appended claims. The novel features believed to be characteristic of the present application, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description. [Brief description of the drawings]

[0024] [Figure 1A] Results of animal feeding studies of selected hydrolysates mixed at 3% in a matrix. Figure 1A shows the results obtained with a corn starch matrix. [Figure 1B] Results of animal feeding studies of selected hydrolysates mixed at 3% in the matrix. Figure 1B shows the results given by the gelatin gel matrix. [Figure 1C] Results of animal feeding studies of selected hydrolysates mixed at 3% in the matrix. Figure 1C shows the results obtained with the autoclaved gel matrix in the study. [Figure 1D] Animal feeding study results of selected hydrolysates mixed at 3% in the matrix. Figure ID shows the feed intake using a gelatin gel matrix containing 20 mM IMP. [Figure 2A] Dose-response curves of test drugs against feline CaSR. Figure 2A shows the dose-response curves of the positive controls CaCl2 and γEVG against feline CaSR. Each assay was performed on a separate day, and each data point represents the average of four replicates in one assay. The mock response of mock cells was from the same day as the second assay. [Figure 2B-1]Dose-response curves of test drugs against feline CaSR. Figure 2B shows the dose-response curves of 14 kokumi peptides against feline CaSR. Two assays were performed for each ligand, each assay was performed on a different day, and each data point represents the average of four replicates in one assay. The response of mock cells was from the same date as the second assay. [Figure 2B-2] Continued from Figure 2B-1 [Figure 2B-3] Continued from Figure 2B-2 [Figure 2B-4] Continued from Figure 2B-3 [Figure 2B-5] Continued from Figure 2B-4 [Figure 2B-6] Continued from Figure 2B-5 [Figure 2B-7] Continued from Figure 2B-6 [Figure 3A] FIG. 3A shows a ribbon diagram of the in silico modeling of the feline CaSR with Asp-Val-Glu. [Figure 3B] FIG. 3B shows a ball-and-stick diagram of in silico modeling of the feline CaSR with Asp-Val-Glu. [Figure 3C] FIG. 3C shows in silico modeling of the feline CaSR using γ-Glu-Val-Gly. [Figure 4-1] Amino acid and nucleotide sequences of the feline calcium-sensing receptor. [Figure 4-2] Figure 4-1 continued [Figure 4-3] The amino acid and nucleotide sequences of the canine calcium-sensing receptor. [Figure 4-4] Figure 4-3 (continued) [Figure 4-5] Amino acid and nucleotide sequences of the human calcium-sensing receptor. [Figure 4-6] Figure 4-5 (continued) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] To date, there continues to be a need for flavor modifiers that can improve and / or enhance the palatability of various pet food products. The present application relates to flavor compositions that include at least one peptide that modulates the activity of the calcium sensing receptor (CaSR). The flavor compositions can be used to improve the palatability and / or enhance or modify the taste of various pet food products, such as nutritionally complete pet foods, and can be added to the pet food products in various delivery system formats. The flavor compositions can further include a combination of multiple compounds.

[0026] 1.Definition The terms used herein generally have their ordinary meaning in the art, within the context of this invention and in the specific context in which each term is used. Certain terms are explained below and elsewhere in this specification to provide further guidance to the practitioner in describing the compositions and methods of the invention and how to make and use them.

[0027] As used herein, when used in conjunction with the term "comprising" in the claims and / or specification, the use of the word "a" or "an" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Furthermore, the terms "having," "including," "containing," and "comprising" are interchangeable, and those skilled in the art will recognize that these terms are open-ended terms.

[0028] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on the limits of how the value is measured or determined, i.e., the measurement system. For example, "about" can mean within or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value.

[0029] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts and structures. The present disclosure also contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0030] As used herein, "taste" refers to a sensation caused by the activation or inhibition of receptor cells in the taste buds of a subject. In certain embodiments, the taste can be selected from the group consisting of sweet, sour, salty, bitter, kokumi, and umami. In certain embodiments, the taste is elicited in the subject by a "tastant." In certain embodiments, the tastant is a synthetic tastant. In certain embodiments, the tastant is prepared from a natural source.

[0031] In certain embodiments, the "taste" can include kokumi. See, e.g., Ohsu et al., J. Biol. Chem., 285(2): 1016-1022 (2010), the contents of which are incorporated herein by reference. In certain embodiments, kokumi is a sensation caused by the activation or inhibition of receptor cells, e.g., CaSR receptors, in a subject's taste buds, and is distinct from other tastes, e.g., sweet, salty, and umami, but can act as a taste enhancer for these tastes.

[0032] As used herein, "taste profile" refers to a combination of one or more tastes, such as sweet, sour, salty, bitter, umami, rich, and free fatty acid tastes. In certain embodiments, a taste profile is generated by one or more tastants present in a composition at the same or different concentrations. In certain embodiments, a taste profile refers to the intensity of a taste or combination of tastes, such as sweet, sour, salty, bitter, umami, rich, and free fatty acid tastes, detected by a subject or by any assay known in the art. In certain embodiments, modifying, changing, or altering the combination of tastants in a taste profile can change the sensory experience of a subject.

[0033] As used herein, a "taste tester" refers to any mammal, such as a human, cat, or dog, that samples a composition or a food or beverage product containing said composition for palatability. In certain embodiments, the taste tester provides feedback on the palatability of the tested composition based on the test parameters and protocol. As used herein, a "flavor" refers to one or more sensory stimuli, such as one or more of taste (gustatory), smell (olfactory), touch (tactile), and temperature (thermometric) stimuli. In certain non-limiting embodiments, the sensory experience of a subject exposed to a flavor can be classified as a characteristic experience for this particular flavor. For example, a subject can identify a flavor as being, but not limited to, a floral, citrus, berry, nutty, caramel, chocolate, pepper, smoky, cheese, meat, and the like flavor. As used herein, a flavor composition can be selected from a liquid, a solution, a dry powder, a spray, a paste, a suspension, and any combination thereof. The flavors can be natural compositions, artificial compositions, nature identical, or any combination thereof.

[0034] As used interchangeably herein, "aroma" and "smell" refer to an olfactory response to a stimulus. For example, and not by way of limitation, an aroma can be produced by an odorant that is perceived by odor receptors in the olfactory system.

[0035] As used herein, "flavor profile" refers to a combination of sensory stimuli, e.g., tastes such as sweet, sour, bitter, salty, umami, kokumi, and free fatty acid tastes, and / or olfactory, tactile, and / or thermosensory stimuli. In certain embodiments, a flavor profile includes one or more flavors that contribute to a subject's sensory experience. In certain embodiments, modifying, changing, or altering the combination of stimuli in a flavor profile can alter the subject's sensory experience.

[0036] As used herein, "admixing", e.g., "admixing the flavor composition or combinations thereof of the present application with a food product", refers to the process of mixing or adding the flavor composition, or individual components of the flavor composition, with or to a finished product, or mixing with some or all of the product's ingredients during the formation of the product or some combination of these steps. When used in the context of admixing, the term "product" refers to the product or any of its components. The admixing step can include a process selected from mixing the flavor composition with the product, spraying the flavor composition onto the product, coating the product with the flavor composition, suspending the product in the flavor composition, applying the flavor composition to the product, applying the flavor composition onto the product, encapsulating the product with the flavor composition, mixing the flavor composition with the product, and any combination thereof. The flavor composition can be a liquid, an emulsion, a dry powder, a spray, a paste, a suspension, and any combination thereof. In certain embodiments, the peptides / compounds of the flavor composition can be generated from precursor compounds present in the pet food product during processing of the pet food product, such as sterilization, retorting, and / or extrusion. In certain embodiments, the peptides / compounds of the flavor composition can be generated during processing of the pet food product and further components of the flavor composition can be added to the pet food product by incorporation.

[0037] As used herein, "ppm" means parts per million and is a weight-related parameter. Parts per million are micrograms per gram, so a component present at 10 ppm is present at 10 micrograms per gram of the mixture as a whole.

[0038] As used herein, "palatability" can refer to an animal's overall willingness to eat a particular food product. Improving the "palatability" of a pet food product can lead to increased enjoyment and acceptance of the pet food by companion animals, thereby ensuring that the animal eats a "healthy amount" of pet food. As used herein, the term "healthy amount" of a pet food refers to an amount that allows a companion animal to maintain or achieve an intake that contributes to its overall health in terms of micronutrients, macronutrients, and calories, as described, for example, in the "Mars Petcare Essential Nutrient Standards." In certain embodiments, "palatability" can refer to an animal's relative preference for one food product over another. For example, if an animal shows a preference for one of two or more food products, the preferred food product is more "palatable" and has "enhanced palatability." The relative palatability of a food product compared to one or more other food products can be determined in a side-by-side free choice comparison, for example by the relative consumption of the food products or other suitable measure of liking that is indicative of palatability. Preference can be determined by standard test protocols in which the animal has equal access to both food products, such as a test called the "two-bowl test" or "versus test." Such preferences can be due to any of the animal's senses, but may in particular relate to taste, aftertaste, odor, mouthfeel, and / or texture.

[0039] The term "pet food" or "pet food product" refers to a product or composition intended for consumption by companion animals, such as cats, dogs, guinea pigs, rabbits, birds, and horses. For example, and without limitation, the companion animal may be a "domestic" cat, such as Felis catus. In certain embodiments, the companion animal may be a "domestic" dog, such as Canis lupus familiaris. "Pet food" or "pet food product" includes food, feed, snacks, dietary supplements, liquids, beverages, treats, toys (chewable and / or consumable toys), and meal replacements or meal substitutes.

[0040] As used herein, "nutritionally complete" refers to a pet food product that contains all known nutrients necessary for the intended recipient of the pet food product in appropriate amounts and ratios based, for example, on the recommendations of recognized authorities or competent authorities in the field of companion animal nutrition, such that such a food can serve as the sole source of dietary intake to sustain life without the addition of supplemental nutritional sources.

[0041] As used herein, "flavor composition" refers to at least one peptide / compound, or biologically acceptable salt thereof, that modulates the taste, odor, flavor, and / or texture of a natural or synthetic tastant, flavoring agent, taste profile, flavor profile, and / or texture profile in an animal or human, including enhancing, multiplying, potentiating, decreasing, suppressing, or inducing. In certain embodiments, a flavor composition comprises a combination of multiple compounds or biologically acceptable salts thereof. In certain embodiments, a flavor composition comprises one or more excipients.

[0042] As used herein, the terms "modulate" or "modify" refer to an increase or decrease in the amount, quality, or effect of a particular activity of a receptor, and / or an increase or decrease in the expression, activity, or function of a receptor. As used herein, a "modulator" refers to any inhibitory or activating compound identified using in silico, in vitro, and / or in vivo assays, e.g., for agonists, antagonists, and homologs (including fragments, variants, and mimetics) thereof.

[0043] As used herein, "inhibitor" or "antagonist" refers to a regulatory compound that reduces, decreases, blocks, prevents, delays, inactivates, desensitizes, or downregulates the biological activity and / or expression of a receptor or pathway of interest.

[0044] As used herein, "inducer," "activator," or "agonist" refers to a regulatory compound that increases, induces, stimulates, opens, activates, promotes, enhances, sensitizes, or upregulates a receptor or pathway of interest.

[0045] In certain embodiments, an "active compound" is a compound / peptide that modulates the calcium-sensing receptor, i.e., has activity at the calcium-sensing receptor. For example, an active compound can have activity at the calcium-sensing receptor as an agonist, antagonist, positive allosteric modulator (PAM), negative allosteric modulator, or by exhibiting a combination of activities, e.g., agonist activity and positive allosteric modulator activity, or agonist activity and negative allosteric modulator activity.

[0046] As used herein, the terms "vector" and "expression vector" refer to a DNA molecule that is linear or circular and can incorporate another DNA sequence fragment of appropriate size. Such one or more DNA fragments can contain additional segments that provide transcription of the gene encoded by the DNA sequence fragment. Such additional segments include, but are not limited to: promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication, and the like. Expression vectors are often derived from plasmids, cosmids, viral vectors, and yeast artificial chromosomes. Vectors are often recombinant molecules that contain DNA sequences from multiple sources.

[0047] As used herein, the terms "nucleic acid molecule" and "nucleotide sequence" refer to a single- or double-stranded covalently linked sequence of nucleotides in which the 3' and 5' ends of each nucleotide are joined by phosphodiester bonds. Nucleic acid molecules can contain deoxyribonucleotide or ribonucleotide bases and can be produced synthetically in vitro or isolated from natural sources.

[0048] The terms "polypeptide," "peptide," "amino acid sequence," and "protein," used interchangeably herein, refer to a molecule formed by the linkage of at least two amino acids. The bond between one amino acid residue and the next is an amide bond, sometimes referred to as a peptide bond. Polypeptides can be obtained by any suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. These terms can apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0049] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs and derivatives can refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to compounds that have a structure that is different from the basic chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0050] The terms "isolated" or "purified," as used interchangeably herein, refer to a nucleic acid, polypeptide, or other biological moiety that has been removed from components with which it is naturally associated. The term "isolated" can refer to a polypeptide that is separate and distinct from the whole organism with which it is found in nature, or that exists in the substantial absence of other biological macromolecules of the same type. The term "isolated," with respect to a polynucleotide, can refer to: a nucleic acid molecule that is completely or partially devoid of sequences with which it is normally associated in nature; or a sequence that exists in nature but has heterologous sequences associated with it; or a molecule that is dissociated from a chromosome.

[0051] As used herein, the term "recombinant" can be used to describe a nucleic acid molecule and refers to a polynucleotide of genomic, RNA, DNA, cDNA, viral, semisynthetic, or synthetic origin that, by its origin or manipulation, is not related in whole or in part to the polynucleotides with which it is associated in nature.

[0052] As used herein, the term "fusion" refers to the joining, by genetic or chemical methods, of different peptide or protein segments, where the joined ends of the peptide or protein segments may be directly adjacent to each other or may be separated by a linker or spacer moiety, such as an amino acid residue or other linking group.

[0053] 2. Calcium-sensing receptor (CaSR) The subject matter of the present disclosure provides calcium-sensing receptors for use in the methods of the present disclosure. The calcium-sensing receptors of the present disclosure can include mammalian calcium-sensing receptors, such as, but not limited to, feline, canine, and human calcium-sensing receptors, for the identification of kokumi-active compounds.

[0054] In certain non-limiting embodiments, the calcium-sensing receptor of the present disclosure is encoded by a nucleic acid described in PCT Application No. PCT / US15 / 55149, filed October 12, 2015, which is incorporated herein by reference in its entirety. In certain non-limiting embodiments, the calcium-sensing receptor of the present disclosure comprises an amino acid sequence described in PCT Application No. PCT / US15 / 55149, filed October 12, 2015.

[0055] In certain non-limiting embodiments, the calcium sensing receptor comprises a feline, canine, or human calcium sensing receptor nucleotide sequence as described in PCT Application No. PCT / US15 / 55149, filed October 12, 2015. In certain non-limiting embodiments, the calcium sensing receptor of the present disclosure is encoded by a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO:2, 4, or 6.

[0056] In certain non-limiting embodiments, the calcium sensing receptor comprises a feline, canine, or human calcium sensing receptor amino acid sequence as described in PCT Application No. PCT / US15 / 55149, filed October 12, 2015. In certain non-limiting embodiments, the calcium sensing receptor of the present disclosure comprises the amino acid sequence set forth in SEQ ID NO: 1, 3, or 5.

[0057] In certain non-limiting embodiments, the calcium-sensing receptor is a feline calcium-sensing receptor comprising the amino acid sequence set forth in SEQ ID NO: 1. In certain non-limiting embodiments, the calcium-sensing receptor is a canine calcium-sensing receptor comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain non-limiting embodiments, the calcium-sensing receptor is a human calcium-sensing receptor comprising the amino acid sequence set forth in SEQ ID NO: 5.

[0058] In certain embodiments, a calcium sensing receptor for use in the presently disclosed subject matter can include a receptor that comprises a nucleotide sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a feline, canine, or human calcium sensing receptor nucleotide sequence.

[0059] In certain embodiments, a calcium sensing receptor for use in the presently disclosed subject matter can include a receptor that includes an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a feline, canine, or human calcium sensing receptor amino acid sequence.

[0060] In certain embodiments, the subject matter of the present disclosure provides the use of isolated or purified calcium sensing receptor and / or variants and fragments thereof. The subject matter of the present disclosure also encompasses the use of sequence variants. In certain embodiments, the variants may occur in either or both of the coding and non-coding regions of the nucleotide sequence of the calcium sensing receptor. Variants can include substantially homologous proteins encoded by the same genetic locus of an organism, i.e. allelic variants. Variants also encompass proteins that have substantial homology to the calcium sensing receptor but originate from another genetic locus of an organism, e.g. a cat, i.e. homologs. Variants can also encompass proteins that have substantial homology to the calcium sensing receptor but originate from another organism, i.e. orthologues. Variants also include proteins that have substantial homology to the calcium sensing receptor that have been produced by chemical synthesis. Variants also encompass proteins that have substantial homology to the calcium sensing receptor that have been produced by recombinant methods.

[0061] The subject matter of the present disclosure also provides a fusion protein comprising a calcium sensing receptor or a fragment thereof. In certain embodiments, the fusion protein of the present disclosure can include a functional group such as a detectable marker, a carrier, a label, a stabilizing sequence, or a mechanism that can detect calcium sensing receptor agonist binding. Non-limiting examples of labels include FLAG tag, His tag, MYC tag, maltose binding protein, and others known in the art. The subject matter of the present disclosure also provides a nucleic acid encoding such a fusion protein, a vector comprising a nucleic acid encoding the fusion protein, and a host cell comprising such a nucleic acid or vector. In certain embodiments, the fusion can be made at the amino terminus (N-terminus) of the calcium sensing receptor, or the carboxy terminus (C-terminus) of the calcium sensing receptor.

[0062] In certain embodiments, the calcium-sensing receptors of the present disclosure, for example when used in the methods of the presently disclosed subject matter, can contain additional amino acids at the N-terminus and / or C-terminus of the sequence, which can aid in immobilization of the polypeptide for screening purposes, or can make the polypeptide part of a fusion protein, as described above, to facilitate detection of biological activity.

[0063] 3. Calcium-sensing receptor modulating peptide The present disclosure relates to flavor compositions comprising at least one compound capable of modulating the activity of calcium sensing receptor (CaSR). The compounds of the present disclosure are identified by in vitro assays that determine the ability of the compounds to activate feline CaSR expressed by cells, and / or by in silico assays that determine the ability of the compounds to bind to CaSR in silico. The flavor compositions can be used to enhance or modulate the palatability, taste, or flavor of pet food products. In certain embodiments, the flavor compositions described herein can be added to pet food product compositions in various delivery system formats.

[0064] In certain embodiments, the CaSR modulating compound is a peptide, such as an oligopeptide. In certain embodiments, the peptide has the following formula: [Negatively charged or polar amino acid] - [Amino acid with molecular mass of 150 daltons or less] - [Negatively charged or polar amino acid] It contains a tripeptide motif according to

[0065] In certain embodiments, the tripeptide motif is: (a) a first amino acid residue at the N-terminus which is a negatively charged amino acid residue or a polar uncharged amino acid residue; (b) a second amino acid residue, which is an amino acid that is not too large; and (c) a third amino acid residue at the C-terminus which is a negatively charged amino acid residue or a polar uncharged amino acid residue; In certain embodiments, the tripeptide motif binds to CaSR and confers kokumi flavor.

[0066] In certain embodiments, the first amino acid residue is a negatively charged amino acid residue. In certain embodiments, the third amino acid residue is a negatively charged amino acid residue. In certain embodiments, the negatively charged amino acid residue is selected from the group consisting of aspartic acid (Asp), β-aspartic acid (β-Asp), glutamic acid (Glu), γ-glutamic acid (γ-Glu), and any phosphorylated amino acid residue. In certain embodiments, the negatively charged amino acid residue is not β-aspartic acid (β-Asp) or γ-glutamic acid (γ-Glu). In certain embodiments, the first amino acid residue is not β-aspartic acid (β-Asp) or γ-glutamic acid (γ-Glu). In certain embodiments, the negatively charged amino acid residue is phosphorylated serine (pSer), phosphorylated tyrosine (pTyr), or phosphorylated threonine (pThr).

[0067] In certain embodiments, the first amino acid residue is a polar uncharged amino acid residue. In certain embodiments, the third amino acid residue is a polar uncharged amino acid residue. In certain embodiments, the polar uncharged amino acid residue is selected from the group consisting of cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asp), serine (Ser), tyrosine (Tyr), and threonine (Thr).

[0068] In certain embodiments, the second amino acid residue has a molecular mass of about 200 daltons or less. In certain embodiments, the second amino acid residue has a molecular mass of about 150 daltons or less, about 140 daltons or less, about 130 daltons or less, about 120 daltons or less, about 110 daltons or less, about 100 daltons or less, about 90 daltons or less, or about 80 daltons. In certain embodiments, the second amino acid residue has a molecular mass of about 50 daltons to about 200 daltons, about 50 daltons to about 150 daltons, about 60 daltons to about 150 daltons, about 60 daltons to about 140 daltons, about 60 daltons to about 130 daltons, or about 60 daltons to about 120 daltons. In certain embodiments, the second amino acid residue is selected from the group consisting of lysine (Lys), isoleucine (Ile), leucine (Leu), alanine (Ala), methionine (Met), proline (Pro), valine (Val), aspartic acid (Asp), glutamic acid (Glu), cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asn), serine (Ser), and threonine (Thr). In certain embodiments, the second amino acid residue is alanine (Ala), valine (Val), or glutamic acid (Glu).

[0069] In certain embodiments, the peptide is a tripeptide selected from the group consisting of Asp-Val-Glu, Glu-Val-Asp, Asp-Glu-Glu, pSer-Glu-pSer, pSer-Val-pSer, pSer-Val-Glu, Ser-Glu-Ser, Cys-Val-Cys, pTyr-Glu-pTyr, pThr-Glu-pThr, Asp-Ala-Glu, Glu-Val-Glu, Asp-Val-Asp, and any combination thereof.

[0070] In certain embodiments, the peptide is selected from the group consisting of Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln, Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln-Ala, Glu-Ile-Val-Pro-Asn-pSer-Ala-Glu-Glu, Asp-Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln-Ala, and any combination thereof.

[0071] In certain embodiments, the peptide is 2+ It can form a chelating agent. In certain embodiments, the peptide can activate feline CaSR. In certain embodiments, the tripeptide motif of the peptide binds to CaSR and imparts kokumi flavor. In certain embodiments, the EC50 of the peptide for activating CaSR is about 100 mM or less, about 90 mM or less, about 80 mM or less, about 70 mM or less, about 60 mM or less, about 50 mM or less, about 40 mM or less, about 30 mM or less, about 20 mM or less, about 10 mM or less, or about 5 mM or less.

[0072] In certain embodiments, the peptides are included in a flavor composition without other palatability enhancers, hi certain embodiments, the peptides are included in one or more flavor compositions with one or more additional palatability enhancers, such as nucleotides, nucleotide derivatives, amino acids, furanones, fatty acid receptor activating compounds, and umami receptor activating compounds described herein.

[0073] In certain embodiments, the peptide can interact with (e.g., bind to) the Venus Flytrap (VFT) domain of CaSR. In certain embodiments, such interaction with the VFT domain of CaSR agonizes CaSR. In other embodiments, the peptide acts synergistically with other CaSR agonists or modulators to modulate CaSR activity. In yet other embodiments, interaction with the VFT domain of CaSR antagonizes CaSR. In certain embodiments, the peptide enhances the ability of a CaSR agonist to activate the receptor (i.e., the peptide functions as a positive allosteric modulator). In certain embodiments, the tripeptide motif of the peptide binds to the VFT domain of CaSR to impart kokumi flavor.

[0074] In certain embodiments, the peptide interacts with one or more amino acids of the VFT domain, such as one or more of Pro39, Asn64, Arg66, Gly67, Arg69, Trp70, Asn102, Thr145, Gly146, Ser147, Gly148, Tyr167, Ala168, Ser169, Ser170, Ser171, Ile187, Tyr218, Ser271, Ser272, Glu297, Ala298, Trp299, Ala300, Ser301, Ser302, Ile416, and any combination thereof. Thus, in certain embodiments, calcium-sensing receptor modulating peptides can be identified and / or defined based on their interaction with one or more of these residues.

[0075] In certain embodiments, the CaSR agonists and / or modulators of the present disclosure include salts of the CaSR agonists and / or modulators, such as, but not limited to, acetate or formate salts. In certain embodiments, the CaSR agonist and / or modulator salts contain a cation (+) (such as, but not limited to, Al 3+ , Ca 2+ , Na + , K + , Cu 2+ , H + , Fe3+ , Mg 2+ , NH4 + and H3O + ) bound by an ionic bond to an anion (-) (such as, but not limited to, Cl - , O 2- , CO3 2- , HCO3 - , O.H. - , NO3 - , PO4 3- , SO4 2- , CH3COO - , H.C.O.O. - and C2O4 2- In other embodiments, the CaSR agonist salt comprises a cation (+) bound by an ionic bond to an anion (-). In certain embodiments, the peptides of the present disclosure comprise sodium or potassium salts of the above peptides.

[0076] In certain embodiments, the CaSR agonists and / or peptides of the present disclosure are included in the flavor composition in an amount of about 0.001% to about 100% w / w, about 0.1% to about 99.9% w / w, about 1% to about 99% w / w, about 1% to about 80% w / w, about 1% to about 50% w / w, about 1% to about 20% w / w, about 50% to about 100% w / w, about 20% to about 80% w / w, or about 30% to about 70% w / w.

[0077] In certain embodiments, the CaSR agonist and / or modulator peptides are produced during the manufacturing process of the food product, for example, by hydrolysis of raw materials.

[0078] 4. Methods for identifying calcium-sensing receptor modulating compounds The present disclosure further provides methods for identifying compounds that modulate calcium-sensing receptor activity and / or expression. For example, and without limitation, the modulators can be agonists or antagonists. The presently disclosed subject matter provides in silico and in vitro methods for identifying compounds that modulate calcium-sensing receptor activity and / or expression as disclosed above.

[0079] 4.1 In silico methods The presently disclosed subject matter further provides in silico methods for identifying compounds that can potentially interact with and / or modulate the activity and / or expression of a calcium sensing receptor, e.g., a feline, canine, or human calcium sensing receptor.

[0080] In certain embodiments, the method can include predicting a three-dimensional (3D) structure of a calcium-sensing receptor; and screening the predicted 3D structure with a putative calcium-sensing receptor modulating compound (i.e., a test compound / peptide). The method can further include predicting whether the putative compound interacts with a binding site of the receptor by analyzing potential interactions of the putative compound with amino acids of the receptor. The method can further include identifying a test compound capable of binding to the calcium-sensing receptor and / or modulating a biological activity of the calcium-sensing receptor by determining whether the 3D structure of the compound fits within the binding site of the 3D structure of the receptor.

[0081] In certain embodiments, a calcium-sensing receptor for use in the methods of the disclosure can have an amino acid or nucleotide sequence set forth in PCT Application No. PCT / US15 / 55149, filed October 12, 2015, or a fragment or variant thereof.

[0082] Non-limiting examples of compounds (e.g., potential calcium-sensing receptor modulators) that can be tested using the methods of the present disclosure include any small molecule compound, or any biological entity, such as peptides, salts, and amino acids, known in the art. In certain embodiments, the test compound can be a small chemical molecule.

[0083] In certain embodiments, a structural model of the calcium-sensing receptor can be constructed using the crystal structure of a closely related GPCR as a template for homology modeling. The X-ray crystallographic structure of the human calcium receptor Venus Flytrap (VFT) domain was recently solved. The structure available in the Protein Databank (PDB, www.rcsb.org) is: PDB ID: 5FBH-bound Gd +3 The crystal structure of the extracellular domain of the human calcium-sensing receptor; PDB ID: 5FBK - Crystal structure of the extracellular domain of the human calcium-sensing receptor; PDB ID: 5K5T - Crystal structure of the inactive form of the extracellular domain of the human calcium-sensing receptor; PDB ID: 5K5S - Crystal structure of the active form of the extracellular domain of the human calcium-sensing receptor (See Geng, et al., Structural mechanism of ligand activation in human calcium-sensing receptor, Elife. 2016 Jul 19;5. pii: e13662; Zhang, et al., Structural basis for regulation of human calcium-sensing receptor by magnesium ions and an unexpected tryptophan derivative co-agonist, Sci Adv. 2016 May; 2(5): e1600241; the disclosures of which are incorporated herein by reference in their entireties.)

[0084] In certain embodiments, model VFT structures can be generated for other species of interest, such as cats and dogs, based on sequence homology to the human VFT.

[0085] 3A-3C show structural models of calcium-sensing receptors that can be used in the in silico methods of the present disclosure. Any modeling software known in the art can be used. In certain embodiments, the Modeller software package (Accelrys, BIOVIA, Dassault Systemes) can be used to generate three-dimensional protein structures.

[0086] In certain embodiments, the in silico method for identifying a compound that binds to a calcium sensing receptor comprises determining whether a test compound interacts with one or more amino acids of a calcium sensing receptor interaction domain as described above.

[0087] Compounds identified by the in silico methods of the present disclosure can be further tested using the in vitro methods of the present disclosure.

[0088] 4.2 Calcium-sensing receptor binding site The present application provides methods of screening for compounds that modulate the activity of a calcium-sensing receptor, e.g., a feline, canine, or human calcium-sensing receptor, where the compound interacts with one or more amino acids of the calcium-sensing receptor. In certain embodiments, the binding site of a calcium-sensing receptor includes amino acids within the Venus Flytrap (VFT) domain of the receptor, and can be identified by generating an interaction map of the receptor using in silico modeling, as described herein. In one non-limiting example, the presence of an amino acid in the interaction map means that the residue is in the vicinity of the ligand binding environment and interacts with the ligand.

[0089] In certain embodiments, the interaction between a compound and one or more amino acids of the calcium-sensing receptor described herein can include one or more hydrogen bonds, covalent bonds, non-covalent bonds, salt bridges, physical interactions, and combinations thereof. The interaction can also be any interaction characteristic of a ligand-receptor interaction known in the art. Such interactions can be determined, for example, by site-directed mutagenesis, X-ray crystallography, X-ray or other spectroscopy, nuclear magnetic resonance (NMR), crosslinking evaluation, mass spectrometry or electrophoresis, cryomicroscopy, displacement assays based on known agonists, structure determination, and combinations thereof. In certain embodiments, the interaction is determined in silico, for example, by theoretical means, such as docking a compound to the feline or canine calcium-sensing receptor described herein, for example, by molecular docking, molecular modeling, molecular simulation, or other means known to those skilled in the art.

[0090] In certain embodiments, the interaction is a salt-bridge interaction.

[0091] In certain embodiments, the interaction is a hydrogen bonding interaction.

[0092] In certain embodiments, the interaction is a hydrophobic interaction.

[0093] In certain embodiments, the interaction is a ring-stacking interaction.

[0094] In certain embodiments, a compound identified according to the methods described herein that modulates calcium sensing receptor activity interacts with one or more amino acids in the Venus Flytrap (VFT) domain of the calcium sensing receptor. In certain embodiments, the amino acids that the compound interacts with are Pro39, Asn64, Arg66, Gly67, Arg69, Trp70, Asn102, Thr145, Gly146, Ser147, Gly148, Tyr167, Ala168, Ser169, Ser170, Ser171, Ile187, Tyr218, Ser271, Ser272, Ser273, Ser274, Ser275, Ser276, Ser277, Ser278, Ser279, Ser280, Ser281, Ser282, Ser283, Ser284, Ser285, Ser286, Ser287, Ser288, Ser289, Ser290, Ser291, Ser292, Ser293, Ser294, Ser295, Ser296, Ser297, Ser298, Ser299, Ser200, Ser201, Ser202, Ser203, Ser204, Ser205, Ser206, Ser207, Ser208, Ser209, Ser210, Ser211, Ser212, Ser213, Ser214, Ser215, Ser216, Ser217, Ser218, Ser220, Ser221, Ser222, Ser223, Ser224, Ser225, Ser226, Ser227, Ser230, Ser231, Ser232, Ser233, Ser234, Ser235, Ser236, Ser237, Ser238, Ser239, Ser240, Ser241, Ser242, Ser243, Ser24 72, Glu297, Ala298, Trp299, Ala300, Ser301, Ser302, Ile416, and any combination thereof, or functionally equivalent amino acids of the canine calcium sensing receptor or the human calcium sensing receptor.

[0095] In a particular embodiment, a method for identifying a composition that modulates the activity of a feline calcium sensing receptor includes the steps of: (a) contacting a test agent with a calcium sensing receptor, for example a feline calcium sensing receptor comprising the amino acid sequence of SEQ ID NO: 1; (b) contacting the test agent with any of the following amino acids in the VFT domain: Pro39, Asn64, Arg66, Gly67, Arg69, Trp70, Asn102, Thr145, Gly146, Ser147, Gly148, Tyr167, Ala168, Ser169, Ser200, Ser170, Ser171, Ser172, Ser173, Ser174, Ser175, Ser176, Ser177, Ser178, Ser179, Ser204, Ser179, Ser179, Ser205, Ser206, Ser207, Ser208, Ser209, Ser210, Ser211, Ser212, Ser214, Ser215, Ser216, Ser217, Ser218, Ser220, Ser225, Ser226, Ser227, Ser228, Ser230, Ser231, Ser232, Ser233, Ser234, Ser235, Ser236, Ser237, Ser238, Ser239, Ser240, Ser241, Ser242, Ser243, Ser244, Ser245, Ser246, Ser247, Ser248, Ser250, Ser251, Ser252, Ser253, Ser254, Ser255, Ser256, Ser257, Ser258, Ser259, Ser260, Ser261, Ser262, Ser263, Ser264, Ser265, Ser266, Ser267, Ser268, and (c) detecting an interaction between one or more amino acids in the interaction site of the calcium-sensing receptor selected from the group consisting of Ser170, Ser171, Ile187, Tyr218, Ser271, Ser272, Glu297, Ala298, Trp299, Ala300, Ser301, Ser302, Ile416, and any combination thereof; and (b) selecting a test agent for the composition that interacts with one or more of the amino acids.

[0096] In certain embodiments, the method further comprises: determining activity of the calcium sensing receptor after step (a); and selecting a test agent for the composition that enhances activity of the calcium sensing receptor.

[0097] In certain embodiments, the method further comprises the steps of: contacting the calcium sensing receptor with a ligand, e.g., an agonist; and selecting for the composition a test agent that improves or potentiates the ability of the agonist to activate the calcium sensing receptor.

[0098] 4.3 In vitro methods The presently disclosed subject matter further provides in vitro methods for identifying compounds capable of modulating calcium-sensing receptor activity and / or expression.

[0099] A calcium sensing receptor for use in the methods of the present disclosure can include an isolated or recombinant calcium sensing receptor, or a cell expressing a calcium sensing receptor, as disclosed herein. In certain embodiments, a calcium sensing receptor for use in the methods of the present disclosure can have an amino acid or nucleotide sequence set forth in PCT Application No. PCT / US15 / 55149, filed October 12, 2015, or a fragment or variant thereof.

[0100] In certain embodiments, the method of identifying a compound that modulates calcium-sensing receptor activity and / or expression comprises measuring the biological activity of the calcium-sensing receptor in the absence and / or presence of a test compound. In certain embodiments, the method can include measuring the biological activity of the calcium-sensing receptor in the presence of various concentrations of the test compound. The method can further include identifying a test compound that results in modulation of calcium-sensing receptor activity and / or expression when compared to calcium-sensing receptor activity and / or expression in the absence of the test compound.

[0101] In certain embodiments, a compound identified according to the methods described herein increases the biological activity of the calcium sensing receptor by at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more, when compared to the biological activity of the calcium sensing receptor in the absence of the compound. In certain embodiments, a compound identified according to the methods described herein increases the biological activity of the calcium sensing receptor by at least about 30%, when compared to the biological activity of the calcium sensing receptor in the absence of the compound.

[0102] In certain embodiments, the method can further comprise analyzing two or more, three or more, or four or more test compounds in combination. In certain embodiments, the two or more, three or more, or four or more test compounds can be from different classes of compounds, such as amino acids and small molecule compounds. For example, but not by way of limitation, the method can comprise analyzing the effect of one or more small molecule test compounds on calcium sensing receptor biological activity and / or expression in the presence of one or more amino acid test compounds. In certain embodiments, the method for identifying the effect of a compound on calcium sensing receptor activity and / or expression comprises analyzing the effect of a test compound on calcium sensing receptor biological activity and / or expression in the presence of one or more nucleotide or nucleotide derivative test compounds.

[0103] In certain embodiments, a method for identifying a compound that modulates calcium sensing receptor activity and / or expression comprises determining whether the compound directly modulates the receptor, e.g., as an agonist or antagonist, hi certain embodiments, the method comprises determining whether the compound indirectly modulates the activity of the receptor by enhancing or reducing the effect of other compounds on activating or inhibiting receptor activity (e.g., as an allosteric modulator).

[0104] In certain embodiments, a method for identifying a compound that modulates calcium sensing receptor activity and / or expression comprises: expressing the calcium sensing receptor in a cell line; and measuring the biological activity of the receptor in the presence and / or absence of a test compound. The method can further comprise identifying a test compound that modulates the activity of the receptor by determining whether there is a difference in receptor activation in the presence of a test compound as compared to the activity of the receptor in the absence of the test compound. In certain embodiments, the selectivity of a putative calcium sensing receptor agonist and / or modulator can be assessed by comparing its effect on other GPCRs or taste receptors, such as umami, GPR120, T1R, etc.

[0105] Activation of the receptor in the disclosed methods can be detected by the use of a labeling compound and / or a labeling agent. In certain embodiments, activity of the calcium-sensing receptor can be determined by detection of a second messenger, such as, but not limited to, cAMP, cGMP, IP3, DAG, or calcium. In certain embodiments, activity of the calcium-sensing receptor can be determined by detection of intracellular calcium levels. Monitoring can be by detection of luminescence or fluorescence, such as by calcium-sensitive fluorescent dyes. In certain embodiments, intracellular calcium levels can be detected using a cell dye, e.g., a fluorescent calcium indicator, such as calcium 4. In certain embodiments, intracellular calcium levels can be detected by measuring the level of calcium binding to a calcium-binding protein, e.g., calmodulin. Alternatively and / or additionally, activity of the calcium-sensing receptor can be determined by detection of phosphorylation, transcription levels, and / or protein levels of one or more downstream protein targets of the calcium-sensing receptor.

[0106] The cell lines used in the methods of the present disclosure can include any cell type capable of expressing a calcium-sensing receptor. Non-limiting examples of cells that can be used in the methods of the present disclosure include HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), Xenopus oocytes, HEK-293 cells, and mouse 3T3 fibroblasts. In certain embodiments, the methods can include expressing the calcium-sensing receptor in CHO-K1 cells. In certain embodiments, the methods can include expressing the calcium-sensing receptor in HEK-293 cells. In certain embodiments, the methods can include expressing the calcium-sensing receptor in COS cells. In certain embodiments, the cells constitutively express the calcium-sensing receptor. In another embodiment, the expression of the calcium-sensing receptor by the cells is inducible.

[0107] In certain embodiments, the cell expresses a calcium-binding photoprotein, which emits light when bound to calcium. In certain embodiments, the calcium-binding photoprotein comprises the protein clytin. In certain embodiments, the clytin is recombinant clytin. In certain embodiments, the clytin comprises an isolated clytin, such as clytin isolated from Clytia gregarium. In certain embodiments, the calcium-binding photoprotein comprises the protein aequorin, such as recombinant aequorin or isolated aequorin, such as aequorin isolated from Aequorea victoria. In certain embodiments, the calcium-binding photoprotein comprises the protein obelin, such as recombinant obelin or isolated obelin, such as obelin isolated from Obelia longissima.

[0108] In certain embodiments, expression of the calcium sensing receptor in a cell can be achieved by introducing into the cell a nucleic acid encoding the calcium sensing receptor. For example, but not limited to, a nucleic acid having a nucleotide sequence as set forth in PCT Application No. PCT / US15 / 55149, filed October 12, 2015, or a fragment thereof, can be introduced into the cell. In certain embodiments, introduction of the nucleic acid into the cell can be achieved by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. Numerous techniques are known in the art for the introduction of heterologous genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92 (1985); the disclosures of which are incorporated herein by reference in their entireties) and can be used in accordance with the subject matter of the present disclosure. In certain embodiments, the techniques can provide for the stable transfer of nucleic acid to a cell, such that the nucleic acid is expressible by the cell and heritable and expressible by its progeny. In certain embodiments, the techniques can provide for the transient transfer of nucleic acid to a cell, such that the nucleic acid is expressible by the cell, such that heritability and expressibility are reduced in subsequent generations of the cell's progeny.

[0109] In certain embodiments, the method can include identifying a compound that binds to a calcium sensing receptor. The method can include: contacting the calcium sensing receptor with a test compound; and measuring binding between the compound and the calcium sensing receptor. For example, but not by way of limitation, the method can include: providing an isolated or purified calcium sensing receptor in a cell-free system; and contacting the receptor with a test compound in the cell-free system to determine whether the test compound binds to the calcium sensing receptor. In certain embodiments, the method can include: contacting a calcium sensing receptor expressed on the surface of a cell with a test compound; and detecting binding of the test compound to the calcium sensing receptor. The binding can be measured directly, for example, using a labeled test compound, or can be measured indirectly. In certain embodiments, the detection can include detecting a physiological event in the cell caused by binding of the compound to the calcium sensing receptor, for example, an increase in intracellular calcium levels. For example, but not by way of limitation, the detection can be performed by fluorescence detection, such as a calcium-sensitive fluorescent dye, luminescence detection, or any other detection method known in the art.

[0110] In certain non-limiting embodiments, the in vitro assay includes cells expressing a calcium-sensing receptor that is native to the cell. Examples of such cells expressing a native calcium-sensing receptor include, but are not limited to, canine and / or feline taste cells (e.g., primary taste receptor cells). In certain embodiments, canine and / or feline taste cells expressing a calcium-sensing receptor are isolated from a canine and / or feline and cultured in vitro. In certain embodiments, taste receptor cells can be, e.g., immortalized, such that cells isolated from, e.g., a canine and / or feline, can be expanded in culture.

[0111] In certain embodiments, expression of the calcium sensing receptor in a cell can be induced by gene editing, for example using a CRISPR gene editing system, to integrate the calcium sensing receptor gene into the genome of the cell, or by editing or modifying the calcium sensing receptor gene so that it is native to the cell.

[0112] In certain embodiments, an in vitro method for identifying a compound that binds to a calcium-sensing receptor comprises determining whether a test compound interacts with one or more amino acids of a calcium-sensing receptor interaction domain, as described herein.

[0113] In certain embodiments, compounds identified as agonists and / or modulators of the calcium sensing receptor can be tested in other analytical methods, including but not limited to in vivo assays, to confirm or quantitate their modulatory activity.

[0114] In certain embodiments, the methods described herein may include determining whether the calcium-sensing receptor modulator is a calcium-sensing taste enhancing compound, such as a calcium-sensing receptor agonist.

[0115] In certain embodiments, methods for identifying calcium sensing receptor agonists and / or modulators can include comparing the effect of a test compound on a calcium sensing receptor agonist. For example, a test compound that enhances the activity of the receptor when compared to the activity of the receptor when contacted with a calcium sensing receptor agonist can be selected as a calcium sensing receptor modulating compound (e.g., as an agonist).

[0116] In certain embodiments, a method for identifying a calcium sensing receptor modulator can include determining whether a test compound modulates the activity of the receptor when the receptor is contacted with an agonist, or whether the test compound can modulate the activity of a positive allosteric modulator (PAM). Test compounds that increase or decrease the effect of the agonist or PAM on the receptor can be selected as calcium sensing receptor modulating compounds (e.g., as allosteric modulators).

[0117] 5. Flavor composition In certain embodiments, the flavor compositions of the present disclosure can be used to improve the palatability of pet food products, such as cat food products. The flavor compositions can include combinations of compounds and can be added to the pet food product in a variety of delivery systems.

[0118] In certain embodiments, the present disclosure relates to a method for modulating the kokumi taste (e.g., calcium-sensing receptor activity) and / or palatability of a pet food product, the method comprising: a) providing at least one pet food product, or a precursor thereof; and b) forming an enhanced pet food product by combining the pet food product or a precursor thereof with at least one flavor composition, e.g., comprising one or more active compounds, or edible acceptable salts thereof, in at least a kokumi taste modulating amount.

[0119] In certain embodiments, the flavor compositions of the present disclosure can enhance the activity of a calcium sensing receptor and / or the palatability of a pet food product, such as a pet food product including a wet pet food product, a dry pet food product, a moist pet food product, a pet beverage product, and / or a snack-type pet food product.

[0120] In certain embodiments, one or more of the flavor compositions of the present disclosure can be added to a pet food product in an amount effective to modify, enhance, or otherwise change the taste or taste profile of the pet food product. Modification can include, for example, increasing or enhancing the palatability of the pet food product, as determined by procedures known in the art, by animals, e.g., cats and / or dogs, or, in the case of formulation testing, by a panel of animal taste testers, e.g., cats and / or dogs. In certain embodiments, the CaSR agonist and / or modulator peptides of the flavor composition are generated during the manufacturing process of the food product, e.g., by hydrolysis of raw materials.

[0121] In certain embodiments of the present disclosure, a pet food product can be produced that contains a sufficient amount of at least one flavor composition described herein, including, for example, a peptide, to produce a pet food product having a desired taste, e.g., kokumi.

[0122] In certain embodiments of the present disclosure, pet food products can be produced that contain a sufficient amount of a flavor composition that includes at least one, two, three, four, five, six, or more peptides.

[0123] In certain embodiments, a calcium-sensing receptor modulating amount of one or more of the flavor compositions of the present disclosure can be added to a pet food product such that said pet food product has improved palatability as compared to a pet food product prepared without the flavor composition, said palatability being determined by procedures known in the art, e.g., by animals, e.g., cats and / or dogs, or, in the case of formulation testing, by a panel of animal taste testers.

[0124] In certain embodiments of the present disclosure, the flavor composition is added to a pet food product in an amount effective to increase, enhance, and / or modify the palatability of said pet food product.

[0125] The concentration of a flavor composition that is blended with a pet food product to modulate and / or improve the palatability of the pet food product may vary depending on variables such as: the specific type of pet food product; what taste modulating compounds / peptides are already present in the pet food product, and their concentrations; and the effectiveness of a particular flavor composition as an enhancer for such taste modulating compounds / peptides.

[0126] A wide range of concentrations of the flavor composition can be employed to provide such modified palatability. In certain embodiments of the present application, the flavor composition is blended with the pet food product, and the flavor composition is present in an amount of about 0.001 ppm to about 1,000 ppm. For example, and without limitation, the flavor composition may be present in an amount of about 0.001 ppm to about 750 ppm, about 0.001 ppm to about 500 ppm, about 0.001 ppm to about 250 ppm, about 0.001 ppm to about 150 ppm, about 0.001 ppm to about 100 ppm, about 0.001 ppm to about 75 ppm, about 0.001 ppm to about 50 ppm, about 0.0 0.01 ppm to about 25 ppm, about 0.001 ppm to about 15 ppm, about 0.001 ppm to about 10 ppm, about 0.001 ppm to about 5 ppm, about 0.001 ppm to about 4 ppm, about 0.001 ppm to about 3 ppm, about 0.001 ppm to about 2 ppm, about 0.001 ppm to about 1 ppm, about 0.01 ppm to about 1,000 ppm, about 0.1 ppm pm~1,000ppm, approximately 1ppm~1,000ppm, approximately 2ppm~approximately 1,000ppm, approximately 3ppm~approximately 1,000ppm, approximately 4ppm~approximately 1,000ppm, Approximately 5ppm to approximately 1,000ppm, approximately 10ppm to approximately 1,000ppm, approximately 15ppm to approximately 1,000ppm, approximately 25ppm to approximately 1,000ppm, approximately 50ppm to approximately 1 It can be present in an amount of about 1,000 ppm, about 75 ppm to about 1,000 ppm, about 100 ppm to about 1,000 ppm, about 150 ppm to about 1,000 ppm, about 250 ppm to about 1,000 ppm, about 250 ppm to about 1,000 ppm, about 500 ppm to about 1,000 ppm, about 750 ppm to about 1,000 ppm, or any value therebetween.

[0127] In certain embodiments of the present application, the flavor composition is admixed with a pet food product, and the flavor composition is present in an amount of from about 0.001 ppm to about 500 ppm, from about 0.01 ppm to about 500 ppm, from about 0.1 ppm to about 500 ppm, from about 1 ppm to about 500 ppm, or any value therebetween.

[0128] In certain embodiments of the present application, the flavor composition is admixed with a pet food product, wherein the flavor composition is present in an amount of from about 0.01 ppm to about 100 ppm, from about 0.1 ppm to about 100 ppm, from about 1 ppm to about 100 ppm, or any value therebetween.

[0129] In certain embodiments, the flavor composition is present in the pet food product in an amount of greater than about 0.001 ppm, greater than about 0.01 ppm, greater than about 0.1 ppm, greater than about 1 ppm, greater than about 2 ppm, greater than about 3 ppm, greater than about 4 ppm, greater than about 5 ppm, greater than about 10 ppm, greater than about 25 ppm, greater than about 50 ppm, greater than about 75 ppm, greater than about 100 ppm, greater than about 250 ppm, greater than about 500 ppm, greater than about 750 ppm, greater than about 1000 ppm, or any value therebetween.

[0130] In certain embodiments, the peptides of the present disclosure are present in a food product in an amount sufficient to modulate, activate, and / or enhance a calcium-sensing receptor. For example, and without limitation, the peptides may be present in a food product at concentrations of about 1 nM to about 1 M, about 1 μM to about 1 M, about 1 mM to about 1 M, about 10 mM to about 1 M, about 100 mM to about 1 M, about 250 mM to about 1 M, about 500 mM to about 1 M, about 750 mM to about 1 M, about 0.001 μM to about 1 M, about 0.001 μM to about 750 mM ... It can be present in an amount of about 500 mM, about 0.001 μM to about 250 mM, about 0.001 μM to about 100 mM, about 0.001 μM to about 50 mM, about 0.001 μM to about 25 mM, about 0.001 μM to about 10 mM, about 0.001 μM to about 1 mM, about 0.001 μM to about 100 μM, about 0.001 μM to about 10 μM, or any value therebetween.

[0131] In certain embodiments, the peptides of the present disclosure are present in the food product in an amount sufficient to modulate, activate, and / or enhance the calcium-sensing receptor. For example, and without limitation, the peptide may be present in a food product in an amount of about 1 nM to about 10 M, about 1 nM to about 1 M, about 1 μM to about 1 M, about 1 mM to about 1 M, about 10 mM to about 1 M, about 100 mM to about 1 M, about 250 mM to about 1 M, about 500 mM to about 1 M, about 750 mM to about 1 M, about 1 μM to about 1 M, about 1 μM to about 750 mM, about 1 μM to about 500 mM, about 1 μM to about 250 mM, about 1 μM to about 100 mM, about 1 μM to about 50 mM, about 1 μM to about 25 mM, about 1 μM to about 10 mM, about 1 μM to about 1 mM, about 1 μM to about 100 μM, about 1 μM to about 10 μM, or values ​​between these.

[0132] In certain embodiments of the present application, the flavor composition is admixed with a pet food product, and said flavor composition is present in an amount of about 10 nM to about 0.5 M, about 1 nM to about 0.5 M, about 0.1 nM to about 0.5 M, or any value therebetween.

[0133] In certain embodiments of the present application, the flavor composition is admixed with a pet food product, wherein the flavor composition is present in an amount of about 10 nM to about 0.1 M, about 1 nM to about 0.1 M, about 0.1 nM to about 0.1 M, or any value therebetween.

[0134] In certain embodiments of the present application, the flavor composition is blended with a food product, and the flavor composition is present in an amount of about 0.0001 to about 10% weight / weight (w / w) of the food product. For example, and without limitation, the flavor composition can be present in an amount of about 0.0001% to about 10%, about 0.0001% to about 1%, about 0.0001% to about 0.1%, about 0.0001 to about 0.01%, about 0.0001% to about 0.001%, about 0.001% to about 10%, about 0.001% to about 1%, about 0.01% to about 1%, about 0.1% to about 1%, or any value between these amounts.

[0135] In certain embodiments of the present application, the flavor composition is admixed with a food product, and said flavor composition is present in an amount of about 0.0001% to about 5%, about 0.001% to about 5%, about 0.01% to about 5% w / w, about 0.1% to about 5% w / w, or any value therebetween.

[0136] In certain embodiments of the present application, the flavor composition is admixed with a food product, wherein the flavor composition is present in an amount of about 0.0001% to about 1%, about 0.001% to about 1%, about 0.01% to about 1% w / w, about 0.1% to about 1% w / w, or any value therebetween.

[0137] In certain embodiments of the present application, the flavor composition is admixed with a food product, said flavor composition being present in an amount of about 0.001% to about 10% w / w.

[0138] 6. Delivery system In certain embodiments, the flavor compositions of the present application can be incorporated into a delivery system for use in a pet food product. The delivery system can be a non-aqueous liquid, solid, or emulsion. The delivery system is generally adapted to the needs of the flavor composition and / or the pet food product in which the flavor composition is incorporated.

[0139] The flavor composition can be used in a non-aqueous liquid form, a dry form, a solid form, and / or as an emulsion. When used in a dry form, suitable drying means such as spray drying can be used. Alternatively, the flavor composition can be encapsulated or absorbed into a water-insoluble material. The actual techniques for the preparation of such dry forms are known in the art and are applicable to the subject matter of the present disclosure.

[0140] The flavor compositions of the presently disclosed subject matter can be used in a number of distinct physical forms known in the art to provide: an initial burst of taste, flavor, and / or texture; and / or a long-lasting sensation of taste, flavor, and / or texture, including, but not limited to, free forms such as spray-dried, powdered, beaded, and encapsulated forms, and mixtures thereof.

[0141] In certain embodiments, the compounds / peptides of the flavor composition may be generated from precursor compounds present in the pet food product during processing of the pet food product, such as sterilization, retorting, and / or extrusion.

[0142] In certain embodiments, the flavor system can be modified using encapsulation techniques, as described above. In certain embodiments, flavor compounds, flavor ingredients, or entire flavor compositions can be fully or partially encapsulated. The choice of encapsulation material and / or technique can determine the type of modification of the flavor system.

[0143] In certain embodiments, the encapsulation material and / or technique is selected to improve the stability of the flavor compound, flavor ingredient, or flavor composition, while in other embodiments, the encapsulation material and / or technique is selected to modify the release profile of the flavor composition.

[0144] Suitable encapsulating materials include, but are not limited to: hydrocolloids such as alginate, pectin, agar, guar gum, cellulose, proteins, polyvinyl acetate, polyethylene, cross-linked polyvinylpyrrolidone, polymethylmethacrylate, polylactic acid, polyhydroxyalkanoates, ethylcellulose, polyvinyl chloride phthalate, polyethylene glycol esters, methacrylic acid comethylmethacrylate, ethylene-vinyl acetate (EVA) copolymers, and the like, and combinations thereof. Suitable encapsulation techniques include, but are not limited to, spray coating, spray drying, spray chilling, absorption, adsorption, inclusion complexation (e.g., formation of a flavor composition / cyclodextrin complex), coacervation, fluidized bed coating, or other processes that can be used to encapsulate an ingredient with an encapsulating material.

[0145] The encapsulation delivery system for flavoring or sweetening agents can include a hydrophobic matrix of fat or wax surrounding the sweetening or flavoring agent core. The fat can be selected from any number of conventional materials, such as fatty acids, glycerides or polyglycerol esters, sorbitol esters, and mixtures thereof. Examples of fatty acids include, but are not limited to, hydrogenated and partially hydrogenated vegetable oils, such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil, and combinations thereof. Examples of glycerides include, but are not limited to, monoglycerides, diglycerides, and triglycerides.

[0146] The wax may be selected from the group consisting of natural and synthetic waxes and mixtures thereof, non-limiting examples of which include paraffin wax, petrolatum, carbowax, microcrystalline wax, beeswax, carnauba wax, candelilla wax, lanolin, bayberry wax, sugar cane wax, spermaceti wax, rice bran wax, and mixtures thereof.

[0147] The fats and waxes can be used individually or in combination in various amounts ranging from about 10% to about 70%, or from about 30% to about 60% by weight of the encapsulation system. When used in combination, the fats and waxes can be present in a ratio of about 70:10 to about 85:15, respectively.

[0148] Exemplary encapsulated flavor compositions, flavoring or sweetening agent delivery systems are disclosed in U.S. Pat. Nos. 4,597,970 and 4,722,845, the disclosures of which are incorporated herein by reference in their entireties.

[0149] Liquid delivery systems include, but are not limited to, dispersions of the flavor compositions of the present application, such as in carbohydrate syrups and / or emulsions. Liquid delivery systems also include extracts of compounds and / or flavor compositions solubilized in a solvent. Solid delivery systems can be formed by spray coating, spray drying, spray chilling, fluidized bed drying, absorption, adsorption, coacervation, complexation, or any other standard technique. In some embodiments, the delivery system can be selected to be compatible with or function in the edible composition. In certain embodiments, the delivery system will include an oleaginous material, such as a fat or oil. In certain embodiments, the delivery system will include a confectionery fat, such as cocoa butter, a cocoa butter substitute, a cocoa butter replacement, or a cocoa butter equivalent.

[0150] When used in a dry form, any suitable drying means, such as spray drying, can be used. Alternatively, the flavor composition can be adsorbed or absorbed onto a substrate, such as a water-insoluble material, or can be encapsulated. The actual techniques for preparing such dry forms are known in the art.

[0151] 7.Pet food products The flavor compositions of the presently disclosed subject matter can be used in a variety of pet food products. Non-limiting examples of suitable pet food products include wet food products, dry food products, moist food products, pet dietary supplements (e.g., vitamins), pet beverage products, snacks, and treats, as described herein.

[0152] Combining one or more of the flavor compositions of the presently disclosed subject matter with a pet food product and optional ingredients, as desired, provides a flavoring agent that has an unexpected taste and imparts, for example, a rich sensory experience, for example, by increasing the activity of calcium-sensing receptors. The flavoring composition of the present disclosure can be added before, during, or after the formulation, processing, or packaging of the pet food product, and the components of the flavoring composition can be added sequentially or simultaneously. In certain embodiments, the compounds / peptides of the flavoring composition can be generated from precursor compounds present in the pet food product during the processing of the pet food product, for example, sterilization, retorting, and / or extrusion.

[0153] In certain embodiments, the pet food product is a complete nutrition dry food product. Dry or low moisture complete nutrition pet food products can have less than about 15% moisture and contain about 10% to about 60% fat, about 10% to about 70% protein, and about 30% to about 80% carbohydrate, including dietary fiber and ash.

[0154] In a particular embodiment, the pet food product is a complete nutrition wet food product. A wet or high moisture complete nutrition pet food product can contain more than about 50% moisture. In a particular embodiment, the wet pet food product contains about 40% fat, about 50% protein, and about 10% carbohydrate, such as dietary fiber and ash.

[0155] In a particular embodiment, the pet food product is a complete nutritional moist food product. Moist, e.g. semi-moist or semi-dry or soft dry or soft moist or medium or moderate moisture content complete nutritional pet food products contain from about 15% to about 50% moisture.

[0156] In certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits, and sweet products.

[0157] In certain embodiments, the protein source may be derived from a plant source, such as lupin protein, wheat protein, soy protein, and combinations thereof. Alternatively, or in addition, the protein source may be derived from a variety of animal sources. Non-limiting examples of animal proteins include beef, pork, chicken, lamb, or fish, including muscle, meat by-products, meat meal, or fish meal.

[0158] 8. Methods for measuring taste attributes In certain embodiments of the present disclosure, the taste, flavor and / or palatability attributes of a pet food product can be modified by blending a flavor composition with the food product or produced under multiple food preparation conditions, as described herein. In certain embodiments, one or more attributes can be enhanced or reduced by increasing or decreasing the concentration of a flavor composition blended with or produced with the food product. In certain embodiments, the taste attributes of the modified food product can be evaluated as described herein, and the concentration of a flavor composition blended with or produced with the food product can be increased or decreased based on the results of this evaluation.

[0159] In certain embodiments of the present disclosure, taste and / or palatability attributes can be measured using an in vitro assay, in which the ability of a compound (e.g., a peptide) to activate a feline calcium-sensing receptor expressed by cells in vitro at various concentrations is measured. In certain embodiments, improved activation of the receptor correlates with improved taste and / or palatability attributes of the compound. In certain embodiments, the composition is measured alone or in combination with other compounds. In certain embodiments, the in vitro assay includes the in vitro assays described in the Examples section of this application.

[0160] In certain embodiments of the present disclosure, taste and / or palatability attributes can be measured using an in silico model, where the ability of a compound to interact with amino acid residues in the binding site of the calcium-sensing receptor is determined in silico. In certain embodiments, the ability of a compound to modulate the feline calcium-sensing receptor correlates with the degree to which the compound binds to an in silico model of the receptor. In certain embodiments, compositions are measured alone or in combination with other compounds. In certain embodiments, the in silico model comprises the in silico model described in the Examples section of this application.

[0161] In certain embodiments of the present disclosure, taste and / or palatability attributes can be measured using a panel of taste testers. For example and without limitation, the panel can include a plurality of cat panelists. In certain embodiments, the panel can include a plurality of dog panelists. In certain embodiments, the palatability of a pet food product can be determined by the sole consumption (e.g., one bowl test, unary ranking) of a pet food product containing a flavor composition. In certain embodiments, the palatability of a pet food product can be determined by preferential consumption (e.g., two bowl test to test preference, discrimination, and / or choice) of a pet food product containing a flavor composition of the present disclosure over a pet food product that does not contain the flavor composition or that contains a different flavor composition.

[0162] In certain embodiments, the palatability and / or kokumi of a flavor composition can be determined by preferential consumption (e.g., two-bottle test) of an aqueous solution containing a flavor composition of the present disclosure versus an aqueous solution that does not contain the flavor composition or that contains a different flavor composition. For example, a panel of solutions can be used to compare the palatability of a range of concentrations of the compound in a single exposure. In certain embodiments, the solution can contain a palatability enhancer, e.g., L-histidine, as an ingestible / positive tastant, which can improve baseline solution intake and allow potential adverse effects of the test compound to be identified.

[0163] The intake rate of each pet food product or emulsion can be determined by measuring the amount of a food consumed and dividing it by the total consumption. The consumption of a food can be compared to other foods by calculating the consumption ratio (CR) to determine the preferential consumption of a food product or emulsion over another. Alternatively, or in addition, the difference in intake (g) can be used to evaluate the mean intake difference between two emulsions in a two-bottle test or between two pet food products in a two-bowl test at a selected significance level, such as a 5% significance level to determine the mean intake difference with a 95% confidence interval. However, any significance level can be used, such as a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 50% significance level. In certain embodiments, a percentage preference score may also be calculated, for example the percentage preference for an emulsion or food product is the percentage that emulsion or food product represents of all emulsions or food products consumed during the test.

[0164] 9. Manufacturing method In certain embodiments, the compounds (e.g., peptides) of the present disclosure can be produced using standard chemical synthesis processes. In certain embodiments, the chemical synthesis processes provide compounds with a purity of at least 99.999%, or at least 99%, or at least 95%, or at least 90%, or at least 85%, or at least 80%. In certain embodiments, the compounds can be prepared using standard hydrolysis processes, such as those using acids, enzymes, or a combination of acids and enzymes.

[0165] In certain embodiments, the compounds of the present disclosure can be produced under food preparation conditions, for example, during the manufacture of the pet food product. For example, but not by way of limitation, the compounds of the present disclosure can be produced from precursor compounds present in the pet food product during thermal processing of the food, for example, sterilization, retorting, and / or extrusion. In certain embodiments, liquid and / or powdered palatants can be added to enhance the taste of the pet food, for example, to dry pet food products, to improve the palatability of the pet food. The palatants can be meat (e.g., liver) digests and / or vegetable digests, and can optionally include other palatants known in the art. In certain embodiments, the compounds can be mixed with or produced in the liquid and / or powdered palatants prior to addition to the pet food product. Alternatively, or in addition, the compounds can be mixed with or produced in the liquid and / or powdered palatants after addition to the pet food product.

[0166] 10. Non-limiting examples of the methods of the present disclosure In certain non-limiting embodiments, the present disclosure provides a method of increasing the palatability of a pet food product, the method comprising blending the pet food product with a flavor composition comprising a peptide as described herein, wherein the peptide is present in the blend at a concentration of from about 1 nM to about 10 M, or from about 1 nM to about 1 M.

[0167] In certain non-limiting embodiments, the present disclosure provides a method of improving the palatability of a pet food product, the method comprising producing the pet food product with a flavor composition comprising a peptide as described herein, wherein the peptide is present in the product at a concentration of from about 1 nM to about 10 M, or from about 1 nM to about 1 M.

[0168] In certain non-limiting embodiments, the present disclosure provides a method of improving the fullness of a pet food product, for example by improving activity of a calcium-sensing receptor, the method comprising blending the pet food product with a flavor composition comprising a peptide as described herein, wherein the peptide is present in the blend at a concentration of about 0.001 ppm to about 1,000 ppm.

[0169] In certain non-limiting embodiments, the present disclosure provides a method of enhancing the palatability of a pet food product, the method comprising blending the pet food product with a flavor composition comprising a peptide as described herein, wherein the flavor composition is present in the blend at a concentration of about 0.001 ppm to about 1,000 ppm.

[0170] In certain non-limiting embodiments, the present disclosure provides a method of improving the fullness of a pet food product, for example by improving activity of a calcium-sensing receptor, the method comprising blending the pet food product with a flavor composition comprising a peptide as described herein, the flavor composition being present in the blend at a concentration of from about 0.0001% to about 10% w / w, or from about 0.001% to about 5% w / w, or from about 0.01% to about 1% w / w.

[0171] In certain non-limiting embodiments, the present disclosure provides a method of increasing the palatability of a pet food product, the method comprising blending the pet food product with a flavor composition comprising a peptide as described herein, wherein the flavor composition is present in the blend at a concentration of from about 0.0001% to about 10% w / w, or from about 0.001% to about 5% w / w, or from about 0.01% to about 1% w / w. EXAMPLES

[0172] The subject matter of the present disclosure will be better understood with reference to the following examples, which are provided by way of illustration and not by way of limitation of the invention.

[0173] Example 1 - Preparation and testing of caseinate hydrolysates This example investigated the use of a protein hydrolysate palatable system for wet cat food.

[0174] Milk proteins (caseinates) were hydrolyzed under different enzymatic conditions. A total of 14 hydrolysates were produced. The degree of hydrolysis of the bulk samples ranged from 8% to 35% and the dry matter ranged from 4 to 8%. All bulk samples were of food grade quality.

[0175] Five different hydrolysates and one control casein hydrolysate were selected for animal feeding studies. The conditions for each hydrolysate are listed in Table 1.

[0176] [Table 1]

[0177] An animal feeding study was performed in which each hydrolysate was mixed at 3% in different matrices (corn starch, gelatin, and autoclave gel). Similar food intake patterns were observed across the different hydrolysates in all three matrices, as shown in Figure 1A-1C. The feeding study was repeated with gelatin gel containing 20 mM IMP to boost intake. Similar food intake patterns were observed, as shown in Figure 1D. Hydrolysate T648 was chosen for further study as it was the most highly ingested across the different matrices.

[0178] Example 2 - Isolation and identification of bioactive compounds from hydrolysates This example illustrates the identification of potentially taste active peptides from hydrolysates that show improved palatability in wet cat food.

[0179] The hydrolysate T648 was analyzed by activity-guided fractionation (AGF) using standard methods known in the art. Briefly, isolation of putative bioactive compounds (BCs) from the hydrolysate was performed by a combination of different separation techniques, such as medium pressure liquid chromatography (MPLC), size exclusion chromatography (SEC), and high performance liquid chromatography (HPLC). Twelve named putative BCs were isolated from the hydrolysate, of which seven were structurally elucidated by NMR and / or peptide mapping using LC-ESI-MS / MS. The resulting sequences are shown in Table 2.

[0180] Aliquots of the isolated samples were tested for their agonist activity in the feline taste receptor assay (f-CaSR). A summary of the activities obtained in the assay are listed in Table 2.

[0181] [Table 2]

[0182] Example 3 - In silico modeling to identify compounds that interact with CaSR This example describes computational modeling of the feline calcium-sensing receptor (CaSR) to identify putative agonists.

[0183] A computational approach was used to analyze the three-dimensional structure of CaSR to identify polypeptide regions that can be utilized to selectively activate the receptor. A structural homology model of the Venus flytrap domain and the cysteine-rich domain of CaSR was generated based on the crystal structure of human CaSR (Geng, et al. 2016; Zhang, et al. 2016). The homology model was constructed using the Discovery Studio (DS) program package from Accelrys. Specifically, the Modeler program from DS was used (see Eswar et al., Current Protocols in Bioinformatics, Supplement 15:5.6.1-5.6.30 (2006); the contents of which are incorporated herein by reference in their entirety). An "in silico" screen was used to identify compounds that interact with the structural domains of CaSR.

[0184] The GPCR group C family of proteins includes T1R1, T1R2, T1R3, CaSR, GabaB, and mGlu proteins. Group C proteins have: (1) a large ectodomain called the Venus Flytrap (VFT) domain; (2) seven transmembrane (7TM) domains; and (3) a cysteine-rich domain connecting the VFT and 7TM domains. A homology model of the VFT and cysteine-rich domains of the feline CaSR receptor was generated based on the latest crystal structure of hCaSR (Geng, et al. 2016; Zhang, et al. 2016), which is currently available from the Protein Data Bank (PDB, www.rcsb.org). Using the docking program BioDock from BioPredict, compounds containing Asp-Val-Glu and γ-Glu-Val-Gly were docked in silico into the active site of the VFT domain of CaSR, as shown in Figures 3A-3C.

[0185] Residues that anchor the active site of the feline CaSR Venus Flytrap domain include: Pro39, Asn64, Arg66, Gly67, Arg69, Trp70, Asn102, Thr145, Gly146, Ser147, Gly148, Tyr167, Ala168, Ser169, Ser170, Ser171, Ile187, Tyr218, Ser271, Ser272, Glu297, Ala298, Trp299, Ala300, Ser301, Ser302, and Ile416. In particular, Arg66, Trp70, Thr145, Ser147, Ala168, Ser170, Tyr218, Ser272, Glu297, and Ile416 functioned in the homology model by forming salt bridges, hydrogen bonds, and hydrophobic interactions to coordinate the negatively charged head group and polar moieties of the compound binding to the active site.

[0186] Example 4 - Identification of the active motif of CaSR active peptides Based on the structural analysis of the kokumi-active peptides identified in Example 2, and the in silico modeling described in Example 3, the following tripeptide motif was predicted as a "kokumi-active motif" capable of activating the CaSR receptor: [negatively charged or polar amino acid]-[amino acid with a molecular mass of 150 daltons or less]-[negatively charged or polar amino acid] Based on this activity motif, 12 additional peptides were predicted to activate CaSR. A complete list of these 12 peptides, along with the two active peptides Asp-Val-Glu and Asp-Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Ala, is listed in Table 3.

[0187] [Table 3-1]

[0188] [Table 3-2]

[0189] [Table 3-3]

[0190] Example 5 - In vitro testing of putative CaSR active peptides The 14 peptides listed in Table 3, as well as a number of control peptides and compounds, were tested in vitro to assess their ability to activate the feline CaSR.

[0191] method The cells used for the assay were HEK293-derived HEK T-Rex / natClytin-fCaSR cells. For the assay, cells were seeded onto 386-well culture plates. The plates had a clear bottom to read luminescence in the wells. The assay setup for a standard assay on the FlexStation was as follows: the wells containing the cells contained 20 μl of calcium-free Tyrode's buffer at the start of the assay. 20 μl of each ligand at the indicated concentration was injected into the cells and the cell response was measured at 1.94 s intervals for 90 s. The resulting curves were analyzed and reduced using the software provided by Molecular Devices, SoftMax Pro (version 5.4.1).

[0192] Each peptide was assayed at least in duplicate, and each concentration was applied to the cells in quadruplicate. In parallel, identical tests were performed on a mock cell line carrying a mock vector to confirm the specificity of any signals measured.

[0193] The data obtained from the FlexStation was used to trace the dose-response curves of each ligand. These graphs were plotted using GraphPad Prism 7.03 software using a [agonist] vs. response-variable slope (4-parameter) plot. The same formula was used to calculate EC50 values ​​with associated standard errors. Each plot contains the average data points with the SEM calculated by the software for each data point.

[0194] One concern during the planning of these assays was that some of these peptides may bind divalent calcium cations, causing a non-specific response from the receptor. To ensure that interactions between the receptor and peptides, and not with calcium, were measured, the peptides were synthesized under conditions that ensured that they were calcium-free. Similarly, all assays were performed using calcium-free reagents.

[0195] result 1. All predicted kokumi-active peptides activate the feline CaSR in vitro Positive controls for this assay were CaCl2 and the kokumi peptide γ-Glu-Val-Gly mentioned above (Figure 2A). In all graphs, two separate assays are shown along with one mock cell assay (no mock cell responses were recorded for any of the ligands). All other ligands were assayed in a similar manner. The data obtained are detailed in Figure 2B and Table 4.

[0196] [Table 4]

[0197] All 12 predicted kokumi peptides activate the feline CaSR receptor at concentrations in the millimolar range. Although slight differences exist in the apparent affinities of the same ligand from assay to assay, these differences are minor and within acceptable limits. The affinities of all peptides are detailed in Table 4. For some of these peptides, the models used to calculate EC50 values ​​provided estimates rather than exact data, and therefore no standard error was associated with these values.

[0198] Although the subject matter of the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the present invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods, and steps described herein. Those skilled in the art will readily appreciate from the disclosure of the subject matter of the present disclosure that existing or future developed processes, machines, manufacture, compositions of matter, means, methods, or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the subject matter of the present disclosure. It is therefore intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0199] Throughout this application, patents, patent applications, publications, product descriptions, and protocols are cited, the disclosures of which are incorporated by reference into this application in their entireties for all purposes. Preferred embodiments of the present invention will be described below in detail. EMBODIMENT 1 1. A flavor composition comprising an oligopeptide comprising a tripeptide motif, said tripeptide motif comprising: (a) a first amino acid residue at the N-terminus which is a negatively charged amino acid residue or a polar uncharged amino acid residue; (b) a second amino acid residue having a molecular mass of 150 daltons or less; and (c) a third amino acid residue at the C-terminus which is a negatively charged amino acid residue or a polar uncharged amino acid residue; Including, The tripeptide binds to the calcium sensing receptor (CaSR) and provides a flavorful taste to companion animals. EMBODIMENT 2 2. The flavor composition of embodiment 1, wherein the first amino acid residue is a negatively charged amino acid residue. EMBODIMENT 3 3. The flavor composition according to embodiment 1 or 2, wherein the third amino acid residue is a negatively charged amino acid residue. EMBODIMENT 4 The flavor composition according to any one of embodiments 1 to 3, wherein the negatively charged amino acid residue is selected from the group consisting of aspartic acid (Asp), glutamic acid (Glu), and any phosphorylated amino acid residue. EMBODIMENT 5 The flavor composition according to any one of embodiments 1 to 4, wherein the negatively charged amino acid residue is phosphorylated serine (pSer), phosphorylated tyrosine (pTyr), or phosphorylated threonine (pThr). EMBODIMENT 6 The flavor composition according to any one of embodiments 1 and 3 to 5, wherein the first amino acid residue is a polar uncharged amino acid residue. EMBODIMENT 7 The flavor composition according to any one of embodiments 1, 2, and 4 to 6, wherein the third amino acid residue is a polar uncharged amino acid residue. EMBODIMENT 8 The flavor composition according to any one of embodiments 1 to 7, wherein the polar uncharged amino acid residue is selected from the group consisting of cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asp), serine (Ser), tyrosine (Tyr), and threonine (Thr). EMBODIMENT 9 The flavor composition according to any one of embodiments 1 to 8, wherein the second amino acid residue is selected from the group consisting of lysine (Lys), isoleucine (Ile), leucine (Leu), alanine (Ala), methionine (Met), proline (Pro), valine (Val), aspartic acid (Asp), glutamic acid (Glu), cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asn), serine (Ser), and threonine (Thr). EMBODIMENT 10 The flavor composition according to any one of embodiments 1 to 9, wherein the second amino acid residue is alanine (Ala), valine (Val), or glutamic acid (Glu). EMBODIMENT 11 The flavor composition according to any one of embodiments 1 to 10, wherein the oligopeptide is a tripeptide selected from the group consisting of Asp-Val-Glu, Glu-Val-Asp, Asp-Glu-Glu, pSer-Glu-pSer, pSer-Val-pSer, pSer-Val-Glu, Ser-Glu-Ser, Cys-Val-Cys, pTyr-Glu-pTyr, pThr-Glu-pThr, Asp-Ala-Glu, Glu-Val-Glu, Asp-Val-Asp, and any combination thereof. EMBODIMENT 12 12. The flavor composition according to any one of embodiments 1 to 11, wherein the oligopeptide is selected from the group consisting of Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln, Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln-Ala, Glu-Ile-Val-Pro-Asn-pSer-Ala-Glu-Glu, Asp-Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln-Ala, and any combination thereof. EMBODIMENT 13 13. The flavor composition according to any one of embodiments 1 to 12, wherein the companion animal is a cat or a dog. EMBODIMENT 14 14. The flavor composition according to any one of embodiments 1 to 13, wherein the companion animal is a cat. EMBODIMENT 15 15. The flavor composition according to any one of embodiments 1 to 14, wherein the oligopeptide is produced during a manufacturing process of a food product. EMBODIMENT 16 A food product comprising the flavor composition according to any one of embodiments 1 to 15, The food product, wherein the flavor composition is present in an amount effective to enhance kokumi (body flavor) of the food product as determined by a panel of taste testers. EMBODIMENT 17 A food product comprising the flavor composition according to any one of embodiments 1 to 15, The food product, wherein the flavor composition is present in an amount effective to enhance the palatability of the food product as determined by a panel of taste testers. EMBODIMENT 18 18. The food product of embodiment 16 or 17, wherein the flavor composition is present in the food product at a concentration of from about 1 nM to about 1 M, from about 1 μM to about 1 M, from about 0.0001% to about 10% w / w, from about 0.001% to about 5% w / w, or from about 0.01% to about 1% w / w. EMBODIMENT 19 19. The food product of any one of embodiments 16-18, wherein the food product comprises a pet food product. EMBODIMENT 20 20. The food product of embodiment 19, wherein the pet food product is a cat pet food product or a dog pet food product. EMBODIMENT 21 The food product of embodiment 19, wherein the pet food product is a wet pet food product. EMBODIMENT 22 The food product of embodiment 19, wherein the pet food product is a dry pet food product. EMBODIMENT 23 23. The food product of any one of embodiments 16-22, wherein the flavor composition is generated during a manufacturing process of the food product. EMBODIMENT 24 1. A method for improving kokumi intensity of a food product, comprising: The method includes a step of mixing the food product with the flavor composition according to any one of embodiments 1 to 14, The method, wherein the flavor composition is present in an amount effective to enhance kokumi (body flavor) of the food product as determined by a panel of taste testers. EMBODIMENT 25 25. The method of embodiment 24, wherein the flavor composition is present in the admixture at a concentration of from about 1 nM to about 1 M, from about 1 μM to about 1 M, from about 0.0001% to about 10% w / w, from about 0.001% to about 5% w / w, or from about 0.01% to about 1% w / w.

Claims

1. A flavor composition comprising a tripeptide that binds to a calcium-sensing receptor (CaSR) to impart a rich flavor to a companion animal, the tripeptide comprising: (a) a first amino acid residue at the N-terminus that is a negatively charged amino acid residue or a polar uncharged amino acid residue; (b) a second amino acid residue having a molecular mass of 150 daltons or less; and (c) a C-terminal third amino acid residue that is a negatively charged amino acid residue or a polar uncharged amino acid residue; The flavor composition, wherein the tripeptide is selected from the group consisting of Asp-Val-Glu, Glu-Val-Asp, Asp-Glu-Glu, pSer-Glu-pSer, pSer-Val-pSer, pSer-Val-Glu, Ser-Glu-Ser, Cys-Val-Cys, pTyr-Glu-pTyr, pThr-Glu-pThr, Asp-Ala-Glu, Glu-Val-Glu, Asp-Val-Asp, and any combination thereof.

2. The flavor composition described in claim 1, wherein the companion animal is a cat or a dog.

3. A flavor composition described in claim 1 or 2, wherein the companion animal is a cat.

4. A food product comprising a flavor composition described in any one of claims 1 to 3.

5. The food product described in claim 4, wherein the flavor composition is present in the food product at a concentration of 1 nM to 1 M, 1 μM to 1 M, 0.0001% to 10% w / w, 0.001% to 5% w / w, or 0.01% to 1% w / w.

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