WO2022224061A1 - Methods of identifying modulators of receptors over-expressed in circumvallate taste papillae in animals such as cats, methods of using such modulators in food, and foods made by such methods - Google Patents

Abstract

Methods identify compounds that modulate the activity and/or expression of one or more receptors over-expressed in circumvallate taste papillae of an animal such as a cat. Also disclosed are methods of using such compounds to modulate palatability of food, and foods made by such methods. The compound can be used in a food further comprising at least one of macronutrient or a micronutrient; for example, the compound can be used in a coating on dry cat kibble. Optionally the compound can modulate the activity and/or expression of one or more receptors GPR55, CCRL2, GPR15, DRD3, GPR137B, GPR137, CXCR4, CHRM1, GPR107, ADRA2A, CNR2, GPR161, LGR6, GPR132, ADORA3, GPR84, PTAFR, P2RY8, CHRM3, GPR35, CCR7, GPR65, ADRB1, TACR1, GPR39, GPR25, ADGRF3, GPR174, GPR18, ADGRE1, ADGRF1, GPR171, GPR160, GPR34, P2RY14, GPRC5A, GPR180 or GPR108.

Description

TITLE

METHODS OF IDENTIFYING MODULATORS OF RECEPTORS OVER-EXPRESSED IN CIRCUMVALLATE TASTE PAPILLAE IN ANIMALS SUCH AS CATS, METHODS OF USING SUCH MODULATORS IN FOOD, AND FOODS MADE BY SUCH

METHODS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/ 177,648 filed April 21, 2021, the disclosure of which is incorporated in its entirety herein by this reference.

TECHNICAL FIELD

[0002] The present disclosure relates generally to methods of identifying compounds that modulate the activity and/or expression of one or more receptors over-expressed in circumvallate taste papillae in animals such as cats. The present disclosure also relates generally to methods of using such compounds to modulate palatability of food; and foods made by such methods. For example, identification of the sensory receptors which are expressed in the feline oral cavity and responsible for the detection of ingredients that drive preference behavior in cats can be advantageously used to design cat foods.

BACKGROUND

[0003] The sense of taste is essential for animals to reach an efficient nutrient recognition and ingestion and avoid the potential of toxic substances consumption. Most mammals can sense the five basic taste modalities: sweet, bitter, umami, sour and salty. Taste chemical molecules are released in saliva when food compounds are consumed and interact with taste receptors expressed in gustatory papillae on the surface of sensory cells on the tongue.

[0004] Three types of taste sensing papillae have been identified: fungiform papillae on the tip of the tongue, foliate papillae on the sides of the tongue, and circumvallate papillae. Taste sensitive sensory cells are packed in onion-shaped structures called “taste buds” distributed in the epithelium of taste sensitive papillae. Taste bud cells can be classified as one of Types I, II and III according to their morphology and their pattern of expressed genes. Taste-sensitive cells are specialized polarized neuro-epithelial cells converting chemical signal into an electrical signal. Taste receptors are localized in the microvilli on the apical side projected into the taste bud pore in contact with saliva. Type II cells are specialized cells expressing G protein-coupled receptors (GPCRs) involved in sweet, umami and bitter perception. Sour and salty perception mechanisms are not fully understood but are believed to be transduced through the stimulation of type III cells. Type I cells are not involved in the sensing of taste molecules but act rather as support cells.

[0005] The basic taste modalities are highly conserved among mammals to ensure a proper source of calories and avoiding toxic substances. However, expression of taste genes is believed to have evolved according to species environment and feeding behaviors. One case is the taste function evolution of the cat, an obligate carnivore. It might be expected that the evolution of its taste function might have been strongly driven by its diet made only of animal preys. For most mammals, sugar is one of the main sources of calories and drives a strong preference, while the cat is insensitive to it. Indeed, their sweet taste receptor gene (Taslr2) is a pseudogene and might reflect the unnecessity from their diet to sense sweet as a source of nutrient. Following this same logic, bitter taste functionality in cat should be lower. The Tas2R family coding for bitterness shows differences in number and homology across species, probably as a function of the specificity in toxic substances found in each species’ environment. Bitter compounds are often toxic substances from plants. However, cats have at least seven functional bitter receptors and no correlation has been observed between the number of bitter receptors and the level of meat in the diet of different carnivores.

[0006] From its diet, it might be expected that cat’s taste preference is highly driven by food proteins, peptides, amino acids and fats content. Indeed, cats show taste preference for solutions of proteins, solutions of amino acids (L-alanine and L-proline), and butterfat mixtures. The cTIRl-N-terminus domain part of the potential T1R1/T1R3 umami receptor in cats has shown in vitro the capability to bind L-amino acids. However, these data were difficult to find confirmation in vitro for the full T1R1/T1R3 umami receptor. The taste preference coding mechanism in cats remains poorly explored.

[0007] In conclusion, mechanistic drivers of cat palatability in cat food are poorly understood. In particular, it is difficult to drive a targeted approach to identify palatant ingredients for cat food, which would facilitate a healthy nutrient intake for the cat. SUMMARY

[0008] The present disclosure is generally directed to one or more receptors over-expressed in circumvallate taste papillae in animals such as cats, and the present disclosure is further directed to compounds that modulate such receptors. The one or more receptors can be sweet, bitter, umami, mineral, fatty acid, minerals, and such; both for known tastes, and animal tastes that have not been identified. Stimulation of these receptors could lead to either aversive or preferred taste responses.

[0009] Without being bound by any theory, the present disclosure is based on the recognition that receptors coding for taste preference, among them sweet or umami preference in species such as humans or rodents, or fatty acid, bitter, mineral, metallic, or pyrophosphate (PPi) receptors in felines, are G-protein-coupled-receptors (GPR or GPCR) expressed in taste cells within the taste papillae on the tongue. In this regard, a transcriptome analysis was performed to identify over-expressed GPCR in taste tissue compared to control tongue epithelial tissue. Analysis identified new candidates as taste preference receptors: GPCRs over-expressed in cat taste tissue. This finding enables potential identification of new palatant molecules for cat food. [0010] Additional features and advantages are described herein and will be apparent from the following Detailed Description and the Figures.

BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1 is a table of the 108 GPCRs over-expressed in CV vs. NT in the experimental example disclosed herein.

[0012] FIGS. 2A and 2B is a table of the 76 GPCRs of the 108 GPCRs, which have an FDR < 0.01 (adjusted p-value) in the experimental example disclosed herein.

[0013] FIG. 3 is a table of the 44 GPCRs of the 76 GPCRs having an FDR < 0.01 (adjusted p-value), which are over-expressed with a LogFC (fold change) > 2 in the experimental example disclosed herein.

[0014] FIG. 4 is a table of the top-20 genes for contrast CV vs. NT in the experimental example disclosed herein.

[0015] FIG. 5 is a table of fold-change and significance of specific taste genes in the experimental example disclosed herein. The column comment indicates whether the gene is differentially expressed (DE) or not (non-DE) or filtered out (FO) due to low expression. The other columns were extracted from the differential expression analysis performed in the previous section.

DETAILED DESCRIPTION

[0016] As used in this disclosure and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” or “the compound” includes a single compound and also two or more compounds.

[0017] The words “comprise,” “comprises” and “comprising” are to be interpreted inclusively rather than exclusively. Likewise, the terms “include,” “including” and “or” should all be construed to be inclusive, unless such a construction is clearly prohibited from the context. However, the compositions disclosed herein may lack any element that is not specifically disclosed. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of’ and “consisting of’ the components identified. Similarly, the methods disclosed herein may lack any step that is not specifically disclosed herein. Thus, a disclosure of an embodiment using the term “comprising” includes a disclosure of embodiments “consisting essentially of’ and “consisting of’ the steps identified. Any embodiment disclosed herein can be combined with any other embodiment disclosed herein. [0018] The terms “at least one of’ and “and/or” used respectively in the context of “at least one of X or Y” and “X and/or Y” should be interpreted as “X without Y,” or “Y without X,” or “both X and Y.” Where used herein, the terms “example” and “such as,” particularly when followed by a listing of terms, are merely exemplary and illustrative and should not be deemed to be exclusive or comprehensive.

[0019] All percentages expressed herein are by weight of the total weight of the composition unless expressed otherwise. As used herein, “about” is understood to refer to numbers in a range of numerals, for example the range of -10% to +10% of the referenced number, preferably within -5% to +5% of the referenced number, more preferably within -1% to +1% of the referenced number, most preferably within -0.1% to +0.1% of the referenced number.

[0020] The terms “food,” “food product” and “food composition” mean a product or composition that is intended for ingestion by an animal and provides at least one nutrient to the animal. The term “animal” or “pet” means any animal which could benefit from or enjoy the food compositions and products provided by the present disclosure. The pet can be an avian, bovine, canine, equine, feline, hircine, lupine, murine, ovine, or porcine animal. The pet can be any suitable animal, and the present disclosure is not limited to a specific pet animal. The term “companion animal” means a dog or a cat.

[0021] The term “pet food” means any composition formulated to be consumed by a pet. A “dry” food composition has less than 10 wt.% moisture and/or a water activity less than 0.64, preferably both. A “semi-moist” food composition has 11 wt.% to 20wt.% moisture and/or a water activity of 0.64 to 0.75, preferably both. A “wet” food composition has more than 20 wt.% moisture and/or a water activity higher than 0.75, preferably both.

[0022] “Kibbles” are pieces of dry pet food which can have a pellet shape or any other shape. Non-limiting examples of kibbles include particulates; pellets; pieces of pet food, dehydrated meat, meat analog, vegetables, and combinations thereof; and pet snacks, such as meat or vegetable jerky, rawhide, and biscuits. The present disclosure is not limited to a specific form of the kibbles.

[0023] The term “enhanced palatability” and similar terms mean that a palatant according to the present disclosure improves the palatability of a food composition relative to an identically formulated food composition lacking the palatant. “Palatability” refers to a quality of a comestible composition that makes it appealing or pleasing to one or more of an animal’s senses, particularly the senses of taste and smell. As used herein, whenever an animal shows a preference, for example, for one of two or more foods, the preferred food is more “palatable” and has greater “palatability.” For companion animals and other non-human animals, the relative palatability of one food compared to one or more other foods can be determined, for example, in side-by-side, free-choice comparisons, e.g., by relative consumption of the foods, or other appropriate measures of preference indicative of palatability.

[0024] “Over-expressed in circumvallate taste papillae” means that the amount of the receptor expressed in circumvallate taste papillae is greater than the amount of the receptor expressed in non-taste tongue epithelium. In some embodiments, the over-expressed receptor is expressed in circumvallate taste papillae more than non-taste tongue epithelium with a LogFC (fold change) > 2.

[0025] The methods and compositions and other advances disclosed herein are not limited to particular methodologies, protocols, and reagents because, as the skilled artisan will appreciate, they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only and does not limit the scope of that which is disclosed or claimed.

[0026] Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by one of ordinary skill in the art in the field(s) of the present disclosure or in the field(s) where the term is used. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used, the preferred devices, methods, articles of manufacture, or other means or materials are described herein.

[0027] Preferred embodiments provided by the present disclosure are described hereafter. An aspect of the present disclosure is a method of identifying a palatability modulator (e.g., a palatability enhancer) for pet food, wherein the palatability modulator is identified from one or more test compounds. The method can comprise: (i) expression, in one or more cells, of one or more receptors over-expressed in circumvallate taste papillae; (ii) contacting each of the one or more cells expressing the one or more receptors with a corresponding test compound from the one or more test compounds; (iii) determining activity and/or expression of each of the one or more receptors in each of the one or more cells in the presence of the corresponding test compound as compared to the activity and/or expression of the receptor in the absence of the corresponding test compound; and (iv) identifying each of the one or more test compounds which achieve a threshold activity and/or expression of the receptor (relative to the activity and/or expression of the one or more receptors in the absence of the corresponding test compound) as a palatability modulator.

[0028] The palatability modulator can be incorporated into a pet food such as a cat food, for example dry cat food (e.g., in a coating on dry cat kibble). The one or more receptors can be a cat taste receptor and/or a GPCR. The method can comprise identifying the one or more receptors as over-expressed in circumvallate taste papillae, for example before step (i) or step (ii), preferably by comparing expression of the receptor in circumvallate taste papillae of an animal (e.g., a cat) to expression in non-taste tongue epithelium of the animal (e.g., by differential expression analysis).

[0029] Another embodiment of the method of identifying a palatability modulator (e.g., a palatability enhancer) for pet food can comprise: (i) contacting an agonist or antagonist with one or more receptors over-expressed in circumvallate taste papillae; (ii) determining activity of the one or more receptors contacted by the agonist or antagonist; (iii) contacting the one or more receptors with a test compound; (iv) determining activity of the one or more receptors contacted by the test compound; and (v) identifying the test compound as a palatability modulator when the activity of the one or more receptors contacted by the test compound is (a) within a threshold difference less than the activity of the one or more receptors contacted by the agonist or antagonist or (b) about equal to or greater than the activity of the one or more receptors contacted by the agonist or antagonist.

[0030] The palatability modulator can be incorporated into a pet food such as a cat food, for example dry cat food (e.g., in a coating on dry cat kibble). The one or more receptors can be a cat taste receptor and/or a GPCR. The method can comprise identifying the one or more receptors as over-expressed in circumvallate taste papillae, for example before step (i) or step (ii), preferably by comparing expression of the one or more receptors in circumvallate taste papillae of an animal (e.g., a cat) to expression in non-taste tongue epithelium of the animal (e.g., by differential expression analysis).

[0031] Yet another embodiment of the method of identifying a palatability modulator (e.g., a palatability enhancer) for pet food can comprise: (i) contacting a test compound with one or more receptors over-expressed in circumvallate taste papillae; (ii) detecting an interaction between the test compound and the one or more receptors; and (iii) identifying the test compound as a palatability modulator when the test compound interacts with the one or more receptors.

[0032] The palatability modulator can be incorporated into a pet food such as a cat food, for example dry cat food (e.g., in a coating on dry cat kibble). The one or more receptors can be a cat taste receptor and/or a GPCR. The method can comprise identifying the one or more receptors as over-expressed in circumvallate taste papillae, for example before step (i) or step (ii), preferably by comparing expression of the one or more receptors in circumvallate taste papillae of an animal (e.g., a cat) to expression in non-taste tongue epithelium of the animal (e.g., by differential expression analysis).

[0033] Another aspect of the present disclosure is a process of making a pet food, the process comprising combining a palatability modulator (e.g., a palatability modulator identified by any of the methods disclosed herein) with at least one of a macronutrient or a micronutrient. Yet another aspect of the present disclosure is a pet food comprising palatability modulator (e.g., a palatability modulator identified by any of the methods disclosed herein) and at least one of a macronutrient or a micronutrient.

[0034] In some embodiments of any of the methods disclosed herein, the one or more receptors are expressed by a gene selected from those shown in the table in FIG. 1.

[0035] In some embodiments of any of the methods disclosed herein, the one or more receptors are expressed by a gene selected from those shown in the table in FIGS. 2A and 2B. [0036] In some embodiments of any of the methods disclosed herein, the one or more receptors are expressed by a gene selected from the group consisting of GPR55, CCRL2, GPR15, DRD3, GPR137B, GPR137, CXCR4, CHRM1, GPR107, ADRA2A, CNR2, GPR161, LGR6, GPR132, ADORA3, GPR84, PTAFR, P2RY8, CHRM3, GPR35, CCR7, GPR65, ADRB1, TACR1 and GPR39.

[0037] In some embodiments of any of the methods disclosed herein, the one or more receptors are expressed by a gene selected from the group consisting of GPR25, GPR84, ADGRF3, GPR35, GPR39, GPR174, GPR18, GPR55, GPR132, GPR15, ADGRE1, ADGRF1, GPR171, GPR65, P2RY8, GPR160, LGR6, GPR34, GPR137B, P2RY14, GPRC5A, GPR180, GPR161, GPR137, GPR107 and GPR108.

[0038] In some embodiments of any of the methods disclosed herein, the one or more receptors are expressed by a gene selected from those shown in the table in FIG. 3.

[0039] As noted above, a palatability modulator can be incorporated into a pet food such as a cat food, for example dry cat food. In some embodiments, the pet food can comprise the palatability modulator in an amount effective to increase or decrease taste and/or palatability of the pet food relative to a pet food without the palatability modulator but otherwise identically formulated.

[0040] In another aspect of the present disclosure, an initial pet food formulation comprises a palatant, and the method comprises adjusting the initial pet food formulation to replace at least a portion of the palatant with one or more compounds identified as a palatability modulator (e.g., a palatability modulator identified by any of the methods disclosed herein) that is a different compound than the palatant, and the adjusted dry pet food formulation has about the same amount of the ingredients other than the palatant and the palatability modulator relative to the initial dry pet food formulation. Preferably the method further comprises producing a pet food according to the adjusted pet food formulation, for example cat food such as dry cat food. [0041] The pet foods disclosed herein can be any food formulated for consumption by a pet such as a dog or cat. In an embodiment, the pet food provides complete nutrition as defined by the Association of American Feed Control Officials (AAFCO) and which depends on the type of animal for which the composition is intended (e.g., a dog or a cat).

[0042] The pet food can comprise meat, such as emulsified meat. Examples of suitable meat include poultry, beef, pork, lamb and fish, especially those types of meats suitable for pets. The meat can include any additional parts of an animal including offal. Some or all of the meat can be provided as one or more meat meals, namely meat that has been dried and ground to form substantially uniform-sized particles and as defined by AAFCO. Additionally or alternatively, vegetable protein can be used, such as pea protein, corn protein (e.g., ground corn or corn gluten), wheat protein (e.g., ground wheat or wheat gluten), soy protein (e.g., soybean meal, soy concentrate, or soy isolate), rice protein (e.g., ground rice or rice gluten) and the like.

[0043] The pet foods disclosed herein can comprise one or more of a vegetable oil, a flavorant, a colorant or water. Non-limiting examples of suitable vegetable oils include soybean oil, corn oil, cottonseed oil, sunflower oil, canola oil, peanut oil, safflower oil and the like.

[0044] Non-limiting examples of suitable flavorants include yeast, tallow, rendered animal meals (e.g., poultry, beef, lamb, pork), flavor extracts or blends (e.g., grilled beef), animal digests, and the like. Non-limiting examples of suitable colorants include FD&C colors, such as blue no. 1, blue no. 2, green no. 3, red no. 3, red no. 40, yellow no. 5, yellow no. 6, and the like; natural colors, such as caramel coloring, annatto, chlorophyllin, cochineal, betanin, turmeric, saffron, paprika, lycopene, elderberry juice, pandan, butterfly pea and the like; titanium dioxide; and any suitable food colorant known to the skilled artisan.

[0045] The pet foods disclosed herein can optionally include additional ingredients, such as starches, humectants, oral care ingredients, preservatives, amino acids, fibers, prebiotics, sugars, animal oils, aromas, other oils additionally or alternatively to vegetable oil, salts, vitamins, minerals, probiotic microorganisms, bioactive molecules or combinations thereof.

[0046] Non-limiting examples of suitable starches include a grain such as corn, rice, wheat, barley, oats, potatoes, peas, beans, cassava, and the like, and mixtures of these grains, and can be included at least partially in any flour. Non-limiting examples of suitable humectants include salt, sugars, propylene glycol and polyhydric glycols such as glycerin and sorbitol, and the like. Non-limiting examples of suitable oral care ingredients include alfalfa nutrient concentrate containing chlorophyll, sodium bicarbonate, phosphates (e.g., tricalcium phosphate, acid pyrophosphates, tetrasodium pyrophosphate, metaphosphates, and orthophosphates), peppermint, cloves, parsley, ginger and the like. Non-limiting examples of suitable preservatives include potassium sorbate, sorbic acid, sodium methyl para-hydroxybenzoate, calcium propionate, propionic acid, and combinations thereof.

[0047] Specific amounts for each additional ingredient in the pet food compositions disclosed herein will depend on a variety of factors such as the ingredient included in the first edible material and any second edible material; the species of animal; the animal's age, body weight, general health, sex, and diet; the animal's consumption rate; the purpose for which the food product is administered to the animal; and the like. Therefore, the components and their amounts may vary widely.

[0048] EXAMPLE

[0049] The following non-limiting example further supports the compounds, compositions and methods disclosed herein.

[0050] Introduction

[0051] Cat taste perception mechanisms involved in their preference for certain food or nutrients are poorly understood. The gene expression analysis in taste papillae of cat can provide insight into the key proteins involved in taste transduction mechanism. For example, transcriptome analysis of circumvallate papillae would provide a mapping of genes specifically expressed in taste-sensitive tissue as mechanistic elements for cat preference coding.

[0052] A study investigated gene expression of eleven samples combining two different animals and biopsies from circumvallate (CV) taste papillae and no gustatory tongue epithelium (non-taste: NT) as control tissue. The study identified 9676 genes from the annotated cat genome (v9.0): Felis_catus_9.0 (GCA_000181335.4), as available at https://useast.ensembl.org/Felis_catus/Info/Index, hereby incorporated by reference in its entirety. These genes were differentially expressed between CV taste papillae and NT; and among them 5103 genes were over-expressed. The enrichment of CV biopsies in cells involved in taste transduction was confirmed by the over-expression of genes related to taste-transduction, as TAS1R3, TAS1R1, PLCB2, GNAT3, TRPM5, GNG13 and TAS2R4. One-hundred and eight (108) over-expressed GPCRs in CV can be considered as potential receptors for transduction mechanisms in taste cells.

[0053] Methods

[0054] Animals

[0055] All the biopsies used in the project were collected post-mortem on tongue from animals euthanized for health reasons independent of this project. The tongues were sampled rapidly after the euthanasia. Animals were males of 10-13 years old.

[0056] Isolation of tissue biopsies

[0057] Two different type of biopsies on tongue tissues from two different animals were sampled: Circumvallate papilla (CV) and non-taste lingual tissue (NT). Four samples from a first animal were collected: NT1_1, NT2_1, CV1_1 and CV3_1. Seven samples from a second animal were collected: CV1_2, CV2_2, CV3_2, NTdessusl_2, NTdessous2_2, NTdessous3_2, and NTdessus4_2.

[0058] RNA extraction and RNA-seq library preparation

[0059] Biopsies were rapidly excised form the tongue and washed in PBS then kept in a stabilization solution (RNAlater) at 4 °C until RNA extraction. RNA was extracted using a MiniARN extraction kit (Qiagen). RNA quantification was performed with Ribogreen (Life Technologies) and quality was assessed on a Fragment Analyzer (Advances Analytical). The quality of RNA obtained was heterogeneous within samples. Sequencing libraries were prepared using the TruSeq Stranded Ribo-Zero Gold Kit protocol (Illumina) followed by PCR amplification according to manufacturer recommendations. The optimal number of PCR cycles was assessed by qPCR (Kapa BioSystems): 14 cycles PCR with the KAPA HiFi HotStart ReadyMix (Kapa BioSystems).

[0060] Sequencing

[0061] Libraries were quantified with Picogreen (Life Technologies), and size pattern was controlled with the DNA High Sensitivity Reagent kit on a LabChip GX (Perkin Elmer). Libraries were then pooled at an equimolar ratio (i.e., an equal quantity of each sample library) and clustered at a concentration of 9 pmol on a paired-end sequencing flow cell (Illumina). Sequencing was performed for 2 x 125 cycles on a HiSeq 2500 (Illumina) using the SBS chemistry (Sequencing by Synthesis).

[0062] Sequence analysis [0063] Primary data control was performed during the sequencing run to ensure the optimal flow cell loading (cluster density) and check the quality metrics of the sequencing run (QC30). Sequencing data were demultiplexed, and FastQ files generated. Fastq format raw sequences were mapped to the latest cat genome (v9.0) using STAR v2.5.3a program.

[0064] Reads mapping to the Cat genome v9.0 were quantified. Genes with very low counts are unlikely to be differentially expressed between groups. Therefore, lowly expressed genes were filtered by selecting only genes with at least five reads in at least two samples. The filtering step was applied to the CPM values, which accounts for the library size and corresponds to a threshold of 0.096 on the CPM values. Genes with no annotation were discarded, and the study kept 21105 features following these filtering criteria. The TMM method (Trimmed Mean of M- values) was used for normalization.

[0065] Differential expression analysis between CV and NT was performed using edgeR v3.28 by fitting a negative binomial log-linear model to the normalized counts for each feature. Empirical Bayes methods were used to moderate the degree of overdispersion. The study further extended the negative binomial model with quasi-likelihood method to account for gene-specific variability. Differential expression was tested by quasi-likelihood F-tests and resulting p-values are adjusted for multiple testing using Benjamini and Hochberg method (BH).

[0066] Results

[0067] Samples Distribution

[0068] The study had 11 samples (5 CV and 6 NT) coming from 2 animals. The sampling design was not balanced between both animals, so subject effect was not considered for further analysis.

[0069] Exploratory data analysis

[0070] The dataset contains information on 31493 genes and 11 samples. The study first explored the dataset for potential outliers by plotting the first two components of a principal component analysis (PCA) on the samples. The PCA plot showed no outlier.

[0071] Data filtering and normalization

[0072] Genes with very low counts are unlikely to be differentially expressed between groups. The study filtered lowly expressed genes by selecting only genes with at least five reads in at least two samples. The threshold on the number of samples ensures that the genes are expressed in the smallest group (which contains two samples). The filtering was performed on the CPM values, which accounts for the library size and corresponds to a threshold of 0.096 on the CPM values. The study also discarded genes with no annotation. The study kept 21105 features following these filtering criteria. The study normalized using TMM method. The density plot of each sample and the boxplots confirmed that the normalization performed well. The MDS plot showed that 1/3 sample NT-Above animal 2 was quite far from the other NT samples. Therefore, it was removed before filtering and normalizing, and the dataset was re processed from the beginning.

[0073] Differential Expression analysis

[0074] The study compared CV and NT samples. The model considers the type of tissue and the animal ID. The study detected 9676 genes that are differentially expressed at a 5% significance level on the adjusted p-values (p-values were corrected for multiple testing using Benjamini and Hochberg correction method). There were 5103 over-expressed and 4573 under-expressed genes.

[0075] The table in FIG. 4 represents the top-20 genes over-expressed in CV vs NT ordered by increasing FDR.

[0076] Taste Genes

[0077] Some genes are of particular interest because they correspond to genes involved in taste transduction cascade. These genes were further studied as shown in the table in FIG. 5. For each gene, the study checked whether the gene is detected as differentially expressed (DE) in the analysis performed in the previous section or not (non-DE) or whether it was filtered out (FO) due to low expression. The table reports, when appropriate, the logFC, logCPM, F-statistic, P- value and adjusted p-value (FDR). The genes TAS1R3, TAS1R1, PLCB2, GNAT3, TRPM5, GNG13, TAS2R4, TAS2R39 and TAS2R8 were seen to be DE. The study confirmed, through the over-expression of these genes, the enrichment of CV biopsies in cells involved in taste transduction.

[0078] GPCR

[0079] Many taste receptors are GPCRs, so the study investigated the GPCR over-expressed in CV vs. NT. One-hundred forty-two (142) GPCR were found to be differentially expressed between CV and NT with a 0.05 threshold on the FDR. Of these 142 GPCR, one-hundred eight (108) were over-expressed (FIG. 1). Among these 108 over-expressed GPCR, the already known taste receptors are TAS2R4, TAS2R8, TAS2R39, TAS1R1 and TAS1R3. Of these 108 over-expressed GPCR, the table in FIGS. 2A and 2B identifies the seventy-six (76) have an FDRO.Ol (adjusted p-value). Of the seventy-six (76) GPCRs with an FDRO.Ol (adjusted p-value), the following do not have a well characterized function: GPR25, GPR84, ADGRF3, GPR35, GPR39, GPR174, GPR18, GPR55, GPR132, GPR15, ADGRE1, ADGRFl, GPR171, GPR65, P2RY8, GPR160, LGR6, GPR34, GPR137B, P2RY14, GPRC5A, GPR180, GPR161, GPR137, GPR107 and GPR108.

[0080] Of the seventy-six (76) GPCRs with an FDR<0.01 (adjusted p-value), the table in FIG. 3 identifies the forty-four (44) that are over-expressed with a LogFC (fold change) > 2. [0081] Discussion

[0082] The analysis presented in this study compared gene expression in taste papillae and non-taste papillae cells from two cats. Differential expression analysis found thousands of differentially expressed genes, including some genes known as taste genes.

[0083] The over-expression of known genes involved in taste transduction was confirmed; indeed TAS1R3, TAS1R1, PLCB2, GNAT3, TRPM5, GNG13, TAS2R4, TAS2R39 and TAS2R8 were seen to be over-expressed in CV papillae of cat. In CV papillae of cat, key proteins as PLCB2, GNAT3, TRPM5 and GNG13, involved in the transduction of basic tastes through GPCR activation, was confirmed. The expression of TAS1R3 already described by Li et. al has been also confirmed by the present sequencing, which suggests an active associated taste pathway in cat’s CV. TAS1R1 is also found expressed in cat’s CV, and thus a functional umami TAS1R1/TAS1R3 may be present in cat CV papillae. The functionality of TAS1R1 has been explored in vitro and shows the capability to bind L-amino acids (L-Ala, L-Ile, L-Arg and L- His). However, the functionalities of the cat TAS1R1/TAS1R3 heterodimer are poorly known, even though neurophysiological data and behavioral data show that amino acids are generating a gustatory response in cats.

[0084] From the TAS2R class of bitter receptors, only TAS2R4, TAS2R39 and TAS2R8 were found to be significantly overexpressed in CV, and TAS2R9, TAS2R10, TAS2R38 and TAS2R41 were found expressed in CV with not significant difference compared to NT.

[0085] Seven bitter receptors - TAS2R2, TAS2R4, TAS2R7, TAS2R12, TAS2R38, TAS2R46 and TAS2R67 - from domestic cats have been functionalized in vitro from sequences amplified from genomic DNA. Among these seven receptors, the study confirmed the expression in CV of TAS2R4 and TAS2R38, and the involvement of the five other functionalized TAS2R genes in bitter taste detection in cat remain to be explored. And additional to the seven TAS2R of the ten explored for which a response to bitter compounds was observed, the study found five TAS2Rs expressed in the CV having a potential role in bitter detection in cats.

[0086] In most mammals, the transduction of sweetness, bitter or umami sensations is regulated by GPCRs, which play a key role in the recognition of gustatory molecules. The detection in cats of key nutrients of the diet, such as amino acids, minerals, fatty acids and vitamins, appears to also involve GPCRs. The present study identified one-hundred eight (108) GPCRs significantly overexpressed in CV, among which some might be gustatory receptors. [0087] The GPCRs reported in the table in FIG. 1 to be over-expressed in cat circumvallate papillae constitute potential receptors involved in taste coding in cats and potentially taste driven of preference. The overview of genes expressed in the CV papillae of cat may serve as a ground for the further exploration of elements of the gustatory transduction in cats. Indeed, the study confirms the overexpression of key conserved element of taste transduction cascade involving GPCRs.

[0088] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1. A method of identifying a palatability modulator for pet food, wherein the palatability modulator is identified from one or more test compounds, the method comprising:

(i) expressing, in one or more cells, one or more receptors over-expressed in circumvallate taste papillae;

(ii) contacting each of the one or more cells expressing the one or more receptors with a corresponding test compound from the one or more test compounds;

(iii) determining activity and/or expression of the one or more receptors in each of the one or more cells in the presence of the corresponding test compound as compared to the activity and/or expression of the one or more receptors in the absence of the corresponding test compound; and

(iv) identifying each of the one or more test compounds which achieve a threshold activity and/or expression of the one or more receptors, as compared to the activity and/or expression of the one or more receptors in the absence of the corresponding test compound, as a palatability modulator.

2. A method of identifying a palatability modulator for pet food, the method comprising:

(i) contacting an agonist or antagonist with one or more receptors over-expressed in circumvallate taste papillae;

(ii) determining activity of the one or more receptors contacted by the agonist or antagonist;

(iii) contacting the one or more receptors with a test compound;

(iv) determining activity of the one or more receptors contacted by the test compound; and

(v) identifying the test compound as a palatability modulator when the activity of the one or more receptors contacted by the test compound is (a) within a threshold difference less than the activity of the one or more receptors contacted by the agonist or antagonist or (b) about equal to or greater than the activity of the one or more receptors contacted by the agonist or antagonist.

3. A method of identifying a palatability modulator for pet food, the method comprising:

(i) contacting a test compound with one or more receptors over-expressed in circumvallate taste papillae;

(ii) detecting an interaction between the test compound and the one or more receptors; and

(iii) identifying the test compound as a palatability modulator when the test compound interacts with the one or more receptors.

4. The method of any of Claims 1-3, wherein the one or more receptors are expressed by a gene selected from those shown in the table in FIG. 1.

5. The method of any of Claims 1-3, wherein the one or more receptors are expressed by a gene selected from selected from those shown in the table in FIGS. 2A and 2B.

6. The method of any of Claims 1-3, wherein the one or more receptors are expressed by a gene selected from gene selected from the group consisting of GPR55, CCRL2, GPR15, DRD3, GPR137B, GPR137, CXCR4, CHRM1, GPR107, ADRA2A, CNR2, GPR161, LGR6, GPR132, ADORA3, GPR84, PTAFR, P2RY8, CHRM3, GPR35, CCR7, GPR65, ADRB1, TACR1 and GPR39.

7. The method of any of Claims 1-3, wherein the one or more receptors are expressed by a gene selected from gene selected from the group consisting of GPR25, GPR84, ADGRF3, GPR35, GPR39, GPR174, GPR18, GPR55, GPR132, GPR15, ADGRE1, ADGRF1, GPR171, GPR65, P2RY8, GPR160, LGR6, GPR34, GPR137B, P2RY14, GPRC5A, GPR180, GPR161, GPR137, GPR107 and GPR108.

8. The method of any of Claims 1-3, wherein the one or more receptors are expressed by a gene selected from those shown in the table in FIG. 3.

9. A process of making a pet food, the process comprising: identifying a palatability modulator, wherein the identifying of the palatability modulator comprises at least one method as recited in Claims 1-8; and combining the palatability modulator with at least one of a macronutrient or a micronutrient.

10. The process of Claim 9, wherein the pet food is formulated for administration to a cat, preferably for complete nutrition of the cat.

11. The process of any of Claims 9-10, wherein the pet food is a dry pet food comprising a coating containing at least a portion of the palatability modulator.

12. The process of any of Claims 9-11, wherein the pet food comprises the palatability modulator in an amount effective to increase taste and/or palatability of the pet food relative to a pet food without the palatability modulator but otherwise identically formulated.

13. A pet food formulated for administration to a companion animal, the pet food:

(i) comprising a palatability modulator identified by at least one method as recited in Claims 1-8 and further comprising at least one of a macronutrient or a micronutrient; and/or

(ii) made by at least one process recited in Claims 9-12.

14. The pet food of Claim 13, wherein the pet food is dry cat food comprising a coating on dry kibble, and the coating comprises at least a portion of the palatability modulator.

15. The pet food of any of Claims 13-14, wherein the pet food is formulated for administration to a cat, preferably for complete nutrition of the cat.