Methods of valorizing pomegranate polysaccharides

Abstract

A method for increasing the value of a polysaccharide containing material obtained from pomegranate by generating a mixture of polysaccharide cleavage products. The method comprises (i) reacting a pomegranate starting material containing polysaccharide with a Fenton's reagent containing a peroxide agent and a metal, and (ii) cleaving the reacted polysaccharide with a cleavage agent to generate the mixture of polysaccharide cleavage products. The polysaccharide cleavage products generally contain higher amounts of oligosaccharides, lower average molecular weight, increased solubility and decreased viscosity when dissolved in aqueous solution compared to the polysaccharide containing material obtained from pomegranate. Also provided are compositions and soluble pomegranate fiber comprising mixtures of polysaccharide cleavage products as well as food, beverage and medicinal products comprising soluble pomegranate fiber.

Description

METHODS OF VALORIZING POMEGRANATE POLYSACCHARIDESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 560,946, filed March 4, 2024 which is incorporated herein by reference in its entirety.FIELD

[0002] This invention relates generally to methods for valorizing polysaccharides obtained from pomegranate by depolymerizing polysaccharides to generate mixtures of polysaccharide cleavage products.BACKGROUND OF INVENTION

[0003] Pomegranate (Punica granatum) is a fruit-bearing shrub which is commercially cultivated for its fruit. The fruit is made up of a husk, which is red purple in color, enclosing sarcotestas which contain seeds and juice. The husk comprises two parts: an outer, hard pericarp, and an inner, spongy mesocarp which forms the inner wall of the husk and to which the sarcotestas are attached. Once the juice has been extracted from the fruit, the husk and the seeds remain as residue. This residue is a significant proportion of the whole fruit. For example, pomegranate husk comprises more than 40% of the wet weight of the whole fruit and is currently mostly disposed of as waste.

[0004] Various attempts have been made to valorize the husk and seeds ranging from using them for animal feeds to extraction of phyto-actives such as polyphenols, seed oils, flavonoids, etc. The material is particularly rich in antioxidants, such as polyphenols, especially ellagitannins, hydroxybenzoic acids and flavonoids (Papaioannou et al, 2020). Most attempts to valorize the husk and seed residue have therefore focused on polyphenols and ellagic acid.

[0005] However, these phyto-actives constitute a minor part of the husk and seed residue. Therefore, even when the phyto-actives are extracted and valorized, much of the remaining husk and seed material goes to waste or is used in low value applications such as for animal feeds (Pathak et al., 2017; and Mo et al., 2022).

[0006] A significant amount of the husk and seed residue is made up of dietary fiber. However, the dietary fiber contains long-chain polysaccharides and is not suitable for use in human foods and beverages. The fiber is insoluble, largely unpalatable, having a gritty texture and an unpleasant mouthfeel, and is difficult to formulate. The fiber is reported to be made up of lignin, cellulose, uronic acid, and neutral sugars including xylose, arabinose, and galactose (Hasnaoui et al, 2014). The structural complexity of the fiber makes further processing of the polysaccharides in the fiber difficult.

[0007] One method for improving the functionality of polysaccharides is to depolymerize them into shorter chain polymers such as oligosaccharides. Oligosaccharides are short chains of carbohydrates, usually having lengths in the range of 3 to 30 monomers. Oligosaccharide and oligosaccharide compositions are often soluble, more palatable, and easier to formulate than the original polysaccharides. Commonly two methods of depolymerizing polysaccharides are used, namely (i) applying high pressure and / or temperature, or (ii) enzymatic methods. The method of applying high pressure and / or temperature has the general disadvantage of producing large amounts of mono- and disaccharides which are not fiber. The enzymatic method has the disadvantage that enzymes are highly specific and are generally able to depolymerize only a single type of glycosidic bond. For complex fibers, multiple enzymes would be needed, and these enzymes may not exist commercially.

[0008] Recently chemical methods for the depolymerization of polysaccharides using Fenton’s chemistry followed by cleavage using a base have been described in published International patent applications WO2018236917 and WO2021097138. Additional depolymerization methods using Fenton’s chemistry are described in International patent application WO2023220318. The methods appear to be applicable to a range of polysaccharides. However, neither application describes depolymerization of dietary fibers extracted from complex polysaccharides which are rich in antioxidants, such as those obtained from pomegranate. Further it is also unclear whether depolymerizing polysaccharides obtained from pomegranate will result in valorization of the polysaccharide.

[0009] Thus, there is a need for methods of valorizing polysaccharides obtained from pomegranate.SUMMARY OF THE INVENTION

[0010] In one aspect, this invention provides a method for valorizing polysaccharide containing material obtained from pomegranate, the method comprising (i) reactingpolysaccharide in the polysaccharide containing material with a Fenton’s reagent comprising a peroxide agent and a metal, and (ii) cleaving the reacted polysaccharide with a cleavage agent to generate a mixture of polysaccharide cleavage products. In this regard, the invention provides a method for depolymerizing polysaccharide containing material obtained from pomegranate to generate a mixture of polysaccharide cleavage products. The invention further provides a method for generating a mixture of polysaccharide cleavage products, the method comprising: (i) reacting a pomegranate starting material containing polysaccharide with a Fenton’s reagent comprising a peroxide agent and a metal, and (ii) cleaving the reacted polysaccharide with a cleavage agent to generate the mixture of polysaccharide cleavage products. In embodiments, pomegranate starting material is polysaccharide containing material obtained from pomegranate which is optionally subjected to pre-processing prior to depolymerization as described herein. Polysaccharide containing material obtained from pomegranate can be subjected to cleaning, drying or reduction of water content, pressing, cutting, chopping, grinding or other processes to adjust particle size, removal of juice, freezing, freeze-thawing, lyophilization, or any combination of the above prior to pretreatment as described herein.

[0011] In another aspect, the method for depolymerizing a polysaccharide containing material obtained from pomegranate further comprises pre-treating the polysaccharide containing material prior to depolymerizing the polysaccharide. In an embodiment, pretreatment involves extraction of polyphenols from the polysaccharide containing material to provide a polyphenol-reduced pomegranate starting material containing polysaccharides. In an aspect, the polyphenols are extracted using water or an organic solvent. In embodiments, the organic solvent is an alkyl alcohol, optionally methanol, ethanol, n-propanol, or isopropanol; a ketone, optionally acetone, or an alkyl ester; optionally ethyl acetate. In embodiments, the organic solvent for polyphenol removal is a mixture of chloroform and methanol. In embodiments, the organic solvent is a 1 : 1 (v:v) mixture of chlorofomrmethanol. In embodiments, the organic solvent is acidified, for example by addition of an organic or mineral acid. In embodiments, the organic acid is a mono-, di-, or tricarboxylic acid. In embodiments, the tricarboxylic acid, citric acid, is used to acidify the organic solvent. In embodiments, the acidified organic solvent contains 0.005 to 1% wt / v of the organic acid. In embodiments, the acidified organic solvent is acidified ethanol or acidified propanol. In embodiments, the acidified organic solvent is acidified with 0.01 to 0.1% wt / v citric acid. In embodiments, the acidified organic solvent is acidified with 0.01% to 0.02% wt / v citric acid.In embodiments, the acidified organic solvent is acidified with 0.05% to 0.15% wt / v citric acid. In embodiments, polyphenols are extracted with acidic ethanol or isopropanol containing 0.01% to 0.02% wt / v citric acid. In embodiments, polyphenols are extracted with acidic ethanol or isopropanol containing 0.05% -0.15% wt / v citric acid. In embodiments, polyphenols are extracted with 0.015% wt / v citric acid in ethanol or isopropanol. In embodiments, polyphenols are extracted with 0.1% wt / v citric acid in ethanol or isopropanol.

[0012] In embodiments, pre-treatment involves removal of protein from the polysaccharide containing material obtained from pomegranate. In embodiments, the proteins are removed using one or more proteases. In embodiments, proteins are removed by addition of two or more different proteases.

[0013] In embodiments, pre-treatment involves removal of one or more polysaccharides other than pectin. In embodiments, pre-treatment involves removal of starch and / or cellulose from the polysaccharide containing material obtained from pomegranate. In embodiments, starch is removed using one or more amylase or isoamylase. In embodiments, cellulose is removed using one or more cellulases. In embodiments, pre-treatment involves addition of one or more cellulases to the polysaccharide containing material from pomegranate. In embodiments, pre-treatment involves addition of one or more glucanases to the polysaccharide containing material from pomegranate. In embodiments, pre-treatment involves removal of monosaccharides and / or di saccharides. In embodiments, one or more disaccharidases are added to degrade disaccharides in the polysaccharide containing material obtained from pomegranate. In embodiments, the polysaccharide containing material obtained from pomegranate is treated with one or more of maltase, isomaltase, lactase, sucrase or combinations thereof to generate monosaccharides. In embodiments, monosaccharides present in or generated by enzyme treatment are removed from the polysaccharide containing material obtained from pomegranate. In embodiments, pretreatment involves removal of lipids from the polysaccharide containing material obtained from pomegranate. In embodiments, pre-treatment involves removal of one or more of polyphenols, starch, cellulose, protein, lipid, monosaccharides, or disaccharides. In embodiments, pre-treatment involves removal of polyphenols and one or more of starch, cellulose, protein, monosaccharides, or disaccharides.

[0014] In another aspect, pre-treatment involves extraction of polysaccharide from the polysaccharide containing material obtained from pomegranate, wherein the extracted polysaccharides provide a polysaccharide-enriched pomegranate starting material containingpolysaccharides. In embodiments, pre-treatment involves extraction of pectins and the extracted polysaccharides provide a pectin-enriched pomegranate starting material containing polysaccharides. In embodiments, polysaccharide extraction involves extraction using an acidic aqueous solution (e.g., water to which acid is added). In embodiments, polysaccharide extraction is extraction with water to which a mineral acid (e.g., sulfuric acid or hydrochloride acid) is added. In embodiments, polysaccharide extraction is extraction with water to which an organic acid (e.g., acetic acid or citric acid) is added. In embodiments, polysaccharide extraction employs acidic aqueous solution at pH of 1-3 (+ / -0.5). In embodiments, polysaccharide extraction employs acidic aqueous solution at pH of 1-2 (+ / - 0.5). In embodiments, polysaccharide extraction employs acidic aqueous solution at pH of 1.0-1.5 (+ / -0.1). In embodiments, polysaccharide extraction employs acidic aqueous solution at pH of 1.2 (+ / -0.1). In embodiments, polysaccharide extraction employs an acidic aqueous solution at pH of 1.0-1.5 (+ / -0.1) acidified with sulfuric or hydrochloric acid. In embodiments, polysaccharide extraction employs an acidic aqueous solution at pH of 1.2 (+ / - 0.1) acidified with sulfuric or hydrochloric acid. In embodiments, extraction of polysaccharide separates polysaccharide from one or more of starch, cellulose, lignin, or protein. In embodiments, extraction of polysaccharide separates polysaccharide from one or more of starch, cellulose, hemicellulose, lignin, lipids or protein. In embodiments, extraction of pectin separates pectin from one or more other polysaccharides, such as starch, cellulose or hemicellulose, and / or separates pectin from lignin, lipids or protein.

[0015] In another aspect, the method for depolymerizing a polysaccharide containing material obtained from pomegranate comprises or further comprises purifying the polysaccharide cleavage products to provide a purified mixture of polysaccharide cleavage products. In embodiments, the polysaccharide cleavage products are subjected to ethanol precipitation to remove ethanol-soluble components from the polysaccharide cleavage products. In embodiments, polysaccharide cleavage products are solubilized in appropriate solvent and the solution is filtered to remove residual solids. In embodiments, salts and mono- and disaccharides are removed from the polysaccharide cleavage products by diafiltration.

[0016] The invention further provides mixtures of pomegranate polysaccharide cleavage products having useful properties. In embodiments, the invention provides soluble pomegranate fiber useful in a variety of food, beverage, nutritional and pharmaceutical compositions. In embodiments, the invention provides soluble pomegranate fiber comprisinga mixture of polysaccharide cleavage products, wherein a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 nephelometric turbidity units (NTU) or less and / or a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C. In embodiments, the mixture of polysaccharide cleavage products of the soluble fiber contains 40% or more by weight of polysaccharide cleavage products having molecular weight less than 100 kDa. In embodiments, the mixture of polysaccharide cleavage products of the soluble fiber has a degree of polymerization (DP) of 3 to 100. In embodiments, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 20 NTU or less or a 20 g / L mixture of the soluble pomegranate fiber in water has dynamic viscosity of 2 millipascal-second (mPa.s) or less at 25°C , or both.

[0017] In embodiments, the mixture of polysaccharide cleavage products of the soluble pomegranate fiber is formed by cleavage of a polysaccharide containing material obtained from pomegranate, a pomegranate starting material containing polysaccharides, a polyphenol-reduced pomegranate starting material containing polysaccharides or a pectin- enriched pomegranate starting material containing polysaccharides.

[0018] In related embodiments, the invention provides a composition comprising a mixture of polysaccharide cleavage products, wherein the composition is formed by:(i) reacting one or more polysaccharides in a polysaccharide containing material obtained from pomegranate with a Fenton’s reagent comprising a peroxide agent and a metal to generate reaction products; and(ii) cleaving the reaction products with a cleavage agent to generate the mixture of polysaccharide cleavage products.

[0019] In embodiments, the mixture of polysaccharide cleavage products contains 40% or more by weight of polysaccharides with a molecular weight of less than 100 kDa. In embodiments, the polysaccharide containing material obtained from pomegranate comprises pomace, husk, arils containing seeds, or any combination thereof. In a specific embodiment, the polysaccharide containing material obtained from pomegranate comprises pomegranate pomace.

[0020] In embodiments, the mixture of polysaccharide cleavage products of the forgoing mixture contains 60% or more, by weight, of polysaccharide cleavage products with a molecular weight of less than 100 kDa. In embodiments, each of the one or more polysaccharide cleavage products of the composition have a degree of polymerization (DP) of3 to 100. In embodiments, each of the one or more polysaccharide cleavage products have a degree of polymerization (DP) of 3 to 50. In embodiments, the mixture of polysaccharide cleavage products of the composition in water at a concentration of 20 g / L has turbidity of 40 NTU or less and / or dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C.

[0021] In embodiments of methods, soluble fiber and compositions, the mixture of polysaccharide cleavage products contains 40% or less by weight of polysaccharides with molecular weight of lOOkDa or more. In embodiments, the mixture of polysaccharide cleavage products generated contains 20% or less by weight of polysaccharides with molecular weight of lOOkDa or more. In embodiments, the mixture of polysaccharide cleavage products generated contains 10% or less by weight of polysaccharides with molecular weight of lOOkDa or more. In embodiments, the mixture of polysaccharide cleavage products generated contains less than 5% by weight of polysaccharide having molecular weight of 500 kDa or higher. In embodiments, the mixture of polysaccharide cleavage products generated contains less than 1% by weight of polysaccharide having molecular weight of 500 kDa or higher.

[0022] In an aspect, the polysaccharide containing material obtained from pomegranate is valorized by increasing the solubility of the polysaccharide containing material. In embodiments, the mixture of polysaccharide cleavage products has a solubility in water or aqueous solution of 20 g / L or higher optionally with a turbidity below 20 NTU. In embodiments, the mixture of polysaccharide cleavage products has solubility in water or aqueous solution of 50 g / L or higher optionally with a turbidity below 40 NTU or below 20 NTU. In embodiments, the mixture of polysaccharide cleavage products has solubility in water or aqueous solution of 100 g / L or higher optionally with a turbidity below 40 NTU or below 20 NTU. In embodiments, the mixture of polysaccharide cleavage products has solubility in water or aqueous solution of 200 g / L or higher optionally with a turbidity below 40 NTU or below 20 NTU.

[0023] In an aspect, the polysaccharide containing material obtained from pomegranate is valorized by decreasing the viscosity of the polysaccharide containing material. In embodiments, the mixture of polysaccharide cleavage products of pomegranate polysaccharides has a viscosity of less than 4 millipascal-second (mPa.s) at 25°C when dissolved in water at a concentration of at least 50 g / L (a 5% w / v aqueous solution). In embodiments, the mixture of polysaccharide cleavage products has a viscosity of less than 4 millipascal-second (mPa.s) at 25°C when dissolved in water at a concentration of 80 g / L. Inembodiments, the viscosity of a 20 g / L water solution of the mixture of polysaccharide cleavage products is less than 4 mPa.s at 25°C. In embodiments, the viscosity of a 20 g / L water solution of the mixture of polysaccharide cleavage products is less than 2 mPa.s at 25°C. In embodiments, the mixture of polysaccharide cleavage products from pomegranate exhibits both low viscosity as described above and high solubility with low turbidity as described above.

[0024] In an aspect, the polysaccharide containing material obtained from pomegranate is valorized by increasing the prebiotic properties of the polysaccharide containing material. In embodiments, the mixture of polysaccharide cleavage products has the ability, when consumed, alone or in combination with a food or beverage product, by an animal, to increase the relative abundance of beneficial bacteria in the gastrointestinal tract of the animal. In embodiments, the mixture of polysaccharide cleavage products increases the relative abundance of one of more of Faecalibacterium, B lamia. Actinobacteria, Rumminococcus, Gordonibacler, Lactobacillus plantarum, Lactobacillus rhamnosus GG, Lactobacillus penstosus, o Bifidobacterium pseudocatenulatum in the intestinal microbiota of the animal. In embodiments, the mixture of polysaccharide cleavage products increases the relative abundance of one or more strains of Lactobacillus in the intestinal microbiota of the animal. In embodiments, the mixture of polysaccharide cleavage products increases the relative abundance of one of more bifidobacteria in the intestinal microbiota of the animal.

[0025] In an aspect, the polysaccharide containing material obtained from pomegranate is valorized by improving the organoleptic properties of the polysaccharide containing material or of a food or beverage product containing the polysaccharide containing material. For example, the mixture of polysaccharide cleavage products exhibits organoleptic properties suitable for its use in human beverages or liquid food products. In an embodiment, suitability in human beverages requires that beverage viscosity is not increased by the addition of the mixture of polysaccharide cleavage products to the point that a consumer will reject the product. In an embodiment, suitability in human beverages requires that a beverage does not acquire a gritty or grainy texture as measured by expert or consumer sensory analysis when the mixture of polysaccharide cleavage products is added. In an embodiment, suitability in human beverages requires than beverage clarity is not substantially decreased by the addition of the mixture of polysaccharide cleavage products as measured by turbidity or expert and consumer panels. In embodiments, the mixture of polysaccharide cleavage products exhibits reduced gritty or grainy texture alone or when combined in a beverage, an aqueous solutionor a liquid or solid food product. In embodiments, an aqueous solution or slurry of the mixture of polysaccharide cleavage products exhibits reduced gritty or grainy texture. In embodiments, the mixture of polysaccharide cleavage products exhibits sweet taste. In some embodiments, a sweet taste is defined as having at least 10% of the sweetness of an equivalent amount of sucrose. For use in beverages, aqueous solutions or liquid or solid food products, the amount of the mixture of polysaccharide cleavage products added will vary with the application. In an embodiment, the amount of the mixture of polysaccharide cleavage product added to a beverage, solution or liquid or solid food product ranges from O.lg / L to the 2000 g / L of beverage, solution or liquid or solid food product. In an embodiment, the amount of the mixture of polysaccharide cleavage product added to a beverage, solution or liquid or solid food product ranges from O.lg / L to the 200 g / L of beverage, solution or liquid or solid food product. In embodiments, the amount of the mixture of polysaccharide cleavage products added to a beverage, solution, or liquid or solid food product ranges from 0.5 g / L to the solubility limit of the mixture of polysaccharide cleavage products in the beverage, solution, or liquid or solid food.

[0026] In additional embodiments of methods, compositions and soluble fiber, the polysaccharide cleavage products comprise less than 10% by weight of residual monosaccharides, for example, less than 5% by weight of residual monosaccharide. In an embodiment, the residual monosaccharides comprise greater than 70% by weight of a combination of galacturonic acid, glucose, and galactose. In a further embodiment, the residual monosaccharides further comprise rhamnose, xylose, and / or arabinose.

[0027] In embodiments, the optionally purified polysaccharide cleavage products contain glycosidic linkages comprising one or more of 4-linked galactose, 6-linked galactose, 4- linked xylose, 3, 6-linked glucose, 3, 6-linked galactose, 4-linked rhamnose or 2, 3, 4-linked rhamnose.

[0028] In embodiments, the purified mixture of polysaccharide cleavage products contains less than 10% by weight polyphenols and preferably contains less than 5% by weight polyphenols. In embodiments, the purified mixture of polysaccharide cleavage products contains less than 10% by weight of proteins and preferably contains less than 5% by weight proteins. In embodiments, the purified mixture of polysaccharide cleavage products contains less than 10% by weight of mono- and / or disaccharides and preferably contains less than 5% by weight of mono- and / or disaccharides. In embodiments, the purified mixture of polysaccharide cleavage products contains less than 5% by weight ash andpreferably contains less than 2% by weight ash. In embodiments, the purified mixture of polysaccharide cleavage products contains greater than 50% by weight or greater than 75% by weight of fiber. Preferably the purified mixture of polysaccharide cleavage products contains greater than 85% by weight of fiber. In embodiments, the purified mixture of pomegranate polysaccharide cleavage products contains greater than 30% by weight of soluble fiber. In embodiments, the purified mixture of pomegranate polysaccharide cleavage products contains greater than 50% by weight of soluble fiber. In embodiments, the purified mixture of pomegranate polysaccharide cleavage products contains greater than 75% by weight of soluble fiber. In embodiments, the purified mixture of pomegranate polysaccharide cleavage products contains greater than 85% by weight of soluble fiber.

[0029] The invention is also directed to mixtures of polysaccharide cleavage products obtained from pomegranate containing oligosaccharides prepared by the methods herein and having properties as described herein. In specific embodiments, the invention provides the mixture of polysaccharide cleavage products designated composition CLX 106. In specific embodiments, the invention provides the mixture of polysaccharide cleavage products designated composition CLX 106Cu.

[0030] The invention also provides prebiotic compositions and synbiotic compositions comprising the composition or soluble pomegranate fiber containing the mixture of polysaccharide cleavage products from pomegranate starting material. The prebiotic compositions stimulate growth of one or more beneficial bacteria. The synbiotic compositions comprise one or more beneficial bacteria.

[0031] The invention also provides a food, beverage, or nutritional or medicinal product comprising the compositions or soluble pomegranate fiber described herein. In embodiments, food products include yogurts, frozen yogurts, ice creams, fruit sauces, syrups, chocolate, tomato sauces, ketchups, barbecue sauces, breads, granola bars, energy bars, or breakfast cereals, among others. In embodiments, the beverage products include infant formulas, follow-on formulas, toddler’s beverages, dairy and nondairy milks, fermented milks, fruit and vegetable juices, fruit-based drinks, protein drinks, energy drinks, sports drinks, sparkling waters, vitamin waters, teas and coffee drinks, among others. In embodiments, the medicinal products include dietary supplements or pharmaceutical products. In embodiments, the medicinal products are in the form of a gummy, capsule, pill or tablet.

[0032] Other aspects and embodiments of the invention will be apparent to one of ordinary skill in the art on consideration of the detailed description, non-limiting examples and drawings provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIGs. 1A and IB illustrate example process flow diagrams of processes for valorizing polysaccharide containing material obtained from pomegranate. FIG. 1A is a flow diagram showing steps in a pomegranate fiber production process adapted for production of either pomegranate fiber powder (14a / 15a) or pomegranate fiber syrup (14b / 15b). FIG. IB is an exemplary pilot process flow diagram described in Example 3 where Tables 6 and 7 indicate the stream information.

[0034] FIG. 2A and 2B illustrate the impact of the composition CLX106 and CLX106Cu, respectively, on the growth of two Bifidobacterium pseudocatenulatum strains.

[0035] FIG. 3 illustrates the impact of the composition CLX106Cu on the growth of three lactobacillus strains.

[0036] FIGs. 4A and 4B illustrate the production of lactate (FIG. 4A) and betahydroxybutyrate (FIG. 4B) by two Bifidobacterium pseudocatenulatum strains.

[0037] FIG. 5 illustrates the production of indolelactate by two Bifidobacterium pseudocatenulatum strains.

[0038] FIG. 6 illustrates the reduction in pH in fecal samples fermented with CLX106Cu.

[0039] FIG. 7 illustrates the production of short chain fatty acids in fecal samples fermented with CLX106Cu.

[0040] FIG. 8 illustrates total short chain fatty acid production in multiple fecal samples fermented with CLX106Cu.

[0041] FIG. 9 illustrates the reduction in pH in fecal samples fermented with CLX106 in comparison to the starting polysaccharide.

[0042] FIG. 10 illustrates the increase in abundance of Gordonibacter species in fecal samples fermented with CLX106 in comparison to FOS.

[0043] FIG. 11 illustrates the production of short chain fatty acids in fecal samples fermented with CLX106 in comparison to the starting polysaccharide.

[0044] FIGs. 12A and 12B illustrate the reduction in ellagic acid in fecal samples from 2 donors, respectively, after fermentation with CLX106.

[0045] FIGs. 13A and 13B illustrate the increase in urolithin A in fecal samples from 2 donors, respectively, after fermentation with CLX106.STATEMENTS REGARDING NOMENCLATURE

[0046] In general, the terms and phrases used in this specification have the meaning recognized in the art, which can be found by reference to standard texts, journal references and contexts known to those skilled in the art. For clarity, the following terms have the following meaning unless otherwise specified:

[0047] It will be understood by one of ordinary skill in the art that single numeric values or numeric values in a range, include slight variations or deviations from the stated value which may be used to achieve substantially the same results as the stated value. In cases, where a numeric value is one that is measured, it will be recognized that there is some level of uncertainty in the stated value due to experimental error, which can be determined by one of ordinary skill in the art. In circumstances where this definition cannot be applied to a given stated value, is exceedingly difficult to apply, or wherein an uncertainty value is not specifically recited, then the numeric value has a reasonable deviation from the value, as known to a skilled person in the art. In embodiments, the reasonable deviation for a given value is + / -10% unless otherwise indicated.

[0048] “Ammonium bicarbonate” means solid ammonium bicarbonate, and / or an aqueous solution containing (i) ammonium and bicarbonate; (ii) ammonium, OH', and CO2, (iii) ammonia, H2O, and CO2, or (iv) any of the preceding and their equilibrium products.

[0049] “Ammonium hydroxide” means aqueous ammonium hydroxide and / or a solution containing (i) ammonia and H2O, (ii) ammonium and OH', (iii) ammonia and OH', or (iv) any of the preceding and their equilibrium products.

[0050] “Average molecular weight”, “average molecular mass”, “mean molecular weight”, “mean molecular mass”, or similar terms, refers to weight average molecular weight. Generally, unless otherwise specified, when a polysaccharide or a polysaccharide composition is described herein to have a specified average molecular weight (e.g., the molecular weight distribution of a polysaccharide cleavage product is such that at least 50%of the mass is smaller than 5 kDa), such values can be calculated with the aid of molecular weight analysis as described herein.

[0051] “Base” means a compound or collection of compounds that can accept hydrogen ions from a peroxyl oxidized carbohydrate, water, or non-aqueous solvent. Base can include Lewis bases, non-Arrhenius bases, strong-Arrhenius bases, weak-Arrhenius bases, other molecules that produce hydroxide ions through their decomposition, or other compounds that can accept hydrogen ions from a hydroperoxyl oxidized carbohydrate. Unless otherwise specified, base includes but does not specifically mean exclusively strong-Arrhenius bases (e.g., Na OH; K OH; or Ca+2(OH )2).

[0052] “Biologically relevant increase” means a statistically significant change as measured by parametric or non-parametric tests, generally in reference to the effects of a method comprising administering a polysaccharide composition or formulation thereof to a subject, or in an in vitro context, relative to an otherwise identical method that does not include administering the polysaccharide composition or formulation thereof. In some aspects, a biologically relevant increase can be measured in feces, jejunum, cecum, ileum, stomach, large intestines, duodenum, mouth, respiratory tract, skin, urogenital tract, vaginal tract, or other microbial community. In some aspects, a biologically relevant increase is a 10% increase or a 5x increase or a lOx increase or a 50x increase or a lOOx or l,000x increase or 10,000x or more. In some aspects the biologically relevant increase can be in the absolute or relative amount of a taxa or group of taxa, or an amount or relative amount of a biological species (e.g., a strain of bacterium) or an amount or relative amount of a given chemical species (e.g., short chain fatty acid, GLP-1, etc.). In some aspects, the biologically relevant increase can be the rate that a taxa, group of taxa, or other given species increases in the microbial community or in a subject (or location therein, such as a GI tract). In some aspects, the biologically relevant increase can be in the relative amount of a taxa, group of taxa, or other given species in a microbial community or in a subject (or location therein, such as a GI tract). In some aspects, an increase in abundance refers to the presence of one microbial taxa as compared to another microbial taxa, or one given species compared to another given species. “Biologically relevant decrease,” “biologically relevant change,” “biologically relevant amount,” and similar such terms can be similarly understood.

[0053] “Bronsted-Lowry base” means a compound or atom that can accept or bond to a hydrogen ion (e.g., methanol, formaldehyde, ammonia, etc.).

[0054] “Cleavage agent” or “cleavage reagent” means one or a combination of strong Arrhenius bases, non-Arrhenius and / or weak-Arrhenius bases used to cleave polysaccharides after hydroperoxyl oxidation, e.g., after reaction with a Fenton’s reagent. In certain aspects, a cleavage agent or cleavage reagent breaks glycosidic bonds in the polysaccharide, which bonds may be present between any two saccharides of the polysaccharide. The cleavage reagent may also be, and preferably is, a peroxide quenching reagent, and in either case may be used in combination with an additional compatible peroxide-quenching agent that may or may not also be a cleavage agent. In some aspects, a cleavage reagent may be an enzyme, for example a glycosyl hydrolase, a lytic polysaccharide monooxygenase, a glycosyl transferase, transglycosidase, polysaccharide lyase, carbohydrate binding module, glycoysl transferase, carbohydrate esterase, a cocktail containing two or more of the aforementioned enzymes, or any enzyme that is carbohydrate active. In some aspects, a cleavage reagent may be a solidphase acid catalyst or a solid-phase base catalyst.

[0055] CLX106” means a mixture of polysaccharide cleavage products and / or an oligosaccharide containing composition in which 31% of the mass comprises arabinose, 26% of the mass comprises glucose, 18% of the mass comprises galacturonic acid, 14% of the mass comprises galactose, 3% of the mass comprises xylose, 3% of the mass comprises rhamnose, and 3% of the mass comprises mannose, as measured by hydrolytic monosaccharide compositional analysis. In embodiments, CLX106 includes the composition with components as listed + or - 10% of the listed amounts. In embodiments, CLX106 includes the composition with components as listed + or - 5% of the listed amounts. In embodiments, CLX106 includes the composition with components as listed + or - 1% of the listed amounts. In embodiments the glycosidic linkage composition of CLX106 is as shown in Table 4, where the listed values are + / - 10%. In embodiments, the glycosidic linkage composition of CLX106 comprises 37% 4-linked glucose, 12% 3-linked glucose, 7% terminal glucose, 13% terminal arabinose, and 7% 5-linked arabinose, again where each value is + / - 10%. In embodiments, the glycosidic linkage composition of CLX106 comprises 37% 4-linked glucose, 12% 3-linked glucose, 7% terminal glucose, 13% terminal arabinose, 7% 5-linked arabinose, and 24% other minor linkages as shown in Table 4, again where each value is + / - 10%. The approximate molecular weight distribution of CLX106 comprises, the values set forth in Table 5, as measured by refractive index detection (RID), where each value is + / - 10%. CLX106 is generally derived from pomegranate pomace, amongst other pomegranate residue sources.

[0056] CLX106Cu” means a mixture of polysaccharide cleavage products and / or an oligosaccharide containing compositions in which 47% of the mass comprises galacturonic acid, 23% of the mass comprises glucose, 20% of the mass comprises galactose, 5% of the mass comprises rhamnose, and 2% of the mass comprises xylose, as measured by hydrolytic monosaccharide compositional analysis. In embodiments, CLX106Cu includes the composition with components as listed + or - 10% of the listed amounts. In embodiments, CLX106Cu includes the composition with components as listed + or - 5% of the listed amounts. In embodiments, CLX106Cu includes the composition with components as listed + or - 1% of the listed amounts. In embodiments the glycosidic linkage composition of CLX106Cu is as shown in Table 2, where the listed values are + / - 10%. In embodiments, , the glycosidic linkage composition of CLX106Cu comprises 37% 4-linked glucose, 13% terminal glucose, 4% 3,6-linked glucose, 14% 4-linked galactose, 7% terminal galactose, and 4% 6-linked galactose, again where each value is + / - 10%. In embodiments, the glycosidic linkage composition of CLX106 Cu comprises 37% 4-linked glucose, 13% terminal glucose, 4% 3,6-linked glucose, 14% 4-linked galactose, 7% terminal galactose, 4% 6-linked galactose and 21% other minor linkages as shown in Table 2, again where each value is + / - 10%. The approximate molecular weight distribution of CLX106Cu is shown in Table 3, as measured by refractive index detection (RID), where each value is + / - 10%. CLX106Cu generally is derived from pomegranate pomace, amongst other pomegranate residue sources.

[0057] "Comprising" means including. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

[0058] “Degree of polymerization” or “DP” of an oligosaccharide refers to the total number of monosaccharide units that are part of a particular carbohydrate polymer. For example, a tetra oligosaccharide has a DP of 4. When used to describe a group of polysaccharides (e.g., a polysaccharide composition), DP generally refers to the mean DP of the polysaccharides in the composition. In some aspects, the DP of a polysaccharide is referred to as “DP#”, where “#” corresponds to an integer representing the total number (or average number if used to describe a group of polysaccharides) of sugar monomer units (e.g., “DP3” means a degree of polymerization of 3). For polysaccharides or polysaccharide compositions discussed herein having a DP between the range of 3-9, the recited DP includes a variance of up to ±2 monomer units of the recited value. For oligosaccharides or oligosaccharide compositions having a DP of 10 or greater, the recited DP includes avariance of up to ±20% of the recited value, for example, an oligosaccharide having a DP of 20 may have a DP of 16, 17, 18, 19, 20, 21, 22, 23, or 24.

[0059] “Depolymerize” or “Depolymerization” and variants of this word refer generally to reacting a specific polysaccharide to reduce the number of monomer units therein, typically by cleavage of one or more glycosidic bonds. Such cleavage also results in a decrease in molecular weight of the polysaccharide. Such cleavage can result in generation of one or more polysaccharides of lower molecular weight, one or more oligosaccharides or one or more monosaccharides or combinations thereof. Depolymerization of a mixture of polysaccharides results in a mixture of polysaccharide cleavage products having a lower DP or lower average molecular weight than the original mixture. The result of such cleavage on mixtures of polysaccharides can also be described in terms of a weight % of polysaccharides therein having molecular weight greater than a selected values, e.g., a polysaccharide composition having less than 5% (or 2% or 1%) by weight of polysaccharides having molecular weight greater than 500kDa (or lOOkDa or 50kDa). In an embodiment, depolymerization can be affected by treatment with a Fenton’s reagent followed by cleaving the reacted polysaccharide with a cleavage agent. As is apparent from examples herein, the extent of depolymerization of polysaccharides achieved herein varies with reaction conditions.

[0060] “Fenton’s reagent” means a reagent comprising a peroxide agent and a metal. In certain aspects, the peroxide agent is hydrogen peroxide. In certain aspects, the metal is Fe(II), Fe(III), Cu(I), Cu(II), Mn(II), Zn(II), Ni(II), and Co(II), alkaline earth metals Ca(II) and Mg(II), the lanthanide Ce(IV) or any combination thereof.

[0061] Fiber or dietary fiber is carbohydrate that cannot be digested by a subject, particularly by an animal or human subject. Fiber cannot be broken down into mono- or disaccharides by the subject, and instead it passes through the body undigested by the subject. Fiber may, however, be digested by organism in the microbiome of the subject. In embodiments, the mixture of polysaccharide cleavage products contains fiber. The amount of fiber present in a given composition can be determined by methods that are well known in the art. Fiber can be characterized as soluble or insoluble. Insoluble fiber is not soluble in water and generally is described as passing through the human gastrointestinal tract unchanged. Soluble fiber is generally described as attracting water or being soluble in water in the colon. Soluble fiber can also form a gel or gel-like substance in the colon. Soluble fiber can be degraded by bacteria in the colon. Even though pomegranate dietary fiber may containnominal soluble fiber, it is generally in the form of long-chain polysaccharides and is generally not suitable for use in human foods and beverages. This invention provides soluble pomegranate fiber that exhibits enhanced water solubility with low turbidity and / or low viscosity in water solutions. More specifically, the invention provides soluble pomegranate fiber wherein a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less or dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C, or both. The soluble pomegranate fiber as described herein is generally suitable for use in human foods and beverages as well as in various nutritional and pharmaceutical applications.

[0062] “Free monosaccharide compositional analysis” refers to the method described in Amicucci, Galermo et al. 2019, the invention of which is incorporated by reference herein for all purposes and specifically for a description of that method, with some modifications. The derivatization reaction to produce monosaccharides is performed at the optimized condition of 70°C for 30 minutes. Samples are run on an Agilent 1290 Infinity II ultra-high performance liquid chromatography (UHPLC) system coupled to an Agilent 6490A triple quadrupole (QqQ) mass spectrometer. Separation is carried out on an Agilent InfinityLab Poroshell HPH-C18 column (2.1 mm x 50 mm, 1.9 pm particle size) plus a guard column (5 mm) with the same solvent system described in the paper. With a constant flow rate of 1.2 mL / min, an isocratic gradient of 8.5% B is used for the first 4-minute elution period, followed by 15% B for 0.4 minutes. For the flush period, 97% B was held for 1 minute. The column thermostat is set at 35°C. For the mass spectrometry parameters, the only change from the method described in Amicucci, Galermo et al. 2019 is that the fragmentor voltage is set at 380V. For data analysis, the hydrolysis correction factor is not applied since the samples contain oligosaccharides instead of polysaccharides. In this analysis method, inherently free unpolymerized monosaccharides are calculated by quantifying the concentrations of 14 monosaccharides (glucose, galactose, fructose, xylose, arabinose, fucose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, N- acetylgalactosamine, mannose, allose, ribose) against their individual standard curves. For example, 30% free glucose, as measured by free monosaccharide compositional analysis, means containing 30 g of glucose per 100 g of the sum of all 14 monosaccharides described above.

[0063] Gastrointestinal tract” or “GI tract” means the passageway in the digestive system of a subject (e.g., animal, including humans) that includes all components from the esophagus to the anus (inclusive), as well as everything situated along the passagewayincluding the stomach, intestines, and so forth. Generally, “gastrointestinal tract” is used interchangeably herein with the term “gut.”

[0064] “Glycosidic linkage composition”, “glycosidic linkage analysis”, “permethylated linkage composition analysis” or similar terms, refer to a method described in Galermo, Nandita et al. 2018, incorporated by reference herein in its entirety for all purposes and specifically for a description of such analysis, with some modifications. The permethylation reaction time is 30 minutes. Samples are run on an Agilent 1290 Infinity II UHPLC system coupled to an Agilent 6490A QqQ mass spectrometer. Separation is carried out on an Agilent InfinityLab Poroshell HPH-C18 column (2.1 mm * 100 mm, 1.9 pm particle size) plus a guard column (5 mm) with the same solvent system described in Galermo, Nandita et al.2018. With a constant flow rate of 0.8 mL / min, an isocratic gradient of 14% B is used for the 16-minute elution period, followed by a 2-minute 99% B flush period. The column thermostat is set at 35°C. The glycosidic linkage composition is calculated by integrating the chromatographic peak area of all peaks with the following m / z values: 481.2, 495.2, 509.2, 523.3, 525.2, 537.3, 539.3, 553.3, 567.3, 581.3. For example, 20% 4-galactose, as measured by the permethylated linkage composition analysis, refers to the peak area of 4-galactose being 20% of the sum of the peak area of all linkage peaks with the m / z values listed above.

[0065] “Hydrolytic monosaccharide compositional analysis” means the method described in Amicucci, Galermo et al. 2019, incorporated by reference in its entirety for all purposes, with the following modifications. The hydrolysis reaction to produce monosaccharides is performed at the optimized condition of 100°C for 2 hours. Samples are run on an Agilent 1290 Infinity II ultra-high performance liquid chromatography (UHPLC) system coupled to an Agilent 6490A triple quadrupole (QqQ) mass spectrometer. Separation is carried out on an Agilent InfinityLab Poroshell HPH-C18 column (2.1 mm x 50 mm, 1.9 pm particle size) plus a guard column (5 mm) with the same solvent system described in Amicucci, Galermo et al.2019. With a constant flow rate of 1.2 mL / min, an isocratic gradient of 8.5% B is used for the first 4-minute elution period, followed by 15% B for 0.4 minutes. For the flush period, 97% B is held for 1 minute. The column thermostat is set at 35°C. For the mass spectrometry parameters, the only change from the method described in Amicucci, Galermo et al. 2019 is that the fragmentor voltage is set at 380V. For data analysis, the hydrolysis correction factor is not applied nor needed since the samples contain oligosaccharides instead of polysaccharides. In this analysis method, monosaccharide composition is calculated by quantifying the concentrations of 14 monosaccharides (glucose, galactose, fructose, xylose,arabinose, fucose, rhamnose, glucuronic acid, galacturonic acid, N-acetylglucosamine, N- acetylgalactosamine, mannose, allose, ribose) against their individual standard curves. For example, 30% glucose, as measured by hydrolytic monosaccharide compositional analysis, means containing 30 g of glucose per 100 g of the sum of all 14 monosaccharides described above.

[0066] “Increases abundance of’ or “increasing abundance of’ refers to a biologically relevant increase in the population of a certain bacterial taxa.

[0067] “Lewis base” means a compound or atom that can donate electron pairs (e.g., F’, benzene, H', pyridine, acetonitrile, acetone, urea, etc.).

[0068] “Linkage ratio”, “linkage peak area ratio”, “ratio of linkage” or other similar terms refer to any number of comparisons dependent upon the relationships observed in the glycosidic linkage composition analysis. Peak area for each linkage is calculated on a relative percent basis of the peak area in relationship to the summation of all other linkage peaks areas observed. Peak area ratios are calculated by dividing one contributing linkage by any other linkage of the same monosaccharide within the composition.

[0069] “Microbiota”, “microflora”, “microbiome” and “microbial community” mean a community of living microorganisms that typically inhabits a bodily organ or part, for example the gastro-intestinal organs of complex organisms, such as mammals and humans. In particular, the most dominant members of the gastrointestinal microbiota include microorganisms of the phyla of Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Synergistetes, Verrucomicrobia, Fusobacteria, and Euryarchaeota, at genus level Bacteroides, Faecalibacterium, Bifidobacterium, Roseburia, Alistipes, Collinsella, Blautia, Coprococcus, Ruminococcus, Eubacterium and Dorea, at species level Bacteroides uniformis, Alistipes putredinis, Parabacteroides merdae, Ruminococcus bromii, Dorea longicatena, Bacteroides caccae, Bacteroides thetaiotaomicron, Eubacterium hallii, Ruminococcus torques, Faecalibacterium prausnitzii, Ruminococcus lactaris, Collinsella aerofaciens, Dorea formicigenerans, Bacteroides vulgatus and Roseburia intestinalis . The gastrointestinal microbiota includes the mucosa-associated microbiota, which is located in or attached to the mucous layer covering the epithelium of the gastrointestinal tract, and luminal-associated microbiota, which is found in the lumen of the gastrointestinal tract.

[0070] “Modulate”, “modulating”, “modulation” or other similar terms refer to the ability of a disclosed compound (e.g., polysaccharide composition, or mixture of polysaccharidecleavage products) to alter the amount, degree, or rate of a biological function (including metabolite production), the progression of a disease, or amelioration of a condition. For example, modulating can refer to the ability of a compound to increase or decrease the abundance of a microorganism, increase or decrease production of a metabolite, or elicit a decrease in the inflammation, pain, incidence, or severity of a symptom associated with a particular condition or disease (e.g., associated with the gastrointestinal system). In embodiments, the modulation is a biologically relevant change.

[0071] “Molecular weight analysis” or “SEC -RID” or similar terms, refer to a method in which samples are prepared by reconstituting dried powders into a 10 mg / mL solution in HPLC grade water. Samples are analyzed on an Agilent Infinity II 1260 RID coupled to an Agilent Infinity II 1260 HPLC. Separation is performed on an Agilent AdvanceBio SEC column (7.8 mm x 300 mm, 2.7 um particle size) with a 10 pL injection volume. Chromatographic solvents consist of A: HPLC grade water and B: 95% acetonitrile in water (v / v) with a 50-minuite gradient of 0.0-13.0 min, 0% B; 13.0-14.0 min, 30% B; 14.0-50.0 min 0% B. The flow rate is set to 1.00 mL / min and the column temperature is set at 35 °C. The RID is operated in positive signal polarity mode and a 2.31 Hz peak width. Samples are integrated using Agilent ChemStation data analysis and molecular weights determined using the Agilent Cirrus GPC program along with a set of Dextran standards spanning the resolution of 180-150,000 Da. It will be appreciated by one having skill in the art that SECRID is subject to random, experimental error, and the molecular weights inferred therefrom should therefore be read to encompass reasonable variations from the stated value. Specifically, in some aspects, the molecular weights of polysaccharide compositions described herein include variations of ±20% of the stated molecular weight, or in some aspects, ±10% of the stated molecular weight, or in some aspects, ±5% of the stated molecular weight.

[0072] “Monosaccharide ratio”, “monosaccharide peak area ratio”, “ratio of monosaccharide” or similar terms refer to any number of the comparisons dependent upon the relationships observed in the hydrolytic monosaccharide compositional analysis. Absolute concentrations of each monosaccharide are calculated on a relative percent basis in relation to the summation of all other monosaccharides observed. Monosaccharide ratios are calculated by dividing one contributing monosaccharide by any other monosaccharide within the composition. Monosaccharide ratios are not intended to limit the composition to the listed monosaccharides. For example, a glucose:galactose ratio of 1 : 1 means that there are roughlyequal amounts of glucose subunits and galactose subunits in the composition, but the composition may also comprise mannose subunits, rhamnose subunits, or any other subunit.

[0073] “Nitrogen-based” means a compound that contains at least one nitrogen atom with four substituent groups that can contain any combination of lone pairs of electrons, hydrogens, or carbon atoms (e.g., ammonia, sodium amide, trimethylamine, di ethylamine, N,N-Diisopropylethylamine, urea, pyridine, ammonium hydroxide, ammonium bicarbonate, etc.). Examples of nitrogen-based, peroxide-quenching, polysaccharide-cleavage agents are listed in Table 1. A nitrogen-based reagent may have an unsubstituted or substituted ammonium group and can be present in neutral and / or ionic forms.Table 1. Examples of polysaccharide (PS)-cleavage, and / or peroxide-quenching agents

[0074] “NMR HSQC Analysis”, “1H-13C HSQC NMR”, “HSQC spectra” or other similar terms mean the data generated from two-dimensional spectral analysis of a sample via a Heteronuclear Single Quantum Coherence (HSQC) spin coupling of protons and bonded carbons present in the sample. HSQC experimentation depends on the solvation of samples in a deuterated solvent such as D6-DMSO or D2O. An HSQC spectrum contains a unique peak for each proton attached to the heteronuclear carbon atom being considered, allowing for identification of molecular structure of the analyzed sample. Each experiment is conducted with a Bruker AVANCE 600MHz NMR using heteronuclear single quantum coherence (HSQC) to illustrate the correlation between the 1H and 13C chemical shifts through 1JCH coupling. The resulting FIDs are processed using Bruker TopSpin 4.1.3 and the experimentalchemical shifts are utilized to determine oligosaccharide structures and the anomeric characteristics of the glycosidic bonds with the aid of the CASPER program. Relative ratios between alpha and beta bonds are calculated through examination of the 2D 1H-13C HSQC via examination of signal strength in Hz. These values are then compared to determine the percent abundance of each linkage type among the same carbohydrate. NMR samples are dried via lyophilization, and the resulting material is then dissolved in 0.75mL of dimethyl sulfoxide-d6 (DMS0-d6) with a 0.03% (v / v) TMS internal standard at a concentration of 20mg / mL at a 4.5-6pH range.

[0075] “Non-Arrhenius base” means a compound or atom that can donate electrons (e.g., Lewis Bases), accept protons (e.g., Bronstead-Lowry Bases), or releases hydroxide ions through its decomposition (NH4HCO3), but does not qualify as an Arrhenius base.

[0076] “Oligosaccharide” means an oligomer of monosaccharides, in which the DP of the oligomer is between 2 and 30 monosaccharide units, such as between 3-30, 3-20, 3-15, 3-10, 3-8, 3-6, or 5-15, or any subrange thereof, monosaccharide units. An oligosaccharide can be linear, branched, primarily linear with pendant saccharide monomers, or any combination thereof. An oligosaccharide is an individual oligomer chain.

[0077] “Oligosaccharide composition”, “oligosaccharide pool” or oligosaccharide mixture” means a mixture of two or more oligosaccharides, each of which can be the same or different from one another. Although efforts have been made to consistently use the terms “oligosaccharide” and “oligosaccharide composition” according to their preceding definitions, the intended meaning will be clear from context when such terms are used herein. In embodiments, “one or more oligosaccharides” refers to an oligosaccharide mixture when more than one oligosaccharide is present. In embodiments, “one or more oligosaccharides” refers to one oligosaccharide. In some embodiments, an oligosaccharide composition comprises one or more polysaccharides. In keeping with this aspect, an oligosaccharide composition may comprise up to 60-80% polysaccharides by mass, optionally less than 70% or less than 60% polysaccharides by mass (e.g., between 0.5% and 70% polysaccharides, between 0.5% and 60% polysaccharides, or between 0.5% and 50% polysaccharides). In embodiments, an oligosaccharide composition comprising up to 60-80% polysaccharides has a higher solubility, increased bioactivity, or a combination thereof, as compared to a composition comprising between 80-100% polysaccharides. Mixtures of polysaccharide cleavage products are examples of oligosaccharide compositions.

[0078] “Oligosaccharide analysis” or “oligosaccharide composition analysis” (or similar terms) refer to a HPLC-quadrupole time-of-flight (Q-TOF) method described in Amicucci, Nandita et al. 2020, incorporated by reference in its entirety for all purposes, with some modifications. For sample preparation, oligosaccharides are reduced by incubation with 2.0 M NaBH4 for 1 hour at 65 °C. The oligosaccharides are purified using C-18 cartridge 96- well plates: the plates are washed with 100% ACN, and the oligosaccharides are loaded and eluted with water. The oligosaccharides are subsequently purified using porous graphitized carbon (PGC) 96-well plates: PCG plates are washed with 80% acetonitrile and 0.1% (v / v) TFA in water, and the oligosaccharides from C-18 purification are loaded and washed with water. The oligosaccharides are eluted with 40% acetonitrile with 0.05% (v / v) TFA. Samples are completely dried by evaporative centrifugation and reconstituted for mass spectrometry analysis. Instrumentation is performed on an Agilent 1260 Infinity II HPLC coupled to an Agilent 6530 Q-TOF mass spectrometer. Using the same stationary (plus a 5 mm guard column) and mobile phase as described in Amicucci, Nandita et al. 2020, separation is carried out using the following gradient: 2-15% B, 0-20 minutes; 15-60 % B; 20-45 minutes. The column thermostat is set at 35°C. The fragmentor voltage is set at 75V. In this method, the “oligosaccharide weight %” (or “oligo wt%” or such terms) is calculated by dividing the chromatographic peak area of a particular oligosaccharide by the total peak area of all oligosaccharides identified in that sample during the defined chromatographic period. Generally, when an oligosaccharide composition is described to contain a specified weight percent of oligosaccharides on a dry basis having a degree of polymerization of a specified number (e.g., at least 50 wt% oligosaccharides on a dry basis having a degree of polymerization of between 3 and 50 monosaccharide subunits), such values can be calculated with the aid of the oligosaccharide analysis described above; however, other methods can also aid this determination, such as size exclusion chromatography using a universal detector, or other methods known in the art.

[0079] “Organoleptic” means a quality (such as taste, color, odor, and feel) of a substance that stimulates the sense organs. Organoleptic properties include among others taste (sweetness, sourness, bitterness, saltiness, umami or savory), mouthfeel or texture (the way a food or drink feels when put in the mouth and can include among others hardness / softness, chewy, gummy, slimy, gritty / grainy, crisp, and crunchy), smell or color. Organoleptic properties of a food or beverage are often assessed by expert taste testers or panels of consumers.

[0080] Other minor linkages” means the sum of linkages which are either not entirely annotated or constitute less than 2% of any samples. Therefore, the contributions of these linkages to the sample glycosidic linkage composition are summed into the “other minor linkages” category.

[0081] “Pectin” means a heteropolysaccharide found in the primary lamella, in the middle lamella, and in the cell walls of plants. Pectin is rich in galacturonic acid but may take different forms such as: 1) homogalacturonans which are linear chains of a-(l-4)-linked D- galacturonic acid; 2) branched galacturonans in which the D-galacturonic acid residues in the a-(l-4)-linked chains have branches, such as for example D-xylose branches from a backbone of D-galacturonic acid residues; 3) Rhamnogal acturonan I pectins (RG-I) which have a backbone of the repeating disaccharide: 4)-a-D-galacturonic acid-(l,2)-a-L-rhamnose- (1), often with sidechains of various neutral sugars branching off from many of the rhamnose residues; and 4) rhamnogal acturonan II pectins (RG-II) which are complex, highly branched polysaccharides in which the backbone is made primarily of D-galacturonic acid units. In embodiments, pectins comprise more than one of the forms. In embodiments, pectins comprise all the forms.

[0082] “Peeling reaction” or “peeling” means the sequential alkaline degradation of carbohydrates through a mechanism that releases monomeric units from the reducing end of the polymer.

[0083] “Peroxide agent” means a compound that contain oxygen-oxygen bonds that can produce, natively, with light, temperature, or catalyst (e.g., metals and enzymes), R-O' and / or R-O-O' species, where “R” refers to a hydrogen or carbon group that is attached to the rest of the molecule. In one aspect, a peroxide agent is hydrogen peroxide.

[0084] “Peroxide quenching reagent” means a compound or atom which is not a strong- Arrhenius base, and that can convert hydrogen peroxide, peroxyl radicals, and hydroperoxyl radicals to a less reactive or non-reactive state (e.g., ammonium hydroxide, ammonium bicarbonate, ammonia, etc.). In certain aspects, a peroxide quenching reagent converts hydrogen peroxide as well as radicals produced from hydrogen peroxide to less reactive species (e.g. water). In certain aspects, a peroxide quenching reagent may reduce the hydrogen peroxide concentration to zero, below 5 mg / L, below 10 mg / L, below 25 mg / L, or below 50 mg / L. In certain aspects, a peroxide quenching reagent may form water, hydroxide ions, or oxygen gas. In certain aspects, the peroxide quenching reagent may be an enzyme,for example, a catalase. The enzyme can be of microbial origin, from recombinant origin, or from animal origin, for example from bovine liver. In certain aspects, different enzymes may be mixed to quench the peroxide species.

[0085] “Polysaccharide” means a polymer of monosaccharide units having greater than 30 monosaccharide units, optionally up to hundreds of thousands of monosaccharides in length, or a material comprising such a polymer. The polysaccharide can be linked to other non-carbohydrate moieties (e.g., glycoproteins, proteoglycans, glycopeptides, glycolipids, glycoconjugates, glycosides, or any combination thereof). The polysaccharide can be a linear polymer, branched polymer, primarily linear polymer with pendant saccharide monomers, or any combination thereof.

[0086] “Polysaccharide composition”, “polysaccharide pool” or “polysaccharide mixture” means a mixture of two or more polysaccharides, each of which can be the same or different from one another. Although efforts have been made to consistently use the terms “polysaccharide” and “polysaccharide composition” according to their preceding definitions, the intended meaning will be clear from context when such terms are used herein. In embodiments, “one or more polysaccharides” refers to a polysaccharide mixture when more than one polysaccharide is present. In embodiments, “one or more polysaccharides” refers to one polysaccharide. In some aspects, polysaccharide composition refers to a composition comprising, or consisting of, polysaccharide cleavage products. In methods herein polysaccharides are obtained from pomegranate.

[0087] “Polysaccharide cleavage product” is a product formed from the chemical and / or enzymatic cleavage of a polysaccharide. In some aspects, the polysaccharide cleavage product comprises one or more oligosaccharides. In some aspects, the polysaccharide cleavage product comprises one or more polysaccharides. In some aspects, the polysaccharide cleavage product comprises a mixture of one or more oligosaccharides and one or more polysaccharides. In methods herein polysaccharide cleavage products are generated from polysaccharide containing material obtained from pomegranate. In some aspects, the polysaccharide cleavage product comprises 5% or more oligosaccharides by weight, such as 5% or more, 10% or more, 15% or more, or 20% or more oligosaccharides by weight. In some aspects, the polysaccharide cleavage product comprises 10% or more oligosaccharides by weight, such as 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more oligosaccharides by weight.

[0088] “Pomegranate" means any part of the fruit of the plant Punica granatum and any cultivar thereof Cultivars of interest include, among others, ‘Acco,’ ‘Eversweet,’ ‘Granada,’ ‘Foothill,’ ‘Kazake,’ ‘Laffan,’ ‘Pinl Satin,’ ‘Purple Heart,’ ‘Red Silk,’ ‘Salavatski,’ ‘Surh Anor,’ ‘Texas Pink,’ ‘Tom’s Red,’ and ‘Wonderful.’

[0089] “Pomegranate residue” means by-products of processing of the pomegranate fruit. The residue can be generated by any processing of the fruit, such as roasting, fermentation, or various extraction processes so long as some polysaccharide, and in embodiments pectin, remains. In particular, pomegranate residue is generated as the by-product of juicing. Pomegranate residue can contain any portion of the fruit or a combination of such portions. Non-limiting examples include the husk which has a hard outer rind or exocarp or peel and a spongy mesocarp with attached sarcotestas membranes containing arils, arils containing seeds, pomace, and the like. Pomace can be the residue of juicing or other processing of the arils of the fruit or of juicing any portion of the fruit, combination thereof, or the whole fruit. Pomegranate residue generally contains one or more polysaccharides unless treated to release or extract a given polysaccharide. In specific embodiments, pomegranate residue contains pectin. Pomegranate residue can also contain in addition to polysaccharides, polyphenols, lignin, protein, or lipids, among others. Pomegranate residue is optionally dried (e.g., air dried, oven dried or freeze-dried), chopped and / or processed into a powder and / or formed into pellets. Particles of pomegranate residue can be ground or otherwise processed to have a selected size-range. In embodiments, pomegranate residue provides a polysaccharide containing material obtained from pomegranate.

[0090] “Pomegranate starting material containing polysaccharide” is the starting material used in processes herein and can comprise, consist essentially of or consist of any pomegranate residue. In embodiments, pomegranate starting material is a polysaccharide containing material obtained from pomegranate. In embodiments, pomegranate starting material containing polysaccharide comprises, consists essentially of or consists of polysaccharide. In the methods herein a pomegranate starting material containing polysaccharide is treated to generate polysaccharide cleavage products. More generally the pomegranate starting material containing polysaccharide is treated to depolymerize at least a portion of the polysaccharides therein. The pomegranate starting material of this method can be any form of pomegranate residue and more specifically can be pomegranate pomace or can be pomegranate peel. In embodiments, pomegranate starting material comprises, consistsof or consists essentially of pectin. Pomegranate starting material can also, in addition to polysaccharide, contain, polyphenols, lignin, protein, or lipids, among others.

[0091] Pomegranate starting material can be treated, for example, to reduce the level of polyphenols therein providing a “polyphenol-reduced pomegranate starting material.” In embodiments, the level of polyphenol in the “polyphenol-reduced pomegranate starting material” is reduced by 50% or more by weight compared to pomegranate starting material. In embodiments, the level of polyphenol in the “polyphenol-reduced pomegranate starting material” is reduced by 80% or more by weight compared to pomegranate starting material. In embodiments, the polyphenol level in polyphenol-reduced pomegranate starting material is 15% or less by weight. In embodiments, the polyphenol level in polyphenol-reduced pomegranate starting material is 10% or less by weight. In embodiments, the polyphenol level in polyphenol-reduced pomegranate starting material is 5% or less by weight. Use of the polyphenol-reduced pomegranate starting material in Fenton-based depolymerization methods herein has been found to result in higher yields of polysaccharide cleavage products.

[0092] Pomegranate starting material can be treated to enhance the level of one or more polysaccharides therein. Pomegranate starting material can be treated to extract one or more polysaccharides and the extracted one or more polysaccharides can then be employed as a “polysaccharide-enriched pomegranate starting material.” The level of polysaccharides in the polysaccharide-enriched pomegranate starting material is generally higher than in pomegranate starting material containing polysaccharides. In embodiments, the level of polysaccharides is enhanced by 10% or more by weight compared to pomegranate starting material. In embodiments, the level of polysaccharides is enhanced by 25% or more by weight compared to pomegranate starting material. In embodiments, the level of polysaccharides is enhanced by 50% or more by weight compared to pomegranate starting material. In specific embodiments the polysaccharide that is extracted is pectin and a pectin- enriched pomegranate starting material is provided.

[0093] The amount of polysaccharide and its composition, as well as, the properties of the polysaccharides, including average molecular weight and DP and extent and kind of esterification, in the pomegranate starting material as well as the concentration of other components therein, will vary depending upon the source pomegranate, type of processing, components of the residue, among other variables. In embodiments, the pomegranate starting material contains at least 1% by weight (dry weight) polysaccharides and preferably contains 5%, 7% or 10% or more of polysaccharides. For example, the outer peel of certain cultivarsof pomegranate can contain up to 13% by weight (dry weight) of polysaccharide. Pomegranate polysaccharide can contain pectin, starch, cellulose, galactomannan, xylan, arabinoxylan, xyloglucan and other types of polysaccharide. Polysaccharides in the pomegranate starting material can be esterified, for example by methyl or acetyl groups. The degree of esterification of polysaccharide will vary dependent, at least, upon the source of pomegranate and the pomegranate components in the pomegranate residue.

[0094] Pomegranate starting material is optionally pre-treated, as described herein, prior to treatment with Fenton’s reagent. Pre-treatment can include, among others, removal or extraction of polyphenols; removal of starch, removal of cellulose, removal of lignin, removal of protein, removal of lipids, precipitation steps, and various filtration steps. Pretreatment, can include various extractions (e.g., with organic solvent, with acidic organic solvent, with aqueous acid), various enzyme treatments or combinations of such treatments (e.g., with one or more protease, with one or more endo- or exo-peptidase, with one or more amylase (including isoamylase), with one or more cellulase, with one or more glucanase, with one or more pectinase, with one or more xylanases, with one or more mannanases, or with one or more arabinanses, with one or more disaccharidases (e.g., maltase, isomaltase, lactase, or sucrose)). Pre-treatment can also include deesterification of polysaccharide (e.g., by chemical or enzymatic deesterification). In embodiments, pre-treatment includes a step of removal of polyphenols, particularly by extraction with organic solvent and more specifically by extraction with acidic alcohol. In embodiments, pre-treatment includes a step of extraction of polysaccharides, particularly pectin, from polysaccharide containing material. In embodiments, polysaccharide is extracted from polysaccharide containing material using extraction with an acidic aqueous solution. In embodiments, such polysaccharide extraction can function to separate polysaccharide from protein, lignin, and lipid. In embodiments, pretreatment, includes a step of extraction of polyphenols and a step of extraction of polysaccharides from polysaccharide containing material obtained from pomegranate. In embodiments, pre-treatment does not include pre-treatment with a pectinase. When used with reference to a pre-treatment step the terms “remove” or “removing” refer to extraction and / or enzymatic degradation of a given component (e.g., polyphenols, starch, etc.) and include any measurable decrease in the given component. It will be appreciated that in embodiments, it is preferred to obtain a significant reduction in the component, for example a reduction of 10%, 25% or 50% or more of the component. In some embodiments, the component can be reduced to a non-detectible level. In some embodiments, the component is reduced to a levelthat such that yield of the depolymerization reaction is increased. In some embodiments, the level of polyphenol in the polysaccharide starting material is reduced to a level that such that yield of the depolymerization reaction is increased.

[0095] “Prebiotic” means a selectively fermented composition that results in specific changes in the composition and / or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health. In some aspects, a prebiotic is generally a non-digestible or partially-digestible (i.e., digestible by the subject / human / animal, and does not include digestion by microbes) food ingredient that beneficially affects a host when ingested by selectively stimulating the growth and / or the activity of one or a limited number of microbes in the gastrointestinal tract or other portion of the host. As used herein, the term “prebiotic” refers to the above described non-digestible or partially-digestible food ingredients in their non-naturally occurring states, e.g., after purification, chemical or enzymatic synthesis as opposed to, for instance, in whole human milk. The invention provides prebiotic compositions comprising mixtures of polysaccharide cleavage products generated from polysaccharide containing materials obtained from pomegranate.

[0096] “Purified” means a molecule or composition which is at least partially or substantially purified. A substantially purified molecule or composition has a purity of 95%, optionally for some applications 99%, optionally for some applications 99.9%, optionally for some applications 99.99%, and optionally for some applications 99.999% pure. The terms “purified” and “isolated” are used interchangeably. In embodiments, the mixture of polysaccharide cleavage products is purified to remove ash, unreacted, (e.g., insoluble polysaccharide) or residual non-polysaccharide components, disaccharides or monosaccharides or salts remaining from depolymerization reactions.

[0097] “Reaction mixture” means a mixture comprising reagents which may react chemically to form products which are distinct from the reagents.

[0098] The term “remove,” or “removing” relates to reducing or reduction of a component in a material.

[0099] "Reducing" or any variation of the term such as “reduction” means any measurable decrease to achieve a desired effect. In aspects, “reducing” refers to a biologically relevant decrease as defined herein.

[0100] “Retention factor” means the ratio obtained by dividing the retention time of a given peak observed in an oligosaccharide analysis (e.g., HPLC spectrum) by the first oligosaccharide peak (i.e., the lowest retention time) observed in the oligosaccharide analysis.

[0101] Short chain fatty acid” or “SCFA” includes one or more of butyrate, propionate, beta-hydroxybutyrate, lactate, or acetate.

[0102] “Specified reaction time” or “reaction time” means the time for a reaction to proceed toward an equilibrium state between reagents added and products produced by the reaction of the reagents. In certain aspects, specified reaction time allows sufficient time to reach an equilibrium. In certain other aspects, specified reaction time, while allowing time for the reaction to proceed toward equilibrium, does not provide the time needed to reach equilibrium.

[0103] “Strong- Arrhenius base” means a compound that completely dissociates in water to release one or more hydroxide ions into solution. Examples of a “strong- Arrhenius base” are KOH, NaOH, Ba(OH)2, CsOH, Sr(OH)2, Ca(OH)2, LiOH, and RbOH.

[0104] “Subject” or “patient” generally refer to a living organism suffering from or prone to a disease or condition that can be treated by administration of a compound or pharmaceutical composition, as provided herein. Non-limiting examples include humans, other mammals, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other nonmammalian animals. In some embodiment, a subject is an animal. In some embodiments, a subject is human. In those aspects where the subject is a human, the subject can be a pediatric or adult subject. In some embodiments, a subject is a mammal. In some embodiments, a subject is a mouse. In some embodiments, a subject is an experimental animal. In some embodiments, a subject is a rat. In some embodiments, a subject is a test animal.

[0105] “Substantially commensurate with initiation of peroxide-quenching” means the relationship between the timing of a cleavage reaction and the timing of a peroxide quenching reaction indicating that the initiation of the cleavage reaction and the initiation of the peroxide quenching reaction occur within a short time duration of each other (e.g. on the order of seconds, or on the order of minutes but not more than one day).

[0106] Subunit” (sometimes referred to herein as “unit”) means a species that is covalently bonded to or within an oligomer (e.g., oligosaccharide) or polymer (e.g., polysaccharide). Such species generally can include saccharides (e.g., glucose, galactose, mannose, etc.). For example, when a polysaccharide composition comprises a glucosesubunit, it means that the composition comprises a glucose molecule that is bound to or within a polymer. Therefore, a composition that contains only free monomeric glucose would not contain a glucose subunit. Similarly, when a polysaccharide composition comprises a sum of glucose, galactose, and mannose subunits in an amount of at least 60 wt% based on total weight of saccharide subunits, this means that the mass of all of the glucose subunits, galactose subunits, and mannose subunits are summed, and the subunits of all saccharides are summed, and then the first sum is divided by the second sum. Additionally, when a polysaccharide composition comprises non-terminal galactose subunits, and at least 70 wt% of the non-terminal galactose subunits are specified to have at least one 4-linkage, this feature is calculated by summing the mass of all non-terminal galactose subunits having at least one 4-linkage (and this can include, for example, galactose subunits with 4,6-linkages and 4,3- linkages), and then dividing by the total mass of non-terminal galactose subunits regardless of linkage type. The same concept is applicable to any feature where reference to “at least one X-linkage,” in which X is an integer (e.g., such as “a weight ratio of glucose subunits having at least one 4-linkage to glucose subunits having at least one 3-linkage is between 2: 1 to 4: 1” and other such features). Moreover, in such calculations the actual mass of the subunit is used (i.e., in bound form) rather than the mass of the unit as if it was hydrolyzed (which would add the mass of water). Other features can be calculated similarly. These features can be determined with the aid of the various analytical techniques, such as hydrolytic monosaccharide compositional analysis, oligosaccharide analysis, glycosidic linkage analysis, NMR HSQC Analysis, and so forth, as well as other techniques known in the art.

[0107] “Synthetic composition” means a composition which is artificially prepared and preferably means a composition containing at least one compound that is produced ex vivo chemically and / or biologically, e.g., by means of chemical reaction, enzymatic reaction, recombinantly, or any combination thereof. The synthetic composition typically comprises one or more compounds, including one or more of the polysaccharides described herein. In some aspects, the polysaccharides and polysaccharide compositions can be formulated into a synthetic composition or administered as the polysaccharide alone or the polysaccharide cleavage product alone.

[0108] Treated polysaccharide” means a polysaccharide which has been contacted with at least one reagent capable of reacting with the polysaccharides (e.g. an enzyme or a Fenton’s reagent).

[0109] “Weak- Arrhenius base” means a compound that incompletely dissociates in water to release one or more hydroxide ions into solution, e.g. ammonium hydroxide, H2O, etc. As“weak-Arrhenius base” is used herein, there are no compounds which meet both the definitions used of strong-Arrhenius base and weak-Arrhenius base.

[0110] When the amount of a component is expressed in terms of weight or mole percent, it is intended that the amount is on a dry basis unless otherwise specified. “Dry basis” means in the absence of water or other solvent. For example, when a composition comprises 10 g of glucose, 40 g of xylose, and 50 g of water, it means the composition comprises 25% (mass% or wt.%) glucose on a dry basis, but the glucose is present in the composition at a concentration of 10% (mass% or wt.%).

[0111] Any viscosity measurement or property reported employs water as the solvent, unless specified otherwise.DETAILED DESCRIPTION OF THE INVENTION

[0112] In the following description, numerous specific details of the method of the invention are set forth to provide a thorough explanation of the precise nature of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without these specific details. Although the following description is divided into sections, it is contemplated that each section contains various aspects of the invention and as such invention from within each section and across two or more sections can be combined to form any aspect of the invention. When a table, spectrum, or other data is referred to as representing the features or properties possessed by a particular composition, polysaccharide, or other compound or mixture, unless specified otherwise, the same analysis method and procedure used to obtain the table, spectrum, or other data is to be used to determine the properties of the particular composition, polysaccharide, or other compound or mixture.

[0113] It has been surprisingly found that a dietary fiber suitable for use in foods and beverages can be produced from pomegranate residue by (i) reacting polysaccharide in the pomegranate residue with a Fenton’s reagent comprising a peroxide agent and a metal, and (ii) cleaving the reacted polysaccharide with a cleavage agent to generate a mixture of polysaccharide cleavage products. The cleavage products have improved solubility, improved organoleptics and improved prebiotic properties, while retaining the basic monomer structure of the polysaccharide. The method thus valorizes a major component of pomegranate waste.

[0114] In one aspect, the polysaccharide cleavage products can be produced as broadly disclosed in WO 2018 / 236917, hereby incorporated by reference herein in its entirety for all purposes. This method comprises reacting a pomegranate polysaccharide using a Fenton’s reagent composed of iron (Fe+, Fe2+) or other transition metal (including but not limited to, Cu1+, Co2+, etc.,) and hydrogen peroxide. The reaction can be allowed to proceed, for example, between 10 minutes and 24 hours, more specifically 15 minutes to 4 hours or 30 minutes to 3 hour. The transition metal or alkaline earth metal in the reaction mixture can, for example, be at a concentration of 10 pM to 5M, but generally at a concentration of more than 0.65 mM. If desired, prior to the Fenton reaction, the polysaccharides can be contacted with one or more polysaccharide degrading enzymes, such as an amylase, isoamylase, cellulase, maltase, glucanase, or a combination thereof. If desired, prior to the Fenton reaction, the polysaccharides can be pre-treated as described herein, such as to extract polyphenols or extract polysaccharides. The reaction is subsequently quenched with base (e.g., an Arrhenius base or non- Arrhenius base, such as aqueous sodium hydroxide calcium hydroxide, potassium hydroxide, etc., or any combination thereof).

[0115] In another aspect, the polysaccharide cleavage products can be produced as broadly disclosed in in WO 2021 / 097138, hereby incorporated by reference in its entirety for all purposes. This method comprises a multi-step reaction that includes reacting the pomegranate polysaccharide with a Fenton’s reagent and a then a peroxide- quenching / polysaccharide-cleavage step using either: a polysaccharide-cleavage agent that also functions as a peroxide-quenching agent; or using a polysaccharide-cleavage agent in combination with a compatible peroxide-quenching reagent that does not interfere with the polysaccharide-cleavage reaction. The polysaccharide-cleavage agent may be, for example, a weak-Arrhenius base or non- Arrhenius base. The polysaccharide-cleavage agent preferably also functions as a peroxide-quencher to quench (e.g., sufficiently reduce or eliminate) residual hydrogen peroxide and / or radicals thereof to minimize or eliminate off-target side reactions. If desired, prior to the Fenton reaction, the polysaccharides can be contacted with one or more polysaccharide degrading enzymes, such as an amylase, isoamylase, cellulase, maltase, glucanase, or a combination thereof. If desired, prior to the Fenton reaction, the polysaccharides can be pre-treated as described herein, such as to extract polyphenols or extract polysaccharides. In a specific embodiment, the method, for example, comprises reacting the pomegranate polysaccharide with hydrogen peroxide and a suitable metal or metal ion (e.g., a transition metal, alkaline earth metal, or lanthanide, such as, for example,Fe(II), Fe(III), Cu(I), Cu(II), Ca(II), Mg(II), Mn(II), Zn(II), Ni(II), Ce(IV), Co(II) or other metal ions, or any combination thereof), followed by cleaving the glycosidic linkages in the hydroperoxyl-treated polysaccharides with a high-yield peroxide-quenching / cleavage agent such as ammonium bicarbonate, ammonium hydroxide, ammonia, urea, sodium amide, other ammonium-based reagent, a weak Arrhenius base, a non-Arrhenius base, a Lewis base, a Bronsted-Lowry base, or any combination thereof. This generates high yields of polysaccharide cleavage products from the starting pomegranate polysaccharide, while reducing or eliminating peeling and unwanted side-reactions.

[0116] The polysaccharide-cleavage reagent used in the methods herein can comprise at least one reagent selected from ammonium hydroxide, ammonia, ammonium bicarbonate, urea, etc., or a combination thereof. In some aspects, the polysaccharide-cleavage reagent may comprise the conjugate base of an alcohol or amine. In some aspects, the polysaccharide-cleavage reagent may comprise sodium methoxide, sodium ethoxide, sodium tert-butoxide, or other deprotonated alcohol. In some aspects, the polysaccharide-cleavage reagent may be or comprise one or more relatively “bulky bases” such as tert-butoxide, triethylamine, or other sterically hindered base. In some aspects, the use of such bulky cleavage reagents / bases results in selective cleavage of the accessible glycosidic bonds to provide oligosaccharide profiles unique / specific to the cleavage reagent / base. In some aspects the polysaccharide-cleavage reagent is not a base, / ?er se, but consists of, or comprises one or more reactive agent(s) that react to produce basic conditions and / or decomposition products. In all the methods described herein, the polysaccharide-cleavage reagent (cleavage initiator) can also be, and preferably is, a peroxide-quenching reagent, and in either case may be used in combination with an additional compatible peroxide-quenching agent that may or may not also be a polysaccharide-cleavage agent.

[0117] The use of weak Arrhenius bases and / or non-Arrhenius bases (e.g., ammonium- based peroxide-quenching / polysaccharide-cleavage reagents, etc.) as polysaccharide- cleavage agents not only provides for improved high-yield polysaccharide cleavage product production (relative to the strong Arrhenius bases) but also eliminates the need for costly and time-consuming post-reaction concentration, and desalting steps.

[0118] The cleavage initiator also can, and preferably does, function as a peroxidequencher to quench (sufficiently reduce or eliminate) residual peroxide and / or radicals thereof to reduce or eliminate peeling and unwanted side-reactions. Alternatively, the cleavage agent can be added to the reaction after, or along with addition of a compatibleperoxide-quenching agent (that could also be a cleavage reagent). The peroxide- quenching / cleavage agent can be, and preferably is, selected from one or more nitrogen-based agents as described above. This not only provides high-yield cleavage and residual peroxide- quenching, but also provides for cleavage specificity tailoring (e.g., by replacing nitrogen bound hydrogen with larger moieties to sterically hinder or otherwise modify access by, or activity of the cleavage agent).

[0119] The transition metal or alkaline earth metal in the reaction mixture is at a concentration of at least about 10 pM, for example about 10 pM to about 20 mM. In some aspects, the concentration is at least about 0.65 mM (e.g. at least a value in the range of 0.5 to 0.7 mM). In some aspects, the transition metal or alkaline earth metal in the reaction mixture is at a concentration from 0.65 mM to 500 mM. In some aspects, the peroxide agent (e.g., hydrogen peroxide) in the reaction mixture is at a concentration of at least about 0.02 M (e.g. at least a value in the range of 0.015 to 0.025 M). In some aspects, the peroxide agent (e.g., hydrogen peroxide) in the reaction mixture is at a concentration of from 0.02 M to 1 M, or in some aspects up to 5 M. In some aspects, the peroxide agent (e.g., hydrogen peroxide) in the reaction mixture is at a concentration of from 1 M to 5 M. In some aspects, the cleavage reagent / base is or comprises ammonium hydroxide, ammonia, ammonium bicarbonate, a weak Arrhenius base, a non-Arrhenius base, a Lewis base, and / or a Bronsted-Lowry base. Moreover, combinations of two or more cleavage reagents / bases (e.g., such as the cleavage reagents / bases discussed herein) may be used. In some aspects, strong-Arrhenius bases (e.g., Na+OH', K+0H', or Ca+2(OH )2) can be used in combination with the cleavage reagents / bases discussed herein. In some aspects, ammonia gas can be in contact with the solution through bubbling or as an atmospheric component to act as a cleavage and / or quenching reagent. In some aspects, the cleavage reagent is at a concentration of at least about 0.1 M (+ / - 20%). In some aspects, the cleavage reagent is at a concentration of from 0.1 M-5.0 M. In some aspects the cleavage reagent is present as a saturated solution or insoluble material. In some aspects the cleavage reagent brings the solution to pH 7.5, 8, 9, 10, 12, or higher. In all the methods described herein, the cleavage reagent (cleavage initiator) may also be, and preferably is a peroxide-quenching reagent, and in either case may be used in combination with an additional compatible peroxide-quenching agent that may or may not also be a cleavage agent.

[0120] In some aspects, the pomegranate polysaccharides first undergo initial oxidative treatment with the hydrogen peroxide and a transition metal, alkaline earth metal, orlanthanide catalyst to render the glycosidic linkages more labile. Ammonium hydroxide, ammonium bicarbonate, ammonia, urea, etc., or other weak Arrhenius or non-Arrhenius base is then used for cleavage, which results in a variety of distinctive oligosaccharides (distinctive oligosaccharide profile), or smaller polysaccharides. In some aspects, peroxide-quenching and / or neutralization takes place immediately to reduce unwanted oxidation, or peeling, respectively. In some aspects the treated polysaccharide (e.g., the polysaccharide comprising starting material after treatment with a Fenton’s reagent) is allowed to react with the cleavage reagent at reduced, ambient, or room temperature to facilitate the production of oligosaccharides. In some aspects the cleavage reaction takes place at 4-100°C, 20-80°C, 30- 60°C or about 40°C. In some aspects, cleavage and peroxide-quenching are immediate. In some aspects the cleavage step is conducted for 10-30 minutes, 20-60 minutes, or 60-360 minutes. In some aspects the cleavage step is conducted for 2-6 hours, 3-12 hours, 6-24 hours or longer. In some aspects the cleavage step is conducted overnight. In all the methods described herein, the cleavage reagent (cleavage initiator) may also be, and preferably is a peroxide-quenching reagent, and in either case may be used in combination with an additional compatible peroxide-quenching agent that may or may not also be a cleavage agent.

[0121] In some aspects, the method of cleaving polysaccharides comprises multiple steps. For instance, the method can comprise: a) contacting one or more polysaccharide with a Fenton’s reagent, comprising a peroxide agent and metal ions to form a mixture; b) allowing the Fenton’s reagent to react with the polysaccharide for a specified reaction time; and c) after step b, adding a cleavage agent which may also be a peroxide quenching reagent to the mixture. In such aspect, the steps of contacting the polysaccharide with a Fenton’s reagent (step a) and allowing a specified reaction time to pass (step b) can be performed at the same or different pH wherein the pH is selected from within a range of pH 3 to 8, pH 4 to 7, pH 4.5 to 6.5, and pH 5 to 6. The pH can be any possible value between the specified ranges of pH values. The step of adding a cleavage agent which may also be a peroxide quenching reagent (step c) can be performed at a pH selected from within a range of pH 6 to 11, pH 6.5 to 9.5, pH 7 to 9, and pH 7 to 8. The pH can be any possible value between the specified ranges of pH values. In such aspect, the step of contacting the polysaccharide with a Fenton’s reagent (step a) and passage of the specified reaction time (step b) can be performed at the same or different temperature wherein the temperature is selected from within a range of temperature between 10 and 70 degrees Celsius, between 20 and 60° Celsius, and between 25 and 55°Celsius. The temperature can be any possible value between the specified ranges of temperature values. The step of adding a cleavage agent which may also be a peroxide quenching reagent (step c) can be performed at a temperature selected from within a range of temperature between 10 and 70 degrees Celsius, between 20 and 60 degrees Celsius, and between 25 and 55 degrees Celsius. The temperature can be any possible value between the specified ranges of temperature values.

[0122] In some aspects, if desired, the polysaccharide source material can optionally be treated with one or more polysaccharide-degrading enzyme(s) to reduce the average size or complexity of the polysaccharide before the resulting polysaccharides are treated with the Fenton’s reagent. Non-limiting examples of polysaccharide enzymes that can be used include for example, amylase, isoamylase, cellulase, maltase, glucanase, lactase, xylanase, arabinase, pectinase, mannanase, or a combination thereof.

[0123] In some aspects, the pomegranate polysaccharides first undergo initial oxidative treatment with hydrogen peroxide and a transition metal or alkaline earth metal (e.g., iron(III) sulfate) catalyst to render the glycosidic linkages more labile. A weak-Arrhenius base or nonArrhenius base is then used for base induced cleavage, which results in a variety of oligosaccharides. Immediate neutralization may take place to reduce any peeling reaction. This method can generate large amounts of biologically active oligosaccharides from a variety of pomegranate sources. The initial oxidative treatment can include hydrogen peroxide and a transition metal or an alkaline earth metal. Metals with different oxidation states, sizes, periodic groups, and coordination numbers have been tested and each of the different metals has shown activity in the reaction. The oxidative treatment is followed by a base treatment.

[0124] Prior to reacting the polysaccharide from the polysaccharide containing material with the Fenton’s reagent, the polysaccharide can be purified from the pomegranate containing material, i.e., the pomegranate polysaccharide containing material can be pretreated. In embodiments, the polysaccharide can be enriched in the pomegranate material by removing one or more of polyphenols, lignin, lipids, proteins, starch, salts, monosaccharides, and di saccharides. This can be done using various methods known for purification of polysaccharides from polysaccharide containing material. Polyphenols and lipids can be removed by any suitable extraction technology. Conventional batch extraction or continuous extraction, as well as extraction employing ultrasound or microwave assistance, as understood in the art, can be employed to enhance extraction efficiency. In embodiments, thecontent of polyphenols can be reduced by extraction with a suitable organic solvent (e.g., alcohols (e.g., methanol, ethanol, or isopropanol), ketones (e.g., acetone), esters (e.g., ethyl acetate) or a mixture of organic solvents (e.g., chloroform / methanol). Polyphenols can also be extracted with acidified organic solvent (e.g., acidified alcohol). Methods useful for extraction of polyphenols can also be employed to extract lipids. In embodiments, supercritical fluid extraction, as known in the art, can be used to extract polyphenols as well as lipids.

[0125] In embodiments, the content of polyphenols as well as that of lipids can be reduced by extraction with an acidified solvent comprising a solvent and an acidifying agent. In embodiments, the solvent is an organic solvent rather than an aqueous solvent. In embodiments, the solvent can be any solvent suitable for removing polyphenols. In embodiments, the solvent is preferably one that is suitable for processing foodstuffs, for example ethanol. The acidifying agent can be any suitable acidifying agent, for example a mineral or organic acid. The mineral acid can, for example, include sulfuric or hydrochloric acid. The organic acid can be a carboxylic acid, and more specifically can be a mono-, di- or tricarboxylic acid. In embodiments, the organic acid can be acetic acid, propionic acid or butyric acid. In embodiments, the organic acid can be malonic, succinic glutaric or adipic acid. In embodiments, the organic acid can be citric acid, lactic acid, malic acid, or propane- 1,2, 3 -tricarboxylic acid. In embodiments, the acidifying agent can be a salt of an organic acid.

[0126] In embodiments, the organic solvent can contain about 0.005% to about 1% wt / v of the acidifying agent, for example about 0.01% to about 1% wt / v of the acidifying agent or any subrange thereof. In embodiments, the amount of acidifying agent ranges from 0.01% to 0.5% wt / v. In embodiments, the amount of acidifying agent ranges from 0.01% to 0.2% wt / v. In embodiments, the amount of acidifying agent ranges from 0.01% to 0.05% wt / v. In embodiments, the amount of acidifying agent ranges from 0.01% to 0.02% wt / v. In embodiments, the amount of acidifying agent ranges from 0.05% to 0.5% wt / v. In embodiments, the amount of acidifying agent ranges from 0.05% to 0.2% wt / v. In embodiments, the amount of acidifying agent ranges from 0.05% to 0.1% wt / v. In embodiments, the amount of acidifying agent ranges from 0.1% to 0.2% wt / v. In embodiments, the amount of acidifying agent ranges from 0.1% to 0.5% wt / v. In embodiments, the amount of acidifying agent ranges from 0.1% to 1% wt / v. In embodiments, the amount of acidifying agent ranges from 0.5% to 1% wt / v. In embodiments, the amount ofacidifying agent is 0.013%-0.017 wt / v. In embodiments, the amount of acidifying agent ranges from 0.85 to 1% wt / v. The foregoing embodiments include all subranges of the listed ranges.

[0127] In an embodiment, the extraction can take place at a temperature of ambient room temperature (e.g., 25°C) to 90°C, for example at 35°C to 80°C or at room temperature, or any subrange thereof. It will be appreciated that the temperature at which the extraction is conducted will depend upon the volatility of the solvent employed. In an embodiment, the extraction can take 1 hour to 5 hours, for example, 2 hours to 4 hours, or any subrange thereof. In an embodiment, the ratio (v / w) of acidified solvent to pomegranate material can be 15: 1 to 1 : 1, or any subrange thereof, for example, 10: 1 to 4: 1. After the extraction, the polyphenols and / or lipids can be removed with the liquid phase by filtration; for example, using centrifugation. Extraction can optionally be repeated multiple times. Extraction is optionally accompanied by one or more steps of washing the extracted solid with acidified solvent or solvent.

[0128] In one aspect, the polysaccharide can be enriched by removing the protein and starches from the pomegranate material. This can be achieved by utilizing any suitable protein and / or starch removal technology. An example of a suitable technology for removal is incubation with one or more enzymes followed by separation of peptides, other protein material, and / or monosaccharides, and / or disaccharides. For separation of the protein, the enzyme can be any suitable protease, for example the protease can be an endo- or exopeptidase or a mixture thereof as found in (Flavorzyme® peptidase), or a serine endopeptidase (subtilisinA) as found in Alcalase®, or the like. For separation of the starch, the enzyme can be any suitable amylase, including isoamylase. The polysaccharides can be separated from the peptides, other protein material and / or monosaccharides, and / or disaccharides by causing the polysaccharides to precipitate or solubilizing the peptides, other protein material and / or monosaccharides and / or disaccharides followed by filtration. In one example, the polysaccharides are separated by adding ethanol to precipitate polysaccharide followed by filtration such as centrifugation to separate precipitated polysaccharide. The process may be repeated as necessary. Ordinarily, salts, monosaccharides, and disaccharides will also be removed in the liquid filtrate along with the peptides and other protein material. If desired, an acid extraction can also be used to remove peptides, salts, monosaccharides, and di saccharides.

[0129] In one aspect, polysaccharide can be extracted from polysaccharide containing material obtained from pomegranate using an acidic aqueous solution. This method is particularly useful for extracting pectin containing polysaccharide from polysaccharide containing materials. In embodiments, polysaccharide is extracted from polysaccharide containing material with an aqueous solution having pH of 1 to 3 (+ / -0.5). In embodiments, the aqueous solution used for extraction has pH of 1 to 2 (+ / -0.5). In embodiments, the aqueous solution used for extraction has pH of 1.0 to 1.5 (+ / -0.1). In embodiments, the aqueous solution used for extraction has pH of 1.2 (+ / -0.1). In embodiments, any suitable acidifying agent can be used. In embodiments, the acidifying agent is a mineral acid or an organic acid. In embodiments, the mineral acid is sulfuric or hydrochloric acid. In embodiments, concentrated sulfuric acid or concentrated hydrochloric acid is added to water or an aqueous solution to obtain the desired pH. In embodiments, the organic acid can be a liquid or solid acid. A selected amount of the liquid or solid organic acid is added to water or an aqueous solution to obtain the desired pH. Useful organic acids include, among others, formic acid, acetic acid, propionic acid, butyric acid, malonic, succinic, glutaric, adipic acid, citric acid, lactic acid, malic acid, or propane-1, 2, 3 -tricarboxylic acid. In embodiments, preferred acidifying agents and solvents are those that are suitable for use in processing animal or human food products. In embodiments, the organic acidifying agent is acetic acid or citric acid.

[0130] In an embodiment, the extraction can take place at a temperature of ambient room temperature (e.g., 25°C) to 90°C, for example at 35°C to 80°C or at room temperature, or any subrange thereof. Extraction with acidic aqueous solution can result in acid hydrolysis of polysaccharide. In embodiments, it is preferred to minimize hydrolysis of polysaccharide and maximize extraction of polysaccharide. It will be appreciated by one of ordinary skill in the art that the pH of the acidic aqueous solution employed as well as the temperature and length of extraction time can affect the amount of hydrolysis and extraction. It is preferred to adjust extraction conditions to minimize acid hydrolysis of polysaccharides, and particularly to minimize the hydrolysis of pectin. In an embodiment, the extraction can take 1 hour to 5 hours, for example 2 hours to 4 hours, or any subrange thereof. In an embodiment, the ratio (v / w) of acidified solvent to pomegranate material can be 15: 1 to 1 : 1, or any subrange thereof, for example, 10: 1 to 4:1. Polysaccharide, particularly pectin, is extracted from the polysaccharide containing material into the aqueous phase. After extraction, the aqueous phase is separated from remaining solids by filtration; for example, using centrifugation. Theaqueous phase containing polysaccharide is optionally extracted with a suitable organic solvent and / or subjected to additional purification steps, for example the aqueous solution is optionally treated to remove mono- or disaccharides or salts, and particularly the aqueous phase can be treated by diafiltration. The aqueous phase after any further optional purification is neutralized and solvent can be removed by drying. Alternatively, polysaccharide, particularly pectin, can be precipitated from aqueous solution as is known in the art by addition of suitable organic solvent, more specifically an alkyl alcohol and yet more specifically ethanol. Precipitated polysaccharide is optionally washed one or more times with organic solvent, e.g., ethanol. Extraction can optionally be repeated multiple times.

[0131] In embodiments herein, precipitation of polysaccharide from aqueous solution is referred to as ethanol precipitation. It will be apparent to one of ordinary skill in the art that other organic solvents can be employed to precipitate polysaccharides, such as isopropanol. In embodiments, the precipitating solvent is miscible with water. In embodiments, the precipitating organic solvent (e.g., ethanol or isopropanol) is added to the aqueous solution to achieve a 40%-80% v / v concentration of precipitation solvent to water. In embodiments, the v / v concentration of precipitating solvent to water ranges from 50%-70%. In embodiments, the v / v concentration of precipitating solvent to water ranges from 55%-65%. Optionally, the aqueous solution is subjected to multiple precipitations where the concentration of precipitation solvent added in different precipitations steps is the same or is different.

[0132] In embodiments, polysaccharide containing material obtained from pomegranate is pre-treated prior to depolymerization (e.g., treatment with Fenton’s reagent) to remove starch, for example by treatment with amylase in aqueous solution. Optionally, one or more proteases are added as well to remove protein. Amylase-treated material or amylase / protease treated material is then subjected to ethanol precipitation to collect the insoluble fraction which contains purified polysaccharides from which starch or starch and protein has been removed. Optionally, one or more additional polysaccharide degrading enzymes (e.g., one or more cellulases) are added to the aqueous mixture. In an embodiment, polysaccharide from which starch (and protein and / or other polysaccharides, e.g., cellulose) has been removed is extracted with organic solvent or acidic organic solvent to remove polyphenols. Preferably, pectinase is not added to remove pectin. In embodiments, polysaccharide from which starch has been removed is treated in a separate step with one or more proteases to remove protein and optionally in another separate step is treated to remove cellulose or other polysaccharide other than pectin. In embodiments, pre-treatment does not include treatment with one or morepectinases. In embodiments, one or more steps of ethanol precipitation follow removal of starch, optional removal of protein and optional removal of cellulose or polysaccharide other than pectin. Polysaccharides purified by the forgoing embodiments can be employed as pomegranate polysaccharide starting material.

[0133] In embodiments, dried polysaccharide containing material obtained from pomegranate is extracted with acidified organic solvent to remove polyphenols. In embodiments, the organic solvent is acidified with citric acid. In embodiments, the organic solvent is ethanol. In embodiments, the acidified organic solvent is ethanol acidified with citric acid. Polyphenol reduced polysaccharide material is then subjected to treatment with one or more proteases, one or more amylase or a combination thereof in aqueous medium. In embodiment, polysaccharide in the aqueous medium is subjected to ethanol precipitation to obtain pre-treated pomegranate polysaccharide starting material.

[0134] In an embodiment, dried polysaccharide containing material obtained from pomegranate is treated with amylase in aqueous medium and thereafter subjected to ethanol precipitation. The resulting pre-treated polysaccharide containing material can be used as pomegranate polysaccharide starting material.

[0135] In an embodiments, dried polysaccharide containing material obtained from pomegranate is extracted with acidified organic solvent to remove polyphenols. In embodiments, the organic solvent is acidified with citric acid. In embodiments, the organic solvent is ethanol. In embodiments, the acidified organic solvent is ethanol acidified with citric acid. Polyphenol reduced polysaccharide containing material is subjected to acidic aqueous extraction (pH 1 to 3, preferably pH 1.2), followed by neutralization and treatment with one or more amylases, and optionally one or more proteases. This treatment is followed by ethanol precipitation to obtain solid purified polysaccharides which can be used as polysaccharide containing starting material.

[0136] In an embodiment, dried polysaccharide containing material obtained from pomegranate is treated with one or more amylases in aqueous medium and thereafter subjected to ethanol precipitation to recover polysaccharide from which starch has been removed. This material is then subjected to extraction with organic solvent (e.g., chloroform / methanol / acetone extraction) to remove polyphenols. This pre-treated polysaccharide material can be used as polysaccharide containing starting material.

[0137] In an embodiment, dried polysaccharide containing material obtained from pomegranate is extracted with acidic organic solvent, e.g., acidic ethanol, to remove polyphenols. Polyphenol reduced polysaccharide containing material is then subjected to acidic aqueous extraction (pH 1-3, preferably pH 1.2) to obtain extracted polysaccharide from which polyphenols have been removed. After neutralization, extracted polysaccharide in aqueous medium is then treated with one or more amylases and the aqueous medium is separated form remaining solids, e.g., containing cellulose and protein. The separated aqueous medium is then subjected to ethanol precipitation to precipitate polysaccharide. This precipitated polysaccharide can be used as polysaccharide containing starting material.

[0138] In embodiments, polysaccharide containing material obtained from pomegranate is first treated to extract polyphenols and is thereafter treated to remove starch, optionally to remove protein or other polysaccharides other than pectin. In embodiments, polysaccharide containing material obtained from pomegranate is treated both to extract polyphenols and is treated to extract polysaccharides from polysaccharide containing material obtained from pomegranate, wherein the order of treatment is not critical.

[0139] In additional aspects, polysaccharide cleavage products can be subjected to further processing to purify them. This can be done using various methods known for purification of oligosaccharides.

[0140] In embodiments, the polysaccharide cleavage products can be purified by removing cellulose and other unreacted polysaccharide material. The cellulose and unreacted polysaccharide material can be removed using any suitable technology, for example, filtration such as ultrafiltration, or by precipitation, such as precipitation in ethanol.

[0141] In embodiments, the polysaccharide cleavage products can be purified by removing residual metals, such as iron or copper. In embodiments, the residual metal (e.g., iron and copper) can be removed by suitable resins. In embodiments, the residual metal is removed by suitable nanofiltration membranes. In embodiments, useful nanofiltration membranes have molecular weight cutoffs of 400-600 daltons, 600-800 daltons, or 800-1000 daltons. In embodiments, the polysaccharide cleavage products can be purified by removing remaining hydrogen peroxide. In embodiments, residual hydrogen peroxide can be removed by addition of a quenching agent. In embodiments, hydrogen peroxide can be removed by enzymes, such as one or more catalase, for example Catazyme® catalase (Novozymes A / S, Denmark). In embodiments, the hydrogen peroxide is removed by suitable nanofiltrationmembranes. In embodiments, suitable nanofiltration membranes have molecular weight cutoffs of 400-600 daltons, 600-800 daltons, or 800-1000 daltons.

[0142] In embodiments, the polysaccharide cleavage products can be purified by removing salts. In embodiments, salts are removed by applying a suitable resin material. Suitable resin materials may include anion-exchange, cation-exchange, mixed bed, decolorizing, other chelation resins. For example, suitable resins may include, but are not limited to, lonac NM-60, MBD-10 ULTRA, Thermax Tulsion MB, Cole-Parmer RR-1400, Amberlite MB20, DOWEX Monosphere MR-450, and LSF 973 resin. Two or more resins may be combined to create mixed-bed resins. In embodiments, the salts are removed by suitable nanofiltration membranes. In embodiments, the nanofiltration membranes have molecular weight cutoffs of 400-600 daltons, 600-800 daltons, or 800-1000 daltons. In embodiments, the polysaccharide cleavage products can be purified by removing residual monosaccharide or disaccharides. In embodiments, the residual monosaccharides and disaccharides are removed by suitable nano-filtration membranes. In embodiments, the nanofiltration membranes have molecular weight cutoffs of 400-600 daltons, 600-800 daltons, or 800-1000 daltons.

[0143] In embodiments, the polysaccharide cleavage products may be treated with carbon to remove undesired components (e.g., impurities). The carbon may be activated carbon, charcoal, graphitized carbon, porous graphitized carbon, or any carbon-based material that is added with the goal of purification.

[0144] In embodiments, purified polysaccharide cleavage products contain less than 10% (less than 9%, less than 8%, less than 7%, or less than 6%) by weight of polyphenols. In preferred embodiments, purified polysaccharide cleavage products contain less than 5% (less than 4%, less than 3%, less than 2% or less than 1%) by weight of polyphenols. In embodiments, purified polysaccharide cleavage products contain less than 10% (less than 9%, less than 8%, less than 7%, or less than 6%) by weight of protein. In preferred embodiments, purified polysaccharide cleavage products contain less than 5% (less than 4%, less than 3%, less than 2% or less than 1%) by weight of protein. In embodiments, purified polysaccharide cleavage products contain less than 10% (less than 9%, less than 8%, less than 7%, or less than 6%) by weight of mono-and / or disaccharides. In preferred embodiments, purified polysaccharide cleavage products contain less than 5% (less than 4%, less than 3%, less than 2% or less than 1%) by weight of mono-and / or disaccharides. In embodiments, purified polysaccharide cleavage products contain less than 5% (less than 4%, less than 3%, less than2%) by weight of ash. In embodiments, purified polysaccharide cleavage products contain less than 2% (less than 1.75%, less than 1.5%, less than 1.25%) by weight of ash. In preferred embodiments, purified polysaccharide cleavage products contain less than 1% (less than 0.7%, less than 0.5%, or less than 0.2%) by weight of ash.

[0145] In embodiments, purified polysaccharide cleavage products contain 40% or more (or 50% or more, or 60% or more, or 70% or more, or 80% or more) by weight of fiber (e.g., dietary fiber). In preferred embodiments, purified polysaccharide cleavage products contain 85% or more (or 90% or more, or 95% or more, or 98% or more) by weight of fiber (e.g., dietary fiber).

[0146] In embodiments, any carbohydrate active enzyme or combination thereof can be used to modify the resulting products in the mixture of polysaccharide cleavage products by either adding or removing monomeric units to make a new product.

[0147] FIG. 1A provides an exemplary schematic flow diagram of a process for production of pomegranate fiber powder. FIG. 1A illustrates pre-treatment of pomegranate material containing polysaccharides as pomegranate fiber extraction. The steps of this pretreatment (before polysaccharide cleavage) include extraction with acidified organic solvent (e.g., acidified ethanol, preferably ethanol acidified with citric acid, e.g., absolute ethanol acidified with 0.015% citric acid (w / v)) to removal organic solvent soluble components, particularly polyphenols. The solid remaining after this extraction is filtered (or otherwise separated from extracting solvent) and dried. This polysaccharide-containing material can be designated polyphenol-reduced polysaccharide. Polyphenol-reduced polysaccharide is then optionally, but preferably, treated to remove protein, for example subjected to one or more proteases and / or one or more peptidases. The protein-reduced material is then optionally but preferably subjected to starch removal, for example by treatment with one or more amylases. The protein-reduced (optionally starch-reduced) polysaccharide-containing material (typically an aqueous solution or slurry) is then treated to precipitate polysaccharides, for example, by addition of ethanol. Precipitated polysaccharide from pomegranate starting material is largely pectin. Precipitated pectin is filtered (separated) from the aqueous ethanol medium and dried.

[0148] Pre-treatment is followed by polysaccharide cleavage as generally described herein. In FIG. 1A steps of Fenton activation (treatment with Fenton’s reagent) is followed by base cleavage to generate polysaccharide cleavage products. Any methods ofpolysaccharide cleavage as described herein can be employed. For example, pretreated- polysaccharide enriched pomegranate material is dissolved in water to a selected concentration (e.g., 10-15w / v). A 55 mM ammonium acetate buffer is added, and the pH adjusted to 5.5 with acetic acid. To this mixture 30% v / v hydrogen peroxide is added to a selected final concentration of (e.g., 4-10% v / v) of hydrogen peroxide in the mixture and a catalytic amount of copper (II) sulfate is added (e.g., approx. 0.0005% w / v of the mixture). The mixture is heated and stirred for a selected time at a selected temperature (e.g., 50-60°C for 1-3 hours). The reaction mixture is cooled to RT (approx. 20°C ) and the pH is adjusted to about pH 9 (preferably + / - 0.5 pH units) with the addition of base (e.g., ammonium hydroxide). The basic mixture is then stirred at a selected temperature or a selected time (e.g., 40-50°C for 1-3 hours) to cleave the polysaccharide. The reaction mixture is filtered to remove any remaining solid and the filter permeate (an aqueous solution of polysaccharide cleavage products) containing polysaccharide cleavage products (e.g., including oligosaccharides) is optionally, but preferably, further purified. For example as shown in FIG. 1A, the solution of polysaccharide cleavage products can be subjected to diafiltration, followed by ion exchange (e.g., deionized using a mixed bed resin made of a strong acid cation resin and strong base type 1 anion resins). The resulting purified solution of polysaccharide cleavage products can then be concentrated by removal of a desired amount of water. For example, the polysaccharide cleavage product can be concentrated to a powder which is dried, for example, subjected to spray drying (14a). Alternatively, water can be removed from the aqueous solution of polysaccharide cleavage product, for example by evaporation (14b) to generate a syrup, which contains a selected level of water (e.g., containing 15-40% water by weight of the product). The product can be sterilized, for example as shown in FIG. 1A (15a, 15b), by any appropriate method and packaged.Source Polysaccharides and Source Materials Comprising Polysaccharides

[0149] Any suitable source of pomegranate polysaccharide can be used to prepare the polysaccharide cleavage products. For example, the source of pomegranate polysaccharide can be pomegranate pomace obtained after juice processing of the pomegranates. The pomace comprises mainly parts of the husk, membranes and seeds. The source of pomegranate polysaccharide can be pomegranate press cake obtained by pressing pomegranate residues after juice processing. The source of pomegranate polysaccharide can be any portion of the pomegranate fruit, particularly the peel. The source of pomegranate can be any pomegranate cultivar. It will be appreciated by one having skill in the art that anytoxic portions of the source of pomegranate polysaccharide will be minimized or excluded from the methods, compositions, medicaments, and formulations herein.Polysaccharide cleavage products

[0150] The polysaccharide cleavage products have suitable features, structural characteristics, and other various properties. In embodiments, the polysaccharide cleavage products can have the features as described for CLX106 and CLX106Cu.

[0151] In embodiments, the polysaccharide cleavage products can have a number of oligosaccharide / polysaccharide structures ranging in size from a DP of 3 to 500, or any subrange thereof. The profile of oligosaccharide structures may depend on the polysaccharide source and reaction conditions. The source material and the conditions may be altered to provide the desired profile.

[0152] In embodiments, the polysaccharide cleavage products can have an average DP in the range of 3 to 500, or any subrange thereof, for example, 3 to 500, 9 to 500, 15 to 500, 21 to 500, 27 to 500, 33 to 500, 39 to 500, 45 to 500, 51 to 500, 57 to 500, 63 to 500, 69 to 500, 75 to 500, 81 to 500, 87 to 500, 93 to 500, 99 to 500, 105 to 500, 111 to 500, 117 to 500, 123 to 500, 129 to 500, 135 to 500, 141 to 500, 147 to 500, 153 to 500, 159 to 500, 165 to 500, 171 to 500, 177 to 500, 183 to 500, 189 to 500, 195 to 500, 201 to 500, 207 to 500, 213 to 500, 219 to 500, 225 to 500, 231 to 500, 237 to 500, 243 to 500, 249 to 500, 255 to 500, 261 to 500, 267 to 500, 273 to 500, 279 to 500, 285 to 500, 291 to 500, 297 to 500, 303 to 500, 309 to 500, 315 to 500, 321 to 500, 327 to 500, 333 to 500, 339 to 500, 345 to 500, 351 to 500, 357 to 500, 363 to 500, 369 to 500, 375 to 500, 381 to 500, 387 to 500, 393 to 500, 399 to 500, 405 to 500, 411 to 500, 417 to 500, 423 to 500, 429 to 500, 435 to 500, 441 to 500, 447 to 500, 453 to 500, 459 to 500, 465 to 500, 471 to 500, 477 to 500, 483 to 500, 489 to 500, 495 to 500, 3 to 494, 3 to 488, 3 to 482, 3 to 476, 3 to 470, 3 to 464, 3 to 458, 3 to 452, 3 to 446, 3 to 440, 3 to 434, 3 to 428, 3 to 422, 3 to 416, 3 to 410, 3 to 404, 3 to 398, 3 to 392, 3 to 386, 3 to 380, 3 to 374, 3 to 368, 3 to 362, 3 to 356, 3 to 350, 3 to 344, 3 to 338, 3 to 332, 3 to 326, 3 to 320, 3 to 314, 3 to 308, 3 to 302, 3 to 296, 3 to 290, 3 to 284, 3 to 278, 3 to 272, 3 to 266, 3 to 260, 3 to 254, 3 to 248, 3 to 242, 3 to 236, 3 to 230, 3 to 224, 3 to 218, 3 to 212, 3 to 206, 3 to 200, 3 to 194, 3 to 188, 3 to 182, 3 to 176, 3 to 170, 3 to 164, 3 to 158, 3 to 152, 3 to 146, 3 to 140, 3 to 134, 3 to 128, 3 to 122, 3 to 116, 3 to 110, 3 to 104, 3 to 98, 3 to 92, 3 to 86, 3 to 80, 3 to 74, 3 to 68, 3 to 62, 3 to 56, 3 to 50, 3 to 44, 3 to 38, 3 to 32, 3 to 26, 3 to 20, 3 to 14, or 3 to 8 or any subrange thereof. In embodiments, the polysaccharide cleavageproducts comprise a backbone containing galacturonic acid monomers, wherein each galacturonic acid monomer is optionally bonded in an alternating fashion to a rhamnose monomer, and wherein the total number of monomers in the synthetic oligosaccharide ranges from 3 to 30. For example, the total number of monomers in the synthetic oligosaccharide ranges from 3 to 30, 6 to 30, 9 to 30, 12 to 30, 15 to 30, 18 to 30, 21 to 30, 24 to 30, 27 to 30, 3 to 27, 3 to 24, 3 to 21, 3 to 18, 3 to 15, 3 to 12, 3 to 9, or 3 to 6. In embodiments, the backbone is branched with galactose, arabinose, or xylose monomers. In embodiments, the arabinose branches are further extended with additional arabinose monomers. In embodiments, the galactose branches are further extended with additional galactose monomers. In embodiments, the extended galactose monomers are further branched with arabinose monomers. Such synthetic oligosaccharides can be obtained, for example, by depolymerizing pectin according to the methods described herein. Pectin is known to contain both galacturonic acid and alternating galacturonic acid and rhamnose backbones with arabinose, galactose, and arabinogalactan branches. Arabinose branches contain 3- and 5- linked arabinose monomers. Galactose branches contain 4-linked galactose monomers. Arabinogalactan branches contain 4-linked galactose with terminal arabinose branches. Pectins may be obtained from pomegranates, pomegranate pomace, pomegranate products, pomegranate waste streams, and pomegranate juice.

[0153] In some aspects, the polysaccharide cleavage products have a solubility in water or aqueous solution of between 50 mg / mL and 2000 mg / mL, for example, between 50 mg / mL and 2000 mg / mL, between 100 mg / mL and 2000 mg / mL, between 150 mg / mL and 2000 mg / mL, between 200 mg / mL and 2000 mg / mL, between 150 mg / mL and 1500 mg / mL, between 1000 mg / mL and 2000 mg / mL, between 500 mg / mL and 2000 mg / mL, between 50 mg / mL and 1000 mg / mL, between 100 mg / mL and 1000 mg / mL, between 150 mg / mL and 1000 mg / mL, between 200 mg / mL and 1000 mg / mL, between 150 mg / mL and 500 mg / mL, or between 200 mg / mL and 1000 mg / mL. In some embodiments, the polysaccharide cleavage product has a solubility of at least 200 mg / mL, optionally less than 1000 mg / mL. In further embodiments, the polysaccharide cleavage product has a solubility of at least 200 mg / mL, optionally less than 1000 mg / mL, with a turbidity value of less than 20 NTU, optionally greater than 0.5 NTU. In some embodiments, the polysaccharide cleavage product has a solubility of at least 200 mg / mL, of at least 500 mg / mL, of at least 1000 mg / mL or of at least 2000 mg / mL.

[0154] In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 50 NTU. In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 40 NTU. In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 35 NTU. In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 30 NTU. In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 25 NTU. In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 20 NTU. In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 15 NTU. In embodiments, solutions of the polysaccharide cleavage products in water at 25 °C exhibit turbidity less than 10 NTU. In embodiments, the concentration of the polysaccharide cleavage products in water at 25 °C is 5 g / L or greater. In embodiments, the concentration of the polysaccharide cleavage products in water at 25 °C is 10 g / L or greater. In embodiments, the concentration of the polysaccharide cleavage products in water at 25 °C is 15 g / L or greater. In embodiments, the concentration of the polysaccharide cleavage products in water at 25 °C is 20 g / L or greater. In embodiments, the concentration of the polysaccharide cleavage products in water at 25 °C is 30 g / L or greater.

[0155] In embodiments, a solution of the polysaccharide cleavage products in water at 25 °C at a concentration of 5 g / L or greater has turbidity of 40 NTU or less, optionally 30 NTU or less, optionally 20 NTU or less, or optionally 15 NTU or less. In embodiments, a solution of the polysaccharide cleavage products in water at 25 °C at a concentration of is 10 g / L or greater has turbidity of 40 NTU or less, optionally 30 NTU or less, optionally 20 NTU or less, or optionally 15 NTU or less. In embodiments, a solution of the polysaccharide cleavage products in water at 25 °C at a concentration of 15 g / L or greater has turbidity of 40 NTU or less, optionally 30 NTU or less, optionally 20 NTU or less, or optionally 15 NTU or less. In embodiments, a solution of the polysaccharide cleavage products in water at 25 °C at a concentration of 20 g / L or greater has turbidity of 40 NTU or less, optionally 30 NTU or less, optionally 20 NTU or less, or optionally 15 NTU or less. In embodiments, the concentration of the polysaccharide cleavage products in water at 25 °C is 30 g / L or greater has turbidity of 40 NTU or less, optionally 30 NTU or less, optionally 20 NTU or less, or optionally 15 NTU or less.

[0156] In some aspects, the polysaccharide cleavage products have an average molecular weight of less than 100 kDa, optionally greater than 0.5 kDa, 1 kDa, or 2kDa. For example,the polysaccharide cleavage products have an average molecular weight of about 0.5 kDa to about 100 kDa, about 1 kDa to about 100 kDa, about 2 kDa to about 100 kDa, about 5 kDa to about 100 kDa, about 10 kDa to about 100 kDa, about 15 kDa to about 100 kDa, about 20 kDa to about 100 kDa, about 25 kDa to about 100 kDa, about 30 kDa to about 100 kDa, about 35 kDa to about 100 kDa, about 40 kDa to about 100 kDa, about 45 kDa to about 100 kDa, about 50 kDa to about 100 kDa, about 55 kDa to about 100 kDa, about 60 kDa to about 100 kDa, about 65 kDa to about 100 kDa, about 70 kDa to about 100 kDa, about 75 kDa to about 100 kDa, about 80 kDa to about 100 kDa, about 85 kDa to about 100 kDa, about 90 kDa to about 100 kDa, about 95 kDa to about 100 kDa, about 0.5 kDa to about 95 kDa, about 0.5 kDa to about 90 kDa, about 0.5 kDa to about 85 kDa, about 0.5 kDa to about 80 kDa, about 0.5 kDa to about 75 kDa, about 0.5 kDa to about 70 kDa, about 0.5 kDa to about 65 kDa, about 0.5 kDa to about 60 kDa, about 0.5 kDa to about 55 kDa, about 0.5 kDa to about 50 kDa, about 0.5 kDa to about 45 kDa, about 0.5 kDa to about 40 kDa, about 0.5 kDa to about 35 kDa, about 0.5 kDa to about 30 kDa, about 0.5 kDa to about 25 kDa, about 0.5 kDa to about 20 kDa, about 0.5 kDa to about 15 kDa, about 0.5 kDa to about 10 kDa, about 0.5 kDa to about 5 kDa, about 0.5 kDa to about 2 kDa, or about 0.5 kDa to about 1 kDa. In some aspects, 50% or less (e.g., 40% or less, 30% or less, 20% or less, or 10% or less) of the polysaccharide cleavage products by weight have a molecular weight of about 100 kDa or more. In preferred aspects, 10% or less of the polysaccharide cleavage products by weight have a molecular weight of about 100 kDa or more. In keeping with this aspect, preferred polysaccharide cleavage products comprise between 5% and 10% (such as between 5% and 9%, between 5% and 8%, between 5% and 7%, between 6% and 10%, or any subranges thereof) polysaccharides by weight having a molecular weight of about 100 kDa or more.

[0157] In some aspects, the polysaccharide cleavage products have an average molecular weight ranging from 0.5-50kDa. In some aspects, the polysaccharide cleavage products have an average molecular weight ranging from l-50kDa. In some aspects, the polysaccharide cleavage products have an average molecular weight ranging from 0.5-25kDa. In some aspects, the polysaccharide cleavage products have an average molecular weight ranging from l-25kDa. In some aspects, the polysaccharide cleavage products have an average molecular weight ranging from 5-25kDa, from 5-50kDa, from 10-25 kDa, from 10-15 kDa, from 15-25kDa, from 15-50kDa, or from 20-50kDa.Soluble Fiber

[0158] In an aspect, the invention provides soluble pomegranate fiber comprising, consisting essentially of, or consisting of a mixture of polysaccharide cleavage products from pomegranate, as described herein. In embodiments, the soluble pomegranate fiber exhibits high solubility in water or aqueous solution with low turbidity. In embodiments, solutions of soluble pomegranate fiber in water or aqueous solution exhibit low viscosity. In embodiments, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less, optionally 30 NTU or less, optionally 20 NTU or less or optionally 15 NTU or less. In embodiments, a 20 g / L mixture of the soluble pomegranate fiber in water has a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C, optionally 2 mPa.s or less, optionally 1.5 mPa.s or less, or optionally ImPa.s or less. In embodiments, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less, optionally 30 NTU or less, optionally 20 NTU or less or optionally 15 NTU or less and has a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C, optionally 2 mPa.s or less, optionally 1.5 mPa.s or less, or optionally 1 mPa.s or less.

[0159] In embodiments, the mixture of polysaccharide cleavage products of the soluble pomegranate fiber contains 40% or more, optionally 50% or more, optionally 60% or more, optionally 70% or more, optionally 80 % or more, optionally 90% or more by weight of polysaccharide cleavage products having molecular weight less than 100 kDa. In embodiments, each of the polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the soluble pomegranate fiber has a degree of polymerization (DP) of 3 to 100, optionally of 3 to 75, optionally of 3-50, optionally of 3-25 or optionally of 23-20.

[0160] In embodiments, the mixture of polysaccharide cleavage products of the soluble pomegranate fiber is formed by cleavage of a polysaccharide containing material obtained from pomegranate. In embodiments, the polysaccharide containing material obtained from pomegranate comprises, consists essentially of or consists of pomace, husk, arils containing seeds, or a combination thereof. In a specific embodiment, the polysaccharide containing material obtained from pomegranate comprises, consists essentially of or consists of pomace.

[0161] In embodiments, polysaccharides in the polysaccharide material obtained from pomegranate are depolymerized by any chemical or physical method that will achieve desired level of depolymerization, for example, the specified degree of polymerization or the specified range of molecular weights. In embodiments, the depolymerization method is achemical and / or enzymatic method. In embodiments, the polysaccharide material obtained from pomegranate is first treated to remove polyphenols prior to depolymerization. In embodiments, the mixture of polysaccharide cleavage products is formed by:(i) reacting one or more polysaccharides in a pomegranate starting material containing polysaccharide with a Fenton’s reagent comprising a peroxide agent and a metal to generate reaction products; and(ii) cleaving the reaction products with a cleavage agent to generate the mixture of polysaccharide cleavage products.Uses for Soluble Fiber or Polysaccharide Cleavage Products

[0162] The polysaccharide cleavage products and soluble pomegranate fiber herein have a variety of beneficial uses. In some aspects, the polysaccharide cleavage products and / or soluble pomegranate fiber can be combined with other ingredients to produce pharmaceutical compositions, foodstuffs, food supplements, drinks and beverages, nutritional supplements, medicinal products, including medicinal foods, and the like. In general, the invention provides compositions, including, for example, pharmaceutical compositions, foodstuffs, food supplements, drinks and beverages, nutritional supplements, medicinal products, medicinal foods and the like, comprising or consisting essentially of soluble pomegranate fiber as described herein or optionally purified polysaccharide cleavage products obtained from pomegranate. In general, the invention provides compositions, including, for example, pharmaceutical compositions, foodstuffs, food supplements, drinks and beverages, nutritional supplements, medicinal products, medicinal foods and the like, comprising or consisting essentially of soluble pomegranate fiber or optionally purified polysaccharide cleavage products obtained from pomegranate which is optionally prepared by one or more methods herein.

[0163] In embodiments, the invention provides a food, beverage, or medicinal product comprising the soluble pomegranate fiber. In embodiments, food products include yogurts, frozen yogurts, ice creams, fruit sauces, syrups, chocolates, tomato sauces, ketchups, barbecue sauces, breads, granola bars, energy bars, or breakfast cereals, among others. In embodiments, the beverage products include infant formulas, follow-on formulas, toddler’s beverages, dairy and nondairy milks, fermented milks, fruit and vegetable juices, fruit-based drinks, protein drinks, energy drinks, sports drinks, sparkling waters, vitamin waters, teas and coffee drinks, among others. In embodiments, the medicinal products include dietarysupplements or pharmaceutical products. In embodiments, the medicinal products are in the form of a gummy, capsule, pill or tablet. In embodiments, solid or gel food, beverage or medicinal products contain 1 mg of soluble pomegranate fiber or more / gram of product. In embodiments, solid or gel food, beverage or medicinal products contain 5 mg of soluble pomegranate fiber or more / gram of product. In embodiments, solid or gel food, beverage or medicinal products contain 10 mg of soluble pomegranate fiber or more / gram of product. In embodiments, solid or gel food, beverage or medicinal products contain 20 mg of soluble pomegranate fiber or more / gram of product. In embodiments, liquid food, beverage or medicinal products contain 1 mg of soluble pomegranate fiber or more / mL of product. In embodiments, solid or gel food, beverage or medicinal products contains 5 mg of soluble pomegranate fiber or more / mL of product. In embodiments, solid or gel food, beverage or medicinal products contains 10 mg of soluble pomegranate fiber or more / mL of product. In embodiments, solid or gel food, beverage or medicinal products contain 20 mg of soluble pomegranate fiber or more / mL of product.

[0164] In some aspects, the optionally purified polysaccharide cleavage products can be used as a prebiotic to selectively stimulate growth of one or more beneficial bacteria. In some aspects, the optionally purified polysaccharide cleavage products are consumed by bacteria beneficial in the intestinal microbiome, the vaginal microbiome, and / or to the skin microbiome. In some aspects, the optionally purified polysaccharide cleavage products are consumed by bacteria beneficial in the soil microbiome. In some aspects, the polysaccharide cleavage products can be used to modulate microbial metabolite output. In some aspects, the invention provides methods to selectively stimulate growth of one or more beneficial bacteria, for example, bacteria beneficial in the intestinal microbiome, the vaginal microbiome, and / or beneficial to the skin microbiome employing the optionally purified polysaccharide cleavage products. In some aspects, the invention provides methods to selectively stimulate growth of one or more beneficial bacteria, for example, bacteria beneficial in the soil microbiome employing the optionally purified polysaccharide cleavage products. In some aspects, the optionally purified polysaccharide cleavage products can be used in a method to modulate microbial metabolite output. Use in such methods involves providing optionally purified polysaccharide cleavage products to the environment of the beneficial bacteria. In embodiments, providing optionally purified polysaccharide cleavage products to the environment of the beneficial bacteria includes feeding or otherwise administering the optionally purified polysaccharide cleavage products to a subject.

[0165] In some aspects, the polysaccharide cleavage products can be administered as a prebiotic formulation (i.e., without bacteria) or as a synbiotic formulation (i.e., with one or more desirable bacteria such as bifidobacteria). Bifidobateria are members of the genus Bifidobacterium which are anaerobic bacteria which colonize the human gastrointestinal tract and are believed to provide beneficial health benefits on their host, particularly human hosts. Bifidobacteria are some of the major strains that make up the gut microbiome, the bacteria that reside in the gastrointestinal tract and have health benefits for their hosts (Guarner and Malagelada 2003).

[0166] In general, any food or beverage that can be consumed by humans or animals, or otherwise suitably administered, can be used to make formulations containing the prebiotic and synbiotic containing compositions.

[0167] In some aspects, the polysaccharide cleavage products can be used as bulking- agents. In some aspects, the polysaccharide cleavage products can be used as bulking-agents in reduced sugar food applications. In some aspects, the polysaccharide cleavage products can be used as bulking-agents that do not affect flavor, odor, rheological, and textural properties. In some aspects, the polysaccharide cleavage products are employed in foods, beverages, or medicinal formulations in a manner that affects the rheological and / or textural properties of the foods, beverages, or medicinal formulations.

[0168] In some aspects, the polysaccharide cleavage products are useful as immune modulators, digestion aids, enhancers of the gastrointestinal intestinal barrier, gut motility aids, and the like. The polysaccharide cleavage products can be combined with other ingredients to produce pharmaceutical compositions, foodstuffs, food supplements, drinks, nutritional supplements, medical foods and the like.

[0169] In some aspects, the polysaccharide cleavage products can be used for growth or maintenance of specific microorganism in humans, other mammals, or in the rhizosphere of plants. The polysaccharide cleavage products can contain specific glycosidic linkages not able to be digested by the particular host (e.g., a person, livestock animal, or companion animal) but able to be metabolized by specific groups of commensal microorganism or probiotics. As such, the polysaccharide cleavage products can function as carriers to transport exogenous microorganisms (e.g., probiotic) to a specific niche, or as a nutritional source for microorganisms already present in the host.

[0170] In some aspects, the invention provides optionally purified polysaccharide cleavage products obtained from pomegranate for use in pharmaceutical compositions, nutrition supplements, medicinal products, including medicinal foods and the like for treatments as described herein. In some embodiments, the invention provides medicaments comprising, consisting essentially of or consisting of optionally purified polysaccharide cleavage products for treatments as described herein.Formulations and Administration

[0171] In some aspects, polysaccharide cleavage products can be used to supplement foods and beverages. Examples of such foods and beverages include, without limitation, yogurts, frozen yogurts, ice creams, fruit sauces, syrups, chocolates, tomato sauces, ketchups, barbecue sauces, breads, granola bars, energy bars, or breakfast cereals, infant formulas, follow-on formulas, toddler’s beverages, dairy and nondairy milks, fermented milks, fruit and vegetable juices, fruit-based drinks, protein drinks, energy drinks, sports drinks, sparkling waters, vitamin waters, teas and coffee drinks, among others.

[0172] In some aspects, polysaccharide cleavage products can be used to supplement medical foods intended for the nutritional management of diseases or conditions.

[0173] In some aspects, polysaccharide cleavage products can be formulated into dietary supplements, nutritional supplements or pharmaceutical products such as gummies, pills, tablets or capsules. These forms can include, for example, one or more additional components such as lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. The polysaccharide cleavage products can also be formulated into lozenge forms, gels, and the like.

[0174] The amount of the polysaccharide cleavage products to be administered or consumed can be determined in a conventional manner, based upon factors such as the application, and the person’s health, immune status, body weight and age. In general, the amount of polysaccharide cleavage products delivered is in the range from about 0.5 g to about 20 g per day, in certain embodiments from about 1 g to about 15 g per day, for example about 2 g to about 10 g per day. Appropriate dose regimes can be determined by methods known to those skilled in the art.

[0175] In a first aspect, the invention provides a method for generating a mixture of polysaccharide cleavage products from pomegranate, the method comprising:(i) reacting one or more polysaccharide in a pomegranate starting material containing polysaccharide with a Fenton’s reagent comprising a peroxide agent and a metal to generate reaction products, and (ii) cleaving the reaction products with a cleavage agent to generate the mixture of polysaccharide cleavage products from pomegranate. In embodiments of the first aspect of the invention, the pomegranate starting material comprises pomegranate pomace, pomegranate husk, pomegranate arils containing seed or a combination thereof. In embodiments of the first aspect of the invention, the pomegranate starting material is pomegranate pomace, pomegranate husk, pomegranate arils containing seed or a combination thereof. In embodiments of the first aspect of the invention, the pomegranate starting material comprises pomegranate pomace. In embodiments of the first aspect of the invention, the pomegranate starting material is pomegranate pomace.

[0176] In a first embodiment of the first aspect, the method further comprises pretreating the pomegranate starting material prior to reacting the one or more polysaccharide therein. In further embodiments of the first aspect, pre-treatment comprises extracting polyphenols from the pomegranate starting material containing polysaccharide to generate a polyphenol- reduced pomegranate starting material containing polysaccharide prior to reacting the one or more polysaccharide in the polyphenol -reduced pomegranate starting material. In further embodiments of the first aspect, polyphenols are extracted with water, methanol, ethanol, acetone or ethyl acetate. In further embodiments of the first aspect, polyphenols are extracted with acidified organic solvent. In further embodiments of the first aspect, the polyphenols are extracted with acidified ethanol or acidified isopropanol. In further embodiments of the first aspect, the extraction solvent is acidified by addition of organic acid. In further embodiments of the first aspect, the organic acid is a carboxylic acid. In further embodiments of the first aspect, the carboxylic acid is a mono-, di- or tricarboxylic acid. In further embodiments of the first aspect, the organic solvent is acidified by addition of a carboxylic acid that is optionally a di- or tricarboxylic acid. In further embodiments of the first aspect, the organic acid is citric acid. In further embodiments of the first aspect, the organic solvent is acidified by addition of citric acid.

[0177] In a second embodiment of the first aspect, polyphenol extraction employs ultrasound or microwave assistance. In further embodiments of the second embodiment,extraction is conducted as detailed in the further embodiments of the first embodiment listed above.

[0178] In a third embodiment of the first aspect, polyphenols are extracted using supercritical fluid extraction. In further embodiments of the third embodiment, supercritical fluid extraction can be combined with extraction as detailed in the further embodiments of the first embodiment listed above.

[0179] In a fourth embodiment of the first aspect, pre-treatment comprises or further comprises removal of one or more polysaccharides from the polysaccharide containing material obtained from pomegranate. In further embodiments of the fourth embodiment, pretreatment comprises or further comprises removal of one or more polysaccharides from the pomegranate starting material containing polysaccharide or the reduced-polyphenol pomegranate starting material containing polysaccharide. In further embodiments of the fourth embodiment, pre-treatment comprises or further comprises removal of one or more polysaccharides other than pectin from the polysaccharide containing material obtained from pomegranate, the pomegranate starting material containing polysaccharide or the reduced- polyphenol pomegranate starting material containing polysaccharide. In the forgoing embodiments, the one or more polypeptides are removed by extraction of enzyme treatment. In the forgoing embodiments, the one or more polypeptides other than pectin are removed by extraction of enzyme treatment. In further embodiments, pectin is removed or reduced by treatment with one or more pectinases. In further embodiments, starch is removed by treatment with one or more amylases. In further embodiments of the fourth embodiment, enzyme treatment can be combined with extraction as detailed in the further embodiments of the first, second, and / or third embodiments listed above.

[0180] In a fifth embodiment of the first aspect, pre-treatment comprises or further comprises extracting pectin from the pomegranate starting material containing polysaccharide or the reduced-polyphenol pomegranate starting material containing polysaccharide to generate a pectin-enriched pomegranate starting material prior to reacting the one or more polysaccharide in the pectin-enriched pomegranate starting material. In further embodiments of the fifth embodiment, pectin is extracted with an acidic aqueous solution. In further embodiments of the fifth embodiment, pectin extraction can be combined with extraction or enzyme treatment as detailed in the further embodiments of the first, second, third and / or fourth embodiments listed above.

[0181] In a sixth embodiment of the first aspect, pre-treatment comprises or further comprises removal of protein from the polysaccharide containing material obtained from pomegranate prior to reacting the one or more polysaccharide therein. In further embodiments of the sixth embodiment, pre-treatment comprises or further comprises removal of one or more polysaccharides from the pomegranate starting material containing polysaccharide or the reduced-polyphenol pomegranate starting material containing polysaccharide prior to reacting the one or more polysaccharide therein. In further embodiments, protein is removed by extraction. In further embodiments, protein is removed using one or more proteases. In further embodiments of the sixth embodiment, protein removal can be combined with extraction or enzyme treatment as detailed in the further embodiments of the first, second, third, fourth and / or fifth embodiments listed above.

[0182] In a seventh embodiment of the first aspect, the mixture of polysaccharide cleavage products is purified to provide a purified mixture of polysaccharide cleavage products. In further embodiment, the polysaccharide cleavage products are subjected to filtration or ethanol precipitation followed by filtration to remove ethanol insoluble material from the polysaccharide cleavage products. In further embodiments, the polysaccharide cleavage products are treated to remove protein, In further embodiments, the polysaccharide cleavage products are treated with one or more proteases to remove protein. In further embodiments of the seventh embodiment, purification of polysaccharide cleavage products can be combined with extraction or enzyme treatment as detailed in the further embodiments of the first, second, third, fourth and / or fifth embodiments listed above. In further embodiments of the seventh embodiment, purification of polysaccharide cleavage products can be combined with pre-treatment comprising protein removal as detailed in embodiments of the sixth embodiment listed above.

[0183] In an eighth embodiment of the first aspect, salts and / or mono- and / or disaccharides are removed from the mixture of polysaccharide cleavage products. In further embodiments, salts and / or mono- and di-saccharides are removed from the mixture of polysaccharide cleavage products by nanofiltration and / or treatment with ion exchange resins. In further embodiments of the eighth embodiment, removal of salts and / or mono- and / or disaccharides can be combined with extraction or enzyme treatment as detailed in the further embodiments of the first, second, third, fourth and / or fifth embodiments listed above. In further embodiments of the eighth embodiment, removal of salts and / or mono- and / or disaccharides can be combined with pre-treatment comprising protein removal as detailed inembodiments of the sixth embodiment listed above. In further embodiments of the eighth embodiment, removal of salts and / or mono- and / or disaccharides can be combined with purification of polysaccharide cleavage products as detailed in embodiments of the seventh embodiment above.

[0184] In a ninth embodiment of the first aspect, the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof comprise less than 20% by weight of residual monosaccharides. In a further embodiment, the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof comprise less than 10% by weight of residual monosaccharides.

[0185] In a tenth embodiment of the first aspect , the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof comprise less than 15% by weight of ash. In a further embodiment, the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof comprise less than 10% by weight of ash.

[0186] In an eleventh embodiment of the first aspect, the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof contain 60% or less by weight of polysaccharides with molecular weight of lOOkDa or more. In a further embodiment, the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof contain 40% or less by weight of polysaccharides with molecular weight of lOOkDa or more. In a further embodiment, the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof contain 20% or less by weight of polysaccharides with molecular weight of lOOkDa or more.

[0187] In a twelfth embodiment of the first aspect , each of the one or more polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof have a degree of polymerization (DP) of 3 to 100. In a further embodiment, each of the one or more polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof have a degree of polymerization (DP) of 3-50.

[0188] In a thirteenth embodiment of the first aspect , the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof has solubility in water of 20 g / L or higher at 25°C. In a further embodiment, the mixture of polysaccharide cleavage products produced by the method of the first aspect and anyembodiments thereof has solubility in water of 20 g / L or higher at 25°C. In a further embodiment, the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof has solubility in water of 50 g / L or higher at 25°C. In a further embodiment, the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof has solubility in water of 100 g / L or higher at 25°C.

[0189] In a fourteenth embodiment of the first aspect , the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof has dynamic viscosity of less than 4 millipascal-second (mPa.s) at 25°C when dissolved in water at a concentration of 20 g / L. In embodiments, the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof has dynamic viscosity of less than 2 millipascal-second (mPa.s) at 25°C when dissolved in water at a concentration of 20 g / L.

[0190] In a fifteenth embodiment of the first aspect , a 20 g / L solution of the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof in water has turbidity of 20 NTU or less. In further embodiments, a 20 g / L solution of the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof in water has turbidity of 40 NTU or less.

[0191] In a sixteenth embodiment of the first aspect , the polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof contain glycosidic linkages comprising one or more of the linkages 4-linked galactose, 6-linked galactose, 4-linked xylose, 3, 6-linked glucose, 3, 6-linked galactose, 4-linked rhamnose and 2, 3, 4-linked rhamnose. In further embodiments, each of the one or more polysaccharide cleavage products of the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof comprises one or more glycosidic linkage selected from the group consisting of 4-linked glucose, 4-linked galactose, 6-linked galactose, 4-linked xylose, 3, 6-linked glucose, 3, 6-linked galactose, 4-linked rhamnose, 2, 3, 4-linked rhamnose, and a combination thereof.

[0192] In a seventeenth embodiment of the first aspect, the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof exhibits the ability, when consumed by an animal, to increase the relative abundance of beneficial bacteria in the gastrointestinal tract of the animal. In further embodiments, themixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof exhibits the ability, when consumed by an animal, to increase the relative abundance of any one or more of Faecalibacterium, Blautia, Actinobacteria, Rumminococcus, Gordonibacter, Lactobacillus plantarum, Lactobacillus rhamnosus GG, Lactobacillus penstosus, or Bifidobacterium.

[0193] In an eighteenth embodiment of the first aspect, the organoleptic properties of the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof in a food or beverage product are suitable for consumption of the food or beverage product by a human. In further embodiments, the addition of the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof to a food or beverage product is such that consumers will not reject the product. In further embodiments, the addition of the mixture of polysaccharide cleavage products produced by the method of the first aspect and any embodiments thereof to a food or beverage product does not substantially decrease the clarity of the food or beverage product as measured by turbidity or expert and consumer sensory analysis.

[0194] In a second aspect, the invention provides, soluble pomegranate fiber comprises a mixture of polysaccharide cleavage products from pomegranate. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 20 NTU or less. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has dynamic viscosity of 2 millipascal-second (mPa.s) or less at 25°C. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less and a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less and a dynamic viscosity of 2 millipascal-second (mPa.s) or less at 25°C. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 20 NTU or less and a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C. In further embodiments of the second aspect, a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 20 NTU or less and a dynamic viscosity of 2 millipascal-second (mPa.s) or less at 25°C. In furtherembodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble pomegranate fiber contains 40% or more by weight of polysaccharide cleavage products having molecular weight less than 100 kDa. In further embodiments of the second aspect, each of the polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the soluble pomegranate fiber has a degree of polymerization (DP) of 3 to 100. In further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble pomegranate fiber contains 40% or more by weight of polysaccharide cleavage products having molecular weight less than 100 kDa and each of the polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the soluble pomegranate fiber has a degree of polymerization (DP) of 3 to 100. In further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble pomegranate fiber contains 40% or more by weight of polysaccharide cleavage products having molecular weight less than 100 kDa and / or each of the polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the soluble pomegranate fiber has a degree of polymerization (DP) of 3 to 100 and / or a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less or 20 NTU or less, and / or has a dynamic viscosity of 4 millipascal-second (mPa.s) or less or 2 millipascal-second (mPa.s) or less at 25°C.

[0195] In a first embodiment of the second aspect and each embodiment thereof, detailed above, the polysaccharide cleavage products of the soluble pomegranate fiber are formed by cleavage of a polysaccharide containing material obtained from pomegranate. In further embodiments of the first embodiment of the second aspect, the polysaccharide containing material obtained from pomegranate comprises pomegranate pomace, pomegranate husk, pomegranate arils containing seed or a combination thereof. In further embodiments of the first embodiment of the second aspect, the polysaccharide containing material obtained from pomegranate is pomegranate pomace, pomegranate husk, pomegranate arils containing seed or a combination thereof. In further embodiments of the first embodiment of the second aspect, the polysaccharide containing material obtained from pomegranate is or comprises pomegranate pomace. In further embodiments of the forgoing embodiments of the second aspect and each embodiment thereof detailed above, the polysaccharide cleavage products of the soluble pomegranate fiber are formed by any embodiment of the first, second, third, fourth, fifth, sixth, seventh, eighth, and / or ninth embodiments of the first aspect.

[0196] In a second embodiment of the second aspect, the polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the soluble fiber comprise at least one glycosidic linkage selected from the group consisting of 4-linked glucose, 4-linked galactose, 6-linked galactose, 4-linked xylose, 3,6-linked glucose, 3,6-linked galactose, 4- linked rhamnose, 2, 3, 4-linked rhamnose, and a combination thereof. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 25-50% 4-linked glucose. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 10-20% 4-linked galactose. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 2-10% 6-linked galactose. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 1-10% 4-linked xylose. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 2-7% 3,6- linked galactose.

[0197] In a second embodiment of the second aspect, each of the one or more polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the soluble fiber comprises one or more glycosidic linkage selected from the group consisting of 4-linked glucose, 4-linked galactose, 6-linked galactose, 4-linked xylose, 3,6-linked glucose, 3,6-linked galactose, 4-linked rhamnose, 2, 3, 4-linked rhamnose, and a combination thereof. . In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 15-50% by weight galacturonic acid units. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 20-50% by weight glucose units. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 1-10% by weight xylose units. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 15-20% by weight galacturonic acid units and 20-30% by weight glucose units. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 15-20% by weight galacturonic acid units, 20-30% by weight glucose units and / or 25-35% by weight arabinose units. In a further embodiment of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 15-20% by weight galacturonic acid units, and / or 20-30% by weight glucose units, and / or 25-35% by weight arabinose units, and / or 10-20% by weight galactose units. In a further embodiment of thesecond aspect, the mixture of polysaccharide cleavage products of the soluble fiber comprises 15-20% by weight galacturonic acid units, and / or 20-30% by weight glucose units, and / or 25- 35% by weight arabinose units, and / or 10-20% by weight galactose units, and / or 1-5% by weight rhamnose units.

[0198] In additional embodiments of the second aspect, the soluble fiber comprises 15% or less by weight of ash. In additional embodiments of the second aspect, the soluble fiber comprises less than 10% by weight of mono- and disaccharides. In additional embodiments of the second aspect, the soluble fiber comprises 15% or less by weight of ash and less than 10% by weight of mono- and disaccharides.

[0199] In additional embodiments of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber are purified. In additional embodiments of the second aspect, the mixture of polysaccharide cleavage products of the soluble fiber is less than 10% by weight of residual monosaccharides.

[0200] In additional embodiments of the second aspect, the soluble fiber can be in the form of a powder, a syrup or a solution in an appropriate solvent, for example a solution in water or in an aqueous solution. In additional embodiments, pomegranate fiber syrup contains from 20-40% by weight water. In additional embodiments, pomegranate fiber syrup can contain from 20 to 26% by weight water. In additional embodiments, pomegranate fiber syrup can contain from 22 to 25% by weight water.

[0201] In additional embodiments of the second aspect, the soluble fiber contains 60% or more dry weight of total carbohydrate. In additional embodiments of the second aspect, the soluble fiber contains 65% or more dry weight of total carbohydrate. In additional embodiments of the second aspect, the soluble fiber contains 8% or less dry weight of sodium. In additional embodiments of the second aspect, the soluble fiber contains 6% or less dry weight of sodium. In additional embodiments of the second aspect, the soluble fiber contains 8% or less dry weight of fat. In additional embodiments of the second aspect, the soluble fiber contains 6% or less dry weight of fat. In additional embodiments of the second aspect, the soluble fiber contains 15% or less dry weight of protein. In additional embodiments of the second aspect, the soluble fiber contains 12% or less dry weight of protein. In additional embodiments of the second aspect, the soluble fiber contains 2.5-7.5 dry weight of sodium. In additional embodiments of the second aspect, the soluble fiber contains 2.5-7.5% dry weight of fat. In additional embodiments of the second aspect, thesoluble fiber contains 10-13% dry weight of protein. In additional embodiments of the second aspect, the soluble fiber contains 20% or less dry weight of ash. In additional embodiments of the second aspect, the soluble fiber contains 10% or less dry weight of ash. In additional embodiments of the second aspect, the soluble fiber contains, less than 3% dry weight starch. In additional embodiments of the second aspect, the soluble fiber contains, less than 2% dry weight starch. In additional embodiments of the second aspect, the soluble fiber contains, less than 20% dry weight of sugars. In additional embodiments of the second aspect, the soluble fiber contains, less than 10% dry weight of sugars. In additional embodiments of the second aspect, the soluble fiber contains greater than 30% by weight total non-digestible saccharides (total carbohydrate-sugar-starch). In additional embodiments of the second aspect, the soluble fiber contains greater than 50% by weight total non-digestible saccharides (total carb ohy drate- sugar- starch) .

[0202] In a third aspect, the invention provides a composition comprising a mixture of polysaccharide cleavage products, wherein the composition is formed by:

[0203] (i) reacting one or more polysaccharides in a pomegranate starting material containing polysaccharide with a Fenton’s reagent comprising a peroxide agent and a metal to generate reaction products; and

[0204] (ii) cleaving the reaction products with a cleavage agent to generate the mixture of polysaccharide cleavage products.

[0205] In further embodiments of the third aspect, the mixture of polysaccharide cleavage products formed contains 40% or more by weight of polysaccharides with a molecular weight of less than 100 kDa. In further embodiments of this third aspect, the mixture of polysaccharide cleavage products formed contains 60% or more by weight of polysaccharides with a molecular weight of less than 100 kDa. In further embodiments of this third aspect, each of the one or more polysaccharide cleavage products have a degree of polymerization (DP) of 3 to 100. In further embodiments of this third aspect, each of the one or more polysaccharide cleavage products have a degree of polymerization (DP) between 3 and 50. In further embodiments of the third aspect, pomegranate starting material comprises pomegranate pomace, pomegranate husk, pomegranate arils containing seeds, or a combination thereof. In further embodiments of the third aspect, pomegranate starting material is pomegranate pomace, pomegranate husk, pomegranate arils containing seeds, or a combination thereof. In further embodiments of the third aspect, pomegranate startingmaterial is or comprises pomegranate pomace, pomegranate husk, pomegranate arils containing seeds, or a combination thereof. In further embodiments, a 20 g / L mixture in water of the mixture of polysaccharide cleavage products of this third aspect has turbidity and / or dynamic viscosity as detailed in the second aspect of the invention as detailed above. In a specific embodiment, a 20 g / L mixture of the mixture of polysaccharide cleavage products in water has turbidity of turbidity of 40 NTU or less or 20 NTU or less. In a specific embodiment, a 20 g / L mixture of the mixture of polysaccharide cleavage products in water has dynamic viscosity of 4 millipascal-second (mPa.s) or less or of 2 millipascal-second (mPa.s) or less at 25°C.

[0206] In a first embodiment of the third aspect, the polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the composition comprise at least one glycosidic linkage selected from the group consisting of 4-linked glucose, 4-linked galactose, 6-linked galactose, 4-linked xylose, 3,6-linked glucose, 3,6-linked galactose, 4-linked rhamnose, 2, 3, 4-linked rhamnose, and a combination thereof. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 25-50% 4-linked glucose. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 10-20% 4-linked galactose. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 2-10% 6-linked galactose. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 1-10% 4-linked xylose. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 2-7% 3,6-linked galactose.

[0207] In a second embodiment of the third aspect, each of the one or more polysaccharide cleavage products of the mixture of polysaccharide cleavage products of the composition comprises one or more glycosidic linkage selected from the group consisting of 4-linked glucose, 4-linked galactose, 6-linked galactose, 4-linked xylose, 3,6-linked glucose, 3,6-linked galactose, 4-linked rhamnose, 2, 3, 4-linked rhamnose, and a combination thereof. . In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 15-50% by weight galacturonic acid units. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 20-50% by weight glucose units. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 1-10% by weight xylose units. In a further embodiment of the third aspect, the mixture ofpolysaccharide cleavage products of the composition comprises 15-20% by weight galacturonic acid units and 20-30% by weight glucose units. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 15-20% by weight galacturonic acid units, 20-30% by weight glucose units and / or 25-35% by weight arabinose units. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 15-20% by weight galacturonic acid units, and / or 20-30% by weight glucose units, and / or 25-35% by weight arabinose units, and / or 10-20% by weight galactose units. In a further embodiment of the third aspect, the mixture of polysaccharide cleavage products of the composition comprises 15-20% by weight galacturonic acid units, and / or 20-30% by weight glucose units, and / or 25- 35% by weight arabinose units, and / or 10-20% by weight galactose units, and / or 1-5% by weight rhamnose units.

[0208] In additional embodiments of the third aspect, the composition comprises 15% or less by weight of ash. In additional embodiments of the third aspect, the composition comprises less than 10% by weight of mono- and disaccharides. In additional embodiments of the third aspect, the composition comprises 15% or less by weight of ash and less than 10% by weight of mono- and disaccharides.

[0209] In additional embodiments of the third aspect, the mixture of polysaccharide cleavage products of the composition is purified. In additional embodiments of the third aspect, the mixture of polysaccharide cleavage products of the composition less than 10% by weight of residual monosaccharides.

[0210] In a fourth aspect, the invention provides a prebiotic composition comprising a composition or soluble fiber as detailed in aspects and embodiments above, wherein the prebiotic composition stimulates growth of one or more beneficial bacteria. In embodiments of the fourth aspect, the one or more beneficial bacteria are selected from the group consisting of Faecalibacterium, Blautia, Actinobacteria, Rumminococcus, Gordonibacter , Lactobacillus plantarum, Lactobacillus rhamnosus GG, Lactobacillus penstosus, and Bifidobacterium .

[0211] In a fifth aspect, the invention provides a synbiotic composition comprising a composition or soluble fiber as detailed in aspects and embodiments above, and one or more beneficial bacteria. In embodiments of the fifth aspect, the one or more beneficial bacteria are selected from the group consisting of Faecalibacterium, Blautia, Actinobacteria,Rumminococcus, Gordonibacter , Lactobacillus plantarum, Lactobacillus rhamnosus GG, Lactobacillus penslosus, and Bifidobacterium.

[0212] In a sixth aspect the invention provides a food, beverage, or medicinal product comprising a composition or soluble fiber as detailed in aspects and embodiments above. In embodiments of the sixth aspect, the food product comprises a yogurt, a frozen yogurt, an ice cream, a fruit sauce, a syrup, a chocolate, a tomato sauce, a ketchup, a barbecue sauce, a bread, a granola bar, an energy bar, or a breakfast cereal. In embodiments of the sixth aspect, the food product is a yogurt, a frozen yogurt, an ice cream, a fruit sauce, a syrup, a chocolate, a tomato sauce, a ketchup, a barbecue sauce, a bread, a granola bar, an energy bar, or a breakfast cereal. In embodiments of the sixth aspect, the food product is chocolate, a soft candy, a frozen juice, a frozen popsicle, tomato sauce, fruit sauce, a topping, a syrup, a frozen yogurt, a granola bar, an energy bar, ice cream or yogurt., In embodiments of the sixth aspect, the beverage product comprises an infant formula, a follow-on formula, a toddler’s beverage, a dairy or nondairy milk, a fermented milk, a fruit or vegetable juice, a fruit-based drink, a protein drink, an energy drink, a sports drink, a sparkling water, a vitamin water, a tea, or a coffee drink. In embodiments of the sixth aspect, the beverage product is an energy drink, a sports drink, a fortified beverage, nondairy milk, processed fruit juice, ready to drink tea or coffee, flavored vitamin water, flavored sparkling water, or vegetable juice. In embodiments of the sixth aspect, the beverage product is an infant formula, a follow-on formula, a toddler’s beverage, a dairy or nondairy milk, a fermented milk, a fruit or vegetable juice, a fruit-based drink, a protein drink, an energy drink, a sports drink, a sparkling water, a vitamin water, a tea, or a coffee drink. In embodiments of the sixth aspect, the medicinal product comprises a dietary supplement or a pharmaceutical product. In embodiments of the sixth aspect, the medicinal product comprises is dietary supplement or a pharmaceutical product. In embodiments of the sixth aspect, the medicinal product is in the form of a gummy, capsule, pill or tablet.

[0213] The invention can be further understood by the following non-limiting examples.EXAMPLESEXAMPLE 1: Bench Scale Production of CLX 106Cu, an Example Pomegranate Polysaccharide Cleavage Product

[0214] Pomegranate pomace obtained after juicing was pressed in a dewatering screw press (Vincent Corp.) and stored frozen. The pomace was then thawed and processed in a commercial food processor to a size of about 0.0025 mm (-1 / 16 in) or smaller. The processed pomace was then frozen in 3 kg parcels and stored until ready to use.

[0215] Polyphenols were extracted from the processed pomace using absolute ethanol acidified with 0.015% citric acid (w / v). The acidified ethanol was added to the processed pomace to give a pomace concentration of 0.75% (w / v). The mixture was then heavily agitated at 30°C for 4 hours. The mixture was then subjected to centrifugal filtration using a 3 pm filter to generate a polyphenol-rich ethanol stream (~120g of dried polyphenols is extracted from 1kg of wet pomace) and a solid stream containing polysaccharide (largely pectin), starch, proteins, lignin, and some residual polyphenols.

[0216] Next, the polysaccharide, particularly pectin, was extracted from the solid stream. The solid stream was covered by hot water and the pH adjusted to 1.2 with sulfuric acid. The mixture was heated to 86°C and stirred for 6 hours. This acid extraction is intended to protonate galacturonic acid residues in pectin to disrupt cross-linking and increase pectin solubility. The pH of the mixture was then adjusted to 5.7 with sodium hydroxide and an alpha-amylase enzyme (Termamyl® SC (Novozymes A / S, Denmark) was added to a final concentration of 0.24% w / v. The solution was then stirred for 6 hours before being filtered through a centrifuge with a 3 pm filter to remove remaining solid. The remaining solid retained in the centrifuge, which contained protein, cellulose, and lignin, was rejected as waste. The permeate, which contained pectinaceous material (polysaccharide) and glucose from starch degradation, was then added to cold absolute ethanol at a concentration of 60% v / v (ethanol / water) at 0°C for 6 hours to precipitate polysaccharide (e.g., pectin). Next, the mixture was subjected to centrifugation through a 1 pm filter, separating the ethanol and ethanol soluble material from the precipitated polysaccharide. Once the solid was separated, it was washed with a minimal volume of 80% v / v ethanol / water. The solid was homogenized and dried in a vacuum oven to produce a polysaccharide-rich material with a yield of 10% w / w from the wet starting material. This material is an example of pretreated and / or purified pomegranate starting material containing polysaccharides.

[0217] The pre-treated / purified polysaccharide-rich material was then dissolved in water to a final concentration of 12.5%w / v. A 55 mM ammonium acetate buffer was added, and the pH adjusted to 5.5 with acetic acid. To this mixture 30% v / v hydrogen peroxide was added to a final concentration of 6% v / v (hydrogen peroxide in the mixture) and copper (II) sulfatewas added to a final concentration of 0.000479% w / v of the mixture. The mixture was then stirred at 55°C for 2 hours. Then the reaction mixture was cooled to 20°C and the pH was adjusted to about pH 9 (preferably + / - 0.5 pH units) with the addition of 5.12% v / v 35% w / v ammonium hydroxide. The mixture was then stirred for 2 hours at 45°C to cleave the polysaccharide. On completion, the mixture was clarified using a centrifuge with a 3 pm filter, with remaining solid material being discarded. The filter permeate was deionized using a mixed bed resin made of a strong acid cation resin and strong base type 1 anion resins (e.g., Seplite® MB10IND (Sunresin New Materials Co., Ltd) and dried by lyophilization. The resulting light-yellow powder (designated CLX106Cu) is a mixture of polysaccharide cleavage products containing pomegranate oligosaccharides.

[0218] A glycosidic linkage analysis of CLX106Cu is shown in Table 2. Data are presented in units of peak area%. “Other” refers to linkages making up less than 1%. The notation ” represents a linkage that exists in an amount less than 1% (which can be 0%) of the total oligosaccharide weight. If a linkage is not fully described it is denoted by the monosaccharide, when known, or the type of monosaccharide, either pentose or hexose, followed by multiple or a single “x” denoting the number of branch points and finally the retention time, in parentheses, in the units of minutes.Table 2. Glycosidic Linakage Analysis of CLX106CuTable 3. Refractive index detection (RID) analysis of CLX106Cu. Data are presented in units of peak area%EXAMPLE 2; Bench Scale Production of CLX106, an Example Pomegranate Polysaccharide Cleavage Product

[0219] Pomegranate pomace obtained after juicing was pressed in a dewatering screw press (Vincent Corp.) and stored frozen. The pomace was then thawed and processed in a commercial food processor to a size of 0.0025 mm (-1 / 16 in) or smaller. The processed pomegranate pomace was then dried and stored until ready to use.

[0220] Polysaccharide was extracted from the processed pomace. This was done by hydrating 400g of pomace in 40 mM sodium acetate buffer adjusted to pH 4.9 in a ratio of 1 gram pomace to 10 mL of buffer. Alpha-amylase and cellulase enzymes were added to degrade starch and cellulose and the mixture was stirred at 45°C for 18 hours until a homogenous slurry was obtained. The material was spun down at 7500 rpm for 15 minutes, the supernatant discarded along with soluble sugars, and the resulting pellet was transferred to an 85% aqueous ethanol solution (85% v / v ethanol / water). The resulting slurry was mixed vigorously for 4 hours at 55°C and then spun down at 7500 rpm for 15 minutes. The supernatant was discarded along with ethanol soluble material, and the resulting pellet waswashed with 85% aqueous ethanol. The resulting pellet was dried by lyophilization. This material is an example of pretreated and / or purified pomegranate starting material containing polysaccharides.

[0221] The dried pellet was then purified further by three sequential washings / extractions with hexane, followed by washing / extraction with 1 : 1 (v:v) chlorofomrmethanol and finally with acetone. This step functions to remove polyphenols. Each extraction was conducted at a loading capacity of 50% w / v, and at 35°C for 30 minutes. At the completion of each extraction, the slurry was pelleted by centrifugation at 7500rpm for 15 minutes. Once all extractions were completed, the purified solid material was dried in a vacuum oven resulting 230g of purified polysaccharide, which is another example of a pre-treated and / or purified pomegranate starting material containing polysaccharides.

[0222] The purified polysaccharide was dissolved at 40g / L in 65 mM sodium acetate buffer at pH 5.6. Iron (III) sulfate was added to a final concentration of 1 mM and hydrogen peroxide was added to a final concentration of 7% volume / volume. The reaction mixture was stirred at 55°C for 3 hours. The reaction mixture was then cooled to below room temperature and ammonium hydroxide was added until the pH reached 10. The basic reaction mixture was then heated to 45 °C for two hours.

[0223] Upon completion, the resulting product solution was deionized using a mixed bed ion exchange resin made of a strong acid cation resin and strong base type 1 anion resins. The deionized product solution was then filtered to remove all non-solubilized components. The resulting filtered product solution was lyophilized to provide a solid which is a mixture of pomegranate cleavage products containing pomegranate oligosaccharides.

[0224] A glycosidic linkage analysis of CLX106 is shown in Table 4. Data are presented in units of peak area%. “Other” refers to linkages making up less than 1%. The notationrepresents a linkage that exists in an amount less than 1% (which can be 0%) of the total oligosaccharide weight. If linkage is not fully described it is denoted by the monosaccharide, when known, or the type of monosaccharide, either pentose or hexose, followed by multiple or a single “x” denoting the number of branch points and finally the retention time, in parentheses, in the units of minutes.Table 4. Glycosidic linkage analysis of CLX106Table 5. Refractive index detection (RID) analysis of CLX106. Data are presented in units of peak area%EXAMPLE 3: Example Pilot Scale Production of CLX 106Cu

[0225] The illustrated process is composed of three subprocesses: 1) Polysaccharide extraction, 2) Oligosaccharide production (polysaccharide depolymerization) and 3) separation and purification process. A pilot process flow diagram is shown in FIG. IB and Tables 6 and 7 indicate the stream information.1) Polysaccharide extraction process

[0226] Polysaccharide extraction from pomegranate pomace material begins with removal of polyphenols and ethanol-soluble impurities by acidic ethanol extraction in reactor R-101 (P-1-1). The spent solvent and impurities (S-103 and S-105) are separated from solid products (S-106) by centrifugation CF-101 (P-1-2). This is followed by a washing step by using 80 wt% ethanol (S-104) into CF-101 to wash the solid cake (P-1-2) for removal of residual polyphenols. Following the acidic ethanol extraction, solid products are extracted with an aqueous H2SO4 solution (0.27 weight %) at 90°C in R-101 to isolate the polysaccharide (P-1-3). Upon neutralization with NaOH, amylase enzyme (S-109) is added into R-101 to remove starch (P-1-4). Following the acid extraction and enzymatic hydrolysis treatment, the products are separated into a liquid fraction containing polysaccharide and a solid waste fraction containing cellulose, lignin, and protein using CF-101 (P-1-5). The polysaccharide containing liquid fraction (S-l 11) which also contains soluble starch degradation products is moved to the next reactor (R-102) and cooled down. Polysaccharide is then precipitated (S-l 13) by introducing ethanol into the R-102 at a constant flow rate while mixing (P-1-6). The ethanol and polysaccharide mixture (S-l 14) is centrifuged in CF- 101, followed by an ethanol washing step to further purify the polysaccharide (P-1-7). Then polysaccharide is stored for oligosaccharide production and is optionally dried / lyophilized.2) Oligosaccharide production

[0227] Polysaccharide cleavage products containing oligosaccharides are produced from the pre-treated polysaccharide containing starting material (10 wt% loading) in a reactor (R- 101) by a two-step reaction: Fenton reaction (6% hydrogen peroxide, 55mM buffer and copper catalyst, P-2-1) followed by a base cleavage reaction (NH4OH, P-2-2). After the production of oligosaccharides, the products enter the purification process. The unreacted polysaccharide and other impurities, including protein and the copper catalysts, are removed from the oligosaccharide products using 1 pm microfiltration MF-101 (P-2-3).3) Separation and purification process

[0228] To remove other minor impurities, the filtrate (S-206) is passed through ultrafiltration UF-101 (P-3-1). Next, the ultrafiltration filtrate (S-207) flows into a nonfilter NF-101 to concentrate it (P-3-2). After the concentration step, diafiltration with the introduction of water (S-209) is employed to remove salts and monosaccharides from the product, which is retained during diafiltration (P-3-2). Following diafiltration, the retentate is collected in the feed tank (S-211) and is again concentrated for spray drying (P-3-2). Finally, the concentrated retentate flows (S-211) to the spray dryer SDR-101 and the purified and dried polysaccharide cleavage product (S-214) is collected (P-3-3).Atty Docket: 340220: 83-23 WOTable 6. Example Stream table for POM extraction process (100kg wet POM press cake based)Atty Docket: 340220: 83-23 WOTable 7. Stream table for oligosaccharide production and separation and purification process (100kg dried polysaccharide based)Atty Docket: 340220: 83-23 WO**Oligosaccharide (mixture of polysaccharide cleavage products)* * *Poly saccharideEXAMPLE 4: Effect of Polyphenol Removal on Yield of Polysaccharide Cleavage Products

[0229] Initial attempts to produce polysaccharide cleavage products containing oligosaccharides from pomegranates generated unexpectedly low yield. To increase the yield of polysaccharide cleavage products, several depolymerization and pre-purifications were performed. It was discovered that pre-treatment to remove polyphenols provided major improvements in yield. We hypothesize that the polyphenols, which are anti-oxidants, can at least partially quench the hydrogen peroxide reagent and result in lower amounts of depolymerization of polysaccharide. Pomegranate polysaccharide containing material (3kg of dried pomegranate pomace) was added into 5L of water and the solution was adjusted to pH 5.7 using a 50% (w / v) solution of sodium hydroxide. To remove starch, the solution was treated with 12 mL Termamyl® SC DS (amylase, Novozymes A / S, Denmark) for 6 hours. The resulting solution was added to cold absolute ethanol to create a final 60% v / v (ethanol / water) solution (~ 6.5L) and solids were allowed to precipitate at 0 °C for 2 hours. Next, the mixture was subjected to filter centrifugation with a 1pm filter to separate the ethanol fraction from the solid polysaccharide-containing material. The solid was homogenized and dried via vacuum oven to produce approximately 1.86 kg of dark-brown polysaccharide-rich material. Small sugars, starch and proteins and other components that were soluble in 60% ethanol / water were removed from this polysaccharide containing material. This material was not specifically extracted to remove polyphenols. The dark brown color of the material indicates the presence of polyphenols.

[0230] For Fenton depolymerization, 300g of the starch reduced polysaccharide was mixed in2.4 L of water with 55 mM ammonium acetate adjusted to pH 5.5 and homogenized. To this mixture, 600 mL of 30% v / v aqueous hydrogen peroxide and 479 mg of copper (II) sulfate was added and stirred at 55°C for 2 hours. On completion, the reaction mixture was cooled to 20 °C, and the pH was adjusted with the addition of 162 mL of 35% ammonium hydroxide over the course of 15 min. Once the ammonia hydroxide solution was added, the solution was stirred for 2 hours at 45 °C. On completion, the mixture was clarified via filter centrifugation with a 3um filter. The filtrate was deionized with MB 10 mixed bed ion exchange resin and dried via lyophilization. The dried powder was subjected to further purification via dissolution in 60% aqueous ethanol. The resulting mixture was separated via centrifugation. The supernatant was evaporated via rotary evaporation under reduced pressure resulting in 39g of a dark tan powder containing the polysaccharide cleavage products. The final yield of polysaccharide cleavage products from the Fenton depolymerization wasonly 13%, where yield is based on grams of product / grams of pre-treated or purified starting polysaccharide containing material prior to depolymerization.EXAMPLE 5: The use of Sodium Hydroxide as a Cleavage Reagent

[0231] To purify the pomegranate polysaccharides, 2 kg of dried pomegranate pomace was added into 10 L of absolute ethanol containing 0.1% citric acid and brought to a temperature of 70 °C. The mixture was mixed for 3 hours and filtered via centrifugation with a 10 pm filter. The ethanol soluble fraction, containing polyphenols and lipids, was evaporated via rotary evaporation under reduced pressure and produced 600 g of bright red powder. The ethanol insoluble fraction was dried at 50 °C in a vacuum oven, producing1.4 kg of tan powder. To digest proteins, the dried ethanol insoluble fraction was mixed with 12 L of water containing 50 mL of Flavourzyme® (a mixture of endo- and exo-peptidases, Novozymes A / S, Denmark) and 50 mL of Alcalase® (protease, Novozymes A / S, Denmark), heated to 60 °C and pH adjusted to 7.4 by the addition of 50% aqueous NaOH. The mixture was mixed for 7 hours. For starch digestion, the temperature was adjusted to 86 °C, the pH was reduced to 5.75 with glacial acetic acid, and 84 mL Termamyl® SC DS (amylase, Novozymes A / S, Denmark) was added. After 12 hours, the reaction mixture was poured into 12 L of absolute ethanol producing a 50% v / v aqueous ethanol solution. The solution was allowed to precipitate for 2 hours at 6 °C. Next, the mixture was filtered via centrifugation with a 1 um filter separating the ethanol from the solid polysaccharide. The solid material was homogenized and dried via vacuum oven to produce approximately 1.02 kg of tan polysaccharide-rich material. This material was used as the pre-treated / purified polysaccharide starting material for depolymerization.

[0232] For Fenton-based depolymerization, 500 g of the pre-treated polysaccharide starting material was mixed in 3.7 L of water with 55 mM ammonium acetate adjusted to pH5.5 and homogenized. To this mixture, 1.25 L of 30% v / v hydrogen peroxide was added with 1.88 g of copper (II) sulfate penta hydrate (1.5 mM) and the resulting mixture was allowed to stir at 55 °C for 3 hours. The reaction mixture was then cooled to 20 °C and pH was adjusted with the addition of 50 mL of a 50% w / v sodium hydroxide over the course of 50 min. The mixture was then stirred for 3 hours at 45 °C. At the time of reaction completion, the mixture was clarified via filter centrifugation with a 3 pm filter. The liquid fraction was deionized with MB 10 mixed bed ion exchange resin and dried via lyophilization, resulting in 200 g of adark tan powder. The final yield of polysaccharide cleavage products from the Fenton depolymerization was 40% which is a suitable yield from a manufacturing standpoint.EXAMPLE 6: Ability of CLX 106Cu and CLX106 to stimulate probiotics

[0233] To determine whether CLX106Cu and CLX 106 affect Bifidobacterium and / or Lactobacillus growth, bacterial strains were grown in the presence of either CLX 106Cu or CLX106 as sole carbon source and assayed for growth and metabolic output. The selected Bifidobacterium and Lactobacillus strains included microbes from the American Type Culture Collection and human isolates. Pure cultures were incubated in minimal media containing CLX 106Cu or CLX106 at a final concentration of 2% w / v. Growth was monitored by measuring OD (600 nm) using a microtiter plate-reader (Epoch 2, BioTek).

[0234] Specifically, fresh cultures were generated by transferring a colony (per replicate) into fresh liquid media and incubating in an anaerobic chamber (Anaerobic Chamber Vinyl Type B), using a mix of gas (carbon dioxide 5%, hydrogen 5%, nitrogen balance). De Man, Rogosa and Sharpe media (MRS) was used to grow Bifidobacterium and Lactobacillus species. Cells were harvested at late exponential phase and an experimental inoculum was prepared by washing once with phosphate-buff ered saline (PBS). This was done by centrifuging the culture at 8000g for 5 minutes, discarding the supernatant, and resuspending the cells in the same volume of reduced PBS. Minimal media containing 2% of CLX 106Cu or CLX106 or no carbon source (negative control) were inoculated with 2% of the experimental inoculum. Growth was run 4 times (replicates) and media sterility was tested by incubating non-inoculated media. Growth was determined based on absorbance measurements at 600 nm. Minimal media used for bacterial growth was basal MRS previously described by Ruiz-Moyano et al. (2013).

[0235] As shown in FIGs. 2A and 2B respectively, CLX 106Cu and CLX106 selectively support the growth of two Bifidobacterium pseudocatenulatum strains which are associated with improved intestinal health. Further, as shown in FIG. 3, CLX 106Cu can selectively support the growth of several Lactobacilli species that are currently used as probiotics (Lactobacillus plantarum, Lactobacillus rhamnosus GG and Lactobacillus penstosus strains). Of note, CLX 106Cu supports Lactobacillus rhamnosus GG, which despite being one of the most studied and popular probiotics, does not yet have a commercialized prebiotic pairing.

[0236] The production of bacterial metabolites which potentially impact human health was assessed by metabolomics analysis of the CLX106Cu and CLX106 culture supernatantsof the two B. pseudocatenulatum strains. Short chain fatty acid (SCFA) analysis was performed as follows. The supernatants were diluted with water (1 :20 w / w). Twenty microliters of the supernatant dilutions were added to 20 pL of N-(3-Dimethylaminopropyl)- N'-ethylcarbodiimide hydrochloride (1-EDC HC1) in 5% pyridine. Then 40 pL of 200mM 2- nitrophenylhydrazine (2-NPH) in 80% acetonitrile with 50mM HC1 was added and briefly vortexed prior to incubating for 30 minutes at 40°C. After incubation, samples were diluted with 400 pL of 10% acetonitrile and vortexed. Aliquots of the samples were transferred to 96 well plates for LC-MS / QqQ analysis.

[0237] Short chain fatty acids were analyzed with a 1290 Infinity II LC (Agilent Technologies) equipped with a reverse phase column (Zorbax Eclipse C18 2.1 x50mm; Agilent Technologies) and 6490 Triple Quad LC / MS (Agilent Technologies, Santa Clara, CA). LC separation was performed with 5% acetonitrile with 0.1% formic acid (solvent A) and 95% acetonitrile with 0.1% formic acid (solvent B). A separation gradient was as follows: 5% to 20% B for 2 minutes, then 20% to 40% B for 1 minute, 40-55% B in 1 minute, 55-100% B in 0.1 minute, hold at 100%B for 0.5 minutes, return to 5% B in 0.01 minute, and equilibrate at 5% B for 0.99 minutes. ESLMS conditions were performed in positive mode, and the dynamic multiple reaction monitoring (dMRM) mode was used to monitor the precursor and product ion transitions. Peak areas were quantitated using Agilent Quantitative Analysis software and areas were normalized to internal standards and compared to an external standard curve for quantitation.

[0238] As shown in FIGS. 4A and 4B, B. pseudocatenulatum metabolism of CLX106Cu and CLX106 results in lactate (FIG. 4A) and beta-hydroxybutyrate (FIG. 4B) production. Lactate can exert strong antimicrobial effects against pathogens, and in the gut, lactate can be converted to butyrate and / or propionate by the endogenous microbial community, being both compounds involved in numerous beneficial health related mechanisms. Beta- hydroxybutyrate, a ketone body, is a potential source of energy for muscles and the brain.

[0239] An additional analysis using Negative Mode LC / MS was performed. Supernatants of fecal bacterial growths were diluted 1 :5 with Methanol: Water (1 : 1, v / v) containing a mixture of internal standards for LC-MS / QTOF analysis. Metabolites were analyzed with a 1290 Infinity II LC (Agilent Technologies) equipped with a BEH C18 column (Acquity UPLC BEH C18 1.7pm, 2.1x100mm; Waters) and 6530 LC-MS QTOF (Agilent Technologies). LC separation was performed with Water + 0.01% Formic Acid (solvent A) and Acetonitrile (solvent B). The separation gradient was as follows: 25-40%B in 10 mins,40-70%B in 8 minutes, 100% in 0.1 minutes, 100%B hold 1.4 minutes, return to 25%B in0. Imin, and equilibrate 30%B for 2.4 minutes. The MS conditions were set to negative mode with a scan range set at m / z 50-1200 at 1 spectra / sec scan rate.

[0240] As shown in FIG. 5, the analysis revealed that A pseudocatenulatum produced indole lactic acid (ILA) or indole lactate. ILA can activate different host receptors in the gut such as AhR. Metabolites that can activate AhR may reduce gut inflammation or metabolic syndrome.EXAMPLE 7: Impact of CLX 106Cu on the fecal microbiome and metabolism

[0241] Fecal samples were collected from healthy donors by BioIVT and stored at -80 °C until processing. Aliquots of slurry from the fecal samples were prepared by mixing three parts of fecal samples, one part of glycerol and one part of PBS. The slurries were stored at - 80 °C.

[0242] Static fecal fermentations were conducted in a deep 96-well format, under anaerobic conditions (Anaerobic Chamber Vinyl Type B), using a mix of gas (carbon dioxide 5%, hydrogen 5%, nitrogen balance), using inoculum from either individual donors or a pool of feces (2% of the fermentation mix). Fermentation media was optimized to support diverse microbial taxa and control pH within the range of the colon physiological conditions, containing mineral and vitamin solution, CaCh (10 mg / mL) and basic fermentation medium as described by MacFarlane GT et al (1989). A mix of background sugars (xylan, amylopectin, potato starch, and pectin) were included in the basic fermentation media in low concentration to sustain microbial networks and minimize changes due to lack of nutrients which would confound the experimental results. Samples were taken at different time points (Oh and 24h) for metabolomics analysis.

[0243] To measure the impact of bacterial fermentation of CLX 106Cu on the environmental pH, fermentation media was supplemented with the fluorescent pH indicator 2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), which enabled coupling of pH measurements with optical density (OD) growth measurements.

[0244] Supernatants of fecal bacterial growths were derivatized with 2- nitrophenylhydrazine (2-NPH) for LC-MS / QqQ analysis. In brief, supernatants of fecal bacterial growths were diluted with water (1 :20). Twenty microliters of the supernatantdilutions were added to 20 pL of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (1-EDC HC1) in 5% pyridine. Then 40 pL of 200mM 2-NPH in 80% acetonitrile with 50mM HC1 was added and briefly vortexed prior to incubating for 30 minutes at 40°C. After incubation, samples were diluted with 400 pL of 10% acetonitrile and vortexed. Aliquots of the samples were transferred to 96 well plates for LC-MS / MS analysis.

[0245] Short chain fatty acids were analyzed with a 1290 Infinity II LC (Agilent Technologies) equipped with a reverse phase column (Zorbax Eclipse C18 2.1 x50mm; Agilent Technologies) and 6490 Triple Quad LC / MS (Agilent Technologies, Santa Clara, CA). LC separation was performed with 5% acetonitrile with 0.1% formic acid (solvent A) and 95% acetonitrile with 0.1% formic acid (solvent B). A separation gradient was as follows: 5% to 20% B for 2 minutes, then 20% to 40% B for 1 minute, 40-55% B in 1 minute, 55-100% B in 0.1 minute, hold at 100% B for 0.5 minutes, return to 5% B in 0.01 minute, and equilibrate at 5% B for 0.99 minutes. ESLMS conditions were performed in positive mode, and the dynamic multiple reaction monitoring (dMRM) mode was used to monitor the precursor and product ion transitions. Peak areas were quantitated using Agilent Quantitative Analysis software and areas were normalized to internal standards and compared to an external standard curve for quantitation.

[0246] As shown in FIG. 6, CLX 106Cu significantly lowered the pH of the samples compared to the untreated control. Furthermore, short chain fatty acid analysis revealed that CLX 106Cu is a potent enhancer of microbial short chain fatty acid production. As shown in FIG. 7, CLX 106Cu induced significantly increased amounts of total short chain fatty acids, butyrate, and propionate. Additionally, FIG. 8 illustrates that CLX 106Cu showed a highly robust response where total short chain fatty acids were increased in 100% of donor samples. Testing multiple donors provides further support that these prebiotic effects are not restricted to a specific gut microbiome from a specific donor, but rather, can be fermented by highly diverse microbial communities from a large donor cohort.

[0247] In conclusion, CLX 106Cu shows increased bioactivity and utility and is suitable for many food and supplement applications. CLX106Cu is expected to provide additional health benefits associated with increased butyrate production and improved gut barrier function. These include improved nutrient absorption, improved sleep, improved mood, reduction of constipation, reduction of visceral pain, and reduced consequences of chronic inflammation (e.g., cardiovascular diseases and diabetes).EXAMPLE 8: Impact of CLX 106 on the fecal microbiome and metabolism

[0248] We evaluated the effect of CLX 106 in 8 individual fecal samples from healthy donors, and a pool of samples. The impact of the POM Fiber was compared to press cake polysaccharides and an untreated control.

[0249] Fecal samples were collected from healthy donors by BioIVT and stored at -80 °C until processing. Aliquots of slurry from the fecal samples were prepared mixing three parts of fecal samples, one part of glycerol and one part of PBS. Slurries were stored at -80 °C.

[0250] Static fecal fermentations were conducted in a deep 96-well format, under anaerobic conditions (Anaerobic Chamber Vinyl Type B), using a mix of gas (carbon dioxide 5%, hydrogen 5%, nitrogen balance), using inoculum from either individual donors or a pool of feces (2% of the fermentation mix).

[0251] Fermentation media was optimized to support diverse microbial taxa and control pH within the range of the colon physiological conditions, containing mineral and vitamin solution, CaCh (10 mg / mL) and basic fermentation medium as described by MacFarlane GT et al (1989). A mix of background sugars (xylan, amylopectin, potato starch, and pectin) were included in the basic fermentation media in low concentration to sustain microbial networks and minimize changes due to lack of nutrients which would confound the experimental results. The fermentation media was supplemented with 0.05 mg / mL of ellagic acid to study if CLX 106 promotes the bioconversion of ellagic acid and production urolithin A.

[0252] Multiple samples were taken at different time points (Oh, 6h, 11 h and 20 h) for monosaccharide analysis, oligosaccharide profiling, short chain fatty acid (SCFA) analysis and metabolomics analysis. After 20 hours of fermentation, gDNA was extracted from each well and sent for 16S rRNA sequencing. Three replicates were run for each oligo and untreated control.

[0253] DNA was extracted from fecal slurries using ZymoBIOMICS Kit D4308 and a KingFisher Flex DNA extraction robot. Microbial communities were profiled by sequencing the V4 region of the bacterial 16S rRNA gene amplified using 515F (5’- GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’ GGACTACHVGGGTWTCTAAT-3’) primers. Ilumina MiSeq was used to obtain 300 bp paired end reads. Raw demultiplexed reads were processed using QIIME2 2020.11. Briefly, after quality checking, trimming,filtering and denoising were performed using the "dada2 denoise-paired" plugin in QIIME2. Taxonomic classification of AS Vs was performed with the "q2-feature-classifier" plugin and a Naive Bayes classifier trained on Silva 138 99% OTUs from the 515F / 806R region of 16S rRNA sequences. Finally, alpha diversity and beta diversity analyses were conducted with QIIME2 plugins and subsequently imported into MATLAB, R and Excel for further analysis.

[0254] As shown in FIG. 9, pH measurements revealed that CLX 106 was more fermentable than its starting polysaccharide in all the tested microbial communities.

[0255] Change in gut microbiome in the presence CLX 106 during the fermentation was assessed using 16S rRNA sequencing. In some of the donors, CLX 106 promoted the increase of health-related bacteria such Faecalibacterium, Blautia, Actinobacteria, Rumminococcus and Gordonibacter . For comparative purposes, the fermentation was run again using fecal samples from donors who had Gordonibacter species, and a comparison was made between CLX 106 and FOS (fructo-oligosaccharide). CLX 106 increased the abundance of Gordonibacter species while with FOS, no increase of Gordonibacter species was observed. The results are shown in FIG. 10. As illustrated in FIG. 11, SCFA analysis of supernatants after fermentation indicates that CLX 106 promotes the production of total SCFA, in higher amount than what was observed for the starting polysaccharide. In humans, SCFA production results from prebiotic metabolism in the colon and is associated with various health effects.

[0256] The production of urolithin A by the gut microbiome was also assessed. Urolithin A has been related to restoration of mitochondrial function and it is considered a potent antiaging molecule. Several studies have linked ellagic acid in pomegranate extract and the conversion of this pomegranate ellagic acid into urolithin A by the gut microbiome. The presence of the species Gordonibacter in the gut has been described as being responsible for this activity.

[0257] The presence of urolithin A and ellagic acid before and after fermentation was measured by LC / MS / MS. Supernatants of fecal bacterial growths were diluted 1:5 with Methanol: Water (1 : 1, v / v) containing an internal standard (Taurocholic acid-d4) for LC- MS / QqQ analysis. Metabolites were analyzed with a 1290 Infinity II LC (Agilent Technologies) equipped with reverse phase column (Zorbax Eclipse C18 2.1 x50mm; Agilent Technologies) and 6490 Triple Quad LC / MS (Agilent Technologies). LC separation was performed with Water + 0.01% Formic Acid (solvent A) and Acetonitrile (solvent B). The separation gradient was as follows: 0-100%B in 1.5 mins, 100%B hold 0.5 minutes, return to0%B in O. lmin, and equilibrate 0%B for 1.9 minutes. ESI-MS conditions were performed in negative mode, and the dynamic multiple reaction monitoring (dMRM) mode was used to monitor the precursor and product ion transitions. Peak areas were quantitated using Agilent Quantitative Analysis software and areas were normalized to internal standards and compared to an external standard curve for quantitation.

[0258] The results show that ellagic acid levels decreased significantly in 6 out of 8 donors (75%). Results for 2 donors are illustrated in FIGS. 12A and 12B. These two fecal samples were enriched in Gordonibacter species. The supernatants from these two fecal samples were analyzed and increased concentrations of urolithin A were detected. The results are shown in FIGS. 13A and 13B.EXAMPLE 9: Physical and Chemical Properties of CLX106Cu and CLX106

[0259] Solubility and viscosity are important for formulation of food ingredients, with high solubility and tunable viscosity characteristics being preferred for fluid applications. Generally, polysaccharide rich materials have high viscosity, low solubility, or both, making them unsuitable for fluid applications without significantly affecting the organoleptics of a product. In providing a method to create oligosaccharides from a beneficial polysaccharide source, we provide the ability to deliver high concentrations of beneficial polysaccharide cleavage products to the gut microbiome without significantly modifying the clarity or viscosity of a product.

[0260] Solubility: 1.6 g / 8 oz (6.8 g / L), 2.8 g / 8 oz (11.8 g / L), 5.6 g / 8 oz (23.7 g / L) solutions of CLX 106Cu in water were incubated at 25 °C for 10 minutes with agitation and evaluated for solubility by measuring turbidity. These concentrations were chosen as they exemplify beverages that could use “source of fiber”, “good source of fiber” and “excellent source of fiber” label claims, respectively.

[0261] Viscosity: 1.6 g / 8 oz (6.8 g / L), 2.8 g / 8 oz (11.8 g / L), 5.6 g / 8 oz (23.7 g / L) solutions of CLX 106Cu in water were incubated at 25°C, and the absolute viscosity was measured using a micro VISC-m viscometer from Rheosense in automatic mode. The shear stress was measured across a range of shear rates and verified to be linear (a Newtonian fluid) in the measured regime. The relationship T = - where q is the absolute (dynamic) viscosity (mPa-s), y is the shear rate (s'x), and T is the shear stress (mPa), was used to solve for thedynamic viscosity of the mixture of polysaccharide cleavage products at a given temperature and concentration.

[0262] CLX 106Cu was shown to have substantially low turbidity at all three concentrations, 15.3 NTU (1.6 g / 8 oz), 23.6 NTU (2.8 g / 8 oz), 32.3 NTU (5.6 g / 8 oz) indicating that “source of fiber”, “good source of fiber”, and “excellent source of fiber” claims will not impact beverage clarity.

[0263] CLX 106Cu was shown to have substantially low impact on viscosity at all three concentrations, 1.27 mPa s (1.6 g / 8 oz), 1.28 mPa s (2.8 g / 8 oz), 1.30 mPa s (5.6 g / 8 oz) indicating that “source of fiber”, “good source of fiber”, and “excellent source of fiber” claims, respectively, will not impact beverage viscosity or organoleptics.EXAMPLE 10: Pilot plant production of pomegranate fiber powder and pomegranate fiber syrup.

[0264] The manufacturing processes of pomegranate oligosaccharides (POS) is shown in the flow diagram of FIG. 1A with adaptation for powder or syrup production. Pomegranate pomace is used as the starting material to produce POS. The manufacturing process includes two stages: 1. Polysaccharide extraction / upstream processing from pomegranate pomace, using alpha-amylase, protease and exopeptidase enzymes followed by ethanol precipitation; and 2. Oligosaccharide production process / downstream processing. The pomegranate polysaccharides are depolymerized into an oligosaccharide form. The pomegranate oligosaccharide ingredient is then purified and packaged.

[0265] The first phase of the production of POS entails the enrichment of the polysaccharide fraction by removal of polyphenols, lipids (and other ethanol soluble materials) via ethanol extraction, the removal of starch via amylase hydrolysis, the removal of protein via proteolysis, and the isolation of the pomace polysaccharides by way of ethanol precipitation and centrifugation.

[0266] To produce the soluble POS, the molecular weight of the non-digestible carbohydrate fraction is reduced by way of Fenton depolymerization. During the Fenton depolymerization, copper, hydrogen peroxide, and sodium hydroxide are used to induce carbohydrate structure independent depolymerization. Following the depolymerization, the residual insoluble material is removed via centrifugation and filtration. The remainingmaterial is processed on membranes and resin to reduce ash and copper in the final product and then spray dried or evaporated to a stable syrup.

[0267] The pomegranate fiber ingredient is primarily comprised of low-molecular weight fibers and is low in digestible carbohydrates (<3% starch) and sugars (<20% sugars including glucose, fructose, sucrose, maltose, and lactose), deeming the remaining carbohydrates indigestible by human metabolism. Furthermore, extensive carbohydrate characterization was performed on the pomegranate pomace substrate, the intermediate purified pomegranate fiber polysaccharides, and the final product.

[0268] More specifically, in the context of the flow diagram of FIG. 1A, the process of Example 4 was employed using pomegranate pomace as starting material to produce 6 additional batches of pomegranate fiber. For 3 of the batches, the liquid fraction after depolymerization was deionized with MB 10 mixed bed ion exchange resin and then concentrated by evaporation to obtain a syrup (water content 23-24% by weight). For the remaining 3 batches, the liquid fraction after depolymerization was deionized with MB 10 mixed bed ion exchange resin and then dried (spray-dried) to obtain a powder.EXAMPLE 11: Characterization of Pomegranate Soluble Fiber

[0269] The pomegranate fiber syrup and the pomegranate fiber powder products were characterized. To further understand the composition of the carbohydrate fraction, the pomegranate pomace underwent acid hydrolysis and subsequent analysis of the liberated monosaccharides. The results indicated that the carbohydrate fraction was comprised of roughly 57.75% glucose, 13.55% galacturonic acid, 18.82% fructose, 2.33% arabinose, 3.13% galactose, 2.43% mannose, 1.35% xylose, and less than 1% other low abundance monosaccharides (Table 8), which confirms the high abundance of pectin like polymers in pomegranate pomace. In addition, the pomegranate pomace was subjected to glycosidic linkage analysis. Glycosidic linkage analysis describes the position of the glycosidic linkage between each sugar unit. For example, starch would contain 4-, 4,6-, and terminal (end caps with no linkage) glucose residues, cellulose would contain 4-, and terminal glucose residues, while pomegranate fiber would contain 13.72% terminal arabinose, 8.89% terminal glucose, 0.96% terminal xylose, 1.10% terminal glucose residues, and 0.09% terminal galacturonic acid. Linked galacturonic acid is unique and confirmatory for the presence of pectin-like structure. Abundances of each linkage can be found in Table 9.

[0270] Prior to depolymerizing the pomegranate pomace, the fiber components were purified into intermediate pomegranate polysaccharides by employing enzymatic and ethanol removal of starch, proteins, lipids and polyphenols. The resulting pomegranate polysaccharide had a highly similar monosaccharide profile to pomegranate pomace and showed roughly 14.34% glucose, 38.46% galacturonic acid, 2.09% fructose, 14.31% arabinose, 9.20% galactose, 1.71% mannose, 16.52% xylose, and 3.36% other low abundance monosaccharides (Table 8). The glycosidic linkage analysis revealed that the starch content was lowered to 23.75% 4-linked glucose, and that 4-xylose, and 3,4-xylose were enriched to 28.66% and 10.18%, respectively (Table 9).

[0271] Finally, the final pomegranate fiber syrup product and powder product were characterized. In the syrup, monosaccharide composition was analyzed to demonstrated that 25.60% glucose, 27.05% galacturonic acid, 22.74% arabinose, and 11.77% galactose were the most abundant oligosaccharides (Table 8). Glycosidic linkage analysis showed that there was a reduction in the overall degree of polymerization, indicated by the reduction in linear and bisecting signals and an increase in the terminal signals. Consistent with the monosaccharide analysis, glucose and arabinose and xylose containing linkages were observed in highest abundance. For example, terminal arabinose increased from 14.73% to 32.97% and terminal glucose increased from 2.44% to 3.41%, while the 4-xylose decreased from 28.66% to 24.74%, in the pomace polysaccharide and oligosaccharides, respectively, which indicates a cleavage of glycosidic bonds (Table 9). In the powder, the monosaccharide composition was analyzed to demonstrated that 29.12% glucose, 24.38% galacturonic acid, 25.55% arabinose, and 10.87% galactose were the most abundant oligosaccharides (Table 8). Glycosidic linkage analysis showed that there was a reduction in the overall degree of polymerization, indicated by the reduction in linear and bisecting signals and an increase in the terminal signals. Consistent with the monosaccharide analysis, glucose and arabinose and xylose containing linkages were observed in highest abundance. For example, terminal arabinose increased from 14.73% to 25.69% and terminal glucose increased from 2.44% to 5.07%, while the 4-xylose decreased from 28.66% to 19.13%, in the pomace polysaccharide and oligosaccharides, respectively, which indicates a cleavage of glycosidic bonds (Table 9).Table 8. Monosaccharide analysis of pomegranate fiberTable 9. Glycosidic linkage analysis of pomegranate fiber*ND=not detected

[0272] This detailed analysis of the carbohydrate structures indicates that the detailed molecular structure of the fibers remained the same, except for being shortened during depolymerization. The monosaccharides were measured using the method as described in Amicucci et al. (2019), while the glycosidic linkages were measured using the method as described in Galermo et al. (2018).

[0273] The starting raw material, pomegranate pomace, was composed of roughly 83.15% carbohydrates (of which approx. 69% is non-digestible saccharides, 10.85% is sugar, and 3.3% is starch), approx. 7.31% protein, and 4.52% lipid. Nutritional analysis was performed on the pomegranate fiber powder and pomegranate fiber syrup batches as detailed in Table 10 (powder) and Table 11 (syrup).Table 10. Nutritional data for pomegranate fiber powderTable 11. Nutritional data for pomegranate fiber syrup*residual weight percent is water

[0274] Pomegranate fiber is low in digestible carbohydrates (less than 3% by weight) and low in sugars (less than 20% by weight). Pomegranate syrup of this example contained 23- 24% by weight water.EXAMPLE 12: Additional characterization of pomegranate fiber (powder and syrup)

[0275] The average molecular weight of POS was approximately 0.5 kDa - 50 kDa. Specifications and test methods for the product are presented in Tables 12-16.Table 12. Specifications and available analytical data for pomegranate fiber ingredientsTable 13. Analytical data for three non-consecutive batches of pomegranate fiber ingredients (Powder)Table 14. Analytical data for three non-consecutive batches of pomegranate fiber ingredients (syrup)Table 15. Analytical data of residual processing aids for three non-consecutive batches of pomegranate fiber powder*ND=not detectedTable 16. Analytical data of residual processing aids for three non-consecutive batches of pomegranate fiber syrup*ND=not detected

[0276] The analytical (physical, chemical, and microbiological) results for the POS ingredients summarized in the above tables confirm that the ingredient meets the proposed analytical specifications and demonstrates the consistency of production.

[0277] Long term ambient and accelerated conditions were employed to test the stability of pomegranate fiber powder and syrup, using material from one batch (Batch #9 (powder) and 6 (syrup)) in metalized PET pouches. The mono and disaccharide composition (total sugars, a measure of oligosaccharide degradation) was tested for both the fiber and syrup forms (Tables 16-17). Proposed storage conditions are room temperature in a closed container. Currently, the accelerated stability testing data supports stability of approximately 18 months. The empty cells in Tables 17 and 18 indicate timepoints at which data will be collected in the future.Table 17. Stability testing data for pomegranate fiber powder ingredientTable 18, Stability testing data for pomegranate fiber syrup ingredientEXAMPLE 13: Use of Pomegranate Fiber Powder Product and Syrup Product

[0278] The daily consumption of pomegranate fiber may increase the consumption of dietary fiber as a result of its introduction. The pomegranate fiber will be used in a number of products including beverages and beverage bases, dairy product analogs, fruit and water ices, processed fruits and fruit juices, baked goods and baking mixes, fresh fruits and fruit juices, frozen dairy desserts and mixes, sweet sauces, soft candy, and gravies and sauces (Table 19).Table 19. Pomegranate fiber proposed food uses and use levels

[0279] To determine the intended addition of POS / pomegranate fiber per food, the serving size of each food was estimated using the What We Eat in America (WWEIA) Food and Nutrient Database (FNDDS) (What’s in the Foods You Eat Search Tool, 2024), which relates food codes to serving sizes. In cases where data were not available, analogs were used to determine proportions. For example, food code 11400000 (Yogurt, NFS [not further specified]) was matched in the FNDSS database with a typical serving size of 170 g. However, 11420000 (Yogurt, vanilla, lemon, or coffee flavor) was not. The assumption was made that the consumption of Yogurt, NFS, would be equal to vanilla, lemon, or coffee yogurt variants. By either direct match or definition of a suitable analog, serving sizes were determined for all proposed foods. The grams of pomegranate fiber (for yogurt, 12 g / serving) were divided by the serving size to determine the use level (%). In this case, the use level is estimated as 12 / 170 = 0.0705. This calculation was performed for every food in Table 19.

[0280] Dietary survey data were obtained from WWEIA, the dietary interview portion of NHANES. NHANES is carried out in two-year cycles (biennials) by the Centers for Disease Control and Prevention (CDC), to characterize the general health and nutritional status of children and adults across the US. The dietary intake survey portion of NHANES is a joint effort between the CDC and the US Department of Agriculture (USDA). All NHANES biennials for which dietary intake data were available were included in this analysis (CDC, 2015-2016 and 2017-2018).

[0281] The first day of the WWEIA dietary questionnaire was administered in person, in conjunction with the participants’ interviews and examinations for the other NHANES lifestyle and laboratory assessments. The second day of the survey was collected via a phone interview at some point 3-10 days after the first survey day. Data collected during the dietary interview include foods consumed by the participant, encoded by a food code, and amounts eaten. Respondents who provided complete records for both days were designated reliable by WWEIA; only reliable respondents who also provided body-weight data were considered in this analysis.

[0282] Both days of the NHANES WWEIA dietary interviews from each biennial were analyzed. Participants’ consumption of pomace powder was averaged over the two response days — i.e., (Dayl consumption + Day2 consumption) / 2.

[0283] The 2015-2016 and 2017-2018 NHANES biennial dietary intake data were included in this analysis (CDC, 2015-2016, 2017-2018). The dietary and sample weighting data from the two biennials were combined according to the NHANES analytic guidelines for combining surveys. From the combined data set, we estimated survey-design-weighted descriptive statistics for the population consumption per day. Population statistics were estimated using the “survey” package (Lumley, 2004) in the R environment for statistical computing (R Core Team, 2019), using the appropriate adjustment to sampling weights for combining biennials, and then incorporating survey sampling units and strata from the survey design to ensure that subpopulations and areas were represented correctly. Descriptive statistics (mean, 90th percentile) were calculated for the subset of consumers of pomegranate fiber and for the entire population; these were broken down by age range and body -weight adjustment.

[0284] Table 20 and Table 21, respectively, present the EDIs for pomegranate fiber in grams per day and grams per kilogram body weight per day for the following age groups in the US populations: 2 years and older, 2 to 5 years, 6 to 18 years, and 19 years and older (up to 80 years). The “number of users” refers to the number of survey participants in a given age group who consumed a food item in a given food category. The “percent users” is the percentage of pomace powder users out of the total number of reliable survey participants (users and non-users) belonging to a given age group.Table 20. Estimated daily intake for pomegranate fiber (g / day)Table 21. Estimated daily intake for pomegranate fiber (g / kg-bw / day)

[0285] The total per-user mean and 90th percentile EDIs of pomegranate fiber for the US population ages 2 years and over were determined to be 30.2 and 57.4 g / day (0.459 and 0.887 g / kg body weight / day), respectively.

[0286] It should be noted that this total EDI is based on a theoretical assumption that pomegranate fiber would be added to all proposed foods (i.e. market saturation assumption) and at the maximum proposed use levels. Thus, the actual intake of pomegranate fiber would be expected to be much lower and result in fewer adverse effects.REFERENCES

[0287] All references listed below, or anywhere else throughout this description, are hereby incorporated by reference in their entireties for all purposes:

[0288] Amicucci, M. J., Galermo, A.G., et al. (2019). "A rapid-throughput adaptable method for determining the monosaccharide composition of polysaccharides." International Journal of Mass Spectrometry 438: 22-28.

[0289] Guarner, F. and Malagelada, J.R. (2003). “Gut flora in health and disease.” The Lancet 361(9356): 512-519.

[0290] Galermo, A. G., Nandita, E., et al. (2018). “Liquid chromatography -tandem mass spectrometry approach for determining glycosidic linkages.” Analytical Chemistry 90(21): 13073-13080.

[0291] Hasnaoui N, Wathelet B, & Jimenez- Araujo A., Valorization of pomegranate peel from 12 cultivars: dietary fibre composition, antioxidant capacity and functional properties. Food Chem., (2014), 160:196-203.

[0292] Mo et al, Pomegranate Peel as a Source of Bioactive Compounds: A Mini Review on Their Physiological Functions, Front Nutr., (2022), Jun 9;9.

[0293] Papaioannou EH, Mitrouli ST, Patsios SI, Kazakh M, & Karabelas AJ, Valorization of pomegranate husk - Integration of extraction with nanofiltration for concentrated polyphenols recovery, Journal of Environmental Chemical Engineering (2020),

[0294] Pathak, P. D., Mandavgane, S. A., & Kulkami, B. D., Valorization of pomegranate peels: A biorefinery approach, Waste and Biomass Valorization, (2017), 8(4), 1127-1137.STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

[0295] All references throughout this application, for example patent documents and patent application publications, and non-patent literature documents or other source material, are hereby incorporated by reference in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the invention in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference). All patents and publications mentioned in the specification are indicative of the levels of skill ofthose skilled in the art to which the invention pertains. References cited are incorporated by reference in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employed, if needed, to exclude (e.g., to disclaim) specific embodiments that are in the prior art. For example, when a compound is claimed, it should be understood that compounds known in the prior art, including certain compounds disclosed in the references disclosed (particularly in referenced patent documents), are not intended to be included in the claim.

[0296] This application incorporates each of the following by reference its entirety for all purposes, to the extent not inconsistent with the present invention: PCT Patent Application PCT / US23 / 21949, filed May 11, 2023 (WO2023 / 220318), PCT Patent Application No. PCT / US22 / 29065, filed May 12, 2022 (WO 2022 / 241163 Al), PCT Patent Application No. PCT / US2018 / 038350, filed June 19, 2018 (WO 2018 / 236917 Al), PCT Patent Application No. PCT / US2020 / 035748, filed June 2, 2020 (WO 2020 / 247389 Al), and PCT Patent Application No. PCT / US2020 / 060297, filed November 12, 2020 (WO 2021 / 097138 Al).

[0297] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although this invention has been specifically disclosed by preferred embodiments, example embodiments and optional features, modification and variation of the concepts disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the claims. The specific embodiments provided are examples of useful embodiments of the invention and it will be apparent to one skilled in the art that the invention may be carried out using a large number of variations of the devices, device components, methods steps set forth in the description.

[0298] As used in this specification and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Also, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. The expression “of any of claims XX- YY” (wherein XX and YY refer to claim numbers) is intended to provide a multiple dependent claim in the alternative form, andin some embodiments is interchangeable with the expression “as in any one of claims XX- YY.”

[0299] When a group of substituents is disclosed, it is understood that all individual members of that group and all subgroups, are disclosed separately. When a Markush group or other grouping is used, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the invention. Specific names of compounds are intended to be examples, as it is known that one of ordinary skill in the art can name the same compounds differently.

[0300] Every device, system, formulation, combination of components, or method described or exemplified can be used to practice the invention, unless otherwise stated.

[0301] Whenever a range is given in the specification, for example, a temperature range or composition range, the range is inclusive of the listed end points and all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the invention.

[0302] One of ordinary skill in the art will appreciate that starting materials, biological materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All functional equivalents known in the art, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

We claim:

1. A method for generating a mixture of polysaccharide cleavage products from pomegranate, the method comprising:(i) reacting one or more polysaccharide in a pomegranate starting material containing polysaccharide with a Fenton’s reagent comprising a peroxide agent and a metal to generate reaction products, and (ii) cleaving the reaction products with a cleavage agent to generate the mixture of polysaccharide cleavage products from pomegranate.

2. The method of claim 1, further comprising pretreating the pomegranate starting material prior to reacting the one or more polysaccharide therein.

3. The method of claim 2, wherein pre-treatment comprises extracting polyphenols from the pomegranate starting material containing polysaccharide to generate a polyphenol- reduced pomegranate starting material containing polysaccharide prior to reacting the one or more polysaccharide in the polyphenol -reduced pomegranate starting material.

4. The method of any of claims 2 or 3, wherein pre-treatment comprises or further comprises extracting pectin from the pomegranate starting material containing polysaccharide or the reduced-polyphenol pomegranate starting material containing polysaccharide to generate a pectin-enriched pomegranate starting material prior to reacting the one or more polysaccharide in the pectin-enriched pomegranate starting material.

5. The method of any one of claims 2-4, wherein pre-treatment comprises or further comprises removal of one or more polysaccharides other than pectin from the polysaccharide containing material obtained from pomegranate.

6. The method of any one of claims 2-5, wherein pre-treatment comprises or further comprises removal of protein from the polysaccharide containing material obtained from pomegranate prior to reacting the one or more polysaccharide therein.

7. The method of any one of claims 1-6, further comprising purifying the mixture of polysaccharide cleavage products to provide a purified mixture of polysaccharide cleavage products.

8. The method of any one of claims 1-7, wherein the polysaccharide cleavage products are subjected to filtration or ethanol precipitation followed by filtration to remove ethanol insoluble material from the polysaccharide cleavage products.

9. The method of any one of claims 1-8, wherein the pomegranate starting material comprises pomegranate pomace, pomegranate husk, pomegranate arils containing seed or a combination thereof.

10. The method of any one of claims 1-9, wherein the polysaccharide cleavage products comprise less than 10% by weight of residual monosaccharides, and / or the polysaccharide cleavage products comprise less than 15% by weight of ash.

11. The method of any one of claims 1-10, wherein the mixture of polysaccharide cleavage products contains 40% or less by weight of polysaccharides with molecular weight of lOOkDa or more and / or each of the one or more polysaccharide cleavage products have a degree of polymerization (DP) of 3 to 100, and / or the mixture of polysaccharide cleavage products has solubility in water of 20 g / L or higher at 25°C and / or the viscosity of the mixture of polysaccharide cleavage products is less than 4 millipascal-second (mPa.s) at 25°C when dissolved in water at a concentration of 20 g / L, and / or a 20 g / L mixture of the mixture of polysaccharide cleavage products in water has turbidity of 40 NTU or less.

12. Soluble pomegranate fiber comprising a mixture of polysaccharide cleavage products, from pomegranate, wherein a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less and a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C.

13. The soluble pomegranate fiber of claim 12, wherein the mixture of polysaccharide cleavage products contains 40% or more by weight of polysaccharide cleavage products having molecular weight less than 100 kDa, and / or each of the polysaccharides cleavage products of the mixture of polysaccharide cleavage products has a degree of polymerization (DP) of 3 to 100, and / or a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less, and / or a 20 g / L mixture of the soluble pomegranate fiber in water has dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C.

14. The soluble pomegranate fiber of any one of claims 12-13, where the mixture of polysaccharide cleavage products from pomegranate is formed by cleavage of a polysaccharide containing material obtained from pomegranate and wherein the material obtained from pomegranate comprises pomegranate pomace, pomegranate husk, pomegranate arils containing seeds, or a combination thereof.

15. The soluble fiber of any one of claims 12-14, wherein the polysaccharide cleavage products of the mixture of polysaccharide cleavage products comprise at least one glycosidic linkage selected from the group consisting of 4-linked glucose, 4-linked galactose, 6-linked galactose, 4-linked xylose, 3,6-linked glucose, 3,6-linked galactose, 4-linked rhamnose, 2, 3, 4-linked rhamnose, and a combination thereof.

16. The soluble fiber of any one of claims 12-14, wherein the mixture of polysaccharide cleavage products comprises 25-50% 4-linked glucose and / or wherein the mixture of polysaccharide cleavage products comprises 10-20% 4-linked galactose, and / or wherein the mixture of polysaccharide cleavage products comprises 2-10% 6-linked galactose, and / or wherein the mixture of polysaccharide cleavage products comprises 1-10% 4-linked xylose, and / or wherein the mixture of polysaccharide cleavage products comprises 2-7% 3,6-linked galactose.

17. The soluble fiber of any one of claims 12-16, wherein each of the one or more polysaccharide cleavage products of the mixture of polysaccharide cleavage products comprises one or more glycosidic linkage selected from the group consisting of 4- linked glucose, 4-linked galactose, 6-linked galactose, 4-linked xylose, 3,6-linked glucose, 3,6-linked galactose, 4-linked rhamnose, 2, 3, 4-linked rhamnose, and a combination thereof.

18. The soluble fiber of any one of claims 12-17, wherein the mixture of polysaccharide cleavage products comprises 15-50% by weight galacturonic acid units, and / or wherein the mixture of polysaccharide cleavage products comprises 20-50% by weight glucose units, and / or +wherein the mixture of polysaccharide cleavage products comprises 1-10% by weight xylose units, and / or wherein the mixture of polysaccharide cleavage products comprises 15-20% by weight galacturonic acid units and 20-30% by weight glucose units; and / or wherein the mixture of polysaccharide cleavage products comprises25-35% by weight arabinose units, and / or wherein the mixture of polysaccharide cleavage products comprises 10-20% by weight galactose units, and / or wherein the mixture of polysaccharide cleavage products comprises 1-5% by weight rhamnose units.

19. The soluble fiber of any one of claims 12-18, which comprises 15% or less by weight of ash and / or, wherein the mixture of polysaccharide cleavage products comprises less than 10% by weight of mono- and disaccharides.

20. A food beverage, or medicinal product comprising soluble pomegranate fiber comprising a mixture of polysaccharide cleavage products from pomegranate, wherein a 20 g / L mixture of the soluble pomegranate fiber in water has turbidity of 40 NTU or less and a dynamic viscosity of 4 millipascal-second (mPa.s) or less at 25°C.

21. A food, beverage, or medicinal product comprising the soluble fiber of any one of claims 13-19.

22. The food, beverage, or medicinal product of claim 20 or 21, wherein the food product comprises a yogurt, a frozen yogurt, an ice cream, a fruit sauce, a syrup, a chocolate, a tomato sauce, a ketchup, a barbecue sauce, a bread, a granola bar, an energy bar, or a breakfast cereal.

23. The food, beverage, or medicinal product of claim 20 or 21, wherein the beverage product comprises an infant formula, a follow-on formula, a toddler’s beverage, a dairy or nondairy milk, a fermented milk, a fruit or vegetable juice, a fruit-based drink, a protein drink, an energy drink, a sports drink, a sparkling water, a vitamin water, a tea, or a coffee drink.

24. The food, beverage, or medicinal product of claim 20 or 21, wherein the medicinal product is in the form of a gummy, capsule, pill or tablet.

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