HUMAN DIET EQUIVALENT ANIMAL FEED

CROSS REFERENCE TO RELATED APPLICATIONS

[0001 ] This application claims the priority of US provisional application number 61/841 ,786, filed July 1 , 2013, and US provisional application number

61/869,047, filed August 22, 2013, each of which is hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention provides animal feed compositions comprising a human diet equivalent, and methods for processing human foods into an animal feed ration.

BACKGROUND OF THE INVENTION

[0003] When animal models are employed in the study of human disease, they are frequently selected because of their similarity to humans in terms of genetics, anatomy, and physiology. Also, animal models are often preferable for experimental disease research because of their ease of manipulation. For example, to obtain scientifically valid research, the conditions associated with an experiment must be closely controlled. This often means manipulating only one variable while keeping others constant, and then observing the consequences of that change. In addition, to test hypotheses about how a disease develops, an adequate number of subjects must be used to statistically test the results of the experiment. Therefore, scientists cannot conduct research on just one animal or human, and it is easier for scientists to use sufficiently large numbers of animals (rather than people) to attain significant results.

[0004] Diet and nutritional statues are among the most modifiable determinants of human health. The nutritional value of food is influenced in part by a person's gut microbial community (microbiota) and its component genes (microbiome). Unraveling the interrelations among diet, the structure and operations of the gut microbiota, and nutrient and energy harvest is confounded by variations in human environmental exposures, microbial ecology and genotype. Animal models of the human gut ecosystem have helped to advance our understanding of these complex relationships. Similar advances in animal diets are also needed.

[0005] Historically, studies seeking to understand effects of diet have relied either on traditional animal chow supplemented with one or more nutrients or a cafeteria diet approach (Free Radical Biology and Medicine (2013) 65: 254-261 ;

Physiology and Behavior (1977) 18: 1021 -1026; Obesity (201 1 ) 19: 1 109-1 1 17)., Both approaches fail to reflect human consumption patterns, nor do they rigorously ensure nutritional adequacy and consistency. Thus, there remains a need in the art for animal feed rations that more closely mirror human food consumption patterns in a format that allows for the animal to get a controlled amount and ratio of foods equivalent to a representative diet of a specific human sub-population.

SUMMARY OF THE INVENTION

[0006] In an aspect, the present disclosure encompasses an animal feed composition. The animal feed composition comprises a human diet equivalent. The human diet equivalent consists of 45- 53% by wet weight carbohydrates, 1 - 4% by wet weight total dietary fiber, 20- 25% by wet weight fat, 7- 9% total saturated fatty acids of fat, 83- 88 mg cholesterol/1 OOg total diet (TD), 19- 23% by wet weight protein, 800- 1000 mg sodium/1 OOg TD, 300- 500 mg potassium/1 OOg TD, 4- 8% by wet weight moisture, and 4.6- 4.9 Kcal/1 OOg TD. The human diet equivalent is made from a defined ratio of eight food groups. The eight food groups consist of food items identified by the first three or four digits of an eight digit food code as represented in Table A. Milk and milk products have a food code with a first digit of 1 and are 5- 25% by wet weight. Meat, poultry, fish, egg, nuts, seeds, and legumes have a food code with a first digit selected from the group consisting of 2, 3, and 4 and are 10- 25% by wet weight. Grain products have a food code with a first digit of 5 and are 10- 20% by wet weight. Fruits have a food code with a first digit of 6 and are 0- 1 % by wet weight. Vegetables have a food code with a first digit of 7 and are 0- 1 % by wet weight. Fats and oils have a food code with a first digit of 8 and are 0.5-5.0% by wet weight. Sugars and sweets have a food code with a first digit of 9 and a second digit of 1 and are 0.5-5.0% by wet weight. Beverages have a food code with a first digit of 9 and a second digit of 2 or 3 and are 0.5-5.0% by wet weight.

[0007] In another aspect, the present disclosure encompasses an animal feed composition. The animal feed composition comprises a human diet equivalent. The human diet equivalent consists of 57- 64% by wet weight carbohydrates, 3- 7% by wet weight total dietary fiber, 10- 15% by wet weight fat, 2.0- 3.5% total saturated fatty acids of fat, 35-40 mg cholesterol/1 OOg TD, 13- 17% by wet weight protein, 300- 500 mg sodium/1 OOg TD, 500- 700 mg potassium/1 OOg TD, 4- 8% by wet weight moisture, and 4.0- 4.4 Kcal/100g TD. The human diet equivalent is made from a defined ratio of eight food groups. The eight food groups consist of food items identified by the first three or four digits of an eight digit food code as represented in Table A. Milk and milk products have a food code with a first digit of 1 and are 5- 25% by wet weight. Meat, poultry, fish, egg, nuts, seeds, and legumes have a food code with a first digit selected from the group consisting of 2, 3, and 4 and are 10- 25% by wet weight. Grain products have a food code with a first digit of 5 and are 10- 20% by wet weight. Fruits have a food code with a first digit of 6 and are 10- 25% by wet weight fruits. Vegetables have a food code with a first digit of 7 and are 10- 25% by wet weight. Fats and oils have a food code with a first digit of 8 and are 0.5- 5% by wet weight. Sugars and sweets have a food code with a first digit of 9 and a second digit of 1 and are 0.5- 5% by wet weight sugars and sweets. Beverages have a food code with a first digit of 9 and a second digit of 2 or 3 and are 10- 60% by wet weight beverages.

[0008] In yet another aspect, the present disclosure encompasses a process to prepare pelletized animal feed. The process comprises (i) introducing two or more food items independently selected from the group consisting of frozen food items, refrigerated food items, and room temperature food items into a first mixer and blending the food items, wherein the first mixer is a high shear liquid mixer; (ii) passing and/or recirculating the blended food items into a second mixer to form a homogenous composite, wherein the second mixer is a rotor-stator mixer and the homogenized composite is evenly blended by visual inspection; (iii) freeze drying the homogenous composite and milling the dehydrated composite to a 20 mesh size, wherein the moisture content of the dehydrated composite is less than 5%; and (iv) adding water to the milled composite and extruding into pellets, wherein the moisture content of the composite at the end of the extrusion process is less than 10%.

[0009] In still yet another aspect, the present disclosure encompasses a method of determining the effect of at least one dietary supplement on a subject. The method comprises administering or feeding a composition of any of the compositions described herein further comprising at least one dietary supplement to a subject and determining the presence or absence of a difference in the subject after administering the composition comprising the dietary supplement. The presence or absence of a difference in the subject represents the effect of the dietary supplement on the subject.

[0010] In still yet another aspect, the present disclosure encompasses a method of determining the effect of at least one dietary supplement on a subject. The method comprises (1 ) administering or feeding a first composition to a subject for a first period of time, wherein the first composition is a composition disclosed herein, (2) administering or feeding a second composition to the subject for a second period of time, wherein the second composition consists of the first composition and at least one dietary supplement, and (3) determining the presence or absence of a difference in the subject after administering or feeding the second composition. The presence or absence of a difference in the subject represents the effect of the dietary supplement on the subject.

[001 1 ] In still yet another aspect, the present disclosure encompasses a method of determining the effect of at least one dietary supplement on a subject. The method comprises (1 ) administering or feeding a first composition to a subject for a first period of time, wherein the first composition is a composition disclosed herein and at least one dietary supplement, (2) administering or feeding a second composition to the subject for a second period of time, wherein the second composition consists of the first composition without the at least one dietary supplement, and (3) determining the presence or absence of a difference in the subject after administering or feeding the second composition. The presence or absence of a difference in the subject represents the effect of the dietary supplement on the subject BRIEF DESCRIPTION OF THE FIGURES

[0012] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee

[0013] FIG. 1 depicts a production process flow diagram.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention encompasses methods for creating a human diet equivalent for use in animal feed, animal feed compositions comprising human diet equivalents, and methods for processing human foods into a homogenous animal feed ration. Critically, the processes of the invention that convert human foods into

homogenous animal feed rations provide an even mix of foods in the right quantitative portions while retaining nutrients. This approach also overcomes any taste issues that can be associated with eating particular bitter and sour foods by creating a homogenous composite of foods that is palatable to the animals. As a result, the animal feed compositions accurately model the human diet, deliver known amounts and ratios of nutrients, and can be processed into a structurally stable form. Animal feed

compositions differing in the amounts of one or more food items may be used to test the impact of diet on various facets of a mouse model, and are particularly well suited to test how gut microbes adapt to dietary perturbations. Methods for deriving an animal feed composition from a human diet, animal feed compositions comprising human diet equivalents, and processes for producing said compositions are described in further detail below.

I. METHODS FOR CREATING A HUMAN DIET EQUIVALENT FOR USE IN ANIMAL FEED

[0015] The present invention encompasses methods for creating a human diet equivalent for use in an animal feed ration. Generally speaking, a human diet equivalent is constructed from one or more foods that characterize a particular human diet. Stated another way, a human diet equivalent comprises at least one food item typically consumed by a human. The terms "food" and "food items" are defined below. A method for creating a human diet equivalent typically comprises (i) collecting information on what people eat, (ii) analyzing the data in order to identify one or more subpopulations, (iii) identifying one or more foods that characterize a subpopulation, and (iv) determining the proportion of each characterizing food item to be included in the human diet equivalent.

[0016] In an aspect, the present invention utilizes methods for collecting information on what people eat. Such information is needed in order to determine the foods a person typically eats. Suitable methods for collecting information on what people eat are known in the art, and may include, but are not limited to, 24-hour recalls and food diaries recorded by an individual. A skilled artisan may conduct the food survey or may rely on a previously conducted food survey(s). Such surveys are well known to a skilled artisan and may include, but are not limited to, What We Eat in America (WWEIA), which is the dietary intake interview component of the National Health and Nutrition Examination Survey (NHANES), United States Department of Agriculture (USDA) Nationwide Food Surveys, and World Food Surveys conducted by the Food and Agricultural Organization of the United Nations.

[0017] After data collection, a skilled artisan needs a way to translate the food intake data into a form that can be used for analysis. In some embodiments, the USDA Food and Nutrient Database for Dietary Studies (FNDDS) is used. The USDA FNDDS is a resource that is used to code dietary intakes and to calculate nutrients for WWEIA and other food surveys. The FNDDS is based on nutrient values in the USDA National Nutrient Database for Standard Reference, Release 22 (Agricultural Research Service, Nutrient Data Laboratory, 2009). Other suitable food coding schemes may be used, provided the scheme provides nutrient values and weights for typical food portions and food items.

[0018] The USDA FNDDS provides a unique eight digit food code for specific foods. This food coding scheme provides an outline of the major food groups and subgroups by the first 1 to 4 digits of the food code. The coding scheme is represented in Table A. The first digit in the food code identifies one of nine major food groups: (1 ) milk and milk products, (2) meat, poultry, fish, and mixtures, (3) eggs, (4) legumes, nuts and seeds, (5) grain products, (6) fruits, (7) vegetables, (8) fats, oils and salad dressings, and (9) sugars, sweets and beverages. The second, third and

(sometimes) fourth digits of the food code identify increasingly more specific subgroups within the nine major food groups. Most subgroups are identified by the first three digits, except for some subgroups in the Meat, Poultry, Fish and Mixtures, and Sugar, Sweet and Beverages section. A "food item", as used herein, refers to a food that is

represented by a complete eight-digit FNDDS code or equivalent thereof. The term "food", as used herein, may refer to a food item, a food subgroup (i.e. the second, third, or fourth digit of an eight digit FNDDS food code, or the equivalent thereof), or a major food group (i.e. the first digit of an eight digit FNDDS food code, or the equivalent thereof). The term "food" includes both foods and beverages. Further details regarding the USDA FNDDS may be found in The USDA Food and Nutrient Database for Dietary Studies, 4.1 - Documentation and User Guide (2010. Beltsville, MD: U.S. Department of Agriculture, Agricultural Research Service, Food Surveys Research Group),

incorporated herein by reference in its entirety.

[0019] In an aspect, the present invention utilizes an approach for identifying one or more subpopulations within a total population by characterizing one or more diet patterns of the population. A subpopulation may be identified based on an analysis of the consumption of the total daily intakes of foods. For example, a

subpopulation may be identified based on an analysis of all foods consumed, an analysis of the amount of the types of foods consumed, an analysis of the calories derived from one or more types of food consumed, or a combination thereof. Other suitable subpopulations for analysis will also be apparent to a skilled artisan and are contemplated by the invention. In some embodiments, a subpopulation may be identified based on an analysis of the consumption of at least one food. In other embodiments, a subpopulation may be identified based on an analysis of the

consumption of at least two foods. In still other embodiments, a subpopulation may be identified based on an analysis of the consumption of at least three foods. In yet other embodiments, a subpopulation may be identified based on an analysis of the

consumption of at least four foods. In different embodiments, a subpopulation may be identified based on an analysis of the consumption of at least five foods. In each of the above embodiments, the analysis may occur at the major food group level (i.e. the first digit of an eight digit FNDDS food code, or the equivalent thereof), at a food subgroup level (i.e. the second, third, or fourth digit of an eight digit FNDDS food code, or the equivalent thereof), or at the food item level (i.e. a complete eight-digit FNDDS code, or equivalent thereof).

[0020] Subpopulations may also be identified based on a nutritional analysis of the foods consumed. Suitable nutrients and food associated compounds may include, but are not limited to total fat, saturated fat, monounsaturated fat, polyunsaturated fat, trans saturated fat, cholesterol, carbohydrate, total sugars, dietary fiber, Vitamin A, Vitamin B6, Vitamin B12, Vitamin C, Vitamin D, Vitamin E, Vitamin K1 , Thiamin, Riboflavin, Niacin, Folate, Pantothenic acid, Calcium, Phosphorus,

Magnesium, Manganese, Iron, Zinc, Copper, Selenium, Sodium, Potassium, Fructose, Sucrose, Glucose, Lactose, Maltose, Fatty acid distribution, amino acid distribution, betacarotene, retinol, alphatocopherol, betatocopherol, gammatocopherol,

deltatocopherol, alphatoctrienol, betatoctrienol, gammatocotrienol, deltatocotrienol, apo- 8-carotenal, trans-lycopene, cis-lycopene, trans-beta-carotene, cis-beta-carotene, caffeine, and combinations thereof. In some embodiments, a subpopulation may be identified based on an analysis of the consumption of at least one nutrient. In other embodiments, a subpopulation may be identified based on an analysis of the

consumption of at least two nutrients. In still other embodiments, a subpopulation may be identified based on an analysis of the consumption of at least three nutrients. In yet other embodiments, a subpopulation may be identified based on an analysis of the consumption of at least four nutrients. In different embodiments, a subpopulation may be identified based on an analysis of the consumption of at least five nutrients.

[0021 ] In preferred embodiments, a subpopulation may be identified based on saturated fat intake. In other preferred embodiments, a subpopulation may be identified based on total fruit intake. In still other preferred embodiments, a

subpopulation may be identified based on total vegetable intake. In yet other preferred embodiments, a subpopulation may be identified based on total fruit and vegetable intake. In yet other preferred embodiments, a subpopulation may be identified based on saturated fat intake and either total vegetable intake or total fruit intake. In yet other preferred embodiments, a subpopulation may be identified based on saturated fat intake and total fruit and vegetable intake.

[0022] Once the basis of each grouping has been determined, as described above, the total sample may be divided into two or more subpopulations. For example, the total sample may be divided into groups of two, groups of three, groups of four, groups of five, groups of six, groups of seven, groups of eight, groups of nine, or groups of ten or more. Alternatively, the total sample may be divided into groups of 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more. Groups may or may not be split equally, depending on the type of statistical analysis performed. Suitable methods are well known in the art. The terms "high" and "low" may represent the highest and lowest group in the distribution, or some combination of lowest groups and highest groups in the distribution. In some embodiments, the total sample may be divided into quintiles. In preferred embodiments, the total sample may be divided in tertiles.

[0023] In preferred embodiments, a first subpopulation comprises the highest quintile saturated fat intake and lowest quintile fruit and vegetable intake, and a second subpopulation comprises the lowest quintile saturated fat intake and highest quintile fruit and vegetable intake. In another exemplary embodiment, a first

subpopulation consists of the highest quintile saturated fat intake and lowest quintile fruit and vegetable intake, and a second subpopulation consists of the lowest quintile saturated fat intake and highest quintile fruit and vegetable intake. In an exemplary embodiment, the quintile is a tertile.

[0024] In another aspect, the present invention provides methods for identifying foods that characterize the diet of a subpopulation. The method comprises identifying food groups and subgroups that characterize the diet of a subpopulation (i.e. a "key food group" or "key food subgroup") and then selecting one or more food items that characterize the key food group or key food subgroup.

[0025] Generally speaking, for each subpopulation, key food subgroups may be identified by determining the relative energy intakes by major food group and food subgroup and the proportion of the subpopulation consuming foods within individual food subgroups. Preferably, the following criteria are used to identify key food subgroups: (1 ) Food subgroups providing less than 20 kcal/1000 kcal total diet (i.e., less than 2.0% of energy) are excluded from consideration; (2) Within each food subgroup providing from 20 to 50 kcal/1000 kcal total diet (i.e., 2.0 to 5.0% of energy), the single food subgroup representing the largest source of energy is selected as a key food subgroup; (3) Within each food group providing more than 50 kcal/1000 kcal total diet (i.e., over 5.0% of total energy), all food subgroups meeting at least one of the following two criteria were considered key food subgroups (a) Contributing at least 10% of kcal per 1000 kcal for the food group and consumed by at least 10% of the subpopulation, or (b) Consumed by 20% or more of the subpopulation. Finally, once each key food group or key food subgroup has been identified, the food code with the greatest weighted proportion of members of the subpopulation reporting consumption on the day of recall is selected to be the key food item characterizing the entire food group or key food subgroup.

[0026] In another aspect, the present invention utilizes methods for determining the proportion of each characterizing food to be included in an animal feed composition. The method comprises determining the relative energy contributions from key food subgroups to the total energy intake from the food group (per 1000 kcal total diet) and then determining the weight of the food item (in g) required to deliver the calculated energy contribution from the relevant food group per 1000 kcal total diet.

II. ANIMAL FEED COMPOSITIONS COMPRISING HUMAN DIET EQUIVALENTS

CONSTRUCTED FROM ONE OR MORE FOODS THAT CHARACTERIZE A HISF- LOFV DIET OR LOSF-HIFV DIET

[0027] In another aspect, the present invention encompasses an animal feed composition comprising a human diet equivalent that is constructed from one or more foods that characterize either a high saturated fat, low fruit and vegetable diet (HiSF-LoFV diet) or a low saturated fat, high fruit and vegetable diet (LoSF-HiFV diet). Methods for constructing a human diet equivalent are described above. [0028] A human diet equivalent that is constructed from one or more foods that characterize either a HiSF-LoFV diet or a LoSF-HiFV diet has the right proportions of nutrients for each particular diet. In some embodiments, a human diet equivalent consists of 45- 53% by wet weight carbohydrates, 1 - 10% by wet weight total dietary fiber, 20- 25% by wet weight fat, 7- 9% total saturated fatty acids of fat, 83- 88 mg cholesterol /100g total diet (TD), 19- 23% by wet weight protein, 800- 1000 mg sodium/1 OOg TD, 300- 500 mg potassium/1 OOg TD, 4- 8% by wet weight moisture, and 4.6- 4.9 Kcal/100g TD. In other embodiments, a human diet equivalent consists of 45- 53% by wet weight carbohydrates, 1 - 4% by wet weight total dietary fiber, 20- 25% by wet weight fat, 7- 9% total saturated fatty acids of fat, 83- 88 mg cholesterol/1 OOg TD, 19- 23% by wet weight protein, 800- 1000 mg sodium/1 OOg TD, 300- 500 mg

potassium/1 OOg TD, 4- 8% by wet weight moisture, and 4.6- 4.9 Kcal/100g TD. In still other embodiments, a human diet equivalent consists of 57- 64% by wet weight carbohydrates, 1 - 10% by wet weight total dietary fiber, 10- 15% by wet weight fat, 2.0- 3.5% total saturated fatty acids of fat, 35-40 mg cholesterol/1 OOg TD, 13- 17% by wet weight protein, 300- 500 mg sodium/1 OOg TD, 500- 700 mg potassium/1 OOg TD, 4- 8% by weight moisture, and 4.0- 4.4 Kcal/100g TD. In yet other embodiments, a human diet equivalent consists of 57- 64% by wet weight carbohydrates, 3- 7% by wet weight total dietary fiber, 10- 15% by wet weight fat, 2.0- 3.5% total saturated fatty acids of fat, 35- 40 mg cholesterol/1 OOg TD, 13- 17% by wet weight protein, 300- 500 mg sodium/1 OOg TD, 500- 700 mg potassium/1 OOg TD, 4- 8% by wet weight moisture, and 4.0- 4.4 Kcal/100g TD.

[0029] Preferably, a human diet equivalent of the invention is made from a defined ratio of eight food groups selected from the groups consisting of (i) milk and milk products, (ii) meat, poultry, fish, egg, nuts seeds and legumes, (iii) grain products, (iv) fruits, (v) vegetables, (vi) fats and oils, (vii) sugars and sweets, and (viii) beverages. In some embodiments, the eight food groups consist of food items identified by the first three or four digits of an eight digit food code as represented in Table A, wherein (i) milk and milk products have a food code with a first digit of 1 , (ii) meat, poultry, fish, egg, nuts seeds and legumes have a food code with a first digit selected from the group consisting of 2, 3, and 4, (iii) grain products have a food code with a first digit of 5, (iv) fruits have a food code with a first digit of 6, (v) vegetables have a food code with a first digit of 7, (vi) fats and oils have a food code with a first digit of 8, (vi) sugars and sweets have a first food code with a first digit of 9 and a second digit of 1 , and (h) beverages have a food code with a first digit of 9 and a second digit of 2 or 3. In other

embodiments the eight food groups consist of food items defined by a different food scheme.

[0030] Constructing a human diet equivalent from a defined ratio of the eight food groups insures the nutrients present in the animal feed mirror the proportion of nutrients in the equivalent human diet. In embodiments where a human diet equivalent is constructed from one or more foods that characterize a HiSF-LoFV diet, the ratio of eight food groups may be about 5- 25% by wet weight milk and milk products, about 10- 25% by wet weight meat, poultry, fish, egg, nuts, seeds, and legumes, about 10- 20% by wet weight grain products, about 0- 1 % by wet weight fruits, about 0- 1 % by wet weight vegetables, about 0.5-5.0% by wet weight fats and oils, about 0.5- 5.0% by wet weight sugars and sweets, and about 10- 60% by wet weight beverages. In a preferred embodiment, the ratio of eight food groups may be about 18.9% by wet weight milk and milk products, (b) about 18.0% by wet weight meat, poultry, fish, egg, nuts, seeds, and legumes, (c) about 16.3% by wet weight grain products, (d) about 0.0% by wet weight fruits, (e) about 0.8% by wet weight vegetables, (f) about 0.6% by wet weight fats and oils, (g) about 1 .2% by wet weight sugars and sweets, and (h) about 44.2% by wet weight beverages.

[0031 ] In embodiments where a human diet equivalent is constructed from one or more foods that characterize a LoSF-HiFV diet, the ratio of eight food groups may be about 5- 25% by wet weight milk and milk products, (b) about 10- 25% by wet weight meat, poultry, fish, egg, nuts, seeds, and legumes, (c) 1 about 0- 20% by wet weight grain products, (d) about 10- 25% by wet weight fruits, (e) 1 about 0- 25% by wet weight vegetables, (f) about 0.5- 5% by wet weight fats and oils, (g) about 0.5- 5% by wet weight sugars and sweets, and (h) about 10- 60% by wet weight beverages. In a preferred embodiment, the ratio of eight food groups may be about 10.2% by wet weight milk and milk products, (b) about 15.7% by wet weight meat, poultry, fish, egg, nuts, seeds, and legumes, (c) about 12.7% by wet weight grain products, (d) about 19.5% by wet weight fruits, (e) about 18% by wet weight vegetables, (f) about 0.9% by wet weight fats and oils, (g) about 0.6% by wet weight sugars and sweets, and (h) about 22.2% by wet weight beverages.

[0032] The number of food items that comprise a human diet equivalent can vary provided that the human diet equivalent has the right proportion of nutrients and/or the correct ratio of eight food groups. Generally speaking, a human diet equivalent is comprised of at least 6 food items. For example, a human diet equivalent may be comprised of at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least, 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 food items.

[0033] In a preferred embodiment, an animal feed composition comprises a human diet equivalent, the human diet equivalent consisting of 45- 53% by wet weight carbohydrates, 1 - 4% by wet weight total dietary fiber, 20- 25% by wet weight fat, 7- 9% total saturated fatty acids of fat, 83- 88 mg cholesterol/1 OOg TD, 19- 23% by wet weight protein, 800- 1000 mg sodium/1 OOg TD, 300- 500 mg potassium/1 OOg TD, 4- 8% by wet weight moisture, and 4.6- 4.9 Kcal/100g TD; wherein the human diet equivalent is made from a defined ratio of eight food groups, the eight food groups consisting of food items identified by the first three or four digits of an eight digit food code as represented in Table A, wherein (a) milk and milk products have a food code with a first digit of 1 and are 5- 25% by wet weight; (b) meat, poultry, fish, egg, nuts, seeds, and legumes have a food code with a first digit selected from the group consisting of 2, 3, and 4 and are 10- 25% by wet weight; (c) grain products have a food code with a first digit of 5 and are 10- 20% by wet weight; (d) fruits have a food code with a first digit of 6 and are 0- 1 % by wet weight; (e) vegetables have a food code with a first digit of 7 and are 0- 1 % by wet weight; (f) fats and oils have a food code with a first digit of 8 and are 0.5-5.0% by wet weight; (g) sugars and sweets have a food code with a first digit of 9 and a second digit of 1 and are 0.5-5.0% by wet weight; and (h) beverages have a food code with a first digit of 9 and a second digit of 2 or 3 and are 0.5-5.0% by wet weight.

[0034] In another preferred embodiment, an animal feed composition comprises a human diet equivalent, the human diet equivalent consisting of 57- 64% by wet weight carbohydrates, 3- 7% by wet weight total dietary fiber, 10- 15% by wet weight fat, 2.0- 3.5% total saturated fatty acids of fat, 35-40 mg cholesterol/1 OOg TD, 13- 17% by wet weight protein, 300- 500 mg sodium/1 OOg TD, 500- 700 mg

potassium/1 OOg TD, 4- 8% by wet weight moisture, and 4.0- 4.4 Kcal/100g TD; wherein the human diet equivalent is made from a defined ratio of eight food groups, the eight food groups consisting of food items identified by the first three or four digits of an eight digit food code as represented in Table A, wherein (a) milk and milk products have a food code with a first digit of 1 and are 5- 25% by wet weight, (b) meat, poultry, fish, egg, nuts, seeds, and legumes have a food code with a first digit selected from the group consisting of 2, 3, and 4 and are 10- 25% by wet weight, (c) grain products have a food code with a first digit of 5 and are 10- 20% by wet weight, (d) fruits have a food code with a first digit of 6 and are 10- 25% by wet weight fruits, (e) vegetables have a food code with a first digit of 7 and are 10- 25% by wet weight, (f) fats and oils have a food code with a first digit of 8 and are 0.5- 5% by wet weight, (g) sugars and sweets have a food code with a first digit of 9 and a second digit of 1 and are 0.5- 5% by wet weight sugars and sweets, and (h) beverages have a food code with a first digit of 9 and a second digit of 2 or 3 and are 10- 60% by wet weight beverages.

[0035] In an embodiment, the composition may further comprise a dietary supplement. As used herein, a "dietary supplement" is any component added to the compositions described herein. A dietary supplement may be an amount of a food or food item, as defined herein. Non-limiting examples of a dietary supplement may be milk or milk product, meat, poultry, fish, egg, nuts, seeds, legumes, grains, fruits, vegetables, fats, oils, sugars, sweets, beverages, vitamins, minerals, herbs, botanicals, and amino acids. For example, a dietary supplement may be acai, aloe vera, anabolic steroids, astragalus, bilberry, bitter orange, black cohosh, butterbur, calcium, carnitine, cartilage (bovine and shark), cat's claw, chamomile, chasteberry, chondroitin, chromium, cinnamon, coenzyme Q10, colloidal silver, cranberry, vitamin C, candelion, echinacea, ephedra, essiac/flor-essence, European elder, evening primrose oil, fenugreek, feverfew, fish oil, flaxseed, folate, garlic, ginger, ginkgo, ginseng,

glucosamine, goldenseal, grape seed extract, green tea, hawthorn, hoodia, horse chestnut, iodine, iron, kava, lavender, licorice root, magnesium, melatonin, milk thistle, mistletoe, noni, omega-3 fatty acids, PC-SPES, peppermint oil, red clover, sage, SAMe (S-adenosyl-L-methionine), saw palmetto, selenium, soy, St. John's wort, tea, thunder God vine, turmeric, valerian, vitamin A, vitamin B12, vitamin B6, vitamin C, vitamin D, vitamin E, vitamin K, yohimbe, and zinc. Additionally, a dietary supplement may be derived from a food or food item. For example, a dietary supplement may be derived from cereal grains, legumes, nuts, seeds, fruits and/or vegetables. A dietary supplement may be high in non-digestible carbohydrates and antioxidants. Suitable examples of a dietary supplement high in non-digestible carbohydrates and antioxidants may include agave, chicory, banana, barley, buckwheat, canary seed, chia, cocoa, coffee, corn, fonio, grape, guar, job tears, millet, millet seed, oat, quinoa, rice, rye, sorghum, soy, spelt, teff, tigernut, triticale, wheat bran, acacia and citrus fruits. A dietary supplement may be about 0-10% by wet weight of the compositions described above. For example, a dietary supplement may be about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 % wet weight of the compositions described above. Alternatively, a dietary supplement may be about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10% wet weight of the compositions described above. For a dietary supplement that is a milk or milk product, meat, poultry, fish, egg, nuts, seed, legume, grain, fruit, vegetable, fat, oil, sugar, sweet, and/or beverage, the dietary supplement may be about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10% wet weight of the compositions described above. For a dietary supplement that is a vitamin, mineral, herb, botanical, and/or amino acid, the dietary supplement may be about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 % wet weight of the compositions described above.

[0036] In each of the embodiments above, a composition of the invention may be processed into a form for animal consumption. In preferred embodiments, compositions of the invention are formulated as chow for a laboratory animal. Non- limiting examples of a laboratory animal may include rodents, canines, felines, and non- human primates. In some embodiments, the animal is a mouse. In other embodiments, the animal is a rat. In still other embodiments, the animal is a guinea pig. In yet other embodiments, the animal is a hamster. Methods for processing human foods into an animal feed ration are described below in Section III and further detailed in the

Examples.

III. PROCESSES FOR PRODUCING AN ANIMAL FEED COMPOSITION

COMPRISING A HUMAN DIET EQUIVALENT

[0037] In another aspect, the present invention encompasses processes for producing an animal feed composition comprising a human diet equivalent. Suitable human diet equivalents and animal feed compositions are described above in Section I and II. The process typically comprises (i) introducing one or more food items selected from the group consisting of a frozen food item, a refrigerated food item, a room temperature food item, and a combination thereof into a first mixer and blending the food items, (ii) passing the blended food items into a second mixer to form a

homogenous composite, (iii) dehydrating the homogenous composite and milling the dehydrated composite to a 20 mesh size, and (iv) processing the milled composite into its final form. Step (ii) can be optionally incorporated into step (i) through a recirculation loop that is connected to the vessel carrying out the first mixing step. In certain embodiments, the composition in its final form may then be packaged and/or sterilized by gamma irradiation. Sterilization by gamma irradiation may occur before or after packaging, or a combination thereof.

[0038] A process of the invention generally starts with frozen, refrigerated and/or room temperature food items. Generally, the food items will characterize a diet of a subpopulation and, in total, will provide the right proportions of nutrients for each particular diet. The food items, or the ingredients to make a food item, may be purchased from a grocery store, a farmers market or any other vendor that sells food items or the ingredients to make them. A food item may be used as is or may be prepared from ingredients. Typical preparation methods may include, but are not limited to, peeling, coring, cutting, chopping, dicing, grating, shredding, mincing, de-seeding, kneading, milling, mixing, blending, thawing, boiling, blanching, braising, coddling, infusing, simmering, steaming, steeping, stewing, grilling, broiling, frying, sauteing, microwaving, roasting, searing, pickling, salting, seasoning, marinating, brining, and drying. Recipes for preparing food items that require preparation are known in the art. Food items that require preparation may be prepared as needed, or may be made days, weeks or months in advance, optionally weighed into batches, and frozen until needed.

[0039] Steps (i) and (ii) of the process of the invention comprise intimate mixing. Intimate mixing, as used herein, refers to high shear mixing, homogenization, such as through a homogenizer, sonification, or through ultrasonification. Intimate mixing disperses food items (present as liquid, solid and/or gas) into a main continuous liquid phase, with which it would normally be immiscible. A rotor or impellor, alone or together with a stationary component known as a stator, or an array of rotors and stators, is used either in a tank containing the solution to be mixed, or in a pipe through which the solution passes, to create shear. The end result of steps (i) and (ii) is a homogeneous composite mixture. As used herein, the term "homogenous composite mixture" or "homogenous composite" refers to a mixture that is an emulsion, a suspension, or an emulsion and a suspension. Intimate mixing may or may not occur in combination with other kinds of mixing, for example, static mixing. Non-limiting examples of intimate mixers includes a rotor-stator type homogenizer or mixer, a high pressure homogenizer, a high shear granulator, a batch high-shear mixer, and an impeller mixer (including both an axial flow impeller and a radial flow impeller). In some embodiments, mixing is provided by a high shear mixer. In other embodiments, mixing is provided by a rotor-stator mixer. In still other embodiments, mixing is provided by a homogenizer. In alternative embodiments, mixing is provided by more than one mixer. In preferred embodiments, mixing is provided by a first mixer and a second mixer, wherein the first mixer is a high shear mixer and the second mixer is a rotor-stator type mixer. In an exemplary embodiment, a first mixer is a Breddo mixer and a second mixer is a Silverson Mixer/Pump.

[0040] In some embodiments, a process of the invention comprises introducing one or more food items selected from the group consisting of a frozen food item, a refrigerated food item, a room temperature food item, or a combination thereof into a first mixer. For example, a process of the invention may comprise introducing 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35 or more food items into a first mixer. In embodiments where more than one food item is added to a first mixer, each food item is

independently selected from the group consisting of a frozen food item, a refrigerated food item, and a room temperature food item.

[0041 ] Food items may or may not be added to a first mixer in a particular order. In some embodiments, food items are added to a first mixer in a particular order. In other embodiments, food items are added to a first mixer in a random order. A suitable order for the addition of food items to a first blender for a HiSF-LoFV and LoSF- HiFV diet is provided in the Examples. Those skilled in the art should, however, in light of the present disclosure, appreciate that changes can be made to the order disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0042] In a preferred embodiment, a first mixer is a high shear mixer. For example, a first mixer may be an impeller mixer. The type of impeller can vary, provided the impeller type produces sufficient shear for the blending process to achieve a homogenous emulsion and/or suspension. The size of the impeller can and will vary depending on other parameters. In some embodiments, the impeller diameter is about 5 to about 36 inches. For example, the impeller diameter may be about 5 inches, about 6 inches, about 7 inches, about 8 inches, about 9 inches, about 10 inches, about 1 1 inches, about 12 inches, about 14 inches, about 15 inches, about 16 inches, about 17 inches, about 18 inches, about 19 inches, about 20 inches, about 21 inches, about 22 inches, about 23 inches, about 24 inches, about 25 inches, about 26 inches, about 27 inches, about 28 inches, about 29 inches, about 30 inches, about 31 inches, about 32 inches, about 33 inches, about 34 inches, about 35 inches, or about 36 inches. In other embodiments, the impeller diameter is about 5 to about 10 inches. In still other embodiments, the impeller diameter is about 10 to about 15 inches. In different embodiments, the impeller diameter is about 15 to about 20 inches. In still different embodiments, the impeller diameter is about 20 to about 25 inches. In alternative embodiments, the impeller diameter is about 35 to about 30 inches. In additional embodiments, the impeller diameter is about 30 to about 36 inches. The rotations per minutes (rpm) of the impeller can vary in each of the above embodiments. In some embodiments, the speed ranges from about 1500 rpm to about 3500 rpm. In certain embodiments the speed is 1500 rpm, 1600 rpm, 1700 rpm, 1800 rpm, 1900 rpm, 2000 rpm, 2100 rpm, 2200 rpm, 2300 rpm, 2400 rpm, 2500 rpm, 2600 rpm, 2700 rpm, 2800 rpm, 2900 rpm, 3000 rpm, 3100 rpm, 3200 rpm, 3300 rpm, 3400 rpm, or 3500 rpm. In preferred embodiments, the speed does not exceed 2000 rpm.

[0043] To facilitate blending, food items added to a first mixer may be optionally recirculated or passed through a second mixer until evenly blended by visual inspection. In a preferred embodiment, the second mixer is a rotor-stator type mixer. The size of the rotor can and will vary depending on other parameters. In some embodiments, the rotor diameter is about 1 to about 24 inches. For example, the rotor diameter may be about 1 inch, about 2 inches, about 3 inches, about 4 inches, about 5 inches, about 6 inches, about 7 inches, about 8 inches, about 9 inches, about 10 inches, about 1 1 inches, about 12 inches, about 14 inches, about 15 inches, about 16 inches, about 17 inches, about 18 inches, about 19 inches, about 20 inches, about 21 inches, about 22 inches, about 23 inches, or about 24 inches. In other embodiments, the rotor diameter is about 1 to about 5 inches. In still other embodiments, the rotor diameter is about 5 to about 10 inches. In yet other embodiments, the rotor diameter is about 10 to about 15 inches. In different embodiments, the rotor diameter is about 15 to about 20 inches. In still different embodiments, the rotor diameter is about 20 to about 24 inches. The rotations per minutes (rpm) of the rotor can vary in each of the above embodiments. In some embodiments, the speed ranges from about 1500 rpm to about 3500 rpm. In certain embodiments the speed is 1500 rpm, 1600 rpm, 1700 rpm, 1800 rpm, 1900 rpm, 2000 rpm, 2100 rpm, 2200 rpm, 2300 rpm, 2400 rpm, 2500 rpm, 2600 rpm, 2700 rpm, 2800 rpm, 2900 rpm, 3000 rpm, 3100 rpm, 3200 rpm, 3300 rpm, 3400 rpm, or 3500 rpm. In preferred embodiments, the speed does not exceed 2000 rpm. [0044] In a preferred embodiment, the first mixer is attached through a recirculating loop to the second mixer that is in-line. The recirculating loop may be chosen from any suitable material including, but not limited to, tubing or piping. The second mixer may be optionally employed when, following blending with the first mixer, the mixture comprises a visible distribution of particle sizes in order to create an even particle size distribution.

[0045] Temperature may also be controlled during mixing. The

temperature of the mixture is generally maintained as cool as possible while maintaining the mixture in a liquid phase. Preferably, the maximum temperature during steps (i) and (ii) is about 60°F.

[0046] The residence time from the introduction of the food items to the end of step (ii) may range from about 10 minutes to about 120 minutes, and is dependent, on multiple factors related to process design. In some embodiments, residence time is about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 1 10 minutes, about 1 15 minutes, or about 120 minutes. In other embodiments, residence time is about 10 to about 40 minutes. In still other embodiments, residence time is about 20 to about 50 minutes. In still other embodiments, residence time is about 30 to about 60 minutes. In still other embodiments, residence time is about 40 to about 70 minutes. In still other embodiments, residence time is about 50 to about 80 minutes. In still other

embodiments, residence time is about 60 to about 90 minutes. In still other

embodiments, residence time is about 70 to about 100 minutes. In still other

embodiments, residence time is about 80 to about 1 10 minutes. In still other

embodiments, residence time is about 90 to about 120 minutes. In a preferred embodiment, residence time does not exceed 2 hours.

[0047] A homogenous composite produced by the process of the invention is collected and dehydrated. In certain embodiments, the homogenous composite may be optionally frozen and/or stored prior to dehydration. Suitable dehydration processes are known in the art. In some embodiments, the homogenous composite is freeze dried. Briefly, a homogenous composite is frozen in an appropriate container, finely shredded and placed in a vacuum chamber until available moisture was reduced. The moisture content of the freeze dried composite is generally reduced to less than 10%. For example, the moisture content of the freeze dried composite may be reduced to less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1 %. In some embodiments, the moisture content of the freeze dried composite is about 1 % to about 5%. In other embodiments, the moisture content of the freeze dried composite is about 5% to about 10%. In different embodiments, the moisture content of the freeze dried composite is about 1 % to about 3%. In still different embodiments, the moisture content of the freeze dried composite is about 3% to about 6%. In alternative embodiments, the moisture content of the freeze dried composite is about 6% to about 9%.

[0048] The temperature and pressure of freeze drying can and will vary in order to achieve the described moisture content. In some embodiments, the

temperature is about -20°C to about -60°C. For example, the temperature may be about -20°C, about -21 °C, about -22°C, about -23°C, about -24°C, about -25°C, about -26°C, about -27°C, about -28°C, about -29°C, about -30°C, about -31 °C, about -32°C _i about - 33°C, about -34°C, about -35°C, about -36°C, about -37°C, about -38°C, about -39°C, about -40°C, about -41 °C, about -42 ⁰ C ₅ about -44°C, about -44°C, about -45°C, about - 46°C, about -47°C, about -48°C, about -49°C, about -50°C, about -51 °C, about -52 ⁰ C ₅ about -53°C, about -54°C, about -55°C, about -56°C, about -57°C, about -58°C, about - 59°C, or about -60°C. In some embodiments, the pressure of the vacuum chamber is about 0.001 mbar to about 30 mbar. For example, the pressure of the vacuum chamber may be about 0.001 mbar to about 0.010 mbar, about 0.010 mbar to about 0.100 mbar, about 0.100 mbar to about 1 .000 mbar, about 1 .000 mbar to about 10.000 mbar, or about 10.000 mbar to about 30.000 mbar. In another example, the pressure of the vacuum chamber may be about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 mbar.

[0049] Dehydrated composite may then be milled to a suitable size for processing to a final form. The type of 'grind' (i.e. coarse, medium or fine) plays an important part in press output and quality of final form. Very coarse grinds have the added disadvantage of providing breaking points in the cubes or pellets. Medium and fine grinds generally will result in higher pelleting capacity than a coarse grind. In some embodiments, dehydrated composite is ground to a uniform size. In other embodiments, dehydrated composite is ground to a mixture of grinds of differing fineness. Dehydrated composite of the invention is generally ground to a mesh size of about 5 to about 100. In some embodiments, dehydrated composite is milled to about a 5 mesh size to about a 20 mesh size. In other embodiments, dehydrated composite is milled to about a 15 mesh size to about a 30 mesh size. In still other embodiments, dehydrated composite is milled to about a 25 mesh size to about a 40 mesh size. In yet other embodiments, dehydrated composite is milled to about a 35 mesh size to about a 50 mesh size. In still other embodiments, dehydrated composite is milled to about a 45 mesh size to about a 60 mesh size. In still other embodiments, dehydrated composite is milled to about a 55 mesh size to about a 70 mesh size. In still other embodiments, dehydrated composite is milled to about a 65 mesh size to about a 80 mesh size. In still other embodiments, dehydrated composite is milled to about a 75 mesh size to about a 90 mesh size. In still other embodiments, dehydrated composite is milled to about a 85 mesh size to about a 100 mesh size. In different embodiments, dehydrated composite is milled to at least a 40 mesh size. In alternative embodiments, dehydrated composite is milled to at least a 30 mesh size. In a preferred embodiment, dehydrated composite is milled to at least a 20 mesh size. In another preferred embodiment, dehydrated composite is milled to a 20 mesh size. Methods for milling are well known in the art.

[0050] The milled composite is then processed into a final form. In some embodiments, the milled composite is processed into a liquid. In other embodiments, the milled composite is processed into a dried form. Non-limiting examples of suitable dried forms include powders, agglomerates, granules and pellets. In a preferred embodiment, the milled composite is processed to a granule. In another preferred embodiment, the milled composite is processed to a pellet. When processed into a dried form, moisture may be added to the milled composite to facilitate processing. Generally, the moisture content of the final form is generally less than 10%. For example, the moisture content of the may be less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1 %. In some embodiments, the moisture content of the freeze dried composite is about 1 % to about 5%. In other embodiments, the moisture content of the freeze dried composite is about 5% to about 10%. In different embodiments, the moisture content of the freeze dried composite is about 1 % to about 3%. In still different embodiments, the moisture content of the freeze dried composite is about 3% to about 6%. In alternative embodiments, the moisture content of the freeze dried composite is about 6% to about 9%. In a preferred embodiment, the milled composite is processed to a pellet and the moisture content at the end of the extrusion process is less than 7%. Methods for producing animal feed ration into powder, granules and pellets are known in the art.

[0051 ] In some embodiments, a dietary supplement may be added during the processing of the composition. In an embodiment, a dietary supplement may be added during steps (i) or (ii). In another embodiment, a dietary supplement may be added after step (iii) prior to step (iv). For example, a dietary supplement may be added to a milled composite prior to processing into a final form. In an embodiment where a dietary supplement is added to a milled composite, the dietary supplement may be dehydrated. The dehydration of the dietary supplement may enable homogenous distribution of the dietary supplement and the milled composite. In a preferred

embodiment, a dietary supplement may be added to a milled composite that has been processed into a dried from. In other preferred embodiments, a dietary supplement may be added to a milled composite that has been processed into a powder. The milled composite comprising the dietary supplement may then be processed to a pellet. In an embodiment where a dietary supplement is added to a milled composite that has been processed into a powder, the dietary supplement may be dehydrated and of an equivalent particle size to the particles of the powder. The milled composite comprising the dietary supplement may then be processed to a pellet.

IV. METHODS

[0052] In another aspect, the invention encompasses a method to determine the effect of at least one dietary supplement on a subject. More specifically, the invention encompasses a method to determine the effect of at least one dietary supplement on a gut microbiota of a subject. The method generally comprises evaluating the subject before and after consumption of at least one dietary supplement, wherein the presence or absence of a difference in the subject represents the effect of the dietary supplement on the subject. More specifically, the method generally comprises evaluating the gut microbiota of a subject before and after consumption of at least one dietary supplement, wherein the presence or absence of a difference in the gut microbiota represents the effect of the dietary supplement on the gut microbiota of the subject.

[0053] For example, the method may comprise administering or feeding a composition of the invention comprising at least one dietary supplement to a subject and determining the presence or absence of a difference in the subject after

administering or feeding the composition comprising the dietary supplement(s), wherein the presence or absence of a difference in the subject represents the effect of the dietary supplement(s) on the subject. Alternatively, the method may comprise (1 ) administering or feeding a first composition of the invention to a subject for a first period of time, (2) administering or feeding a second composition to the subject for a second period of time, wherein the second composition consists of the first composition and at least one dietary supplement, and (3) determining the presence or absence of a difference in the subject after administering or feeding the second composition, wherein the presence or absence of a difference in the subject represents the effect of the dietary supplement on the subject. In another alternative, the method may comprise (1 ) administering or feeding a first composition to a subject for a first period of time, wherein the first composition is a composition of the invention further comprising at least one dietary supplement, (2) administering or feeding a second composition to the subject for a second period of time, wherein the second composition consists of the first

composition without the at least one dietary supplement, and (3) determining the presence or absence of a difference in the subject after administering or feeding the second composition, wherein the presence or absence of a difference in the subject represents the effect of the dietary supplement on the subject. As a non-limiting example, the method may comprise comparing the gut microbiota of the subject before and after administration or feeding of the two compositions to determining the presence or absence of a difference, wherein the presence or absence of a difference represents the effect of the dietary supplement(s) on the subject.

[0054] Alternatively, the method may comprise (1 ) administering or feeding to a first subject or first group of subjects a composition comprising at least one dietary supplement and administering or feeding to a second subject or second group of subjects the same composition without said dietary supplement(s), (2) comparing the first subject or groups of subjects to the second subject or group of subjects, and (3) determining the presence or absence of a difference between the first and second subjects or group of subjects, wherein the presence or absence of a difference represents the effect of the dietary supplement(s) on the subject or group of subjects. As a non-limiting example, the method may comprise comparing the gut microbiota of the first subject or groups of subjects to the second subject or group of subjects and determining the presence or absence of a difference between the first and second subjects or group of subjects, wherein the presence or absence of a difference represents the effect of the dietary supplement(s) on the subject.

[0055] The effect of one dietary supplement, or a combination of more than one dietary supplement (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or more supplements), on a subject may be determined by quantifying any measurable change in a subject. Without wishing to be bound by theory, the effect of one or more dietary supplements on a subject may be determined by measuring a change in gut microbiota, a change in weight, a change in body fat, a change in energy consumption, a change in health, a change in inflammation, a change in immune function, a change in metabolism, or a combination thereof. Non-limiting examples of indicators of health may include low birth weight, obesity, arthritis, diabetes, asthma, allergies, high blood pressure, autoimmune disease, heart disease, respiratory disease, cancer incidence, disease resistance, disease susceptibility, pathogen shedding, chronic pain, depression, hospital visits due to injury, foodborne illnesses, mood, mental health, smoking habits, alcohol

consumption, physical exercise habits, and breastfeeding. Methods of measuring indicators of health are known in the art. Further, methods of measuring energy consumption, weight, body fat, inflammation, immune function and metabolism are known in the art. Inflammation or an inflammatory response is achieved by increased movement of plasma and leukocytes from the blood to the region of interest.

Inflammation may be acute or chronic. Signs of acute inflammation may include, but are not limited to, pain, heat, redness, swelling, and loss of function. Signs of chronic inflammation may include, but are not limited to, allergies, hay fever, asthma, periodontitis, atherosclerosis, rheumatoid arthritis and cancer. As such, a change in inflammation may be a change in one or more signs of acute inflammation or a change in one or more signs of chronic inflammation. Alternatively, inflammation may be measured by erythrocyte sedimentation rate or C-reactive protein. Other methods of measuring inflammation are known in the art and may also be used. Immune function refers to the functioning of the immune system. The immune system protects against disease. Disorders of the immune system may result in autoimmune disease, inflammatory diseases and cancer. As such, a change in immune function may be measured as a change in susceptibility to disease or a change in the signs or symptoms associated with disorders of the immune system. Alternatively, immune function may be measure by quantitation of cell-mediated immunity. Other methods of measuring immune function are known in the art and may also be used. Metabolism is enzyme- catalyzed reactions that allow organisms to grow and reproduce, maintain their structures, and respond to their environments. Metabolism may include digestion and the transport of substances into and between different cells. Metabolism may be measured as a metabolic rate. Metabolic rate is the rate of energy expenditure by a subject. As such, a change in metabolism may be measured by a change in digestion, a change in metabolic rate, a change in energy consumption, or a change in energy expenditure. Alternatively, other methods of measuring metabolism are known in the art and may be used.

[0056] The effect of a dietary supplement on a gut microbiota may be measured as change in relative and/or absolute abundance of microbes at any taxonomic level, a change in phylotypic composition, a change in expression of nucleic acids or nucleic acid products, or a change in metabolite profile. Methods to determine the relative or absolute abundance of microbes are known in the art and may include culturing or sequencing. The term "phylotypic composition," as used herein, refers to the composition of a gut microbiota as defined by phylotypes. A phylotype is a biological type that classifies an organism by its phylogenetic, e.g. evolutionary, relationship to other organisms. The term phylotype is taxon-neutral, and therefore, may refer to the species composition, genus composition, class composition, etc. Methods to determine expression of nucleic acids are known in the art and may include northern blot, PCR, RT-PCR, qRT-PCR, array, or microarray. Methods to determine expression of nucleic acid products are known in the art and may include western blot, array, microarray, antibody- or epitope binding agent-based methods, mass spectrometry, or peptide sequencing. Methods of measuring a change in metabolite profile are also known in the art and are common in the field of metabolomics.

[0057] As used herein, "change" means more than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % difference between the subject before and after consumption of the dietary supplement. Alternatively, "change" means more than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 % difference between the subject consuming the dietary supplement compared to the subject not consuming the dietary supplement. A change may be positive or negative (e.g. increase or decrease).

[0058] In the embodiments above, a subject may be a laboratory animal. Non-limiting examples of a laboratory animal may include rodents, canines, felines, and non-human primates. In some embodiments, the animal is a mouse. In other embodiments, the animal is a rat. In still other embodiments, the animal is a guinea pig. In yet other embodiments, the animal is a hamster. The effect of the dietary supplement on a subject may be used as a surrogate to determine the effect of a dietary supplement on a human.

TABLE A. USDA Food and Nutrient Database for Dietary Studies Food Coding Scheme (The USDA Food and Nutrient Database for Dietary Studies, 4.1 - Documentation and User Guide (2010) Beltsville, MD: U.S. Department of

Agriculture, Agricultural Research Service, Food Surveys Research Group)

FOOD CODE

DESCRIPTION

(1 ^st four digits)

Milk and Milk Products 1XXX

Milks and milk drinks 11XX

Milk, human 110X

Milk, fluid (regular; filled; buttermilk; and dry

reconstituted) 111X

Milk, fluid, evaporated and condensed 112X

Milk, fluid, imitation 113X

Yogurt 114X

Yogurt, baby food 1148

Flavored milk and milk drinks, fluid 115X

Milk-based meal replacements, fluid 116X

Infant formulas, fluid, reconstituted concentrate,

reconstituted dry, and ready-to-feed (milk-based

formulas; soy-based formulas; therapeutic

formulas) 117X

Milk, dry, and powdered mixtures with dry milk,

not reconstituted 118X

Creams and cream substitutes 12XX

Sweet dairy cream 121X

Cream substitutes 122X

Sour cream 123X

Milk desserts, sauces, gravies 13XX

Milk desserts, frozen 131X

Puddings, custards, and other milk desserts 132X

Milk desserts baby food 133X

White sauces and milk gravies 134X

Cheeses 14XX

Cheese, NS6 as to type 140X

Natural cheeses 141X

Cottage cheeses 142X

Cream cheeses 143X Processed cheeses and cheese spreads 144X

Imitation cheeses 145X

Cheese mixtures 146X

Cheese soups 147X

Meat, Poultry, Fish, and Mixtures 2XXX

Meat, NS as to type 20XX

Meat, NS as to type 200X

Beef 21XX

Beef, NFS7 210X

Beef steak 211X

Beef oxtails, neckbones, short ribs, head 213X

Beef roasts, stew meat, corned beef, beef brisket,

sandwich steaks 214X

Ground beef, beef patties, beef meatballs 215X Other beef items (beef bacon; dried beef;

pastrami) 216X

Beef baby food 217X

Pork 22XX

Pork, NFS; ground, dehydrated 220X

Pork chops 221X

Pork steaks, cutlets 222X

Ham 223X

Pork roasts 224X

Canadian bacon 225X

Bacon, salt pork 226X

Other pork items (spareribs; cracklings; skin;

miscellaneous parts) 227X

Pork baby food 228X

Lamb, veal, game, other carcass meat 23XX

Lamb, NFS 230X

Lamb and goat 231X

Veal 232X

Game 233X

Lamb or veal baby food 234X

Poultry 24XX

Chicken (breast; leg; drumstick; wing; back; neck or

ribs; misc,) 241X

Turkey 242X

Duck 243X

Other poultry 244X

Poultry baby food 247X

Organ meats, sausages and lunchmeats, and meat

spreads 25XX

Organ meats and mixtures 251X

Liver 2511

Hearts 2512 Kidney 2513 Sweetbreads 2514 Brains 2515 Tongue 2516 Other variety meats 2517 Frankfurters, sausages, lunchmeats, meat spreads 252X Frankfurters 2521 Sausages 2522 Luncheon meats (loaf) 2523 Potted meat, spreads 2524 Fish and shellfish 26XX Finfish 261X Other seafood 262X Shellfish 263X Meat, poultry, fish with nonmeat items 27XX Meat, poultry, fish in gravy or sauce or creamed 271X Beef in gravy or sauce (tomato-based sauce;

gravy; cream, white, or soup-based sauce; soy- based sauce; other sauce; Puerto ican) 2711 Pork with gravy or sauce 2712 Lamb and veal with gravy or sauce 2713 Poultry with gravy or sauce (tomato-based

sauce; gravy; cream, white, or soup-based

sauce; soy-based sauce; other sauces; Puerto

Rican) 2714 Fish, shellfish with gravy or sauce 2715 Miscellaneous meats with gravy or sauce 2716 Meat, poultry, fish with starch item (include white

potatoes) 272X Beef with starch item (potatoes; noodles; rice;

bread; Puerto Rican) 2721 Pork with starch item 2722 Lamb, veal, game with starch item 2723 Poultry with starch item (potatoes; noodles;

rice; bread) 2724 Fish, shellfish with starch item 2725 Miscellaneous meats with starch item 2726 Meat, poultry, fish with starch item and vegetables 273X Beef with starch and vegetable (potatoes;

noodles; rice; bread; Puerto Rican) 2731 Pork with starch and vegetable 2732 Lamb, veal, game with starch and vegetable 2733 Poultry with starch and vegetable (potatoes;

noodles; rice; bread; Puerto Rican) 2734 Fish, shellfish with starch and vegetable 2735 Miscellaneous meats with starch and vegetable 2736 Meat, poultry, fish with vegetables (excluding

white potatoes) 274X

Beef with vegetable, no potatoes 2741

Pork with vegetable, no potatoes 2742

Lamb, veal, game with vegetable, no potatoes 2743

Poultry with vegetables, no potatoes 2744

Fish, shellfish with vegetables, no potatoes 2745 Miscellaneous meats with vegetable, no

potatoes 2746

Sandwiches with meat, poultry, fish 275X

Beef sandwiches 2751

Pork sandwiches 2752

Poultry sandwiches 2754

Fish, shellfish sandwiches 2755 Frankfurters, luncheon meat, potted meat

sandwiches 2756

Hors d'oeuvres, finger sandwiches 2757

Meat, poultry, fish with nonmeat items baby food 276X

Beef mixtures baby food 2761

Poultry mixtures baby food 2764 Frozen and shelf-stable plate meals, soups, and gravies

with meat, poultry, fish base; gelatin and gelatin-based

drinks 28XX

Frozen or shelf-stable plate meals with meat,

poultry, fish as major ingredient 281X

Beef frozen or shelf-stable meals 2811

Pork or ham frozen or shelf-stable meals 2812

Veal frozen or shelf-stable meals 2813

Poultry frozen or shelf-stable meals 2814

Fish, shellfish frozen meals 2815

Miscellaneous meat frozen or shelf-stable meals 2816

Soups, broths, extracts from meat, poultry, fish

base 283X

Beef soups 2831

Pork soups 2832

Lamb soups 2833

Poultry, soups 2834

Fish, shellfish soups 2835

Puerto ican soups 2836

Gelatin and gelatin-based meal supplements 284X

Gravies from meat, poultry, fish base 285X

Eggs 3XXX

Eggs 31XX

Chicken eggs 311X

Other poultry eggs 312X

Egg mixtures 32XX Egg dishes 321X

Egg sandwiches 322X

Egg soups 323X

Meringues 324X

Egg substitutes 33XX

Egg substitute, NS as to form 330X

Egg substitute, from powdered mixture 331X

Egg substitute, from frozen mixture 332X

Egg substitute, from liquid mixture 333X

Frozen plate meals with egg as major ingredient 35XX

Frozen plate meals with egg as major ingredient 350X

Dry Beans, Peas, Other Legumes, Nuts, and Seeds 4XXX

Legumes 41XX

Dried beans 411X

Dried beans mixtures 412X

Dried peas, lentils, and mixtures 413X

Soybean derived products (excluding milks) 414X Frozen plate meals with legumes as major

ingredient 415X

Soups with legumes as major ingredient 416X

Meat substitutes, mainly legume protein 418X

Meat substitute sandwiches 419X

Nuts, nut butters, and nut mixtures 42XX

Nuts 421X

Nut butters 422X

Nut butter sandwiches 423X

Coconut beverages 424X

Nut mixtures 425X

Seeds and seed mixtures 43XX

Seeds 431X

Carob products 44XX

Carob powder, flour 441X

Carob chips, syrup 442X

Grain Products 5XXX

Flour and dry mixes 50XX

Flour and dry mixes 500X

Yeast breads, rolls 51XX

Breads, rolls, NFS 510X

White breads, rolls 511X

Whole wheat breads, rolls 512X

Wheat, cracked wheat breads, rolls 513X

Rye breads, rolls 514X

Oat breads 515X

Multigrain breads, rolls 516X

Other breads 518X

Quick breads 521 Biscuits 52XX Cornbread, corn muffins, tortillas 522X

Other muffins, popovers 523X

Other quick breads 524X

Cakes, cookies, pies, pastries 531 Cakes 53XX

Cookies 532X Pies (fruit pies; pie tarts; cream, custard, and

chiffon pies; miscellaneous pies; pie shells) 533X

Cobblers, eclairs, turnovers, other pastries 534X

Danish, breakfast pastries, doughnuts, granola bars 535X

Coffee cake, not yeast 536X

Cookies and bars, baby food 538X

Crackers and salty snacks from grain products 54XX

Crackers, NS as to type 540X

Sweet crackers 541X

Low sodium crackers 542X

Nonsweet crackers 543X

Salty snacks from grain products 544X

Pancakes, waffles, french toast, other grain products 55XX

Pancakes 551X

Waffles 552X

French toast 553X

Crepes 554X

Flour-water patties 555X

Flour-milk patties 556X

Rice flour cakes 557X

Funnel cakes 558X

Pastas, cooked cereals 56XX

Pastas 561X

Cooked cereals, rice 562X

Cereals, not cooked or NS as to cooked 57XX

Cereal, NS as to cooked 570X

Ready-to-eat cereals 571X

Ready-to-eat cereals 572X

Ready-to-eat cereals 573X

Ready-to-eat cereals 574X

Cereal grains, not cooked 576X

Cereals baby food 578X

Grain mixtures, frozen plate meals, soups 58XX

Mixtures, mainly grain, pasta, or bread 581X

Mixtures, mainly grain, pasta, or bread 582X Frozen plate meals with grain mixture as major

ingredient 583X

Soups with grain product as major ingredient 584X

Grain mixtures baby food 585X

Meat substitutes, mainly cereal protein 59XX

Meat substitutes, mainly cereal protein 590X Fruits 6XXX

Citrus fruits, juices 61XX

Citrus fruits 611X

Citrus fruit juices 612X

Dried fruits 62XX

Dried fruits 621X

Other fruits 63XX

Fruits, excluding berries 631X

Berries 632X

Mixtures of two or more fruits 633X

Mixtures of fruits and nonfruit items 634X

Fruit juices and nectars excluding citrus 64XX

Fruit juices, excluding citrus 641X

Nectars 642X

Vinegar 644X

Fruits and juices baby food 67XX

Fruits and fruit mixtures baby food 671X

Fruit juice and fruit juice mixtures baby food 672X

Fruits with cereal baby food 673X Fruit desserts and fruit-flavored puddings and

yogurt desserts baby food 674X

Fruits with meat or poultry baby food 675X

Fruits and vegetables mixtures baby food 676X

Vegetables 7XXX

White potatoes and Puerto Rican starchy vegetables 71XX

White potatoes, NFS 710X

White potatoes, baked and boiled 711X

White potatoes, chips and sticks 712X White potatoes, creamed, scalloped, au gratin 714

White potatoes, fried 713X White potatoes, fried 714X

White potatoes, mashed, stuffed, puffs 715X

Potato salad 716X

Potato recipes 717X

Potato soups 718X

Puerto Rican starchy vegetables 719X

Dark-green vegetables 72XX

Dark-green leafy vegetables 721X

Dark-green nonleafy vegetables 722X

Dark-green vegetable soups 723X

Deep-yellow vegetables 73XX

Carrots 731X

Pumpkin 732X

Squash, winter 733X

Sweet potatoes 734X

Deep-yellow vegetable soups 735X Tomatoes and tomato mixtures 74XX

Tomatoes, raw 741X

Tomatoes, cooked 742X

Tomato juices 743X

Tomato sauces 744X

Tomato mixtures 745X

Tomato soups 746X

Tomato sandwiches 747X

Other vegetables 75XX

Other vegetables, raw 751X

Raw vegetable mixtures 7514

Other vegetables, cooked 752X

Other vegetable mixtures, cooked 753X Other cooked vegetables, cooked with sauces,

batters, casseroles 754X

Olives, pickles, relishes (excluding tomatoes) 755X

Vegetable soups 756X

Vegetables and mixtures mostly vegetables baby food 76XX

Dark-green vegetables baby food 761X

Deep-yellow vegetables baby food 762X Vegetables other than dark-green, deep-yellow,

and tomato baby food 764X

Vegetables with grain baby food 765X

Vegetables with meat baby food 766X

Vegetables with meat, poultry, fish 77XX

White potato with meat, poultry, fish (mixtures) 771X

Puerto Rican starchy vegetable (viandas) mixtures 772X

Other vegetable mixtures 773X Puerto Rican stews or soups with starchy

vegetables (viandas) 775X

Mixtures mostly vegetables without meat, poultry, fish 78XX

Vegetable and fruit juice blends, 100% juice 781X

Fats, Oils, and Salad Dressings 8XXX

Fats 81XX

Table fats 811X

Cooking fats 812X

Other fats 813X

Oils 82XX

Vegetable oils 821X

Salad dressings 83XX

Regular salad dressings 831X

Low-calorie and reduced calorie salad dressings 832X

Sugars, Sweets, and Beverages 9XXX

Sugars and sweets 91XX

Sugars and sugar-sugar substitute blends 911X

Sugar replacements or substitute 912X Syrups, honey, molasses, sweet toppings 913X

Jellies, jams, preserves 914X

Gelatin desserts or salads 915X

Ices or popsicles 916X

Candies 917X

Chewing gums 918X

Nonalcoholic beverages 92XX

Coffee 921X

Coffee substitutes 922X

Tea 923X

Soft drinks, carbonated 924X

Fruit drinks 925X

Fruit juice drinks and fruit flavored drinks 9251 Group Discontinued as of 12/31/2004;

previously described as "Fruitades and drinks,

low calorie, NS as to vitamin C content" 9252 Fruit juice drinks and fruit flavored drinks with

high vitamin C 9253

Fruit flavored drinks, made from powdered mix 9254 Fruit juice drinks and fruit flavored drinks, low

calorie 9255

Sports drinks and thirst quencher beverages 9256

Beverages, fluid replacement 9257 Fruit juice drinks and fruit flavored drinks,

fortified with calcium 9258

Beverages, nonfruit 926X Beverages, nonfruit, fortified (include energy

drinks) 9265 Group Discontinued as of 12/31/2004; previously

described as "Beverages, noncarbonated, without

vitamin C, made from powdered mixes" 927X

Nonalcoholic beers, wines, cocktails 928X

Beverage concentrates, dry, not reconstituted 929X

Alcoholic beverages 93XX

Beers and ales 931X

Cordials and liqueurs 932X

Cocktails 933X

Wines 934X

Distilled liquors 935X

Water, noncarbonated 940 Water, not bottled 94XX

Water, bottled 941X

Water, bottled, fortified 942X

Note: X represents a place holder, and may be any digit from 1-9. EXAMPLES

[0059] The following examples illustrate various iterations of the invention.

Example 1. Development of Human Food-Based Diets, Based on Two "Typical" U.S. Adult Diets Differing in Saturated Fat and Fruit/Vegetable Consumption

[0060] Dietary recalls collected on Day 1 of the National Health and Nutrition Examination Surveys (NHANES) administered in 2003-2004, 2005-2006 and 2007-2008 were used: 1 ) to identify healthy, non-pregnant, non-lactating American adults, aged 20-65 yrs, who are highly differentiated relative to two aspects of dietary guidance, specifically saturated fat and fruit and vegetable intakes, and 2) to

characterize key foods in the diets for these two populations. The two subpopulations identified within the larger dataset are HiSF-LoFV and LoSF-HiFV (Table 1 ). The HiSF- LoFV (High Saturated Fat, Low Fruit and Vegetable) group included individuals in the highest tertile of saturated fat and lowest tertile of fruit and vegetable intakes. In contrast, the LoSF-HiFV group included individuals in the lowest tertile of saturated fat and highest tertile of fruit and vegetable intakes. Key characterizing foods consumed by the HiSF-LoFV and LoSF-HiFV subpopulations were then identified in a two-step process using eight broad food groups and 63 food subgroups expressed on a kcal food/1000 kcal total diet basis (Table 2). A detailed description of the methodology is provided below.

TABLE 1 . HiSF-LoFV and LoSF-HiFV subpopulations among healthy adults 20-65 yrs of age in the National Health and Nutrition Examination Surveys (NHANES) conducted between 2003-2008 (n=10,190 non-pregnant, non-lactating).

TABLE 2. Characterizing foods consumed by the HiSF-LoFV (A) and LoSF-HiFV (B) subpopulations.

(A) HiSF-LoFV characterizing foods

(B) LoSF-HiFV characterizing foods

[0061 ] Data Source and Sample NHANES utilizes in-person, 24-hr dietary recalls using an automated, multiple-pass method. This method guides the respondent through the dietary intake period more than once and consequently provides multiple opportunities to identify foods and specific details about the foods consumed during the recall period (Raper et al. J Food Compos Anal 2004; 17(3-4): 545-555). Diet recalls were analyzed by the United States Department of Agriculture (USDA) Food Surveys Research Group (FSRG) to estimate the types and amounts of foods, as well as their nutrient composition, consumed by each respondent (CDC 2007, 2008, 2010).

Additional information on foods reported by NHANES respondents in 24-hour recalls has been provided by USDA-FSRG in their Food and Nutrition Database for Dietary Studies (FNDDS); FNDDS data include a food coding scheme, "recipes" used in deriving nutrient composition data, and additional detail on foods categorized by specific food codes. USDA-FSRG has developed a grouping scheme for the survey food codes to aid in reporting food intake estimates. In the USDA-defined food grouping system, NHANES food codes (excluding plain water) are typically grouped into 71 food groups or subgroups.

[0062] Selected data from NHANES Food Frequency Questionnaires (FFQs) administered in 2003-2004 and 2005-2006 were also used in the analyses in order to collect additional information regarding frequency of consumption of caloric beverages. The FFQ, developed by the National Cancer Institute (NCI), is based on the NCI Diet History Questionnaire, a 124-item food frequency instrument that is widely used in nutritional epidemiology research (Subar et al. Am J Epidemiol 2001 ;154:1089- 99).

[0063] Food Categories The 71 USDA-defined food groups and subgroups represent hierarchical groupings of food codes within broad food categories, and consequently the USDA hierarchy provides a food coding scheme that can be used to identify key characterizing foods within groups of similar foods. This food grouping scheme was used to identify key foods consumed by the HiSF-LoFV and LoSF-HiFV subpopulations. In the analyses, the Meat, Poultry and Fish; Eggs; Nuts and Seeds; and Legumes groups were combined into one major food group. The total fluid milk subgroup was used to assess all fluid milk intake and separate subgroups for "total fruit juice drinks and fruit flavored drinks" and "total carbonated soft drinks" were used to assess total intakes of these beverages. After modification, the food grouping system included 63 food subgroups within eight broad food groups.

[0064] Subpopulations Based on Saturated Fat and Total Fruit and Vegetable Consumption Among the total sample (n=10,190), tertiles were determined based on g saturated fat/1000 kcal and g fruit+vegetable/1000 kcal (NHANES food codes starting with 6 for fruit and 7 for vegetables), with >14.1 g saturated fat/1000 kcal and <10.3 g saturated fat/1000 kcal defining the top and bottom tertiles, respectively. For combined fruit and vegetable intakes, < 65 g fruits + vegetables/1000 kcal defined the lowest tertile and >181 g fruits + vegetables/1000 kcal defined the top tertile. Using these tertile cutpoints, 1 1 .8% of individuals were in the HiSF-LoFV group (n=1203) and 14.7% were in Low Saturated Fat, High Fruit and Vegetable (LoSF-HiFV) group

(n=1503).

[0065] Foods that Characterize HiSF-LoFV and LoSF-HiFV Diets Key characterizing foods consumed by the HiSF-LoFV and LoSF-HiFV subpopulations were identified in a two-step process using eight broad food groups and 63 food subgroups expressed on a kcal food/1000 kcal total diet basis.

[0066] In the first step, key food subcategories were identified. Relative energy intakes by major food group and food subgroup, and the proportion of the subpopulation consuming foods within individual food subgroups were used to identify the key food subgroups consumed by the HiSF-LoFV subpopulation, and separately by the LoSF-HiFV subpopulation. The specific criteria used were as follows:

[0067] · Food subgroups providing less than 20 kcal/1000 kcal total diet (i.e., less than 2.0% of energy) were excluded from consideration.

[0068] · Within each food subgroup providing from 20 to 50 kcal/1000 kcal total diet (i.e., 2.0 to 5.0% of energy), the single food subgroup representing the largest source of energy was selected as a key food subgroup.

[0069] · Within each food group providing more than 50 kcal/1000 kcal total diet (i.e., over 5.0% of total energy), all food subgroups meeting at least one of the following two criteria were considered key food subgroups: 1 ) Contributing at least 10% of kcal per 1000 kcal for the food group AND consumed by at least 10% of the subpopulation; 2) Consumed by 20% or more of the subpopulation.

[0070] Coffee and tea were excluded because mice are sensitive to caffeine and because these beverages contribute little to energy intake.

[0071 ] In the second step, food(s) that best characterized each identified key food group/subgroup were selected. For each identified key food group/subgroup, the food code with the highest weight of food consumed per 1000 kcal in the

subpopulation reporting consumption on the day of recall was selected to be the key food characterizing that entire food group/subgroup.

[0072] Proportions of Key Foods in HiSF-LoFV and LoSF-HiFV Diets The proportions of key foods to be included in mouse diets (wet weight) were calculated by first determining the relative energy contributions from key food subgroups to the total energy intake from the USDA-based food group (per 1000 kcal total diet) and then determining the weight of the food (in g) required to deliver the calculated energy contribution from the relevant food group per 1000 kcal total diet (Table 1 ). Once the characterizing foods were identified for the two diets, calculated nutrient intakes were compared to 24 hr recall data as an indicator of how well the characterizing foods reflect reported intakes. Results indicated that cholesterol was low in the LoSF-HiFV diet and therefore egg was added to the LoSF-HiFV diet. Table 3 shows that mean nutrient content of animal chow made from key characterizing foods were similar to intakes reported from the 24-h recall for both the resulting HiSF-LoFV and LoSF-HiFV diets.

TABLE 3. Comparison of nutrient content per 1000 kcal from intake based on a 24-hour diet recall (24 hr Recall); calculated diet based on key characterizing foods (Calculated Diet); or chemical analysis of the Animal Chow (Animal Chow).

Total Polyunsaturated Fat, g 7.4 7.7 8.5 7.7 10.0 9.3

Cholesterol, mg 163 191 179 109 91 84

Carbohydrate, g 105 99 97 142 148 139

Total Sugars, g 48 47 50 66 72 71

Dietary Fiber, g 5.1 3.5 5.7 1 1.0 9.8 13.7

Vitamin A, meg RAE 252 201 188 385 286 370

Vitamin E, mg alpha-toe 2.7 2.5 0.7 1.1 0.9 0.7

Vitamin C, mg 14.3 10.4 8.0 88.5 61 23

Thiamin, mg 0.7 0.8 0.9 0.9 0.7 0.8

Riboflavin, mg 1.1 0.9 0.7 1.1 0.9 0.7

Niacin, mg 10.2 8.9 1 1.4 14.3 16.1 14

Folate, meg DFE 21 1 169 131 331 334 250

Vitamin B6, mg 0.7 0.6 0.4 1.3 1.2 0.9

Vitamin B12, meg 2.6 2.8 2.8 2.4 2.9 3.3

Calcium, mg 480 523 601 418 439 500

Phosphorus, mg 652 672 764 647 613 617

Magnesium, mg 1 13 90 99 171 158 161

Iron, mg 6.5 5.9 8.8 8.7 8.1 8.4

Zinc, mg 6.0 6.2 6.6 5.8 4.5 5.1

Copper, mg 0.5 0.5 0.5 0.8 0.7 0.8

Selenium, meg 52 54 101 55 61 100

Sodium, mg 1676 1975 2021 1621 1279 804

Potassium, mg 1076 899 821 1704 1581 1550

Example 2. Preparing and Processing Animal Chow

[0073] Characterizing foods were processed into dried animal chow pellets using methods to achieve an even mix of foods in quantitative proportions and to retain nutrients. The approach of blending all the diverse food items into one mixture and freeze drying the resulting mixture was experimental. Due to the unique nature of this mixture it was unknown if the freeze dried material would have the correct

physicochemical characteristics to be extruded/molded into a structurally stable granular or pellet form. The blending of all the food items that represent a diet into a

homogeneous mixture distributed the water, fat, oil and low molecular weight solutes (sugars and minerals) into an emulsion and suspension that was amiable to freezing and freeze drying. It is hypothesized that the freezing point of the blended mix was raised compared to some of the individual food items, making the homogenous composite a feasible approach to freeze drying food items that have a much lower freezing points and difficult to freeze dry. Food items with low freezing points and potentially more difficult to freeze dry would be those high in simple sugars, salt and fat (e.g. salad dressing, chocolate, candy). The distribution of the water in this complex food matrix was such that when removed via the freeze drying process the resulting dry powder had the correct characteristics for extrusion and molding into an appropriate form.

[0074] Homogenous food composites for the two diets were prepared as follows. Characterized foods for each diet were purchased from Chicago area stores and/or prepared using standard recipes from the USDA FNDDS or from standard authoritative cookbooks. Mixed dishes (e.g. pizza) were produced over a one week timeframe, weighed into batches, and then frozen until they were thawed prior to compositing. Perishable foods were prepared (e.g., peeled, cored), weighed out the day before blending and stored refrigerated or at room temperature as appropriate. To composite the diets, foods were added in quantitative proportions into a Breddo Likwifer mixer (model LOR 25) and recirculated through a Silverson high energy mixer until evenly blended by visual inspection in single batches to ensure a homogeneous mixture (Tables 4 and 5). Homogenized mixtures were collected in 5 gallon pails and then frozen at -20°C. FIG. 1 illustrates the flow of diet manufacture.

[0075] Homogenous composite formulas were maintained at -20°C.

Frozen blocks of each composite diet were finely shredded and then placed in a vacuum chamber set at 1 mb until available moisture was removed (approximately 27 hours). Freeze dried material was then milled to a 20 mesh size and packed into 40 lb. bags (stored at room temperature). Water was added to the freeze dried formula to reach an acceptable visual viscosity for extrusion into pellets. Samples were collected from the beginning, middle and end of the extrusion process for compositional analysis based on AOAC methodology. The averages of those data are shown in Table 3 and Table 6. Pellets were flushed with nitrogen and vacuum packed. Vacuum packed diets were sterilized via gamma irradiation.

[0076] Recipes: Reference numbers for the following recipes were from the USDA Food and Nutrient Database for Dietary Studies, 4.1 .2010.Beltsville, MD; U.S. Department of Agriculture, Agriculture Research Service, Food Surveys Research Group; and USDA Food and Nutrient Database for Dietary Studies, 3.0.2008. Beltsville, MD; U.S. Department of Agriculture, Agriculture Research Service, Food Surveys Research Group (FNDDS3.0 data used only for Pizza). Pizza, thin crust meat:

58106520, recipe slightly modified, used premade crust and substituted tomato sauce for tomato juice and puree; Double Cheeseburger: 27510330; Egg fried: 31 105000; Tuna Salad: 27450060; Spaghetti with tomato sauce: 581321 10; White Rice:

56205010; Mashed Potato: 71501000; Scrambled Egg: 32105000; Baked Chicken breast: 24122120; Italian Dressing recipe from Joy of Cooking (1975; 23rd printing 1981 ).

TABLE 4. High Saturated Fat Low Fruit Vegetable Processing Protocol

High Saturated Fat Low Fruit and Vegetable Diet

Order Added Process

Food Type to Breddo Adjustment Comments

Breddo at 600rpm

Recirculation split amount by half added second half after

Soft Drink 1 Off ground beef patty addition

Fruit Drink from powder 2

Corn Snack 3

Potato Chip 4

White Roll soft 5

Lite Beer 6

Chocolate Chip Cookie 7

Flour Tortilla 8 Pulse Pulse Breddo at 2200 rpm for a few minutes

Milk Chocolate Candy 9

Processed Cheese American 10 Pulse Pulse Breddo at 2200 rpm for a few minutes

Pulse, Pulse Breddo at 2200 rpm for a few minutes,

Recirculation Recirculation started with Silversonmixer run

Ham sliced 11 On at 3500rpm

Double Cheeseburger w/

tomato on bun 12 Pulse Pulse Breddo at 2200 rpm for a few minutes

Egg Fried 13

Ground Beef Patty 14

Mayonnaise 15

Pizza, thin crust meat 16 Pulse Pulse Breddo at 2200 rpm for a few minutes

Ice Cream 17

Milk 2% 18

Mix for approximately 5 minutes before

collecting mixture TABLE 5. Low Saturated Fat High Fruit Vegetable Diet Processing Protocol

Low Saturated Fat High Fruit and Vegetable Diet

Order Added Process

Food Type to Breddo Adjustment Comments

Recirculation

Soft drink 1 Off : Breddo at 600rpm

Hard Candy 2

Cheese, Cheddar 3

Chicken Breast baked no

skin 4

Potato Chips 5

Corn Snack 6

Ready to Eat Cereal 7 increase Breddo Speed to approx 1,100

Orange Juice 8

Raw Onion 9 Pulse Pulse Breddo at 2200 rpm for a few minutes

Raw Banana 10

Raw Lettuce 11 Pulse iPulse Breddo at 2200 rpm for a few minutes

Raw Tomato 12

Raw Grapes 13

Raw Apple 14 Pulse iPulse Breddo at 2200 rpm for a few minutes

Peanut Butter 15

Chocolate cookie 16

Corn Tortilla 17 Pulse Pulse Breddo at 2200 rpm for a few minutes

Mashed Potatoes 18

White Rice 19 Pulse iPulse Breddo at 2200 rpm for a few minutes

Spaghetti with Tomato Sauce ; 20

Scrambled Egg fat added in i Recirculation ^ Recirculation started with Silversonmixer run cooking 21 On iat 3500rpm

Roll White Soft 22 Pulse iPulse Breddo at 2200 rpm for a few minutes

Apple Juice 23

Ice Cream 24

Milk Skim 25

Soy Milk 26

Beer 27

Powdered Lemonade added to Italian

Lemonade 28 idressing

Italian Dressing vinegar and i

oil 28

Tuna Salad* 29 ;*Stephan processing

; Mix for approximately 5 minutes before i col lecting mixture TABLE 6. Formula Composition after Processing Steps: (A) Starting Weight and Final Weight, (B) Mixing, (C) Freeze Drying, (D) Pellet Extrusion

(A) Starting Weight and Final Weight

(B) After all Mixing

Analysis Units HiSF-LoFV LoSF-HiFV

Moisture % as is 73.14 81.22

Ash % as is 1.28 0.64

Fat. g per 1000 kcal 47.3 43.5

Protein, g per 1000 kcal 57.8 53.5

Carbohydrate, g per 1000 kcal 108.2 175.0

Vit C, mg per 1000 kcal 9.0 63.0

Beta carotene, mg per 1000 kcal 0.2 0.6

(C) After Freeze Drying

Freeze Dried

Analysis Units HiSF-LoFV LoSF-HiFV

Moisture % as is 2.13 2.62

Ash % as is 3.31 3.10

Fat. g per 1000 kcal 45.3 31.5

Protein, g per 1000 kcal 46.4 38.2

Carbohydrate, g per 1000 kcal 101.6 140.8

Vit C, mg per 1000 kcal 1 1.8 43.0

Beta carotene, mg per 1000 kcal 0.1 0.5

(D) Beginning (Beg), Middle (Mid) and End of Pellet Extrusion

HiSF-LoFV LoSF-HiFV

Analysis Units Beg Mid End Beg Mid End

Moisture % as is 5.33 5.25 5.18 7.25 7.70 6.53

Ash % as is 4.34 4.36 4.31 3.06 2.73 3.05

Fat. g per 1000 kcal 47.2 47.4 47.7 32.9 33.0 33.0

Protein, g per 1000 kcal 45.9 46.0 46.1 38.3 38.2 38.1

Carbohydrate, g per 1000 kcal 97.5 97.3 97.2 137.1 136.9 138.9

Vit C, mg per 1000 kcal 8.0 7.8 8.0 22.8 22.5 23.2

Beta carotene, mg per 1000 kcal 0.1 0.1 0.1 0.4 0.4 0.4