Peptide that modulates calcium-sensing receptor activity to modulate kokumi taste and pet food containing the same

Abstract

This application describes flavor compositions comprising at least one peptide that activates or increases the activity of a calcium-sensing receptor that can be used to enhance the kokumi and / or palatability of pet foods. This application also discloses methods for identifying the peptides.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 814,082, filed on March 5, 2019, which is incorporated by reference in its entirety into this application.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on February 27, 2020, is named 069269_0391_SL.txt, and is 41,076 bytes in size. Technical Field

[0005] The presently disclosed subject matter relates to flavor compositions comprising at least one peptide that interacts with a calcium-sensing receptor (CaSR) to modulate kokumi. The flavor compositions can be used to enhance or modify the palatability, taste, and / or flavor of pet foods. The flavor compositions can include combinations of compounds and can be added to pet foods in various delivery systems or generated during the pet food manufacturing process. Background Art

[0006] The taste spectrum of edible compositions includes basic tastes, such as sweet, salty, bitter, sour, umami and kokumi. The compounds that stimulate these tastes are commonly referred to as tastants. Without being bound by theory, it is assumed that taste receptors in the mouth and throat perceive tastants, which transmit signals to the brain, where they record the tastants and the resulting taste spectrum. Taste receptors include calcium-sensing receptors (CaSRs), which are G protein-coupled receptors (GPCRs) that detect changes in extracellular calcium levels and are closely related to T1R1, T1R2 and T1R3 receptors, i.e., sweet and umami receptors. Calcium-sensing receptors have been shown to act as receptors for kokumi.

[0007] Pet food manufacturers have long sought to provide pet foods with high nutritional value. Furthermore, particularly for cats and dogs, pet food manufacturers demand high palatability so that pets can reap the full nutritional benefits of their food. Domestic animals, especially cats, are known for their fickle food preferences and often refuse to eat a pet food they have been receiving for a while, or refuse to consume more than a minimal amount. This phenomenon may be due in part to subtle differences in the sensory characteristics of raw materials, which domestic animals can perceive through their taste and olfactory systems. Consequently, pet owners frequently switch types and brands of pet food to keep their pets healthy and satisfied.

[0008] Despite recent advances in taste and flavor technology, there remains a need for compounds that can enhance or modify the palatability of pet foods by enhancing or modifying their taste, texture, and / or flavor profile. Enhancement or modification can be by increasing the intensity of a desired attribute, replacing a desired attribute that is absent or otherwise lost in the pet food, or reducing the intensity of an undesirable attribute. In particular, it is desirable to increase the intensity of desired tastants in pet foods. Therefore, there remains a need in the art for compositions that enhance the palatability and / or modulate the kokuminess of pet foods. Summary of the Invention

[0009] The presently disclosed subject matter relates to flavor compositions and methods for preparing and improving such compositions in various pet foods. Specifically, the present disclosure relates to compositions comprising one or more peptides that enhance, increase, decrease, and / or modulate the activity of a calcium-sensing receptor (CaSR), thereby modulating kokumi.

[0010] In certain embodiments, the flavor composition comprises an oligopeptide. In certain embodiments, the oligopeptide comprises a tripeptide motif. In certain embodiments, the tripeptide motif comprises:

[0011] (a) a first amino acid residue at the N-terminus, wherein the first amino acid residue is a negatively charged amino acid residue or a polar uncharged amino acid residue;

[0012] (b) a second amino acid residue with a molecular weight not exceeding 150 Daltons; and

[0013] (c) a third amino acid residue at the C-terminus, wherein the third amino acid residue is a negatively charged amino acid residue or a polar uncharged amino acid residue,

[0014] The tripeptide binds to the calcium-sensing receptor (CaSR) to impart kochi taste to companion animals.

[0015] 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).

[0016] 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).

[0017] 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).

[0018] 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.

[0019] 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.

[0020] In certain embodiments, the companion animal is a cat or a dog. In certain embodiments, the companion animal is a cat.

[0021] In certain embodiments, the oligopeptides are produced during the manufacturing process of the food product.

[0022] The presently disclosed subject matter provides a food comprising any flavor composition disclosed herein, wherein the flavor composition is present in an amount that effectively increases the kokumi of the food, as determined by a group of taste testers. The presently disclosed subject matter provides a food comprising any flavor composition disclosed herein, wherein the flavor composition is present in an amount that effectively increases the palatability of the food, as determined by a group of taste testers. In certain embodiments, the flavor composition is present in 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 the food. In certain embodiments, the food comprises pet food. In certain embodiments, the pet food is feline pet food or canine pet food. In certain embodiments, the pet food is wet pet food. In certain embodiments, the pet food is dry pet food.

[0023] The presently disclosed subject matter provides a method for increasing the taste intensity of kokumi in a food product, comprising mixing the food product with any of the flavor compositions disclosed herein, wherein the flavor composition is present in an amount effective to increase the kokumi of the food product, as determined by a panel of taste testers. In certain embodiments, the flavor composition is present in the mixture 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.

[0024] In certain embodiments, the flavor composition is generated during the manufacturing process of the food product.

[0025] The presently disclosed subject matter provides methods of modulating calcium sensing receptor (CaSR) activity comprising contacting the CaSR with any of the flavor compositions disclosed herein.

[0026] The presently disclosed subject matter provides methods for identifying compositions that modulate CaSR activity. In certain embodiments, the methods comprise:

[0027] (a) contacting the test agent with the CaSR,

[0028] (b) detecting an in silico interaction of the test agent with one or more amino acid residues in an interaction site of the Venus flytrap (VFT) domain of the CaSR, the amino acid residues 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, and

[0029] (c) selecting a test agent that interacts with one or more of the amino acids as a composition.

[0030] In certain embodiments, step (b) further comprises detecting an interaction between the test agent and one or more amino acids in the Venus flytrap (VFT) domain interaction site of the CaSR, wherein the amino acids are selected from the group consisting of Asn64, Asn102, Thr145, Ser169, Ser170, Ser272, Ala298, Trp299, Ala300, Ser302, and any combination thereof.

[0031] In some embodiments, the method further comprises determining the activity of CaSR after step (a). In some embodiments, step (c) further comprises selecting a test agent that increases CaSR activity as a composition. In some embodiments, CaSR is expressed by cells, and wherein the test agent is contacted with the cells. In some embodiments, the cells express calcium-binding photoprotein.

[0032] The features and technical advantages of the present application have been outlined in a rather broad manner so that the detailed description below may be better understood. Additional features and advantages of the present application will be described below, which form the subject matter of the claims of the present application. It will be understood by those skilled in the art that the disclosed concepts and specific embodiments may be readily used as a basis for modifying or designing other structures for achieving the same purposes of the present application. It will also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the present application as set forth in the appended claims. Features believed to be novel will better understand the features of the present application, including its organization and method of operation, as well as further objects and advantages, from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figures 1A-1D Depicted are the results of animal feeding tests of selected hydrolysates mixed at 3% in different matrices (maize gravy, gelatine gel and autoclave gel). Figure 1A Results obtained using a corn starch matrix are shown. Figure 1B Results given by a gelatin gel matrix are shown. Figure 1C Results obtained in tests using an autoclave gel are shown. Figure 1D Food intake is shown for a gelatin gel matrix with 20 mM IMP. Figures 2A-2B Dose-response curves for the test agents on the feline CaSR are depicted. Figure 2ADose-response curves for the positive controls CaCl2 and γEVG on the feline CaSR are shown. Each run was performed on a separate day, and each data point represents the average of four replicates in one assay. Mock cell responses were simulated on the same day as run 2. Figure 2B Shown are dose-response curves for 14 savory peptides on the feline CaSR. Two runs were performed for each ligand, each on a separate day, and each data point represents the average of four replicates from a single assay. Mock cell responses were obtained on the same day as Run 2.

[0034] Figures 3A-3C Various aspects of computer simulations of the feline CaSR with Asp-Val-Glu, aspartate, and / or γ-Glu-Val-Gly are depicted. Figure 3A A ribbon diagram of a computer simulation of the feline CaSR with Asp-Val-Glu is shown. Figure 3B Ball-and-stick diagram illustrating computer simulation of the feline CaSR with Asp-Val-Glu. Figure 3C Depicted is a computer simulation of the feline CaSR with γ-Glu-Val-Gly.

[0035] Figure 4 The amino acid and nucleotide sequences of the feline, canine, and human calcium-sensing receptors are depicted. DETAILED DESCRIPTION

[0036] To date, there remains a need for flavor modulators that can increase and / or enhance the palatability of various pet foods. This application relates to flavor compositions comprising at least one peptide that modulates calcium-sensing receptor (CaSR) activity. The flavor compositions can be used to increase the palatability and / or enhance or modify the flavor of various pet foods, such as nutritionally complete pet foods, and can be added to pet foods in various delivery systems. The flavor compositions can also include combinations of compounds.

[0037] 1. Definition

[0038] The terms used in this specification generally have their ordinary meanings in the art, in the context of the present invention, and in the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners when describing the compositions and methods of the present invention and how to make and use them.

[0039] As used herein, when used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" can mean "one," but it is also consistent with the meaning 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 recognize that these terms are open-ended terms.

[0040] 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 how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and 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, and more preferably within 2-fold, of a value.

[0041] As used in this application, the terms "comprise," "include," "have," "have," "may," "include," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional actions or structures. This disclosure also contemplates other embodiments that "comprise," "consist of," and "consist essentially of" the embodiments or elements presented herein, whether explicitly stated or not.

[0042] As used herein, "taste" refers to a sensation caused by activation or inhibition of receptor cells in a subject's taste buds. In certain embodiments, the taste can be selected from the group consisting of sweet, sour, salty, bitter, kokumi, and umami. In certain embodiments, a "tastant" induces a taste sensation in a subject. In certain embodiments, the tastant is a synthetic tastant. In certain embodiments, the tastant is prepared from a natural source.

[0043] In some embodiments, "flavor" can include a kokumi taste. See, for example, Ohsu et al., J. Biol. Chem., 285(2): 1016-1022 (2010), the contents of which are incorporated herein by reference. In some embodiments, kokumi taste is a sensation caused by activation or inhibition of receptor cells (e.g., CaSR receptors) in a subject's taste buds and is different from other tastes (e.g., sweet, salty, and umami), although it can serve as a flavor enhancer for these tastes.

[0044] As used herein, "taste profile" refers to a combination of tastes, such as one or more of sweet, sour, salty, bitter, umami, kokumi, and free fatty acid tastes. In certain embodiments, the taste profile is produced by one or more tastants present in the composition at the same or different concentrations. In certain embodiments, the taste profile refers to the intensity of a taste or combination of tastes, such as sweet, sour, salty, bitter, umami, kokumi, and free fatty acid tastes, as detected by a subject or any known assay. In certain embodiments, modifying, changing, or varying the combination of tastants in the taste profile can alter the sensory experience of the subject.

[0045] As used in this application, "taste tester" refers to any mammal, such as a human, cat or dog, that samples the palatability of a composition or a food or beverage product containing such a composition. In some embodiments, the taste tester provides feedback on the palatability of the tested composition based on test parameters and protocols. As used in this application, "flavor" refers to one or more sensory stimuli, such as, for example, one or more of taste (sense of taste), smell (smell), touch (sense of touch) and temperature (heat) stimulation. In some non-limiting embodiments, the sensory experience of the object exposed to flavor can be classified as the characteristic experience of a specific flavor. For example, the subject can identify flavor as, but not limited to, the flavors such as floral, citrus, berry, nut, caramel, chocolate, spicy (peppery), smoked, cheese, meaty. As used in this application, flavor compositions can be selected from liquids, solutions, dry powders, sprays, pastes, suspensions and any combination thereof. Flavoring agents can be natural compositions, artificial compositions, identical properties or any combination thereof.

[0046] As used interchangeably herein, "aroma" and "smell" refer to an olfactory response to a stimulus. For example, but not limited to, an aroma can be produced by aromatic substances that are sensed by odorant receptors of the olfactory system.

[0047] As used herein, a "flavor profile" refers to a combination of sensory stimuli, such as taste, such as sweetness, sourness, bitterness, saltiness, umami, kokumi, and free fatty acid flavors, and / or olfactory, tactile, and / or thermal 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 varying the combination of stimuli in a flavor profile can alter a subject's sensory experience.

[0048] As used herein, "mixing," e.g., "mixing a flavor composition of the present invention or a combination thereof with a food product," refers to a process in which a flavor composition or individual components of a flavor composition are mixed with or added to a complete product or mixed with some or all of the components of a product during product formation or in some combination of these steps. When used in the context of mixing, the term "product" refers to the product or any component thereof. The mixing step may include a process selected from the group consisting of adding the flavor composition to the product, spraying the flavor composition on the product, coating the flavor composition on the product, suspending the product in the flavor composition, applying the flavor composition to the product, affixing the flavor composition to the product, encapsulating the product with the flavor composition, mixing the flavor composition with the product, and any combination thereof. The flavor composition may be a liquid, an emulsion, a dry powder, a spray, a paste, a suspension, and any combination thereof.

[0049] In certain embodiments, the peptides / compounds of the flavor composition can be generated from precursor compounds present in the pet food during processing of the pet food (e.g., sterilization, retorting, and / or extrusion). In some embodiments, the peptides / compounds of the flavor composition can be generated during processing of the pet food, and additional components of the flavor composition can be added to the pet food by mixing.

[0050] As used herein, "ppm" means parts per million and is a weight-related parameter. Parts per million is microgram per gram, so that a component present at 10 ppm is 10 micrograms of a particular component per 1 gram of aggregate mixture.

[0051] As used herein, "palatability" can refer to an animal's overall willingness to eat a certain food. Increasing the "palatability" of a pet food can result in an increased enjoyment and acceptance of the pet food by a companion animal to ensure that the animal consumes a "healthy amount" of the pet food. As used herein, the term "healthy amount" of a pet food refers to an amount of micronutrients, macronutrients, and caloric intake that enables a companion animal to maintain or achieve an amount that contributes to its overall overall health, such as those listed in the "Mars Petcare Essential Nutrient Standards." In certain embodiments, "palatability" can refer to an animal's preference for one food relative to another. For example, when an animal shows a preference for one of two or more foods, the preferred food is more "palatable" and has "enhanced palatability." In certain embodiments, the relative palatability of a food compared to one or more other foods can be determined, for example, by side-by-side comparison, free choice comparison, for example, by relative consumption of the foods, or other appropriate preference measurements indicative of palatability. Palatability can be determined by a standard test protocol in which animals have equal access to two food items, such as a test known as a "two-bowl test" or a "versus test." This preference may arise from any of the animal's senses, but may be particularly related to taste, aftertaste, smell, mouthfeel, and / or texture.

[0052] The term "pet food" or "pet food" refers to a product or composition intended for consumption by companion animals, such as cats, dogs, guinea pigs, mice, rabbits, birds, and horses. For example, but not limited to, a companion animal may be a "domestic" cat, such as a domestic cat (Feliscatus). In certain embodiments, a companion animal may be a "domestic" dog, such as a domestic dog (Canis lupusfamiliaris). "Pet food" or "pet food" may include any food, feed, snack, food supplement, liquid, beverage, treat, toy (chewable and / or edible toys), meal substitute, or meal replacement.

[0053] As used herein, "nutritionally complete" refers to a pet food that contains all known required nutrients for the intended recipient of the pet food in appropriate amounts and proportions based on, for example, general recognition in the field of companion animal nutrition or the recommendations of competent authorities. Thus, such a food can be used as the sole source of dietary intake to sustain life without the need for the addition of supplemental nutritional sources.

[0054] As used herein, "flavor composition" refers to at least one peptide / compound or biologically acceptable salt thereof that modulates (including enhancing, increasing, potentiating, reducing, inhibiting, or inducing) the taste, smell, flavor, and / or texture of a natural or synthetic tastant, flavoring, taste profile, flavor profile, and / or texture profile in an animal or human. In certain embodiments, the flavor composition comprises a combination of compounds or biologically acceptable salts thereof. In certain embodiments, the flavor composition includes one or more excipients.

[0055] As used herein, the term "modulate" or "improve" refers to an increase or decrease in the amount, quality, or effect of a specific 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 computer simulation, in vitro, and / or in vivo analysis, such as agonists, antagonists, and homologs thereof, including fragments, variants, and mimetics.

[0056] As used herein, "inhibitor" or "antagonist" refers to a modulatory compound that decreases, reduces, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates the biological activity and / or expression of a receptor or pathway of interest.

[0057] As used herein, "inducer," "activator," or "agonist" refers to a modulatory compound that increases, induces, stimulates, opens, activates, promotes, enhances activation, sensitizes, or upregulates a receptor or pathway of interest.

[0058] In certain embodiments, an "active compound" is a compound / peptide that modulates the calcium sensing receptor, i.e., a compound / peptide that is active on the calcium sensing receptor. For example, the active compound can be active on the calcium sensing receptor as an agonist, antagonist, positive allosteric modulator (PAM), negative allosteric modulator, or by exhibiting a mixture of activities, such as agonist activity and positive allosteric modulator activity or agonist activity and negative allosteric modulator activity.

[0059] As used herein, the terms "vector" and "expression vector" refer to a linear or circular DNA molecule into which another DNA sequence fragment of appropriate size may be incorporated. Such one or more DNA fragments may include additional segments that provide for transcription of the gene encoded by the DNA sequence fragment. Additional segments may include, but are not limited to, promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selection markers, origins of replication, and the like. Expression vectors are typically derived from plasmids, cosmids, viral vectors, and yeast artificial chromosomes. Vectors are typically recombinant molecules containing DNA sequences from several sources.

[0060] As used herein, the terms "nucleic acid molecule" and "nucleotide sequence" refer to a single-stranded or double-stranded covalently linked sequence of nucleotides in which the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Nucleic acid molecules can include deoxyribonucleotide bases or ribonucleotide bases and can be synthesized in vitro or isolated from natural sources.

[0061] The terms "polypeptide," "peptide," "amino acid sequence," and "protein" are used interchangeably herein to refer to a molecule formed by the linkage of at least two amino acids. The linkage between one amino acid residue and the next is an amide bond, sometimes also 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. The term can apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0062] 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 in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs and derivatives can refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., a carbon bound to a hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid but function in a manner similar to naturally occurring amino acids.

[0063] The terms "isolated" and "purified" are used interchangeably herein to refer to a nucleic acid, polypeptide, or other biological part that is removed from components with which it is naturally associated. The term "isolated" can refer to a polypeptide that is separate and discrete from the whole organism with which it occurs in nature, or in the substantial absence of other biological macromolecules of the same type. With respect to polynucleotides, the term "isolated" can refer to a nucleic acid molecule that lacks, in whole or in part, sequences that are normally associated with it in nature; or a naturally occurring sequence but with a heterologous sequence associated with it; or a molecule that has been separated from a chromosome.

[0064] As used herein, the term "recombinant" may be used to describe nucleic acid molecules and refers to a polynucleotide of genomic, RNA, DNA, cDNA, viral, semisynthetic, or synthetic origin that, by reason of its origin or manipulation, is not associated with all or part of the polynucleotides with which it is associated in nature.

[0065] As used herein, the term "fusion" refers to the joining of different peptide or protein segments by genetic or chemical methods, wherein 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, for example as amino acid residues or other linking groups.

[0066] Calcium-sensing receptor (CaSR)

[0067] The presently disclosed subject matter provides calcium sensing receptors for use in the disclosed methods. 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 use in identifying kokumi-active compounds.

[0068] In certain non-limiting embodiments, the calcium sensing receptor of the present disclosure is encoded by the nucleic acid described in International Application No. PCT / US15 / 55149, filed on October 12, 2015, the entire contents of which are incorporated herein by reference. In certain non-limiting embodiments, the calcium sensing receptor of the present disclosure comprises the amino acid sequence described in International Application No. PCT / US15 / 55149, filed on October 12, 2015.

[0069] In certain non-limiting embodiments, the calcium sensing receptor comprises a feline, canine, or human calcium sensing receptor nucleotide sequence as described in International Application PCT / US15 / 55149, filed on 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, SEQ ID NO: 4, or SEQ ID NO: 6.

[0070] In certain non-limiting embodiments, the calcium sensing receptor comprises the feline, canine, or human calcium sensing receptor amino acid sequence described in International Application PCT / US15 / 55149, filed on 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, SEQ ID NO: 3, or SEQ ID NO: 5.

[0071] 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.

[0072] In certain embodiments, a calcium sensing receptor for use in the presently disclosed subject matter can include a receptor comprising a nucleotide sequence that is 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% identical to a feline, canine, or human calcium sensing receptor nucleotide sequence.

[0073] In certain embodiments, calcium sensing receptors for use in the presently disclosed subject matter can include receptors comprising an amino acid sequence that is 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% identical to a feline, canine, or human calcium sensing receptor.

[0074] In certain embodiments, the disclosed subject matter provides for the use of isolated or purified calcium sensing receptors and / or variants and fragments thereof. The disclosed subject matter also includes the use of sequence variants. In certain embodiments, the variation can occur in one 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 locus in an organism, i.e., allelic variants. Variants also include proteins derived from other loci in an organism (e.g., felines) but having substantial homology to the calcium sensing receptor, i.e., homologs. Variants can also include proteins that are substantially homologous to the calcium sensing receptor but derived from another organism, i.e., orthologs. Variants also include proteins that are substantially homologous to the calcium sensing receptor produced by chemical synthesis. Variants also include proteins that are substantially homologous to the calcium sensing receptor produced by recombinant methods.

[0075] The disclosed subject matter also provides fusion proteins comprising a calcium sensing receptor or a fragment thereof. In certain embodiments, the fusion proteins of the present disclosure may include a detectable label, a functional group such as a carrier, a label, a stabilizing sequence, or a mechanism that can detect binding of a calcium sensing receptor agonist. Non-limiting embodiments of labels include FLAG tags, His tags, MYC tags, maltose binding protein, and other tags known in the art. The currently disclosed subject matter also provides nucleic acids encoding such fusion proteins, vectors comprising nucleic acids encoding fusion proteins, and host cells comprising such nucleic acids or vectors. In certain embodiments, the fusion may be performed at the amino terminus (N-terminus) of the calcium sensing receptor or at the carboxyl terminus (C-terminus) of the calcium sensing receptor.

[0076] In certain embodiments, the calcium sensing receptors disclosed herein may include additional amino acids at the N-terminus and / or C-terminus of the sequence, for example, when used in the methods of the disclosed subject matter. In certain embodiments, the additional amino acids may help immobilize the polypeptide for screening purposes, or allow the polypeptide to become part of a fusion protein, as disclosed above, to facilitate detection of biological activity.

[0077] 3. Calcium-sensing receptor regulatory peptide

[0078] The present disclosure relates to flavor compositions comprising at least one compound that modulates calcium-sensing receptor (CaSR) activity. The compounds disclosed herein are identified through in vitro assays to determine their ability to activate feline CaSRs expressed in cultured cells and / or in silico assays to determine their ability to bind to the CaSR. Flavor compositions can be used to enhance or modify the palatability, taste, or flavor of pet foods. In certain embodiments, the flavor compositions described herein can be added to pet food compositions in various delivery systems.

[0079] In certain embodiments, the CaSR modulating compound is a peptide, such as an oligopeptide. In certain embodiments, the peptide comprises a tripeptide motif according to the formula:

[0080] [Negatively charged or polar amino acid]-[Amino acid with a molecular weight not exceeding 150 Daltons]-[Negatively charged or polar amino acid].

[0081] In certain embodiments, the tripeptide motif comprises:

[0082] (a) the first amino acid residue at the N-terminus, which is a negatively charged amino acid residue or a polar uncharged amino acid residue;

[0083] (b) a second amino acid residue that is not too large an amino acid; and

[0084] (c) a third amino acid residue at the C-terminus that is a negatively charged amino acid residue or a polar uncharged amino acid residue. In certain embodiments, the tripeptide motif binds to the CaSR to impart kokumi.

[0085] 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).

[0086] 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 cysteine ​​(Cys), glycine (Gly), glutamine (Gln), asparagine (Asp), serine (Ser), tyrosine (Tyr) and threonine (Thr).

[0087] In some embodiments, the second amino acid residue has a molecular weight that is no more than about 200 daltons. In some embodiments, the second amino acid residue has a molecular weight that is no more than about 150 daltons, no more than about 140 daltons, no more than about 130 daltons, no more than about 120 daltons, no more than about 110 daltons, no more than about 100 daltons, no more than about 90 daltons or no more than about 80 daltons. In some embodiments, the molecular weight of the second amino acid residue is between about 50 daltons and about 200 daltons, between about 50 daltons and about 150 daltons, between about 60 daltons and about 150 daltons, between about 60 daltons and about 140 daltons, between about 60 daltons and about 130 daltons or between about 60 daltons and 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).

[0088] 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.

[0089] 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.

[0090] In certain embodiments, the peptide is capable of forming a Ca 2+Chelating agents. In certain embodiments, the peptide is capable of activating the feline CaSR. In certain embodiments, the tripeptide motif of the peptide binds to the CaSR to impart a kokumi flavor. In certain embodiments, the EC50 of the peptide for activating the CaSR is no more than about 100 mM, no more than about 90 mM, no more than about 80 mM, no more than about 70 mM, no more than about 60 mM, no more than about 50 mM, no more than about 40 mM, no more than about 30 mM, no more than about 20 mM, no more than about 10 mM, or no more than about 5 mM.

[0091] In certain embodiments, the peptide is included in a flavor composition without other palatability enhancers. In certain embodiments, the peptide is 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.

[0092] In certain embodiments, the peptide can interact (e.g., bind) with the Venus flytrap (VFT) domain of CaSR. In certain embodiments, this interaction with the VFT domain of CaSR excites CaSR. In other embodiments, the peptide acts synergistically with other CaSR agonists or modulators to regulate the activity of CaSR. Still in other embodiments, the interaction with the VFT domain of CaSR antagonizes CaSR. In certain embodiments, the peptide enhances the ability of CaSR agonists to activate receptors (i.e., the peptide acts as a positive allosteric modulator). In certain embodiments, the tripeptide motif of the peptide binds to the VFT domain of CaSR to impart a kokumi.

[0093] In certain embodiments, the peptide interacts with one or more amino acids in the VFT domain, such as one or more of Pro39, Asn64, Arg66, Gly67, Arg69, Trp70, Asn102, Thr145, Gly146, Ser147, Gly148, Tyrl67, Ala168, Ser169, Ser170, Ser171, Ile187, Tyr218, Ser271, Ser272, Glu297, Ala298, Trp299, Ala300, Ser301, Ser302, and Ile416, and any combination thereof. Thus, in certain embodiments, a calcium sensing receptor modulating peptide can be identified and / or defined based on its interaction with one or more of these residues.

[0094] In certain embodiments, the CaSR agonists and / or modulators of the present disclosure comprise salts of the CaSR agonists and / or modulators, such as, but not limited to, acetates or formates. In certain embodiments, the CaSR agonist and / or modulator salt comprises a CaSR agonist and / or modulator salt that is ionically bonded to a cation (+) (such as, but not limited to, Al 3+ , Ca 2+ 、Na + , K + 、Cu 2+ 、H + 、Fe 3+ Mg 2+ NH4 + and H3O + ) bonded anions (-) (such as but not limited to Cl - , O 2- 、CO3 2- 、HCO3 - OH - 、NO3 - PO4 3- 、SO4 2- 、CH3COO - HCOO - and C2O4 2- ). In other embodiments, the CaSR agonist salt comprises a cation (+) bonded to an anion (-) by an ionic bond. In certain embodiments, the peptides of the present disclosure comprise a sodium salt or a potassium salt of the peptide.

[0095] In certain embodiments, the CaSR agonists and / or peptides disclosed herein are included in a 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.

[0096] 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.

[0097] 4. Methods for Identifying Calcium Sensing Receptor Modulating Compounds

[0098] The present disclosure further provides methods for identifying compounds that modulate the activity and / or expression of a calcium sensing receptor. For example, but not by way of limitation, a modulator can be an agonist or an antagonist. The presently disclosed subject matter provides computer simulation and in vitro methods for identifying compounds that modulate the activity and / or expression of the calcium sensing receptor disclosed above.

[0099] 4.1 Computer Methods

[0100] 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, such as a feline, canine, or human calcium sensing receptor.

[0101] In certain embodiments, the method can include predicting the 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 also include predicting whether the putative compound will interact with the receptor binding site by analyzing potential interactions with the putative compound and the receptor amino acids. The method can further include identifying a test compound that can bind to and / or modulate the biological activity of the calcium sensing receptor by determining whether the compound's 3D structure fits within the binding site of the receptor's 3D structure.

[0102] In certain embodiments, the calcium sensing receptor used in the disclosed methods can have the amino acid or nucleotide sequence described in International Application No. PCT / US 15 / 55149, filed October 12, 2015, or a fragment or variant thereof.

[0103] Non-limiting examples of compounds (e.g., potential calcium sensing receptor modulators) that can be tested using the disclosed methods include any small chemical 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.

[0104] In certain embodiments, a crystal structure of a closely related GPCR can be used as a template for homology modeling to build a structural model of a calcium sensing receptor. The X-ray crystal structure of the flytrap domain (VFT) of the human calcium receptor has recently been solved. The structure available in the Protein Data Bank (PDB, www.rcsb.org) is:

[0105] PDB ID: 5FBH-bound Gd +3 The crystal structure of the extracellular domain of the human calcium-sensing receptor;

[0106] PDB ID: 5FBK - Crystal structure of the extracellular domain of the human calcium-sensing receptor;

[0107] PDB ID: 5K5T - Crystal structure of the inactive form of the human calcium-sensing receptor ectodomain;

[0108] PDB ID: 5K5S - Crystal structure of the extracellular domain of the human calcium-sensing receptor in its active form (see Geng et al., Structural mechanism of ligand activation in human calcium-sensing receptor, Elife. 2016 Jun 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).

[0109] In certain embodiments, model VFT structures can be generated for other species of interest, such as cats and dogs, based on sequence homology to human VFT.

[0110] Figures 3A-3C A structural model of a calcium sensing receptor that can be used in the disclosed computer methods is depicted. Any suitable modeling software known in the art can be used. In certain embodiments, the Modeller software package (Accelrys, BIOVIA, Dassault Systèmes) can be used to generate a three-dimensional protein structure.

[0111] In certain embodiments, a computer 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.

[0112] Compounds identified by the disclosed in silico methods can be further tested using the in vitro methods disclosed herein.

[0113] 4.2 Calcium-sensing receptor binding site

[0114] The present application provides a method for screening compounds that modulate the activity of a calcium sensing receptor (e.g., a feline, canine, or human calcium sensing receptor), wherein the compound interacts with one or more amino acids of the calcium sensing receptor. In certain embodiments, the binding site of the calcium sensing receptor comprises amino acids within the Venus flytrap (VFT) domain of the receptor and can be identified by generating an interaction map of the receptor using computer simulation, 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.

[0115] In certain embodiments, the interaction between the compound and one or more amino acids of the calcium sensing receptor described herein may include one or more hydrogen bonds, covalent bonds, non-covalent bonds, salt bridges, physical interactions, and combinations thereof. The interaction may 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 spectroscopic methods, nuclear magnetic resonance (NMR), cross-linking assessment, mass spectrometry or electrophoresis, cryomicroscopy, displacement analysis 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 the compound into the binding pocket of a feline or canine calcium sensing receptor as described herein, for example, using molecular docking, molecular modeling, molecular simulation, or other means known to those of ordinary skill in the art.

[0116] In certain embodiments, the interaction is a salt bridge interaction.

[0117] In certain embodiments, the interaction is a hydrogen bonding interaction.

[0118] In certain embodiments, the interaction is a hydrophobic interaction.

[0119] In certain embodiments, the interaction is a loop stacking interaction.

[0120] In certain embodiments, the compounds that modulate the activity of the calcium sensing receptor identified according to the methods described herein interact with one or more amino acids in the Venus flytrap (VFT) domain of the calcium sensing receptor. In certain embodiments, the amino acids that interact with the compounds include Pro39, Asn64, Arg66, Gly67, Arg69, Trp70, Asn102, Thr145, Gly146, Ser147, Gly148, Tyr167, Ala168, Ser169, Ser170, Ser171, Ile187, Tyr218, Ser271, Ser272, Glu2 97, Ala298, Trp299, Ala300, Ser301, Ser302 and Ile416, and any combination thereof, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or more of a calcium sensing receptor, such as a calcium sensing receptor comprising a feline calcium sensing receptor, or a functionally equivalent amino acid of a canine calcium sensing receptor or a human calcium sensing receptor.

[0121] In certain embodiments, the method for identifying a composition that modulates the activity of a feline calcium sensing receptor comprises (a) contacting a test agent with a calcium sensing receptor, such as a protein comprising SEQ ID NO: NO:1, (b) detecting the interaction between the test agent and one or more amino acids in the interaction site of the calcium sensing receptor, wherein the amino acids are selected from the group consisting 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 and Ile416 in the VFT domain and any combination thereof, and (c) selecting a test agent that interacts with one or more of the amino acids as a composition.

[0122] In certain embodiments, the method further comprises determining the activity of a calcium sensing receptor after step (a), and selecting a test agent as the composition that increases the activity of the calcium sensing receptor.

[0123] In certain embodiments, the method further comprises contacting the calcium sensing receptor with a ligand, such as an agonist, and selecting a test agent as a composition that increases or enhances the ability of the agonist to activate the calcium sensing receptor.

[0124] 4.3 In vitro methods

[0125] The presently disclosed subject matter further provides in vitro methods for identifying compounds that modulate the activity and / or expression of a calcium sensing receptor.

[0126] The calcium-sensing receptor used in the methods of the present disclosure may include an isolated or recombinant calcium-sensing receptor or a cell expressing the calcium-sensing receptor disclosed herein. In certain embodiments, the calcium-sensing receptor used in the disclosed methods may have an amino acid or nucleotide sequence as described in International Application No. PCT / US15 / 55149, filed October 12, 2015, or a fragment or variant thereof.

[0127] In certain embodiments, a method for identifying a compound that modulates the activity and / or expression of a calcium sensing receptor 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 comprise measuring the biological activity of the calcium sensing receptor in the presence of varying concentrations of the test compound. The method can further comprise identifying a test compound that results in modulation of the activity and / or expression of the calcium sensing receptor compared to the activity and / or expression of the calcium sensing receptor in the absence of the test compound.

[0128] In certain embodiments, a compound identified according to the methods described herein increases the biological activity of a 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, 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 a calcium sensing receptor by at least about 30%, compared to the biological activity of the calcium sensing receptor in the absence of the compound.

[0129] In certain embodiments, the method may 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 may be from different classes of compounds, such as amino acids and small molecules. For example, but not by way of limitation, the method may comprise analyzing the effect of one or more small chemical molecule test compounds on the biological activity and / or expression of a calcium sensing receptor in the presence of one or more amino acid test compounds. In certain embodiments, the method for identifying the effect of a compound on the activity and / or expression of a calcium sensing receptor comprises analyzing the effect of a test compound on the biological activity and / or expression of a calcium sensing receptor in the presence of one or more nucleotide or nucleotide derivative test compounds.

[0130] In certain embodiments, methods for identifying a compound that modulates calcium sensing receptor activity and / or expression include determining whether the compound modulates the receptor directly, e.g., as an agonist or antagonist. In certain embodiments, the methods include determining whether the compound modulates receptor activity indirectly (e.g., as an allosteric modulator), e.g., by enhancing or reducing the effects of other compounds that activate or inhibit receptor activity.

[0131] In certain embodiments, the method for identifying a compound that modulates the activity and / or expression of a calcium sensing receptor comprises expressing a calcium sensing receptor in a cell line in the presence and / or absence of a test compound and measuring the biological activity of the receptor. The method may also comprise identifying a test compound that modulates receptor activity by determining whether there is a difference in receptor activation in the presence of a test compound 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 effects on other GPCRs or taste receptors, such as umami, GPR120, T1R, and the like.

[0132] Activation of the receptor in the disclosed methods can be detected by using labeled compounds and / or reagents. In certain embodiments, the activity of the calcium sensing receptor can be determined by detecting secondary messengers such as, but not limited to, cAMP, cGMP, IP3, DAG, or calcium. In certain embodiments, the activity of the calcium sensing receptor can be determined by detecting intracellular calcium levels. Monitoring can be performed by luminescence or fluorescence detection, such as by using calcium-sensitive fluorescent dyes. In certain embodiments, intracellular calcium levels can be determined using cell dyes, such as fluorescent calcium indicators, such as calcium 4. In certain embodiments, intracellular calcium levels can be determined by measuring the level of calcium bound to calcium-binding proteins, such as calmodulin. Alternatively and / or in addition, the activity of the calcium sensing receptor can be determined by detecting phosphorylation, transcript levels, and / or protein levels of one or more downstream protein targets of the calcium sensing receptor.

[0133] The cell lines used in the disclosed methods can include any cell type capable of expressing a calcium sensing receptor. Non-limiting examples of cells useful in the disclosed methods include HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), Xenopus oocytes, HEK-293 cells, and murine 3T3 fibroblasts. In certain embodiments, the method can include expressing a calcium sensing receptor in CHO-K1 cells. In certain embodiments, the method can include expressing a calcium sensing receptor in HEK-293 cells. In certain embodiments, the method can include expressing a 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.

[0134] In some embodiments, the cells express a calcium-binding photoprotein, wherein the photoprotein emits light when bound to calcium. In some embodiments, the calcium-binding photoprotein comprises the protein clytin. In some embodiments, the clytin is recombinant clytin. In some embodiments, the clytin comprises isolated clytin, such as clytin isolated from the ovary of the jellyfish Clytia gregarium. In some embodiments, the calcium-binding photoprotein comprises aequorin, such as recombinant aequorin or isolated aequorin, such as aequorin isolated from Aequorea victoria. In some embodiments, the calcium-binding photoprotein comprises the protein obelin, such as recombinant obelin or isolated obelin, such as obelin isolated from Obelia longissima.

[0135] In certain embodiments, expression of a calcium-sensing receptor in a cell can be achieved by introducing a nucleic acid encoding the calcium-sensing receptor into the cell. For example, but not limitation, a nucleic acid having a nucleotide sequence as described in International Application No. PCT / US15 / 55149, filed on October 12, 2015, or a fragment thereof, can be introduced into the cell. In certain embodiments, the nucleic acid can be introduced into the cell by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or phage vector containing the nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. Many techniques for introducing exogenous genes into cells are known in the art (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 disclosed subject matter. In certain embodiments, the techniques can provide for the stable transfer of nucleic acids into cells such that the nucleic acids are expressible by the cells and heritable and expressible by their progeny. In certain embodiments, the techniques can provide for the transient transfer of nucleic acids into cells such that the nucleic acids are expressible by the cells, wherein the heritability and expressibility decrease in subsequent generations of the progeny of the cells.

[0136] In certain embodiments, the method may include identifying a compound that binds to a calcium-sensing receptor. The method may 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 may 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 may 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. Binding may be measured directly, for example, by using a labeled test compound, or may be measured indirectly. In certain embodiments, detection includes detecting a physiological event in the cell resulting from binding of the compound to the calcium-sensing receptor, such as an increase in intracellular calcium levels. For example, but not by way of limitation, detection may be performed by fluorescence detection, such as with a calcium-sensitive fluorescent dye, by luminescence detection, or by any other detection method known in the art.

[0137] 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 that express a native calcium-sensing receptor include, for example, but are not limited to, dog (canine) and / or cat (feline) taste cells (e.g., primary taste receptor cells). In certain embodiments, dog and / or cat taste cells expressing a calcium-sensing receptor are isolated from dogs and / or cats and cultured in vitro. In certain embodiments, taste receptor cells can be immortalized, for example, so that cells isolated from dogs and / or cats can be propagated in culture.

[0138] In certain embodiments, expression of a calcium-sensing receptor in a cell can be induced by gene editing, for example, by integrating a calcium-sensing receptor gene into the genome of the cell using the CRISPR gene editing system, or editing or modifying a calcium-sensing receptor gene inherent to the cell.

[0139] 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.

[0140] In certain embodiments, compounds identified as calcium sensing receptor agonists and / or modulators can be further tested in other assays, including but not limited to in vivo assays, to confirm or quantify their modulatory activity.

[0141] In certain embodiments, the methods described herein can include determining whether a calcium sensing receptor modulator is a calcium-sensitive taste-enhancing compound, such as a calcium sensing receptor agonist.

[0142] In certain embodiments, methods for identifying calcium sensing receptor agonists and / or modulators can include comparing the effects of a test compound with those of a calcium sensing receptor agonist. For example, a test compound that increases receptor activity 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).

[0143] In certain embodiments, methods for identifying calcium sensing receptor modulators can include determining whether a test compound modulates the activity of a receptor when the receptor is contacted with an agonist, or whether a 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).

[0144] 5. Flavor ingredients

[0145] In certain embodiments, the flavor compositions of the present disclosure can be used to increase the palatability of pet foods, such as cat foods. The flavor compositions can include a combination of compounds and can be added to pet foods in various delivery systems.

[0146] In certain embodiments, the present disclosure is directed to methods for modulating the kokumi (e.g., calcium sensing receptor activity) and / or palatability of a pet food, comprising: a) providing at least one pet food or a precursor thereof, and b) combining the pet food or a precursor thereof with at least a kokumi-modulating amount of at least one flavor composition, e.g., comprising one or more active compounds or edible salts thereof, to form an enhanced pet food.

[0147] 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, such as, for example, a pet food including wet pet food, a dry pet food product, a moist pet food, a pet beverage product, and / or a treat pet food.

[0148] In certain embodiments, one or more flavor compositions of the present disclosure can be added to pet food in an amount effective to improve, enhance, or modify the taste or taste profile of the pet food. Improvements can include, for example, increasing or enhancing the palatability of the pet food, as determined by animals (e.g., cats and / or dogs) using procedures known in the art, 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 produced during the manufacturing process of the food, for example, by hydrolysis of raw materials.

[0149] In certain embodiments of the present disclosure, pet foods can be produced that include a sufficient amount of at least one flavor composition described herein (eg, including a peptide) to produce a pet food having a desired taste, such as kokumi.

[0150] In certain embodiments of the present disclosure, pet foods can be produced that include a sufficient amount of a flavor composition comprising at least one, two, three, four, five, six, or more peptides.

[0151] In certain embodiments, a calcium sensing receptor modulating amount of one or more flavor compositions of the present disclosure may be added to a pet food such that the pet food has increased palatability compared to a pet food prepared without the flavor composition, as determined by animals (e.g., cats and / or dogs), or in the case of formulation testing, by a panel of animal taste testers using procedures known in the art.

[0152] In certain embodiments of the present disclosure, the flavor composition is added to the pet food in an amount effective to increase, enhance, and / or improve the palatability of the pet food.

[0153] The concentration of the flavor composition that is mixed with the pet food to modulate and / or improve the palatability of the pet food can vary depending on variables such as, for example, the specific type of pet food, what taste-modulating compounds / peptides are already present in the pet food and their concentrations, and the potentiating effect of the specific flavor composition on those taste-modulating compounds / peptides.

[0154] A wide range of concentrations of the flavor composition can be used to provide this palatability improvement.In certain embodiments of the present application, the flavor composition is admixed with the pet food, wherein the flavor composition is present in an amount of from about 0.001 ppm to about 1000 ppm. For example, and not by way of limitation, the flavor composition can 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 0.001 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 1000 ppm ppm to about 1000 ppm, about 1 ppm to 1000 ppm, about 2 ppm to about 1000 ppm, about 3 ppm to about 1000 ppm, about 4 ppm to about 1000 ppm, about 5 ppm to about 1000 ppm, about 10 ppm to about 1000 ppm, about 15 ppm to about 1000 ppm, about 25 ppm to about 1000 ppm, about 50 ppm to about 1000 ppm, about 75 ppm to about 1000 ppm, about 100 ppm to about 1000 ppm, about 150 ppm to about 1000 ppm, about 250 ppm to about 1000 ppm, about 250 ppm to about 1000 ppm, about 500 ppm to about 1000 ppm, or about 750 ppm to about 1000 ppm, and values ​​therebetween.

[0155] In certain embodiments of the present application, a flavor composition is mixed with a pet food, wherein the flavor composition is present in an amount of about 0.001 ppm to about 500 ppm, or about 0.01 ppm to about 500 ppm, about 0.1 ppm to about 500 ppm, or about 1 ppm to about 500 ppm, and values ​​therebetween.

[0156] In certain embodiments of the present application, a flavor composition is mixed with a pet food, wherein the flavor composition is present in an amount of about 0.01 ppm to about 100 ppm, or about 0.1 ppm to about 100 ppm, or about 1 ppm to about 100 ppm, and values ​​therebetween.

[0157] In certain embodiments, the flavor composition is present in the pet food in an amount 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, or greater than about 1000 ppm, and values ​​therebetween.

[0158] In certain embodiments, the peptides of the present disclosure are present in a food in an amount sufficient to modulate, activate and / or enhance a calcium sensing receptor. For example, and not limitation, the peptides can be present in an amount 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, about 0.001 μM to about 500 mM, or about 1 μM to about 1 M. , 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 or about 0.001 μM to 10 μM and values ​​therebetween.

[0159] In certain embodiments, the peptides of the present disclosure are present in foods in an amount sufficient to modulate, activate and / or enhance the calcium sensing receptor. For example, and not limitation, the peptides can be present in foods 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, or about 1 μM to about 10 μM, and values ​​therebetween.

[0160] In certain embodiments of the present application, a flavor composition is mixed with a pet food, wherein the flavor composition is present in an amount of about 10 nM to about 0.5 M, or about 1 nM to about 0.5 M, or about 0.1 nM to about 0.5 M, and values ​​therebetween.

[0161] In certain embodiments of the present application, a flavor composition is mixed with a pet food, wherein the flavor composition is present in an amount of about 10 nM to about 0.1 M, or about 1 nM to about 0.1 M, or about 0.1 nM to about 0.1 M, and values ​​therebetween.

[0162] In certain embodiments of the present application, the flavor composition is mixed with a food product, wherein 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 not 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%, or about 0.1% to about 1%, and values ​​therebetween.

[0163] In certain embodiments of the present application, the flavor composition is mixed with a food product, wherein the flavor composition is present in an amount of about 0.0001% to about 5%, or about 0.001% to about 5%, about 0.01% to about 5% w / w, or about 0.1% to about 5% w / w, and values ​​therebetween.

[0164] In certain embodiments of the present application, the flavor composition is mixed with a food product, wherein the flavor composition is present in an amount of about 0.0001% to about 1%, or about 0.001% to about 1%, about 0.01% to about 1% w / w, or about 0.1% to about 1% w / w, and values ​​therebetween.

[0165] In certain embodiments of the present application, the flavor composition is mixed with a food product, wherein the flavor composition is present in an amount of about 0.001% to about 10% w / w.

[0166] 6. Delivery System

[0167] In certain embodiments, the flavor compositions of the present application can be incorporated into a delivery system for pet food. The delivery system can be a non-aqueous liquid, a solid, or an emulsion. The delivery system is generally adapted to suit the flavor composition and / or the edible composition into which the flavor composition is to be incorporated.

[0168] The flavor composition can be used in a non-aqueous liquid form, in a dry form, in 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 onto a water-insoluble material. Actual techniques for preparing such dry forms are well known in the art and can be applied to the presently disclosed subject matter.

[0169] The flavor compositions of the present disclosure can be used in many different physical forms known in the art to provide an initial burst of taste, flavor, and / or texture; and / or a prolonged sensation of taste, flavor, and / or texture. Without limitation, such physical forms include free forms, such as spray-dried, powdered, and beaded forms, and encapsulated forms, and mixtures thereof.

[0170] In certain embodiments, the compounds / peptides of the flavor composition can be produced from precursor compounds present in the pet food during processing of the pet food, such as sterilization, retorting, and / or extrusion.

[0171] In certain embodiments, as described above, encapsulation techniques can be used to improve flavor systems. In certain embodiments, flavor compounds, flavor ingredients, or entire flavor compositions can be fully or partially encapsulated. Encapsulation materials and / or techniques can be selected to determine the type of improvement in a flavor system.

[0172] In certain embodiments, encapsulation materials and / or techniques are selected to enhance the stability of the flavor compound, flavor ingredient, or flavor composition; while in other embodiments, encapsulation materials and / or techniques are selected to modify the release profile of the flavor composition.

[0173] Suitable encapsulating materials may include, but are not limited to, hydrocolloids such as alginates, pectins, agar, guar gum, cellulose, etc., proteins, polyvinyl acetate, polyethylene, cross-linked polyvinyl pyrrolidone, polymethyl methacrylate, polylactic acid (polylactidacid), polyhydroxyalkanoates, ethylcellulose, polyvinyl acetate phthalate (poly vinylacetatephthalate), polyethylene glycol esters, methacrylic acid-co-methyl methacrylate, ethylene-vinyl acetate (EVA) copolymers, etc. and combinations thereof. Suitable encapsulation techniques may include, but are not limited to, spray coating, spray drying, spray cooling, absorption, adsorption, inclusion complexation (e.g., to produce flavor / cyclodextrin complexes), coacervation, fluidized bed coating, or other processes that can be used to encapsulate ingredients with encapsulating materials.

[0174] The encapsulated delivery system for flavoring or sweetening agents can comprise a hydrophobic matrix of fat or wax around the sweetening or flavoring 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.

[0175] The wax may be selected from the group consisting of natural and synthetic waxes and mixtures thereof. Non-limiting examples include paraffin wax, petrolatum, carbowax, microcrystalline wax, beeswax, palm wax, candelilla wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax, rice wax and mixtures thereof.

[0176] The fat and wax can be used alone or in combination in an amount of about 10% to about 70%, or about 30% to about 60%, by weight of the encapsulation system. When used in combination, the fat and wax can be present in a ratio of about 70:10 to about 85:15, respectively.

[0177] Typical encapsulated flavor compositions, flavoring agents, or sweetener delivery systems are disclosed in US Pat. Nos. 4,597,970 and 4,722,845, the disclosures of which are incorporated herein by reference in their entireties.

[0178] In some embodiments, the delivery system can include but is not limited to a system with a dispersion of the flavor composition of the present application, for example in carbohydrate syrup and / or emulsion. The liquid delivery system can also include an extract, in which compound and / or flavor composition are dissolved in a solvent. The solid delivery system can be produced by spray drying, spray coating, spray cooling, fluidized bed drying, absorption, adsorption, cohesion, complexation or any other standard technology. In some embodiments, the delivery system can be selected to be compatible with edible compositions or to work in edible compositions. In certain embodiments, the delivery system will include an oily material, for example fat or oil. In certain embodiments, the delivery system will include confectionery fat, for example cocoa butter, cocoa butter substitute, cocoa butter substitute or cocoa butter equivalent.

[0179] When used in a dry form, suitable drying means, such as spray drying, may be used. Alternatively, the flavoring composition may be adsorbed or absorbed onto a substrate, such as a water-insoluble material, and may be encapsulated. The actual techniques for preparing such dry forms are well known in the art.

[0180] 7. Pet Food

[0181] The flavor compositions of the presently disclosed subject matter can be used in a variety of pet foods. Non-limiting examples of suitable pet foods include wet foods, dry foods, moist foods, pet food supplements (e.g., vitamins), pet beverage products, treats, and treats as described herein.

[0182] When desired, the flavor compositions of the present disclosure, combined with pet foods and optional ingredients, provide flavors that have an unexpected flavor and impart, for example, a kokumi sensory experience, for example, by increasing the activity of calcium-sensing receptors. The flavor compositions disclosed herein can be added before, during, or after the formulation, processing, or packaging of the pet food, and the components of the flavor composition can be added sequentially or simultaneously. In certain embodiments, the compounds / peptides of the flavor composition can be generated from precursor compounds present in the pet food during processing of the pet food, such as during sterilization, cooking, and / or extrusion.

[0183] In certain embodiments, the pet food is a nutritionally complete dry food. A dry or low-moisture nutritionally complete pet food may contain less than about 15% moisture and include about 10% to about 60% fat, about 10% to about 70% protein, and about 30% to about 80% carbohydrates, such as dietary fiber and ash.

[0184] In some embodiments, the pet food is a nutritionally complete wet food. A wet or high-water content nutritionally complete pet food may contain greater than about 50% water. In some embodiments, the wet pet food includes about 40% fat, about 50% protein, and about 10% carbohydrates, such as dietary fiber and ash.

[0185] In certain embodiments, the pet food is a nutritionally complete moist food. A moist (e.g., semi-moist or semi-dry or soft-dry or soft-moist or intermediate or medium-moist) nutritionally complete pet food comprises about 15% to about 50% moisture.

[0186] In certain embodiments, the pet food is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy snacks, cereal bars, snacks, cookies, and sweet products.

[0187] In certain embodiments, the protein source can be derived from a plant source, such as lupin protein, wheat protein, soy protein, and combinations thereof. Alternatively or additionally, the protein source can be derived from a variety of animal sources. Non-limiting examples of animal protein include beef, pork, poultry, lamb, or fish, including, for example, muscle meat, meat by-products, meat meal, or fish meal.

[0188] 8. Methods for measuring taste attributes

[0189] In certain embodiments of the present disclosure, the taste, flavor, and / or palatability attributes of a pet food can be improved by mixing a flavor composition with the food, or produced under 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 the flavor composition mixed with or produced by the food. In certain embodiments, the taste attributes of the improved food can be evaluated as described herein, and the concentration of the flavor composition mixed with or produced by the food can be increased or decreased based on the evaluation results.

[0190] In certain embodiments of the present disclosure, taste and / or palatability attributes can be measured using in vitro assays, wherein the ability of a compound (e.g., a peptide) to activate a feline calcium sensing receptor expressed by a cell in vitro at varying concentrations is measured. In certain embodiments, an increase in receptor activation is associated with an increase in the 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 assays include the in vitro assays described in the Examples section of the application.

[0191] In certain embodiments of the present disclosure, taste and / or palatability attributes can be measured using a computer model in which the ability of a compound to interact with amino acid residues in the calcium sensing receptor binding site is determined in silico. In certain embodiments, the ability of a compound to modulate the feline calcium sensing receptor is correlated with the degree of binding of the compound to the in silico model of the receptor. In certain embodiments, the composition is measured alone or in combination with other compounds. In certain embodiments, the computer model includes the computer models described in the Examples section of this application.

[0192] In certain embodiments of the present disclosure, taste and / or palatability attributes can be measured using a panel of taste testers. For example, but not limitation, the panel can include members of a feline panel. In certain embodiments, the panel can include members of a canine panel. In certain embodiments, the palatability of a pet food can be determined by consuming a pet food containing only a flavor composition (e.g., a one bowl test, monadic ranking). In certain embodiments, the palatability of a pet food can be determined by preferential consumption of a pet food containing a flavor composition disclosed herein relative to a pet food containing no flavor composition or another flavor composition (e.g., a two bowl test for testing preferences, differences, and / or selection).

[0193] In certain embodiments, the palatability and / or kokumi of a flavor composition can be determined by preferential consumption of an aqueous solution comprising a flavor composition disclosed herein relative to an aqueous solution without a flavor composition or comprising a different flavor composition (e.g., a two-bottle test). For example, a solution panel can be used to compare the palatability of a compound at a range of concentrations in a single exposure. In certain embodiments, a solution can include a palatability enhancer, such as L-histidine, as an intake / positive tastant to increase baseline solution intake, thereby enabling identification of potential negative effects of a test compound.

[0194] The intake ratio of each pet food or emulsion can be determined by measuring a kind of ration consumed divided by the total consumption.Then the consumption rate (CR) can be calculated to compare the consumption of a kind of ration with the consumption of another kind of ration to determine the preferential consumption of a kind of food or emulsion over another kind.Alternatively or additionally, the intake (g) difference can be used to evaluate the average difference of intake between two emulsions in a double bottle test or between two pet foods in a double bowl test at a selected significance level, for example, the average difference of intake is determined at a significance level of 5%, with a confidence interval of 95%.However, any significance level can be used, for example, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 50% significance level.In certain embodiments, percentage preference scores can also be calculated, for example, the percentage preference of an animal to a kind of emulsion or food is the percentage of the total emulsion or food that this emulsion or food accounts for intake during the test.

[0195] 9. Manufacturing method

[0196] In certain embodiments, the compounds (e.g., peptides) of the present disclosure can be manufactured using standard chemical synthesis methods. In certain embodiments, chemical synthesis methods provide compounds having 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, compounds can be prepared using standard hydrolysis processes, such as those using an acid, an enzyme, or a combination of an acid and an enzyme.

[0197] In certain embodiments, the compounds of the present invention can be manufactured under food preparation conditions, such as in the production process of pet food. For example, but not limited to, the compounds of the present invention can be produced from precursor compounds present in pet food during hot food processes such as sterilization, cooking and / or extrusion. In certain embodiments, liquid and / or powdered palatants can also be added to enhance the taste of pet food, for example, added to dry pet food, and increase the palatability of pet food. The palatant can be a digest of meat (such as liver) and / or a digest of vegetables, and can optionally include other palatants known in the art. In certain embodiments, before the compound is added to the pet food, the compound can be mixed with a liquid and / or powdered palatant or produced therein. Alternatively, or in addition, after the compound is added to the pet food, the compound can be mixed with a liquid and / or powdered palatant or produced in a liquid and / or powdered palatant.

[0198] 10. Non-limiting examples of the disclosed methods

[0199] In certain non-limiting embodiments, the present disclosure provides a method of increasing the palatability of a pet food, comprising mixing the pet food with a flavor composition comprising a peptide as described herein, wherein the peptide is present in the mixture at a concentration of about 1 nM to about 10 M, or about 1 nM to about 1 M.

[0200] In certain non-limiting embodiments, the present disclosure provides a method for increasing the palatability of a pet food, comprising producing a pet food having a flavor composition comprising a peptide as described herein, wherein the peptide is present in the product at a concentration of about 1 nM to about 10 M, or about 1 nM to about 1 M.

[0201] In certain non-limiting embodiments, the present disclosure provides a method for increasing the kokumi taste of a pet food, for example by increasing the activity of a calcium sensing receptor, comprising mixing the pet food with a flavor composition comprising a peptide as described herein, wherein the peptide is present in the mixture at a concentration of about 0.001 ppm to about 1000 ppm.

[0202] In certain non-limiting embodiments, the present disclosure provides a method of increasing the palatability of a pet food, comprising mixing the pet food with a flavor composition comprising a peptide described herein, wherein the flavor composition is present in the mixture at a concentration of about 0.001 ppm to about 1000 ppm.

[0203] In certain non-limiting embodiments, the present disclosure provides a method for increasing the kokumi taste of a pet food, for example by increasing the activity of a calcium-sensing receptor, comprising mixing the pet food with a flavor composition comprising a peptide as described herein, wherein the flavor composition is present in the mixture at a concentration of about 0.0001% to about 10% w / w, or about 0.001% to about 5% w / w, or about 0.01% to about 1% w / w.

[0204] In certain non-limiting embodiments, the present disclosure provides a method of increasing the palatability of a pet food, comprising mixing the pet food with a flavor composition comprising a peptide described herein, wherein the flavor composition is present in the mixture at a concentration of about 0.0001% to about 10% w / w, or about 0.001% to about 5% w / w, or about 0.01% to about 1% w / w.

[0205] Example

[0206] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration and not limitation of the invention.

[0207] Example 1 - Production and testing of caseinate hydrolysates

[0208] This example investigates the use of a protein hydrolysate palatant system in wet cat food.

[0209] Milk protein (caseinate) was hydrolyzed under different enzymatic conditions. A total of 14 hydrolysates were produced. The degree of hydrolysis of the bulk samples varied between 8% and 35%, and the dry matter content varied between 4% and 8%. All bulk samples were of food grade quality.

[0210] Five different hydrolysates and a control casein hydrolysate were selected for animal feeding experiments. The conditions of each hydrolysate are listed in Table 1.

[0211] Table 1

[0212]

[0213] Animal feeding trials were conducted in which each hydrolysate was mixed at 3% in different matrices (maize gravy, gelatin and autoclave gel). Figures 1A to 1C As shown in Figure 2, similar food intake patterns were observed in all three matrices for the different hydrolysates. The feeding test was repeated in gelatin gel containing 20 mM IMP to increase intake. Figure 1DAs shown, similar food intake patterns were observed. Hydrolyzate T648 was selected for further testing because it had the highest intake across the different matrices.

[0214] Example 2 - Isolation and identification of bioactive compounds from hydrolysates

[0215] This example describes the identification of potential taste-active peptides from casein hydrolysate that showed increased palatability in wet cat food.

[0216] 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 using a combination of different separation techniques, such as medium pressure liquid chromatography (MPLC), size exclusion chromatography (SEC), and high pressure liquid chromatography (HPLC). Twelve 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.

[0217] Aliquots of the isolated samples were tested for agonist activity in the feline tasting receptor assay (f-CaSR). The summary activities obtained in the assay are listed in Table 2.

[0218] Table 2

[0219]

[0220] Example 3 - In silico modeling for identifying compounds that interact with CaSR

[0221] This example describes computational modeling of the feline calcium-sensing receptor (CaSR) to identify putative agonists.

[0222] Computational methods are used to analyze the three-dimensional structure of CaSR to identify polypeptide regions that can be used to selectively activate the receptor. Structural homology models of the cysteine-rich domains of Venus flytrap and CaSR were generated based on the crystal structure of human CaSR (Geng et al., 2016; Zhang et al., 2016). Homology models were constructed using Accelrys' Discovery Studio (DS) program suite. Specifically, the Modeller program from DS was used (see Eswar et al., Current Protocols in Bioinformatics, Supplement 15: 5.6.1-5.6.30 (2006), which is incorporated herein by reference in its entirety). "Computer" screening was used to identify compounds that interact with the CaSR domain.

[0223] The GPCR C group protein family includes T1R1, T1R2, T1R3, CaSR, GabaB and mGlu proteins. C group proteins have (1) a large external domain called the Venus Flytrap (VFT) domain, (2) 7 transmembrane (7TM) domains and (3) a cysteine-rich domain connecting the VFT and 7TM domains. Based on the recent crystal structure of hCaSR now available from the Protein Data Bank (PDB, www.rcsb.org) (Geng et al. 2016; Zhang et al. 2016), a homology model of the VFT and cysteine-rich domain of the feline CaSR receptor was generated. The docking program BioDock from BioPredict was used to dock compounds (including Asp-Val-Glu and γ-Glu-Val-Gly) into the active site of the VFT domain of the CaSR in silico, as shown in Figure 2. Figures 3A to 3C shown.

[0224] The feline residues lining the active site of the CaSR 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 play a role in the homology model by forming salt bridges, hydrogen bonds, and hydrophobic interactions to coordinate the negatively charged head group and polar part of the compound binding to the active site.

[0225] Example 4 - Identification of the active motif of CaSR active peptide

[0226] Based on the structural analysis of the kokumi active peptides identified in Example 2 and the computer modeling described in Example 3, the following tripeptide motif was predicted to be the "kokumi active motif" capable of activating the CaSR receptor: [negatively charged or polar amino acid]-[amino acid with a molecular weight of no more than 150 Daltons]-[negatively charged or polar amino acid].

[0227] Based on this active motif, 12 additional peptides were predicted to activate the CaSR. Table 3 describes the complete list of these 12 peptides individually as well as the two active peptides Asp-Val-Glu and Asp-Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Ala.

[0228] Table 3

[0229]

[0230]

[0231]

[0232]

[0233] Example 5 - In vitro testing of predicted CaSR-active peptides

[0234] The 14 peptides listed in Table 3, along with a number of control peptides and compounds, were tested in vitro to assess their ability to activate the feline CaSR.

[0235] method

[0236] The cell for measuring is the HEK293 cell that is derived from HEK T-Rex / natClytin-fCaSR.For measuring, the cell is placed on the 386-well culture plate with transparent bottom, for reading the luminescence in the hole. The detection setting for standard determination on Flex workstation (FlexStation) is as follows. At the beginning of the determination, the well containing the cell has 20 μ l calcium-free Tyrode's buffer (Tyrode's buffer). Every kind of ligand of 20 μ l specified concentration is injected on the cell, and the reaction of the cell is measured at intervals of 1.94 seconds for 90 seconds. Use the software SoftMax Pro (5.4.1 version) provided by Molecular Devices to analyze and simplify the obtained curve.

[0237] Each peptide was tested on at least two separate occasions, with each concentration applied to the cells in quadruplicate. In parallel, the same assay was run on a mock cell line using a mock vector to confirm the specificity of any signal measured.

[0238] The data obtained from FlexStation are used to track the dose-response curves of each ligand. These figures are [agonist] relative to (vs.) response--variable slope (four parameters) figures drawn using GraphPad Prism 7.03 software. EC50 values ​​with associated standard errors are calculated using the same formula. Each figure contains average data points, wherein the average data points have SEM, which is calculated by the software for each data point.

[0239] One concern in planning these assays is that some of these peptides may bind divalent calcium cations and elicit nonspecific responses in the receptor. To ensure that the interaction between the receptor and the peptide is measured, rather than the receptor's interaction with calcium, the peptides are synthesized under calcium-free conditions. Similarly, all assays are performed using calcium-free reagents.

[0240] result

[0241] 1. All predicted tasting peptides activated feline CaSR in vitro

[0242] Positive controls for this assay were CaCl2 and the previously described hamami peptide γ-Glu-Val-Gly ( Figure 2AOn all graphs, two individual runs and one mock cell run (no mock cell responses were recorded for any ligand) are shown. All other ligands were analyzed in a similar manner, and the data obtained are Figure 2B and detailed in Table 4.

[0243] Table 4

[0244] Compound Molecular weight Maximum concentration EC50±standard error CaCl2 110.9 15mM 1.6±0.06mM MgCl2 95.2 30mM 6.72±0.13mM GSH 307.3 30mM 6.71±0.57mM γ-Glu-Val-Gly 303.3 15mM 5.78±2.41mM Asp 133.1 20mM ≈6.1mM Glu 147.1 20mM ≈5.9mM Ile-Gly-pSer-Glu-pSer-Thr-Glu-Asp-Gln 1124.3 15mM ≈1.6mM Asp-Val-Glu 361.1 15mM 3.3±0.15mM Glu-Val-Asp 361.1 15mM 3.32±0.77mM Asp-Glu-Glu 391.1 15mM 2.59±0.09mM pSer-Glu-pSer 481.1 15mM 3.04±0.21mM pSer-Val-pSer 451.1 15mM 2.63±0.04mM pSer-Val-Glu 413.1 15mM 3.04±0.03mM Ser-Glu-Ser 321.1 15mM 3.56±0.11mM Cys-Val-Cys 323.1 30mM ≈12.56mM pTyr-Glu-pTyr 633.2 15mM 2.22±0.22mM pThr-Glu-pThr 509.1 15mM 1.98±0.07mM Asp-Ala-Glu 333.3 15mM 3.54±0.05mM Glu-Val-Glu 375.2 15mM 3.54±0.05mM Asp-Val-Asp 347.1 15mM 3.41±0.05mM γ-Glu-Val 246.3 30mM ≈3.9mM γ-Glu-Met 278.3 30mM ≈6.7mM γ-Glu-Phe 294.3 30mM ≈7.8mM γ-Glu-Tyr 310.3 30mM ≈6.9mM

[0245] All 12 predicted umami peptides activated the feline CaSR receptor at concentrations in the millimolar range. Although the apparent affinities of the same ligands varied slightly between runs, the differences were small and within acceptable limits. The affinities of all peptides are detailed in Table 4. For some peptides, the model used to calculate the EC50 values ​​provided estimated data rather than exact data, so these values ​​were not associated with standard errors.

[0246] ***

[0247] 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 modifications may be made without departing from the spirit and scope of the present invention as defined by the appended claims. In addition, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, compositions of matter, devices, methods and steps described in the specification. Those of ordinary skill in the art will readily understand from the disclosure of the disclosed subject matter that, according to the subject matter of the present disclosure, existing or to-be-developed processes, machines, manufactures, compositions of matter, devices, methods or steps that perform substantially the same functions as the corresponding embodiments described herein or achieve substantially the same results may be used. Therefore, the appended claims are intended to include such processes, machines, manufactures, compositions of matter, devices, methods or steps within their scope.

[0248] Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Claims

1. A flavor composition comprising an oligopeptide, wherein the oligopeptide is a tripeptide comprising a first amino acid residue, a second amino acid residue, and a third amino acid residue, wherein the first amino acid residue is selected from the group consisting of Asp, pSer, Ser, Cys, pTyr, and pThr; wherein the second amino acid residue is selected from the group consisting of Ala, Val and Glu; wherein the third amino acid residue is selected from the group consisting of Asp, Glu, pSer, Ser, Cys, pTyr and pThr; and wherein the tripeptide is selected from the group consisting of pSer-Glu-pSer, pSer-Val-pSer, pSer-Val-Glu, Ser-Glu-Ser, Cys-Val-Cys, pTyr-Glu-pTyr, pThr-Glu-pThr, Asp-Ala-Glu, Asp-Val-Asp, and any combination thereof, and The tripeptide binds to a calcium-sensing receptor and imparts a kokumi taste to a companion animal.

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

3. The flavor composition of claim 1 or 2, wherein the companion animal is a cat.

4. The flavor composition according to claim 1 or 2, wherein the oligopeptide is produced during the manufacturing process of the food product.

5. The flavor composition according to claim 3, wherein the oligopeptides are produced during the manufacturing process of the food product.

6. A food product comprising the flavor composition according to any one of claims 1 to 5, wherein the flavor composition is present in an amount effective to increase the kokumi of the food product.

7. A food product comprising the flavor composition according to any one of claims 1 to 5, wherein the flavor composition is present in an amount effective to increase the palatability of the food product.

8. The food product of claim 6 or 7, 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.

9. The food product according to claim 6 or 7, wherein the food product comprises pet food.

10. The food product of claim 8, wherein the food product comprises pet food.

11. The food product of claim 9, wherein the pet food is a feline pet food or a canine pet food.

12. The food product of claim 10, wherein the pet food is a feline pet food or a canine pet food.

13. The food product of claim 9, wherein the pet food is a wet pet food.

14. The food product of claim 10, wherein the pet food is a wet pet food.

15. The food product of claim 9, wherein the pet food is a dry pet food.

16. The food product of claim 10, wherein the pet food is a dry pet food.

17. The food product according to claim 6 or 7, wherein the flavor composition is generated during the manufacturing process of the food product.

18. The food product of claim 8, wherein the flavor composition is generated during the manufacturing process of the food product.

19. The food product of claim 9, wherein the flavor composition is generated during the manufacturing process of the food product.

20. The food product of claim 10, wherein the flavor composition is generated during the manufacturing process of the food product.

21. The food product of claim 11, wherein the flavor composition is generated during the manufacturing process of the food product.

22. The food product of claim 12, wherein the flavor composition is generated during the manufacturing process of the food product.

23. The food product of claim 13, wherein the flavor composition is generated during the manufacturing process of the food product.

24. The food product of claim 14, wherein the flavor composition is generated during the manufacturing process of the food product.

25. The food product of claim 15, wherein the flavor composition is generated during the manufacturing process of the food product.

26. The food product of claim 16, wherein the flavor composition is generated during the manufacturing process of the food product.

27. A method of increasing the taste intensity of kokumi in a food product, the method comprising admixing the food product with the flavor composition of any one of claims 1-3, wherein the flavor composition is present in an amount effective to increase the kokumi of the food product.

28. The method of claim 27, wherein the flavor composition is present in the mixture 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.

29. Use of Asp-Val-Glu for binding to a calcium-sensing receptor to impart a kosher taste to a companion animal.

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