Composition of TRI-substituted starch and method for preparing and using the same

Abstract

This specification discloses compositions containing modified starch. [Solution] Starch is modified to contain ester bonds to multiple short-chain fatty acids of different lengths, and in particular to bonds to acetic acid, propionic acid, and butyric acid. A method for producing and using such starch esters is a composition containing starch esters for nutritional effects and health benefits.

Description

Technical Field

[0001] This application is related to U.S. Provisional Patent Application No. 63 / 034,144, filed on June 3, 2020, which is hereby incorporated by reference in its entirety, and claims the benefit of priority.

[0002] Disclosed herein is a starch-containing composition comprising a starch material modified to form ester bonds with a plurality of short-chain fatty acids (SCFAs) of different lengths. The present invention relates to a triple-modified starch composition useful for nutritional applications. The present invention further relates to methods of making and using the composition.

Background Art

[0003] Short-chain fatty acids (SCFAs), particularly acetic acid, propionic acid, and butyric acid, are the major end products resulting from the gut microbial fermentation of dietary fiber and resistant starch (Bajka et al., Nutrition Research, 2010, 30(6);427-34). Deficiencies of SCFAs in plasma and colon are associated with metabolic and autoimmune disorders. For example, inhibition of SCFA synthesis by antibiotics can cause diarrhea (Binder H.J., AnnuRevPhysiol., 2010;72:297-313). Maintaining optimal levels of colonic SCFAs can prevent and counteract such disorders and related diseases such as obesity, type 2 diabetes, inflammation, inflammatory bowel disease, irritable bowel syndrome, diarrhea, atherosclerosis, Crohn's disease, and ulcerative colitis.

[0004] Digestive-tolerant carbohydrates (dietary fiber) modified to be either acetylated or butyrylated have been shown to provide protection from autoimmune disease in NOD mice (see WO2018 / 027274, published February 15, 2018). Furthermore, acetylated starch molecules have been shown to increase the acetate pool in the rat intestine (ARBird et al., Food Hydrocollocies, 20(2006):1135-40). However, these synthetic reactions typically require the use of dimethyl sulfoxide or DMSO to attach the acetyl or butyryl moiety. Since it is generally desirable to limit the amount of DMSO or other solvents present in the preparation for human consumption, there is a need to provide such modified molecules prepared in the absence of DMSO or other solvents, for example, for dietary or nutritional applications. In some applications, it is preferable that such preparations be completely, and especially, DMSO-free.

[0005] In addition to modifications made to starch by covalently bonding a single type or variety of SCFAs, SCFAs also act when esterifying multiple single types or varieties of SCFAs onto a starch molecule. See, for example, U.S. Patent No. 5,587,412 issued on December 24, 1996. Corn starch was esterified with acetic acid and propionic acid or butyric acid. However, these molecules required a large degree of substitution of at least about 1.8 to provide particles with good dimensional stability and physical properties.

[0006] An improved health composition is needed that provides superior levels of SCFA to the user's colon. [Overview of the Initiative]

[0007] This disclosure relates to modified starch compositions useful in nutritional preparations. In particular, this disclosure relates to compositions comprising starch esters, i.e., starch that has been substituted or modified to bond to a plurality of short-chain fatty acids of different lengths. The starch esters may contain at least three different species of SCFAs. In some embodiments, such starch esters are obtained from starch trisubstituted with acetic acid, propionic acid, and butyric acid. This disclosure also relates to methods for preparing and using the compositions.

[0008] Disclosed herein are modified starches for incorporating multiple SCFAs of different lengths. In some embodiments, the starch ester comprises at least three different SCFAs. Such a starch ester may provide a vehicle for delivering one or more SCFAs.

[0009] In certain exemplary embodiments, the present invention comprises a composition comprising a starch ester, the starch ester being a product of starch modification using acetic acid, propionic acid, and butyric acid. In certain embodiments, such a trisubstituted starch ester composition may result in the production of different SCFAs by colon microorganisms.

[0010] In certain embodiments, the present invention includes a method for producing polysubstituted starch esters by mixing starch with short-chain fatty acids, adding an esterification catalyst, and mixing the compositions. In certain embodiments, the method is carried out in the absence of DMSO.

[0011] In certain embodiments, the present invention includes nutritional or pharmaceutical preparations comprising starch esters. In certain embodiments, the present invention includes the use of starch esters for the treatment of many gastrointestinal disorders.

[0012] In certain embodiments, the present invention includes a method for achieving increased short-chain fatty acid concentrations in a user's colon using the compositions described herein. In certain embodiments, the present invention includes a method for treating an autoimmune or metabolic disorder in a subject by administering a therapeutically effective amount of the compositions or formulations described herein to the subject. [Brief explanation of the drawing]

[0013] The accompanying drawings illustrate, describe, and point to novel features applicable to various embodiments, but it will be understood that various omissions, substitutions, and modifications can be made to the form and details of compositions and methods without departing from the spirit of this disclosure. The figures herein are illustrative in nature and are not intended to limit. As will be recognized, certain embodiments described herein may be embodied in a manner that does not provide all of the features and benefits described herein, since some features may be used or practiced separately from others. [Figure 1] This shows a gel permeation chromatography of natural corn starch used as a base material. [Figure 2] This is a gel permeation chromatograph of experimental sample 1, showing starch esters prepared with starch having a degree of substitution of 0.2 after treatment with acetic anhydride. [Figure 3] The gel permeation chromatography of experimental sample 2 is shown, which is a starch ester prepared by treating starch with acetic acid and having a degree of substitution of 0.2. [Figure 4] The gel permeation chromatograph of experimental sample 3 shows that it is a starch ester prepared from starch treated with acetic acid and having a degree of substitution of 0.2. [Figure 5A] Figure 5B shows the 1H NMR analysis of a dry blend of monosubstituted starch esters: a mixture of starch acetic acid, propionic acid, and starch butyric acid, compared with Figure 5B, which shows the 1H NMR analysis of trisubstituted starch substituted with acetic acid, propionic acid, and butyric acid. [Figure 5B]Figure 5B shows the 1H NMR analysis of a dry blend of monosubstituted starch esters: a mixture of starch acetic acid, propionic acid, and starch butyric acid, compared with Figure 5B, which shows the 1H NMR analysis of trisubstituted starch substituted with acetic acid, propionic acid, and butyric acid. [Figure 6] Micrographs of unmodified base corn starch (HYLON® VII) and trisubstituted HYLON® VII corn starch containing acetic acid, propionic acid, and butyric acid are shown. These images show starch granules both before and after treatment with DMSO. [Figure 7A] This shows the measured acetic acid levels 6 hours after incubation of starch esters in an in vitro colon model study. The effect of starch acetate (HAMSA6, HYLON® VII esterified with acetic acid) is compared to the effect of both trisubstituted starch (HAMSABP6, HYLON® VII esterified with acetic acid, propionic acid, and butyric acid) and a dry blend of monosubstituted starch esters: starch acetate, starch propionic acid, and starch butyrate (a dry blend, a mixture of HYLON® VII esterified with acetic acid, HYLON® VII esterified with butyric acid, and HYLON® VII esterified with propionic acid). Samples were prepared using stock concentrations of 10 mM (low), 20 mM (medium), or 40 mM (high). [Figure 7B] This shows the acetate levels measured during a 20-hour incubation in an in vitro model colon study. [Figure 8A]This shows the measured butyrate levels 6 hours after incubation of starch esters in an in vitro colon study. The effect of starch butyrate (HAMSB6, HYLON® VII esterified with butyrate) is compared to the effects of both trisubstituted starch (HAMSABP6, HYLON® VII esterified with acetate, propionic acid, and butyrate) and a dry blend of monosubstituted starch esters, starch acetate, starch propionic acid, and butyrate (a dry blend, a mixture of HYLON® VII esterified with acetate, HYLON® VII esterified with butyrate, and HYLON® VII esterified with propionic acid). Samples were prepared using stock concentrations of 10 mM (low), 20 mM (medium), or 40 mM (high). [Figure 8B] This shows butyrate levels measured during a 20-hour incubation in an in vitro colon model study. [Figure 9A] This report shows the measured propionic acid levels 6 hours after incubation of starch esters in an in vitro colon study. The effect of starch propionic acid (HAMSP6, HYLON® VII esterified with propionic acid) is compared to that of both trisubstituted starch (HAMSABP6, HYLON® VII esterified with acetate, propionic acid, and butyric acid) and a dry blend of monosubstituted starch esters (starch acetate, starch propionate, starch butyrate) (Dry Blend ABP; the dry blend is a mixture of HYLON® VII esterified with acetate, HYLON® VII esterified with butyric acid, and HYLON® VII esterified with propionic acid). Samples were used from stock concentrations of 10 mM (low), 20 mM (medium), or 40 mM (high). [Figure 9B] This shows propionic acid levels measured during a 20-hour incubation in an in vitro colon study. [Figure 10A]Shows the measured acetic acid levels in an in vitro model colon study after incubation with starch acetate compositions prepared by different processes, measured over a 6-hour incubation. The effect of starch acetate prepared using the anhydrous method is compared to the effect of starch acetate prepared using the aqueous method. Samples were used from stock concentrations of 10 mM (low), 20 mM (medium), or 40 mM (high). [Figure 10B] Shows the acetic acid levels measured in a 20-hour incubation with starch acetate prepared by anhydrous or aqueous preparation in an in vitro colon study.

Mode for Carrying Out the Invention

[0014] The technology is also not limited to the aspects described herein, which are intended as examples of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations of the technology can be made without departing from its spirit and scope. Functionally equivalent methods within the scope of the technology will be apparent to those skilled in the art from the foregoing description in addition to those enumerated herein. Such modifications and variations are intended to be included within the scope of the appended claims. It should be understood that the technology is, of course, not limited to a method, complex, reagent, compound, or composition that can vary. All methods described herein can be carried out in any suitable order, unless otherwise described herein or otherwise clearly inconsistent with the context. It should also be understood that the terms used herein are for the purpose of describing only aspects and are not intended to be limiting. Therefore, this specification is intended to be regarded only as an illustration of the breadth, scope, and spirit of the technology, which is indicated only by the appended claims, their definitions, and any equivalents thereof.

[0015] All publications, patent applications, granted patents, and other documents (e.g., journals, articles, and / or textbooks) referenced herein are incorporated by reference in the same manner as each individual publication, patent application, granted patent, or other document is specifically and individually indicated to be incorporated herein by reference in whole. Definitions contained in the text incorporated by reference are excluded to the extent that they conflict with the definitions in this disclosure. In the event of any conflict between the definitions in this disclosure and the cited references, this disclosure shall prevail.

[0016] The embodiments described herein as exemplary may be suitably practiced in a context that is not limited by any element(s) or limitation(s) not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted broadly and not limitingly. Additionally, the terms and expressions used herein are for illustrative purposes only and not limiting, and there is no intention to exclude any equivalents of any features or parts thereof shown and described in the use of such terms and expressions, although it should be recognized that various modifications are possible within the scope of the claimed technology. Furthermore, the phrase “essentially consisting of” should be understood to include those elements specifically listed, as well as additional elements that do not substantially affect the fundamental and novel features of the claimed technology. The phrase “consisting of” excludes any elements not specified.

[0017] As will be understood by those skilled in the art, for any and all purposes, and particularly from the perspective of providing the specifically described explanations, all ranges disclosed herein also include any and all possible sub-ranges and combinations of those sub-ranges. When used throughout, a range is used as an abbreviation to describe each value within that range and all values. Any value within a range may be selected as the end of the range. All turns of phrase such as "maximum", "at least", "greater than", and "less than" include the recited numbers and refer to ranges that can then be broken down into sub-ranges as described above.

[0018] The use of "about" to modify a number herein means including the recited number ±10%. A legally acceptable recitation of a value in a claim generally means that value. The use of "about" in the claims or the specification is not intended to limit the full scope of equivalents to the subject.

[0019] The terms "short-chain fatty acid" or "SCFA" may be used interchangeably and may refer to a fatty acid containing less than 6 carbon atoms or having a molecular backbone containing less than 6 carbons. Different SCFAs can be described as having different numbers of carbons or different lengths. For example, acetic acid has a molecular backbone containing 2 carbons, propionic acid has a molecular backbone containing 3 carbons, and butyric acid has a molecular backbone containing 4 carbons. Each of these three SCFAs contains a different number of carbons in its chemical or molecular structure and can thus be considered to have different lengths from each other.

[0020] The term "starch ester" may refer to any starch containing an ester group, a natural starch or a modified starch derivative of any origin, or a combination thereof. In some embodiments, such a starch ester includes a starch modified to contain one or more types of SCFAs. The ester groups of the starch esters of the present invention contain or consist of fatty acids having carbon chains of six or fewer carbon atoms. For example, the ester groups may include formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and mixtures thereof.

[0021] The terms “trisubstituted starch ester” or “trimodified starch ester” may be used interchangeably and may refer to starch esters containing at least three SCFAs of different lengths. For example, a trisubstituted starch ester may contain starch molecules having ester bonds to acetic acid, propionic acid, and butyric acid molecules. Individual starch molecules in a starch material may contain zero, one, two, or three ester bonds to SCFA substituents, while aggregated starch materials contain ester bonds to acetic acid, propionic acid, and butyric acid molecules. Each individual glucose unit in any starch molecule may contain zero, one, two, or three ester bonds to SCFA substituents. If an individual glucose unit contains multiple SCFA substituents, those substituents may be the same or different SCFAs.

[0022] The term "monosubstituted starch ester" may refer to a starch ester modified to contain a single species or type of SCFA, while "disubstituted starch ester" may refer to a starch ester modified to contain two SCFAs of different lengths.

[0023] The "degree of substitution" (DS) of a polymer may refer to the average number of substituents attached per base or monomer unit. A starch molecule may contain a chain of attached glucose sugars. In esterified starch or starch esters, the DS may represent the average number of SCFA-substituted hydroxyl groups on each glucose backbone molecule of the starch.

[0024] The terms “crystalline granular structure” or “crystalline structure” may be used interchangeably and may refer to the appearance of starch or starch granules having a highly ordered structure. Such structures may be identified by microscopic examination, for example, by optical microscopy and scanning electron microscopy, as described in the section on starch. (Practical guide in FoodIndustry, pp. 13-15, by David J. Thomas and William A. Atwell (Eagan Press 1999).)

[0025] The term "gelatinization temperature" for starch molecules can refer to the temperature at which the molecular order within the starch molecule is disrupted, resulting in irreversible changes in properties including loss of crystalline structure, loss of birefringence, and viscosity.

[0026] Terms such as “treatment” and “to treat” are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in relation to completely or partially preventing a disease or its symptoms, and / or therapeutic in relation to partially or completely curing the disease and / or adverse effects resulting from the disease. As used herein, the term “treatment” encompasses any treatment of a disease in a subject and includes (a) preventing the disease from occurring in a subject to which the disease may be pre-positioned; (b) inhibiting the disease, i.e., preventing its occurrence; (c) alleviating the disease, i.e., regressing the disease; and (d) reducing the symptoms associated with the disease.

[0027] The terms “effective dose” and “pharmaceutically effective dose” refer to a quantity of a drug sufficient to produce a desired biological effect. This effect may be a reduction and / or mitigation of signs, symptoms, or causes of disease or impairment, or any other desired change in a biological system. The appropriate effective dose in any individual case can be determined by a person skilled in the art using routine experiments. “Therapeutic effective dose” refers to a quantity that provides a therapeutic effect for a given condition and administration regimen. In particular, “therapeutic effective dose” means a quantity that is effective in preventing, reducing, or improving the symptoms of a disease, or in extending the lifespan of the subject being treated, whether human or non-human animal. Determining the therapeutic effective dose is within the scope of the art of the art.

[0028] The term “nutritional preparation” means any composition that meets the nutritional requirements of a particular subject or supplements a particular diet. Such nutritional preparations can promote general health in many ways, such as reducing autoimmune and inflammatory responses, enhancing immune strength, promoting weight loss or appetite suppression, improving overall gut health, improving weight maintenance or weight gain, and managing chronic diseases such as diabetes, hypertension, and digestive disorders. By promoting health, the uptake of vitamins, minerals, proteins, amino acids, or other substances as needed can promote general health in many ways in many populations.

[0029] The term "digestion-resistant" may refer to foods or food components, or parts thereof, that pass through the digestive tract without being digested or absorbed by the digestive tract. Certain polysaccharides and carbohydrates, including certain natural and modified starches and fibers, are partially or completely unaffected by the digestive enzymes and chemicals found in the stomach, small intestine, and large intestine. Digestion-resistant foods and carbohydrates of particular interest include starch esters that are resistant to digestion in different parts of the gastrointestinal tract, such as the small and large intestines. Some embodiments of the technology of the present invention relate to compositions comprising digestion-resistant starch modified to contain short-chain fatty acids (SCFAs) and ester bonds. Digestion-resistant carbohydrates of particular interest include starches that are resistant to digestion in different parts of the gastrointestinal tract, such as the small or large intestine. Such modified starches are useful in nutritional formulations that can deliver SCFAs to the colon of interest.

[0030] While not bound by theory, SCFAs released from bacterial fermentation of dietary fiber in the colon are thought to promote gut health in many ways. For example, these fatty acids are thought to be important for maintaining visceral function by increasing blood flow, and also contribute to improved electrolyte and fluid absorption during diarrhea, maintaining a low colonic pH to limit the growth of intestinal pathogens, and regulating colonic muscle activity. These properties may be achieved when a starch-containing composition with one or more types of SCFAs is delivered to digestive system cells, such as cells present in the colon.

[0031] In certain embodiments, digestion-tolerant carbohydrates may include polysaccharides and oligosaccharides, such as cellulose, hemicellulose, pectin, arabinoxylan, xylongan, glucomannan, galactomannan, galactan, β-glucan, pectide polysaccharides (homogalacturonan, rhamnogalacturonan-I, and rhamnogalacturonan-II), tolerant maltodextrin, fructooligosaccharides, inulin, galactooligosaccharides, mannaoligosaccharides, arabinooligosaccharides, and xylooligosaccharides. Such digestion-tolerant carbohydrates may provide a source of dietary fiber that can be fermented by colonic microbial cells to produce short-chain fatty acids.

[0032] In certain embodiments, digestion-resistant carbohydrates may contain starch, and may also contain digestion-resistant starch. The starch materials used herein may be any of several starches or mixtures thereof. The starch may be natural starch or starch modified by any process including, but not limited to, chemical, enzymatic, and physical treatments, as described below, for example: Starches: Practical Guide for the Food Industry by David J. Thomas and William A. Atwell (Eagan Press 1999).

[0033] Some embodiments relate to trisubstituted starch esters or compositions containing trisubstituted starch esters, which are modified starch molecules bonded to at least three SCFAs of different lengths. In some embodiments, the starch ester may comprise three or more SCFAs selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. In some embodiments, SCFAs with high bioavailability may be selected. In some embodiments, the modified starch ester may provide starch molecules having ester bonds to acetic acid, propionic acid, and butyric acid molecules. In some embodiments, the three or more SCFAs consist of acetic acid, propionic acid, and butyric acid.

[0034] SCFAs are important for gastrointestinal health. They can be naturally provided by microbial fermentation of digestible foods in the small or large intestine. SCFAs can arise as terminal products of such bacterial carbohydrate fermentation of digestible foods. It would be desirable to provide trisubstituted starch esters to deliver SCFAs at increased levels sufficient to provide the therapeutic effects described herein.

[0035] In some embodiments, the starch ester may comprise glucose molecules having one or more hydroxyl groups substituted with various SCFAs, where the unmodified starch molecule may have zero degree of substitution. In the modified starch molecule, each glucose subunit may have one or more hydroxyl groups substituted with substituents. In such a modified starch ester, the starch ester may have a degree of substitution of about 0.01 to about 0.6, or about 0.01 to about 0.4, or about 0.1 to about 0.6, or about 0.1 to about 0.5, or about 0.1 to about 0.4, or about 0.1 to about 0.3, or about 0.2 to about 0.3.

[0036] Certain embodiments provide a composition comprising a digestion-resistant carbohydrate, wherein the digestion-resistant carbohydrate comprises a starch ester. In certain embodiments, the present invention comprises a composition comprising a starch ester, wherein the starch ester is a product of esterification of starch molecules with acetic acid, propionic acid, and butyric acid. In certain embodiments, the present invention comprises a composition comprising a starch ester, wherein the starch ester is modified to contain acetic acid, propionic acid groups, and butyric acid groups.

[0037] In certain embodiments, the present invention comprises a composition containing a starch ester, the starch ester being a product of esterification of starch with acetic acid, propionic acid, and butyric acid, and the composition contains less than 0.01% dimethyl sulfoxide (DMSO). In some embodiments, the starch ester or composition may contain less than 1%, less than 0.1%, or less than 0.01% dimethyl sulfoxide, or may not contain dimethyl sulfoxide at all.

[0038] Starch may have a granular, crystalline, non-granular, amorphous, or both form.

[0039] As used herein, modified starch is intended to include, but is not limited to, crosslinked starch, heat-suppressed starch, stabilized starch, acetylated and organically esterified starch, hydroxyrecylated and hydroxypropylated starch, phosphorylated and non-organically esterified starch, cationic, anionic, nonionic, and zwitterionic starch, as well as succinates and substituted succinic acid derivatives of starch. For example, starch may be oxidized, thinned, and / or crosslinked. Starch may also be reacted with cationic, anionic, amphoteric, and / or nonionic agents.

[0040] Starches include, but are not limited to, those derived from any plant source, including, corn or corn starch, pea starch, legume starch, potato starch, wheat starch, oat starch, rice starch, rye starch, sago starch, tapioca starch, wheat starch, waxy corn starch, high-amylose corn starch, waxy potato starch, waxy rice starch, sorghum starch, and mixtures thereof. The starch can be selected from waxy starches such as corn, peas, potatoes, wheat, oats, rice, rye, sago, tapioca, wheat, waxy corn, waxy potatoes, and waxy rice, sorghum, and high-amylose corn (starch having high-amylose starch, i.e., starch having an amylose content of at least 40% by weight, more specifically at least 65% by weight), and any derivatives or combinations thereof. The amylose content of such high-amylose corn starch may be at least about 70% by weight relative to the starch. Starch powder can also be used. In certain embodiments, the starch may be natural starch such as HYLON® (registered trademark) VII, corn starch (Ingredion, Westchester, IL).

[0041] In certain embodiments, digestion-resistant carbohydrates may include carbohydrates that resist or escape digestion and absorption in the small intestine. In some embodiments, digestion-resistant carbohydrates may include one or more of digestion-resistant oligosaccharides (e.g., carbohydrates with a degree of polymerization of 3 to 10), resistant starches, and non-starch polysaccharides. In certain embodiments, the carbohydrate may be VERSAFIBE®, dietary fiber (Ingredient, Westchester, IL). In certain embodiments, digestion-resistant carbohydrates may include cellulose, hemicellulose (consisting of various heteropolysaccharides including arabinoxylan), β-glucans, and non-starch polysaccharides such as pectin.

[0042] A starch-containing composition can be induced in an in vitro model colon cell assay to induce model colon cells to produce or provide one or more SCFAs over a period of time. A composition containing a trisubstituted starch ester can induce model colon cells to produce one or more SCFAs in different amounts compared to a composition containing unesterified starch or a monosubstituted starch ester containing only one short-chain fatty acid. In some embodiments, the monosubstituted starch ester contains one of the SCFAs contained in the starch ester. In some embodiments, the monosubstituted starch ester has a similar degree of substitution as the trisubstituted starch ester.

[0043] In some embodiments, model colon cells are exposed to or incubated with starch esters for approximately 0.5 hours, 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, 20 hours, 24 hours, or 48 hours to induce SCFA production by the model colon cells.

[0044] In some embodiments, starch esters can be used to achieve one or more increased amounts of SCFA in the user's colon. Further embodiments describe formulations comprising the starch esters described herein.

[0045] Generally, starch esters can be produced by reacting starch with one or more SCFAs in the presence of a catalyst in the solvent of the starch ester. In some embodiments, the esterification catalyst may comprise two or more catalysts. Some embodiments relate to a method for producing or producing starch esters, the method comprising a) mixing starch with one or more short-chain fatty acids, b) adding an esterification catalyst to provide a mixture, c) mixing the mixture, and d) optionally heating the mixture, the method being carried out in the absence of DMSO.

[0046] In some embodiments, the method may incorporate one or more short-chain fatty acids, selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. In some embodiments, the method may incorporate one, two, three, four, five, six, or more different SCFAs. In some embodiments, the method may incorporate at least acetic acid, propionic acid, and butyric acid.

[0047] Dimethyl sulfoxide (DMSO) is widely available as a solvent, but its medical use is restricted by the FDA. Adverse reactions to DMSO are common but usually few in number and are related to the concentration of DMSO in the drug solution. Furthermore, for compositions ingested by individuals, the use of solvents such as DMSO is undesirable to some consumers. Methods for detecting DMSO in solution are generally known to those skilled in the art. In some preferred embodiments, the compositions of the present invention contain less than 0.01% DMSO. In some embodiments, the compositions contain, are made with, or do not contain dimethyl sulfoxide at less than 1%, less than 0.1%, or less than 0.01% dimethyl sulfoxide.

[0048] In some embodiments, the method can provide starch esters having a granular, crystalline, non-granular, amorphous, or both structure.

[0049] In some embodiments, the method can provide starch esters having a degree of substitution of about 0.01 to about 0.6, or about 0.01 to about 0.4, or about 0.1 to about 0.6, or about 0.1 to about 0.5, or about 0.1 to about 0.4, or about 0.1 to about 0.3, or about 0.2 to about 0.3.

[0050] The esterifying agent may refer to any organic anhydride. Organic anhydrides may include, but are not limited to, acetic anhydride, propionic anhydride, butyric anhydride, hexanoic anhydride, maleic anhydride, phthalic anhydride, succinic anhydride, hexenyl succinic anhydride, octenyl succinic anhydride, dodecenyl succinic anhydride, hexadecenyl succinic anhydride, and mixtures thereof.

[0051] The esterification process may involve the use of a catalyst. The catalyst may include any material capable of catalyzing the esterification reaction. The catalyst may be organic or inorganic, acidic or basic. Acidic catalysts may include, but are not limited to, sulfuric acid, perchloric acid, hydrochloric acid, methanesulfonic acid, dodecylbenzenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, strong acid ion exchange resins, phosphoric acid, and mixtures thereof. Basic catalysts may include, but are not limited to, sodium hydroxide, sodium acetate, sodium carbonate, sodium bicarbonate, pyridine, and mixtures thereof. In one embodiment, sulfuric acid is used. The amount of catalyst should be sufficient to catalyze the esterification reaction.

[0052] In some embodiments, the method can use esterification catalysts such as sulfuric acid, perchloric acid, hydrochloric acid, methanesulfonic acid, dodecylbenzenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, strong acid ion exchange resins, phosphoric acid, and mixtures thereof. In some embodiments, the method can use esterification catalysts in amounts of about 0.1 to about 1.0 mol%, about 0.1 to about 0.8 mol%, about 0.1 to about 0.6 mol%, about 0.1 to about 0.5 mol%, about 0.2 to about 1.0 mol%, about 0.2 to about 0.8 mol%, or about 0.25 to about 0.5 mol%.

[0053] In some embodiments, the method may require one or more SCFAs selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. In some embodiments, three or more SCFAs are selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. In some embodiments, three or more SCFAs include acetic acid, propionic acid, and butyric acid. In some embodiments, three or more SCFAs consist of acetic acid, propionic acid, and butyric acid.

[0054] Further embodiments describe starch esters that can be obtained by one or more of the methods described herein, or that are obtained by such methods. In some embodiments, the formulations may include, for example, nutritional formulations, pharmaceutical formulations, and formulations for therapeutic or medical use.

[0055] The starch esters provided by the methods described herein can provide delivery of SCFA to the colon of a subject. SCFA can be provided to an individual requiring treatment by any number of means known to those skilled in the art. For example, in some embodiments, SCFA is provided in a pharmaceutical formulation for oral, topical, or systemic administration, as described herein. In some embodiments, as described herein, the pharmaceutical formulation is adapted for delivery of SCFA to the large intestine, more specifically, to the colon of an individual. In some embodiments, the formulation may comprise a composition containing the starch ester described herein.

[0056] Alternatively, starch esters may be added to an individual's diet to provide SCFA to the individual, thereby providing SCFA to contact the cells of the gastrointestinal tract during digestion of the food preparation in a desired area of ​​the gastrointestinal tract. In some embodiments, the food preparation provides the release of SCFA in the colon, as described herein. Alternatively, SCFA may be provided to an individual as an adjunct to the individual's diet, thereby providing SCFA to contact the cells of the gastrointestinal tract during digestion of the nutritional supplement in a desired area of ​​the gastrointestinal tract. In some embodiments, the nutritional supplement provides the release of SCFA in the colon, as described herein.

[0057] In certain embodiments, the composition induces more acetic acid after 6 hours of incubation in an in vitro model colon assay compared to the amount of acetic acid induced by the same concentration of starch ester having the same DS but esterified to contain only a single SFCA such as acetic acid. In certain embodiments, the composition induces a large amount of propionic acid after 6 hours of incubation in an in vitro model colon assay compared to the same concentration of starch ester having a similar DS but esterified with only a single SFCA such as propionic acid. In certain embodiments, the composition induces a large amount of propionic acid after 6 hours of incubation in an in vitro model colon assay compared to the same concentration of starch ester having a similar DS but esterified with only a single SFCA such as butyric acid. In certain embodiments, the composition induces a large amount of propionic acid after 20 hours of incubation in an in vitro model colon assay compared to the same concentration of starch ester having a similar DS but esterified with only a single SFCA such as acetic acid, butyric acid, or propionic acid.

[0058] In certain embodiments, the objective is the use of a composition comprising starch esters to achieve an increase in the concentration of one or more short-chain fatty acids in the colon of a user. Such an increase in SCFAs to the colon of a target may function as a treatment. In certain embodiments, the use may include formulating a pharmaceutical or nutritional preparation comprising the disclosed trisubstituted starch and taking such preparation. In some embodiments, the use may include a concentrated preparation for the target, such as a pill or powder form of the disclosed trisubstituted starch.

[0059] In some embodiments, starch esters may have specific surface area qualities. A different amount of energy may be required to gelatinize one gram of a composition containing starch esters compared to the amount of energy required to gelatinize one gram of the unesterified version of starch esters. A different amount of energy may be required to gelatinize one gram of a composition containing trisubstituted starch esters compared to monosubstituted starch esters. For example, to gelatinize 1 gram of a composition containing starch esters, it may be necessary to use approximately 0.1 to 10.0 J / g, or approximately 0.5 to 10.0 J / g, or approximately 0.5 to 5.0 J / g, or approximately 0.5 to 4.0 J / g, or approximately 1.0 to 10.0 J / g, or approximately 1.0 to 5.0 J / g, or approximately 1.0 to 4.0 J / g, or approximately 2.0 to 4.0 J / g, or approximately 3.0 to 5.0 J / g, compared to the amount of unesterified control starch molecules.

[0060] In certain embodiments, the formulation may contain the starch esters described herein.

[0061] Certain embodiments may provide a method for providing a nutritional supplement to support intestinal health in a subject, the method comprising administering an effective amount of the composition to the subject. In certain embodiments, the composition comprising starch esters is a nutritional preparation. The nutritional preparation may be nutritionally complete and contain suitable types and amounts of free amino acids, lipids, carbohydrates, vitamins, and minerals. In certain embodiments, the nutritional preparation may be in the form of a liquid, powder, gel, paste, solid, tablet, capsule, concentrate, suspension, or an enteral preparation, oral preparation, infant preparation, pediatric preparation, child preparation, and / or adult preparation in ready-to-use form. In certain embodiments, the nutritional preparation may be a liquid (ready-to-use or concentrated) or a powder. In certain embodiments, beneficial preparations comprising novel starch esters include supplements, nutritional beverages, nutritional bars, and easily dispersed powders. If the nutritional preparation is a liquid, the shelf life of the nutritional preparation is at least 18 months. If the nutritional preparation is a powder, the shelf life of the nutritional preparation is at least 24 months.

[0062] In certain embodiments, starch esters may be added to one or more standard infant formulas, hydrolyzed protein infant formulas, lactose-free infant formulas, soy protein infant formulas, hydrolyzed soy protein infant formulas, or any nutritional preparation that requires the benefit of delivering SCFA to the colon.

[0063] In certain embodiments, starch esters constitute 0.5 to 50% of the nutritional preparation. In certain embodiments, starch esters constitute 0.5 to 30% of the nutritional preparation. In certain embodiments, starch esters constitute 0.5 to 20% of the nutritional preparation. In certain embodiments, starch esters constitute 0.5 to 10% of the nutritional preparation. In certain embodiments, starch esters constitute 0.5 to 5% of the nutritional preparation.

[0064] The methods of the present invention are useful for the prevention and / or treatment of any disease, thereby resulting in an increase in the autoimmune inflammatory response in one or more areas of the body. Therefore, the methods of the present invention are useful for treating diseases associated with dysfunctional / ineffective regulatory T cell function, enlarged autoreactive T effector cells, and / or B cell dysfunction. Diseases that can be prevented and / or treated according to the present invention include, but are not limited to, autoimmune diseases, including, for example, autoimmune diseases selected from the group consisting of type 1 diabetes, psoriasis, rheumatoid arthritis, inflammatory bowel disease, Kelliak disease, autoimmune hepatitis, myocarditis, lupus nephritis, primary biliary cirrhosis, and multiple sclerosis.

[0065] In certain embodiments, the object comprises a method for treating an autoimmune or metabolic disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a starch ester. In certain embodiments, the disorder is selected from obesity, diabetes, inflammation, inflammatory bowel disease, irritable bowel syndrome, diarrhea, atherosclerosis, Crohn's disease, and ulcerative colitis.

[0066] This technology is further described in the following illustrative embodiments and is not intended to limit the entire scope of the claims and their equivalents.

[0067] The subject matter intended by this disclosure is described in the following numbered embodiments. 1. A starch ester comprising starch and at least three short-chain fatty acids selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, Preferably, at least three short-chain fatty acids are starch esters containing acetic acid, propionic acid, and butyric acid. 2. The starch ester has a degree of substitution of approximately 0.01 to approximately 0.6, or approximately 0.01 to approximately 0.4, or approximately 0.1 to approximately 0.6, or approximately 0.1 to approximately 0.5, or approximately 0.1 to approximately 0.4, or approximately 0.1 to approximately 0.3, or approximately 0.2 to approximately 0.3. Preferably, the starch ester according to Embodiment 1 has a degree of substitution of about 0.1 to about 0.6. 3. Starch ester containing less than 1%, less than 0.1%, or less than 0.01% of dimethyl sulfoxide, or not containing dimethyl sulfoxide. Preferably, the starch ester is the starch ester according to Embodiment 1 or 2, containing less than 0.01% dimethyl sulfoxide. 4. Starch esters contain a crystalline granular structure, Preferably, the starch ester includes acetic acid, propionic acid, and butyric acid. Preferably, the starch is selected from corn starch, pea starch, legume starch, potato starch, wheat starch, oat starch, rice starch, rye starch, sago starch, tapioca starch, wheat starch, waxy corn starch, high-amylose corn starch, waxy potato starch, waxy rice starch, and sorghum starch, and mixtures thereof, and optionally the starch is high-amylose corn starch, as described in any one of Embodiments 1 to 3. 5. The starch ester according to any one of Embodiments 1 to 4, wherein the starch ester provides a first amount of short-chain fatty acids greater than a second amount of short-chain fatty acids provided by the control composition in an in vitro model colon assay, and the control composition comprises a monosubstituted or disubstituted starch ester. 6. Starch esters were incubated in an in vitro model colon assay for approximately 0.5 hours, 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, 20 hours, 24 hours, or 48 hours. Preferably, the starch ester is incubated for about 6 hours or 24 hours in an in vitro model colon assay. Preferably, the starch ester according to Embodiment 5, wherein the monosubstituted starch ester or disubstituted starch ester has a degree of substitution similar to that of the starch ester. 7. Compared to unesterified control starch molecules, starch esters require approximately 0.1 to 10.0 J / g, or approximately 0.5 to 10.0 J / g, or approximately 0.5 to 5.0 J / g, or approximately 0.5 to 4.0 J / g, or approximately 1.0 to 10.0 J / g, or approximately 1.0 to 5.0 J / g, or approximately 1.0 to 4.0 J / g, or approximately 2.0 to 4.0 J / g, or approximately 3.0 to 5.0 J / g to gelatinize 1 gram of starch ester. Preferably, the starch ester according to any one of Embodiments 1 to 6, wherein the starch ester is required in an amount of about 0.5 to about 5.0 J / g. 8. Use of the starch ester described in any one of Embodiments 1 to 7 to achieve an increase in short-chain fatty acids in the user's colon. 9. A method for producing starch esters, wherein the method is: a) Mixing starch with one or more short-chain fatty acids, b) Adding an esterification catalyst to provide a mixture, c) Mixing the mixture, d) optionally, including heating the mixture, A method that is carried out in the absence of DMSO. 10. One or more fatty acids are selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. Preferably, the method according to Embodiment 9, wherein one or more short-chain fatty acids include acetic acid, propionic acid, and butyric acid. 11. The method according to any one of embodiments 9 to 10, wherein the method is carried out under anhydrous conditions. 12. The starch ester has a degree of substitution of approximately 0.05 to approximately 0.5, approximately 0.1 to approximately 0.4, approximately 0.1 to approximately 0.3, or approximately 0.2 to approximately 0.3. Preferably, the degree of substitution is about 0.1 to about 0.6. Preferably, the method according to any one of Embodiments 9 to 11, wherein the starch ester has a crystalline granular structure. 13. The esterification catalyst is selected from sulfuric acid, perchloric acid, hydrochloric acid, methanesulfonic acid, dodecylbenzenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, strong acid ion exchange resin, phosphoric acid, and mixtures thereof, and preferably the esterification catalyst is sulfuric acid. The esterification catalyst is used in an amount of about 0.1 to about 1.0 mol%, about 0.1 to about 0.8 mol%, about 0.1 to about 0.6 mol%, about 0.1 to about 0.5 mol%, about 0.2 to about 1.0 mol%, about 0.2 to about 0.8 mol%, or about 0.25 to about 0.5 mol%, preferably the esterification catalyst is used in an amount of about 0.1 to about 0.5 mol%, according to any one of Embodiments 9 to 12. 14. The starch ester contains at least three short-chain fatty acids selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. Preferably, the method according to any one of Embodiments 9 to 13, wherein at least three short-chain fatty acids include acetic acid, propionic acid, and butyric acid. 15. A starch ester obtained by the method described in any one of Embodiments 9 to 14. 16. A composition comprising a starch ester as described in any one of Embodiments 1 to 7 and Embodiment 15, preferably a composition comprising a nutritional preparation or a pharmaceutical preparation. 17. A method for providing a nutritional supplement to support intestinal health in a subject, comprising administering an effective amount of a starch ester described in any one of Embodiments 1 to 7 and Embodiment 15 to the subject. 18. A method for treating an autoimmune or metabolic disorder in a subject, comprising administering an effective amount of a starch ester described in any one of Embodiments 1 to 7 and Embodiment 15 to the subject.

[0068] Preferably, the disorder is selected from obesity, diabetes, inflammation, inflammatory bowel disease, irritable bowel syndrome, diarrhea, atherosclerosis, Crohn's disease, and ulcerative colitis.

[0069] Examples This technology is further illustrated by the following illustrative examples and is not intended to limit the entire scope of the claims or their equivalents.

[0070] Example 1: Preparation of starch acetate under anhydrous conditions. 61 mL of glacial acetic acid was added to a round-bottom flask equipped with a magnetic stirring rod. The bottom of the flask was immersed in a dish filled with water on top of a stirring plate. Next, 50 g of anhydrous corn starch (HYLON® VII, corn starch, Ingredion Inc.) was added under light stirring and mixed for 10-15 minutes.

[0071] The catalyst was prepared by slowly mixing 10 ml of glacial acetic acid with a small amount of sulfuric acid (0.1-0.5 mol%). The mixture was then slowly added to a round-bottom flask containing glacial acetic acid and starch to achieve a final sulfuric acid concentration of 0.1-0.5 mol%. Once the temperature stabilized, the flask was transferred to an oil bath and heated to 85°C. The mixture was reacted at 80°C for 2-8 hours. The product was then slowly transferred to 500 mL of water and mixed using a stirring plate. The residual product was collected and quantitatively transferred using an additional 400 mL of water.

[0072] To dehydrate the product, the mixture was poured through a Buchner funnel containing a filter. The product starch cake was washed three times with 400 mL of water, or until a pH of 5–6 was achieved. The cake was crushed and dried overnight in a hood.

[0073] Example 2: Preparation of monosubstituted starch under anhydrous conditions. To achieve a molar ratio of 0.81:1 acetic acid to starch, the desired amount of anhydrous starch was measured and transferred to a 400 mL Griffin beaker equipped with an overhead stirring motor and a Teflon paddle. Next, using a graduated cylinder and an Eppendorf pipette, the appropriate amounts of acetic acid and sulfuric acid catalysts were added to a 25 mL addition funnel (24 / 40 junction and stopcock for fine adjustment). Then, while gently stirring the starch, the acetic acid and sulfuric acid mixture was slowly added dropwise to the Griffin beaker at a rate of approximately 1 mL / min over 10 minutes. The temperature was monitored during the addition step to ensure that a stable temperature was maintained. Next, the starch and acid mixture was transferred in 4-5 marbles to a 32 ounce Nalgene tumbler jug ​​(rotating to create additional mechanical / shearing). The tumbler jug ​​was placed in a PO tumbler, set to the reaction temperature, and allowed to react for the desired time. At the end of the reaction, the product was slowly transferred to a 1000 part water mixture on a stirring plate. An additional 800 parts water was used to ensure complete product transfer. The starch was dehydrated using a Buchner funnel and flask. The starch cake was then washed three times (3 times) with 800 parts water using a filter. The pH of the cake was tested, and if it was not neutral (e.g., pH 5-6), washing was continued until a neutral pH was achieved. The product cake was crushed and dried overnight in a hood.

[0074] Example 3: Preparation of monosubstituted or polysubstituted starches under aqueous conditions. Anhydrous starch was slowly added to a container containing 170 parts of water to adjust the water content of the starch. The addition was carried out with stirring. The pH was adjusted to 8.0–8.5 with a 3% NaOH solution. Next, 3–4 drops of 30% hydrogen peroxide were added. The slurry was then pumped into a clamped 5-neck / 5L reaction flask in a water bath maintained at 75–80°F. Acid anhydride reagents (which may be a single SCFA or multiple acid anhydrides for multiple SCFAs intended to be added) were added to the slurry. For example, acetic anhydride is added if acetic acid binds to the starch, while acetic anhydride, propionic acid, and butyric acid are used if they bind to the starch at a rate of 1.2 mL / min while stirring and maintaining a pH of 8.0–8.5 with a 3% NaOH solution. After the reagents had been completely added, the reaction pH was maintained for a further 1 hour. Next, the starch was dehydrated using a Buchner funnel and flask. The cake was then re-slurred with 170 parts of water and adjusted to pH 5.5 with 3 NHCl solution. The material was filtered using a Buchner funnel and flask. The starch cake was washed with 3 × 100 parts of water (on the filter). The starch cake was crushed and dried overnight in a hood.

[0075] Example 4: Characterization of the physical properties of trisubstituted starch. Gel permeation chromatography (GPC), 1 The structures of trisubstituted starches prepared by aqueous or anhydrous methods were characterized by 1H NMR chromatography and degree of substitution (DS) titration.

[0076] GPC was performed using an AllianceGPCV2000 instrument equipped with a refractive index detector with the following parameters: Column set = phenotype gel 10 μm, 100 Å, 103 Å, 105 Å; Injection volume = 102 μL in one injection, two replications per sample; Mobile phase = DMSO + 0.03 M NaNO3; Run time = 50 min; Flow rate = 1 mL / min; Column temperature = 80 °C; Detector temperature = 80 °C; Sample autoloader temperature = 80 °C; Standard = Pullulan (180 Da ~ 642 kDa); Sample concentration = 20 mg in 10 mL of mobile phase; Sample and standard preparation = Samples were rotated overnight (15 hours) in a headspace vial with the mobile phase at room temperature, heated in boiling water for 1 hour, and cooled to ambient temperature. The samples were then filtered into a 4 mL LC vial using a 2 μm GMF syringe filter.

[0077] GPC of natural HYLON®VII, corn starch, exhibited three distinct populations, with the highest molecular weight population having a retention time of 15 minutes and a molecular weight of 5.6 MDa (see Figure 1). HYLON®VII starch acetic acid prepared by an aqueous process (prepared using experimental sample 1 and the protocol cited in Example 3) also exhibited three distinct populations with nominally the same retention time and molecular weight (see Figure 2). Differences were observed in the peak percentage of the highest molecular weight population (14% and 6%, respectively) where natural HYLON®VII had a higher molecular weight density population than starch acetic acid. There were two prototypes of HYLON®VII acetic acid prepared by an anhydrous process with equivalent degrees of substitution, but with different amounts of sulfuric acid catalyst used to prepare the samples. Samples prepared with a larger amount of catalyst (Experimental Sample 2, prepared using the protocol from Example 1 with modification using 0.5 mol% H2SO4 catalyst, 80°C, 2 hours) showed two distinct populations with a clear shift to a lower molecular weight population (determined by a shift to a longer retention time) (see Figure 3). The maximum recorded average molecular weight was 114,000 Da. Samples prepared with a smaller amount of catalyst and a longer reaction time (Experimental Sample 3, prepared using the protocol from Example 1, including modification using 0.25 mol% catalyst and incubation at 80°C for 4 hours) showed four distinct molecular weight populations (see Figure 4). The retention time was slightly shifted to a longer time compared to the natural starch. However, compared to samples with higher catalyst concentrations, the maximum recorded average molecular weight was 730,000 Da, which indicates that smaller amounts of catalyst exhibit surprisingly favorable esterification with minimal degradation of the molecular weight profile of the treated starch.

[0078] Nuclear magnetic resonance (NMR) spectroscopy was performed using a Bruker 500 MHz instrument, as shown in Figures 5A-5B. 1The 1H NMR spectrogram shows the results obtained from NMR treatment of a dry blend of monosubstituted starch esters (Figure 5A, the dry blend is a mixture of HYLON® VII esterified with acetic acid, HYLON® VII esterified with butyric acid, and HYLON® VII esterified with propionic acid, providing a combination of mixed starch acetic acid, propionic acid starch and butyric acid starch) compared to a single trimodified starch containing acetic acid, propionic acid, and butyric acid moieties (Figure 5B).

[0079] Here, the degree of substitution was determined by titration. 1 gram of starch was transferred to a 600 mL low-molecular-weight beaker and 200 mL of deionized water was added. The solution was heated in a boiling water bath for 30 minutes, with continuous stirring for the first 3 minutes. Next, the solution was cooled to at least 60°C and 3-4 drops of phenolphthalein were added. The mixture was titrated with 0.1N NaOH to achieve a pink endpoint. Then, 75 mL of 0.1N NaOH was added, the solution was covered with Parafilm and transferred to an incubator, incubated at 60°C for 24 hours, and then the solution was titrated with 0.1N HCl until the endpoint was achieved. This data was recorded. 1-2 mL of 0.1N HCl was added in excess. The solution was covered with Parafilm and returned to the incubator at 60°C for 1 hour. Then, the solution was back-titrated with 0.1N NaOH to achieve a pink endpoint. This data was recorded. Calculations were performed according to the following procedure.

[0080]

number

[0081] The total degree of esterification and the net degree of esterification were used to determine the DS.

[0082] Example 5: Effect of DMSO on starch esterified with SCFA. Trisubstituted HYLON® VII, corn starch (prepared by an aqueous process), and natural HYLON® VII starch (the starch base used to produce the trisubstituted starch) were treated with DMSO according to the protocol described in U.S. Patent Publication No. 2019 / 0167615, published on June 6, 2019. Micrographs of natural HYLON® VII and trisubstituted HYLON® VII were taken before and after treatment of the starch with DMSO. A 1% dispersion of each product was prepared in deionized water. Next, 20 μL of the dispersion was applied to a microscope slide using a microglass coverslip. The samples were evaluated using an optical microscope equipped with a polarizer filter. Starch present in granular form is known to exhibit birefringence and Malteser cross-diffraction patterns when viewed under polarized light, as described by Thomas and Atwell, pp. 14-15 (1999). Similarly, the non-granular form of starch refers to any starch or starch derivative whose natural granular structure has been destroyed or removed.

[0083] Microscopic images show the heating of starch in DMSO using the conditions described in U.S. Patent Publication No. 2019 / 0167615 (see Figure 6). Loss of the maltoscross effect was also observed (not shown). These results indicate a loss of crystallinity and integrity in these starch granules.

[0084] In vitro effects of DMSO treatment on the physical structure of starch products suggest that DMSO treatment reduces or impairs the functional performance of starch. Rats supplied with a combination of acetylated high-amylose corn starch in the presence of DMSO and unmodified corn starch (LAHAMS+MS) were compared with rats supplied with acetylated high-amylose corn starch in the absence of DMSO and unmodified corn starch (IAHAMS+MS). The LAHAMS+MS group had both lower body weight (180 g vs. 201 g, respectively) and lower wet weight (2.80 g vs. 3.72 g, respectively) of Caecal contents than rats given DMSO-treated starch (IAHAMS+MS) (see Birdetal, Food Hydrocolloids, 2006, 20:1135-40). While not theoretically bound, these results may be due to DMSO-induced loss of crystallinity from the starch products.

[0085] Example 6: Differential scanning calorimetry (DSC) test for granular integrity. To characterize the effect of the anhydrous process on the granular integrity of natural starch bases, differential scanning calorimetry was performed on natural HYLON® VII, HYLON® VII acetate prepared using the aqueous process of the present invention, and HYLON® VII acetate prepared using the anhydrous process of the present invention. The results are reported in Table 1. The start temperature is the temperature at which gelatinization begins. The enthalpy of starch gelatinization can be described as the amount of energy required to gelatinize or heat 1 gram of starch. The maximum temperature is the highest temperature reading during the gelatinization process.

[0086] The gelatinization onset and maximum temperatures of all denatured samples are lower than those of natural HYLON® VII. While not theoretically bound, this may occur due to the weakening of hydrogen bonds caused by the introduction of short-chain fatty acids, thus lowering the gelatinization temperature. The data show the difference in enthalpy between the denatured and natural HYLON® VII.

[0087] [Table 1]

[0088] Example 7: Characteristic evaluation of short-chain fatty acid release in an in vitro colon model. To evaluate the beneficial effect of trisubstituted starch on the delivery of individual SCFAs to the colon, in vitro colon studies were conducted to measure the amount of individual SCFAs produced at different time points when colonic microorganisms or cells were incubated with a single dose or trisubstituted starch ester. The starch ester was prepared using the aqueous method described in Example 3.

[0089] A pool of human fecal samples (n=6) from adults aged 20–65 years was used as inoculum for model colon microbial fermentation. Fecal inoculum was prepared by mixing 0.1% frozen pooled fecal material with model colon intestinal medium. The intestinal medium used for model colon fermentation was adapted from a previously published colonic medium (Macfarlane et al., Microbial Ecology, 1998, 35:180-187), and contained experimental starch, 4.0 g / L mucin (porcine stomach type III), 3.0 g / L casein, 5.0 g / L peptone water, 0.5 g / L tryptone, 0.4 g / L bile salt number 3, 4.5 g / L yeast extract, and 0.005 g / L Fe Distilled water contains SO4·7H2O, 4.5 g / L of NaCl, 4.5 g / L of KCl, 0.5 g / L of KH2PO4, 1.25 g / L of MgSO4·7H2O2, 0.15 g / L of CaCl2·6H2O, 1.5 g / L of NaHCO3, 0.8 g / L of cysteine, 0.05 g / L of hemein, and 1.0 g / L of Tween80.

[0090] Starch products such as trisubstituted starch were applied at stock concentrations of 10 mM (low), 20 mM (medium), or 40 mM (high). 100 μl of starch solution was added to 900 μl of intestinal medium containing fecal samples. The assay was performed in double cycles. Incubation was performed for 20 hours, and the starch fibers were resuspended after 6 hours of fermentation. Samples were taken at 6 and 20 hours, and the levels or amounts of different SCFAs present in the assay were measured by placing them at solution organic acid concentrations using high-performance liquid chromatography (HPLC). The 6-hour time point represents a short transit time in the digestive system (e.g., in patients with diarrhea). The 20-hour time point represents a longer transit time in the digestive system. The results are shown in Figures 7-10.

[0091] Figures 7–10 show the average or mean levels of individual SCFAs produced under various experimental conditions. Figures 7–9 show the amount of SCFA produced in the experimental sample, adjusted for starch content, with black bars in each column. Figures 7–9 compare the effects of monosubstituted starch esters, trisubstituted starch esters, and mixtures of acetylated, butylated, and propionated starches in a model colon assay with the various starch esters listed in Table 2. HAMSA is acetylated high-amylose corn starch. HAMSB is butylated high-amylose corn starch. HAMSP is propionated high-amylose corn starch. HAMSABP is acetylated, butylated, and propionated high-amylose corn starch.

[0092] [Table 2]

[0093] Figure 7 shows the effects of various treatments on the amount of acetic acid produced in model colon microorganisms when microorganisms were incubated in the presence of low, medium, and high concentrations of mixtures of acetylated starch, trisubstituted starch, or monosubstituted starch esters. As shown in Figure 7A, after 6 hours of incubation, colon microorganisms cultured in the presence of acetylated starch (HAMSA6, made from HYLON® VII esterified with acetic acid) produced acetic acid levels (see column) slightly higher than the amount of acetic acid bound to the starch ester (see attached bar). In comparison, the presence of trisubstituted starch (HAMSABP6, made from HYLON® VII esterified with acetic acid, propionic acid, and butyric acid) induced acetic acid levels (column) higher than the amount of acetic acid bound to the trisubstituted starch ester (see attached bar). A dry blend of monosubstituted starch esters (dry blend 6) resulted in acetic acid levels similar to or lower than those present in acetylated starch (HAMSA6).

[0094] As shown in Figure 7B, after 20 hours of incubation, all three compounds induced amounts of acetic acid exceeding the amount of acetic acid present in the treated compound. However, the trisubstituted starch ester (HAMSABP20) induced a greater amount of acetic acid than the other compounds (HAMSA20 and dry blend 20). In summary, Figures 7A–7B show that treatment with the trisubstituted starch ester induced higher levels of acetic acid than the amount of acetic acid provided by the trisubstituted starch ester itself. While some of the measured acetic acid may be produced as a degradation product of the starch ester added to the model colon assay, these results suggest that at least some of the acetic acid levels in the assay are a result of the assay being produced by microorganisms. Although not theoretically bound, the increase in the amount of acetic acid may be due to the trisubstituted starch ester inducing acetic acid production in the model colon microorganisms.

[0095] Table 3 shows the distribution of acetic acid in model colon assays at various time points, where acetic acid may remain bound to the starch ester, or it may be released from the starch ester into the intestinal-like medium after the fecal inoculum has been exposed to starch denatured only with acetic acid, trisubstituted starch, or a blend of monosubstituted starch esters (starch acetate, starch propionate, and starch butyrate). To calculate the bound acetic acid, 1 First, 1H NMR analysis was used to determine the individual mass percentages of starch base and acetic acid. Next, GC-FIID was used to quantify the released acetic acid. The difference was calculated by subtracting the bound amount from the released amount.

[0096] [Table 3]

[0097] Figures 8A–8B show the effects of various treatments on the amount of butyrate produced by colonic microorganisms when incubated in the presence of low, medium, and high concentrations of mixtures of butylated starch, trisubstituted starch, or monosubstituted starch esters. As shown in Figure 8A, after 6 hours of incubation, colonic microorganisms incubated in a mixture of butylated starch (HAMBA6 made from HYLON® VII esterified with butyrate) or monosubstituted starch ester (dry blend 6) produced moderate amounts of butyrate exceeding the level of butyrate bound to the starch ester (see attached bar). At the highest concentration of added starch ester, the model colonic microorganisms showed a net loss of butyrate. In comparison, the presence of low, medium, and high levels of trisubstituted starch (HAMSABP6 prepared from HYLON® VII esterified with acetic acid, propionic acid, and butyric acid) induced butyric acid levels (column) that were much higher than the levels of butyric acid bound to the trisubstituted starch esters (attached bar). As shown in Figure 8, after 20 hours of incubation, Table 4 shows the distribution of acetate in model colon assays at various time points, where butyrate may remain bound to the starch ester, or it may be released from the starch ester into the intestinal-like medium after the fecal inoculum has been exposed to starch denatured only with butyrate, trisubstituted starch, or a blend of monosubstituted starch esters (starch acetate, starch propionate, and starch butyrate). To calculate the bound butyrate, 1 First, 1H NMR analysis was used to determine the individual mass percentages of starch base and butyric acid. Next, GC-FIID was used to quantify the released butyric acid. The difference was calculated by subtracting the bound amount from the released amount.

[0098] [Table 4] * The release values ​​are calculated based on the difference between butyric acid produced by the control (unmodified Hylon VII) and experimentally modified samples (monosubstituted and trisubstituted Hylon VII). Negative values ​​indicate that the control sample produced more butyric acid.

[0099] Figures 9A–9B show the effects of various treatments on the amount of propionic acid produced by model colon microorganisms when microorganisms were incubated in the presence of low, medium, and high concentrations of mixtures of propionated starch, trisubstituted starch, or monosubstituted starch esters. As shown in Figure 9A, after 6 hours of incubation, colon microorganisms incubated in a mixture of propionated starch (HAMBP6 made from HYLON® VII esterified with propionic acid) or monosubstituted starch ester (dry blend 6) showed a net decrease in propionate beyond the level of propionate bound to the starch ester, except for the lowest level of dry blend which showed a limiting increase in propionate levels (see attached bars). In comparison, the presence of low, medium, and high levels of trisubstituted starch (HAMSABP6 prepared from HYLON® VII esterified with acetic acid, propionic acid, and butyric acid) induced propionate levels (column) that were much higher than the levels of propionic acid bound to the trisubstituted starch ester (attached bar). As shown in Figure 9B, the effect of trisubstituted starch was not significant compared to the effects of other comparators after 20 hours of incubation.

[0100] Some of the measured propionic acid may be produced as degradation products of starch esters added to the model colon assay, and these results suggest that at least some of the propionic acid levels are a result of propionic acid production by microorganisms in the assay. While not theoretically bound, these results may be due to trisubstituted starch esters inducing propionic acid production in model colon microorganisms.

[0101] To investigate the effect of starch esterification procedures on SCFA release in an in vitro model colon assay, acetate-substituted starch molecules prepared by either an aqueous method or an anhydrous process were applied to the in vitro model colon assay. The results are shown in Figure 10. After 6 hours, starch acetate produced by the anhydrous process yielded a higher amount of acetate than starch acetate produced by the aqueous process.

Claims

1. A starch ester comprising starch and at least three short-chain fatty acids selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid, Preferably, the at least three short-chain fatty acids are starch esters containing acetic acid, propionic acid, and butyric acid.

2. The starch ester has a degree of substitution of about 0.01 to about 0.6, or about 0.01 to about 0.4, or about 0.1 to about 0.6, or about 0.1 to about 0.5, or about 0.1 to about 0.4, or about 0.1 to about 0.3, or about 0.2 to about 0.

3. Preferably, the degree of substitution is about 0.1 to about 0.6, the starch ester according to claim 1.

3. The starch ester contains less than 1%, less than 0.1%, or less than 0.01% of dimethyl sulfoxide, or does not contain dimethyl sulfoxide. Preferably, the starch ester contains less than 0.01% dimethyl sulfoxide, according to claim 1 or 2.

4. The starch ester comprises a crystalline granular structure, Preferably, the starch ester comprises acetic acid, propionic acid, and butyric acid. Preferably, the starch is selected from corn starch, pea starch, legume starch, potato starch, wheat starch, oat starch, rice starch, rye starch, sago starch, tapioca starch, wheat starch, waxy corn starch, high-amylose corn starch, waxy potato starch, waxy rice starch, and sorghum starch, and mixtures thereof, and optionally the starch is high-amylose corn starch, according to any one of claims 1 to 3.

5. The starch ester according to any one of claims 1 to 4, wherein the starch ester provides a first amount of short-chain fatty acids greater than a second amount of short-chain fatty acids provided by the control composition in an in vitro model colon assay, and the control composition comprises a monosubstituted or disubstituted starch ester.

6. The starch ester is incubated in an in vitro model colon assay for approximately 0.5 hours, 1 hour, 3 hours, 6 hours, 12 hours, 18 hours, 20 hours, 24 hours, or 48 hours. Preferably, the starch ester is incubated for about 6 hours or 24 hours in an in vitro model colon assay. Preferably, the starch ester according to claim 5, wherein the monosubstituted starch ester or disubstituted starch ester has the same degree of substitution as the starch ester.

7. Compared to unesterified control starch molecules, the starch ester requires approximately 0.1 to approximately 10.0 J / g, or approximately 0.5 to approximately 10.0 J / g, or approximately 0.5 to approximately 5.0 J / g, or approximately 0.5 to approximately 4.0 J / g, or approximately 1.0 to approximately 10.0 J / g, or approximately 1.0 to approximately 5.0 J / g, or approximately 1.0 to approximately 4.0 J / g, or approximately 2.0 to approximately 4.0 J / g, or approximately 3.0 to approximately 5.0 J / g to gelatinize 1 gram of the starch ester. Preferably, the starch ester according to any one of claims 1 to 6, wherein the amount of the starch ester is about 0.5 to about 5.0 J / g.

8. Use of the starch ester according to any one of claims 1 to 7 to achieve an increase in the amount of short-chain fatty acids in the colon of the user.

9. A method for producing starch esters, wherein the method is: e) Mixing starch with one or more short-chain fatty acids, f) Adding an esterification catalyst to provide a mixture, g) Mixing the above mixture, h) optionally heating the mixture, The method described above is carried out in the absence of DMSO.

10. The one or more fatty acids mentioned above are selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. Preferably, the method according to claim 9, wherein the one or more short-chain fatty acids include acetic acid, propionic acid, and butyric acid.

11. The method according to any one of claims 9 to 10, wherein the method is carried out under anhydrous conditions.

12. The starch ester has a degree of substitution of about 0.05 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, or about 0.2 to about 0.

3. Preferably, the degree of substitution is about 0.1 to about 0.

6. Preferably, the method according to any one of claims 9 to 11, wherein the starch ester has a crystalline granular structure.

13. The esterification catalyst is selected from sulfuric acid, perchloric acid, hydrochloric acid, methanesulfonic acid, dodecylbenzenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, strong acid ion exchange resin, phosphoric acid, and mixtures thereof, and preferably the esterification catalyst is sulfuric acid. The esterification catalyst is used in an amount of about 0.1 to about 1.0 mol%, about 0.1 to about 0.8 mol%, about 0.1 to about 0.6 mol%, about 0.1 to about 0.5 mol%, about 0.2 to about 1.0 mol%, about 0.2 to about 0.8 mol%, or about 0.25 to about 0.5 mol%, preferably the esterification catalyst is used in an amount of about 0.1 to about 0.5 mol%, according to any one of claims 9 to 12.

14. The starch ester comprises at least three short-chain fatty acids selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. Preferably, the starch ester according to any one of claims 9 to 13, wherein the at least three short-chain fatty acids include acetic acid, propionic acid, and butyric acid.

15. A starch ester obtained by the method according to any one of claims 9 to 14.

16. A composition comprising a starch ester according to any one of claims 1 to 7 and claim 15, wherein the composition preferably comprises a nutritional preparation or a pharmaceutical preparation.

17. A method for providing a nutritional supplement that supports intestinal health in a subject, the method comprising administering an effective amount of the starch ester described in any one of claims 1 to 7 and claim 15 to the subject.

18. A method for treating an autoimmune or metabolic disorder in a subject, the method comprising administering an effective amount of the starch ester described in any one of claims 1 to 7 and claim 15 to the subject, Preferably, the disorder is selected from obesity, diabetes, inflammation, inflammatory bowel disease, irritable bowel syndrome, diarrhea, atherosclerosis, Crohn's disease, and ulcerative colitis.

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