Pet food compositions and methods of using the same

Abstract

Described herein is a pet food composition comprising at least one fermentable fiber; betaine; carnitine; tryptophan; at least one fruit, vegetable, or extract thereof comprising phytochemicals; and at least two omega-3 polyunsaturated fatty acids. Also disclosed herein are methods of treating or preventing anxiety and / or an inflammation or allergy in a companion animal by administering the pet food composition to the companion animal.

Description

Attorney Docket No.: 14405-00-WO-01-HL PET FOOD COMPOSITIONS AND METHODS OF USING THE SAME CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 614956, filed 27 December 2023, the contents of which are hereby incorporated herein by reference in their entirety. BACKGROUND

[0002] An animal’s diet can affect many aspects of the animal’s daily life, including the overall health and behavior of the animal. Consequently, changes to the animal’s diet may seek to improve the animal’s health and / or behavior, including, for example, the animal’s immune system or negative behaviors associated with, for example, hyperactivity, advanced age, or anxiety.

[0003] Canine anxiety may be characterized as a response to fear and agitation, or by apprehension when the canine anticipates a threat or fearful situation. Some individual dogs experience disproportionate levels of anxiety. Anxiety can develop into an anxiety disorder and can lead to behavioral and other issues. Some dogs experience a generalized anxiety, in which the fearful reaction is displayed in a wide range of situations to which a non-anxious pet would be unlikely to react. Anxiety can take the form of one of various anxiety disorders such as generalized anxiety disorders, excessive stimulus anxiety, separation anxiety, confinement, and noise phobias, among others.

[0004] Causative factors of anxiety may include genetic components, prenatal and neonatal stressors, maternal separation, lack of socialization, unfamiliarity, or a previous unpleasant outcome during encounters with the stimulus (or similar stimuli). The most common causes are fear, separation and aging. Fear-related anxiety can be caused by loud noises, strange people or animals, visual stimuli, new or strange environments, and specific situations, among others. Age- related anxiety affects older dogs and can be associated with cognitive dysfunction syndrome (CDS). Separation anxiety is a specific anxiety arising from an inability of the pet to find comfort when separated from family members. About 14% of dogs have separation anxiety. Some separation anxiety may be the result of dysfunctional attachment as a puppy ages and matures. In some cases, separation anxiety may arise in cases involving a change in household or daily routine, while in other cases, separation anxiety is associated with an underlying state of anxiety along with other behavioral issues such as phobias.Attorney Docket No.: 14405-00-WO-01-HL

[0005] Anxiety may lead to destructive behavior (particularly at exits or toward owner possessions), distress vocalization, house-soiling, salivation, pacing, restlessness, inability to settle, anorexia, and repetitive or compulsive behaviors. In some instances, anxiety may play a role in aggressive behavior.

[0006] There is a need for methods and compositions for treating or reducing the severity of elevated levels of anxiety in companion animals such as dogs.

[0007] In addition to anxiety and / or stress, inflammation and its associated proinflammatory substances may be part of an animal’s immunological response to such challenges as disease or invading pathogens, or may be a result of an allergic response to a non-pathogen. Inflammation, which can be internal, external, or both, sometimes occurs persistently and / or at levels that negatively impact the health of the animal. At times, the sustained and / or elevated production of proinflammatory substances such as cytokines may cause inflammation to work against the body’s tissues and cause damage. Inflammation can be classified as either acute or chronic. Acute inflammation is an initial response to a harmful stimuli and is achieved by the increased movement of plasma and leukocytes from the blood into the injured tissues. Chronic inflammation, or prolonged inflammation, leads to a progressive shift in the type of cells that are present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. An animal’s cytokine level may be indicative of its present inflammatory state, and management and / or reduction of proinflammatory substances such as cytokines in an animal may be beneficial to that animal’s health. There remains, therefore, a need for new or alternative methods and compositions for treating or preventing an inflammatory condition.

[0008] While pet food compositions may be desirable to help treat or prevent anxiety in an animal or to treat or prevent inflammatory conditions, the pet food composition, for maximum efficacy, should also be palatable to the animal. Enhancing the palatability of an animal food is a continuing endeavor by food manufacturers. Addition of palatants to the food is desirable as a means to increase acceptance by the animals, resulting in, for example, overall improved health of the animal, maintenance of weight, and increased weight gain. Accordingly, there is a need for pet food compositions to offer palatability enhancing properties, while also offering enhanced health benefits, such as treating or preventing anxiety or treating or preventing inflammatory conditions.Attorney Docket No.: 14405-00-WO-01-HL BRIEF SUMMARY

[0009] Some embodiments of the present disclosure provide a pet food composition comprising at least one fermentable fiber (e.g., scFOS); betaine; carnitine; tryptophan; at least one fruit, vegetable or extract thereof comprising phytochemicals (e.g., spinach, broccoli, citrus pulp); and at least two omega-3 polyunsaturated fatty acids.

[0010] In certain embodiments, the at least one fermentable fiber is selected from short-chain fructooligosaccharides (scFOS), for example scFOS present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 5 wt% to about 15 wt%, about 8 wt% to about 12 wt%, or about 10 wt%, based on the total weight of the pet food composition.

[0011] In certain embodiments, the betaine is present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 5 wt% to about 15 wt%, about 8 wt% to about 12 wt%, or about 10 wt%, based on the total weight of the pet food composition, and in certain embodiments, the carnitine is present in the pet food composition in an amount ranging from about 0.001 wt% to about 5 wt%, such as from about 0.01 wt% to about 2 wt%, about 0.1 wt% to about 1 wt%, about 0.2 wt% to about 0.9 wt%, or about 0.8 wt%, based on the total weight of the pet food composition. In still further embodiments, the tryptophan is present in the pet food composition in an amount ranging from about 0.001 wt% to about 5 wt%, such as from about 0.01 wt% to about 2 wt%, about 0.1 wt% to about 1.5 wt%, about 0.2 wt% to about 1.2 wt%, or about 1 wt%, based on the total weight of the pet food composition.

[0012] In certain embodiments, the at least one fruit, vegetable, or extract thereof comprising phytochemicals is selected from broccoli, spinach, or citrus pulp, and in certain embodiments, the at least one fruit, vegetable, or extract thereof comprising phytochemicals is a combination of broccoli, spinach, and citrus pulp. In certain embodiments, the citrus pulp is present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 2 wt% to about 12 wt%, about 5 wt% to about 10 wt%, or about 7 wt%, based on the total weight of the pet food composition. In still further embodiments, the broccoli is present in the pet food composition in an amount ranging from about 1 wt% to about 30 wt%, such as from about 2 wt% to about 25 wt%, about 3 wt% to about 20 wt%, about 4 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 6 wt% to about 7 wt%, or about 6 wt%, based on the total weight of the pet food composition, and in certain embodiments, the spinach is presentAttorney Docket No.: 14405-00-WO-01-HL in the pet food composition in an amount ranging from about 1 wt% to about 30 wt%, such as from about 2 wt% to about 25 wt%, about 5 wt% to about 20 wt%, about 6 wt% to about 15 wt%, about 8 wt% to about 12 wt%, about 9 wt% to about 11 wt%, or about 10 wt%, based on the total weight of the pet food composition.

[0013] In certain embodiments, the at least two omega-3 polyunsaturated fatty acids are selected from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and in certain embodiments, the pet food composition comprises fish oil comprising EPA and DHA. According to certain embodiments, the fish oil is present in the pet food composition in an amount ranging from about 0.5 wt% to about 10 wt%, about 1 wt% to about 5 wt%, about 2 wt% to about 4 wt%, about 2.5 wt% to about 3.5 wt%, or about 3 wt%, based on the total weight of the pet food composition.

[0014] In certain embodiments, there is disclosed a pet food composition comprising scFOS, betaine, carnitine, tryptophan, citrus pulp, spinach, broccoli, and fish oil.

[0015] In certain embodiments, the pet food composition disclosed herein is in the form of a supplement.

[0016] Also disclosed herein are methods of treating or preventing anxiety in a companion animal comprising administering to the companion animal an effective amount of the pet food composition disclosed herein. In certain embodiments, disclosed herein are methods of reducing one or more anxiety biomarkers in a companion animal comprising administering to the companion animal an effective amount of the pet food composition disclosed herein. In certain embodiments, the one or more anxiety biomarkers are selected from pantothenate, acetylcarnitine (C2), N-acetylphenylalanine, isobutyrlcarnitine (C4), beta-hydroxyisovalerate, triglycarnitine (C5:1-DC), 3-hydroxy-3-methylglutarate, beta-hydroxyisovaleroylcarnitine, 4- hydroxyphenylacetate, 5-hydroxymethyl-2-furoic acid, glycerophosphorylcholine (GPC), 2- aminoadipate, 3-hydroxykynurenine, guanidinosuccinate, malonylcarnitine, xanthurenate, N- acetylisoleucine, N-formylmethionine, 2-hydroxyphenylacetate, gamma-CEHC sulfate, pyridoxal, N2,N6-diacetyllysine, N-acetylglutamine, alpha-CEHC glycine, and / or glutamate.

[0017] In certain aspects of the method of reducing one or more anxiety biomarkers, the method comprises first obtaining a biological sample from the companion animal and measuring the one or more biomarkers after and optionally before administering the pet food composition to the companion animal.Attorney Docket No.: 14405-00-WO-01-HL

[0018] Further embodiments of the present disclosure provide a method of treating or preventing inflammation or an allergy in a companion animal comprising administering to the companion animal an effective amount of the pet food composition disclosed herein. Also disclosed herein is a method of reducing levels of at least one inflammatory cytokine in a companion animal comprising administering to the companion animal an effective amount of the pet food composition disclosed herein, wherein the at least one inflammatory cytokine is selected from MCP-1, IL-6, TNF-alpha, IL-15, IL-7, and IL-18. In certain embodiments, the method further comprises increasing levels of cytokine interferon-gamma (IFN-gamma) in the companion animal, and in certain embodiments, the method further comprises reducing levels of symmetrical dimethylarginine (SDMA) in the companion animal. According to certain embodiments, the method further comprises obtaining a biological sample from the companion animal and measuring the one or more inflammatory cytokines after and optionally before administering the pet food composition.

[0019] In accordance with certain embodiments, the biological sample is selected from blood, urine, or saliva, and in certain embodiments, the companion animal is a canine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The features, and advantages of the disclosure will be apparent from the following more detailed description of certain embodiments and as illustrated in the accompanying drawings in which:

[0021] FIG. 1 is a graph showing the concentration of 25 different urinary metabolites for both anxious and non-anxious dogs prior to treatment, as disclosed in Example 2.

[0022] FIG.2 is a graph showing the concentration of 25 different urinary metabolites for anxious dogs after treatment with the control and test compositions, as disclosed in Example 2.

[0023] FIG. 3 depicts a plot showing the difference in consumption time between the control and test compositions for various dogs, as disclosed in Example 3.

[0024] It should be understood that the various aspects are not limited to the compositions, arrangements, and instrumentality shown in the figure.Attorney Docket No.: 14405-00-WO-01-HL DETAILED DESCRIPTION

[0025] The following description of various embodiments is merely exemplary in nature and is in no way intended to limit the disclosure, its application, or its uses. The description of illustrative embodiments is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description. The following description of the preferred embodiment(s) is merely exemplary in nature and is in no way intended to limit the invention. The description of illustrative embodiments is intended to be read in connection with the accompanying drawings, which are to be considered part of the entire written description.

[0026] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context dictates otherwise. The singular form of any class of the ingredients refers not only to one chemical species within that class, but also to a mixture of those chemical species; for example, the term “protein” in the singular form, may refer to a mixture of compounds each of which is also considered a protein. The terms “a” (or “an”), “one or more” and “at least one” may be used interchangeably herein. The terms “comprising”, “including”, and “having” may be used interchangeably. The term “include” should be interpreted as “include, but are not limited to.” The term “including” should be interpreted as “including, but are not limited to.”

[0027] As used herein, the words “preferred” and “preferably” refer to embodiments that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.

[0028] As used herein, the term “food” may refer not only to a food product which typically provides most, if not all, the nutrient value for a companion animal, but may also refer to such items as a snack, treat, supplement, and the like.

[0029] A “nutritionally complete diet” may be a diet that may include sufficient nutrients for maintenance of normal health of a healthy animal on the diet. In certain aspects, the pet food composition(s) disclosed herein may be a nutritionally complete diet, and in certain embodiments, the pet food composition may not be a nutritionally complete diet, but may be used to supplement an animal’s otherwise nutritionally complete diet, e.g., as a treat or a supplement. In certainAttorney Docket No.: 14405-00-WO-01-HL embodiments, the pet food composition may be blended with a nutritionally complete diet and / or balanced food diet.

[0030] For example, a nutritionally complete and balanced pet (e.g., dog, cat, etc.) food composition may comprise: about 0 to about 90%, preferably about 5% to 60%, by weight of carbohydrates; about 5% to about 70%, preferably about 10% to about 60%, more preferably about 20% to about 50%, by weight of protein: about 1% to about 50%, preferably about 2% to about 40%, more preferably about 3% to about 15%, by weight of fat; about 0.1% to about 40%, preferably about 1% to about 30%, more preferably about 15% to about 50%, by weight of total dietary fiber; about 0 to about 15%, preferably about 2% to about 8%, by weight of vitamins and minerals, antioxidants, and other nutrients which support the nutritional needs of the animal.

[0031] Unless stated otherwise, all percentages of composition components given in this specification are by weight based on a total composition or formulation weight of 100%.

[0032] The term “effective amount” as used herein means that the amount of the composition may be of sufficient quantity to achieve the intended purpose, such as, for example, to induce or cause increased food intake in the subject. Such effective activity may be achieved, for example, by administration of compositions of the present disclosure to an animal. An effective amount may be based on several factors, including an animal’s ideal weight, the metabolizable energy of the composition, and frequency of feeding the animal one or more compositions of the present disclosure, e.g., once, twice, or three times daily, and other compositions fed to the animal.

[0033] The compositions and formulations as provided herein may be described and claimed with reference to their ingredients, as is usual in the art. As would be evident to one skilled in the art, the ingredients may in some instances react with one another, so that the true composition of the final formulation may not correspond exactly to the ingredients listed. Thus, it should be understood that the compositions of the present disclosure extend to the product of the combination of the listed ingredients.

[0034] The phrase “companion animal” refers to a domesticated or domestic-bred animal whose physical, emotional, behavioral and social needs can be readily met as companions in a home, or in close daily relationship with one or more humans. Under one embodiment, species included in the definition of a companion animal include dogs, canines, cats, felines, horses, rabbits, ferrets, guinea pigs, and select other small mammals. Under another embodiment, species included in theAttorney Docket No.: 14405-00-WO-01-HL definition of companion animal include dogs, cats, horses, rabbits, ferrets, guinea pigs, select other small mammals, birds, small reptiles, fish, and domestic-bred animals.

[0035] The definition of the term “dog” includes a companion dog, a guard dog, a hunting dog, a herding dog, and a working dog.

[0036] Any member in a list of species that are used to exemplify or define a genus may be mutually different from, or overlapping with, or a subset of, or equivalent to, or nearly the same as, or identical to any other member of the list of species. Further, unless explicitly stated, such as when reciting a Markush group, the list of species that define or exemplify the genus is open, and it is given that other species may exist that define or exemplify the genus just as well as, or better than, any other species listed.

[0037] As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. In addition, all references cited herein are hereby incorporated by reference in their entireties. In the event of a conflict in a definition in the present disclosure and that of a cited reference, the present disclosure controls.

[0038] Unless otherwise defined, all technical and scientific terms and associated acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Further, it should be understood that the present disclosure is not limited to any particular method, protocol, and reagent described herein. The described methods, protocols, and reagents are merely examples and for illustration purposes only. Pet food compositions

[0039] Disclosed herein is a pet food composition that may be used, inter alia, in a method of improving an immune response in companion animals, wherein the addition of certain components, e.g., one or more of short-chain fructooligosaccharides (scFOS), betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp, and / or fish oil may reduce inflammation. The immune response improvement may be achieved by feeding the animal the pet food compositions disclosed herein. Further disclosed herein is a pet food composition that may be used, inter alia, in a method of treating or reducing anxiety in companion animals, wherein the addition of certain components, e.g., one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp, and / or fish oil may reduce anxiety or reduce symptoms of anxiety in the companion animal. The pet food compositions disclosed herein may further have enhanced palatability, including enhancedAttorney Docket No.: 14405-00-WO-01-HL palatability over a pet food composition that does not include the addition of one or more of the following components: short-chain fructooligosaccharides, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp, and / or fish oil.

[0040] In certain embodiments, the pet food composition disclosed herein comprises fermentable fibers. “Fermentable fibers” may be plant parts or carbohydrates resistant to digestion and absorption in the small intestine and may be fermented by colonic microorganisms. Examples of fermentable fibers may include simple carbohydrates such as short-chain fructo-oligosaccharides (scFOS), galacto-oligosaccharides (GOS), oligo-fructans and complex polysaccharides such as long chain inulin, beta-glucans, pectin, resistant starch as well as fruit or vegetable extracts rich in fiber, such as apple pomace, tomato pomace, cranberry pomace, beet pulp and citrus pulp.

[0041] In certain embodiments, the fermentable fibers may be selected from scFOS, GOS, oligo- fructans, and complex polysaccharides such as long chain inulin, beta-glucans, pectin, and resistant starch, as well as fruit or vegetable extracts rich in fiber, such as apple pomace, tomato pomace, cranberry pomace, beet pulp, and citrus pulp. In certain embodiments, the fermentable fibers may be a single species of fermentable fiber. In further embodiments, the fermentable fibers may be mixed. In certain embodiments, the composition may include 0.05 wt% to 50 wt% of the fermentable fibers, based on the total weight of the composition. In certain embodiments, the composition may include 5 wt% to 45 wt% of the fermentable fibers, based on the total weight of the composition.

[0042] In certain embodiments, the pet food composition disclosed herein comprises at least one fermentable fiber, and in certain embodiments, the pet food composition disclosed herein comprises at least two or at least three fermentable fibers. In certain embodiments, the fermentable fibers comprise scFOS, and in certain embodiments, the fermentable fibers comprise citrus pulp. In certain embodiments, the fermentable fibers comprise a mixture of scFOS and citrus pulp.

[0043] In embodiments of the disclosure wherein the fermentable fiber comprises scFOS, the scFOS may be present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 5 wt% to about 15 wt%, about 8 wt% to about 12 wt%, or about 10 wt%, based on the total weight of the pet food composition. In certain embodiments, scFOS may be present in the pet food composition in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, orAttorney Docket No.: 14405-00-WO-01-HL about 15 wt%, based on the total weight of the pet food composition. In embodiments of the disclosure wherein the fermentable fiber comprises citrus pulp, the citrus pulp may be present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 2 wt% to about 12 wt%, about 5 wt% to about 10 wt%, or about 7 wt%, based on the total weight of the pet food composition. In certain embodiments, citrus pulp may be present in the pet food composition in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, or about 15 wt%, based on the total weight of the pet food composition. In certain embodiments, the fermentable fiber comprises a mixture of scFOS and citrus pulp, and the mixture is present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 2 wt% to about 20 wt%, about 12 wt% to about 18 wt%, or about 17 wt%, based on the total weight of the pet food composition. In certain embodiments, mixture of scFOS and citrus pulp may be present in the pet food composition in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, or about 25 wt%, based on the total weight of the pet food composition.

[0044] In certain embodiments of the disclosure, the pet food composition comprises glycine betaine. Glycine betaine, or simply “betaine,” is a trimethylglycine or an adduct thereof. Glycine betaine is also known as trimethyl glycine, 2-trimethylammonioacetate, or N,N,N- trimethylglycine, and may be abbreviated as TGM. Glycine betaine is a derivative of the amino acid glycine. Glycine betaine is an N-trimethylated amino acid, wherein the quaternary ammonium exists as the zwitterion at neutral pH. Glycine betaine has the formula (CH3)3N+–CH2–COO-.

[0045] Glycine betaine also includes an adduct of the compound glycine betaine, such as a hydrate, acid, or salt thereof. Examples of hydrates include monohydrate, dihydrate, trihydrate, and the like.

[0046] In certain embodiments, the pet food compositions disclosed herein may comprise betaine in an amount ranging from about 1 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 2 wt% to about 15 wt%, about 8 wt% to about 12 wt%, or about 10 wt%, based on the total weight of the pet food composition. In certain embodiments, betaine may be present in theAttorney Docket No.: 14405-00-WO-01-HL pet food composition in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, or about 15 wt%, based on the total weight of the pet food composition.

[0047] In certain embodiments, the pet food composition disclosed herein may comprise carnitine. Carnitine is β-hydroxy-γ-N-trimethylaminobutyric acid, or 3-hydroxy-4-N,N,N- trimethylaminobutyrate, and has the formula Me3N+–CH2–CHOH–CH2–COO-. Carnitine is a quaternary ammonium compound involved in metabolism in most mammals, plants, and some bacteria. Under one embodiment, carnitine as used in the present disclosure is the L-enantiomer.

[0048] Acetyl-L-carnitine is an acylated form of L-carnitine and has the formula Me3N+–CH2– C*H(O–C(=O)-Me–CH2–COO-. Propionyl-L-carnitine, under certain embodiments, is equivalent to one or more of the following: 3-propanoyloxy-4-(trimethylazaniumyl) butanoate, 3-(1-oxo- propoxy)-4-trimethyl ammonium chloride butanoic acid, glycine propionyl-L-carnitine, L- carnitine propionyl, LPC, PLC, GPLC, and propionylcarnitine.

[0049] In certain embodiments of the disclosure, carnitine may be present in the pet food composition in an amount ranging from about 0.001 wt% to about 5 wt%, such as from about 0.01 wt% to about 2 wt%, about 0.1 wt% to about 1 wt%, about 0.2 wt% to about 0.9 wt%, or about 0.8 wt%, based on the total weight of the pet food composition. In certain embodiments, carnitine may be present in the pet food composition in an amount of about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, or about 1.5 wt%, based on the total weight of the pet food composition.

[0050] In certain embodiments of the disclosure, the pet food composition may comprise tryptophan. Tryptophan is an indispensable amino acid that may to be supplied by dietary protein. Apart from its incorporation into body proteins, tryptophan is the precursor for serotonin, an important neuromediator, and for kynurenine, an intermediary metabolite of a complex metabolic pathway ending with niacin, CO2, and kynurenic and xanthurenic acids. Tryptophan metabolism within different tissues is associated with numerous physiological functions. The liver regulates tryptophan homeostasis through degrading tryptophan in excess. Tryptophan degradation into kynurenine by immune cells plays a role in the regulation of immune response during infections,Attorney Docket No.: 14405-00-WO-01-HL inflammations and pregnancy. Serotonin is synthesized from tryptophan in the gut and also in the brain, where tryptophan availability is known to influence the sensitivity to mood disorders.

[0051] In certain embodiments, tryptophan may be present in the pet food composition disclosed herein in an amount ranging from about 0.001 wt% to about 5 wt%, such as from about 0.01 wt% to about 2 wt%, about 0.1 wt% to about 1.5 wt%, about 0.2 wt% to about 1.2 wt%, or about 1 wt%, based on the total weight of the pet food composition. In certain embodiments, tryptophan may be present in the pet food composition in an amount of about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, or about 1.5 wt%, based on the total weight of the pet food composition.

[0052] In certain embodiments of the disclosure, the pet food composition may comprise at least one phytochemical. Phytochemicals, also known as phytonutrients, can include, for example, carotenoids and polyphenols. Phytochemicals are biologically active compounds found in various plants, including fruit and vegetable plants, such as citrus, spinach and broccoli plants, and extracts thereof. The biological activity of phytochemicals may include, for example, DNA damage protection, antioxidant activity, hormonal regulation, immune function (e.g., anti-inflammatory properties), and / or antimicrobial properties. In certain embodiments, the pet food compositions disclosed herein comprise a fruit or vegetable plant, or extracts of a fruit or vegetable plant, comprising phytochemicals. In certain embodiments, the phytochemicals are selected from tomato, beet, carrots, peppers, squash, sweet potatoes, fruits (e.g., peaches, mangoes, melons, berries, citrus fruits), and dark leafy vegetable such as spinach, broccoli, kale, bok choy, chard, and lettuce. In certain embodiments, the pet food composition disclosed herein comprises at least one source of a phytochemical selected from broccoli, spinach, citrus pulp, or extracts thereof.

[0053] In certain embodiments of the disclosure, the pet food composition may comprise spinach (e.g., spinach powder) comprising phytochemicals. Spinach may be present in the pet food compositions disclosed herein in an amount ranging from about 1 wt% to about 30 wt%, such as from about 2 wt% to about 25 wt%, about 5 wt% to about 20 wt%, about 6 wt% to about 15 wt%, about 8 wt% to about 12 wt%, about 9 wt% to about 11 wt%, or about 10 wt%, based on the total weight of the pet food composition. In certain embodiments, spinach is present in the pet food composition in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12Attorney Docket No.: 14405-00-WO-01-HL wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, or about 20 wt%, based on the total weight of the pet food composition.

[0054] In embodiments of the disclosure, the pet food composition may comprise citrus pulp comprising phytochemicals. The citrus pulp may be present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 2 wt% to about 12 wt%, about 5 wt% to about 10 wt%, or about 7 wt%, based on the total weight of the pet food composition. In certain embodiments, citrus pulp may be present in the pet food composition in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, or about 15 wt%, based on the total weight of the pet food composition.

[0055] In certain embodiments of the disclosure, the pet food composition may comprise broccoli (e.g., broccoli powder) comprising phytochemicals. Broccoli may be present in the pet food compositions disclosed herein in an amount ranging from about 1 wt% to about 30 wt%, such as from about 2 wt% to about 25 wt%, about 3 wt% to about 20 wt%, about 4 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 6 wt% to about 7 wt%, or about 6 wt%, based on the total weight of the pet food composition. In certain embodiments, broccoli is present in the pet food composition in an amount of about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 6 wt%, about 7 wt%, about 8 wt%, about 9 wt%, about 10 wt%, about 11 wt%, about 12 wt%, about 13 wt%, about 14 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, or about 20 wt%, based on the total weight of the pet food composition.

[0056] In certain embodiments, the pet food composition comprises at least one fruit or vegetable or extract thereof comprising phytochemicals. In certain embodiments, the total amount of fruit or vegetable or extract thereof comprising phytochemicals present in the pet food composition ranges from about 1 wt% to about 40 wt%, such as from about 5 wt% to about 30 wt%, about 10 wt% to about 28 wt%, about 15 wt% to about 25 wt%, or about 20 wt% to about 25 wt%, based on the total weight of the pet food composition. In certain embodiments, the at least one fruit or vegetable or extract thereof comprising phytochemicals is present in the pet food composition in an amount of about 5 wt%, about 10 wt%, about 15 wt%, about 16 wt%, about 17 wt%, about 18 wt%, about 19 wt%, about 20 wt%, about 21 wt%, about 22 wt%, about 23 wt%, about 24 wt%, about 25 wt%,Attorney Docket No.: 14405-00-WO-01-HL about 26 wt%, about 27 wt%, about 28 wt%, about 29 wt%, or about 30 wt%, based on the total weight of the pet food composition.

[0057] In one aspect, the present disclosure provides pet food compositions comprising at least two omega-3 polyunsaturated fatty acids. The at least two omega-3 polyunsaturated fatty acids typically have at least one aliphatic tail of 14 or more carbons. In some instances, the aliphatic tail of the at least two omega-3 polyunsaturated fatty acids may have at least one tail of 16 or more carbons, 17 or more carbons, 18 or more carbons, or 20 or more carbons. For example, the at least two omega-3 polyunsaturated fatty acid may have at least one tail of 16 to 24 carbons, 17 to 24 carbons, 18 to 24 carbons, 19 to 24 carbons, 20 to 24 carbons; 16 to 23 carbons, 17 to 23 carbons, 18 to 23 carbons, 19 to 23 carbons, 20 to 23 carbons; 16 to 22 carbons, 17 to 22 carbons, 18 to 22 carbons, 19 to 22 carbons, or 20 to 22 carbons. Preferably, the pet food composition comprises at least two omega-3 polyunsaturated fatty acids, wherein each omega-3 polyunsaturated fatty acids has at least one aliphatic tail of 18 to 22 carbons. In at least one embodiment, each of the at least two omega-3 polyunsaturated fatty acids comprise an unbranched aliphatic tail having at least 20 carbons.

[0058] The at least two omega-3 polyunsaturated fatty acid may comprise linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid, docosahexaenoic acid, or a combination of two or more thereof. In some cases, the at least two omega-3 polyunsaturated fatty acids may be selected from eicosapentaenoic acid, docosahexaenoic acid, linolenic acid, and a combination of two or more thereof. In certain embodiments, the at least two omega-3 polyunsaturated fatty acids comprise eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

[0059] Various omega-3 polyunsaturated fatty acid are useful within embodiments of the disclosure. In certain embodiments, such fatty acids are long chain fatty acids having at least 20 carbons. In certain embodiments, the omega-3 polyunsaturated fatty acids are selected from eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and mixtures thereof. Marine-based fish and fish oil are well-known sources of n-3 polyunsaturated fatty acids (PUFAs), namely, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In certain embodiments, the fatty acids are derived from fish oil. Although the omega-3 polyunsaturated fatty acid may be obtained from an animal source, the omega-3 polyunsaturated fatty acid may be obtained from a plant source in some embodiments. Examples of plant sources for deriving or obtaining the fatty acids include,Attorney Docket No.: 14405-00-WO-01-HL e.g., flaxseed, algae, avocado, hemp seeds, pumpkin seeds, sunflower seeds, walnuts, soya, or combinations of two or more thereof.

[0060] The at least two omega-3 polyunsaturated fatty acids may be present in the pet food composition in an amount of from about 0.01 wt % to about 10 wt%, about 0.05 wt% to about 5 wt%, about 0.1 wt% to about 3 wt%, about 0.5 to about 1.5 weight %, or about 0.8 wt% to about 1.2 wt%, based on the total weight of the pet food composition. For example, the at least two omega-3 polyunsaturated fatty acids may be present in an amount of about 0.01 wt%, about 0.1 wt%, about 0.2 wt%, about 0.3 wt%, about 0.4 wt%, about 0.5 wt%, about 0.6 wt%, about 0.7 wt%, about 0.8 wt%, about 0.9 wt%, about 1 wt%, about 1.1 wt%, about 1.2 wt%, about 1.3 wt%, about 1.4 wt%, about 1.5 wt%, or any range or subrange therebetween, based on the total weight of the pet food composition. In another example, the pet food composition may comprise fish oil comprising the at least two omega-3 polyunsaturated fatty acids, and the fish oil may be present in the pet food composition in an amount ranging from about 0.5 wt% to about 10 wt%, about 1 wt% to about 5 wt%, about 2 wt% to about 4 wt%, or about 2.5 wt% to about 3.5 wt%, based on the total weight of the pet food composition. In another example, the fish oil may be present in an amount ranging from about 0.5 wt%, about 1 wt%, about 1.5 wt%, about 2 wt%, about 2.5 wt%, about 3 wt%, about 3.5 wt%, about 4 wt%, about 4.5 wt%, or about 5 wt%, based on the total weight of the pet food composition.

[0061] In some cases, the pet food composition has a weight ratio of eicosapentaenoic acid and docosahexaenoic acid (EPA:DHA) of from about 1:5 to about 5:1, about 1:4 to about 5:1, about 1:3 to about 5:1, about 1:2 to about 5:1, about 1:1 to about 5:1; about 2.5:5 to about 5:1, about 2.5:4 to about 5:1, about 2.5:3 to about 5:1; about 1:5 to about 2.5:1, about 1:4 to about 2.5:1, about 1:3 to about 2.5:1, about 1:2 to about 2.5:1, about 1:1 to about 2.5:1; about 1:5 to about 4:1, about 1:4 to about 3:1, about 1:3 to about 2:1, about 1:4 to about 1:1.

[0062] In addition to the one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp, and / or fish oil (e.g., DHA and EPA) as discussed above, or as part of the one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp, and / or fish oil (e.g., DHA and EPA) ingredients, the pet food compositions may contain protein, fat, carbohydrate, dietary fiber, and / or nutritional balancing agents, e.g., additives, minerals, vitamins, amino acids, carotenoids, antioxidants, fatty acids, glucose mimetics, probiotics, prebiotics, and others. Specific suitable amounts for each component in a composition will depend on a variety of factors, such asAttorney Docket No.: 14405-00-WO-01-HL the species of animal consuming the composition, the particular components included in the composition, the age, weight, general health, sex, and diet of the animal, the animal’s consumption rate, and the like. Thus, the component amounts may vary widely and in certain instances may deviate from the proportions set forth herein.

[0063] Protein may be supplied by any of a variety of sources known by those skilled in the art, including plant sources, animal sources, or both. Animal sources may include, for example, meat, meat by-products, seafood, dairy and eggs. Meats may include, for example, the flesh of poultry, fish, and mammals (e.g., cattle, pigs, sheep, goats, and the like). Meat by-products may include, for example, lungs, kidneys, brain, livers, and stomachs and intestines (freed of all or essentially all their contents). The protein can be intact, almost completely hydrolyzed, or partially hydrolyzed. The amount of “crude protein” in a composition disclosed herein may be determined based on the amount of nitrogen in the composition according to methods familiar to one of skill in the art. As contemplated herein, the pet food compositions may comprise from about 5 wt% to about 70 wt% crude protein, from about 10 wt% to about 60 wt% crude protein, from about 20 wt% to about 50 wt% crude protein, from about 25 wt% to about 40 wt% crude protein, or about 25 wt% crude protein. In certain embodiments, the pet food composition may comprise more than about 16 wt% crude protein, such as more than about 17 wt%, more than about 18 wt%, more than about 19 wt%, more than about 20 wt%, more than about 21 wt%, more than about 22 wt%, more than about 23 wt%, or more than about 24 wt% crude protein, based on the total weight of the pet food composition.

[0064] In certain embodiments, the pet food compositions disclosed herein may comprise fat. Sources of fat for the compositions can be supplied by any of a variety of sources known by those skilled in the art, including meat, meat by-products, fish oil, and plants. Plant fat sources may include wheat, flaxseed, rye, barley, rice, sorghum, corn, oats, millet, wheat germ, corn germ, soybeans, peanuts, and cottonseed, as well as oils derived from these and other plant fat sources. As contemplated herein, the pet food compositions may comprise from about 1 wt% to about 20 wt% fat, from about 2 wt% to about 18 wt% fat, from about 3 wt% to about 17 wt% fat, from about 10 wt% to about 15 wt%, from about 7 wt% to about 14 wt% fat, or from about 9 wt% to about 12% fat. In certain embodiments, the pet food composition may comprise more than about 10 wt% fat, such as more than about 11 wt%, more than about 12 wt%, more than about 13 wt%, or more than about 14 wt% fat, based on the total weight of the pet food composition.Attorney Docket No.: 14405-00-WO-01-HL

[0065] In certain embodiments, the pet food compositions disclosed herein may comprise fat and carbohydrate. The fat and carbohydrate food ingredient may be obtained from a variety of sources such as animal fat, fish oil, vegetable oil, meat, meat by-products, grains, other animal or plant sources, and mixtures thereof. Grains may include wheat, corn, barley, and rice.

[0066] In certain embodiments, the pet food compositions disclosed herein may comprise fiber. The fiber food ingredient may be obtained from a variety of sources such as vegetable fiber sources, e.g., cellulose, beet pulp, peanut hulls, and soy fiber.

[0067] The compositions may further contain additives known in the art. Preferably, such additives may be present in amounts that do not impair the purposes and effects disclosed herein. Examples of contemplated additives may include, for example, substances that may be functionally beneficial to weight management, substances with a stabilizing effect, processing aids, substances that enhance palatability, coloring substances, and substances that provide nutritional benefits.

[0068] Contemplated substances that may provide a benefit for weight management may include, for example, nonfermentable fiber, carnitine, chrominium-picolinate, and the like.

[0069] Contemplated stabilizing substances may include, for example, substances that tend to increase the shelf life of the composition. Potentially suitable examples of such substances may include, for example, preservatives, antioxidants, synergists and sequestrants, packaging gases, stabilizers, emulsifiers, thickeners, gelling agents, and humectants. Examples of emulsifiers and / or thickening agents may include, for example, gelatin, cellulose ethers, starch, starch esters, starch ethers, and modified starches.

[0070] Contemplated additives for coloring, palatability, and nutritional purposes may include, for example, colorants; iron oxide, sodium chloride, potassium citrate, potassium chloride, and other edible salts; vitamins; minerals; and flavoring. The amount of such additives in a pet food composition typically may be up to about 5 wt%, based on the total weight of the pet food composition. Other additives can include antioxidants, omega-3 fatty acids, omega-6 fatty acids, glucosamine, chondroitin sulfate, vegetable extracts, herbal extracts, etc.

[0071] In certain embodiments, the pet food composition comprises vitamins and minerals in amounts required to avoid deficiency and maintain health. These amounts are readily available in the art. The Association of American Feed Control Officials (AAFCO) provides recommended amounts of such ingredients for dogs and cats (see Association of American Feed ControlAttorney Docket No.: 14405-00-WO-01-HL Officials. Official Publication, pp.126-140 (2003)). Vitamins could as an example include vitamin A, vitamin B1 (thiamine or related sources such as thiamine mononitrate), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid or related sources such as calcium pantothenate), vitamin B6 (pyridoxine or related sources such as pyridoxine hydrochloride), vitamin B8 (folic acid), vitamin B12, vitamin C (ascorbic acid), vitamin D (such as a vitamin D3 supplements), vitamin E, vitamin H (biotin), vitamin K, acetate, choline and choline related sources such as choline chloride, and inositol. Minerals and trace elements could as an example include calcium, phosphorus, sodium, potassium, magnesium, copper, zinc, choline, and iron salts. Mineral sources can include, for example, sodium selenite, monosodium phosphate, calcium carbonate, potassium chloride, ferrous sulfate, zinc oxide, manganese sulfate, copper sulfate, manganous oxide, potassium iodide, and / or cobalt carbonate.

[0072] Examples of fatty acid ingredients, in addition to or as part of the at least two omega-3 fatty acids discussed above, may comprise arachidonic acid, alpha-linolenic acid, gamma linolenic acid, linoleic acid, EPA, DHA, and / or fish oils as a source of EPA and / or DHA. Sources of glucose mimetics may comprise glucose anti-metabolites including 2-deoxy D-glucose, 5-thio-D-glucose, 3-O-methylglucose, anhydrosugars including 1,5-anhydro-D-glucitol, 2,5-anhydro-D-glucitol, and 2,5-anhydro-D-mannitol, mannoheptulose, and / or avocado extract comprising mannoheptulose.

[0073] Still other ingredients may include beef broth, brewers dried yeast, egg, egg product, flax meal, DL methionine, amino acids, leucine, lysine, arginine, cysteine, cystine, aspartic acid, polyphosphates, sodium pyrophosphate, sodium tripolyphosphate; zinc chloride, copper gluconate, stannous chloride, stannous fluoride, sodium fluoride, triclosan, glucosamine hydrochloride, chondroitin sulfate, green lipped mussel, blue lipped mussel, methyl sulfonyl methane (MSM), boron, boric acid, phytoestrogens, phytoandrogens, genistein, diadzein, Lcarnitine, chromium picolinate, chromium tripicolinate, chromium nicotinate, acid / base modifiers, potassium citrate, potassium chloride, calcium carbonate, calcium chloride, sodium bisulfate; eucalyptus, lavender, peppermint, plasticizers, colorants, flavorants, sweeteners, buffering agents, slip aids, carriers, pH adjusting agents, natural ingredients, stabilizers, biological additives such as enzymes (including proteases and lipases), chemical additives, coolants, chelants, denaturants, drug astringents, emulsifiers, external analgesics, fragrance compounds, humectants, opacifying agents (such as zinc oxide and titanium dioxide), antifoaming agents (such as silicone),Attorney Docket No.: 14405-00-WO-01-HL preservatives (such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), propyl gallate, benzalkonium chloride, EDTA, benzyl alcohol, potassium sorbate, parabens and mixtures thereof), reducing agents, solvents, hydrotropes, solubilizing agents, suspending agents (non-surfactant), solvents, viscosity increasing agents (aqueous and non-aqueous), sequestrants, and / or keratolytics.

[0074] The probiotic component may comprise any suitable bacteria, yeast, microorganisms, and / or mixtures of any thereof. Various probiotic microorganisms are known in the art. In certain embodiments, the probiotic component may comprise bacteria of the order Lactobacillales; bacteria of the genus Bacillus, Bacteroides, and / or Bifidobacterium; yeast of the order Saccharomycetales including the genus Saccharomyces and Candida; and / or mixtures of any thereof. The probiotic may or may not form a spore.

[0075] In certain embodiments, the pet food composition may include polyphenols. In some embodiments, the polyphenol source comprises a phenolic compound selected from ellagic acid; gallic acid; protocatechuic acid; p-hydroxybenzoic acid; catechin; and a combination of two or more thereof. In some embodiments, the polyphenol source comprises pecan shells, or any other component of the pecan nut. Examples of further sources of polyphenols may comprise tea extract, rosemary extract, rosemarinic acid, coffee extract, pecan shells, caffeic acid, turmeric extract, blueberry extract, grape extract, grapeseed extract, and / or soy extract.

[0076] The pet food composition may be determined by any of the variety of methods for feed analysis known by one skilled in the art. Feed analysis may be done to measure any of the nutritional content listed herein including moisture, protein, fiber, carbohydrate, energy, vitamin, mineral, energy, fat, and ash content.

[0077] Protein content may be measured and reported in any of the variety of methods known to one skilled in the art. Protein may be reported as crude protein (CP) to measure both true protein content and non-protein nitrogen. Crude protein content may be further differentiated between degradable intake protein (DIP), undegradable intake protein (UIP) and metabolizeable protein (MP). In certain embodiments, protein content may be differentiated to include heat damaged protein or insoluble crude protein (ICP), adjusted crude protein (ACP), and digestible protein (DP).

[0078] Fiber content may be measured and reported in any of the variety of methods known to one skilled in the art. Fiber content may be reported as total dietary fiber (TDF, a combination of soluble and insoluble fiber) crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiberAttorney Docket No.: 14405-00-WO-01-HL (ADF) and / or acid detergent lignin (ADL). Crude fiber is generally known to estimate the indigestible portion of plant material found in pet food compositions. ADF measures cellulose and lignin, components of plant cell walls. NDF measures the total material found in plant cell walls and includes hemicellulose in addition to the fiber content measured as ADF. ADL measures only the lignin portion of a plant cell wall.

[0079] Energy content may be measured and reported in any of the variety of methods known to one skilled in the art. Energy content may be reported as digestible energy (DE), metabolizable energy (ME), net energy (NE), total digestible nutrient (TDN), ether extract (EE), relative feed value (RFV), and relative forage quality (RFQ).

[0080] As disclosed herein, in certain embodiments, the pet food composition may be in the form of a supplement. Supplements may include, for example, a feed used with another feed to improve the nutritive balance or performance of the total food intake of an animal. Contemplated supplements may include compositions that may be fed undiluted as a supplement to other feeds, offered free choice with other parts of an animal’s diet that may be separately available (e.g., such as an edible treat), or diluted and mixed with an animal’s regular feed to produce a complete feed. The Association of American Feed Control Officials (AAFCO), for example, provides a discussion relating to supplements in the American Feed Control Officials, Inc. Official Publication, p. 220 (2003). Supplements may be in various forms including, for example, powders, liquids, syrups, pills, etc. In the certain embodiments, the supplement comprising the pet food composition disclosed herein may constitute at least about 1% of the total daily diet of the animal, such as at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% of the animal’s total daily diet. In certain embodiments, the supplement comprising the pet food composition disclosed herein may constitute from about 1% to about 20%, such as from about 2% to about 18%, from about 5% to about 15%, from about 8% to about 12%, from about 9% to about 11%, or about 10% of the animal’s total daily diet. Methods of Making a Pet Food Composition

[0081] The methods to manufacture a pet food composition including one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) may be provided. In certain embodiments, the pet food composition may be in solid or liquid form.Attorney Docket No.: 14405-00-WO-01-HL In certain embodiments, the pet food composition may be in wet form, such as a wet loaf, or in a dry form, such a kibble. In certain embodiments, the moisture content of the pet food compositions disclosed herein may range from less than about 1 wt% to more than about 90 wt%, such as from about 1 wt% to about 50 wt%, from about 5 wt% to about 25 wt%, from about 7 wt% to about 15 wt%, from about 8 wt% to about 12 wt%, or from about 9 wt% to about 10 wt%, based on the total weight of the pet food composition.

[0082] Pet food compositions disclosed herein may be prepared in a canned or wet form using conventional pet food processes. In one contemplated embodiment, ground animal and poultry proteinaceous tissues may be mixed with the other ingredients, including fish oils, cereal grains, other nutritionally balancing ingredients, special purpose additives (e.g., vitamin and mineral mixtures, inorganic salts, cellulose and beet pulp, bulking agents, and the like); and water sufficient for processing may also be added. These ingredients may preferably be mixed in a vessel suitable for heating while blending the components. Heating of the mixture may be effected using any suitable manner, such as, for example, by direct steam injection or by using a vessel fitted with a heat exchanger. Following the addition of the last ingredient, the mixture may be heated to a temperature range of from about 50 °F to about 212 °F. Temperatures outside this range may be acceptable, but may be commercially impractical without the use of other processing aids. When heated to the appropriate temperature, the material will typically be in the form of a thick liquid. The thick liquid may be filled into cans. A lid may be applied, and the container may be hermetically sealed. The sealed can may then be placed into conventional equipment designed to sterilize the contents. This may be usually accomplished by heating to temperatures of greater than about 230 °F for an appropriate time, which may be dependent on, for example, the temperature used and the composition.

[0083] Pet food compositions disclosed herein may be prepared in a dry form using conventional processes. In one contemplated embodiment, dry ingredients, including, for example, animal protein sources, plant protein sources, grains, etc., may be ground and mixed together. Moist or liquid ingredients, including fats, oils, animal protein sources, water, etc., may then be added to and mixed with the dry mix. The mixture may be then processed into kibbles or similar dry pieces. Kibble is often formed using an extrusion process in which the mixture of dry and wet ingredients may be subjected to mechanical work at a high pressure and temperature, and forced through small openings and cut off into kibble by a rotating knife. The wet kibble may then be dried andAttorney Docket No.: 14405-00-WO-01-HL optionally coated with one or more topical coatings which may include, for example, flavors, fats, oils, powders, and the like. Kibble also can be made from the dough using a baking process, rather than extrusion, wherein the dough may be placed into a mold before dry-heat processing.

[0084] In certain embodiments, the one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) may be included in animal treats. Treats may include compositions that may be given to an animal to entice the animal to eat during a non- meal time, e.g., dog bones for canines. Treats may be nutritional wherein the composition may include one or more nutrients, and may have a composition as described above for food. Non- nutritional treats encompass any other treats that may be nontoxic. Any one of the scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) may be coated onto the treat, incorporated into the treat, or both.

[0085] In certain embodiments, the one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) may be included in animal toys. Toys may include chewable toys such as artificial bones. The one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) can form a coating on the surface of the toy or on the surface of a component of the toy, be incorporated partially or fully throughout the toy, or both. In one embodiment, the one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) may be orally accessible by the intended user. There are a wide range of suitable toys currently marketed, e.g., U.S. Pat. No. 5,339,771, U.S. Pat. No. 5,419,283, and references disclosed therein. The composition may be provided as partially consumable toys, e.g., toys including plastic components, or fully consumable toys, e.g., rawhides and various artificial bones. Further, the composition may be provided as toys for both human and non-human use, particularly for companion, farm, and zoo animal use, and particularly for dog, cat, or bird use.

[0086] In preparing the compositions, the components may be adjusted so that the total amount of the one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) may be present in the pet food composition at a concentration of at least 10 wt%, preferably from about 20 wt% to about 80 wt%, most preferably from about 40 wt% to about 60 wt%, based on the total weight of the composition. The one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) may be incorporated into the composition during the processing of the formulation, such as during and / orAttorney Docket No.: 14405-00-WO-01-HL after mixing of other components of the composition. Distribution of these components into the composition may be accomplished by conventional means. Methods of Diagnosing a Disease or Condition

[0087] Disclosed herein are methods of treating or preventing anxiety in a companion animal. The methods to treat or prevent the symptoms of anxiety in a companion may include administering an effective amount of the pet food composition disclosed herein (e.g., including one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil) to the oral cavity of a companion animal in need thereof. In certain embodiments, methods to treat or prevent anxiety in a companion animal may include administering an effective amount of the pet food composition including one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) to the oral cavity of a companion animal in need thereof, wherein the companion animal may be a canine or feline. In certain embodiments, methods to treat or prevent anxiety may include administering an effective amount of the pet food composition including one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., EPA and DHA) to the oral cavity of a companion animal in need thereof, wherein the pet food composition is in the form of a supplement.

[0088] It is recognized that there is no “gold standard” by which canine anxiety may be assessed in large groups of canines, making it more difficult to assess changes in behavior over time in the absence of repeated assessments by a trained animal behaviorist. Accordingly, described herein is a novel method for assessing anxiety in canines, which assessment solves the problem of needing to perform rapid assessments of anxiety-related traits repeated in a large group of canines, as multiple assessments by a trained behaviorist may not be practical under certain conditions.

[0089] Further disclosed herein are methods of improving immune health in a companion animal. In certain embodiments, method of improving immune health may comprise methods of treating or preventing inflammation or an allergy in the companion animal. The methods to improve immune health in a companion animal may include administering an effective amount of the pet food composition disclosed herein (e.g., scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil) to the oral cavity of a companion animal in need thereof. In certain embodiments, methods to improve immune health in a companion animal may include administering an effective amount of the pet food composition including one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil to the oral cavity of aAttorney Docket No.: 14405-00-WO-01-HL companion animal in need thereof, wherein the companion animal may be a canine or feline. In certain embodiments, methods to improve immune health in a companion animal may include administering an effective amount of the pet food composition including one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil to the oral cavity of a companion animal in need thereof, wherein the pet food composition is in the form of a supplement.

[0090] In a particular embodiment, the methods to improve food intake of a companion animal may be provided. The method may include administering an effective amount of the pet food composition of any of the compositions described herein to the oral cavity of a companion animal in need thereof. In a particular embodiment, a method for improving food intake of a companion may be provided, and may include administering an effective amount of the pet food composition of any of the compositions described herein to a companion animal. In certain embodiments, the method uses a solid or liquid pet food composition. In certain embodiments, the companion animal may be a canine or feline.

[0091] In certain embodiments, there is disclosed herein a method of identifying or diagnosing a condition in an animal, such as a method of identifying or diagnosing anxiety in an animal, such as a canine or a feline. Methods disclosed herein of identifying or diagnosing a condition in an animal may comprise, inter alia, analyzing a biological sample obtained from the animal or from a control animal or animals.

[0092] As used herein, the term “biological sample” may be used interchangeably with the terms “biosample,” “sample,” “specimen,” “biomaterial” and “biological material.” A biological sample refers to any organic material obtained from a pet including bodily fluids such as blood, saliva, and urine; tissue samples such as from a biopsy or fur; and other clinical specimens such as exhaled breath condensate. A biological sample could be obtained in a noninvasive and / or invasive manner. For example, a biological sample may be provided in such noninvasive ways as via a swabbing of a mouth, a collection of fur, or a urination. In other examples, a biological sample may be provided in such invasive ways as via a taking of blood via a needle or a removal of tissue via a biopsy.

[0093] In certain embodiments, the biological sample may further comprise one or more excipients. Excipients may be added to the biological sample at any time. For example, an excipient may be added to the biological sample during collection, transportation, preparation and / or analysis of the sample.Attorney Docket No.: 14405-00-WO-01-HL

[0094] The addition of excipients are well known in the art. Such excipients should be present in amounts that do not impair the purpose and effect provided by the invention. An excipient may be included as a stabilizer, preservative, processing aid, pH buffer, bulking agent, diluent, color reagent and dye. For example, ethylenediaminetetraacetic acid (EDTA) may be added to a biological sample during collection to preserve the biological sample.

[0095] Examples of excipients may include boric acid and derivatives thereof, dimethyl sulfoxide (DMSO), ethanol, polyethylene glycol, ethylenediaminetetraacetic acid (EDTA), formic acid and derivatives thereof, protease inhibitors, sodium salts such as sodium citrate and sodium metabisulfate, and protease inhibitors.

[0096] A biological sample (e.g., blood, saliva, urine, exhaled breath condensate, tissue, etc.) may be used as a diagnostic tool in the methods disclosed herein. For example, a biological sample (such as a biofluid) may be used as a diagnostic tool due to its ability to correlate to a health status and / or condition of an animal (such as a companion animal or a person). The health condition may relate to a disease, disorder, or other condition, such as, for example, anxiety or immune health (e.g., inflammation or allergy). For example, a biological sample may be used to diagnose and / or determine existence of a disease or condition, an onset of a disease or condition, progression of the disease or condition, and / or treatment progress of the disease or condition. A biological sample may be used as a diagnostic fluid in a noninvasive and / or invasive manner. For example, in some examples a biological sample may be provided in such noninvasive ways as via a swabbing of a mouth, a spitting of saliva from the mouth, or a urination. In other examples a biological sample may be provided in such invasive ways as via a taking of blood via a needle, a removal of tissue, etc. Detection (e.g., rapid detection) of antibodies in biological sample (such as saliva) may be performed rapidly and via compact tools (such as via a toothbrush, mouthguard, patch, etc.). The detection of a disease, disorder, or other condition via a biological sample may enable point-of- care diagnosis for diseases, disorders, or other conditions. Although the disclosure may provide many examples relating to biological sample, it should be understood that such examples are for illustration purposes only. The examples may extend to any biological sample, including a biofluid as well as a biological tissue, for example.

[0097] Methods to quantify one or more biomarkers within a biological sample are well known in the art (e.g. colorimetric reporting, nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy, mass spectrometry, etc.). Although the disclosure may provide many examplesAttorney Docket No.: 14405-00-WO-01-HL relating to blood, saliva, or urine, it should be understood that such examples are for illustration purposes only. The examples may extend to any biological sample, including a bodily fluid or biological tissue.

[0098] As used herein, the term “biomarker” may be used interchangeably with “biological marker” and is used to refer to any measurable substance that could be used to examine organ function or any other biological state or condition. In certain embodiments, a biomarker may include a protein such as an immunoglobulin, a polynucleotide such as DNA and RNA, and a metabolite. In certain embodiments, a biomarker may be a cytokine including, but not limited to, a colony stimulating factor (CSF), interferon (IFN), interleukin (IL), and tumor necrosis factor (TNF). In certain embodiments, the biomarker is a blood cell, such as any combination, amount, or ratio of neutrophils, lymphocytes, basophils, and / or eosinophils. In certain embodiments, the biomarker is at least one cytokine selected from MCP-1, IL-6, TNF-alpha, IL-15, IL-7, IL-18, and IFN-gamma. In certain embodiments, the biomarker is symmetrical dimethylarginine (SDMA).

[0099] In certain embodiments, the biomarker is any combination of at least one metabolite selected from pantothenate, acetylcarnitine (C2), N-acetylphenylalanine, isobutyrlcarnitine (C4), beta-hydroxyisovalerate, triglycarnitine (C5:1-DC), 3-hydroxy-3-methylglutarate, beta- hydroxyisovaleroylcarnitine, 4-hydroxyphenylacetate, 5-hydroxymethyl-2-furoic acid, glycerophosphorylcholine (GPC), 2-aminoadipate, 3-hydroxykynurenine, guanidinosuccinate, malonylcarnitine, xanthurenate, N-acetylisoleucine, N-formylmethionine, 2- hydroxyphenylacetate, gamma-CEHC sulfate, pyridoxal, N2,N6-diacetyllysine, N- acetylglutamine, alpha-CEHC glycine, and / or glutamate.

[0100] Detection and quantitation of biomarkers in a sample (such as urine or blood) may be performed at any time after collection. For example, quantification of a biomarker within a biological sample may be performed within a short time period (e.g. within 2-minutes, within 5- mintes, etc.) or after a longer storage, transportation, preservation, or incubation phase (e.g. within 6 hours, within, 72 hours, etc.). In certain embodiments, the biological sample is stored, transported, or preserved at temperatures ranging about from about -200 °C to about 30 °C, e.g., - 80 °C. For another example, a tissue biopsy is preferably stored at -20 °C for short term storage and -80 °C for long term storage. It should be understood that these examples are for illustration purposes only and that the proper temperatures depend on the type of biological sample used inAttorney Docket No.: 14405-00-WO-01-HL the present disclosure. The biological sample should be stored at the proper temperature for the specific type of biological sample as commonly understood by one of ordinary skill in the art.

[0101] One or more biomarkers within a sample may be used as a diagnostic tool for to any type of disease, disorder, or other condition. For example, one or more biomarkers may indicate a propensity (e.g., likelihood of developing) a companion animal may have for a disease, disorder, and / or condition. In a preferred embodiment, one or more biomarkers are used to diagnose or identify the presence of anxiety in a canine. In another preferred embodiment, one or more biomarkers are used to diagnose or identify the presence of inflammation in a canine.

[0102] In some embodiments, the pet food composition disclosed herein may be used to treat any of the disease states or conditions described herein. In a preferred embodiment, disclosed herein is a method for treating anxiety in canines comprising administering a pet food composition as disclosed herein. In a preferred embodiment, disclosed herein is a method for improving immune health, including a method of treating or preventing inflammation or an allergy, in a companion animals comprising administering a pet food composition as disclosed herein. Preferably, the pet food composition comprises one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil (e.g., DHA and EPA). In certain embodiments, the pet food composition comprises at least two of, at least three of, at least four of, at least five of, at least six of, at least seven of, or all eight of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil. In one embodiment, the pet food composition comprises one or more of scFOS, betaine, carnitine, tryptophan, broccoli, spinach, citrus pulp and / or fish oil and is administered to the companion animal daily (e.g., once, twice, three, or four times daily) for at least 5 days, such as at least 10 days, at least 2 weeks, at least one month, at least 6 weeks, at least 2 months, at least 12 weeks, at least 3 months, at least 4 month, at least 5 months, or at least 6 weeks.

[0103] Embodiments of the present disclosure will now be further described by way of the following, non-limiting, examples. EXAMPLES Example 1

[0104] Two pet food compositions in the form of a treat / supplement were prepared as disclosed below in Table 1. The Test composition comprised scFOS, betaine, carnitine, tryptophan, broccoli,Attorney Docket No.: 14405-00-WO-01-HL spinach, citrus pulp and / or fish oil, while the Control composition did not contain any of the aforementioned ingredients. The nutrient profile of both the Test and the Control compositions is provided in Table 2 below.

[0105] Table 1 – Composition of the treat / supplement for Control and Test Compositions Ingredient Control Test (% wt) (% wt)Attorney Docket No.: 14405-00-WO-01-HL Food gross energy, 3571 3578 kcal / kgpplement for Control and Test Compositions Nutrient Proximate analysis Control TestExample 2 –Reducing Anxiety-Related Urinary Biomarkers in Dogs

[0107] In this example, dogs’ behaviors and personalities were scored using pet personality and temperament questionnaires to identify traits related to anxiety. Dogs were phenotyped as anxious dogs and non-anxious dogs by using a composite index score derived from three tools. Namely, dogs were phenotyped as high in neuroticism / fearfulness / sensitivity or low in neuroticism / fearfulness / sensitivity by using a composite index. The composite index was calculated as the average of the Z-scores of the Monash Neuroticism domain, the Dog Personality Questionnaire (“DPQ”) Fearfulness domain, and the Highly Sensitive Dog “HSD” total score, i.e., hereinafter “the temperament score,” which score is a proxy measure in lieu of a behaviorist’s assessment of anxiety.

[0108] These Monash Neurotocism domain, DPQ fearfulness factor, and HSD total score were selected because together they may reflect characteristics associated with anxiety. Although a single questionnaire could be used according to embodiments disclosed herein, it was believed that consistently high scores on all 3 questionnaires would provide a consistent assessment of traits associated with anxiety. This questionnaire scoring approach is designed to identify dogs that are highly neurotic, fearful and sensitive and reflects the input of care technicians who work with the dogs and are able to observe them under a variety of conditions. However, this approach has not been validated against a behaviorist’s assessment.Attorney Docket No.: 14405-00-WO-01-HL

[0109] In the DPQ questionnaire, one of the questions that contributes to the score in the fearfulness domain is “Dog is anxious,” supporting the use of the DPQ fearfulness domain as a measure of traits associated with anxiety. Previous research reported in Posluns, J.A., Behaviour differences in companion dogs in response to unfamiliar conspecific contact and personality measurements, Thesis submitted to Memorial University of Newfoundland, Oct (2017), has shown that the DPQ fearfulness and the Monash neuroticism domains are highly correlated supporting the validity of the characteristic being measured.

[0110] Urine metabolites were then compared between the dogs to identify metabolites that differentiated the anxious from the non-anxious dogs (i.e., high or low on the temperament score described above for neuroticism / fearfulness / sensitivity). In a separate study, it was shown that a dietary intervention resulted in changes to the identified urinary metabolite markers that are characteristic of anxious and non-anxious phenotypes (i.e., high or low on the temperament score described above for neuroticism / fearfulness / sensitivity).

[0111] A feeding study was conducted on 20 dogs to evaluate the effect of the test composition as disclosed in Example 1 in reducing these metabolites. Dogs were fed maintenance food for 15 days during the washout period. For the next 150 days of the treatment period, dogs received nutritionally complete food (defined by 90% of their caloric requirements from the same maintenance food) and 10% of their caloric requirements from either the test or the control composition as disclosed in Example 1 in treat / supplement form.

[0112] A composite normalized temperament score was created as one-third times the sum of the z-scores (score minus overall mean divided by standard deviation) of a score from each of 3 questionnaires. The tertiles of the population were determined for the temperament score and labeled as: (1) low neuroticism / fearfulness / sensitivity (hereinafter “non-anxious”); (2) moderate neuroticism / fearfulness / sensitivity (hereinafter “moderately anxious”); and (3) high neuroticism / fearfulness / sensitivity (hereinafter “highly anxious”). Metabolomics were conducted on urine samples collected at the end of the washout period and on day 150 of the treatment period. Urine metabolites that were elevated in highly anxious dogs were identified by comparing the urine metabolite profiles of non-anxious dogs with those of the highly anxious dogs.

[0113] The questionnaires helped to identify 18 highly anxious dogs and 18 non-anxious dogs. Metabolomics data showed that a set of urinary markers were differentially elevated in anxious dogs compared to non-anxious dogs, as shown in Figure 1. In the feeding study conducted toAttorney Docket No.: 14405-00-WO-01-HL evaluate the effect of the test composition, the consumption of the test composition led to a significant reduction in the levels of the set of urine metabolites that were elevated in the anxious dogs as compared to the non-anxious dogs, as shown in Figure 2.

[0114] The urinary metabolites were compared using MANOVA analysis. 25 metabolites were identified that were significantly higher in highly anxious dogs as compared to non-anxious dogs (p < 0.0001). The 25 identified metabolites were as follows: (1) pantothenate; (2) acetylcarnitine (C2); N-acetylphenylalanine; (4) isobutyrlcarnitine (C4); (5) beta-hydroxyisovalerate; (6) triglycarnitine (C5:1-DC); (7) 3-hydroxy-3-methylglutarate; (8) beta-hydroxyisovaleroylcarnitine; (9) 4-hydroxyphenylacetate; (10) 5-hydroxymethyl-2-furoic acid; (11) glycerophosphorylcholine (GPC); (12) 2-aminoadipate; (13) 3-hydroxykynurenine; (14) guanidinosuccinate; (15) malonylcarnitine; (16) xanthurenate; (17) N-acetylisoleucine; (18) N-formylmethionine; (19) 2- hydroxyphenylacetate; (20) gamma-CEHC sulfate; (21) pyridoxal; (22) N2,N6-diacetyllysine; (23) N-acetylglutamine; (24) alpha-CEHC glycine; and (25) glutamate.

[0115] As shown in Figure 2, the MANOVA analysis showed that the test composition led to significant reductions in the set of 25 urinary metabolites (P<0.0001). The least squared means (Y- axis) of the 25 metabolites on the X-axis are shown in Figure 2, with each number on the X-axis representing the 25 metabolites as set forth above. Example 3 – Enhancing Palatability

[0116] Treats or supplements containing active ingredients may be rejected by companion animals. The test composition disclosed herein containing scFOS, citrus pulp, broccoli powder, spinach, betaine, carnitine, tryptophan, and fish oil was tested to determine the acceptance of the composition in companion animals.

[0117] A cross-over treat acceptance test was performed on 24 dogs, of which 5 had a relatively longer prehension time (1 second and longer) before the consumption of a control and / or a test treat. The test treat contained scFOS, citrus pulp, broccoli powder, spinach, betaine, carnitine, tryptophan, and fish oil, as disclosed in Example 1, and the control treat (see Example 1) did not contain any of the above-mentioned ingredients. A kibble from each composition was provided to each dog twice. The time each dog took to start consuming the test or the control composition (prehension time) was longer for the 5 dogs compared to other dogs. Although most dogs are interested in treats, some dogs may have decreased enthusiasm for treats due to taste, health, orAttorney Docket No.: 14405-00-WO-01-HL behavioral conditions. The test composition was shown to increase treat acceptance by dogs that are less eager to consume treats.

[0118] The change in treat consumption time when dogs were offered the treats for the second time compared to the first time (delta consumption) was calculated for each dog when given the test or the control treat. The test treat delta consumption time was significantly shorter than the control treat delta consumption time (p = 0.021), as shown in Figure 3. The results suggest that the specific composition and nutrient profile of the test composition allows increased acceptance of nutritional actives by dogs. Example 4 – Supporting Immune Health

[0119] The test composition disclosed herein can be used to provide immune support to dogs having reduced immune defense, chronic inflammation, and / or allergic reactions. In this example, a total of 40 dogs were fed a washout / maintenance food for 11 days and were randomly assigned to consume 10% of their caloric requirements from either the test composition or the control composition and 90% of their caloric requirements from the maintenance food for 150 days. The composition and nutrient profiles of the test and control compositions are provided in Table 1 and 2 in Example 1 above.

[0120] Blood cell counts and chemical tests were performed on Day 53 and Day 150 after the washout period. Unpaired t-test was used to compare the mean blood measurements after consumption of the control and the test compositions. It was demonstrated that the test composition increased the percentages of neutrophils significantly compared to the control composition (P=0.006). Although not statistically significant, the test composition also increased the percentage of lymphocytes (P=0.079). The ratio of neutrophils to lymphocytes, an inflammatory marker associated with various diseases, including acute and chronic inflammation, tended to be lower after dogs consumed the test composition, implying a less inflammatory status. The test composition led to a significant reduction in the percentages of cells involved in allergy and inflammatory symptoms, including basophils (P=0.002) and eosinophils (P=0.007). The absolute number of basophils (P=0.006) and eosinophils (P=0.01) was also significantly reduced after dogs consumed the test composition.

[0121] The test composition also led to a significant reduction in several inflammatory cytokines, including MCP-1, IL-6, TNF-alpha, IL-15, IL-7, and IL-18. The test composition increased levels of cytokine interferon-gamma (IFN-gamma), which is believed to be reduced in stress and is alsoAttorney Docket No.: 14405-00-WO-01-HL correlated to anti-inflammatory and antiviral effects. The results are shown in Tables 3 and 4 below.

[0122] Table 3 – Change in Neutrophils, Basophils, and Eosinophils Blood cell measured Control Test P-value (Mean ± SD) (Mean ± SD)g y y Cytokine Control Test P-value measured (Mean ± SE) (Mean ± SE)

[0124] Furthermore, it is known that increased levels of symmetrical dimethylarginine (SDMA) in blood have been associated with renal disease, cardiovascular disease, and mortality. High protein consumption has been shown to increase markers of kidney disease. Although the test composition contained a higher percentage of protein as compared to the control composition, it nonetheless led to a significant reduction in blood SDMA (P=0.006). Dogs had a lower concentration of blood SDMA after they consumed the test composition (12.8 ± 1.7) as compared to the control composition (13.8 ± 1.7). The results indicate that the test composition provides a significant benefit to dogs’ overall health by reducing the blood concentration of SDMA and supporting the immune system.

[0125] While the present disclosure has been described with reference to several embodiments, which embodiments have been set forth in considerable detail for the purposes of making a complete disclosure, such embodiments are merely exemplary and are not intended to be limiting or represent an exhaustive enumeration of all aspects of the disclosure. The scope of the disclosure is to be determined from the claims appended hereto. Further, it will be apparent to those of skillAttorney Docket No.: 14405-00-WO-01-HL in the art that numerous changes may be made in such details without departing from the spirit and the principles of the disclosure.

Claims

Attorney Docket No.: 14405-00-WO-01-HL CLAIMS What Is Claimed Is:

1. A pet food composition comprising: at least one fermentable fiber; betaine; carnitine; tryptophan; at least one fruit, vegetable, or extract thereof comprising phytochemicals; and at least two omega-3 polyunsaturated fatty acids.

2. The pet food composition of claim 1, wherein the at least one fermentable fiber is selected from short-chain fructooligosaccharides (scFOS).

3. The pet food composition according to claim 2, wherein the scFOS is present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 5 wt% to about 15 wt%, about 8 wt% to about 12 wt%, or about 10 wt%, based on the total weight of the pet food composition.

4. The pet food composition according to any one of the preceding claims, wherein the betaine is present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 5 wt% to about 15 wt%, about 8 wt% to about 12 wt%, or about 10 wt%, based on the total weight of the pet food composition.

5. The pet food composition according to any one of the preceding claims, wherein the carnitine is present in the pet food composition in an amount ranging from about 0.001 wt% to about 5 wt%, such as from about 0.01 wt% to about 2 wt%, about 0.1 wt% to about 1 wt%, about 0.2 wt% to about 0.9 wt%, or about 0.8 wt%, based on the total weight of the pet food composition.

6. The pet food composition according to any one of the preceding claims, wherein the tryptophan is present in the pet food composition in an amount ranging from about 0.001 wt% toAttorney Docket No.: 14405-00-WO-01-HL about 5 wt%, such as from about 0.01 wt% to about 2 wt%, about 0.1 wt% to about 1.5 wt%, about 0.2 wt% to about 1.2 wt%, or about 1 wt%, based on the total weight of the pet food composition.

7. The pet food composition according to any one of the preceding claims, wherein the at least one fruit, vegetable or extract thereof comprising phytochemicals is a selected from broccoli, spinach, and citrus pulp.

8. The pet food composition according to claim 7, wherein the citrus pulp is present in the pet food composition in an amount ranging from about 0.05 wt% to about 50 wt%, such as from about 1 wt% to about 25 wt%, about 2 wt% to about 12 wt%, about 5 wt% to about 10 wt%, or about 7 wt%, based on the total weight of the pet food composition.

9. The pet food composition according to claim 7 or 8, wherein the broccoli is present in the pet food composition in an amount ranging from about 1 wt% to about 30 wt%, such as from about 2 wt% to about 25 wt%, about 3 wt% to about 20 wt%, about 4 wt% to about 10 wt%, about 5 wt% to about 8 wt%, about 6 wt% to about 7 wt%, or about 6 wt%, based on the total weight of the pet food composition.

10. The pet food composition according to any one of claims 7-9, wherein the spinach is present in the pet food composition in an amount ranging from about 1 wt% to about 30 wt%, such as from about 2 wt% to about 25 wt%, about 5 wt% to about 20 wt%, about 6 wt% to about 15 wt%, about 8 wt% to about 12 wt%, about 9 wt% to about 11 wt%, or about 10 wt%, based on the total weight of the pet food composition.

11. The pet food composition according to any one of the preceding claims, wherein the at least two omega-3 polyunsaturated fatty acids are selected from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

12. The pet food composition according to claim 11, wherein the pet food composition comprises fish oil comprising the EPA and DHA.Attorney Docket No.: 14405-00-WO-01-HL 13. The pet food composition according to claim 12, wherein the fish oil is present in the pet food composition in an amount ranging from about 0.5 wt% to about 10 wt%, about 1 wt% to about 5 wt%, about 2 wt% to about 4 wt%, about 2.5 wt% to about 3.5 wt%, or about 3 wt%, based on the total weight of the pet food composition.

14. A method of treating or preventing anxiety in a companion animal comprising administering to the companion animal an effective amount of the pet food composition according to any one of claims 1-13.

15. The method of reducing one or more anxiety biomarkers in a companion animal comprising administering to the companion animal an effective amount of the pet food composition according to any one of claims 1-13, wherein the one or more anxiety biomarkers are selected from pantothenate, acetylcarnitine (C2), N-acetylphenylalanine, isobutyrlcarnitine (C4), beta- hydroxyisovalerate, triglycarnitine (C5:1-DC), 3-hydroxy-3-methylglutarate, beta- hydroxyisovaleroylcarnitine, 4-hydroxyphenylacetate, 5-hydroxymethyl-2-furoic acid, glycerophosphorylcholine (GPC), 2-aminoadipate, 3-hydroxykynurenine, guanidinosuccinate, malonylcarnitine, xanthurenate, N-acetylisoleucine, N-formylmethionine, 2- hydroxyphenylacetate, gamma-CEHC sulfate, pyridoxal, N2,N6-diacetyllysine, N- acetylglutamine, alpha-CEHC glycine, and / or glutamate.

16. The method of claim 15, further comprising obtaining a biological sample from the companion animal and measuring the one or more biomarkers after administering the pet food composition, and optionally further comprising measuring the one or more biomarkers before administering the pet food composition.

17. The method of claim 15 or 16, wherein the biological sample is selected from blood, urine, or saliva.

18. A method of treating or preventing inflammation or an allergy in a companion animal comprising administering to the companion animal an effective amount of the pet food composition according to any one of claims 1-13.Attorney Docket No.: 14405-00-WO-01-HL 19. A method of reducing levels of at least one inflammatory cytokine in a companion animal comprising administering to the companion animal an effective amount of the pet food composition according to any one of claims 1-13, wherein the at least one inflammatory cytokine is selected from MCP-1, IL-6, TNF-alpha, IL-15, IL-7, and IL-18.

20. The method of claim 19, wherein the method further comprises increasing levels of cytokine interferon-gamma (IFN-gamma) in the companion animal.

21. The method according to any one of claims 18-20, wherein the method further comprises reducing levels of symmetrical dimethylarginine (SDMA) in the companion animal.

22. The method according to any one of claims 18-21, further comprising obtaining a biological sample from the companion animal and measuring the one or more inflammatory cytokines after administering the pet food composition, and optionally further comprising measuring the one or more inflammatory cytokines before administering the pet food composition.

23. The method according to any one of claims 14-22, wherein the companion animal is a canine.

24. The method according to any one of claims 14-23, wherein the pet food composition comprises at least about 2%, such as at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, or at least about 10% of the total daily diet of the companion animal.

25. A pet food composition comprising: short-chain fructooligosaccharides; betaine; carnitine; tryptophan; citrus pulp; spinach;Attorney Docket No.: 14405-00-WO-01-HL broccoli; and fish oil.

26. The pet food composition according to any one of claims 1-13 and 25, wherein the pet food composition is a supplement.

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