WO2025029589A2

WO2025029589A2 - Compositions having beet preparations and resistant starch for gut health and metabolic health, and related methods

Info

Publication number: WO2025029589A2
Application number: PCT/US2024/039585
Authority: WO
Inventors: Charitharth Vivek LAL
Original assignee: Resbiotic Nutrition, Inc.
Priority date: 2023-07-28
Filing date: 2024-07-25
Publication date: 2025-02-06
Also published as: WO2025029589A3

Abstract

This disclosure relates to compositions including a beet preparation, a resistant starch, and optionally a nutritionally active additive, for gut health and metabolic health and other purposes. Also, the disclosure relates to methods for preparing and for using a composition of this invention.

Description

COMPOSITIONS HAVING BEET PREPARATIONS AND RESISTANT STARCH FOR GUT HEALTH AND METABOLIC HEALTH, AND RELATED METHODS

TECHNICAL FIELD

[0001] This disclosure relates to compositions including a beet preparation, a resistant starch, and optionally a nutritionally active additive, for gut health and metabolic health and other purposes. Also, the disclosure relates to methods for preparing and for using a composition of this invention.

BACKGROUND

[0002] The gut microenvironment is the nexus of health in the human body, hosting a complex microbiota that breaks down and synthesizes nutrients, mounts immune responses, and regulates mood and energy. Various products contain multitudes of vitamins, minerals, or other substances that may be helpful in promoting gut health, however, the substances may not be present in quantities that are physiologically relevant or may not work well together, sometimes even providing an antagonistic or inhibitor effect.

[0003] Beets are known for their high nutritional value and have been widely studied for a variety of health benefits. Beetroot contains a class of compounds called betalains that have extensive antioxidant properties (Zielihska-Przyjemska, M., et al., In vitro effects of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals. Phytother Res. 2009;23(l):49-55; El Gamal AA., et al., Beetroot (Beta vulgaris L.) extract ameliorates gentamicin-induced nephrotoxicity associated oxidative stress, inflammation, and apoptosis in rodent model. Mediators Inflamm. 2014;2014:983952; Kanner, J., et al., A new class of dietary cationized antioxidants. J Agric Food Chem. 2001;49(l 1): 5178-85) as well as indications of antitumor and chemoprotective activity (Kapadia G.J., et al., Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract. Cancer Lett. 1996; 100(1 -2):211-4; Zhang, Q., et al., Beetroot red (betanin) inhibits vinyl carbamate- and benzo(a)pyrene-induced lung tumorigenesis through apoptosis. Mol Carcinog. 2013;52(9):686-91 ; Kapadia G.J , et al. Chemoprevention of DMBA- induced UV-B promoted, NOR- 1 -induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot. Pharmacol Res. 2003;47(2): 141-8). Beet powder or juice also contains nitric oxide, a critical component required for oxygenation of the blood (Premont R.T., et al., Role of Nitric Oxide Carried by Hemoglobin in Cardiovascular Physiology: Developments on a Three-Gas Respiratory Cycle. Circ Res. 2020; 126(1): 129-58). Beet root powder has potent antioxidant and anti-inflammatory characteristics via its nitric oxide increasing activity in the body. Hemoglobin carrying nitric oxide dilates the microvasculature, increasing oxygen delivery to local tissues. Due to the resultant increased blood flow, beet products are commonly consumed to boost muscle recovery after physical exercise (Ferguson S.K., et al. Impact of dietary nitrate supplementation via beetroot juice on exercising muscle vascular control in rats. J Physiol. 2013;591(2):547-57; Clifford T., et al., The effects of beetroot juice supplementation on indices of muscle damage following eccentric exercise. Eur J Appl Physiol. 2016;l 16(2):353-62; Hoon M.W., et al., The effect of nitrate supplementation on muscle contraction in healthy adults. Eur J Sport Sci. 2015; 15(8):712-9 ) . Additionally, beet powder may reduce blood pressure (Hobbs D.A., et al., Acute ingestion of beetroot bread increases endothelium-independent vasodilation and lowers diastolic blood pressure in healthy men: a randomized controlled trial. J Nutr. 2013;143(9):1399-405; Tropea T., et al., Beetroot juice lowers blood pressure and improves endothelial function in pregnant eNOS(- /-) mice: importance of nitrate-independent effects. J Physiol. 2020;598(18):4079-92) and reduce pro-inflammatory biomarkers such as c-reactive protein (CRP) and tumor necrosis factor alpha (TNF-alpha) in subjects with hypertension (Asgary, S., et al., Improvement of hypertension, endothelial function and systemic inflammation following short-term supplementation with red beet (Beta vulgaris L.) juice: a randomized crossover pilot study. J Hum Hypertens. 2016;30(10):627-32).

[0004] Resistant starch is a form of starch that is not easily digested by the body, instead passing through the digestive tract and providing a source of fuel to intestinal microbiota. For this reason, resistant starch, in particular certain resistant starches, can be classified as prebiotic, affecting the composition of the microbiome and supporting the growth and metabolic output of probiotic strains by providing fuel for the existing bacteria to convert into metabolites (Yang, X., et al., Resistant Starch Regulates Gut Microbiota: Structure, Biochemistry and Cell Signalling. Cell Physiol Biochem. 2017;42(l):306-l 8). Microbes ferment prebiotic materials like resistant starch and dietary fiber, breaking them down into metabolites such as short chain fatty acids (SCFAs) that can mediate distal immune response and inflammation (Arpaia, N., et al., Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature. 2013;504(7480):451-5; Iraporda, C., et al., Lactate and short chain fatty acids produced by microbial fermentation downregulate proinflammatory responses in intestinal epithelial cells and myeloid cells. Immunobiology. 2015;220(10):l 161 -9; Trompette, A., et al., Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nature Medicine. 2014;20(2): 159-66).

[0005] Consumption of resistant starch is associated with improved insulin resistance, heightened short chain fatty acid production, and reduced inflammatory biomarker levels. In clinical trials, starches from potato, maize, and tapioca have shown benefits for reducing diarrhea and constipation, lowering blood glucose levels, and improving insulin sensitivity (Bush JR, Baisley J, Harding SV, Alfa MJ. Consumption of Solnul(™) Resistant Potato Starch Produces a Prebiotic Effect in a Randomized, Placebo-Controlled Clinical Trial. Nutrients. 2023;15(7); Alfa, M.J., et al., A Randomized Placebo Controlled Clinical Trial to Determine the Impact of Digestion Resistant Starch MSPrebiotic(®) on Glucose, Insulin, and Insulin Resistance in Elderly and MidAge Adults. Front Med (Lausanne). 2017;4:260; Peterson, C M., et al., Effect of 12 wk of resistant starch supplementation on cardiometabolic risk factors in adults with prediabetes: a randomized controlled trial. Am J Clin Nutr. 2018;108(3):492-501; Gower, B.A., et al., Baseline insulin sensitivity affects response to high-amylose maize resistant starch in women: a randomized, controlled trial. Nutr Metab (Lond). 2016; 13:2; Robertson, M.D., et al., Insulin-sensitizing effects of dietary resistant starch and effects on skeletal muscle and adipose tissue metabolism. Am J Clin Nutr. 2005;82(3):559-67; Astina, J., et al., Attenuation of glycaemic and insulin responses following tapioca resistant maltodextrin consumption in healthy subjects: a randomised cross-over controlled trial. J Nutr Sci. 2020;9:e29). Preclinical data suggests that resistant starch isolated from kudzu root shows similar insulin-reducing properties in animal models of diabetes ( Song, X., et al., Kudzu Resistant Starch: An Effective Regulator of Type 2 Diabetes Mellitus. Oxid Med Cell Longev. 2021;2021 :4448048). Similar to other resistant starches, arrowroot starch shows prebiotic effects in in vitro models of gut digestion (Samedi, L., et al., Viability of 4 Probiotic Bacteria Microencapsulated with Arrowroot Starch in the Simulated Gastrointestinal Tract (GIT) and Yoghurt. Foods. 2019;8(5)).

[0006] Nutrients and microbial metabolites stimulate gut cells to produce key hormones that regulate functions such as gastrointestinal motility. The glucagon-like peptide (GLP) family of hormones stimulate insulin secretion and are key targets when addressing metabolic disorders such as type 2 diabetes (Carlstrbm, M., Nitric oxide signaling in kidney regulation and cardiometabolic health. Nature Reviews Nephrology. 2021 ; 17(9):575-90). Resistant starch in particular has been found to increase GLP-1 production in preclinical models in part due to the fermentation of SCFAs (Huang, P.L., et al. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature. 1995;377(6546):239-42; Huang, P.L., eNOS, metabolic syndrome and cardiovascular disease. Trends in Endocrinology & Metabolism. 2009;20(6):295-302).

SUMMARY

[0007] The present invention is directed to compositions specifically formulated to target gut health and thereby to support energy and metabolic health. Compositions of the present invention comprise a beet preparation and a resistant starch, and optionally a nutritionally active additive such as Vitamin B 12. The present invention is also directed to a method of preparing a composition of this invention, including preparing a beet preparation such as a beet powder and/or a resistant starch as needed. The present invention is also directed to a method of promoting gut health and/or promoting metabolic health in a subject, and also to a method of increasing bacterial growth in vitro or in a subject’s gut after administration. The present invention is also directed to a method of increasing desirable bacterial growth in vitro or in a subject’s gut after administration, for instance by increasing desirable bacterial growth such as Lactobacillus or Bifidobacterium, with less or minimal E. coli growth. The present invention is also directed to increasing nitric oxide production by bacteria in vitro or in a subject’s gut (e.g. Lactobacillus or B fidobacterium) .

[0008] In an embodiment, the beet preparation, resistant starch, and optionally a nutritionally active additive together in a subject provide a statistically significant and/or synergistic result relating to the subject’s gut health or metabolic health, including for instance increasing bacterial growth in the subject’s gut. In an embodiment, the beet preparation, resistant starch, and optionally nutritionally active additive operate with statistical significance and/or synergy in relation to each other and/or bacteria naturally found in the gut microbiome.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows three bar graphs (A, B, C) demonstrating that a resistant starch of the present invention derived from potato (C only), tapioca, com, kudzu, or arrowroot significantly increased the growth of Lactobacillus bacteria strains as measured by optical density [OD600 nm] after (A) 4 hours and (B, C) 24 hours of cell culture. (Asterisk(s) indicate statistical significance; Control represented in left column: Bacteria grown without resistant starch (“Lacto”)). [0010] FIG. 2 shows two bar graphs (A, B) demonstrating that a resistant starch of the present invention derived from tapioca, com, kudzu, or arrowroot significantly increased the growth of Lactobacillus bacteria strains as measured by colony count [CFU/ml] x 10⁸, after (A) 4 hours and (B) 24 hours of culture. (Asterisk(s) indicate statistical significance; Control represented in left column: Bacteria grown without resistant starch (“Lacto”)).

[0011] FIG. 3 shows three bar graphs (A, B, C) demonstrating that a resistant starch of the present invention derived from potato (C only), tapioca, corn, kudzu, or arrowroot significantly increased the growth of Bifidobacterium bacteria strains as measured by optical density [OD600 cells/mL] after (A) 4 hours and (B, C) 24 hours of cell culture. (Asterisk(s) indicate statistical significance; Control represented in left column: Bacteria grown without resistant starch (“Bifido”)).

[0012] FIG. 4 shows two bar graphs (A, B) demonstrating that a resistant starch of the present invention derived from tapioca, corn, kudzu, or arrowroot increased the growth of Bifidobacterium bacteria strains as measured by colony count [CFU/ml] x 10⁸, after (A) 4 hours and (B) 24 hours of culture. (Asterisk(s) indicate statistical significance; Control represented in left column: Bacteria grown without resistant starch (“Bifido”)).

[0013] FIG. 5 is a bar graph demonstrating that a composition of the present invention comprising beet powder and prebiotic potato starch (“prebeet®”) significantly and synergistically increased the growth of a Lactobacilli blend by 2-fold compared with beet powder or potato starch alone after 4 hours of in vitro culture.

[0014] FIG. 6 is a bar graph showing that prebeet® (mixture of beet powder and prebiotic potato starch) significantly and synergistically increased the growth of Lactobacilli blend by 2-fold compared with beet powder or potato starch alone after 4 hours of in vitro culture.

[0015] FIG. 7 shows two bar graphs (A, B) demonstrating that prebeet® (mixture of beet powder and prebiotic potato starch) significantly and synergistically increased the growth of Bifidobacterium blend by 4-fold compared with beet powder or potato starch alone after 4 hours and 2-fold after 24 hours of in vitro culture.

[0016] FIG. 8 is a bar graph showing that prebeet® (mixture of beet powder and prebiotic potato starch) significantly and synergistically increased the growth of Bifidobacterium blend by 3-fold compared with beet powder or potato starch alone after 4 hours of in vitro culture. [0017] FIG. 9 shows two bar graphs (A, B) demonstrating that prebeet®, beet powder alone, and potato starch alone do not cause overgrowth of E. coli after 4 hours as measured by (A) optical density (OD) and (B) colony forming units (CFU).

[0018] FIG. 10 is a bar graph showing that prebeet® significantly increased Lactobacillus growth and at similar levels as individual resistant starches after 4 hours of culture (as measured by OD). [0019] FIG. 11 is a bar graph showing that prebeet® significantly increased Bifidobacterium growth more than any individual starch or beet powder after 4 hours of culture (as measured by OD).

[0020] FIG. 12 is a bar graph showing that prebeet® significantly increased the production of nitric oxide (as measured by nitrate) by the Lactobacillus blend after 4 hours in culture.

[0021] FIG. 13 is a bar graph showing that prebeet® significantly increased the production of nitric oxide (as measured by nitrate) by the Bifidobacterium blend after 4 hours in culture.

[0022] FIG.14 is a bar graph showing that prebeet® significantly increased production of GLP-1 protein in Caco-2 human intestinal epithelial cells after 4 hours of in vitro culture.

[0023] FIG. 15 is a bar graph showing that prebeet® significantly increased production of GLP-1 protein in Caco-2 human intestinal epithelial cells after 24 hours of in vitro culture.

DETAILED DESCRIPTION

[0024] The foregoing and other features and advantages of the invention are apparent from the following detailed description of exemplary embodiments, read in conjunction with the accompanying drawings. The detailed description and drawings are merely illustrative of the invention rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof.

[0025] The present invention is directed in part to a composition formulated for gut and metabolic health, and methods of use thereof. Although beet powder and resistant starch have their individual benefits, their combination produces an increased probiotic effect. Beet powder and starch, taken together, stimulate growth of beneficial bacteria in the gut, such as Lactobacillus and Bifidobacterium found in a typical gut microenvironment. These bacterial species break down organic material into metabolites that can reduce inflammation and affect immune response throughout the body. [0026] Without being bound by theory, nitric oxide plays or may play a key role in regulating cardiometabolic health. Nitrate/ni trite is processed in the gastrointestinal system and sent into circulation as NO. Impaired nitric oxide signaling and reduced NO bioavailability in preclinical models have been shown to drive hypertension and symptoms of metabolic syndrome. Boosting nitrate levels through supplementation or production (as shown in Figures 12 and 13) has been shown to improve metabolic function via pathways modulating oxidative stress, mitochondrial function, AMP-activated protein kinase (AMPK) signaling, and insulin sensitivity.

[0027] In an embodiment, the present invention is a composition comprising a beet preparation and a resistant starch, and optionally a nutritionally active additive such as Vitamin Bl 2. In an embodiment, the beet preparation and resistant starch, and optionally nutritionally active additive, operate in a statistically significant manner and/or with synergy with each other. In an embodiment, the beet preparation and resistant starch, and optionally nutritionally active additive, operate in a statistically significant manner and/or with synergy in relation to bacteria naturally found in the gut microbiome.

[0028] A “composition” of this invention comprises a beet preparation and a resistant starch and optionally a nutritionally active additive. In an embodiment, a composition of the present invention comprises 1 g beet root powder, 3.5 g resistant starch (i.e./e,g, potato resistant starch), and lOOOug Vitamin B12 (methylcobalamin). Said composition may be in a dosage form for daily administration. Said amounts may be provided in relevant proportions in other compositions of this invention, as well. In an embodiment, a composition of this invention is designated as prebeet®. According to this invention, in the present Examples, prebeet® is a blend of 1 g beet root powder (IFF - The Dalles, OR, USA, discussed further below) and 3.5 g potato resistant starch (Solnul-Carberry, Manitoba, Canada, discussed further below). 60 mg of the blend was added to 6 mL MRS broth to create a stock concentration of 10 mg/mL for the experiments. 2 mL of the stock solution was used per sample. In an embodiment, the beet root powder and the potato resistant starch may be in the same ratio but in larger or smaller total amounts. In an embodiment, the potato resistant starch may be substituted with another resistant starch of this invention.

[0029] In an embodiment, a composition of this invention is formulated for oral administration, for instance as a capsule, microcapsule, tablet, granule, powder, troche, pill, suspension, solution, or syrup. In an embodiment, a composition of this invention is a dietary supplement, a food product, or a nutraceutical composition. In an embodiment, a composition of the present invention is a blend of beet powder and resistant starch.

[0030] A “dietary supplement” according to the present invention refers to a composition of this invention which is orally administered as an addition to a subject’s diet, which is not a natural or conventional food, which when administered maintains or improves gut or metabolic health, increases bacterial growth of desirable bacteria in the gut such as Lactobacillus o Bifidobacterium, minimizes the growth of undesirable gut bacteria in the gut such as E. coli, and/or increases bacterial production of nitric oxide. In an embodiment, the dietary supplement is administered daily; in an embodiment, the dietary supplement is administered daily for instance for at least 1 day, 2 days, 5 days, 1 week, 1 day to 1 week, 1 week to 26 weeks, or chronically for at least 1 day to 3 months, 6 months, 9 months, or 1 year or more, or for another period of time according to the present invention, or for any period of time within 1 day to 26 weeks or more. A dietary supplement may be formulated into various forms, as discussed throughout this application.

[0031] A composition of this invention includes at least two ingredients: a beet preparation and also as a second ingredient, a different plant preparation in the form of a resistant starch. The composition may optionally include, as a third and separate ingredient from the beet preparation and resistant starch, a nutritionally active additive.

[0032] In an embodiment, a composition of this invention is prepared by a conventional method, such as a method currently known in the art. Similarly, in an embodiment, a beet preparation and/or resistant starch, and/or other additive or ingredient of a composition of this invention, is prepared by a conventional method, such as a method currently known in the art.

[0033] In an embodiment, a “beet preparation” of this invention includes a preparation made from a beet plant. In an embodiment, “beet” according to this invention may include the stems and leaves of the plant, and “beetroot” refers only to the root. In an embodiment, stems and leaves are excluded from a beet preparation of this invention. Beet powder according to this invention may include stem and leaf material, or may include beetroot alone. In an embodiment, “beet powder” of this invention is derived from only the root. In an embodiment, beet juice is juiced from the root. In an embodiment, a beet preparation is made from the “bulb” or “root” of the beet plant, which is typically the portion that would grow underground or mostly underground if grown in the ground, and that may provide a color such as red, purple, yellow, or white (i.e. not leaves, or not leave or stems). Hydroponic beets or beets not grown underground are also available to make a beet preparation according to the present invention. In an embodiment, a beet preparation may include the bulb or root of the beet plant, and also, other parts of a beet plant such as stems and leaves. In an embodiment, a beet preparation is prepared so that the beet plant part of the preparation is 100% or at least 99%, 95%-99%, 90%-95%, 85%-90%, 80%-85%, 75%-80%, 70%- 75%, 65-70%, 60-65%, 55-60%, 50-55%, and/or 40-50% from the bulb or root of the beet plant, with the remainder of the preparation from beet plant stems or leaves. In an embodiment, a beet preparation of this invention is a beet, or beet root, powder.

[0034] In an embodiment, a beet preparation of the present invention is “Beet Powder” purchased from IFF - The Dalles, OR, USA (Product Code AZ316-A1016; PowderPure™ by IFF). Said Beet Powder was used in the prebeet® compositions in the in vitro Examples described below, and is a beetroot powder. According to the Product Specification Sheet accompanying said Beet Powder, the ingredient of the Beet Powder is Beet (Beta vulgaris). No processing aids were used in its manufacture. The Beet Powder has a Kosher status of Kosher, KOF-K, Parve; is made from conventional beets, compliant with GMO regulations; Gluten-Free; made in the USA; sold in a 15kg increment; recommended storage is under cool conditions less than 75F; recommended handling is 40% or less relative humidity to prevent clumping; recommended to be used within 3 years of date of manufacture. The Beet Powder used in the below Examples had a purple-red color, fresh beet aroma, and fresh beet flavor. The Beet Powder water activity maximum is 0.350 partial vapor pressure (Test Method AO AC 978.18). The Beet Powder had a particle size target of 40 Mesh, centered on sieve (Test Method: Rotap). The Beet Powder had a moisture content target of 5% and maximum of 10% (Test Method: Powderpure SOP 2026). The Beet Powder’s microbiological properties were as follows: less than 10,000 CFU/g standard plate count (AO AC 2015.13); less than 10 CFU/g (Negative) for Coliform (AO AC 991.14); less than 10 CFU/g (Negative) for /■/ coli (PCR(BAM Chp 4A)); negative for Salmonella (CFU/375g, PCR(BAM Chp 5:2007); Negative for Listeria spp. (CFU/375g, PCR(BAM Chp 10); less than 100 CFU/g yeast and mold (AO AC 2014.05).

[0035] A “resistant starch” of this invention is a form of starch that is not easily digested by the body, instead passing through the digestive tract and providing a source of fuel to intestinal microbiota. A resistant starch is not fully digested by the time said starch reaches the large intestine. In an embodiment, a resistant starch of this invention has a high amylose content; without being bound by theory, starches with a high amylose content are typically more resistant to digestion. In an embodiment, a resistant starch of this invention is a prebiotic starch, i.e. a resistant starch with prebiotic qualities or activity, i.e. a prebiotic resistant starch. In an embodiment, a resistant starch of this invention is prepared from a potato, tapioca, com, kudzu, and/or arrowroot. In an embodiment, a resistant starch of this invention is from a natural source, such as those discussed above, or for instance from cassava. In an embodiment, a resistant starch of this invention may be chemically modified and substituted for a similar effect within the formulation. In an embodiment, a resistant starch of this invention is from a specific source such as resistant potato starch, resistant banana starch, and/or resistant oat starch. In an embodiment, all resistant starches identified in this application are prebiotic resistant starches.

[0036] In an embodiment, a resistant starch of the present invention is Solnul™ Resistant Potato Starch purchased from MSP Starch Products Inc., Carberry, Manitoba, Canada (Item Number SOLIOO). Said Resistant Potato Starch was used in the prebeet® compositions in the in vitro Examples described below. The US Specification Sheet accompanying said Resistant Potato Starch states as follows: 100% Resistant Potato Starch, Solanum tuberosum. Used Parts: Tuber. Manufactured in Canada. Characteristics: appearance: free-flowing powder, color: white, taste: neutral, mouthfeel: smooth. Typical Composition and acceptable ranges: greater than 80% total carbohydrate (method: calculation); 60% or more resistant starch (as is basis, Method: SOP-050- 20, Reference AO AC 2002.02); greater than 63% total dietary fiber (as is basis (Method: AO AC 2009.01)). Analytical specifications: moisture: 20% or less (SOP-050-06, AOAC 930.15), pH 6.0- 8.0 (SOP-050-07, CRA B-44-1); less than 0.5% Ash (SOP-050-10, CRA M-4). Allergens: Gluten less than 5ppm (SOP-050-23, AOAC 051101). Other Allergens: None. Microbiological: APC less than 1,000 CFU/g (SOP-050-09, AOAC Method 990.12); Yeast less than 100 CFU/g (SOP-050- 09, AOAC Method 997.02); Mold less than 100 CFU/g (SOP-050-09, AOAC Method 997.02); Coliform/E.coli (Absent in 10g, SOP-050-09, USP Method <2022>); Salmonella absent in 10g (SOP-050-38, USP Method <2022>). Other Information: UPC 829068 00005 6, Packaging 25kg, 3 ply kraft polyethylene lined bags, quantity per pallet: 1,000kg; labeling: six digit lot number followed by date of manufacture; shelflife: 36 months from date of manufacture; storage: store in dry ambient environment, optimum temperature range is 45-80F. Nutritional Profile Amounts are (per 100g): Moisture: 18.5g (AOAC 950-468), Energy: 324Kcal (calculated), Protein: less than 0.10g (AOAC 992.15), Fat:0.005g (AOAC 996.06 - this method applied for all of the following unless marked otherwise) - saturated fat 0.004g, Trans Fatty Acid less than 0.001g, Polyunsaturated Fat less than 0.001g, Monounsaturated Fat less than 0.001g, Omega 3 less than 0.001g, Omega 6 less than 0.001g, Cholesterol less than Img (cholesterol analysis method AOAC 976.26, AO AC 994.10). Total Carbohydrate: 811g (calculated) -total dietary fiber: 67.1g (AOAC 2009.01), resistant starch: 61.7g (AOAC 2002.02), sugars: less than 0.4g (AOAC 980.13). Ash: 0.4g (AOAC 923.03) - using AOAC 984.27 (mod) for the following 5 minerals: Sodium (8mg), Potassium (less than lOmg), Phosphorus (60mg), Calcium (32mg), Iron (0.3mg). Also, Vitamin A: less than 50 IU (AOAC 992.04, AOAC 992.06), Vitamin C less than 0.5mg (CFIA-LCAQ- 001-09), Riboflavin less than O.lmg (AOAC 970.65), Niacin less than 1 mg (AOAC 944.13), thiamine less than O.lmg (AOAC 942.23), Folic Acid less than 0.2 micrograms (AOAC 2004.05), Vitamin D less than 0.25 micrograms (AOAC 982.29). (US Specification Sheet Version 6, effective June 14, 2022).

[0037] In an embodiment, a resistant starch of this invention is one or more of Tapioca (root) (white powder, from Bob’s Red Mill (Milwaukie, OR, US); Kudzu (root) (white powder, from Frontier Co-op (Norway, IA, USA); Arrowroot (root) (white powder, Bob’s Red Mill (Milwaukie, OR, US); Corn (kernel) (white powder, Frontier Co-op (Norway, IA, US); potato (root) (white powder, Solnul (Carberry, Manitoba, Canada).

[0038] In an embodiment, a resistant starch is a prebiotic fiber. A resistant starch of this invention may be a prebiotic fiber, however, a prebiotic fiber is not necessarily a resistant starch. Dietary fiber may be soluble or insoluble, with both having prebiotic properties. In an embodiment, a resistant starch of this invention behaves like soluble fiber. In an embodiment, a composition of the present invention may include one or more resistant starch.

[0039] In an embodiment, a composition of this invention includes a “nutritionally active additive”. Additional vitamins and minerals can supplement the health benefits provided by a composition of the present invention, comprising beet preparation and resistant starch. An “additive” of the present invention refers to a substance independently added to a composition of this invention, in addition to the beet preparation and the resistant starch, and is not for instance an inherent component of a beet preparation or resistant starch.

[0040] Vitamin B12 (in the form of methylcobalamin) is a water-soluble vitamin that supports healthy central nervous system development and function, red blood cell production, and DNA synthesis. A double-blinded, placebo-controlled clinical trial suggests that Vitamin B12 may be useful for managing pain, improving quality of life, and improving neurological scores in patients with diabetic neuropathy (Didangelos T, Karlafti E, Kotzakioulafi E, Margariti E, Giannoulaki P, Batanis G, et al. Vitamin B12 Supplementation in Diabetic Neuropathy: A 1-Year, Randomized, Double-Blind, Placebo-Controlled Trial. Nutrients. 2021;13(2):395).

[0041] In an embodiment, a nutritionally active additive of this invention is a vitamin, a micronutrient, a mineral, a fatty acid, an amino acid, or any combination thereof.

[0042] In an embodiment, the nutritionally active additive is a vitamin, and the vitamin is ascorbic acid (vitamin C), vitamin D, vitamin E, vitamin A, vitamin K, thiamine (vitamin Bl), riboflavin (vitamin B2), niacin (vitamin B3), pantothenic acid (vitamin B5), vitamin B6, vitamin B7, vitamin B9, vitamin Bl 2, or any combination thereof.

[0043] In an embodiment, the nutritionally active additive is a mineral, and the mineral is at least one of the group consisting of sulfur, potassium, chlorine, sodium, calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, chromium, molybdenum, selenium, and cobalt. In an embodiment, the nutritionally active additive is one or more of sulfur, potassium, chlorine, sodium, calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, chromium, molybdenum, selenium, and cobalt, including any combination thereof. Additional nutritionally active additives can support gut health directly while also having a distal effect on organs such as the heart, brain, and lungs. In an embodiment, the nutritionally active additive is a plant extract. In an embodiment, the nutritionally active additive is a plant extract, and the extract is at least one of the group consisting of ginger, acerola cherry, carrot, turmeric, holy basil, vasaka, chicory, and artichoke. In an embodiment, the nutritionally active additive is powdered ginger or a mushroom powder.

[0044] In an embodiment, a composition of this invention supports health, such as gut and/or metabolic health, in a subject to whom the composition has been administered. In an embodiment, this invention is a method of preparing a composition of this invention, including preparing a beet preparation such as a beet powder and/or a resistant starch as needed, and combining a beet preparation and a resistant starch for instance for oral administration. In an embodiment, this invention is directed to a method of promoting gut health and/or promoting metabolic health in a subject, and also to a method of increasing bacterial growth in vitro or in a subject’s gut after administration, for instance as described in the attached Figures. In an embodiment, this invention is directed to a method of increasing desirable bacterial growth in vitro or in a subject’s gut after administration, for instance by increasing desirable bacterial growth such as Lactobacillus or Bifidobacterium, with less or minimal E. coli growth. In an embodiment, this invention is directed to a method of increasing nitric oxide production by bacteria in vitro or in a subject’s gut (e g. Lactobacillus or Bifidobacterium). These methods are supported by information provided in the attached Figures and as described throughout this application.

[0045] In the present invention, “administering”, “administration”, and the like refer to providing a composition of the present invention to a subject so that the composition (and/or components thereof) reaches the subject’s gut including intestinal tracts and/or portions of the gastrointestinal system where bacterial growth is desirable or desired, so that the composition (and/or its components) increase bacterial growth of desired or desirable bacteria, such as Lactobacillus or Bifidobacterium as described herein. In an embodiment, the growth of E. coli is not increased as much as the growth of desirable or desired bacteria; E. coli growth may be only minimally altered by the present composition. Administration may be by the subject or by another. Administration to the subject may be oral, for instance in the form of a dietary supplement, and/or in a solid pharmaceutical dosage form, preferably in a discrete dose unit, such as a tablet, hard gelatin capsule, soft gelatin capsule, etc. Administration may also be through parenteral and other physiologically acceptable routes. In an embodiment, a dose unit of the present invention comprises 1g beet powder, 3.5g prebiotic (resistant) starch, and lOOOug Vitamin B12 (methylcobalamin). In an embodiment, a dose unit of the present invention comprises about Img-lOg beet powder and about Img to about 10g resistant starch, including for instance 300- 3000mg beet powder and about 1.5-7 g prebiotic (resistant) starch. In an embodiment, a dose unit of the present invention comprises 500-2000ug Vitamin B 12. In an embodiment, one, two, three, four, five, or more dose units is/are administered daily to a subject. In an embodiment, a dose unit is for human consumption. In an embodiment, the separate administration of a beet preparation such as beet powder and a resistant starch such as a resistant prebiotic starch within 0-3 hours of the other is equivalent to the administration of a composition of the present invention, wherein the substances are administered orally at the same time.

[0046] In an embodiment, a “subject” according to this invention is a human; in another embodiment, the subject is a mouse, rat, dog, horse, or any mammal.

[0047] In an embodiment, a composition of the present invention may be used to treat (for instance improve, reduce, or eliminate symptoms of) and/or prevent pulmonary hypertension, type 2 diabetes, metabolic syndrome, and/or chronic kidney disease, and/or other conditions, disorders, diseases or the like including in particular those related to cardiometabolic function and disease. Without being bound by theory, the effect of compositions of this invention on nitric oxide are discussed above. A composition of this invention may act by administering a composition of this invention to a subject, in an embodiment orally, said composition and/or components thereof entering the blood stream and/or other bodily tissues being distributed throughout the body, affecting nitric oxide levels and providing treatment/prevention thereby, without being bound by theory. In an embodiment, the present invention is directed to a method of treating and/or preventing any of the above-discussed conditions/diseases/disorders therefore, comprising the step of providing a composition and administering the composition to a subject. [0048] In an embodiment, the present invention is directed to a method of treating and/or preventing metabolic conditions in a subject, for example a metabolic condition responsive to GLP-1 modulation of the metabolic condition, without being by bound by theory. In an embodiment, the present invention is directed to a method comprising administering the compositions of the invention in a therapeutically or nutritionally effective amount. In an embodiment, the present invention is directed to a method comprising orally administering the compositions of the invention in a therapeutically or nutritionally effective amount. In an embodiment, the present invention is directed to a method comprising administering the compositions of the invention in a therapeutically or nutritionally effective amount. In an embodiment, the present invention is directed to a method comprising measuring the metabolic response of a subject treated with any of the compositions of the present invention. In an embodiment, the present invention is directed to a method comprising measuring the metabolic response of a subject treated with any of the compositions of the present invention, wherein the metabolic response of the subject is measured by measuring GLP-1 production in the subject.

[0049] Embodiments of the invention will now be described with reference to the Figures, wherein like numerals reflect like elements throughout. The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive way, simply because it is being utilized in conjunction with detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several novel features, no single one of which is solely responsible for its desirable attributes or which is essential to practicing the invention described herein. [0050] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0051] In the below Examples, prebeet® is a blend of 1 g beet root powder (IFF - The Dalles, OR, USA, discussed above) and 3.5 g potato resistant starch (Solnul-Carberry, Manitoba, Canada, discussed above). 60 mg of the blend was added to 6 mL MRS broth to create a stock concentration of 10 mg/mL for the experiments. 2 mL of the stock solution was used per sample.

[0052] Optical Density Growth Measurements 600nm (OD600) & Nitric Oxide Measurements: prebeet®.

[0053] Example 1 (See Figures 5-9)

[0054] N = 6

[0055] Materials:

Lactobacillus species: 1 : 1: 1 ratio (ATCC; Manassas, VA, USA)

• Lactiplantibacillus plantarum Lp-115

• Lactobacillus acidophilus La- 14

• Lacticaseibacillus rhamnosus GG

Bifidobacterium species: - 1 : 1 : 1 : 1 ratio (ATCC; Manassas, VA, USA)

• Bifidobacterium infantis Bi-26

• Bifidobacterium longum BL05

• Bifidobacterium breve Bb- 18

• Bifidobacterium lactis Bi-07

MRS growth media Beet extract powder prebeet® powder Solnul - Potato starch

[0056] OD Protocol:

[0057] Probiotics

[0058] 3 mL of MRS was inoculated with each of the two bacterial blends and incubated at 37°C (175rpm) for 24 hours. Each experimental compound was weighed out to 60 mg and added to 6 mL of MRS, to a final concentration of 10 mg/mL. 2 mL of each compound stock was then aliquoted into 3 separate incubation tubes: Lactobacillus, Bifidobacterium, and blank. 100 uL of each bacterial stock was then added to their respective incubation tubes, shown below in Table 1.

[0059] TABLE 1

[0060] Each incubation tube was then incubated at 37°C (175rpm) for 24 hours. During the incubation period the OD600 was measured at 4 hours and at 24 hours.

[0061] E. coli

[0062] 3 mL of LB broth was inoculated with E. coli and incubated at 37°C (175rpm) for 24 hours. Each experimental compound was weighed out to 60 mg and added to 6 mL of LB, to a final concentration of 10 mg/mL. 2 mL of each compound stock was then aliquoted into 2 separate incubation tubes: E. coli and blank. 100 uL of each bacterial stock was then added to their respective incubation tubes, shown below in Table 2.

[0063] TABLE 2

[0064] Each incubation tube was then incubated at 37°C (175rpm) for 24 hours. During the incubation period the OD600 was measured at 4 hours. [0065] CFU Count/Bacterial Plating Protocol:

[0066] 3 mL of MRS was inoculated with each of the two bacterial blends and incubated at 37°C (175rpm) for 24 hours. Each experimental compound was weighed out to 60 mg and added to 6 mL of MRS, to a final concentration of 10 mg/mL. 2 mL of each compound stock was then aliquoted into 3 separate incubation tubes: Lactobacillus, Bifidobacterium, and blank. Each stock was incubated at 37°C (175rpm) for 24 hours, and then culture stocks were measured for optical density to determine approximate CFU/mL concentrations. Each stock was then standardized to 1.0 x E8 cells/mL and then serial diluted to a final concentration of 100 cells/mL. Each stock was then incubated at 37°C (175rpm) for 4 hours. After the 4 hour incubation, each stock was then plated onto a MRS/Agar plate (n=3). Plating volumes were at 50 uL of each stock, and then diluted at 1 : 10 in fresh MRS media to be plated again at a second concentration, also a plating volume of 50 uL. The bacterial plates were then incubated anaerobically at 37°C for 72 hours and then counted.

[0067] Example 2 (See Figures 1-4, 10, 11)

[0068] N = 8

[0069] Materials:

Lactobacillus species: 1 : 1: 1 ratio (ATCC; Manassas, VA, USA)

• Lactiplantibacillus plantarum Lp-115

• Lactobacillus acidophilus La- 14

• Lacticaseibacillus rhamnosus GG

Bifidobacterium species: - 1 : 1 : 1 : 1 ratio (ATCC; Manassas, VA, USA)

• Bifidobacterium infantis Bi-26

• Bifidobacterium longum BL05

• Bifidobacterium breve Bb- 18

• Bifidobacterium lactis Bi-07

MRS growth media Tapioca starch Corn starch Kudzu starch Arrow root starch

[0070] Protocol :

[0071] 3 mL of MRS was inoculated with each of the two bacterial blends and incubated at 37°C (175rpm) for 24 hours. Each experimental compound was weighed out to 60 mg and added to 6 mL of MRS, to a final concentration of 10 mg/mL. 2 mL of each compound stock was then aliquoted into 3 separate incubation tubes: Lactobacillus, Bifidobacterium, and blank. 100 uL of each bacterial stock was then added to their respective incubation tubes, shown below in Table 3.

[0072] TABLE 3

[0073] Each incubation tube was then incubated at 37°C (175rpm) for 24 hours. During the incubation period the OD600 was measured at 4 hours and at 24 hours.

[0074] Example 3 Nitric Oxide Assay Protocol: (See Figures 12 and 13)

[0075] N = 6

[0076] After OD600 measurements were taken at the 24 hour timepoint, the incubation tubes were spun down at 8000 x g for 5 minutes and the supernatant was collected for Nitric Oxide measurements. Nitric oxide measurements were carried out in accordance with Griess Reagent Kit for Nitrite Determination (Cat. No. G-7921) available from Molecular Probes, Inc. (Eugene, Oregon).

[0077] Statistics

Kruskal-Wallis tests with multiple comparisons were run on all data. *P<0.05, **P<0.01, ***p<0.001, ****P<0.0001. [0078] Example 4 GLP-1 ELISA Protocol (See Figures 14 and 15)

[0079] N= 4

[0080] Caco-2 human intestinal epithelial cells (80% confluent in 24-well plates) were incubated at 37degC for 4 hours or 24 hours with 10 mg/mL prebeet® 100 100 pL of cell culture supernatant was isolated and tested according to the manufacturer’s protocol. The ELISA protocol was performed in accordance with Invitrogen Human GLP-1 ELISA Kit (Cat. No. BMS2194) available from ThermoFisher Scientific (Waltham, Massachusetts).

EXAMPLE - IN VIVO STUDY

[0081] Example 5 prebeet®//? vivo Study entitled Effect of a Novel Prebiotic and Beet Formula on Digestion and Quality of Life. Clinical Phase: supplement. Name of product/dose and method of administration are respectively prebeet® Energy + powder mixed in water, oral. Endpoints: the primary endpoints are digestive quality of life, stool consistency /frequency, and exercise capacity. Secondary endpoints are complete metabolic blood panel, general health and activity status, complete blood count, gut microbiome, and serum biomarkers. Study population are healthy adults. The study design is a double-blind, placebo-controlled, randomized control study of a nutritional supplement in healthy adults. More than one site is enrolling subjects where the total enrollment of subjects is 200 (two hundred). The test product, dose and mode of administration are respectively, active prebeet® Energy + powder, 1 scoop (6 g) in 8-12 oz of water daily, and placebo is maltodextrin + flavoring (6 g) in 8-12 oz of water administered daily. The study duration is 28 days/4 weeks.

[0082] The study subject population inclusion criteria are: provide voluntary signed and dated informed consent; be in good health as determined by medical history and routine blood chemistries; be aged between 18 and 50 years (inclusive); have a body Mass Index of 18.5-29.9 (inclusive); have a body weight of at least 120 pounds; have normal or acceptable to the investigator vital signs (blood pressure and heart rate) and normal or acceptable to the investigator physical exam findings (if applicable) at screening; have a normal seated, resting heart rate (<90 per minute); the subject agrees to maintain existing dietary and physical activity patterns throughout the study period; the subject is willing and able to comply with the study protocol; female subjects of childbearing potential must agree to practice a medically acceptable form of birth control throughout the study; male subjects of reproductive potential must agree to use condoms or other medically acceptable methods to prevent pregnancy throughout the study; complete abstinence from heterosexual intercourse is also acceptable; subjects must agree to refrain from treatments listed in Section 6.5 in the defined time frame; and subjects must be willing and able to agree to the requirements and restrictions of this study, be willing to give voluntary consent, be able to understand and read the questionnaires, and carry out all study-related procedures.

[0083] The study exclusion criteria are having a history of unstable or new-onset cardiovascular/cardiorespiratory, liver, or renal conditions; having a history of diabetes or endocrine disorder; having a history of use of medications or dietary supplements known to confound the study or its endpoints; having a history of alcohol consumption (more than 2 standard alcoholic drinks per day or more than 10 drinks per week) or drug abuse or dependence within the past 6 month; being current smokers or smoking within the past month; having a history of hyperparathyroidism or an untreated thyroid condition; having a history of malignancy in the previous 5 years except for non-melanoma skin cancer (basal cell cancer or squamous cell cancer of the skin; having a history of other known gastrointestinal or metabolic conditions that might impact nutrient absorption or metabolism, e.g., short bowel syndrome, Irritable bowel syndrome (IBS), diarrheal illnesses, history of colon resection, gastroparesis, Inborn-Errors-of-Metabolism (such as PKU); having a chronic inflammatory condition (e.g., rheumatoid arthritis, Crohn's, ulcerative colitis, Lupus, HIV/AIDS, etc.); having a previous medical diagnosis of asthma, gout, or fibromyalgia, pregnant women, women trying to become pregnant, women less than 120 days postpartum or nursing women; women who participate in this study must agree to the use of contraception for the duration of the study and any woman that is sexually active will have to take a negative pregnancy test prior to enrolling; women of childbearing age (any woman prior to menopause) regardless of their use of contraception will be provided a urine pregnancy test kit at their screening visit and take the test in private using the female lavatory after signing an informed consent; having known sensitivity to any ingredient in the test formulations as listed in the product label; subject is currently participating in another research study with an investigational product or have been in another research study in the past 30 days; having any other conditions that, in the opinion of the medical staff, could confound the primary endpoints or place the subject at increased risk of harm if they were to participate. [0084] The study procedures are as follows. Screening: subjects will be screened for eligibility to participate in the study within 7 days of being randomized (Day 0). Baseline (Day 0): Subjects will be randomized for either placebo or active. Study Drug Administration: all subjects will receive prebeet® after screening at Visit 1. Participants will self-administer prebeet® at home for 4 weeks and complete a study diary to track compliance. For study visits during the Treatment Period subjects will conduct on-site visits for screening, randomization, 2 weeks, and closeout.

[0085] The study criteria for evaluation are as follows. Primary criteria are: first digestive measures (time frame is 28 days) a Digestive Quality of Life Questionnaire (DQLQ) utilized to assess the effect of the interventions on participant's digestive health. Scores range from 0 to 9 with a higher score indicating worse digestion associated with quality of life. Second criteria are evaluation of stool consistency and frequency (time frame is 28 days) using a Bristol Stool Scale (score 1-7) questionnaire that allows participants to identify how loose or hard their stool consistency is. 100mm Visual Analog Scale (VAS) to assess subjective ratings of stool consistency and frequency of bowel movements. Third criteria are Exercise Tolerance (time frame is 28 days) as determined by a cardiopulmonary exercise test (CPET) o determine the subject’s exercise capacity and energy levels before and after taking prebeet®. Secondary criteria for evaluation are first performing a Complete Metabolic Blood Panel (time frame is 28 days) measuring serum concentrations of sodium, potassium, carbon dioxide, chloride, albumin, total protein, glucose, and calcium., ALT, AST, bilirubin, BUN, creatinine. Second criteria is determining overall activity of the subject (time frame is 28 days) by performing a Duke Activity Status Index (DASI) to assess the effect of the intervention on a patient's overall health and energy. Third criteria is performing a complete Blood Count (time frame is 28 days) measuring circulating concentrations of white blood cells, red blood cells, hemoglobin, and hematocrit. Fourth criteria is evaluating the gut microbiome (time frame is 28 days) by examining changes in the gut microbiome structure and function measured by shallow shotgun metagenomics. Also, to examine enrichment of taxa and metabolic pathways in the gut microbiome as a result of dietary supplementation. And fifth criteria is to evaluate any change in serum biomarkers (time frame is 28 days) by measuring any change in inflammatory cytokines (IL- 113, IL-6, IL-8 & TNF-a) and short chain fatty acids (SCFA). The study is summarized in Table 4 below.

[0086] TABLE 4 - Study protocol summary

[0087] In conclusion, it is expected that a study performed according to the principles of the above example will clearly demonstrate statistically significant improvements as measured by one or more of DQLQ score, Bristol Stool Scale score, or CPET score. In addition, serum biomarkers will be measured and it is expected that the serum levels of certain inflammatory cytokines will be decreased.

[0088] Results

[0089] FIG. 1 shows three bar graphs demonstrating that a resistant starch of the present invention derived from potato, tapioca, com, kudzu, or arrowroot significantly increased the growth of Lactobacillus bacteria strains (blend of L. plantarum, L. acidophilus, L. rhamnosus) as measured by optical density [OD600 nm] after (A) 4 hours and (B, C) 24 hours of culture. N=8. Asterisk(s) indicate statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Kruskal-Wallis test; Control represented in left column: Bacteria grown without resistant starch (“Lacto”)). (Charts A and B, x-axis left-to-right: Lacto, Lacto +Tapioca Starch, Lacto +Com Starch, Lacto +Kudzu Starch, Lacto +Arrowroot Starch; y-axis: OD600nm; Chart C x-axis: Lacto, Lacto +Potato Starch, Lacto +Tapioca Starch, Lacto +Corn Starch, Lacto +Kudzu Starch, Lacto +Arrowroot Starch; y- axis: OD600 Cells/mL).

[0090] FIG. 2 shows two bar graphs (A, B) demonstrating that a resistant starch of the present invention derived from tapioca, com, kudzu, or arrowroot significantly increased the growth of Lactobacillus bacteria strains (blend of L. plantarum, L. acidophilus, L. rhamnosus) as measured by colony count [colony forming units CFU/ml] x 10⁸, after (A) 4 hours and (B) 24 hours of culture. (N=8. Asterisk(s) indicate statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****p<0 0001 by Kruskal -Wallis test; Control: Bacteria grown without resistant starch (“Lacto”)). (Charts A and B, x-axis left-to-right: Lacto, Lacto +Tapioca Starch, Lacto +Com Starch, Lacto +Kudzu Starch, Lacto +Arrowroot Starch; y-axis: Colony Count [CFU/mL]xlO⁸).

[0091] FIG. 3 shows three bar graphs (A, B, C) demonstrating that a resistant starch of the present invention derived from potato (C only), tapioca, corn, kudzu, or arrowroot significantly increased the growth of Bifidobacterium bacteria strains (blend of B. breve, B. infantis, B. lactis, B. longum) as measured by optical density [OD600 cells/mL] after (A) 4 hours and (B, C) 24 hours of cell culture. (N=8. Asterisk(s) indicate statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****p<0 0001 by Kruskal-Wallis test; Control represented in left column: Bacteria grown without resistant starch (“Bifido”)). (Charts A and B, x-axis left-to-right: Bifido, Bifido+Tapioca Starch, Bifido+Corn Starch, Bifido+Kudzu Starch, Bifido+ Arrowroot Starch; y-axis: OD600 Cells/mL; Chart C x-axis: Bifido, Bifido+Potato Starch, Bifido+Tapioca Starch, Bifido+Corn Starch, Bifido+Kudzu Starch, Bifido+ Arrowroot Starch; y-axis: OD600 Cells/mL).

[0092] FIG. 4 shows two bar graphs (A, B) demonstrating that a resistant starch of the present invention derived from tapioca, corn, kudzu, or arrowroot increased the growth of Bifidobacterium bacteria strains (blend of B. breve, B. infantis, B. lactis, B. longum) as measured by colony count [colony forming units CFU/ml] x 10⁸, after (A) 4 hours and (B) 24 hours of culture. (N=8. Asterisk(s) indicate statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Kruskal-Wallis test; Control represented in left column: Bacteria grown without resistant starch (“Bifido”)). Asterisk(s) indicate statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****p<0 0001 by Kruskal-Wallis test. (Both charts, x-axis left-to-right: Bifido, Bifido+Tapioca Starch, Bifido+Corn Starch, Bifido+Kudzu Starch, Bifido+ Arrowroot Starch; y-axis: Colony Count [CFU/mL]xlO⁸). [0093] FIG. 5 is a bar graph demonstrating that a composition of the present invention comprising beet powder and prebiotic potato starch (“prebeet®”) significantly and synergistically increased the growth of a Lactobacilli blend by 2-fold after 4 hours of in vitro culture, prebeet® increased OD (optical density as a marker of probiotic growth) more than beet powder or potato starch alone at 4 hours.

[0094] FIG. 6 is a bar graph demonstrating that prebeet® (mixture of beet powder and prebiotic potato starch) significantly and synergistically increased the growth of a Lactobacilli blend by 2- fold after 4 hours of in vitro culture, prebeet® increases CFU (colony forming units as a marker of probiotic growth) more than beet powder or potato starch alone.

[0095] FIG. 7 shows two bar graphs (A, B) demonstrating that prebeet® (mixture of beet powder and prebiotic potato starch) significantly and synergistically increased the growth of Bifidobacterium blend by 4-fold after 4 hours and 2-fold after 24 hours of in vitro culture, prebeet® increased OD (optical density as a marker of probiotic growth) more than beet powder or potato starch alone at (A) 4 hours and as much as potato starch at (B) 24 hours.

[0096] FIG. 8 is a bar graph showing that prebeet® (mixture of beet powder and prebiotic potato starch) significantly and synergistically increased the growth of Bifidobacterium blend by 3-fold after 4 hours of in vitro culture, prebeet® increased CFU (colony forming units) as a marker of probiotic growth) more than beet powder or potato starch alone.

[0097] FIG. 9 shows two bar graphs (A, B) demonstrating that prebeet®, beet powder alone, and potato starch alone do not cause overgrowth of E. coli after 4 hours as measured by (A) optical density (OD) and (B) colony forming units (CFU).

[0098] The words proximal and distal are applied herein to denote specific ends of components of the instrument described herein. A proximal end refers to the end of an instrument nearer to an operator of the instrument when the instrument is being used. A distal end refers to the end of a component further from the operator and extending towards the surgical area of a patient and/or the implant.

[0099] FIG. 10 is a bar graph showing that prebeet® significantly increased Lactobacillus growth and at similar levels as individual prebiotic resistant starches after 4 hours of culture (as measured by OD). (X-axis, left to right: Lacto, Lacto+ prebeet®, Lacto+Beet alone, Lacto+Potato Starch, Lacto +Tapioca Starch, Lacto+Corn Starch, Lacto+Kudzu Starch, Lacto+Arrowroot Starch; y- axis: OD600 cells/ml). [0100] FIG. 11 is a bar graph showing that prebeet® significantly increased Bifidobacterium growth more than any individual starch or beet powder after 4 hours of culture (as measured by OD). (X-axis, left to right: Bifido, Bifido+ prebeet®, Bifido+Beet alone, Bifido+Potato Starch, Bifido+Tapioca Starch, Bifido+Com Starch, Bifido+Kudzu Starch, Bifido+ Arrowroot Starch; y- axis: OD600 cells/ml).

[0101] FIG. 12 is a bar graph showing that prebeet® significantly increased the production of nitric oxide (as measured by nitrate) by the Lactobacillus blend after 4 hours in culture.

[0102] FIG. 13 is a bar graph showing that prebeet® significantly increased the production of nitric oxide (as measured by nitrate) by the Bifidobacterium blend after 4 hours in culture.

[0103] Unless indicated otherwise, Asterisk(s) indicate statistical significance: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Kruskal-Wallis test.

[0104] FIG. 14 is a bar graph showing that prebeet® increases the production of GLP-1 protein in human intestinal epithelial cells (Caco-2) as measured by ELISA after 4 hours in culture.

[0105] FIG. 15 is a bar graph showing that prebeet® increases the production of GLP-1 protein in human intestinal epithelial cells (Caco-2) as measured by ELISA after 24 hours in culture.

[0106] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. It will be further understood that the terms “comprises,” “comprising,” “includes,” and/or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0107] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The word “about,” when accompanying a numerical value, is to be construed as indicating a deviation of up to and inclusive of 10% from the stated numerical value. The use of any and all examples, or exemplary language (“e.g.” or “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention. [0108] References to “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment, although they may.

[0109] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0110] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc ), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

[OHl] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0112] While the invention has been described in connection with various embodiments, it will be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles of the invention, and including such departures from the present disclosure as, within the known and customary practice within the art to which the invention pertains.

Claims

CLAIMS What is claimed is:

1. A composition comprising a beet preparation and a resistant starch.

2. The composition of claim 1 comprising a beet preparation such as a beet powder, resistant starch such as a prebiotic resistant starch, and optionally a nutritionally active additive.

3. The composition of claims 1-2, wherein said composition is a dietary supplement, food product, or nutraceutical.

4. The composition of claims 1-3, in which the resistant starch comprises or is a prebiotic fiber.

5. The composition of claims 1-4, in which the resistant starch comprises or is resistant potato starch, resistant banana starch, resistant oat starch, or any combination thereof.

6. The composition of claims 1-5, in which the at least one nutritionally active additive is selected from the group consisting of a vitamin, a micronutrient, a mineral, a fatty acid, an amino acid, a plant preparation such as powdered ginger and/or a plant extract such as a ginger extract such as ginger extract powder, or any combination thereof.

7. The composition of claims 1-6, in which the at least one vitamin is ascorbic acid (vitamin C), D, E, A, E, K, thiamine (Bl), riboflavin (B2), niacin (B3), pantothenic acid (B5), B6, B7, B9, Bl 2, or any combination thereof.

8. The composition of claims 1-7, in which the at least one mineral is sulfur, potassium, chlorine, sodium, calcium, phosphorus, magnesium, iron, zinc, manganese, copper, iodine, chromium, molybdenum, selenium, cobalt, or any combination thereof.

9. The composition of claims 1-8, wherein said composition is formulated for oral administration.

10. The composition of claims 1-9, wherein the formulation such as for oral administration is a capsule, microcapsule, tablet, granule, powder, troche, pill, suspension, solution, or syrup.

11. The composition of claims 1-10, further comprising a nutritionally active ingredient.

12. The composition of claims 1-11, wherein said composition is a dose unit formulated for administration such as oral administration and comprises 1 g beet powder, 3.5 g prebiotic (resistant) starch, and lOOOug Vitamin B12 (methylcobalamin).

13. A composition comprising the subject matter of any of the above claims, in any combination.

14. A method of preparing a composition comprising the step of combining a beet preparation such as a beet powder and a resistant starch such as a resistant prebiotic starch, and optionally a nutritionally active additive.

15. The method of claim 14, wherein said beet preparation such as beet powder and said resistant starch such as resistant prebiotic starch are blended together.

16. A method of improving or maintaining gut and/or metabolic health in a subject comprising the steps of (a) providing a composition of claims 1-13 to the subject, and (b) administering such as orally administering the composition to the subject to improve or maintain gut and/or metabolic health.

17. A method of increasing desirable bacterial growth in a subject’s gut preferably while decreasing or minimizing undesirable bacterial growth, comprising the steps of (a) providing a composition of claims 1-13 to the subject, and (b) administering such as orally administering the composition to the subject to improve or maintain gut and/or metabolic health.

18. A method of increasing nitric oxide production in desirable bacteria in a subject’s gut, comprising the steps of (a) providing a composition of claims 1-13 to the subject, and (b) administering such as orally administering the composition to the subject to improve or maintain gut and/or metabolic health.

19. A method of treating and/or preventing pulmonary hypertension; type 2 diabetes; metabolic syndrome; chronic kidney disease; other conditions, disorders, diseases, syndromes, symptoms or the like including in particular those related to cardiometabolic function, comprising the step of providing a composition of claims 1-13 to the subject and then administering such as orally administering the composition for the above purpose(s).

20. The methods of claims 16-19, wherein at least Img to 10g, for instance 300-3000 mg beet preparation such as beet powder and at least Img to 10g, for instance 1.5-7 g resistant starch such as resistant prebiotic starch is administered to the subject.

21. The methods of claims 16-20, further comprising administration of a nutritionally active additive.

22. The methods of claims 16-21, wherein 1g beet powder, 3.5 g resistant starch, and lOOOug Vitamin B 12 are administered to the subject, or where more than these amounts are administered to the subject.

23. A method of assessing metabolic response in a subject treated with a composition according to the methods of claims 16-22, comprising the step of providing a composition of claims 1-13, administering the composition to a subject, and measuring the metabolic response in the subject.

24. The method of claim 23 wherein the composition is orally administered to the subject in a therapeutically or nutritionally effective amount.

25. The method of claim 23 or 24 wherein the metabolic response is measured by GLP-1 production in the subject.