Methods of improving microvascular health

Abstract

Described here are methods for assessing status of microvascular function in a subject by measuring levels of biomarkers in a biological sample obtained from the subject. Also described are methods of improving microvascular function in a subject identified as having an impaired microvascular function by providing a legume-based snack food. Also described are methods of improving microvascular function in a subject by providing a legume-based snack food. Further described are methods of alleviating an inflammatory lung disease in a subject by providing a legume-based snack food.

Description

[0001] METHODS OF IMPROVING MICRO VASCULAR HEALTH

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 519,013, filed on August 11, 2023, the contents of which are incorporated by reference herein in its entirety.

[0004] FIELD OF THE INVENTION

[0005] The presently disclosed subject matter relates to methods of improving microvascular health with legume-based food products.

[0006] BACKGROUND OF THE INVENTION

[0007] Dietary intake patterns comprising high calorie foods and snacks that are low in essential nutrients contribute to a high prevalence of obesity across diverse populations. The public health ramifications of these are significant, as evidenced by the observation that the prevalence of obesity for children 6-19 years of age in the United States is 19% [1, 2], Specifically, studies in California middle school children report overweight and obesity prevalence ranging from 26% to as high as 52% with disproportionate impacts for certain race / ethnic groups and those living in urban areas [3-5], Lifestyle factors that can attribute to obesity during childhood include excessive intakes of energy dense snacks. For example, children 6-18 years of age consume two or more snacks per day providing on average 550 kcal, with a majority of these calories coming from energy dense, salty, sweet, or dessert type snacks [6], Moreover, a recent National Health and Nutrition Examination Survey (NHANES) estimates the daily intake of “unhealthy” snacks at 17.5% of energy for children 2-19 years of age [7],

[0008] Similar to adults, overweight and obesity in young children and adolescents is associated with an increase in cardiovascular (CV) risk factors [8], which includes promotion of systemic inflammation that aggravates marginal micronutrient deficiencies

[0014] , Moreover, in large longitudinal studies in Europe and the US, the presence of CV risk factors in childhood has been associated with an increased risk for vascular dysfunction in adulthood [9, 10], Vascular dysfunction, an early indicator of atherosclerotic disease development, can occur in childhood but could improve with diet [11-13], Thus, one targeted strategy for reducing obesity in children could involve replacing energy-dense and nutrient-poor snacks with value-added products aimed at improving nutritional status.

[0009] Concurrent to looming obesity crisis is the lack of food security for some children who have limited access to affordable nutrient-dense foods containing essential nutrients

[0014] , In developing countries, lack of proper nutrition is partially driven by a diet poor in protein and the poor quality of available proteins. Optimal growth and cognitive development particularly in children depends on protein quality and key micronutrient (bio)availability. Thus, there is a recognized need to develop snacks that can be part of a healthy dietary pattern for both over nourished and under nourished children [15, 16], Legumes are part of the cultural food repertoire of many countries. They have many health benefits associated with high quality protein and complex carbohydrate content, making them a suitable choice food for incorporating plant-based proteins into the diet of over nourished and under nourished children. In addition to protein, legumes are an excellent source of fiber, and micronutrients with increased intakes associated with lower blood pressure in adults and improved cardiometabolic health in obese children [17-19], Thus, there is a need in the art for new compositions and methods that alleviate obesity and lack of proper nutrition in children by improving vascular health.

[0010] SUMMARY

[0011] In one embodiment of the presently disclosed subject matter, there is provided a method of assessing a status of microvascular function in a subject. The method comprises, obtaining a biological sample, measuring in the biological sample, an amount of one or more biomarkers associated with the microvascular function, to obtain a test value for each of the one or more biomarkers, and comparing the test value with a reference value for the one or more biomarkers, wherein a change in the test value as compared to the reference value indicates an improved or an impaired microvascular function.

[0012] In another embodiment, the biological sample is a blood sample, a tissue sample, a saliva sample, a urine sample, or a feces sample.

[0013] In another embodiment, the biomarker is selected from the group consisting of metabolites, cytokines, lipids, sugars, cholesterols, bile acids, high-density lipoproteins, an insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP), and derivatives thereof.

[0014] In another embodiment, a decrease in at least one of biomarkers, IL-ip, IL-4, IL-22, IL-23, IL-31, F2-isoprostane, 11-HETE, and 12-HETE, and / or an increase in urosodeoxycholic acid (UDCA) compared to a reference value indicates an improved microvascular function.

[0015] In another embodiment, the method comprises measuring in the biological sample, an amount of an insulin-like growth factor (IGF) and an amount of an insulin-like growth factor binding protein (IGFBP), calculating a ratio of the IGF to IGFBP to obtain a test ratio, and comparing the test ratio with a reference ratio, wherein an increase in the test ratio compared to the reference ratio indicates improved microvascular function. In some embodiments, the IGF is IGF-1 or IGF-2. In some embodiments, the IGFBP is IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, and IGFBP6. In other embodiments, the IGFBP is IGFBP3, IGFBP5, or a combination thereof.

[0016] In another embodiment, the method comprises measuring in the biological sample, an amount of a cholesterol and an amount of a high-density lipoprotein (HDL), calculating a ratio of the amount of the cholesterol to the amount of the HDL to obtain a test ratio, comparing the test ratio with a reference ratio, wherein a decrease in the test ratio compared to the reference ratio indicates improved microvascular function.

[0017] In another embodiment, the method further comprises providing to a subject identified as having an impaired microvascular function, a legume-based snack food comprising, at least one extruded first legume and a first additive comprising at least one vitamin, at least one mineral, or a combination thereof, and optionally, a creamy layer comprising at least one extruded second legume and a second additive comprising at least one vitamin, at least one mineral, or a combination thereof, wherein consuming the snack food improves microvascular function.

[0018] In another embodiment, the at least one first legume and the at least one second legume are independently selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean, soya bean, and combinations thereof. In some embodiments, the first legume is yellow pea. In some embodiments, the second legume is yellow pea. In some embodiments, the first legume and the second legume are both yellow pea.

[0019] In yet another embodiment, the first additive and the second additive are independently selected from the group consisting of Vitamin B 1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B 12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof. In some embodiments, the first additive and the second additive are independently, a Vitamin B6, a Vitamin B12, a Vitamin D, zinc, iron, copper, magnesium, or manganese, or a combination thereof.

[0020] In other embodiments, the snack food further comprises flour from at least one first legume and / or at least one second legume. In yet other embodiments, the snack food further comprises one or more edible oils.

[0021] In some embodiments, he legume-based snack food is consumed for at least 4 weeks.

[0022] In yet another embodiment, the snack food is provided daily for a total daily calorie intake from about 200 kcal to about 250 kcal.

[0023] In yet another embodiment, the snack food is provided to the subject daily, or twice daily.

[0024] In yet another embodiment, the snack food is provided to the subject in an amount from about 10 g / serving to about 30 g / serving.

[0025] In yet another embodiment, the snack food is provided to the subject in an amount of 26g / serving.

[0026] In yet another embodiment, the snack food comprises a legume protein content from about 0.1 g protein to about 0.5 g of protein, per gram of snack food.

[0027] In yet another embodiment, the snack food delivers an amount of the legume protein from about 0.7 g to about 1.0 g per day, per kg body weight of the subject.

[0028] In yet another embodiment, the subject is suffering from overweightness, poor nutrition, obesity, or a cardiometabolic disorder.

[0029] In yet another embodiment, the subject is a pediatric subject.

[0030] In one embodiment of the presently disclosed subject matter, there is provided a method of improving microvascular function in a subject. The method comprises, providing to a subject, a legume-based snack food comprising at least one extruded first legume, and a first additive comprising at least one vitamin, at least one mineral, or a combination thereof, wherein consuming the snack food improves microvascular function.

[0031] In another embodiment, the snack food further comprises a creamy layer comprising at least one extruded second legume, and a second additive comprising at least one vitamin, at least one mineral, or a combination thereof.

[0032] In yet another embodiment, the method further comprises calculating a first Framingham reactive hyperemia index (fRHI) value for the subj ect before consuming the snack food and calculating a second fRHI value after consuming the snack food, wherein a higher second fRHI value compared to the first fRHI value indicates improved microvascular function.

[0033] In another embodiment, the at least one first legume and the at least one second legume are independently selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean, soya bean, and combinations thereof. In yet another embodiment, the first additive and the second additive are independently selected from the group consisting of Vitamin B 1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B 12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof.

[0034] In other embodiments, the snack food further comprises flour from at least one first legume and / or at least one second legume.

[0035] In yet other embodiments, the snack food further comprises one or more edible oils.

[0036] In yet another embodiment, the snack food is provided daily for a total daily calorie intake from about 200 kcal to about 250 kcal.

[0037] In yet another embodiment, the snack food is provided to the subject daily, or twice daily.

[0038] In yet another embodiment, consuming the snack food for at least 4 weeks improves microvascular function.

[0039] In yet another embodiment, the snack food is provided to the subject in an amount from about 10 g / serving to about 30 g / serving.

[0040] In yet another embodiment, the snack food is provided to the subject in an amount of 26g / serving.

[0041] In yet another embodiment, the snack food comprises a legume protein content from about 0.1 g protein to about 0.5 g of protein, per gram of snack food.

[0042] In yet another embodiment, the snack food delivers an amount of the legume protein from about 0.7 g to about 1.0 g per day, per kg body weight of the subject.

[0043] In some embodiments, the method causes a weight loss in the subject.

[0044] In some embodiments, the method improves cardiometabolic health in the subject.

[0045] In yet another embodiment, the subject is a pediatric subject.

[0046] In one embodiment of the presently disclosed subject matter, there is provided a method of alleviating an inflammatory lung disease in a subject. The method comprises, providing to a subject, a snack food comprising at least one extruded first legume wherein consuming the snack food improves microvascular function thereby alleviating the inflammatory lung disease.

[0047] In another embodiment, the inflammatory lung disease is allergy or asthma.

[0048] In yet another embodiment, the subject is undergoing treatment for the inflammatory lung disease. In yet another embodiment, providing the legume-based snack food to the subject reduces inflammation thereby alleviating the inflammatory lung disease.

[0049] In yet another embodiment, consuming the snack food increases levels of urosodeoxycholic acid (UDCA) compared to a reference value, thereby alleviating the inflammatory lung disease in the subject.

[0050] In yet another embodiment, consuming the snack food reduces levels of at least one cytokine compared to a reference value, thereby alleviating the inflammatory lung disease in the subject.

[0051] In yet another embodiment, consuming the snack food reduces a level of IL-23 or IL-4 or a combination thereof.

[0052] In yet another embodiment, the snack food further comprises a first additive comprising at least one vitamin, at least one mineral, or a combination thereof.

[0053] In yet another embodiment, the snack food further comprises a creamy layer comprising at least one extruded second legume, and optionally, a second additive comprising at least one vitamin, at least one mineral, or a combination thereof.

[0054] In another embodiment, the at least one first legume and the at least one second legume are independently selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean, soya bean, and combinations thereof.

[0055] In another embodiment, the first additive and the second additive are independently selected from the group consisting of Vitamin B 1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B 12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof.

[0056] In other embodiments, the snack food further comprises flour from at least one first legume and / or at least one second legume.

[0057] In other embodiments, the snack food further comprises one or more edible oils.

[0058] In some embodiments, the snack food is provided daily for a total daily calorie intake from about 200 kcal to about 250 kcal.

[0059] In some embodiment, the snack food is provided to the subject daily, or twice daily.

[0060] In some embodiments, the snack food is consumed for at least 4 weeks.

[0061] In some embodiments, the snack food is provided to the subject in an amount from about 10 g / serving to about 30 g / serving. In other embodiments, the snack food is provided to the subject in an amount of 26g / serving.

[0062] In yet another embodiment, the snack food comprises a legume protein content from about 0.1 g protein to about 0.5 g of protein, per gram of snack food.

[0063] In yet another embodiment, the snack food delivers an amount of the legume protein from about 0.7 g to about 1.0 g per day, per kg body weight of the subject.

[0064] In yet another embodiment, the method improves cardiometabolic health in the subject. In yet another embodiment, the method alleviates poor nutrition or obesity in the subject In yet another embodiment, the subject is a pediatric subject.

[0065] BRIEF DESCRIPTION OF THE FIGURES

[0066] Figure 1 shows nutrient compositions for exemplary legume-based snack products.

[0067] Figure 2 shows a flowchart of study participant selection for the various study groups.

[0068] Figure 3 presents a summary of baseline characteristics for study participants.

[0069] Figure 4 provides a summary of baseline dietary intake.

[0070] Figure 5 illustrates exemplary significant interactive effects for change in outcome measures after 4 weeks of legume-based snack product intake.

[0071] Figures 6A-6D show exemplary effects of legume-based snack products on microvascular function and plasma cholesterol. Figure 6A is a graph showing changes in the Framingham Reactive Hyperemia Index (fRHI) for the groups receiving fortified and nonfortified legume-based products. Figure 6B is a graph showing changes in the Reactive Hyperemia Index (RHI) for the groups receiving fortified and non-fortified legume-based products. Figure 6C is a graph showing changes in the total cholesterol :HDL ratios for the groups receiving fortified and non-fortified legume-based products. Figure 6D is a graph showing changes in the total cholesterol :HDL ratios for the groups receiving legume-based products comprising 6g protein and 12g protein.

[0072] Figure 7 shows exemplary 4-week changes from baseline for all children and overweight / obese children.

[0073] Figure 8 shows exemplary 4-week changes from baseline by protein intake levels for all children and overweight / obese children.

[0074] Figure 9 shows exemplary differences in several variables between the study groups.

[0075] Figure 10 shows exemplary correlations of fRHI and Cholesterol :HDL ratio.

[0076] Figures 11A-11D show exemplary top predictors of fRHI and cholesterol :HDL ratio in all children with a Variable Importance in Projection (VIP) score greater than 1. Figure 11 A shows a graph of the top predictors of fRHI response in all children. Figure 1 IB shows a graph of predictors of cholesterol :HDL ratios in all children. Figure 11C shows a graph of predictors of fRHI response in overweight / obese children. Figure 1 ID shows a graph of predictors of cholesterol :HDL ratios in overweight / obese children.

[0077] The figures constitute a part of this specification and include illustrative embodiments of the presently disclosed subject matter and illustrate various objects and features thereof. Further, the figures are not necessarily to scale, some features can be exaggerated to show details of particular components. In addition, any measurements, specifications, and the like shown in the figures are intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the presently disclosed subject matter.

[0078] DETAILED DESCRIPTION OF THE INVENTION

[0079] The presently disclosed subject matter provides methods for assessing and improving microvascular function in a subject by proving a legume-based snack food. Also provided are methods of alleviating an inflammatory lung disease, by providing a subject, a legume-based snack food.

[0080] For clarity of description, and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0081] 1. Definitions;

[0082] 2. Products;

[0083] 3. Methods; and

[0084] 4. Examples.

[0085] 1. DEFINITIONS

[0086] As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. Abbreviations used herein have their conventional meaning within the chemical and biological arts.

[0087] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases “in one embodiment” and “in some embodiments” as used herein do not necessarily refer to the same embodiment s), though it can. Furthermore, the phrases “in another embodiment” and “in some other embodiments” as used herein do not necessarily refer to a different embodiment, although it can. Thus, as described below, various embodiments of the presently disclosed subject matter can be readily combined, without departing from the scope or spirit of the presently disclosed subject matter.

[0088] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0089] As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to “a compound” includes mixtures of compounds.

[0090] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, z.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value.

[0091] In addition, as used herein, the term “or” is an inclusive “or” operator and is equivalent to the term “and / or,” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for additional factors not described unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on”.

[0092] As used herein, the term “substantially free” means less than 5%.

[0093] As used herein, the term “free” means 0%.

[0094] As used herein, the term “organoleptic properties” includes the flavor display, texture, and sound of a food that are experienced by the eater of said food when said food is eaten.

[0095] As used herein, the term “single serving” means any quantity of food sold, marketed, described, advertised, or implied to be equivalent to a single serving size or unit. For example, in the U.S., single serving sizes for foods are defined in the FDA Labeling Rules as contained in 21 CFR § 101.12, which is incorporated herein by reference in its entirety. As used herein, the term “fat” refers to the total amount of digestible, partially digestible and nondigestible fats or oils that are present in the embodiments of the disclosed subject matter. As used herein, the terms “lipid”, “fat” and “oil” are synonymous.

[0096] As used herein, the term “carbohydrate” refers to the total amount of sugar alcohols, monosaccharides, disaccharides, oligosaccharides, digestible, partially digestible, and nondigestible polysaccharides; and lignin or lignin like materials that are present in the embodiments of the presently disclosed subject matter.

[0097] As used herein, the term “legume” means a fruit or seed of leguminous plants.

[0098] As used herein, the phrase “legume-based flour” means a powder formed from milling one or more legumes.

[0099] As used herein, the terms “legume-based snack food”, “legume-based snack food product”, “snack food”, “snack food product”, “legume-based product” and “food” are used interchangeably unless specifically stated otherwise and refer to the legume-based snack food described herein.

[0100] As used herein, the term “grain” means edible seeds of grasses. The term “grain” and “cereal” are used interchangeably herein. Non-limiting examples of grains include rice, teff, sorghum, com, millet, wheat, quinoa, and farro.

[0101] As used herein, the phrase “grain-based flour” means a powder formed from milling grains.

[0102] As used herein, the term “pulse” means the dried seed of a legume.

[0103] As used herein, the term “ready-to-eat” when used to describe a food, means that after manufacture and packaging, the food product requires no additional processing, including but not limited to cooking, baking, microwaving, boiling, frying; or combination with components outside of the product’s packaging to achieve the novel combination of balanced nutrition and taste that are disclosed. However, this does not rule out that one or all of the parameters of the disclosed subject matter, for example, balanced nutrition, convenience, and taste, can be improved when said compositions are processed further or combined with other foods.

[0104] In embodiments, a product is determined to be “crispy” by texture measurements using an Instron texture analyzer or equivalent. In other embodiments, a product is determined to be “crispy” based on organoleptic properties and / or mouthfeel. In yet other embodiments, a product is determined to be “crispy” based on an acoustic peak count of at least 10 as determined by the Acoustic Crispiness Test Procedure detailed herein. In yet other embodiments, a product is determined to be “crispy” based on a peak fracture force of at least 1,200 grams as determined by the Force Crispiness Test Procedure detailed herein. As used herein, a product is determined to be “creamy” based on viscosity measurements using a Brookfield viscometer and / or a stress-controlled rheometer equipped with a tribology probe or equivalent. In other embodiments, a product is determined to be creamy based on organoleptic properties and / or mouthfeel.

[0105] As used herein, the “shelf life” of a product is determined based on the change in organoleptic properties of a food or drink in barrier packaging over time.

[0106] As used herein, the term “seasoning” means salt, herbs and / or spices added to a food product to enhance flavor. In some embodiments, the average particle size of the “seasoning” is reduced to 1 micron to 1000 microns via milling or other size reduction process.

[0107] As used herein, the phrase “water content” or “moisture content” of a product means the wt. % of water in the product, on a wet basis.

[0108] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0109] Unless otherwise noted, all component or composition levels are in reference to the active level of that component or composition, and are exclusive of impurities, for example, residual by-products, which can be present in commercially available sources.

[0110] As used herein, the phrase “providing to a subject a snack food”, “provided... a snack food to a subject” is meant to imply that the subject has been provided the snack food for consumption as instructed.

[0111] The term “ratio” or “ratios” as used herein when referring to relative amounts of two or more agents refers to relative amounts of these agents not limited to mole ratios (e.g., mole / mole), weight ratios (w / w, e.g., nanogram / nanogram, pg / pg, ng / pg), volume ratios (v / v, e.g., mL / mL), or weight / volume (w / v, e.g., pg / pL, mg / mL).

[0112] As used herein the terms “calorie” or “calories” are equivalent to “C”, “Calorie”, “Calories”, “kilocalorie”, “kilocalories”, “kcal” and “kcals”. For example, “a caloric density of about 1 to about 9 calories per gram of the product” is equivalent to “a caloric density of about 1 to about 9 kcal per gram of the product.”.

[0113] As used herein, the phrase “cardiometabolic health” refers to the overall health of the cardiovascular system which encompasses multiple factors including, but not limited to body mass index (BMI), blood sugar regulation, cholesterol, primary and fatty acid metabolism, innate immunity, blood pressure, risks for heart attack and stroke.

[0114] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either endpoint of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 can include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0115] A “subject” can be a human, or a non-human animal not limited to a non-human primate, a dog, a cat, a horse, a rodent, a cow, a goat, a rabbit, and a mouse. A human subject can be an infant (e.g., 1 month-1 year), a child (e.g. 1 year-12 years), an adolescent subject (e.g., 13 years - 17 years), a pediatric subject (e.g., 1 month - 17 years), , or an adult (e.g., over 18 years).

[0116] As used herein, the term “weight percent” is meant to refer to the quantity by weight of a constituent or component, for example, in the pet food composition as a percentage of the overall weight of the pet food composition. The term “weight percent” can also refer to the quantity by weight of a constituent or component, for example, in the hydrolysate composition as a percentage of the overall weight of the hydrolysate composition. The terms “weight percent,” “wt-%,” “wt.%”, and “wt%” are used interchangeably.

[0117] The terms “alleviating,” or “alleviate,” and other grammatical equivalents as used herein include abating, ameliorating, or relieving a disease, condition or symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, complementing other forms of treatment to achieve a therapeutic benefit, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition. The terms further include achieving a therapeutic benefit and / or a prophylactic benefit. Therapeutic benefit is meant to indicate eradication or amelioration of an underlying disorder. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient can still be afflicted with the underlying disorder.

[0118] 2. PRODUCTS

[0119] The present disclosure relates to ready-to-eat, shelf stable legume-based nutrient dense snack food products having a crispy texture. The present disclosure also relates to ready-to-eat, shelf stable legume-based nutrient dense snack food products in the form of a filled pillow having a legume-based outer shell and an inner filling of legume-based cream. In certain embodiments, the legume-based filled pillow products have a crispy outer shell and a creamy inner filling. In certain embodiments, the legume-based snack food products are crispy snacks with no inner filling. Nonlimiting examples of such snack food products are disclosed in U.S. 11,540, 553, the contents of which are incorporated herein by reference in its entirety.

[0120] In embodiments, the presently disclosed subject matter is a plurality of products, wherein each product comprises an outer shell and an inner filling. The outer shell comprises at least one first legume and a first additive selected from the group consisting of vitamins and minerals, wherein the at least one additive is not a part of the at least one first legume, and the inner filling comprises at least one second legume and a second additive selected from the group consisting of vitamins and minerals, wherein the second additive is not a part of the at least one second legume. In particular embodiments, at least one of the outer shell and the inner filling is substantially free of grain-based flour. In certain embodiments, the product is a ready-to-eat food. In particular embodiments, the outer shell is crispy and the inner filling is creamy.

[0121] In embodiments, the presently disclosed subject matter is a plurality of crispy products with no inner filling. In particular embodiments, the crispy product comprises at least one first legume, and a first additive selected from the group consisting of vitamins and minerals, wherein the at least one additive is not a part of the at least one first legume. In particular embodiments, the crispy product is substantially free of grain-based flour. In certain embodiments, the product is a ready-to-eat food. In other embodiments, the crispy product comprises at least one first legume and at least one second legume.

[0122] In one aspect of the presently disclosed subject matter, the snack food is in a ready-to- eat, shelf stable product. The snack food can have any geometric shape including, but not limited to circular, triangular, rectangular, square, cube and rhomboid. In some embodiments, the shape of the product can include a closed pillow with rounded sides and flattened closed ends. In some embodiments, the seasoning is visible in the outer shell. In other embodiments, the seasoning is not visible in the outer shell.

[0123] In some embodiments, the snack food has a crispy texture comprising a first legume and a first additive. In some embodiments, the snack food is in the form of a pillow with an extruded first legume and first additive forming an outer shell of the pillow, and an extruded second legume-based creamy layer comprising the second additive. In some embodiments, the snack food is crispy throughout with no outer shell or creamy layer. In some embodiments, the snack food remains crispy when exposed to high moisture environments over time. In some embodiments, the snack food, when consumed, provide a significant amount of high quality protein, fiber, and nutrients.

[0124] In other embodiments, the outer shelling and / or the inner filling includes at least one grain-based flour or cereal. In yet other embodiments, the at least one additive in the outer shell and / or filling is at least one cereal. In some embodiments, the cereal can include, but is not limited to, rice, teff, sorghum, corn, millet, wheat, quinoa, and / or farro. In some embodiments, the grain-based flour or cereal is added to reduce the density of the products. In some embodiments, the outer shell is substantially free of grain-based flour. In other embodiments, the inner filling is substantially free of grain-based flour. In other embodiments, the outer shell and / or inner filling is free of grain-based flour. In other embodiments, the product that is crispy throughout is free of grain-based flour.

[0125] In some embodiments, the snack food comprises flour from at least one first legume and / or at least one second legume. In certain embodiments, a first legume is any extruded legume known in the art. In other embodiments, a second legume can be used. A combination of two or more legumes can also be used. In some embodiments, the first legume and the second legume are the same. In other embodiments, the first legume and the second legume are different. Non-limiting examples of such legumes include, yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean and soya bean. In certain embodiments, the first legume is yellow pea. In an alternative embodiment, the first legume and the second legume are both yellow pea.

[0126] In certain embodiments, a first additive can comprise at least one vitamin, derivatives thereof or analogs thereof. In certain embodiments, the first additive can comprise at least one mineral. In certain embodiments, a second additive can be used, or alternatively two or more additives. In particular embodiments, the first additive and the second additive are the same. In some embodiments, the first additive and the second additive are different. Non-limiting examples of such additives include, Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B 12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, and chromium. A combination of vitamins and minerals can also be used. For example, in one embodiment, the first additive and the second additive independently comprise, one or more of Vitamin A, Vitamin C, Niacin, Thiamine, Riboflavin, Vitamin B6, Vitamin B12, Vitamin A, and Vitamin D, zinc, iron, copper, magnesium, or manganese.

[0127] In certain embodiment, the product further includes an oil layer and wherein the oil layer coats at least a portion of the outside of the product. In some embodiments, the oil layer coats the entire outside of the product. In some embodiments, the oil layer coats less than 50% of the outer surface area of the product. In other embodiments, the oil layer comprises a vegetable oil. In some embodiments, the vegetable oil is sunflower oil, palm oil, peanut oil, canola oil, or mixtures thereof. In certain embodiments the snack food further comprises one or more edible oils. In some embodiments, the oil is rich in polyunsaturated fatty acids (PUFA). In particular embodiments, the oil is a n-3 PUFA (omega-3 PUFA) rich oil. Non-limiting examples of PUFA rich oils include sunflower oil, flax seed oil, corn oil, soyabean oil and safflower oil, or mixtures thereof.

[0128] In an embodiment, the outer shell coats 100% of the inner filling. In other embodiments, the outer shell coats less than 100% of the inner filling. In particular embodiments, the snack product has an outer shell with no inner filling. In particular embodiments, the snack product is crispy throughout with no outer shell or inner filling.

[0129] In some embodiments, the crispy portion is about 80 wt. % to about 100 wt. % of the product. In some embodiments, the crispy portion is about 40 wt. % to about 80 wt. % of the product. In yet other embodiments, the outer shell is about 40 wt. % to about 60 wt. % of the product. In yet other embodiments, the outer shell is about 40 wt. % to about 50 wt. % of the product. As an example and not by way of limitation, the crispy portion is about 100 wt. % of the product.

[0130] In yet other embodiments, the inner filling is about 0 wt. % (no inner filing) to about 60 wt. % of the product. In yet other embodiments, there is no inner filling and therefore the inner filing is about 0 wt. % of the product. In yet other embodiments, the inner filling is about 1 wt. % to about 10 wt. % of the product. In yet other embodiments, the inner filling is about 10 wt. % to about 30 wt. % of the product. In yet other embodiments, the inner filling is about 20 wt. % to about 50 wt. % of the product. In yet other embodiments, the inner filling is about 30 wt. % to about 60 wt. % of the product. As an example and not by way of limitation, there is no inner filling in the product.

[0131] In particular embodiments, the product includes about 80 wt. % to about 100 wt. % of the crispy portion, with no inner filling, about 5 wt. % to about 10 wt. % seasoning and about 5 wt. % to about 10 wt. % oil, wherein the seasoning and the oil form a coating on the outside of the product. In particular embodiments, the product includes about 40 wt. % to about 50 wt. % outer shell, about 40 wt. % to about 50 wt. % inner filling, about 5 wt. % to about 10 wt. % seasoning and about 5 wt. % to about 10 wt. % oil, wherein the seasoning and the oil form a coating on the outer shell of the product. In embodiments, the product includes about 40 wt. % to about 45 wt. % outer shell, about 40 wt. % to about 45 wt. % inner filling, about 5 wt. % to about 8 wt. % seasoning and about 5 wt. % to about 8 wt. % oil, wherein the seasoning and the oil form a coating on the outer shell of the product. In embodiments, the product includes about 43 wt. % to about 45 wt. % outer shell, about 43 wt. % to about 45 wt. % inner filling, about 6 wt. % to about 7 wt. % seasoning and about 6 wt. % to about 7 wt. % oil, wherein the seasoning and the oil form a coating on the outer shell of the product.

[0132] In certain embodiments, the product has a shelf-life of at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months. In particular embodiments, the product has a shelf-life of greater than 12 months.

[0133] In certain embodiments, the product has a caloric density of about 1 to about 9 calories per gram of the product. In some embodiments, the product has a caloric density of about 2 to about 9 calories per gram, about 3 to about 9 calories per gram about 4 to about 9 calories per gram about 5 to about 9 calories per gram, about 6 to about 9 calories per gram, about 7 to about 9 calories per gram, or about 8 to about 9 calories per gram. Alternatively, the product has a caloric density of about 1 to about 8 calories per gram, about 1 to about 7 calories per gram, about 1 to about 6 calories per gram about 1 to about 5 calories per gram, about 1 to about 4 calories per gram, about 1 to about 3 calories per gram, or about 1 to about 2 calories per gram. In other embodiments, the product has a caloric density of about 2 to about 9 calories per gram, about 3 to about 8 calories per gram, about 4 to about 7 calories per gram, or about 5 to about 6 calories per gram of the product. In particular embodiments, the product has a caloric density of about 3.8 to about 4.8 calories per gram of the product.

[0134] In some embodiments, the calorie content of the product ranges from about 200 calories to about 700 calories per 100 grams of the product. In some embodiments, the calorie content of the product ranges from about 300 calories to about 700 calories, about 400 calories to about 700 calories, about 500 calories to about 700 calories, about 600 calories to about 700 calories, about 200 calories to about 600 calories, about 200 calories to about 500 calories, about 200 calories to about 400 calories, about 300 calories to about 600 calories, or about 400 calories to about 500 calories per 100 grams of the product. In particular embodiments, the calorie content of the product ranges from about 384 calories to about 480 calories per 100 grams of the product.

[0135] In some embodiments, the protein content of the product ranges from about 5 grams to about 50 grams per 100 grams of the product. In some embodiments, the protein content of the product ranges from about 5 grams to about 40 grams, about 5 grams to about 35 grams, about 5 grams to about 30 grams, about 5 grams to about 25 grams, about 5 grams to about 20 grams, about 5 grams to about 15 grams, or about 5 grams to about 10 grams per 100 grams of the product. In some embodiments, the protein content of the product ranges from about 10 grams to about 50 grams, about 10 grams to about 40 grams, about 15 grams to about 40 grams, about 20 grams to about 40 grams, about 25 grams to about 40 grams, about 30 grams to about 40 grams, about 35 grams to about 45 grams, about 20 grams to about 50 grams, or about 20 grams to about 25 grams per 100 grams of the product. In particular embodiments, the protein content of the product ranges from about 23 grams to about 46 grams per 100 grams of the product.

[0136] 3. METHODS

[0137] Subjects

[0138] The presently disclosed subject matter includes various methods of assessment and / or alleviation of a health condition as disclosed below. In certain embodiments, the subject is a human. The human subject can be an adult or can be a pediatric subject. In specific embodiments, the human subject is a pediatric subject aged about one month to about 17 years. In certain embodiments, the subject is a child aged from about 1 year to about 12 years. In certain embodiments, the subject is an adolescent aged from about 13 years to about 17 years. In alternative embodiments, the subject is an adult aged over 17 years.

[0139] The presently disclosed subject matter can be used in various human subject having specific health-related conditions. Exemplary health conditions, which can be alleviated by improving microvascular function include, but are not limited to, poor nutrition, cardiometabolic health, overweightness, allergy, asthma, diabetes, and inflammatory diseases.

[0140] In certain embodiments, the subject is suffering from lack of proper nutrition. In certain embodiments, the subject is overweight when compared with others within the subject’s age group. In alternative embodiments, the subject is undergoing pharmacological or non- pharmacological treatment for an inflammatory disease.

[0141] Assessing Status of Microvascular Function

[0142] In one embodiment of the presently disclosed subject matter, there is provided, a method of assessing a status of microvascular function in a subject comprising obtaining a biological sample from a subject, measuring in the biological sample, an amount of one or more biomarkers associated with microvascular function, to obtain a test value for each of the one or more biomarkers, and comparing the test value with a reference value for the one or more biomarkers, wherein a change in the test value as compared to the reference value indicates an improved or an impaired microvascular function.

[0143] In certain embodiments, the subject is any subject in need of alleviation of a health condition by improving microvascular function. Exemplary health conditions, which can be alleviated by improving microvascular function include, but are not limited to, poor nutrition, cardiometabolic health, overweightness, allergy, asthma, diabetes, and inflammatory diseases.

[0144] In certain embodiments, non-limiting examples of a biological sample include, a blood sample, a tissue sample, a saliva sample, a urine sample, or a fecal sample.

[0145] In certain embodiments, the biomarker is any biomolecule from a biological sample, which can be measured to assess the status of microvascular function in the subject. Nonlimiting examples of such biomolecules include, metabolites, cytokines, lipids, sugars, cholesterols, bile acids, high-density lipoproteins, an insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP), and derivatives thereof. In some embodiments, a combination of biomarkers are measured to assess status of microvascular function in the subject.

[0146] In some embodiments, in an exemplary method of assessing microvascular function, a decrease in at least one of IL-ip, IL-4, IL-22, IL-23, IL-31, F2-isoprostane, 11- hydroxy eicosatetraenoic acid (11- HETE), and 12-HETE compared to a reference value, and / or an increase in urosodeoxycholic acid (UDCA) compared to a reference value, indicates an improved microvascular function. In some embodiments, in another example, the method comprises measuring in the biological sample, an amount of an insulin-like growth factor (IGF- 1 or IGF-2) and an amount of an insulin-like growth factor binding protein (IGFBP), calculating a ratio of IGF to IGFBP to obtain a test ratio, and comparing the test ratio with a reference ratio, wherein an increase in the test ratio compared to the reference ratio indicates improved microvascular function. In certain embodiments, the IGF being measured is one or more of IGF-1 or IGF-2. In certain embodiments, the IGFBP being measured is one or more of IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5 or IGFBP6. In some embodiments, the IGFBP being measured is IGFBP3, IGFBP5, or a combination of these.

[0147] In some embodiments, in yet another example, the method comprises measuring in the biological sample, an amount of a cholesterol and an amount of a high-density lipoprotein (HDL), calculating a ratio of the amount of the cholesterol to the amount of the HDL to obtain a test ratio, and comparing the test ratio with a reference ratio, wherein a decrease in the test ratio compared to the reference ratio indicates improved microvascular function.

[0148] In any of the embodiments discussed above, in one aspect, the reference value or reference ratio is determined from a different time point, for example, before the subject has begun consuming a legume-based product. In another aspect, the reference value or reference ratio is determined by using a control product that delivers the same calories but not the legumebased product. In a third aspect, the reference value or reference ratio is obtained from a database of reference ranges.

[0149] Administration of Food Products

[0150] Further to these embodiments, the method comprises providing to a subject identified as having an impaired microvascular function, a legume-based snack food product described above in Section 2, wherein consuming the snack food product improves microvascular function. For example, in some embodiments, the first legume is yellow pea, in some embodiments, the second legume is yellow pea, in other embodiments, both the first and the second legume are yellow pea. Also, in some embodiments, the first additive and the second additive are independently one or more of Vitamin B6, Vitamin B12, Vitamin D, zinc, iron, copper, magnesium, or manganese.

[0151] In some embodiments, the legume-based snack food is provided to the subject in an amount from about 5 grams / serving to about 30 grams / serving. For example, in some embodiments the legume-based snack food is provided to the subject in an amount of about 26 grams / serving. In some embodiments, the legume-based snack food is provided to the subject in an amount from about 5 grams / serving to about 30 grams / serving. In one non -limiting embodiment, the legume-based snack food is provided to the subject in an amount of about 26 grams / serving.

[0152] In some embodiments, the legume-based snack food product has a caloric density of about 2 calories per gram of the product to about 8 calories per gram of the product. In some embodiments, the product has a caloric density of about 4 calories per gram of the product to about 6 calories per gram of the product. In one non-limiting embodiment, the product has a caloric density of about 4.8 calories per gram.

[0153] In some embodiments, the protein content of the product ranges from about 20 grams to about 50 grams per 100 grams of the product. In some embodiments, the protein content of the product ranges from about 20 grams to about 30 grams per 100 grams of the product. In some embodiments, the protein content of the product ranges from about 30 grams to about 50 grams per 100 grams of the product. In some embodiments, the protein content of the product ranges from about 40 grams to about 50 grams per 100 grams of the product. In one nonlimiting embodiment, the protein content of the product is about 23 grams per 100 grams of the product.

[0154] In some embodiments, the snack food is provided daily for a total daily calorie intake from about 200 kcal to about 250 kcal. In other embodiments, the snack food is provided daily for a total daily calorie intake from about 165 kcal to about 250 kcal. In yet other embodiments, the subject is instructed to consume the snack food once daily, twice daily, thrice daily, or four times daily such that the total calorie intake from the legume-based snack food product does not exceed about 250 calories per day. For example, consuming one serving of about 26 gram of the product per day delivers a daily calorie density of about 125 calories. Similarly, consuming two servings of about 26 gram of the product per day delivers a daily calorie density of about 250 calories.

[0155] In some embodiments, the subject is provided the snack food from about 1 week to about 4 weeks. In some embodiments, the subject is provided the snack food from about 2 weeks to about 4 weeks . In some embodiments, the subject is provided the snack food from about 3 weeks to about 4 weeks.

[0156] In some embodiments, the subject is provided the snack food from about 4 weeks to about 8 weeks. In some embodiments, the subject is provided the snack food from about 5 weeks to about 8 weeks. In some embodiments, the subject is provided the snack food from about 6 weeks to about 8 weeks. In some embodiments, the subject is provided the snack food from about 7 weeks to about 8 weeks.

[0157] In some embodiments, the subject is provided the snack food from about 1 month to about 6 months. In some embodiments, the subject is provided the snack food from about 2 months to about 6 months. In some embodiments, the subject is provided the snack food from about 3 months to about 6 months. In some embodiments, the subject is provided the snack food from about 4 months to about 6 months. In some embodiments, the subject is provided the snack food from about 5 months to about 6 months.

[0158] In some embodiments, the subject is provided the snack food from about 6 months to about 12 months. In some embodiments, the subject is provided the snack food from about 7 months to about 12 months. In some embodiments, the subject is provided the snack food from about 8 months to about 12 months. In some embodiments, the subject is provided the snack food from about 9 months to about 12 months. In some embodiments, the subject is provided the snack food from about 10 months to about 12 months. In some embodiments, the subject is provided the snack food from about 11 months to about 12 months. In some embodiments, the subject is instructed to consume the snack food for 4 weeks. In some embodiments, the subject is instructed to consume the snack food for at least 4 weeks.

[0159] Improving Microvascular Function

[0160] In another embodiment of the presently disclosed subject matter, there is provided, a method of improving microvascular function in a subject comprising, providing to a subject, a legume-based snack food as disclosed herein comprising at least one extruded first legume and a first additive comprising at least one vitamin, at least one mineral, or a combination thereof, wherein consuming the snack food improves microvascular function.

[0161] In certain embodiments, the subject is any subject in need of an intervention for a health condition by improving microvascular function. In some embodiments, the method causes a weight loss in the subject. In some embodiments, the method improves cardiometabolic health in the subject.

[0162] In certain embodiments, the legume-based snack food product has a caloric density and protein content as disclosed herein. Also in certain embodiments, the serving size and frequency of consumption of the legume-based snack food is disclosed above. For example, in one embodiment, the subject is provided the snack food for about 4 weeks. In other embodiments, the subject is provided the snack food in an amount of about 52 grams / day, for at least about 4 weeks, which delivers a total daily calorie intake of about 250 calories.

[0163] In certain embodiments, the method further comprises, calculating a first Framingham reactive hyperemia index (fRHI) value for the subject before consuming the snack food and calculating a second fRHI value after consuming the snack food, wherein a higher second fRHI value compared to the first fRHI value indicates improved microvascular function.

[0164] In certain embodiments, the fRHI is calculated from the reactive hyperemia index (RHI), which measured by peripheral arterial tonometry (PAT) as the ratio of the average of pulse wave amplitude (PWA) during a 1 min period following 90s of reactive hyperemia to the average PWA during a 3.5 min baseline period. fRHI is then calculated as the natural logarithmic transformation of the RHI ratio during the data collection from 90s to 120s after release of occlusion.

[0165] Alleviating Inflammatory Luns Diseases

[0166] In yet another embodiment of the presently disclosed subject matter, there is provided a method of alleviating an inflammatory lung disease in a subject comprising, providing to a subject, a snack food comprising at least one extruded first legume wherein consuming the snack food improves microvascular function thereby alleviating the inflammatory lung disease.

[0167] In some embodiments, the subject is undergoing treatment for an inflammatory lung disease with a prescription therapy, over-the-counter (OTC) therapy, or a non-pharmacological therapy, and consuming the snack food complements the prescription therapy and / or the OTC therapy, thus alleviating the inflammatory lung disease.

[0168] In certain embodiments, the method comprises providing to the subject a legume-based snack food product described above, wherein consuming the snack food alleviates inflammatory lung disease.

[0169] In certain embodiments, the legume-based snack food product has a caloric density and protein content as disclosed herein. Also, the serving size and frequency of consumption of the legume-based snack food is disclosed above. For example, in one embodiment, the subject is provided the snack food for 4 weeks. In other embodiments, the subject is provided the snack food in an amount of about 52 grams / day, for at least 4 weeks, which delivers a total daily calorie intake of about 250 calories. In some embodiments, the subject is instructed to consume the snack food once daily, twice daily, thrice daily, or four times daily. In an exemplary embodiment, the subject is instructed to consume one serving of an about 26 gram snack food twice daily, which delivers a total daily calorie intake of about 250 calories.

[0170] In some embodiments, the inflammatory lung disease includes, but is not limited to allergy and asthma.

[0171] In some embodiments, consuming the legume-based snack food reduces inflammation thereby alleviating the inflammatory lung disease.

[0172] In some embodiments consuming the snack food increases levels of urosodeoxycholic acid (UDCA) compared to a reference value, thereby alleviating the inflammatory lung disease in the subject.

[0173] In some embodiments, consuming the snack food reduces levels of at least one cytokine compared to a reference value, thereby alleviating the inflammatory lung disease in the subject. In some embodiments the cytokine is an inflammatory cytokine. Non-limiting examples of inflammatory cytokines include IL-ip, IL-4, IL-22, IL-23, IL-31, or a combination of these.

[0174] In some embodiments, the snack food reduces levels of IL-23, IL-4, or a combination of these. In other embodiments snack food consumption increases UDCA levels compared to a reference value and reduces levels of at least one inflammatory cytokine compared to a reference value, thereby alleviating the inflammatory lung disease in the subject.

[0175] In any of the embodiments discussed above, in one aspect, the reference value is determined from a different time point, for example, before the subject has begun consuming of a legume-based product. In another aspect, the reference value is determined by using a control product that delivers the same calories but not the legume-based product. In a third aspect, the reference value is obtained from a database of reference ranges.

[0176] In any of the above embodiments, in non-limiting examples, the subject is instructed to consume the snack food for about 1-week, about 2-weeks, about 3 -weeks, about 4-weeks, about 1 -month, from about 1 -month to about 6-months, or from about 1 -month to about 12-months. In one specific example, the subject is instructed to consume the snack food for 4-weeks, which complements prescription and / or OTC therapy for inflammatory lung disease.

[0177] 4. EXAMPLES

[0178] The following examples are provided to further illustrate some embodiments of the presently disclosed subject matter but are not intended to limit the scope of the subject matter; it will be understood by their exemplary nature that other procedures, methodologies, or methods known to those skilled in the art can alternatively be used.

[0179] 4.1 METHODS

[0180] 4.1.1 Product

[0181] The development goals for the legume-based snack products were that they be nutrient dense, vegetarian, and low cost to produce. The final product aimed to address poor nutrition along with associated deficiencies of iron, vitamin B12 and folate amongst others, or to examine the influence of protein intake or micronutrient fortification on outcomes of cardiometabolic health.

[0182] 4.1.2 Participants

[0183] School aged children (9-13 years of age) from the greater Sacramento, California Metropolitan area were recruited through the University of California (UC) Davis Health Clinical Studies website. Children who were free of self-reported disease, serious illness and not currently under acute medical care, with no known allergy or intolerance to peas, legumes, pulses, and soy, dairy or wheat / gluten products were eligible for enrollment. Children taking supplements, multivitamins and / or multimineral supplements or, prescription medications other than asthma or seasonal allergy medications were excluded from the study. Parents and children who agreed to participate in this trial signed informed consent and assent documents respectively, which were approved along with the study protocol by the Institutional Review Board of UC Davis.

[0184] 4.1.3 Study Design

[0185] Prior to start of the study, a clinical screening was performed for each participant to confirm eligibility. This screening visit also served to familiarize the participants to the study procedures, which included measurement of blood pressure and peripheral arterial tonometry (PAT). Eligible children were randomized into a double-blind parallel-arm probe study examining the effects of a legume-based snack food on metabolism and CV health. The children were randomized to one of four intervention products providing 6g or 12g of extruded yellow pea with micronutrient fortification (+F) or without micronutrient fortification (NF) as shown in exemplary Figure 1. The 6g and 12g products provided 20% and 40% of the recommended daily allowance of protein respectively

[0021] , The products were snack puffs and were provided in 26g coded packaged servings. Ingredients of note included yellow pea flour, as well as palm and sunflower oils. The products were manufactured and provided by Mars International India Pvt. Ltd.

[0186] Randomization was performed following a predetermined plan formulated via a webbased random number generator, and stratified based on sex, BMI (i.e., < or > 85 percentile for age and sex) and Pubertal Development Score (PDS, i.e., score < or > 2), assessed using a questionnaire

[0022] , The children were instructed to consume two 26g packages of the products, provided in a salsa or salt and pepper flavoring, every day for 4 weeks. The children were encouraged to consume this snack in place of similar amounts of “minimal nutritional value” snack foods (e.g., cookies, potato chips, etc.) that they typically consume.

[0187] All children participated in two study day visits scheduled before and after 4 weeks of product intake. Study participants arrived at the facility after an overnight fast. Upon arrival, they provided a urine sample, and after anthropometric measurements, the children rested in a seated position for 15 min in a quiet, temperature-controlled room for blood pressure measurements. During this resting period, the children participated in a tablet based nutrition education program that provided lessons on the nutrition facts label and included activities that prompted the participants to choose a healthier snack option

[0023] , After a PAT measurement, a registered nurse collected blood samples for the outcomes outlined below, along with a comprehensive metabolic and lipid panel, and a complete blood count analyzed. For each study visit, the child and / or the child’s parents completed a web-based automated self-administered 24 h dietary recall (ASA24, administered via the NIH website) with the assistance of trained researchers or a registered dietitian. At completion of the first study visit, the study participants were provided with enough product for the 4 week intervention period. The children and their parents were asked to save and return all used and unused product packaging and to maintain a daily product intake log. At the end of the 4 week period, the returned packages were counted, and any leftover product weighed. The total percent consumed was calculated based on a total of 1456g of product provided. The average product consumed per day per kg body weight was also calculated.

[0188] 4.1.1 Microvascular function

[0189] Digital microvascular function was assessed by PAT (Endo-PAT® 2000; Itamar Medical, Caesarea, Israel). After blood pressure measurements, all participants rested in a supine position for 10 min. Following a single supine blood pressure measurement, the PAT measurement was performed, which involved a minimum of a 6 min baseline recording, followed by a 5 min occlusion period to eliminate pulsatile blood flow, induced by inflating a blood pressure cuff placed on the proximal forearm to approximately 20-30 mm Hg above systolic blood pressure. After 5 min, the pressure was released, and the resulting reactive hyperemia response was recorded for an additional 5 min. The system software automatically calculated the reactive hyperemia index (RHI), which is the ratio of the average of pulse wave amplitude (PWA) during a 1 min period following 90s of reactive hyperemia to the average PWA during a 3.5 min baseline period. Additionally, the Framingham Reactive Hyperemia Index (fRHI) was calculated, which uses the natural logarithmic transformation of the RHI ratio during the data collection from 90s to 120s after release of the occlusion. The fRHI is strongly correlated with CV risk factors in adults

[0024] ,

[0190] 4.1.2 Blood Pressure

[0191] For blood pressure measurements, each child was fitted with an appropriate size pediatric or small adult blood pressure cuff and instructed to sit for 15 min prior to blood pressure measurement initiation. Three blood pressure measurements were recorded, each 5 min apart, using an automated oscillometer blood pressure unit (Vital Spot, VSM 300™, Welch Allyn).

[0192] 4.1.3 Cytokines, Growth Factors and Adipokines

[0193] Luminex methodology with bead immunoassays (BioRad, Hercules, CA) was employed to measure circulating plasma levels of inflammatory TH17 cytokines; IL-ip, IL-4, IL-6, IL- 10, IL- 17a, IL-17f, IL-21, IL-22, IL-23, IL-31, IL-33, Interferon g (IFNg), Tumor Necrosis Factor a (TNF a), and growth factors including Insulin Like Growth Factor-1 (IGF- 1), IGF -2 and the IGF binding proteins (IGFBP), IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7. Briefly, analyte-specific antibody conjugated beads were incubated with a 25 pL assay buffer and 25mL of plasma on an orbital shaker in the dark. Following incubation, the fluid was gently aspirated by vacuum manifold and the beads washed. Detection antibodies (25pL) were then added to each well and incubated on an orbital shaker for Ih at room temperature. Subsequently, 25pl of Streptavidin-Phycoerythrin was added to each well and incubated on a plate shaker for 30 min at room temperature. The plate was then washed 3 times, 150 l of lx Sheath fluid (Bio-Rad Laboratories Inc., Hercules, CA) was added to each well, and the fluorescence measured on a Bio-Plex 200 System (Bio-Rad Laboratories Inc., Hercules, CA). Hepcidin, and the adipokines, leptin and adiponectin were assessed via enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). All inter-assay percent coefficient of variability (CV) were at or below 10%, while all intra-assay CV were below 10%.

[0194] 4.1.4 Plasma Micronutrients

[0195] Heparinized plasma was analyzed for iron, zinc, magnesium, manganese, and copper by the UC Davis Interdisciplinary Center for Inductively Coupled Plasma (ICP) Mass Spectrometry (MS), with a quadrupole ICP-MS (Agilent Technologies). Sample volumes were determined during transfer to 2mL digestion tubes. The samples were rinsed with 0.25 mL Milli-Q® Water and transferred to the digestion tubes. The original tubes were subsequently rinsed with 120 mL of trace metals grade concentrated nitric acid, which was transferred to the digestion tubes. An additional 180 mL of trace metals grade concentrated nitric acid was then added to the digestion tubes. Samples and digestion controls were heated at 95 °C for 2 h, after which 1.2 mL 30% hydrogen peroxide was added incrementally over 1 h and the samples were heated for an additional 1.5 h at 95 °C. The volume of all samples and digestion controls was adjusted to 1.5 mL using Milli-Q® water. Samples were then transferred to 15 mL tubes, and the volume adjusted to 5 mL using Milli-Q® water, which resulted in a 6% nitric acid matrix. Small amounts of solids remained for the heparinized plasma samples. Samples and digestion controls were filtered using 0.45 mm syringe filters prior to analysis by ICP-MS. Soluble transferrin receptor (sTfR) was measured with a human quantikine IVC Elisa Kit (R&D systems). Chemiluminescent determination of vitamin B12, ferritin, and homocysteine was performed on an Immulite® 1000 system (Siemens), with inter-assay CV controls below 5%.

[0196] 4.1.5 Lipid Mediators

[0197] Bile acids were quantified by liquid chromatography mass spectrometry (LC-MS) using modifications of a published method

[0025] , Separation of bile acids were achieved by reversed- phase capillary ultra-performance liquid chromatography (UPLC) using a 2.1 x 100 mm, 1.7 Waters™ Acquity BEH Cl 8 column (Waters, Milford, MA) and a 16 min gradient (Solvent A= 0.1% formic acid in water; Solvent B = 0.1% formic acid in acetonitrile) on a Waters™ Acquity UPLC (Waters Corp., Milford MA) and detected on an API 4000™ QTrap (Sciex, Framingham, MA) by multiple reaction monitoring in negative mode electrospray ionization.

[0198] Oxylipins were isolated and quantified using modifications of published protocols [25, 26]. Briefly, plasma aliquots (100 mL) were spiked with deuterated oxylipin surrogates and extracted with 60 mg Oasis HLB (Waters) solid phase extraction cartridges. Solvents were removed under vacuum. Residues were reconstituted in methanol containing an internal standard, filtered at 0.1 mm, and analyzed by UPLC-MS / MS. Analytes were separated on a 2.1 x 150 mm, 1.7 mm Waters™ Acquity BEH column on an Waters™ Acquity UPLC, ionized by electrospray ionization and detected by multi -reach on monitoring on an API 4000™ QTRAP (Sciex, Framingham, MA). Oxylipins and N-acylethanolamides, monoacylglycerols, and lipoamino acids were analyzed in independent injections and ionized in negative and positive modes, respectively.

[0199] Residues were quantified against 7 point curves of authentic standards purchased from Steraloid Inc. (Newport, RI), Sigma-Aldrich (St. Louis, MO), and Medical Isotopes, Inc. (Pelham, NH), Cayman Chemical (Ann Arbor, MI) and Larodan Fine Lipids (Malmo, Sweden) using internal standard methodologies

[0026] , Data was processed in MultiQuant™ Version 3.0 (Sciex, Framingham, MA). All auto integrations were manually evaluated and modified as appropriate.

[0200] 4.1.6 Serum and Urine Metabolomics

[0201] Serum was prepared for metabolomics by filtering 350 mL through an Amicon® Ultra- 0.5 mL centrifugal filter unit with an Ultracel®-3 membrane (EMD Millipore, Billerica, MA, USA) that was pre-washed (5x) with purified water and dried with a Kim wipe to remove lipids and proteins. Samples were centrifuged (60 min, 4 °C, 14 krcf), and 207 pL of filtrate was combined with 23 pL of internal standard containing 5 mM 3 -(trimethylsilyl)- 1- propanesulfonic acid-d6 (DSS-d6), Na s, and D2O (Chenomx, Edmonton, AB, Canada). Sample pH was adjusted to 6.8 ± 0.1 using 1 N HC1 or NaOH, and 180 pL of sample was transferred to a 3 mm NMR tube and stored at 4 °C until NMR data were acquired (within 24h of sample preparation). Urine samples were thawed on ice and prepared for analysis by centrifuging (1 min, 4 °C, 14 krcf), and tested for protein using Multistix® Urinalysis Reagent Test Strips (Siemens Healthineers, Erlangen, Germany). Protein-free urine was centrifuged (15 min, 4 °C, 14 krcf), an aliquot of 207 pL was combined with 23 pL of internal standard, and the sample prepared as described above. All samples were stored at 4 °C until NMR data were acquired (within 24h of sample preparation).

[0202] NMR spectra were acquired at 298 K using the ‘NOESYPR1D’ experiment on a Bruker Avance™ 600 MHz NMR equipped with a SampleJet autosampler (Bruker BioSpin, Germany). The acquisition parameters were: 12 ppm sweep width, 2.5 s acquisition time, 2.5 s relaxation delay, and 100 ms mixing time. Water saturation was applied during the relaxation delay and mixing time. The resulting spectra were zero-filled to 128,000 data points and an exponential apodization function corresponding to a line broadening of 0.5Hz was applied. The acquired spectra were processed using Chenomx NMR Suite v8.43 (Chenomx Inc., Edmonton, Canada), which uses the concentration of a known reference signal (in this case disodium-2,2- dimethyl-2-silapentane-5-sulfonate (DSS)) to determine the concentration of individual compounds. Metabolite quantitation was achieved using the 600 MHz library as previously described [27-29],

[0203] 4.1. 7 Statistical Analysis

[0204] The primary outcome was microvascular function as measured by PAT. Based on the data from adolescents in a dietary intervention trial

[0013] , with a mean between group difference in RHI response of 0.52 and a standard deviation of 0.78, an n =13 per group was determined as sufficient for a 4 group analysis with ANOVA at an a =0.05 at 80% power. To account for potential attrition, the initial recruitment goals were for 20 participants per group.

[0205] Data were analyzed by multiple imputation to address missing data using the multivariate by chained equations (MICE) approach (using R, Version 1.1.463)

[0030] , A total of 350 variables were collected at each time point. Of these, 157 variables represented complete cases (no missing data). The remaining 193 variables were imputed on raw values using 35 imputed models (m = 35). Variables used as predictors met the following criteria: absolute correlative value of <0.70 (mincor = 0.70) and minimum proportion of usable cases >85% (minpuc = 0.85, i.e. <15% missing data). The imputed data sets were combined and histograms of variables from the imputed and original data set were generated to ensure the two were comparable. The current report focuses on 258 variables from 11 classes (Table 1).

[0206] Differences at baseline between normal weight and overweight / obese children were assessed using t-test or Wilcoxon / Kruskal-Wallis test as appropriate. Individual response to the intervention was calculated as the change from baseline prior to product intake (i.e., Variable4weeks - Variablebaseline), with the resulting values used for all statistical tests (analyzed using JMP Pro 15). Non-normal variables were transformed prior to analysis using the Johnson transformation. Potential between group differences at baseline were assessed using one-way analysis of variance (ANOVA) with Tukey post hoc. Univariate analysis was conducted on the 4 week change values using protein and fortification level as fixed effects, and subject as random effect, in a standard least squares model with Tukey as the post hoc test. Variables at the p<0.1 level are presented as the least square means ± SEM. A sub-group analysis in those children who were overweight or obese (BMI% for age and sex >85) was also performed and reported.

[0207] Presented variables were further subject to predictive modeling with partial least square regression using NIPALs method with k-fold cross validation (PLS-R, JMP Pro, vl5). As discriminate variable reduction was necessary to increase model predictive power, an ensemble of partial least square regression (PLS-R), bootstrapped forest, and boosted tree techniques were used to reduce the number of discriminate variables considered. Specifically, variables were initially selected from: 1) a stepwise linear regression with a Bayesian information criterion (BIC) cut-off and false discovery rate (FDR) correction

[0031] ; and 2) bootstrapped forest and boosted tree models that account for >1% of the explained model. Variables meeting two out of the three conditions were used in the final PLS-R model. Metabolites with variable importance in projection (VIP) scores >1.0 were further considered in a Pearson’s correlation analysis, with p < 0.2 considered significant after FDR adjustment.

[0208] 4.2 STUDY GROUPS

[0209] Of the 68 children enrolled into the study, 60 children were assigned to one of the four legume-based intervention products. Of the eight children not assigned to the intervention, four could not continue due to difficulty in obtaining a blood sample, with the remainder deciding not to participate prior to start of the study. In addition, five children assigned to the study did not complete the full course of the study and were not included in the final data analysis. Figure 2 provides a flow chart demonstrating how the participants were selected for the various study groups. For the remaining 55 children (29 males, 26 females) assigned to the study, the mean age was 11.0 ± 1.3 years, with a mean PDS score of 2.0 ± 0.7 (Figure 3). Nineteen children (35%) self-reported as multiracial, which included eight (15%) children identifying as both African American or Black and Caucasian or White; five (9%) as Asian and Caucasian or White; three (6%) as American Indian or Alaskan Native and Asian; and two (4%) American Indian or Alaskan Native and Caucasian or White. For the remaining children, 25 (45%) were Caucasian or White, 2 (4%) American Indian or Alaskan Native, and 5 (9%) Asian. Twenty one (38%) of the children identified as Hispanic or Latino. Four parents did not report their child’s race and ethnicity (Table 2). Table 2 also provides additional descriptive characteristics of the children assigned to each dietary group assignment. Of the 55 participants, 13 were assigned 12g protein without micronutrient fortification (12gNF), 12 were assigned to 6g protein fortified with micronutrients (6g+F), 15 were assigned to 12g protein fortified with micronutrients (12+F), and 15 were assigned to 6g protein without micronutrient fortification (6gNF). The ratio of male to females was similar in three of the four study groups; however, there were three fewer females assigned to the 12gNF group. There was an equal distribution of children between BMI classifications in the non-fortified (NF) groups; however, the micronutrient-fortified groups had approximately 50% fewer children with a normal BMI. One child each in the 12g+F and 12gNF groups reported the use of seasonal allergy medications as needed, with one child prescribed a daily allergy medication in the 12gNF group. In the 12gNF group, one child was prescribed daily asthma medication, and one child was prescribed asthma medication on an as needed basis. For the 4 week intake period, those assigned to product 12gNF consumed 86 ± 14 % (n =13) of the product, with a percent intake of 80 ± 17 % (n =12), 81 ± 24 % (n =15), and 80 ± 28 % (n =15) recorded for the 6g+F, 12g+F and 6gNF groups, respectively. The difference in percent product intake between groups was not significant (p =0.79).

[0210] Figure 4 presents the dietary intake of the children at baseline prior to legume-based product intake. On an average, the children met or exceeded daily recommended or adequate intakes except for dietary fiber, vitamin D, and potassium. There was a significantly greater intake of protein, niacin, and vitamin B6 in the 12gNF group compared to the 6g+F group. After 4 weeks of product intake, children who consumed the 6g products had a significantly increased the percentage of protein in their diet, as well as increased intakes of magnesium, thiamin, and niacin compared to those that consumed the 12g products (Table 3). Children who were overweight or obese and consumed the +F products significantly increased their intake of zinc as well as vitamins B6 and D compared to those who consumed the NF products. The intake of vitamin D and dietary fat as a percent of energy (kcal) was increased in those children who were overweight or obese and consumed the 6g protein products compared to those who consumed the 12g products (Table 3).

[0211] At baseline, the children’s average percent BMI for their age and sex (BMI%) was 77.5 ± 27 % with 21 of the children having a normal BMI% (i.e. <85%). Children who were overweight or obese, were taller, had lower high-density lipoprotein (HDL) cholesterol, along with higher total cholesterol to HDL cholesterol ratio (Cholesterol :HDL), non-high-density lipoprotein (non-HDL) cholesterol, and triglyceride levels. Correspondingly, these children had higher fasting insulin, leptin, and high-sensitivity C-reactive protein (hsCRP) levels, along with a trend for reduced RHI and fRHI compared to normal weight children (Figure 3).

[0212] A non-significant trend for higher BMI% or z-score was observed in those children consuming the 12g+F products for 4 weeks. Percent diastolic blood pressure based on age and sex (DBP%) was significantly greater in children consuming the 6g+F products compared to those consuming the 6gNF products, with a similar trend in overweight or obese children (Figure 5). Significant differences between group effects were not observed after 4 weeks of product intake for fasting blood glucose, insulin, leptin, hsCRP, and systolic blood pressure measures. However, 4 weeks of product intake did significantly influence microvascular function, and several markers related to inflammation and metabolism as described below.

[0213] Table 2. Descriptive statistics, by study group

[0214] Vanable

[0215] (n6g=+1F2) (n12=g+1F5) (n6g=N1F5) (1n2=gN13F)

[0216] Age (Years) 11.2 ± 1.6 11.3 ± 1.0 11.6 ± 1.3 11.7 ± 1.7

[0217] Sex

[0218] Female 6 (50.0) 7 (46.7) 8 (53.3) 5 (38.5)

[0219] Male 6 (50.0) 8 (53.3) 7 (46.7) 8 (61.5)

[0220] Protein level

[0221] 6 grams 12 (100.0) 0 (0.0) 15 (100.0) 0 (0.0)

[0222] 12 grams 0 (0.0) 15 (100.0) 0 (0.0) 13 (100.0)

[0223] Micronutrient Fortification

[0224] Yes 12 (100.0) 15 (100.0) 0 (0.0) 0 (0.0)

[0225] No 0 (0.0) 0 (0.0) 15 (100.0) 13 (100.0)

[0226] Weight Class

[0227] Normal weight 4 (33.3) 4 (26.7) 7 (46.7) 6 (46.2)

[0228] Overweight / obese 8 (66.7) 11 (73.3) 8 (53.3) 7 (53.8)

[0229] Self-Reported Ethnicity

[0230] Hispanic or Latino 3 (25.0) 7 (46.7) 6 (40.0) 5 (38.5)

[0231] Not Hispanic or Latino 9 (75.0) 7 (46.7) 7 (46.7) 7 (53.9)

[0232] Unknown 0 (0.00) 1 (6.7) 2 (13.3) 1 (7.7)

[0233] Self-Reported Race

[0234] American Indian or Alaskan Native 1 (8.33) 0 (0.00) 1 (6.67) 0 (0.00)

[0235] Asian 1 (8.33) 0 (0.00) 1 (6.67) 3 (23.1)

[0236] Native Hawaiian or Other Pacific 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)

[0237] Islander

[0238] Black or African American 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)

[0239] Caucasian or White 8 (66.7) 5 (33.3) 6 (40.0) 6 (46.2)

[0240] More than One Race 1 (8.33) 8 (53.3) 7 (46.7) 3 (23.1)

[0241] Unknown 1 (8.33) 2 (13.3) 0 (0.00) 1 (7.69)

[0242] Values are mean ± SD or values (percent of study group). 12gNF, 12g of protein, not micronutrient fortified; 6g+F, 6g of protein, micronutrient fortified; 12g+F, 12g of protein, micronutrient fortified, 6gNF, 6g of protein, not micronutrient fortified.

[0243] Table 3. Four week change in dietary intake1,2

[0244] V

[0245] Variab bl le (n6g=+1F2) (n12=g+1F 6gNF 12gNFP value 5) (n = 15) (n = 13 )P 1

[0246] Iron (mg) 2.99 ± 1.97 -1.87 ± 1.93 0.08

[0247] Overweight or Obese (P = 19) (n = 15)

[0248] Fat (%kcal) -4.13 ± 2.23 2.14 ± 2.48 0.07

[0249] Sugar (g) 0.28 ± 0.24 -0.36 ± 0.26 0.08

[0250] Vitamin B6 0.29 ± 0.25 -0.52 ± 0.28 0.04

[0251] (mg) -

[0252] Vitamin D 0.20 ± 0.21 -0.55 ± 0.23 0.02

[0253] (mcg)

[0254] Iron (mg) 2.89 ± 2.53 -3.68 ± 2.82 0.09

[0255] Zinc (mg) 1.27 ± 1.78 -4.26 ± 1.99 0.047

[0256] Variable Protein 6 g Protein 12 g P-value

[0257] Energy (kcal) 0.35 ± 0.19 -0.16 ± 0.19 0.06

[0258] Protein (%kcal) 2.1 ± 1.1 -2.3 ± 1.0 0.01

[0259] Magnesium 0.30 ± 0.22 -0.33 ± 0.21 0.04

[0260] (mg) -

[0261] Thiamin (mg) 0.36 ± 0.18 -0.36 ± 0.18 0.01

[0262] Niacin (mg) 0.40 ± 0.17 -0.36 ± 0.17 0.003

[0263] Sodium (mg) -0.29 ± 0.20 0.24 ± 0.20 0.07

[0264] Overweight or Obese (n = \6) (n = \5)

[0265] Fat (%kcal) -5.74 ± 2.40 3.76 ± 2.32 0.01

[0266] Vitamin D 0.23 ± 0.22 -0.58 ± 0.22 0.01

[0267] (mcg) -

[0268] Niacin (mg) 0.31 ± 0.22 -0.31 ± 0.21 0.06

[0269] Thiamin (mg) 0.29 ± 0.26 -0.41 ± 0.25 0.06

[0270] ’Dietary intake represents the values obtained from 24 hour recalls, with the average daily intake of product added.2Values are the Least Square Mean ± SEM and represent the 4 week change from baseline (e.g. Variable4weeks - Variablebaseime); data that were not normally distributed were Johnson transformed priorto analysis with Least Square Means. Results shown forthose variables that are P < 0. 1 for the main or interactive effects of protein and fortification level. 12gNF, 12g of protein, not micronutrient fortified; 6gF, 6g of protein, micronutrient fortified; 12gF, 12g of protein, micronutrient fortified, 6gNF, 6g of protein, not micronutrient fortified.

[0271] 4.3 RESULTS 4.3.1 Intake of fortified protein products increased microvascular function.

[0272] There were no significant differences between the four legume-based products for the 4-week change in either the RHI (p=0.14) or the fRHI (p=0.09). The amount of protein consumed did not significantly effect vascular function. On the other hand, children who consumed the fortified products (+F) showed significant increases in the primary outcome measures of microvascular function. This included an increase in fRHI (Figure 6A, Figure 7) and a trend for increased RHI (Figure 6B, Figure 7). Similar trends in fRHI following fortified product consumption were observed for both females and males (p=0.048 and p=0.06, respectively), whereas RHI response to fortified product intake appeared to be influenced by improvements in vascular response in male children only (male: p=0.06; female: p=0.47). For overweight / obese children, fRHI increased with fortified product intake (Figure 7).

[0273] 4.3.2 Amounts of protein and fortification improved plasma cholesterol concentrations.

[0274] After consuming the four intervention products for 4 weeks, there were no significant changes between groups for total cholesterol (p=0.7), HDL (p=0.6), non-HDL (p=0.6) and LDL-cholesterol (p=0. 4). However, children who consumed the fortified (+F) products had a significant reduction in LDL and non-HDL levels compared to those that consumed the NF products (Figure 7). Also, at baseline, 11 children had a Cholesterol :HDL >4. After 4 weeks of product intake, Cholesterol :HDL was lower in children in the fortified product group (Figure 7, Figure 6C) and 12 g protein product group (Figure 6D), with similar trends observed in children who were overweight or obese (Figure 7).

[0275] 4.3.3 Effect of legume-based product intake on fasting plasma micronutrient concentrations.

[0276] There were no significant changes for plasma iron, sTfR, copper, manganese, and vitamin B12 after 4 weeks of product intake. On the other hand, after 4 weeks, zinc levels showed an increasing trend in the 6g+F group compared to the 6gNF group in all children and in children who were overweight or obese (Figure 5). For all children, there was an increasing trend for magnesium levels for the 6g protein group relative to the 12g protein group (Figure 4), with the intracellular iron storage protein, ferritin, significantly increased in those children consuming the 12g protein products compared to those in the 6g product group (Figure 8).

[0277] 4.3.4 Effect of short-term intake of fortified protein products on circulating IGFBP levels.

[0278] While IGF-1 and IGF -2 did not change during short-term product intake (data not shown), the 6gNF group showed a trend for higher IGFBP4 levels compared to the other three groups (Figures 5). IGFBP5 levels were increased in all children in the +F product groups, and for those who were overweight or obese (p =0.02 and p=0.02, respectively, (Figure 7). As an assessment of “bioavailable” IGF-1, the ratios of IGF-1 to IGFBP3 and IGF-1 to IGFBP5 was calculated

[0032] , Compared to the 12g legume-based products, intake of the 6g legume-based products significantly increased IGF-1 / IGFBP3 in all children and in children who were overweight or obese (p=0.004, and p=0.01, respectively, Figure 8). A similar trend was observed for IGF-1 / IGFBP5 in all children (p=0.05) and in children who were overweight or obese (p=0.07, Figure 8).

[0279] 4.3.5 Fortified product intake decreased circulatins proinflammatory cytokines.

[0280] At baseline, children assigned to the 12gNF group had significantly higher levels of Thl7 cytokines IL-22 (vs 6g+F and 12g+F group), IL-23 (vs all groups) and IL-31 (vs 12g+F Figure 9), compared to other groups. After 4 weeks of legume-based product intake, the 12gNF group showed reduced levels of IL-23 relative to the 6gNF group , with similar trends observed for IL-22, IL-31, and IL-33 (Figures 5). Within the 12gNF group, overweight and obese children showed significantly lower levels of IL-4 relative to those in the 6gNF group, whose IL-4 levels were higher compared to the 6g+F group (Figures 5). The intake of fortified products significantly reduced IL-ip compared to the non-fortified products, with a similar trend observed in those children who were overweight or obese (Figure 7). Also, in the overweight / obese children subgroup, IL-4 was significantly reduced in those consuming the fortified products and the 12g products relative to those consuming the non-fortified and 6g products, respectively (Figure 7 and Figure 8).

[0281] 4.3.6 Fortified legume-based products reduced lipid mediators and epoxide hydrolase metabolites.

[0282] Compared with the non-fortified group, the fortified group exhibited a trend (p=0.07) for reduced levels of the cytochrome P450 (CYP) derived linoleic acid metabolite, 9, 10- epoxy octadecenoic acid (9, 10-EpOME or leukotoxin). Moreover, its corresponding soluble epoxide hydrolase (sEH) metabolite, 9, 10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) was reduced in children who are overweight or obese (Figure 7). All children, including overweight / obese children showed a significantly increase in 19,20- dihydroxy docosapentaenoic acid (19, 20-DiHDoPA) (a sEH metabolite of docosahexaenoic acid) after consuming the 12gNF product compared to children consuming the 12g+F product (Figure 4). Correspondingly, 19, 20-DiHDoPA levels were significantly lower in the fortified product group compared to non-fortified product group for all children, and overweight / obese children (Table 3). Four week intake of the fortified (+F) products also reduced the arachidonic acid (AA)-derived dihydroxyeicosatetraenoic acids (DiHETrE), 5,6-DiHETrE, 11,12- DiHETrE, and 14,15- DiHETrE (Figure 7). In all children and the overweight / obese group, those consuming the fortified products showed significantly reduced lipoxygenase (LOX)- derived 12-HETE, and cyclooxygenase-derived 11-HETE (Figure 7). The lipid oxidation product, F2-isoprostane was also significantly reduced in overweight or obese children who consumed the fortified products compared to the non-fortified products (Figure 7).

[0283] 4.3. 7 Effect of legume-based product intake on bile acids and endocannabinoids.

[0284] The glycine conjugated primary bile acid glycochenodeoxycholic acid (GCDCA) and the secondary glycine conjugated bile acid, glycodeoxycholic acid (GDCA), were significantly increased in the 12g protein group compared to the 6g protein group (p=0.02 and p=03, respectively; Figure 8). In addition, the taurine conjugated primary bile acid, taurocholic acid (TCA), was increased in the group provided with the 12g protein compared to the 6g protein group and in overweight or obese children (p =0.04 and p=0.049, respectively; Figure 8). Compared to children consuming the non-fortified products, children in the fortified product group had higher circulating levels of ursodeoxycholic acid (UDCA, p=0.049; Figure 7). Among the overweight / obese children, UDCA levels were higher for the 6g+F group compared to the 12g+F group (Figure 5). The effect of legume-based product consumption on circulating mediators of the endocannabinoid system, a class of molecules linked to states of metabolic dysfunction [26, 33] were also assessed. Significant changes to endocannabinoids were observed in children who were overweight or obese. These include significantly lower levels of docosatetraenoylethanolamide (DEA, adrenoyl ethanolamide) (Figure 7) following intake of the fortified products relative to non-fortified products, and significantly lower levels of N- oleoyl glycine (NO-GLY) following intake of the 6g products compared to the 12g products (Figure 8).

[0285] 4.3.8 Effect of 4 weeks of legume-based product consumption on serum and urinary metabolites.

[0286] Several metabolites of amino acid metabolism were significantly influenced by product intake. This included tyrosine, which was increased with the intake of the fortified products in all children including the overweight / obese subgroup (Table 2). In addition, in children from the overweight / obese subgroup, the 4-week change in serum tyrosine levels increased following 6g+F or 12g+F intake relative to 6gNF intake (p=0.02, Figure 3). Moreover, in the overweight / obese children, there was increased urinary excretion of alanine, glycine and 2- aminoadipate for the fortified group compared to the non-fortified group (Figure 7) with, urinary excretion of glycine increased in the overweight / obese group consuming the 12g products (Figure 5). In all children, 3-hydroxyisovalerate levels were greater following intake of the 12g protein products compared to the 6g protein products (Figure 8). Metabolites of glucose metabolism and the tricarboxylic acid (TCA) cycle that were significantly changed after 4 weeks of product intake included an increase in lactate levels for the fortified product (+F) groups including the overweight / obese subgroup (Figure 7). In all children and in the overweight / obese subgroup, consuming the 12g protein products resulted in increased urinary cis-aconitate compared to those consuming the 6g protein products (Figure 7 and Figure 8). Urinary excretion of glucose was also significantly reduced in the group consuming the 12g protein product (Figure 8). The fatty acid metabolite, glycolate, was higher in all children consuming the 12g+F product compared to the 12gNF product (Figures 5), and in overweight / obese children consuming the fortified (+F) products compared to the non-fortified (NF) products (Figure 7). Additionally, the fatty acid metabolite, O-acetyl carnitine, was present at higher levels in serum from the 12g protein groups (Figure 8), and in urine from overweight / obese children consuming the 6g+F product (Figure 7). Glycerol levels were significantly reduced in children consuming the fortified (+F) products (Figure 7). Among coproducts of host and microbial metabolism, 4-hydroxyphenyl lactate, was higher in the fortified (+F) product groups compared to the non-fortified (NF) product groups (Figure 7); whereas higher dimethyl sulfone levels were associated with intake of the 12g products compared to the 6g products (Figure 8). Those who consumed the 12g products also had a significant increase in circulating levels of hippurate (glycine conjugate of benzoic acid) as reflected by a significant increase in urinary excretion of hippurate in all children, and in the overweight / obese subgroup, (Figure 8). Among other metabolites, mannose was lower in overweight / obese children consuming the fortified products (Figure 7), whereas 5, 6- dihydrothymine was higher in the fortified product groups compared to non-fortified product groups (Figure 7).

[0287] 4.3.9 Metabolites associated with the 4-week change in microvascular function and the Cholesterol :HDL ratio.

[0288] Figure 10 summarizes correlation of fRHI and Cholesterol :HDL ratio. Figure 11A shows a graph of the top predictors of fRHI response in all children, with a Variable Importance in Projection (VIP) score of > 1.0 and representing 61% of the explained variability in response. It is noteworthy that the significant predictors IGFBP5 and UDCA were both positively associated with one another (Figure 10) and increased in those children consuming the fortified (+F) products for 4 weeks (Figure 7). A reduced inflammatory burden was negatively associated with IGFBP5 response, to include most measured cytokines, the lipid mediators, 11- HETE and 12-HETE, and oleic acid (C18: ln9%). In addition, IGFBP5 was positively associated with plasma zinc and 2-aminoadipate excretion (Figure 10, Groups A and B). In overweight / obese children, in addition to IGFBP5 and UDCA, positive predictors of fRHI response include dietary iron, serum and urinary levels of alanine, urinary tyrosine, and the microbial derived metabolite dimethyl sulfone. Significant negative predictors include IGFBP4, serum creatinine and as a percent of total fatty acids; Figure 11C). In contrast to IGFBP5, IGFBP4 was strongly and negatively associated with IGFBP5, while strongly and positively associated with cytokines and 12-HETE (Figure 10, Group B). Dietary intake of iron was strongly and positively associated with caloric and sugar intake, along with dietary intake of niacin, thiamin, vitamins B6 and D, and zinc. In addition, dietary iron was positively associated with serum dimethyl sulfone levels (Figure 10, Group B). Ascorbate was negatively associated with IGFBP5 and n-3 polyunsaturated fatty acid (omega-3 PUFA), while positively associated with IGFBP4 and the proinflammatory cytokines (Figure 10, Group B). Urinary excretion of alanine was negatively associated with creatinine, C18: ln9 %, C22:5n3, 12-HETE, and 14, 15-DiHETrE, while positively associated with formate, glutamine, glycine and glycolate excretion (Figure 10, Group B).

[0289] Specific predictors of Cholesterol :HDL response in all children are presented in Figure 11B, with the inflammatory cytokines IL-4 and IL-31, along with urinary theophylline, as significant positive predictors, and chloride and IGFBP5 as negative predictors. Apart from the previously discussed trends between IGFBP4 and IGFBP5, IL-ip and IL-4 were negatively associated with the urinary excretion of alanine and choline, while IL-4 was positively associated with ascorbate and TMAO (Figure 10, Group C). In children who were overweight or obese, positive predictors of Cholesterol :HDL were urinary creatine, glucose, and WBC, with the bile acids GCDCA and TCA, and dimethyl sulfone as negative predictors. At the False Discovery Rate (FDR) cutoff of <0.2, these predictors had very few significant associations with the other measured metabolites and included significant positive associations between GCDCA and TCA and GDCA (Figure 10, Group D). In addition, GCDCA was negatively associated with dietary Protein (% kcal), whereas TCA was negatively associated with dietary thiamin intake. White blood cell count (WBC) was positively associated with the 4-week change in circulating lymphocytes. Creatine excretion was positively associated with serum butyrate and urinary formate, glucose was positively associated with the theophylline excretion, while the 4-week change in serum dimethyl sulfone had no significant associations with any of the measured metabolites (Figure 10, Group D).

[0290] 4.4 CONCLUSION

[0291] The above data support snack food products designed to observe current guidelines of incorporating plant-based protein

[0034] , while at the same time providing additional micronutrients into the diet can indeed reduce low-grade inflammation and improve microvascular function in a population of apparently healthy, well-nourished children. The data suggest that children with apparently adequate dietary intakes, but who were predominately overweight or obese, can benefit from additional micronutrient supplementation. Multiple physiological stressors, including diabetes and hypertension, can induce inflammation and acute phase responses that can lead to secondary micronutrient deficiencies

[0035] ,

[0292] As shown above, daily intake of legume-based snack foods providing both protein and micronutrient fortification reduced inflammatory cytokines and lipid mediators (IL-1 beta, IL- 4, 11-HETE, and 12-HETE), and products of auto-oxidation (e.g., F2-isoprostanes). Furthermore, these changes were associated with increased IGFBP5 concentrations and improved microvascular function as measured by fRHI, an index of microvascular function associated with increased CV risk burden in adults

[0024] , In overweight or obese children, metabolic mediators positively associated with vascular response include tyrosine and the gluconeogenic substrates, alanine, and glycine. The enhanced excretion or circulating levels of these metabolites in those consuming the +F products suggest an overall shift in metabolism with product intake that was associated with vascular response.

[0293] Obesity predisposes individuals to low-grade inflammation and metabolic dysregulation, which increase CV risk

[0036] , The growth hormone / IGF axis is a complex system of binding proteins and proteases that provide both endocrine and paracrine signaling to regulate metabolism and vascular physiology in support of growth

[0037] , which can be dysregulated in obese children [38, 39], IGF-1 levels are influenced by protein intake

[0040] , and certain micronutrients, including zinc, can influence IGF binding to IGFBPs and its receptors [40, 41], IGFBP binding of IGF-1 and IGF-2 prolongs IGF half-life in the circulation and regulates binding to IGF receptors. IGF-1 is predominately bound to IGFBP3, the ratio of which serves as an index of “bioavailable or free” IGF-1

[0032] , In animal models, an increase in the IGF1 / IGFBP3 ratio demonstrates positive vascular effects that include reduced reactive oxygen species (ROS) and increased NO availability [42, 43], with a lower ratio associated with endothelial dysfunction and increased CV risk

[0032] , The above data show an increase in both the IGF1 / IGFBP3 and IGF1 / IGFBP5 ratios with intake of the 6g protein products. However, both microvascular function and IGFBP5 were significantly increased only in those children consuming the fortified products relative to non-fortified products, with IGFBP5 identified as a significant predictor of vascular response. These data suggest a potentially independent effect of IGFBP5 from IGF. Evidence that IGFBP5 can influence vascular health comes from data demonstrating elevated circulating levels in patients with disease

[0044] , yet increased IGFBP5 gene expression has been observed in the endothelium under normal, laminar, blood flow conditions

[0045] , Overall, defining the role of IGFBP in both health and disease has been daunting, as it can be conditionally regulated [44, 46], It is noteworthy that in this group of children, IGFBP5 was associated with a reduced inflammatory burden indicated by negative associations with 11-HETE and 12-HETE, and the cytokines IL-ip, IL-4, IL-22, IL-23, IL-31, and IL-33 (Figure 10).

[0294] Circulating levels of 11-HETE can be the result of COX metabolism or non-enzymatic oxidation by ROS [47, 48], The 12-LOX AA metabolite, 12-HETE, is known for its role in the initiation and progression of inflammation, increased ROS production from NADPH oxidases, and cytokine induction to include IL-1 beta [49-52], and can serve as a vasoconstrictor

[0053] , Levels of 12-HETE are known to have complex impacts on insulin and insulin signaling, to include inhibition of the translocation of nuclear factor erythroid 2-related factor 2 (NRF2), a zinc-dependent transcription factor involved in antioxidant enzyme regulation [54, 55], In the current study, both 12- and 11-HETE were positively associated with IL-ip and IL-4, both positive predictors of Cholesterol :HDL (Figure 10B and Figure 11, Group A). Metabolic dyslipidemia associated with obesity is characterized by increased inflammation and disruption of cholesterol transport and trafficking to the bile and feces [36, 56], The nod-like receptor containing a pyrin domain (NLRP3) inflammasome promotes metabolic inflammation through the cleavage of pro-IL-ip, reportedly primed by saturated fatty acids, oxidized LDL, and microbial products, to its active form IL-ip form

[0036] , A potent driver of the inflammatory response, IL-ip, is produced by a number of cell types including neutrophils, monocytes, macrophages, and vascular smooth muscle and endothelial cells, where it promotes proliferation and adhesion molecule expression

[0057] , Allergic rhinitis and asthma, often associated with obesity, can induce a Th2 cytokine profile by IL-4

[0058] , This is suggested by the above data, which shows that in addition to IL-4, children consuming the fortified, 12g protein product had similar trends for improved IL-23, IL-31 and IL-33 levels, a cytokine profile often associated with both asthma and development of atherosclerosis [57, 59],

[0295] Consuming the fortified 12g products had positive impacts on the Cholesterol :HDL ratio as inferred by normalization of cholesterol levels in overweight / obese children. Of interest is that this group of children had significantly higher intakes of vitamins B6 and D, as well as zinc (Table 3) after consumption of fortified products, which is consistent with reports of improved cholesterol levels with either increased intake or supplementation of these nutrients in children and adults [60-62], It is also likely that positive changes in bile acid synthesis and excretion influenced cholesterol levels. In the liver, cholesterol 7a-hydroxylase, also known as CYP7A1, synthesizes bile acids from cholesterol. After conjugation with taurine or glycine, the resulting bile salts are stored in the gall bladder and released into the gut with food intake to emulsify dietary fats and aid in both fat and fat-soluble vitamin absorption. Approximately 95% of the bile acids are enterohepatic recirculated with the remaining excreted in the feces. Dietary fiber and polyphenols can sequester conjugated bile acids, limiting their absorption, and increase their accessibility for bacterial deconjugation in the colon, thus producing more hydrophobic bile acids with limited absorption and higher fecal excretion. For those that are absorbed, additional conjugation in the liver and kidney produces secondary / tertiary bile acids

[0063] , Therefore, increased circulating bile acids, such as observed with consumption of the 12g products could represent an increase synthesis of bile acids and subsequent elimination of cholesterol. It is also noteworthy that the secondary bile acid, UDCA was both a predictor of fRHI and associated with IGFBP5 response. UDCA, is known to have a cytoprotective and anti-inflammatory role, and has been reported to reduce the vasoconstrictor endothelin-2 in individuals with primary biliary cirrhosis

[0064] , with improved vascular function in heart failure and coronary heart disease patients [65, 66],

[0296] Taken together, the above data revealed that continued consumption of the legumebased snack products disclosed here is associated with improved microvascular function and plasma lipids that is driven in part by positive changes in IGFBP5 and reduced inflammation. The above results support the ability of protein / micronutrient fortified legume-based snack products in reducing an individual’s short term, and potentially long-term, risk for vascular disease. Such products could also be effective in populations with food insecurity

[0069]

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[0367] * * *

[0368] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the disclosed subject matter as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0369] Patents, patent applications, publications, product descriptions and protocols are cited throughout this application the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of assessing a status of microvascular function in a subject comprising:(i) obtaining a biological sample from a subject;(ii) measuring in the biological sample, an amount of one or more biomarkers associated with microvascular function, to obtain a test value for each of the one or more biomarkers; and(iii) comparing the test value with a reference value for the one or more biomarkers, wherein a change in the test value as compared to the reference value indicates an improved or an impaired microvascular function.

2. The method of claim 1 , wherein the subj ect is suffering from overweightness, poor nutrition, obesity, or a cardiometabolic disorder.

3. The method of claim 1, wherein the biomarker is selected from the group consisting of metabolites, cytokines, lipids, sugars, cholesterols, bile acids, high-density lipoproteins, an insulin-like growth factor (IGF) and insulin-like growth factor binding protein (IGFBP), and derivatives thereof.

4. The method of claim 3, wherein a decrease in at least one of biomarkers, IL- ip, IL-4, IL-22, IL-23, IL-31, F2-isoprostane, 11-HETE, and 12-HETE compared to a reference value, and / or an increase in urosodeoxycholic acid (UDCA) levels compared to a reference value, indicates an improved microvascular function.

5. The method of claim 3, the method comprising: measuring in the biological sample, an amount of an insulin-like growth factor (IGF) and an amount of an insulin-like growth factor binding protein (IGFBP); calculating a ratio of the IGF to IGFBP to obtain a test ratio; and comparing the test ratio with a reference ratio, wherein an increase in the test ratio compared to the reference ratio indicates improved microvascular function.

6. The method of claim 3, wherein the IGF is IGF-1 or IGF-2.

7. The method of claim 3, wherein the IGFBP is IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, and IGFBP6, or a combination thereof.

8. The method of claim 3, wherein the IGFBP is IGFBP3, IGFBP5, or a combination thereof.

9. The method of claim 3, the method comprising: measuring in the biological sample, an amount of a cholesterol and an amount of a high-density lipoprotein (HDL); calculating a ratio of the amount of the cholesterol to the amount of the HDL to obtain a test ratio; comparing the test ratio with a reference ratio, wherein a decrease in the test ratio compared to the reference ratio indicates improved microvascular function.

10. The method of claim 1, wherein the biological sample is a blood sample, a tissue sample, a saliva sample, a urine sample, or a feces sample.

11. The method of claim 1, further comprising providing to a subject identified as having an impaired microvascular function: a legume-based snack food, wherein consuming the legume-based snack food improves microvascular function; wherein the legume-based snack food comprises: at least one extruded first legume and a first additive comprising at least one vitamin, at least one mineral, or a combination thereof; and optionally, a creamy layer comprising at least one extruded second legume and a second additive comprising at least one vitamin, at least one mineral, or a combination thereof.

12. The method of claim 11, wherein the legume-based snack food further comprises flour from at least one first legume.

13. The method of claim 11, wherein the legume-based snack food further comprises one or more edible oils.

14. The method of claim 11, wherein the at least one first legume and the at least one second legume are independently selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean, soya bean, and combinations thereof.

15. The method of claim 11, wherein the first legume is yellow pea and / or the second legume is yellow pea.

16. The method of claim 11, wherein the first additive and the second additive are independently selected from the group consisting of Vitamin B 1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof.

17. The method of claim 11, wherein the first additive is a Vitamin B6, a Vitamin B12, a Vitamin D, zinc, iron, copper, magnesium, or manganese, or a combination thereof, and / or the second additive is a Vitamin B6, a Vitamin B 12, a Vitamin D, zinc, iron, copper, magnesium, or manganese, or a combination thereof.

18. The method of claim 11, wherein the legume-based snack food further comprises flour from at least one second legume.

19. The method of claim 11, wherein the legume-based snack food further comprises one or more edible oils.

20. The method of claim 11, wherein the legume-based snack food is consumed for at least 4 weeks.

21. The method of claim 11, wherein the legume-based snack food is provided daily for a total daily calorie intake from about 200 kcal to about 250 kcal.

22. The method of claim 11, wherein the legume-based snack food is provided to the subject daily, or twice daily.

23. The method of claim 11, wherein the legume-based snack food is provided to the subject in an amount from about 10 g / serving to about 30 g / serving.

24. The method of claim 23, wherein the legume-based snack food is provided to the subject in an amount of 26g / serving.

25. The method of claim 11, wherein the legume-based snack food comprises a legume protein content from about 0.1 g protein to about 0.5 g of protein, per gram of snack food.

26. The method of claim 25, wherein the legume-based snack food delivers an amount of the legume protein from about 0.7 g to about 1.0 g per day, per kg body weight of the subject.

27. The method of claim 1, wherein the subject is a pediatric subject.

28. A method of improving microvascular function in a subject, the method comprising: providing to a subject, a legume-based snack food comprising: at least one extruded first legume; and a first additive comprising at least one vitamin, at least one mineral, or a combination thereof; wherein consuming the legume-based snack food improves microvascular function.

29. The method of claim 28, further comprising: calculating a first Framingham reactive hyperemia index (fRHI) value for the subj ect before consuming the legume-based snack food and calculating a second fRHI value after consuming the legume-based snack food, wherein a higher second fRHI value compared to the first fRHI value indicates improved microvascular function.

30. The method of claim 28, wherein the legume-based snack food further comprises flour from at least one first legume.

31. The method of claim 28, wherein the legume-based snack food further comprises one or more edible oils.

32. The method of claim 28, wherein the first legume is selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean, soya bean, and combinations thereof.

33. The method of claim 32, wherein the first legume is yellow pea.

34. The method of claim 28, wherein the first additive is selected from the group consisting of Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof.

35. The method of claim 28, wherein the first additive is a Vitamin B6, a Vitamin B12, a Vitamin D, zinc, iron, copper, magnesium, or manganese, or a combination thereof.

36. The method of claim 28, the legume-based snack food further comprising: a creamy layer comprising: at least one extruded second legume; and a second additive comprising at least one vitamin, at least one mineral, or a combination thereof.

37. The method of claim 36, wherein the legume-based snack food further comprises flour from at least one second legume.

38. The method of claim 36, wherein the legume-based snack food furthercomprises one or more edible oils.

39. The method of claim 36, wherein the second legume selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin, soya, and combinations thereof.

40. The method of claim 39, wherein the second legume is yellow pea.

41. The method of claim 36, wherein the second additive is selected from the group consisting of Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof.

42. The method of claim 28, wherein the legume-based snack food is provided daily for a total daily calorie intake from about 200 kcal to about 250 kcal.

43. The method of claim 28, wherein the legume-based snack food is provided to the subject daily, or twice daily.

44. The method of claim 28, wherein consuming the legume-based snack food for at least 4 weeks improves microvascular function.

45. The method of claim 28, wherein the legume-based snack food is provided to the subject in an amount from about 10 g / serving to about 30 g / serving.

46. The method of claim 45, wherein the legume-based snack food is provided to the subject in an amount of 26g / serving47. The method of claim 28, wherein the legume-based snack food comprises a legume protein content from about 0.1 g protein to about 0.5 g of protein, per gram of snackfood.

48. The method of claim 47, wherein the legume-based snack food delivers an amount of the legume protein from about 0.7 g to about 1.0 g per day, per kg body weight of the subject.

49. The method of claim 28, wherein the method causes a weight loss in the subject.

50. The method of claim 28, wherein the method improves cardiometabolic health in the subject.

51. The method of claim 28, wherein the subject is a pediatric subject.

52. A method of alleviating an inflammatory lung disease in a subject comprising: providing to a subject, a legume-based snack food comprising at least one extruded first legume wherein consuming the legume-based snack food improves microvascular function thereby alleviating the inflammatory lung disease.

53. The method of claim 52, wherein the inflammatory lung disease is allergy or asthma.

54. The method of claim 52, wherein the subject is undergoing a treatment for the inflammatory lung disease.

55. The method of claim 52, wherein providing the legume-based snack food to the subject reduces inflammation thereby alleviating the inflammatory lung disease.

56. The method of claim 52, wherein consuming the legume-based snack food increases levels of urosodeoxycholic acid (UDCA), compared to a reference value, thereby alleviating the inflammatory lung disease in the subject.

57. The method of claim 52, wherein consuming the legume-based snack foodreduces levels of at least one cytokine compared to a reference value, thereby alleviating the inflammatory lung disease in the subject.

58. The method of claim 52 wherein consuming the legume-based snack food reduces a level of IL-23 or IL-4 or a combination thereof.

59. The method of claim 52, wherein the legume-based snack food further comprises flour from at least one first legume.

60. The method of claim 52, wherein the legume-based snack food further comprises one or more edible oils.

61. The method of claim 52, wherein the at least one first legume is selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean, soya bean, and combinations thereof.

62. The method of claim 52, wherein the first legume is yellow pea.

63. The method of claim 52 wherein the legume-based snack food further comprises a first additive comprising at least one vitamin, at least one mineral, or a combination thereof.

64. The method of claim 63, wherein the first additive is selected from the group consisting of Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof.

65. The method of claim 63, wherein the first additive is a Vitamin B6, a Vitamin B12, a Vitamin D, zinc, iron, copper, magnesium, or manganese, or a combination thereof, and / or the second additive is a Vitamin B6, a Vitamin B 12, a Vitamin D, zinc, iron, copper,magnesium, or manganese, or a combination thereof.

66. The method of claim 52, wherein the legume-based snack food further comprises a creamy layer comprising: at least one extruded second legume; and optionally a second additive comprising at least one vitamin, at least one mineral, or a combination thereof.

67. The method of claim 66, wherein the legume-based snack food further comprises flour from at least one second legume.

68. The method of claim 66, wherein the legume-based snack food further comprises one or more edible oils.

69. The method of claim 66, wherein the extruded second legume is selected from the group consisting of yellow pea, chickpea, green pea, black eyed peas, pinto bean, kidney bean, black bean, mung bean, adzuki bean, fava bean, edamame, green lentil, red lentil, black lentil, lupin bean, soya bean, and combinations thereof.

70. The method of claim 66, wherein the extruded second legume is yellow pea.

71. The method of claim 66„ wherein the second additive is selected from the group consisting of Vitamin Bl (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), Vitamin B5 (pantothenic acid), Vitamin B6 (pyridoxine), Vitamin B7 (biotin), Vitamin B9 (folate), Vitamin B12 (cobalamin), Vitamin C (ascorbic acid), Vitamin A, Vitamin D, Vitamin E, Vitamin K, Thiamin, Niacin, choline, calcium, sulfur, phosphorus, magnesium, potassium, sodium, iron, zinc, Iron, Copper, Manganese, zinc, iodine, selenium, boron, molybdenum, cobalt, fluorine, chromium, and combination thereof.

72. The method of claim 66, wherein the second additive is a Vitamin B6, a Vitamin B 12, a Vitamin D, zinc, iron, copper, magnesium, or manganese, or a combination thereof, and / or the second additive is a Vitamin B6, a Vitamin B 12, a Vitamin D, zinc, iron, copper, magnesium, or manganese, or a combination thereof.

73. The method of claim 52, wherein the legume-based snack food is provided daily for a total daily calorie intake from about 200 kcal to about 250 kcal.

74. The method of claim 52, wherein the legume-based snack food is provided to the subject daily, or twice daily.

75. The method of claim 52, wherein the snack food is consumed for at least 4 weeks.

76. The method of claim 52, wherein the legume-based snack food is provided to the subject in an amount from about 10 g / serving to about 30 g / serving.

77. The method of claim 76, wherein the legume-based snack food is provided to the subject in an amount of 26g / serving78. The method of claim 52, wherein the legume-based snack food comprises a legume protein content from about 0.1 g protein to about 0.5 g of protein, per gram of snack food.

79. The method of claim 78, wherein the legume-based snack food delivers an amount of the legume protein from about 0.7 g to about 1.0 g per day, per kg body weight of the subject.

80. The method of claim 52, wherein the subject is a pediatric subject.

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