Fiber-bound polyphenol compositions and methods

Abstract

Compositions and methods for dietary fiber materials containing a plurality of distinct dietary fiber materials derived from distinct plant materials. At least some of the dietary fiber materials comprise fiber-bound polyphenols. Advantageously, the materials presented herein increase, upon oral administration, short chain fatty acid concentration, microbial biodiversity, and / or microbial abundance in the gut of the subject, even when compared to administration of a weight-equivalent dosage of inulin or psyllium.

Description

FIBER-BOUND POLYPHENOL COMPOSITIONS AND METHODS

[0001] This application claims priority to our copending US provisional application with the serial number 63 / 648,074, which was filed 5 / 15 / 2024, and which is incorporated by reference herein in its entirety.Sequence Listing

[0002] The content of the XML file of the sequence listing named 100700.0084PCT.xml, which is 3KB in size was created on May 11, 2025 and electronically submitted via Patent Center along with the present application, and is incorporated by reference in its entirety.Field of the Invention

[0003] The field of the invention is compositions and methods for nutritional supplements, especially as it relates to fiber-containing supplements that include fiber-bound polyphenols.Background of the Invention

[0004] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0006] The human gastrointestinal (GI) tract has an estimated surface of more than 200 square meters and represents the interface between the body and the external environment, hosting a complex polymicrobial ecology that includes bacteria, archaea, fungi, protists, and viruses. The population of human gut microorganisms is estimated at approximately 1013-1014, and thus, outnumber the somatic cells of the host by over 10 times. Therefore, it is not unexpected thatthe intestinal microbiome can directly beneficially affect or adversely interfere with human health and disease.

[0007] While highly abundant, a significant amount of the microbial flora is physically separated from the intestinal epithelium by a mucosal layer, which may form a nutrient source for the microbiome. Under healthy conditions, bacteria will only exceptionally cross the mucosal layer to specifically interact with epithelial cells. In addition, the microbiome is also able to communicate with its host via several metabolic products (postbiotic), including shortchain fatty acids (SCFAs) that may originate from the host’s diet and dietary' fiber degradation. It is generally thought that close interaction between the gut bacteria and the host generates many benefits through the control of nutrient uptake and metabolism, strengthening intestinal integrity, preventing pathogen propagation, promoting immunological tolerance to antigens, and regulating host immunity'.

[0008] The Human Microbiome Project and MetaHit have led to an improved overvieyv of the human-associated microbial repertoire. The compiled data from these studies revealed that the human microbiota comprises twelve different phyla, of yvhich 93.5% belong to Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, with Firmicutes and Bacteroidetes dominating the gut microbiota in healthy subjects. The Lachnospiraceae family is a phylogenetically and morphologically heterogeneous taxon belonging to the clostridial cluster XlVa of the phylum Firmicutes. Within Lachnospiraceae, Blautia, Coprococcus, Dorea, Lachnospira, Oribacterium, Roseburia. and L-Ruminococcus are the main genera that have been detected in the human intestine by metagenomics analyses.

[0009] In general, it has also been observed that microbial abundance and diversity in healthy subjects is significantly larger than abundance and diversity' in unhealthy populations. Likeyvise, lifestyle and diet can have a profound influence on the gut microbiome, which in turn is often associated with various diseases. For example, the gut microbiome has been thought to play a role in the etiology of various diseases, including inflammatory' bowel disease, type 2 diabetes, hypertension, and colorectal cancer. Additionally, individual clinical blood markers, such as those for diabetes and cholesterol, have been found to be associated with abundances of certain gut bacteria {Nature Communications 2020 11:5206). In this context, more recent studies established that certain prebiotic dietary fibers can act as carbon sources for primary and secondary fermentation pathways in the colon, and support digestive health inmany ways, and such prebiotics include fructooligosaccharides (FOS), xylooligosaccharides (XOS). galactooligosaccharides (GOS), and inulin (see e.g, Curr Dev Nutr 2018; 2:nzy005.).

[0010] Not surprisingly, numerous nutritional approaches can be taken to positively affect the gut microbiome, and commonly used compositions include various soluble and insoluble fibers such as methylcellulose (e.g., CITRUCEL®), psyllium (e.g.. METAMUCIL®), and inulin (e.g, FIBER CHOICE®). However, while effective to relieve to at least some degree certain intestinal symptoms such as congestion or diarrhea, use of such fibers is often associated with bloating, flatulence, and / or cramping. Worse yet, and particularly with extended use, bowel irritation and even inflammation have been reported.

[0011] In other known approaches, a combination of cocoa polyphenols and soluble dietary fiber was used in the treatment or prevention disorders associated with an above-normal number of granulocytes in a tissue as is described in US 2019 / 0008824. In still further known approaches, US 6,087.092 describes the use of hemicelluloses (preferably from Cam: .spec.) in combination with certain polyphenols to decrease presence of harmful bacteria such as Clostridium spec, in the gastrointestinal tract, and US 10,441,602 describes various human gastrointestinal microbiome modulating compositions in which a combination of fermentable fiber, beta glucans, and phenolics from certain berry’ pomaces are administered to increase the ratio of Bacteroidetes to Firmicutes.

[0012] US 2019 / 0008186 teaches a combination of a high solubility' fiber source and a low solubility fiber source and a polyphenol source to so deliver the polyphenols to the lower gastrointestinal tract for immune optimization, and US 2002 / 0168429 teaches compositions in which juice is expressed from a fruit, concentrated, and then combined with the pomace of the fruit from which the juice was expressed to so obtain a juice infused pomace. W02008 / 052990 teaches use of certain fruit pomaces with high polyphenol content (>60%) as a feed additive to animal feed to so reduce use of antibiotics. While these and other composition may provide selected benefits to at least some degree, all or almost all of them have either a relatively moderate effect on the microbiome or may produce undesirable side effects such as bloating and flatulence, particularly at higher doses.

[0013] Thus, even though various systems and methods of dietary fibers are known in the art, all or almost all of them suffer from several drawbacks. Therefore, there remains a need for improved compositions and methods for dietary fiber that beneficially affects the intestinalmicrobiome, and especially compositions and methods that promote gentle fermentation (i.e., avoid excessive bloat / gas) and that have a significant prebiotic and postbiotic effect.Summary of The Invention

[0014] The inventive subject matter is directed to various compositions and methods of improved dietary fibers that include fiber-bound polyphenols. Notably, such improved dietary fibers are shown to be effective to increase the short chain fatty acid concentration, microbial biodiversity, and / or microbial abundance in the gut of the subject as compared to administration of a weight-equivalent dosage of inulin and / or psyllium, all while producing no or only minimal pH changes and / or gas.

[0015] In one aspect of the inventive subject matter, the inventors contemplate a dietary fiber composition that comprises a plurality of distinct dietary fiber materials, optionally in combination with a nutritionally acceptable carrier, wherein each of the dietary fiber materials is derived from distinct plant materials, and wherein each of the dietary fiber materials comprises fiber-bound polyphenols; and wherein the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to a subject at an effective dosage, increase short chain fatty acid concentration, microbial biodiversity, and / or microbial abundance in a gut of the subject as compared to administration of a weight-equivalent dosage of inulin and / or psyllium. Most typically, the distinct dietary fiber materials will provide distinct dietary fiber types to so provide a prebiotic blend that can be utilized by a variety of microbial species throughout the lower gastrointestinal tract. In some embodiments, it is contemplated that the inventive subject matter would include a nutritionally acceptable carrier in combination with a plurality of the distinct blend of dietary7fiber materials described above.

[0016] Most typically, the distinct plant materials comprise edible fruit materials and / or edible vegetable materials, which may or may not be processed plant materials (e.g. comminuted plant materials, expressed plant materials, and / or extracted plant materials). In some embodiments, the distinct dietary7fiber materials are dehydrated fiber materials. Typically, but not necessarily, the fiber-bound polyphenols in the dietary fiber materials occur naturally in the plant materials from which the dietary fiber materials are derived. Where desired, at least one of the distinct dietary fiber materials may further comprise a soluble saccharide and / or a non-polyphenol flavor component. In further embodiments, the fiber-bound polyphenols are present in the dietary fiber materials in an amount of at least 0.1 wt%. It is still furthercontemplated that some or all of the distinct dietary fibers are comminuted (e g., to an average particle size of equal or less than 500 pm). Typically, but not necessarily, the effective dosage is equal or less than 10,000 mg, for example, between 250 mg and 6,000 mg, and / or the composition is formulated as a powder.

[0017] In some aspects the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the short chain fatty acid concentration in the gut of the subject. In further aspects, the distinct dietary7fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subj ect at an effective dosage, increase the microbial biodiversity (as evidenced by total count of unique taxa) in the gut of the subject. In still further aspects, the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the microbial abundance in the gut of the subject.

[0018] Therefore, the inventors also contemplate a dietary fiber composition that comprises a nutritionally acceptable carrier in combination with a plurality7of distinct dietary fiber materials, wherein each of the dietary7fiber materials is derived from distinct plant materials, and wherein each of the dietary fiber materials comprises fiber-bound polyphenols; and wherein the distinct dietary fibers and fiber-bound polyphenols are dehydrated and ground to have an average particle size of equal or less than 500 pm, thereby enhancing, upon oral administration to a subject, short chain fatty7acid concentration, microbial biodiversity7, and / or microbial abundance in a gut of the subject as compared to corresponding unground dietary fiber materials on a weight-equivalent basis.

[0019] With respect to the plant and fiber materials, the same considerations as noted above apply. In addition, it is contemplated that the dehydrated fiber materials are air-dried materials, heat-dried materials, vacuum dried materials, infrared dried materials, microwave-dried materials, or freeze-dried materials. Most typically, the dehydrated fiber materials will have a residual water content of equal or less than 10 wt%. Where desired, at least one of the distinct dietary fiber materials further comprises a soluble saccharide and / or a non-polyphenol flavor component, and / or the fiber-bound polyphenols can be present in the dietary fiber materials in an amount of at least 0. 1 wt%. In further embodiments, the distinct dietary fibers and fiberbound polyphenols have an average particle size of equal or less than 500 pm (e.g, average particle size of between 100 pm and 500 pm). Most typically, the effective dosage is equal or less than 10,000 mg, such as between 250 mg and 6,000 mg.

[0020] In some aspects the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the short chain fatty acid concentration in the gut of the subject. In further aspects, the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the microbial biodiversity in the gut of the subject. In still further aspects, the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the microbial abundance in the gut of the subject (e.g., relative to inulin and / or psyllium). For example, contemplated compositions may enhance the short chain fatty acid concentration, the microbial biodiversity, and / or the microbial abundance by at least 10% as compared to corresponding unground dietary fiber materials on a weight-equivalent basis.

[0021] Viewed from a different perspective, the inventors also contemplate a dietary7fiber composition that comprise a nutritionally acceptable carrier in combination with a plurality of distinct dietary fiber materials, wherein each of the dietary fiber materials is derived from distinct plant materials, and wherein at least one of the dietary fiber materials is impregnated with one or more polyphenols.

[0022] Most preferably, the distinct plant materials comprise edible fruit materials and / or edible vegetable materials, which may or may not be processed plant materials. In certain embodiments, at least one fiber material may be impregnated with a single type of polyphenol while in other embodiments at least one fiber material is impregnated with multiple and distinct types of polyphenols. Similarly, in some aspects the one or more polyphenols are from a plant from which at least one of the distinct plant materials are derived from, whereas in other aspects the at least one of the dietary fiber materials is thermally or chemically impregnated with the at least one or more polyphenols.

[0023] Therefore, the inventors also contemplate a dietary fiber composition that comprises a dietary fiber material that is impregnated with one or more polyphenols. As noted above, the one or more polyphenols may be from a single plant type or from multiple and distinct plant types.

[0024] Consequently, the inventors also contemplate a method of preparing a dietary fiber composition that includes the steps of providing a plurality of distinct dietary fiber materials, wherein each of the dietary fiber materials is derived from distinct plant materials; rapidlydrying, or freezing and subsequently drying, the distinct dietary fiber materials to so produce respective dehydrated dietary fiber materials, wherein each of the dehydrated dietary fiber materials comprises fiber-bound polyphenols; and comminuting and combining the dehydrated dietary fiber materials to thereby form the dietary fiber composition.

[0025] As will be readily appreciated the step of rapidly drying is performed under a protocol that prevents microbial spoilage and is typically performed to achieve a residual water content of equal or less than 10 wt%. Moreover, it is contemplated that the step of rapidly drying may use air-drying, pressure drying, heat- drying, vacuum drying, infrared drying, microwave drying, and / or freeze- drying.

[0026] Thus, the inventors also contemplate a method of preparing a dietary fiber composition that includes the steps of providing a dietary7fiber material; rapidly dehydrating, or freezing and subsequently drying, the distinct dietary fiber materials in the presence of one or more exogenously added polyphenols to so produce a dehydrated dietary fiber material with fiberbound polyphenols; and optionally comminuting the dehydrated dietary fiber material with fiber-bound polyphenols.

[0027] Furthermore, the inventors also contemplate a method of preparing a dietary supplement that includes a step of formulating the dietary fiber composition as presented herein into liquid or solid to so form an article suitable for oral ingestion.

[0028] For example, where the article is a solid article, contemplated articles include a capsule, a ready-to-mix powder, a snack bar, an energy bar, a chew, a gummy, a confectionary article, a baked good, or a cereal product. In another example, where the article is liquid article, suitable articles include an energy7drink, a juice beverage, a coffee, a fermented beverage, a tea, or a soda.

[0029] In still another aspect of the inventive subject matter, the inventors also contemplate a method of increasing short chain fatty7acid concentration, microbial biodiversity7, and / or microbial abundance in a gut of a subject that includes the steps of administering to the subject a plurality of distinct dietary fiber materials: wherein each of the dietary fiber materials is derived from distinct plant materials, and wherein each of the dietary fiber materials comprises fiber-bound polyphenols; and wherein the distinct dietary7fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase short chain fatty acid concentration, microbial biodiversity, and / or microbial abundance in thegut of the subject. For example, the plurality of distinct dietary fiber materials as presented herein may be administered at dosage between 250 mg and 6.000 mg.

[0030] Various objects, features, aspects, and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.Brief Description of The Drawing

[0031] FIG.1A is a graph depicting exemplary results for changes in pH in the M-SHIME model for individual donors and average from the donors with an exemplary non-organic fiber composition.

[0032] FIG.1B is a graph depicting exemplary results for changes in gas production in the M- SHIME model for individual donors and average from the donors with an exemplary non- organic fiber composition.

[0033] FIG.2A is a graph depicting exemplary results for changes in total SCFA in the M- SHIME model for individual donors and average from the donors with an exemplary non- organic fiber composition.

[0034] FIG.2B is a graph depicting exemplary results for changes in lactate in the M-SHIME model for individual donors and average from the donors with an exemplary non-organic fiber composition.

[0035] FIG.2C is a graph depicting exemplary results for changes in branched SCFA in the M-SHIME model for individual donors and average from the donors with an exemplary non- organic fiber composition.

[0036] FIG.2D is a graph depicting exemplary results for changes in ammonium nitrogen in the M-SHIME model for individual donors and average from the donors with an exemplary non-organic fiber composition.

[0037] FIG.3A is a graph depicting exemplary results for absolute taxa composition in the luminal compartment of the M-SHIME model for individual donors and average from the donors with an exemplary non-organic fiber composition.

[0038] FIG.3B is a graph depicting exemplary results for absolute taxa composition in the mucosal compartment of the M-SHIME model for individual donors and average from the donors with an exemplary non-organic fiber composition.

[0039] FIG.4A is a graph depicting exemplary results for absolute abundance in the luminal compartment at the phylum and family level after 24 hours of the M-SHIME model with an exemplary non-organic fiber composition.

[0040] FIG.4B is a graph depicting exemplary results for absolute abundance in the luminal compartment at the phylum and family level after 48 hours of the M-SHIME model with an exemplary non-organic fiber composition.

[0041] FIG.4C is a graph depicting exemplary results for absolute abundance in the mucosal compartment at the phylum and family level after 48 hours of the M-SHIME model with an exemplary non-organic fiber composition.

[0042] FIG.5A is a graph depicting exemplary results for alpha diversity indices in the luminal compartment at the phylum and family level after 48 hours of the M-SHIME model with an exemplary non-organic fiber composition.

[0043] FIG.5B is a graph depicting exemplary' results for alpha diversity' indices in the mucosal compartment at the phylum and family level after 48 hours of the M-SHIME model with an exemplary non-organic fiber composition.

[0044] FIG.6A is a graph depicting exemplary' results for changes in pH in the M-SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0045] FIG.6B is a graph depicting exemplary results for changes in pH in the M-SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0046] FIG.7 is a graph depicting exemplary' results for average changes in pH in the M- SHIME model over time from the individual donors of FIG.6A and FIG.6B with the exemplary organic fiber composition.

[0047] FIG.8A is a graph depicting exemplary' results for changes in gas production in the M- SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0048] FIG.8B is a graph depicting exemplary' results for changes in gas production in the M- SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0049] FIG.9 is a graph depicting exemplary results for average changes in gas production in the M-SHIME model over time from the individual donors of FIG.8 A and FIG.8B with the exemplary organic fiber composition.

[0050] FIG.10A is a graph depicting exemplary results for changes in lactate in the M-SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0051] FIG.10B is a graph depicting exemplary' results for changes in lactate in the M-SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0052] FIG.11 is a graph depicting exemplary' results for average changes in lactate in the M- SHIME model over time from the individual donors of FIG.10A and FIG. 10B with the exemplary organic fiber composition.

[0053] FIG.12A is a graph depicting exemplary' results for changes in total SCFA in the M- SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0054] FIG.12B is a graph depicting exemplary' results for changes in total SCFA in the M- SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0055] FIG.13 is a graph depicting exemplary' results for average changes in total SCFA in the M-SHIME model over time from the individual donors of FIG. 12A and FIG. 12B with the exemplary organic fiber composition.

[0056] FIG.14A is a graph depicting exemplary results for changes in acetate in the M-SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0057] FIG.14B is a graph depicting exemplary results for changes in acetate in the M-SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0058] FIG.15 is a graph depicting exemplary results for average changes in acetate in the M- SHIME model over time from the individual donors of FIG.14A and FIG.14B with the exemplary organic fiber composition.

[0059] FIG.16A is a graph depicting exemplary results for changes in propionate in the M- SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0060] FIG.16B is a graph depicting exemplary7results for changes in propionate in the M- SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0061] FIG.17 is a graph depicting exemplary7results for average changes in propionate in the M-SHIME model over time from the individual donors of FIG.16A and FIG. 16B with the exemplary organic fiber composition.

[0062] FIG. ISA is a graph depicting exemplary7results for changes in buty rate in the M- SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0063] FIG.18B is a graph depicting exemplary7results for changes in buty rate in the M- SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0064] FIG.19 is a graph depicting exemplary7results for average changes in butyrate in the M-SHIME model over time from the individual donors of FIG. 18A and FIG. 18B with the exemplary organic fiber composition.

[0065] FIG.20A is a graph depicting exemplary' results for changes in branched SCFA in the M-SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0066] FIG.20B is a graph depicting exemplary' results for changes in branched SCFA in the M-SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0067] FIG.21 is a graph depicting exemplary' results for average changes in branched SCFA in the M-SHIME model over time from the individual donors of FIG.20A and FIG.20B with the exemplary organic fiber composition.

[0068] FIG.22A is a graph depicting exemplary results for changes in ammonium-N in the M- SHIME model over time for individual donors from individual donors with an exemplary organic fiber composition.

[0069] FIG.22B is a graph depicting exemplary' results for changes in ammonium-N in the M- SHIME model over time for individual donors from further individual donors with an exemplary organic fiber composition.

[0070] FIG.23 is a graph depicting exemplary' results for average changes in ammonium-N in the M-SHIME model over time from the individual donors of FIG.22A and FIG.22B with the exemplary organic fiber composition.

[0071] FIG.24 is a graph depicting the biomass density in the reactors at experimental start for the individual donors.

[0072] FIG.25 is a graph depicting the alpha diversity in the reactors at experimental start for the individual donors.

[0073] FIG.26 is a graph depicting the microbial community composition (%) of the donor fecal samples at the bacterial phylum level.

[0074] FIG.27 is a graph depicting the microbial community composition (%) of the donor fecal samples at the bacterial family level.

[0075] FIG.28 is a graph depicting the microbial community7composition (%) of the donor fecal samples at the bacterial genus level.

[0076] FIG.29 is a graph depicting bacterial biomass (Logl0(cells / mL)) across donors in the various treatments after 24h of incubation in the luminal environment.

[0077] FIG.30 is a V olcano plot graph showing differences in biomass between each treatment and negative control at 24h of incubation in the luminal environment.

[0078] FIG.31 is a graph depicting bacterial biomass (LoglO(cells / mL)) across donors in the various treatments after 48h of incubation in the luminal environment.

[0079] FIG.32 is a Volcano plot graph, showing differences in biomass between each treatment and negative control at 48h of incubation in the luminal environment.

[0080] FIG.33 is a graph depicting bacterial diversity' in the luminal environment, expressed by four different diversity indices ((A) observed and Chaol (genus richness), (B) Shannon and Simpson (genus evenness)), in the various treatments 24h after start of incubation.

[0081] FIG.34 is a graph depicting bacterial diversity' in the luminal environment, expressed by four different diversity indices ((A) observed and Chaol (genus richness), (B) Shannon and Simpson (genus evenness)), in the various treatments 48h after start of incubation.

[0082] FIG.35 is a graph depicting bacterial diversity' in the mucosal environment, expressed by four different diversity indices ((A) observed and Chaol (genus richness), (B) Shannon and Simpson (genus evenness)), in the various treatments 48h after start of incubation.

[0083] FIG.36 is a graph depicting beta-diversity in form of hierarchical clustering and a DAPC (discriminant analysis of principal components) plot for the various treatment conditions 24h after start of incubation in the luminal environment.

[0084] FIG.37 is a graph depicting beta-diversity' in form of hierarchical clustering and a DAPC (discriminant analysis of principal components) plot for the various treatment conditions 48h after start of incubation in the luminal environment.

[0085] FIG.38 is a graph depicting beta-diversity' in form of hierarchical clustering and a DAPC (discriminant analysis of principal components) plot for the various treatment conditions 48h after start of incubation in the mucosal environment.

[0086] FIG.39 is a summary table illustrating changes in bacterial taxa in the luminal and mucosal compartment at 24 and 48 hrs.

[0087] FIG.40 is a graph depicting relative abundances of selected genera in the luminal compartment for the various treatments after 24h.

[0088] FIG.41 is a is a graph depicting relative abundances of selected genera in the luminal compartment for the various treatments after 48h.

[0089] FIG.42 is a is a graph depicting relative abundances of selected genera in the mucosal compartment for the various treatments after 48h.

[0090] FIG.43 is a graph depicting comparative changes between the non-organic composition of Example and the organic composition of Example 2 with respect to taxa as expressed in OTU in the luminal compartments after 48 hours of the various treatment.Detailed Description

[0091] The inventors have discovered various compositions and methods for improved dietary fiber supplements that beneficially modulate the gut microbiome in a way that is superior to existing, best-in-class options and while also avoiding common side effects often associated with such fibers. Particularly preferred dietary fiber compositions are prepared from a plurality of a blend of distinct plant materials, at least some of which contain fiber-bound polyphenols. Moreover, it is generally preferred (but not necessary) that the enhanced fiber compositions will be comminuted not only to generate a larger surface area available for microbial fermentation but also help expose the fiber-bound polyphenols to the microbiome.

[0092] In this context, it should be appreciated that the term “fiber-bound polyphenol” as used herein refers to a polyphenol that is associated with a dietary fiber in a manner such that the polyphenol remains tightly associated with the dietary fiber even after washing the dietary fiber with the fiber-bound polyphenol with a solvent mixture commonly used to extract polyphenols (water / ethanol mix at 70 / 30 vol% ratio with pH between 4-7, ratio of solvent mixture to dietary fiber with the fiber-bound polyphenol 10: 1 by weight, 25 °C temperature, 10 minutes per wash). Without wishing to be bound by any theory or hypothesis, such tight binding may be due to steric encasement, ionic or electrostatic binding (e.g., via divalent cations bridging vicinal diols of the fiber and the polyphenol), covalent binding. As also used herein, the term “short chain fatty acid” includes all fatty acids that are metabolites of microbial fermentation and that are shorter than 10 carbon atoms. Moreover, such short chain fatty acids may be unsubstituted or substituted with one or more hydroxy, keto, or carboxylic acid groups.

[0093] Moreover, it is contemplated that the fiber-bound polyphenols are gradually released in the gut upon microbial fermentation to so produce a timed-release of the polyphenols from the fiber. Most notably, and regardless of the manner of binding, the polyphenol release from the dietary fibers presented herein will substantially exclusively (i.e., at least 95%) occur in the alkaline intestinal milieu of the small intestine and colon, and substantially not (i.e., less than 5%) occur in the neutral milieu of the oral compartment and the acidic milieu of the gastric compartment. Viewed from a different perspective, it should be appreciated that the fiberbound polyphenol will be protected from the upper gastrointestinal compartment and be selectively available to the lower gastrointestinal compartment to so become selectively available to the microbiome, which will at the same time also benefit from the prebiotic effect of the dietary fiber to which the polyphenols were previously bound. As such, an at least additive or even synergistic effect between the fiber-bound polyphenol and the dietary fiber are specifically contemplated, where such effect includes an increase in the short chain Patty acid concentration, an increase in microbial biodiversity7, and / or an increase in microbial abundance in the gut of the subject as is also described in more detail below.

[0094] For example, a typical dietary fiber composition contemplated herein will include two or more (e.g., three or four, or five, or even more) dietary fiber materials, wherein at least one or two (or more, or each) of the dietary fiber materials are derived from respective plant materials, and wherein at least one (and more typically at least two, three or more, or each) of the dietary7fiber materials comprises fiber-bound polyphenols. Most ty pically, but not necessarily, the fiber-bound polyphenols will be polyphenols that are naturally present in the respective plant materials, but additional polyphenols may also be included.

[0095] However, with regard to suitable dietary fiber materials it should be recognized that the number and t pe of material is not limiting to the inventive subject matter, and that any number of distinct fibers are deemed suitable for use herein. Thus, contemplated dietary fiber compositions will include at least one dietary fiber material that comprises a fiber-bound polyphenol. However, it is generally preferred that the dietary' fiber composition will ty pically include at least two, or at least three, or at least four, or at least five dietary7fiber materials are derived from respective plant materials. Such materials will generally be from a nutritionally acceptable plant component, and more typically from an edible plant material such as from a fruit, a seed, a berry, a vegetable, a root, an herb, etc.

[0096] Therefore, and viewed from a different perspective, contemplated fiber materials will provide soluble and / or insoluble fibers such as pectins, mucilages, beta-glucans, as well as cellulose, hemi-cellulose, and / or lignins. Without wishing to be bound by any specific theory or hypothesis, it is contemplated that the variety of diverse fiber materials, especially in the ratios contemplated below along with the bound polyphenols, will stimulate the intestinal microbiome across a large variety of commensal species, will promote production of SCFA and acetate across a variety of bacterial species to so maximize fermentative production of desirable metabolites with the least (or significantly reduced) amount of gas production.

[0097] In this context, it should be further appreciated that in especially contemplated combinations of different fiber materials the fraction (by weight) of total insoluble fiber is larger than the fraction (by weight) of soluble fiber materials. For example, insoluble fiber will make up at least 50 wt%, or at least 60 wt%, or at least 70 wt%, or at least 80 wt% of total fiber in contemplated products. Thus, insoluble fiber may be present in a range of between 50-65 wt%, or between 60-75 wt%, or between 50-80 wt%, and even higher. In further examples of contemplated formulations, it is generally preferred that the total fiber comprises at least two or at least three different fiber types, and particularly preferred fiber types include pectins, hemi-celluloses, starches, celluloses, and lignins. While not limiting to the inventive subject matter, and considering the distinct fiber types, it is also contemplated that the predominant fraction by weight of fiber types will be celluloses and pectins. For example, celluloses and pectins may account for at least 50 wt%, or at least 60 wt%, or at least 70 wt%, or at least 80 wt% of total fiber in contemplated products. Thus, celluloses and pectins may be present in a range of between 50-70 wt%, or between 60-80 wt%, or between 70-90 wt%, and even higher, and in at least some examples celluloses will be the predominant fraction by weight. Thus, starches and hemicelluloses combined will typically be present at quantities of less than 50 wt%, and more typically less than 40 wt%, or less than 30 wt%, or less than 20 wt%, or less than 10 wt%. In addition, it should be appreciated that contemplated fiber products may also include oligosaccharides in a total amount of between 0. 1-1.5 wt%, or 1-5 wt%, or 3-10 wt% of total fiber. Viewed from a different perspective, in some embodiments, cellulose may be present in an amount of between 10-20 wt%, or between 15-25 wt%, or between 20-30 wt%, or between 30-40 wt%, or between 40-50 wt%, or between 50-70 wt%, or between 10-40 wt%, or between 20-60 wt%, or between 30-80 wt% of the total fiber content, while pectins may be present in an amount of between 10-20 wt%, or between 15-25 wt%, or between 20-30 wt%, or between 30-40 wt%, or between 40-50 wt%, or between 50-70 wt%, or between 10-40 wt%,or between 20-60 wt%, or between 30-80 wt% of the total fiber content. Similarly, starches may be present in an amount of between 0.1-2 wt%, or between 1-3 wt%, or between 3-10 wt%, or between 7-20 wt%, or between 10-25 wt%, or between 15-30 wt%, or between 1-15 wt%, or between 5-25 wt%, or between 10-30 wt% of the total fiber content, while hemicelluloses may be present in an amount of between 0.1 -2 wt%, or between 1 -3 wt%, or between 3-10 wt%, or between 7-20 wt%, or between 10-25 wt%, or between 15-30 wt%, or between 1-15 wt%, or between 5-25 wt%. or between 10-30 wt% of the total fiber content.

[0098] In most embodiments, the plant materials will have been processed to obtain at least one other value component. For example, processing may include comminuting, pressing, shredding, extracting, etc., and the removed value component may therefore be a juice, an extract, a select portion of a plant. Consequently, especially contemplated processed plant materials include pomaces, peels, extracted plant components, etc. As will be readily appreciated, and depending on the degree of processing, the plant materials suitable for use herein may therefore also include unbound polyphenols and other desirable components, however, the presence of such unbound polyphenols and other components is not required. Viewed from a different perspective, contemplated plant materials may be purpose-produced or may be waste streams of other plant processing operations (e.g., juicing operation, extraction, peeling, etc.). In addition, it is also contemplated that the processed plant materials may be washed, or otherwise treated to remove unbound polyphenols and other components. However, it is also contemplated that the compositions presented herein may include raw plant materials (e.g, leaves from kale, collard, spinach, mustard, turnip, etc.) that may or may not be previously pressed or otherwise processed to yield an at least partially dehydrated material.

[0099] For example, the fiber materials and / or source materials for the fiber materials can be provided as washed (with water or water alcohol mic, e.g. , at least once with equal weight wash fluid) pomace or as raw or partially pressed whole plant material (which may or may not be further washed), but can also be provided in other forms from plant materials such as extracts, peels, etc. However, it is typically preferred that at least some (e.g. , at least one or at least two) of the plant materials are processed such that the processed plant material has a fiber content of at least 20 wt%, or more typically at least 25 wt%, or at least 30 wt%, or at least 35 wt%, or at least 40 wt%, or at least 45 wt%, or at least 50 wt%, or at least 60 wt%, or at least 70 wt%, or at least 80 wt% (e.g.. between 25-40 wt%. or between 30-60 wt%, or between 40-70 wt%. or between 50-80 wt%, or between 20-60 wt%, or between 30-80 wt% or between 40-90 wt%).Thus, suitable fiber preparations include those where the preparation is enriched to increase the fiber fraction (at compared to the source material) to the desired and above noted degree.

[0100] While a large variety of source materials for dietary fiber compositions is contemplated, particularly contemplated source materials include a variety of stone fruits and berries such as apple, pear, peach, nectarines, plums, mangoes, cherries, grape, apricots, coconut, blackberries, blueberries, cranberry, mulberries, etc. Also contemplated source materials are vegetables and leafy- greens such as kale, spinach, carrot, broccoli, artichokes, sweet potatoes, various legumes (beans, lentils, peas, etc.), and various seaweed and algae are also contemplated. Viewed from a different perspective, contemplated source materials may numerous edible fruits that include simple fruits that develop from a single ovary, like peaches, plums, and apples, aggregate fruits that are formed from multiple ovaries of a single flower, like strawberries and raspberries, multiple fruits that result from the fusion of ovaries of multiple flowers, like pineapples and blackberries, fleshy fruits such as berries, pomes, and drupes, where the flesh is edible, and dry fruits that have a hard, dry pericarp (outer layer), like legumes. Similarly, contemplated source materials may include numerous edible vegetables and therefore include root vegetables such as edible roots, like carrots and beets, stem vegetables such as edible stems, like celery and asparagus, tuber vegetables such as edible underground stems (e.g. potatoes), bulb vegetables such as edible bulbs (e.g, onions, garlic), leafy vegetables such as lettuce and spinach, flower vegetables with edible flower heads such as broccoli and cauliflower, fruity vegetables such as tomatoes, cucumbers, and peppers, pod vegetables such as green beans and peas, seed vegetables such as beans and peas, and various fungi (mushrooms). Thus, contemplated source materials include turmeric, ginger, citrus (all nutritionally acceptable types), berries (all nutritionally acceptable types), aronia, black currant, cacao, coffee fruit, tomato, spinach, cherry, haskap, pomegranate, celery, chard, cucumber, collard greens, romaine, parsley, broccoli, brussels sprouts, beet, wheatgrass, spirulina, alfalfa, chlorella, buckwheat, and algae.

[0101] In still further contemplated aspects, it is generally preferred that the dietary fiber materials will be at least partially dehydrated so as to reduce or even entirely avoid microbial spoilage. Therefore, the at least partially dehydrated fiber materials may have a residual water content of no more than 30 wt%. or no more than 25 wt%, or no more than 20 wt%, or no more than 15 wt%. or no more than 10 wt%, or no more than 5 wt%. As will be readily appreciated, there are numerous manners of drying known in the art, and it is generally preferred that thedehydration is performed within equal or less than 48 hours, or equal or less than 24 hours, or equal or less than 18 hours, or equal or less than 12 hours from provision or preparation of the (processed) plant materials. Alternatively, the plant materials may also be frozen and after thawing then subject to dehydration in the above time frame. Suitable dehydration methods include, among other methods, air-drying, heat- dry ing, pressure drying, vacuum drying, infrared drying, microwave drying, and freeze- drying. Similarly, it should be appreciated that the plant materials may also be comminuted to a desired particle size range prior to drying, which may further assist in (quick) drying and / or extraction of other desired components.

[0102] While not wishing to be bound by any theory or hypothesis, it is contemplated that the process of drying the distinct dietary fiber materials may "set7the polyphenols to the distinct dietary fiber materials in a process similar to the dying process of cotton fibers using natural dyes. As such, it is contemplated that residual or externally added polyphenols may be added to the dietary' fiber materials prior to the drying process (e.g., via increase of reactive group concentrations in the remaining liquid phase). Alternatively, at least some of the fiber-bound polyphenols will be derived from polyphenols that were present in unbound form (e.g, in residual liquid before drying or via added polyphenols in liquid). Viewed from a different perspective, the process of dry ing the dietary fiber materials may contribute to the formation of at least a fraction of the fiber-bound polyphenols.

[0103] As will be readily appreciated, the drying conditions will vary considerably depending on the particular dry ing method employ ed. However, it is generally contemplated that the fiber materials will be dried within less than 6 hours, or less than 4 hours, or less than 2 hours, or less than 1 hour or less than 30 minutes, at temperatures below 120 °C, or below 1 10 °C, or below- 100 °C, or below' 90 °C, or below 70 °C, or below 60 °C, or below 50 °C, or below' 40 °C, and even lower. Thus, dry ing may be performed at pressures below' atmospheric pressure (e.g., below 600 mbar, or below 400 mbar, or below' 200 mbar. or below 100 mbar, etc.). As such, the plant source materials may be provided as frozen materials, optionally comminuted, and then processed in a freeze-drying operation to remove water to a desired degree.

[0104] Preferably, but not necessarily, the amount of fiber-bound polyphenols in the (dried) dietary fibers will be at least 0.001 wt%, or at least 0.005 wt%, or at least 0.01 wt%, or at least 0.05 wt%, or at least 0.1 wt%, or at least 0.5 wt%, or at least 1.0 wt%, or at least 2.5 wt%, or at least 5.0 wt%, or at least 7.0 wt%, and even higher. For example, fiber-bound polyphenolsin the (dried) dietary fibers may be present overall in an amount of between 0.1 wt% to 0.5 wt%. or between 0.5 wt% to 1.5 wt%, or between 1.0 wt% to 3 wt% of the composition or the total fiber content, and in some cases even higher (especially where additional polyphenols were added prior to drying). Of course, it should also be noted that the exact quantity and chemical nature of the fiber-bound polyphenols may vary7between the different types of fibers.

[0105] Once the fiber materials are dried (which can be performed individually for each of the distinct dietary fibers, or as a mixture), it is generally preferred that the dried materials are comminuted to an average particle size of equal or less than 1,000 pm, or equal or less than 900 pm, or equal or less than 800 pm, or equal or less than 700 pm, or equal or less than 600 pm, or equal or less than 500 pm. or equal or less than 400 pm, or equal or less than 300 pm, or equal or less than 200 pm, or equal or less than 100 pm, or equal or less than 50 pm, or equal or less than 25 pm, or equal or less than 10 pm, or equal or less than 5 pm, and even smaller. For example, suitable particle size ranges include average particle sized of between 50 pm to 250 pm, or between 200 pm and 600 pm, or between 400 pm and 1,000 pm, or between 600 pm and 2,000 pm. Viewed from a different perspective, the fiber particles of contemplated fiber compositions may have an average particle size of 20-50 mesh, or 50-100 mesh, or 330- 150 mesh.

[0106] Preferred particles sizes especially include those that are smaller than is generally obtained by mastication as such smaller particle sizes will significantly increase the available surface area. In this context, it should be recognized that increasing the surface area of the fiber material will lead to a variety of beneficial effects. For example, comminution may lead to an improved release or releasability of the bound polyphenols, may facilitate microbial fermentation (e.g., by way of enhanced access to the fiber), as well as the presentation and release of the fiber-bound polyphenols to the microbiome. As such, any manner of increasing the surface area of the fiber materials is expressly contemplated herein, and suitable comminution methods include grinding, milling, sonication, etc.

[0107] With respect to the polyphenols, it should be recognized that all dietarily acceptable polyphenols are contemplated herein, whether or not such polyphenols naturally occur in the processed plant materials. Thus, it should be appreciated that the dietary fiber materials may also include non-natural or synthetic polyphenols (e.g, apple pomace may include added green tea catechins). Viewed from another perspective, contemplated polyphenols include a large variety of phenolic acids (e.g., variousJE»-coumaric acids, caffeic acids, ferulic acids, and / orsinapic acids), flavonoids (e.g, various flavonols, flavones, flavanones, flavanols, anthocyanins and / or isoflavones), stilbenes (e.g., resveratrol, piceatannol, pterostilbene), and lignans (e.g, artigenin, enterodiol, enterolactone, sesamin, syringaresinol, medioresinol, (-)- matairesinol, (-)-secoisolariciresinol, (+)-lariciresinol and (+)-pinoresinol). Moreover, it should be appreciated that the polyphenols will typically be present as a heterogenous mixture of polyphenols. Indeed, contemplated compositions will comprise at least 50. or at least 60, or at least 70, or at least 80. or at least 90 chemically different fiber-bound polyphenols. However, isolated and purified polyphenols are also deemed suitable for use herein. In addition, it should be appreciated that the dietary fiber compositions may further include other components that may or may not be tightly bound to the dietary fiber materials, and especially contemplated components include soluble saccharides, colorants, and non-polyphenol flavor components such as terpenoids or other volatile aromatic components.

[0108] Indeed, it is contemplated that the fiber-bound polyphenols will be protected by the fiber portion during their passage through the gastrointestinal system by virtue of their tight association with the fiber, while at the same time the combination of polyphenols and dietary fiber produces an enhanced prebiotic effect on the gut microbiome. Viewed from a different perspective, it should be appreciated that the so-prepared dietary fibers from edible fruits and vegetables with fiber-bound polyphenols has unexpectedly exhibited a greater prebiotic effect than isolated and purified fibers as is also shown in more detail below, including one designed specifically to maximize prebiotic effect. Moreover, due to the presence of multiple and distinct dietary fibers from edible fruits and vegetables with fiber-bound polyphenols, the fiber composition will surprisingly exhibit a longer and more gentle fermentation profile than isolated and purified fibers as is also shown in more detail below. Thus, it is contemplated that the complexity and diversity of fibers will increase the likelihood of microbiome interaction, while fiber-bound polyphenols will also interact with the gut microbiome. Such may be particularly observable where the plant materials from which the dietary fibers are derived from is already similar to the ordinary diet of a subject (e.g., fiber from apple as plant material is more common than fiber from artichoke). As such, the existing microbiome will not be metabolically ‘shocked’ by high concentrations of single isolated and purified fiber materials such as inulin or psyllium.

[0109] In still further contemplated aspects, and due to its mixed composition and fiberbound polyphenols, it should be appreciated that various prebiotic effects of contemplatedmaterials are substantially improved without generating undesirable side effects such as bloating and flatulence. For example, and as shown in the examples below, the compositions presented herein will generally have a desirable prebiotic effect and will increase the short chain fatty acid concentration, the microbial biodiversity, and / or microbial abundance in a gut of a subject upon ingestion of an effective dose as compared to a weight-equivalent dosage of inulin (or psyllium). Exemplary’ results for such improvements are provided below. Most typically, the effective dosage will be between 50 mg and 25.000 mg, such as for example, between 50 mg and 250 mg, or between 250 mg and 500 mg, or between 500 mg and 1,000 mg, or between 1,000 mg and 2,500 mg, or between 2,500 mg and 5,000 mg, or between 5,000 mg and 10,000 mg, and even higher.

[0110] As one will appreciate, suitable dosages can be formulated for oral administration in a number of manners, including solid formulations and liquid formulation. For example, solid dosages can be formulated as a capsule, a ready-to-mix powder, a snack bar, an energy bar, a chew, a gummy, a confectionary article, a backed good, or a cereal product, whereas liquid dosages can be formulated as an energy drink, a juice beverage, a coffee, a fermented beverage, a tea, a smoothie, or a soda. Moreover, it is noted that the dosages may be formulated to deliver an effective dosage in one serving, or that effective dosages may be provided in multiple services. Moreover, it is also contemplated that the final fiber product may include additional ingredients such as at least partially isolated and purified prebiotics, including psyllium husk, inulin, fructooligosaccharides (FOS), xylooligosaccharides (XOS), galactooligosaccharides (GOS), isomaltooligosaccharides (IMO), lactulose, etc. In addition, or alternatively, the final fiber product may also include one or mredietary ingredients or blends demonstrated or believed to advantageously affect the brain-gut axis, heart-gut axis, gut-skin axis, gut-HPA (hypothalamus-pituitary-adrenal) axis, and gut-pancreas-liver axis, as well as advantageously affect (or maintain) cognition, mood, immunity, endocrine system function, cardiac function, etc.Examples

[0111] In the following examples, the inventor used a model system for the human gastrointestinal tract, M-SHIME (Mucsal-Simulator of the Human Intestinal Microbial Ecosystem), which is an art recognized human equivalent model system. This system has been validated for human in vivo conditions and is engineered to, among other things, gradually change pH to align with in vivo conditions. In this system, the inventors used three humanmicrobiome samples that were separately isolated from healthy adult fecal donor samples. These samples include the fecal microbiome, and the model represents both the luminal and mucosal gut microbiome.

[0112] The study intervention materials included (1) Control, (2) two different exemplary dietary fiber compositions ’NatureKnit" (used in Example 1) and “NatureKnit - Organic” (used in Example 2). The “NatureKnit” composition comprised comminuted and dried carrot pomace, comminuted and dried apple pomace, comminuted and dried blueberry pomace, comminuted and dried cranberry' pomace, and comminuted and dried whole spinach, while the “NatureKnit - Organic” comprised comminuted and dried organic apple pomace, comminuted and dried organic grape pomace, comminuted and dried organic blueberry pomace, comminuted and dried organic carrot pomace, and comminuted and dried organic spinach. (3) commercially available isolated and purified inulin, and (4) commercially available isolated and purified psyllium. The studies were designed to be ‘Tiber-matched”, so each sample was dosed at the equivalent level to represent 1 gram of fiber (e.g., 2 grams of “NatureKnit” containing 50% fiber). In this context, it should be appreciated that the exemplary dietary fiber compositions used herein are merely illustrative of the concept of use of multiple and distinct fiber types from multiple and distinct source materials, each fiber ty pe comprising plant fibers with fiber-bound polyphenols. Thus, one or more specific source material can be readily replaced by another source material comprising alternative fiber materials with fiber-bound polyphenols (e.g., using pear pomace, broccoli pomace, tomato pomace, whole parsley, etc. Moreover, while the exemplary' dietary' fiber composition in the examples below included dietary fiber materials derived from five distinct plant materials, alternative compositions may be derived from more or less than five plant materials, such as from two, three, four, six, seven, etc.

[0113] EXAMPLE 1

[0114] Test Products

[0115] The fruit and vegetable fiber blend (“NatureKnit”) comprised a blend of apple pomace (about 45 wt%), carrot pomace (about 40 wt%), cranberry pomace (about 5 wt%), blueberry- pomace (about 5 wt%), and whole spinach (about 5 wt%), each washed, dried, and comminuted. This blend contained greater than 50 wt% dietary fiber and naturally occurringfiber-bound polyphenols of 1 to 1.5%. Two common purified fibers, inulin and psyllium, were used for comparison in the short-term colonic simulations.

[0116] Short-Term Colonic Simulation

[0117] Fresh human fecal samples were collected from three healthy adults (male, n = 1; female, n = 2). Healthy donors were characterized as having a healthy body mass index (18.5 to 24.9), no diagnosed diseases which could result in a dysbiosed gut microbiota (e.g., inflammatory bowel disease, irritable bowel syndrome, Parkinson’s disease, diabetes), had not taken antibiotics in the four months prior to fecal sample collection, and aged between 20 and 45 years. Fecal materials were collected and used as approved by the Ethics Committee of the University Hospital Ghent (reference number ONZ-2022-0267).

[0118] A short-term single-stage colonic M-SHIME experiment was carried out to explore the microbial metabolic activity and community composition following the fermentation of the test products in a simulated colon model. This model has been validated for representativeness of the human in vivo situation in multiple studies and has been widely used to evaluate the effects of test products / conditions on the human gut microbiota. At the beginning of the shortterm colonic incubation, either no test product (negative control) or one of the test products (NatureKnit, inulin, psyllium; fiber-matched so that each test condition received 1.667 g fiber / L) were added to 63 mL fresh carbohydrate-depleted medium representative for the colonic environment (nutritional medium PD01; ProDigest, Gent, Belgium). The amount of fiber used in the study was calculated to represent the physiological equivalent of 1 g fiber in a human. Fresh fecal inoculum from one of three healthy donors was then added (7 mL) to simulate a metabolically active colonic microbial community. The mucosal layer was simulated by inserting five mucus-coated carriers into each colonic reactor. The reactors were made anaerobic by flushing with nitrogen gas. Incubations were caried out for 48 h (37 °C with shaking [90 rpm]). All conditions were performed in triplicate to account for biological variation.

[0119] Samples were collected at 0 h, 6 h, 24 h, and 48 h for assessment of pH, gas pressure, SCFA, lactate, and ammonium (fermentation profiling and microbial metabolic activity). Samples for assessment of the luminal microbial community composition were collected at 0 h (negative control only), 24 h, and 48 h. The mucosal microbial community composition was only assessed at 48 h.

[0120] Fermentation Profiling and Microbial Metabolic Activity'

[0121] pH changes were measured using a Senseline F410 pH meter (ProSense, Oosterhout, The Netherlands). Gas pressure was measured using a hand-held pressure indicator (CPH6200: Wika, Echt, The Netherlands). The methods of De Weirdt et al. were used to measure SCFAs (acetate, propionate, and butyrate) and branched SCFAs

[0050] , An Enzytec™ kit was used to measure lactate concentrations according to the manufacturer’s instructions (R- Biopharm, Darmstadt, Germany). Ammonium levels were measured according to the method of Tzollas et al. (Int. J. Environ. Anal. Chem. 2010, 90, 115-126).

[0122] DNA Extraction and 16S rRNA Sequencing

[0123] Total DNA was isolated as described by Duysburgh et al. (Int. J. Pharm. X 2019, 1, 100021). 16S-targeted sequencing was accomplished using primers spanning two hypervariable regions (V3-V4) of the 16S rRNA gene (341F, 5'-CCTACGGGNGGCWGCAG-3’; 785R, 5'-GACTACHVGGGTATCTAAKCC-3'). Using a pair-end sequencing approach, sequencing of 2 x 250 bp resulted in 424 bp amplicons (LGC Genomics GmbH, Berlin, Germany). Fragments of this size are taxonomically more informative than smaller ones. The Schloss lab MiSeq SOP was used for read assembly and cleanup. Briefly, mothur (v. 1.44.3) was used to assemble reads into contigs, perform alignment-based quality fdtering (alignment to the mothur-reconstructed SILVA SEED alignment, vl38), remove chimeras (vsearch v2.13.3), assign taxonomy using a naive Bayesian classifier and SILVA NR vl38_l, and cluster contigs into Operational Taxonomic Units (OTUs) at 97% sequence similarity. All sequences that could not be classified and those that were classified as Eukaryota, Archaea, chloroplasts, and mitochondria were removed. The most abundant sequence within an OTU was used as the representative.

[0124] Quantification of Total Bacterial Cells in the Lumen Samples

[0125] Flow cy tometry' was used to determine the total number of bacterial cells in the luminal samples. This allowed for the conversion of the metagenomics data from relative abundances to absolute abundances by multiplying relative abundances in a sample with the total cell count. Samples were analyzed using a BD Accuri C6 Plus Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using the high flow rate setting. A threshold level of 700 on the SYTO channel was used to separate bacterial cells from medium debris and signal noise.

[0126] Statistical Analysis

[0127] Differences in pH, gas pressure, SCFA, ammonium, and branched SCFA were determined over the course of the entire colonic incubation (i.e., between 0 h and 48 h incubation). For lactate, differences were determined over the course of the initial time interval (i.e., between 0 h and 6 h incubation). Statistically significant differences between different test conditions were determined using a paired student’s t-test (p < 0.05). Alpha diversity was analyzed using three common indices: Shannon (species richness and evenness), Inverse Simpson (species richness and evenness, giving more weight to common or dominant species), and observed taxa (species richness).

[0128] Results

[0129] Fermentation Profile

[0130] Changes in pH and gas over time for individual donors and overall (average of all donors) are shown in FIG.1A and FIG. IB. Test product administration resulted in a significantly greater pH reduction compared with the negative control both at the individual donor level and for the average of all donors (FIG.1A). The strongest overall decrease was observed with NatureKnit, followed by the purified fibers inulin and psyllium. Additionally, the pH decrease was continuous over the course of the 48 h period with NatureKnit, while inulin had a sharp pH decrease between 0 h and 6 h, followed by a subsequent increase between 6 h and 24 h. Overall gas production increased over time for the negative control and with the test product and was significantly greater with the test products versus negative control (FIG.1B). The increase in gas pressure was similar with NatureKnit and inulin, and lower for psyllium.

[0131] More specifically, FIGS. 1 A and IB depict changes in pH (FIG.1A) and gas pressure (FIG.1B) over time following test product administration in M-SHIME short-term colonic incubations. Incubations included the negative control (colonic incubation blank medium), NatureKnit (1.667 g fiber / L, 3.333 g / L total), inulin (1.667 g fiber / L), and psyllium (1.667 g fiber / L). Donors A. B, and C represent three individual healthy human fecal donors, and average donor represents the average of the three donors. Incubations were performed in triplicate (n = 3) and the results are presented as mean ± standard deviation. Statistical analysis was performed over the course of the entire colonic incubation phase (i.e., between 0 h and 48 h). Paired student’s t-tests were used to compare changes observed for the test products versusnegative control. A p-value of <0.05 was considered statistically significant. Different letters above the bars indicate statistically significant differences between test conditions, while no significant differences were observed between test conditions that share the same letter. (M- SHIME is Mucosal Simulator of the Human Intestinal Microbial Ecosystem).

[0132] Microbial Metabolic Activity’

[0133] Changes in microbial metabolic activity following test product administration are shown in FIG.2A and FIG.2B. Changes in total SCFA levels w ere most pronounced between 6 h and 24 h (FIG.2A). The changes were significantly greater with each of the test products compared to the negative control with all individual donors (except inulin with Donor C) and for the average of all donors (FIG.2A). The greatest change in SCFA levels w as observed with NatureKnit (change in total SCFA: 0 h to 6 h, +14.2 mM; 6 h to 24 h, +29.2 rnM; 24 h to 48 h, +6.0 mM), which was significantly greater (p < 0.05) than with either inulin (change in total SCFA: 0 h to 6 h. +16.9 rnM; 6 h to 24 h. +22.0 mM; 24 h to 48 h, +3.4 mM) or psyllium (change in total SCFA: 0 h to 6 h, +10.1 mM; 6 h to 24 h, +22.4 mM; 24 h to 48 h, +5.5 mM). The difference in change in total SCFA production between NatureKnit and the purified fibers was numerically greatest between 6 h to 24 h. Acetate levels followed a similar pattern as total SCFA, though the difference between inulin and psyllium tended to be more pronounced. Propionate levels were significantly enhanced with each test product versus the negative control and for each individual donor and overall, but the differences between the test products were less pronounced than for total SCFA. Butyrate levels were less affected by the test products compared with the other SCFAs and appeared to be highly donor-dependent; significant differences between the negative control and the test products were not observed for the average of all donors. Overall, NatureKnit resulted in greater production of total SCFAs and individual SCFAs as compared to the purified fibers inulin and psyllium.

[0134] Lactate levels were increased between 0 h and 6 h and then decreased between 6 h and 24 h with the negative control and test products (FIG.2B). These changes were most pronounced with inulin, followed by NatureKnit and psyllium. The changes in lactate levels with inulin administration were donor-dependent, being greatest with Donor A compared with Donors B and C.

[0135] For all test conditions, including the negative control, branched SCFA levels had very' little increase between 0 h and 6 h; the levels increased greatly between 6 h and 24 h, andfurther still, though to a lesser extent, between 24 h and 48 h. The overall level of branched SCFA increase tended to be greater with the negative control compared with the test products, though across donors the difference versus the negative control only reached significance with inulin (FIG.2C). For some individual donors, levels were significantly lower versus the negative control for NatureKnit (Donors A and C), inulin (Donor A and B), and psyllium (Donor A). For all test conditions, the greatest increase in ammonium was observed between 0 h and 24 h; the level continued to increase but to a lesser extent between 24 h and 48 h (FIG.2D). Overall, ammonium levels were significantly lower for all test products versus the negative control for each individual donor and for the average of all donors. For change in ammonium level, the greatest difference between test product and negative control was observed with inulin.

[0136] More particularly, FIGS.2A-D depict changes in total SCFA (FIG.2A), lactate (FIG.2B), branched SCFA (FIG.2C), and ammonium-N over time (FIG.2D) following test product administration in M-SHIME short-term colonic incubations. Incubations included the negative control (colonic incubation blank medium), NatureKnit (1.667 g fiber / L, 3.333 g / L total), inulin (1.667 g fiber / L), and psyllium (1.667 g fiber / L). Donors A, B, and C represent three individual healthy human fecal donors, and average donor represents the average of the three donors. Incubations were performed in triplicate (n = 3) and the results are presented as mean ± standard deviation. Statistical analysis was performed over the course of the entire colonic incubation phase (i.e., between 0 h and 48 h) or over the course of the initial time interval (i.e., between 0 h and 6 h; lactate only). Paired student’s t-tests were used to compare changes observed for the test products versus negative control. A p-value of <0.05 was considered statistically significant. Different letters above the bars indicate statistically significant differences between test conditions, while no significant differences were observed between test conditions that share the same letter. M-SHIME = Mucosal Simulator of the Human Intestinal Microbial Ecosystem; SCFA =short chain fatty acid.

[0137] Microbial Community Composition

[0138] The bacterial community composition at the phylum level is shown in FIG.3A and FIG.3B. In the luminal compartment, the absolute bacterial abundance was increased with all test products relative to the negative control (FIG.3A). For all donors, the strongest effect was observed for NatureKnit (+170% on average, p = 1 x 108). followed by psyllium (+142% on average, p = 5 x 105). and inulin (+121% on average, p = 1 x 105). Firmicutes andBacteroidota were the most abundant phyla, along with Actinobacteriota for Donor A and Proteobacteria for Donors B and C. Assessment of relative abundance in the mucosal compartment revealed that the main phyla for all conditions were Firmi cutes, Bacteroidota, and Actinobacteriota (FIG.3B)

[0139] More specifically, FIG.3A depicts stacked bar plots showing absolute phyla abundances (cells / mL) in the lumen compartment and FIG.3B depicts relative phyla abundances in the mucosal compartment. Incubations included the negative control (colonic incubation blank medium), NatureKnit (1.667 g fiber / L, 3.333 g / L total), inulin (1.667 g fiber / L), and psyllium (1.667 g fiber / L). Donors A, B, and C represent three individual healthy human fecal donors. Incubations were performed in triplicate (n = 3). Flow cytometry was used to determine the total number of bacterial cells in the luminal samples.

[0140] Absolute abundance in the lumen compartment and relative abundance in the mucosal compartment at the phylum and family level are shown in FIGS.4A-4C. At the 24 h timepoint, the Bacteroidota phylum was enriched in the lumen compartment following the addition of NatureKnit and psyllium; the opposite was observed with inulin (FIG.4A). The Bacteroidota enrichment was linked to the Bacteroidacea family. The Proteobacteria phylum was upregulated in all conditions and was mainly linked to an enrichment of the Enterobacteriaceae family. An enrichment of members of the Firmicutes phylum and the Lachnospiraceae family specific to NatureKnit was observed. NatureKnit and inulin supported an increase in the Actinobacteria phylum that was linked mainly to enrichments of the Coriobacteriaceae family in both conditions and the Bifidobacteriaceae family with inulin. The findings at 48 h were largely similar to those at 24 h with a few exceptions (FIG.4B). In the mucosal compartment (48 h), the Firmicutes phylum was enriched with NatureKnit and inulin, but not with psyllium; this was mainly attributed to an increase in members of the Lachnospiracea family (FIG.4C).

[0141] More specifically. Jitter plots showing (FIG.4A) the absolute abundance (cells / mL) of the top 20 most abundant phyla and families in the lumen compartment after 24 h, (FIG.4B) the absolute abundance (cells / mL) of the top 20 most abundant phyla and families in the lumen compartment after 48 h are provided, and FIG.4C shows the relative abundance of the top 20 most abundant phyla and families in the mucosal compartment after 48 h. Incubations included the negative control (colonic incubation blank medium), NatureKnit (1.667 g fiber / L, 3.333 g / L total), inulin (1.667 g fiber / L), and psyllium (1.667 g fiber / L). Incubations were performedfor each donor in triplicate (per donor, n = 3; total, n = 9). Each dot represents the average across donors.

[0142] Results for the three alpha diversity indexes are shown in FIG.5A and FIG.5B. In the lumen compartment, the observed taxa index showed a tendency for increased bacterial richness with NatureKnit and psyllium that was significant with NatureKnit at 48h across donors (negative control, 496 observed taxa; NatureKnit, 680 observed taxa; p < 0.05) (FIG.5A). The Shannon and inverse Simpson indices indicated that diversity tended to decrease for all conditions in both colonic compartments after 24h and 48 h (FIG.5A). In the lumen compartment, diversity as measured by the Shannon index, was significantly decreased with inulin relative to the negative control for each individual donor and across all donors at 24h and 48h (FIG.5A).

[0143] In more detail, the effect of the test products on alpha diversity as calculated by the observed taxa, Shannon, and inverse Simpson indices are shown in the lumen compartment at 24h and 48 h (FIG.5A) and mucosal compartment at 48 h (FIG.5B). Incubations included the negative control (colonic incubation blank medium), NatureKnit (1.667 g fiber / L, 3.333 g / L total), inulin (1.667 g fiber / L), and psyllium (1.667 g fiber / L). Donors A, B, and C represent three individual healthy human fecal donors. Incubations were performed in triplicate (n = 3). Paired student’s t-tests were used to compare each test product the negative control. A p-value of <0.05 was considered statistically significant; the asterisk represents a significant difference versus negative control.

[0144] As will be readily appreciated, the study of Example 1 used a short-term single- stage colonic M-SHIME model to investigate changes in microbial metabolism and community composition following administration of NatureKnit, a proprietary blend of diverse fruit and vegetable fibers rich in naturally occurring bound polyphenols, compared with inulin or psyllium, both purified fibers. All test products were fermented by the colonic microbiota of three different donors as evidenced by changes in pH and gas pressure over time, though the kinetics of these changes differed among the products. Further, SCFA production was significantly increased compared with the negative control for all test products, but this was most notable for NatureKnit. Measures of microbial community composition demonstrated a shift in the makeup of the microbiota following the addition of the test products. Absolute bacterial abundance increased in the lumen compartment with all test products and was more pronounced with NatureKnit than wi th the purified fibers. The shift in community compositionincluded an increase in the absolute and relative abundances of families capable of producing SCFAs. Additionally, NatureKnit tended to support an increase in bacterial richness, with a significantly greater number of unique taxa in the lumen compartment at 48 h compared to the negative control.

[0145] The pattern of slower decrease in pH and slower increase in gas production over time with NatureKnit compared with the isolated fibers suggests a more gradual fermentation with a proprietary blend of natural fruit and vegetable fibers. Slower fermentation of fibers is generally considered beneficial as it may allow for the delivery of dietary fibers to both the proximal and distal colon. Rapid fermenting fibers are generally fully metabolized in the proximal colon, meaning that the resulting beneficial metabolites, such as SCFAs. are limited to that area of the colon. Undigested proteins are often fermented by proteolytic bacteria in the distal colon. For this reason, metabolites of proteolytic fermentation such as branched SCFAs and ammonia are present at a higher concentration in the distal versus proximal colon. The presence of dietary fibers in the distal colon may benefit the host by supporting the growth of saccharolytic bacteria, potentially reducing the activity of proteolytic bacteria. The slower fermentation profile observed upon administration of NatureKnit could also in part be related to the gradual release of the bound polyphenols in the colonic environment. Indeed, the fiberpolyphenol bonds can be broken by the action of the gut microbial community, allowing both compounds to exert their potential beneficial effects.

[0146] The overall increase in gas pressure for each test product was within the acceptable range of <100 kPa. Psyllium had the lowest increase in gas pressure, followed by NatureKnit and Inulin. The amount of fiber used in this study was well below the amount that would be expected to produce a sufficiently high level of gas pressure to result in discomfort in humans; however, higher doses of purified, rapidly fermenting fibers have been associated with increased bloating and flatulence.

[0147] SCFA levels were significantly increased relative to the negative control with all test products; the SCFA level was significantly higher with NatureKnit than with inulin or psyllium. This, together with the fact that inulin is a well-established prebiotic, confirms the prebiotic effects of NatureKnit and suggests a potential additional benefit of naturally occurring polyphenols. The increased production of SCFAs may be explained by the changes observed in the microbial community composition. For example, members of the Bifidobacteriaceae family are know n to produce acetate and the absolute abundance of the Bifidobacteriaceae wasincreased in the lumen compartment, with inulin and NatureKnit (to a lesser extent). The absolute abundance of the Bacteroidaceae family, containing bacterial species that are able to produce acetate and / or propionate, was also enhanced in the lumen environment with NatureKnit and psyllium. NatureKnit also supported an increase in the abundance of the Lachnospiraceae family in the lumen and mucosal environment. Members of this bacterial family are able to produce acetate, propionate, and / or butyrate. While butyrate was not significantly increased relative to the negative control across all donors, it was significantly increased when looking at the levels for each individual donor. Given that SCFAs have several benefits to human health, including anti-diabetes, anti-obesity, anti-inflammatory, anticancer, immunoregulatory, cardioprotective, neuroprotective, and hepatoprotective activities, the increase in SCFAs following the addition of NatureKnit. inulin, or psyllium suggests the test products may have health benefits for humans. Our findings are in line with previous studies showing that exposure to both inulin and psyllium increases SCFA levels in humans and newly demonstrates the ability7of a fruit and vegetable fiber blend, rich in naturally occurring bound polyphenols, to increase SCFA levels.

[0148] Compared with the negative control, there was a tendency for the test products to reduce the production of branched SCFAs and ammonium, which are byproducts of proteolytic fermentation and are generally considered to have a negative effect on host health. For example, high levels of branched SCFAs are associated with depression and cortisol levels and it has been suggested that branched SCFAs may play a role in regulating glucose and lipid metabolism. Prolonged exposure to branched SCFAs, ammonium, and other protein fermentation products can damage colonic epithelial cells. The reduction in branched SCFA with NatureKnit reached significance for two of the three donors but not with the average of all donors, which was similar to the findings for inulin except the latter did reach significance across donors. With all the test products, ammonium was significantly reduced versus the negative control, but the effect was strongest with inulin. In addition to altering the composition of the microbial community, each of the test products stimulated a significant increase in the absolute abundance of gut bacteria. An increased total bacterial abundance can potentially contribute to improved donor health. For example, differences in fecal microbial load were previously observed between diseased and healthy individuals, with a 100-fold lower fecal microbial load being observed in patients with Crohn's disease as compared to the healthy control group. There was also a tendency for an increased bacterial richness with NatureKnitand psyllium that was significant for NatureKnit at 48 h. Decreased bacterial richness is associated with a number of diseases, disorders, and negative health outcomes.

[0149] In view of the data and observations from Example 1, the inventors contemplate that NatureKnit was well-fermented by the colon microbiota and had several prebiotic effects, including an increase in SCFA production, a shift in the microbial community composition, and an increase in bacterial richness. The prebiotic effects of NatureKnit were either similar to or significantly greater than those observed with inulin, one of the most well-established prebiotic fibers. Additionally, the pH and gas pressure results suggest a slower fermentation with NatureKnit, a blend of natural fruit and vegetable fibers, compared with purified prebiotic fibers. Thus, the prebiotic effect of NatureKnit may be more significant and gentler than that of inulin and psyllium alone.

[0150] EXAMPLE 2

[0151] In this study, an organic fiber blend “NatureKnit - Organic”, w as tested using the short-term colonic simulation model substantially recapitulating the study design of Example 1 for the “NatureKnit” fiber blend a noted above. The aim of the short-term colonic simulation was to assess the effects of the product on the microbial community composition and microbial fermentation activity for healthy adults. To be able to assess the impact on the luminal and mucosal microbiome, the short-term colonic simulation was extended with a mucosal simulation. Two comparative products (i.e., organic inulin and organic psyllium) were tested for comparison. In contrast to Example 1, the microbiome of nine donors was tested in single to also be able to assess interindividual variability of the effects. The main endpoints of the experiment were related to assessing the gut microbial activity by measurement of lactate, (branched) short-chain fatty' acids ((b)SCFA), ammonium nitrogen (NH4-N), pH, and gas production. In addition, and as in Example 1, the gut microbial community composition was investigated by means of 16S rRNA gene profiling.

[0152] As is demonstrated in more detail below; NatureKnit - Organic as well as the competitor products organic inulin and organic psyllium were well fermented by the gut microbial communities of the different donors, as indicated by (significant) reductions in pH and increases in gas, total SCFA and acetate production compared to the negative control, with strongest effects being observed upon administration of NatureKnit - Organic and organic inulin. Furthermore, the initially enhanced lactate concentrations resulted in boosted cross-feeding interactions between primary' substrate degraders and secondary metabolite producers, as seen by the increased conversion of lactate to propionate and butyrate following product supplementation. Administration of the different test products also showed a limited proteolytic protection. Upon comparison of the different test products, it was overall concluded that administration of NatureKnit - Organic significantly increased total SCFA production by the gut microbial communities of the different adult human donors as compared to the other products, with enhanced propiogenic and butyrogenic properties being observed, potentially governing a more positive effect on human health.

[0153] These shifts in metabolic activity’ yvere accompanied by shifts in the gut microbial community composition. Treatment-induced bacterial enrichments were overall most pronounced after 24h of incubation in the luminal environment. Supplementation yvith NatureKnit - Organic and organic inulin stimulated the groyvth of species belonging to the Bifidobacterium genus, while administration of organic psyllium was associated with a significant enrichment of Bacteroides. Further, shifts in Firmicutes genera were observed in response to all three treatments, i.e., different genera belonging to the Lachnospiraceae and / or Oscillospiraceae family were enriched folloyving 24h of incubation. After 48h of incubation, supplementation of organic psyllium still exhibited particularly strong Bacteroidogenic effects, while enrichment of Bifidobacterium upon NatureKnit - Organic and organic inulin administration was diminished. Nevertheless. NatureKnit - Organic treatment still promoted the groyvth of various genera yvithin the Firmicutes and Bacteroidota phyla, yvith the most pronounced effects being observed for Monoglobus, Lachnospiraceae ND3007 group, and Lachnospiraceae NK4A136 group. Enrichment of species belonging to the Eisenbergiella, Marvinbryantia, and Monoglobus genera was still observed upon administration of organic psyllium, yvhile none of these species were enriched upon administration of organic inulin. Finally, in the mucosal colonic environment, no significant effects yvere observed upon administration of NatureKnit - Organic, while enrichment of (i) Bifidobacterium species and (ii) species belonging to the Lachnospiraceae ND3007 group and Eisenbergiella were observed upon administration of organic inulin and organic psyllium, respectively.

[0154] Test Products

[0155] The fruit and vegetable fiber blend (‘'NatureKnit - Organic’’) comprised a blend of organic apple pomace (about 65 wt%), organic grape pomace (about 24 wt%), organic blueberry pomace (about 6 wt%), organic carrot (about 2.5 wt%), and organic whole spinach(about 2.5 wt%), each washed, dried, and comminuted. This blend contained greater than 50 wt% dietary fiber and naturally occurring fiber-bound polyphenols of 1 to 1.5%. Two common purified fibers, inulin and psyllium, were used for comparison in the short-term colonic simulations.

[0156] Short-Term Colonic Simulation

[0157] Fresh human fecal samples were collected from nine healthy adults (male, n = 5; female, n = 4), no diagnosed diseases which could result in a dysbiosed gut microbiota (e.g., inflammatory’ bowel disease, irritable bowel syndrome, Parkinson’s disease, diabetes), had not taken antibiotics in the four months prior to fecal sample collection, and aged between 20 and 45 years. The remainder of the short-term colonic simulation was performed substantially as described in Example 1 above.

[0158] Fermentation profiling and microbial metabolic activity-, DNA extraction and 16s rRNA sequencing, quantification of total bacterial cells in the lumen samples, and statistical analysis were performed substantially as described in Example 1 above.

[0159] Results

[0160] Microbial metabolic activity

[0161] Change in pH: Monitoring the pH during a short-term colonic incubation provides a good indication of the production of SCFA, lactate, and ammonium. In general, a pH drop is observed during the first 24h of incubation due to the formation of SCFA and / or lactate. This pH drop is often folloyved by a pH increase during the last 24h (24h - 48h) of incubation due to (i) proteolytic fermentation, which results amongst others in the production of ammonium, and (ii) conversion of stronger acids into yveaker acids through cross- feeding (for instance lactate-to-propionate / butyrate conversion). Results are presented as the change in pH of the different test conditions for each individual donor (FIG.6A and FIG.6B) as well as for the average donor (FIG.7).

[0162] More particularly, FIGS.6A and 6B depict pH changes (-) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01). and organic psyllium (OP)), and a negative control (NC). Results have been included forthe nine individual healthy human donors (FIG.6A, donors A-E; FIG.6B, donors F-I). Each condition was examined in single replicate (n=l). FIG.7 depicts pH changes (-) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the pH change over the entire colonic incubation phase (i.e.. between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05). Based on these data, the following observations were made:

[0163] For the negative control, it was concluded that the microbial communities of all nine donors were able to decrease the pH between Oh and 48h of incubation. Depending on the donor, the overall pH change ranged between -0. 11 and -0.04 pH units, indicating that the microbial communities of the different donors were able to ferment the substrates which were available in the simulated colonic medium. Moreover, the strongest pH decrease was obtained during the first 6h of incubation, indicating a fast to moderate fermentation rate.

[0164] Addition of the test products (i.e., NatureKnit - Organic, organic inulin, or organic psyllium) resulted in a stronger overall decrease of the environmental pH, reaching significance as compared to the negative control across donors. The gut microbial communities of the different doners were thus able to use these test products as substrates. Moreover, the strongest pH decrease was again generally obtained during the first 6h of incubation.

[0165] The strongest overall pH decrease was observed upon administration of the NatureKnit - Organic test product (-0.38 pH units on average), followed by organic inulin (- 0.32 pH units on average), and organic psyllium (-0.22 pH units on average), reaching significance across donors. While NatureKnit - Organic and inulin showed a rather similar pH change over the entire incubation period, the results of the separate time intervals indicated a different fermentation profile. Indeed, addition of the NatureKnit - Organic test product generally showed a more continuous pH decrease over the entire incubation period, while a strong decrease during the first 6h of incubation upon administration of organic inulin was often followed by a subsequent increase between 6h and 48h of incubation. The NatureKnit - Organic test product was thus likely more gradually fermented. Nevertheless, this differencein fermentation profile could also be attributed to a strongly boosted conversion of primary to secondary metabolites upon administration of inulin.

[0166] Gas Production: Gas production is a measure of the microbial activity, and thus the rate of substrate fermentation. This is a very relevant parameter as rapid fermentation causes a high initial gas production, which can potentially result in discomfort related to bloating and flatulence. A low to moderate initial rate of fermentation would thus be desirable to minimize such effects. Results are presented as the change in gas pressure of the different test conditions for each individual donor (FIG.8A and FIG.8B) as well as for the average donor (FIG.9).

[0167] In particular, FIG.8A and FIG.8B depict gas pressure changes (kPa) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the nine individual healthy’ human donors (Figure 8A, donors A-E; Figure 8B, donors F-I). Each condition was examined in single replicate (n=l). FIG.9 shows gas pressure changes (kPa) during the different time intervals of the short-term colonic incubations (i.e., Oh- 611, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the gas pressure change over the entire colonic incubation phase (i.e., between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05). Based on these data, the following observations were made:

[0168] The overall gas production (i.e., between Oh and 48h of incubation) of the negative control ranged between 24.6 and 31.1 kPa, depending on the specific donor. This overall gas pressure increase confirms that the gut microbiota of the different donors were indeed able to ferment the substrates which were available in the simulated colonic medium. For the individual donors as well as for the average donor, the strongest gas pressure increase was observed during the initial 24h of incubation, indicating a moderate fermentation rate.

[0169] Administration of NatureKnit - Organic and organic inulin resulted in a significantly enhanced gas production as compared to the negative control across donors, indicating that the gut microbiota of the different donors were able to use these administeredsubstrates for their metabolic activity. The overall gas pressure across donors also increased upon administration of organic psyllium, but significance as compared to the negative control was not reached. As for the negative control, the strongest gas production was again observed during the initial 24h of incubation, indicating a moderate fermentation rate.

[0170] Comparison of the different test products indicated that the highest overall gas pressure increase was observed upon addition of NatureKnit - Organic (47.4 kPa), followed by organic inulin (41.6 kPa), and organic psyllium (34.2 kPa). Discomfort related to bloating and flatulence would thus be less likely upon administration of organic psyllium. Nevertheless, the overall gas pressure increase upon administration of organic inulin and NatureKnit - Organic was still within an acceptable range (< 100 kPa).

[0171] Lactate production and consumption: The human intestine harbors both lactate- producing and lactate-utilizing bacteria. Lactate is produced by lactic acid bacteria during primary substrate degradation and decreases the pH of the environment. Especially at low pH values, lactate can exert strong antimicrobial effects against pathogens. Another beneficial effect of lactate results from its conversion to butyrate and / or propionate. As different microbial species thus produce and convert lactate, an increase of the lactate concentration can both result from an increased production and decreased conversion. Results are presented as the change in lactate concentration of the different test conditions for each individual donor (FIG.10A and FIG.10B) as well as for the average donor (FIG.ll). Based on these data, the following observations were made:

[0172] For the negative control, lactate accumulation occurred during the first six hours of incubation, reaching lactate concentrations between 0.35 and 1.78 mM, depending on the donor. During the subsequent 6h-24h time interval, a reduction of the lactate concentration w as observed, while (almost) no further changes w ere observed during the final 24h of incubation. The reduction in lactate concentrations observed during the 6h-24h time interval can be related to a more limited production of lactate and / or a strong conversion of lactate to secondary metabolites such as propionate and / or butyrate due to the establishment of cross- feeding interactions. This means that metabolites produced by a certain (group of) bacterial species can be used as substrates by another (group of) bacterial species.

[0173] A similar trend was observed upon addition of the test products, i.e., the initial lactate accumulation between Oh and 6h of incubation w as follow ed by a subsequent decreasebetween 6h and 24h of incubation. Nevertheless, for NatureKnit - Organic and organic inulin, both the initial increase and subsequent decrease were more pronounced as compared to the negative control, indicating that the cross-feeding interactions were stimulated upon administration of those test products. Indeed, significance as compared to the negative control was reached across donors. For organic inulin, similar results were observed as for the negative control.

[0174] The lactate accumulation between Oh and 6h was highly boosted upon addition of organic inulin (3.03 mM on average) and NatureKnit - Organic (2.33 mM on average), while the lactate accumulation in the presence of organic psyllium was much more limited (0.70 mM on average). Statistical significance between the difference test products was reached across donors. Nevertheless, it should be noted that the lactate production (and subsequent conversion) upon administration of the test products was clearly donor-dependent (with strongest effects observed for donor D).

[0175] SCFA production: SCFA production results from the carbohydrate metabolism in the colon and is related with various health effects. The most abundantly produced SCFAs consist of acetate, propionate, and butyrate. Whereas acetate can be used as an energy source for the host and as a potential substrate for lipid synthesis in the body, propionate reduces cholesterol and fatty acid synthesis in the liver (beneficial effect on metabolic homeostasis). Butyrate, on the other hand, is a major energy source for colonocytes and induces differentiation in these cells (related to cancer prevention).

[0176] First, the total SCFA levels reflect the overall fermentation by the different gut microbiota. Results are presented as the change in total SCFA concentration of the different test conditions for each individual donor (FIG.12A and FIG.12B) as well as for the average donor (FIG.13). More specifically FIG.12A and FIG.12B depict total SCFA concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e.. 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP)), and a negative control (NC). Results have been included for the nine individual healthy human donors (Figure 10A, donors A-E; Figure 10B, donors F-I). Each condition was examined in single replicate (n=l). FIG.13 shows total SCFA concentration changes (mM) during the different time intervals of the shortterm colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP)),and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the total SCFA concentration change over the entire colonic incubation phase (i.e.. between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05).). Based on these data, the following observations were made:

[0177] For the negative control, the overall total SCFA production (i.e., between Oh and 48h of incubation) ranged between 28.1 and 35.0 mM, with the strongest production being observed during the first 24h of incubation.

[0178] Administration of the different test products resulted in a significantly enhanced total SCFA production as compared to the negative control across donors. The additional substrates provided upon administration of NatureKnit - Organic, organic inulin, and organic psyllium thus clearly resulted in a boosted carbohydrate mechanism, especially during the first 24h of incubation. Indeed, as SCFAs are generally produced as a result of carbohydrate metabolism of the gut microbial community, this observation indicates that the provided carbohydrates got more and more depleted during the final 24h of incubation, which typically results in a shift of the gut microbial community towards proteolytic breakdown.

[0179] Comparison of NatureKnit - Organic with the two competitor products showed that the highest overall total SCFA production was obtained upon administration of NatureKnit - Organic (48.2 mM on average), followed by organic inulin (42.6 mM on average), and organic psyllium (38.7 mM on average), reaching significance across donors.

[0180] Acetate production: Acetate (FIG.14A and FIG.14B, FIG.15) is mostly one of the key metabolites formed during primary substrate fermentation and can be produced by a wide range of gut microbes including Bacteroides spp. (phylum Bacteroidota) and Bifidobacterium (phylum Actinobacteriota). In particular, FIG.14A and FIG.14B depict acetate concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (OI). and organic psyllium (OP)), and a negative control (NC). Results have been included for the nine individual healthy human donors (FIG.14A, donors A-E; FIG.14B, donors F-I). Each condition was examined in single replicate (n=l). FIG.15 shows acetate concentration changes (mM) during the different time intervals of the short-termcolonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the acetate concentration change over the entire colonic incubation phase (i.e., between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05). Based on these data, the following observations were made:

[0181] The overall acetate production (i.e., between Oh and 48h of incubation) of the negative control incubations ranged between 16.8 and 21.6 mM. The vast majority was produced during the initial 24h of incubation, which can be linked with acetate being formed during primary substrate fermentation.

[0182] For the average donor, administration of the different test products showed a significantly enhanced acetate production over the 48h interval. Similar as for the total SCFA production, this indicated that the carbohydrate metabolism was boosted upon administration of the different test products.

[0183] Comparison of NatureKnit - Organic with the other test products showed that the highest overall acetate production was obtained upon administration of NatureKnit - Organic (28.9 mM on average), followed by organic inulin (27.5 mM on average), and organic psyllium (21.8 mM on average), reaching significance across donors.

[0184] Propionate production: Like acetate, propionate (FIG.16A and FIG.16B, FIG.17) can be produced by a wide range of gut microbes, with the most abundant propionate producers being Bacteroides spp. (phylum = Bacteroidota), Akkermansia muciniphila (phylum = Verrucomicrobiota), and Veillonellaceae (phylum = Firmicutes). More specifically, FIG.16A and F1G.16B show propionate concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP)), and a negative control (NC). Results have been included for the nine individual healthy human donors (FIG.16A, donors A-E; FIG.16B, donors F-I). Each condition was examined in single replicate (n=l). FIG.17 depicts Propionate concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic(ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the propionate concentration change over the entire colonic incubation phase (i.e., between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05). Based on these data, the following observations were made:

[0185] For the negative control, the propionate production over the entire course of the colonic incubation (i.e., between Oh and 48h) ranged between 5.8 and 9.6 mM. As for the total SCFA and the acetate production, the strongest propionate increase was observed during the first 24h of incubation, indicating that the cross-feeding interactions between the different (groups) of bacterial species to convert primary substrates to propionate were rather rapidly established.

[0186] Administration of the different test products showed a significantly enhanced propionate production over the 48h interval as compared to the negative control across donors. The strongest propionate production was observed between 6h and 24h of incubation, which coincided with a decrease of the lactate concentrations. The stimulated production and subsequent conversion of lactate to secondary metabolites upon administration of the different test products was thus likely (at least in part) responsible for the increased propionate levels.

[0187] When focusing on the average donor, it was concluded that NatureKnit - Organic resulted in significantly higher propionate levels (12.2 mM) as compared to organic psyllium (10.4 mM) and organic inulin (9.7 mM).

[0188] Butyrate Production: Butyrate (FIG.18A and FIG.18B, FIG.19) is produced by members of the Clostridium clusters IV and XlVa (phylum = Firmicutes). In a process called cross-feeding, these microbes convert acetate and / or lactate (along with other substrates) to the health-related butyrate. More specifically, FIG.18A and FIG.18B depict butyrate concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP)), and a negative control (NC). Results have been included for the nine individual healthy human donors (FIG.18A, donors A-E; FIG.18B, donors F-I). Each condition was examined in single replicate (n=l). FIG.19 depicts butyrate concentration changes (mM) during the different timeintervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the butyrate concentration change over the entire colonic incubation phase (i.e., between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05). Based on these data, the following observations were made:

[0189] The overall butyrate production (i.e., between Oh and 48h of incubation) of the negative control ranged between 2.0 and 4.6 mM, depending on the specific donor. Similar as for acetate and propionate, the strongest butyrate production was observed during the first 24h of incubation. The cross-feeding interactions involved in the production of butyrate were thus again rather rapidly established. Moreover, the strong butyrate production between 6h and 24h of incubation again coincided with the decreased lactate concentrations within this specific time interval, so lactate was (at least in part) converted to butyrate by secondary metabolites producers.

[0190] Upon assessing the effects of the different test products across donors it was observed that butyrate production significantly enhanced as compared to the negative control upon administration of NatureKnit - Organic and organic inulin. For organic psyllium, butyrate levels also slightly increased, though significance as compared to the negative control was not obtained.

[0191] When focusing on the average donor, it was concluded that NatureKnit - Organic resulted in higher butyrate levels (4.3 mM on average) than observed upon administration of organic inulin (4 mM on average) and organic psyllium (3.4 mM on average), with statistical significance being reached for organic psyllium as compared to both other test products. Altogether, the results indicated that NatureKnit - Organic tended to result in the strongest butyrogenic effect as compared to the other test products.

[0192] Branched SCFA: Branched SCFA (bSCFA; FIG.20A and FIG.20B, FIG.21) are a result of proteolytic microbial activity, which is also associated with the production of toxic compounds such as p-cresol. Therefore, high bSCFA levels have been associated with detrimental health effects in the colon. Reduced levels are thus considered beneficial. Inparticular, FIG.20A and FIG.20B and depict bSCFA concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the nine individual healthy human donors (FIG.20A, donors A-E; FIG.20B, donors F-I). Each condition was examined in single replicate (n=l). FIG.21 shows bSCFA concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)), and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the bSCFA concentration change over the entire colonic incubation phase (i.e., between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05). Based on these data, the following observations were made:

[0193] For the negative control, the overall bSCFA production (i.e., between Oh and 48h of incubation) ranged between 0.99 and 3.89 mM, depending on the specific donor. The gut microbiota of the different donors were thus able to ferment the proteins available in the simulated colonic environment. The strongest increases were observed during the 6h-24h or 24h-48h time interval, depending on the specific donor. This confirms that the microbial activity of the gut microbiota indeed shifted more towards proteolytic breakdown when the incubation proceeded.

[0194] Administration of NatureKnit - Organic and organic psyllium resulted in a (slightly) increased overall bSCFA production as compared to the negative control, while organic inulin resulted in a decrease. However, significance as compared to the negative control was only reached upon administration of organic inulin. Focusing on the average donors, the strongest bSCFA production was generally observed during the 24h-48h time interval, indicating a more delayed shift from carbohydrate degradation to proteolytic fermentation.

[0195] The lowest overall bSCFA production was observed for organic inulin (1.31 mM on average), followed by NatureKnit - Organic (2.62 mM on average), and organic psyllium (2.90 mM on average). Statistical significance was, however, only reached for NatureKnit - Organic as compared to organic inulin.

[0196] Ammonium production: As bSCFA, ammonium production (measured as NFU-N (ammonium-nitrogen); FIG.22A and FIG.22B, FIG.23) is also a result of the proteolytic microbial activity and thus associated with the production of toxic compounds such as p- cresol. Therefore, high ammonium levels can be indicative of a detrimental proteolytic activity in the colon. Reduced levels are thus considered beneficial. More specifically, FIG.22A and FIG.22B depict ammonium concentration changes (mg / L) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP)), and a negative control (NC). Results have been included for the nine individual healthy human donors (FIG.22A, donors A-E; FIG.22B, donors F-I). Each condition was examined in single replicate (n=l). FIG.23 depicts ammonium concentration changes (mM) during the different time intervals of the short-term colonic incubations (i.e., 0h-6h, 6h-24h, and 24h-48h) upon administration of the different test products (i.e., NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP)), and a negative control (NC). Results have been included for the average donor. Results are presented as mean ± stdev (n=9) and statistical analysis was performed for the ammonium concentration change over the entire colonic incubation phase (i.e., between Oh and 48h). Statistically significant differences between the different test conditions were indicated by means of different letters (p<0.05).

[0197] Changes in microbial community composition

[0198] Treatment impact on microbial community composition was assessed using 16S rRNA gene targeted sequencing, which provides resolution at the bacterial genus level. It thus allows to evaluate how a treatment modifies microbial community composition, and as such to explain alterations in metabolite production.

[0199] Bacterial composition of the fecal inocula used to inoculate the reactors: An assessment was made of gut microbial community composition of the nine healthy adult donors that were used to inoculate the experiment. To do so, samples were collected from the control reactors at experimental start (TOh), and biomass density, alpha-diversity and community composition were determined.

[0200] Bacterial biomass: Biomass density in the reactors at experimental start is given in FIG.24, for the various donors. Bacterial biomass in the original suspensions varied between 1.10 x 109(or 9.04 log; donor B) and 2.31 x 109(or 9.36 log; donor G) bacterial cells / mL withan average of 1.86 x 109(or 9.34 log) cells / mL across donors. Taking into account the stool concentrations in these suspensions (7.5% (m / v), inoculated at 10% (v / v)), these bacterial densities correspond with respectively 1.47 x 1011(donor B) and 3.08 x 1011(donor G) bacterial cells per gram of wet stool. These fecal densities, and respective inter-donor variations, are in line with what is typically observed for the adult gut microbiota (i.e., IO10- 1011bacterial cells per gram of wet stool). FIG.24 depicts the bacterial biomass density (LoglO(total bacterial cells / mL)) of the fecal suspensions of the nine healthy adult donors (donor A-I).

[0201] Alpha Diversity: Alpha-diversity in the reactors at experimental start (thus representing the original fecal samples) is shown in FIG.25, panel A and B, for the various donors. Here, alpha-diversity is shown in the control reactors (n=9) at experimental start, thus representing the donors’ original fecal samples. Four indices are shown, representing genus richness (Observed and Chaol indices) (Panel A) and genus evenness (Simpson and Shannon indices) (Panel B). The observed and Chaol indices express the number of bacterial genera in a sample (genus richness), whereas the Shannon and Simpson indices are measures for genus evenness, or the distribution of the various taxa in a sample, with the Simpson index giving more weight to higher abundant genera. Genus richness ranged from 49 to 79, with an average of 62 genera across donors (observed index). Genus evenness, expressed by the Shannon index, ranged from 2.48 to 3.24 with an average of 2.80, and the Simpson index ranged from 0.85 to 0.93 with an average of 0.88 across donors. Genus richness and evenness of the nine microbiota are in line with what is generally observed in a healthy adult gut microbiota. The microbiota of donor C and I were characterized by the highest diversity, while donors E and F were characterized by an overall lower microbial diversity.

[0202] Microbial Community Composition: Gut microbial community composition of the nine healthy adult donors is shown as relative abundances (%) in FIG.26, FIG.27, and FIG.28. In particular, FIG.26 shows microbial community composition (%) of the nine donors’ fecal samples at the bacterial phylum level. FIG.27 shows microbial community composition (%) of the nine donors’ fecal samples at the bacterial family level. The top 20 most abundant families are shown. All remaining families are classified as ‘Others’, and FIG.28 shows Microbial community composition (%) of the nine donors’ fecal samples at the bacterial genus level. The top 20 most abundant genera are shown. All remaining genera are classified as ‘Others’. As can be seen from the data, Firmicutes, Bacteroidota, and Actinobacteriota were the dominant phyla across donors, with relative abundances consistent with literature,characteristic for healthy adults. At lower phylogenetic level (family and genus), the microbiota displayed more interindividual differences in terms of distribution. For instance, Rikenellaceae was represented in donors B and I, while Bifidobacteriaceae was more abundant in donors G and H. Such interindividual differences are expected at this level, as gut microbial community composition not only depends on the health status of the host, but on many other factors, including amongst others diet, genetics, age, and environment, finally resulting in a highly personalized nature of the gut microbiome.

[0203] Community composition 24h and 48h post-treatment: Treatment impact on microbial community composition was assessed using 16S rRNA gene targeted sequencing, which provides resolution at the bacterial genus level. An assessment was made of treatment impact on the gut microbial communities of the donors selected for the experiment, by analyzing samples collected 24h and 48h after start of incubation in de luminal environment and after 48h incubation in mucosal environment. Treatment effects on biomass in the luminal environment and alpha- and beta-diversity and taxa abundance (differential abundance analysis and redundancy analysis) in both the luminal and mucosal environment were assessed.

[0204] Effects on bacterial biomass: Treatment impact on bacterial biomass across donors is shown in FIG.29 and FIG.31 (box plot) and FIG.30 and FIG.32 (volcano plot). More specifically, FIG.29 shows a box plot showing bacterial biomass (Logl0(cells / mL)) across donors in the various conditions after 24h of incubation in the luminal environment. NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP) were tested on the fecal microbiota of nine healthy adults, and a negative control (NC) was included for each donor as a reference. Statistical significance as compared to the negative control is indicated with ‘ ’ (p <=0.05). FIG.30 depicts a Volcano plot, showing differences in biomass between each treatment (i.e., NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP)) and the negative control at 24h of incubation in the luminal environment. Statistical significance (-loglO(p-value)) is plotted in function of fold change (log2(treatment / control)). Value 1.3 on the y-axis (red dotted line) corresponds with p-value 0.05, thus indicating the level above which a difference between treatment and control is statistically significant across the donors. The zero value on the x-axis marks the separation between elevated biomass in treatment versus control (right) or lower biomass than control (left). This classifies conditions into one of four categories: non-significantly lower biomass than reference (bottom left),significantly lower biomass than reference (top left), non-significantly higher biomass than reference (bottom right), and significantly higher biomass than reference (top right).

[0205] FIG.31 depicts a box plot showing bacterial biomass (LoglO(cells / mL)) across donors in the various conditions after 48h of incubation in the luminal environment. NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP) were tested on the fecal microbiota of nine healthy adults, and a negative control (NC) was included for each donor as a reference. Statistical significance as compared to the negative control is indicated with (p <=0.05). FIG.32 shows a Volcano plot, showing differences in biomass between each treatment (i.e., NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP)) and the negative control at 48h of incubation in the luminal environment. Statistical significance (-loglO(p-value)) is plotted in function of fold change (log2(treatment / control)). Value 1.3 on the y-axis (red dotted line) corresponds with p-value 0.05, thus indicating the level above which a difference between treatment and control is statistically significant across the donors. The zero value on the x-axis marks the separation between elevated biomass in treatment versus control (right) or lower biomass than control (left). This classifies conditions into one of four categories: non-significantly lower biomass than reference (bottom left), significantly lower biomass than reference (top left), non-significantly higher biomass than reference (bottom right), and significantly higher biomass than reference (top right).

[0206] After 24h of incubation (FIG.29, FIG.30), NatureKnit - Organic and organic psyllium tended to increase biomass production in nearly all donors, with a significant increase observed across donors following organic psyllium treatment. In contrast, the impact of organic inulin treatment on biomass production was more donor-dependent, with an increase observed in approximately 50% of the donors. In addition, after 48h incubation (FIG.31, FIG.32), treatment with NatureKnit - Organic and organic psyllium significantly increased the biomass production in all donors, except for donor I upon organic psyllium treatment. In contrast, organic inulin treatment decreased biomass production in nearly all donors. As a result, supplementation of Natureknit - Organic and organic psyllium significantly increased biomass across donors whereas organic inulin significantly lowered biomass production, compared to the negative control.

[0207] Effects on alpha diversity: Effects on alpha-diversity at genus level are shown in FIG.33 to FIG.35. After 24h of incubation (FIG.33), supplementation with organic inulin decreased genus richness (observed and Chao 1 indices) in the luminal environment whereasthe number of bacterial taxa was not altered by treatment with NatureKnit - Organic and organic psyllium. However, genus evenness was significantly reduced by all treatments, as indicated by the Shannon index. This suggests that treatment with Natureknit - Organic and organic psyllium benefited growth of a select number of bacteria, without losing the diversity in the sample. These findings were confirmed by the Simpson index for all treatments except organic psyllium, which could mean a less strong impact on the higher abundant genera (indeed, the Simpson index gives more weight to more abundant genera). A reduction in bacterial evenness is inherent to the definition of a prebiotic, i.e., a substrate that selectively stimulates growth of specific bacteria within a community. The selective enrichment of health- beneficial bacteria creates a more competitive environment for pathogenic organisms to proliferate. It is important to note that, although all treatments lowered genus evenness, the impact remained within ranges that are generally considered healthy.

[0208] FIG.33 depicts bacterial diversity, expressed by four different diversity indices ((A) observed and Chaol (genus richness), (B) Shannon and Simpson (genus evenness)), in the various conditions 24h after start of incubation in the luminal environment. NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP) were tested on the fecal microbiota of nine healthy adults, and a negative control (NC) was included for each donor as a reference. Statistical significance as compared to the negative control is indicated with (p <=0.05).

[0209] After 48h of incubation in the luminal environment (FIG.34), supplementation with Natureknit - Organic resulted in a significant reduction in genus evenness, indicated by the Simpson index, whereas for organic inulin and organic psyllium, no significant differences in genus richness or genus evenness could be observed compared to the negative control. This suggests that the impact of the treatments were largely diminished upon incubation time. FIG.34 depicts Bacterial diversity, expressed by four different diversity indices ((A) observed and Chaol (genus richness), (B) Shamion and Simpson (genus evenness)), in the various conditions 48h after start of incubation in the luminal environment. NatureKnit - Organic (ON), organic inulin (OI), and organic psyllium (OP) were tested on the fecal microbiota of nine healthy adults, and a negative control (NC) was included for each donor as a reference. Statistical significance as compared to the negative control is indicated with (p <=0.05).

[0210] In the mucosal environment (FIG.35), genus richness (observed and Chaol indices) was significantly decreased after 48h supplementation with organic inulin, but significantlyincreased upon organic psyllium treatment, compared to the untreated control. However, none of the treatments altered genus evenness.

[0211] FIG.35 depicts Bacterial diversity, expressed by four different diversity indices ((A) observed and Chaol (genus richness), (B) Shannon and Simpson (genus evenness)), in the various conditions 48h after start of incubation in the mucosal environment. NatureKnit - Organic (ON), organic inulin (01), and organic psyllium (OP) were tested on the fecal microbiota of nine healthy adults, and a negative control (NC) was included for each donor as a reference. Statistical significance as compared to the negative control is indicated with ‘*’ (p <=0.05).

[0212] Effect of Beta-Diversity: Beta-diversity analysis provides a holistic view on how treatments affect the gut microbial community composition, and on how their impact relates to that of other treatments. Impact on beta-diversity is visualized using hierarchical clustering (FIG.36, Panel A, FIG.37, Panel A, and FIG.38, Panel A) and a DAPC scatter plot (FIG.36, Panel B, FIG.37, Panel B, and FIG.38, Panel B).

[0213] After 24h and 48h incubation in the luminal environment, each treatment impacted gut microbial community composition, as indicated by the consistent segregation of treatments and untreated control (NC) (FIG.36, Panel A-B, FIG.37, Panel A-B). Supplementation of Natureknit - Organic and organic psyllium resulted in the strongest shifts in microbiota composition, as demonstrated in the hierarchical clustering and DAPC plots (strongest shift along the LD1 axis, which explains most of the variation). After 48h incubation, conditions with NatureKnit - Organic and organic psyllium clustered more together, indicating that these treatments had a highly comparable impact on community composition. In contrast, organic inulin showed less pronounced segregation from the negative control, suggesting a less pronounced effect on the gut microbiota of the nine healthy adult donors.

[0214] FIG.36 depicts beta-diversity in the various conditions (i.e., NatureKnit - Organic (ON), organic inulin (OI), organic psyllium (OP), and negative control (NC)), 24h after start of incubation in the luminal environment, represented by hierarchical clustering (Panel A) and DAPC (Panel B). Panel A shows dissimilarities in community composition between the various conditions are expressed in the dendrogram, where the sum of the horizontal lines separating two conditions is a measure for dissimilarity in terms of community composition betweenrespective conditions. Panel B shows LD1 and LD2 in the DAPC plot are Linear Discriminants, and each dot represents one of the nine donors.

[0215] FIG.37 depicts Beta-diversity in the various conditions (i.e., NatureKnit - Organic (ON), organic inulin (01), organic psyllium (OP), and negative control (NC)), 48h after start of incubation in the luminal environment, represented by hierarchical clustering (Panel A) and DAPC (Panel B). Panel A shows dissimilarities in community composition between the various conditions are expressed in the dendrogram, where the sum of the horizontal lines separating two conditions is a measure for dissimilarity in terms of community composition between respective conditions. Panel B shows LD1 and LD2 in the DAPC plot are Linear Discriminants, and each dot represents one of the nine donors.

[0216] FIG.38 depicts Beta-diversity in the various conditions (i.e., NatureKnit - Organic (ON), organic inulin (01), organic psyllium (OP), and negative control (NC)). 48h after start of incubation in the mucosal environment, represented by hierarchical clustering (Panel A) and DAPC (panel B). Panel A shows dissimilarities in community composition between the various conditions are expressed in the dendrogram, where the sum of the horizontal lines separating two conditions is a measure for dissimilarity in terms of community composition between respective conditions. Panel B shows LD 1 and LD2 in the DAPC plot are Linear Discriminants, and each dot represents one of the nine donors.

[0217] After 24h incubation with organic psyllium, shifts were mostly along the LD1 axis, while for the other treatments, the shift was also along the LD2-axis. Although shifts along the LD2 axis are less impactful as compared to shifts along the LD1 axis (12.04% versus 79.67%), this does indicate that supplementation with Natureknit - Organic and organic inulin resulted in a different community shift as compared to the negative control. However, after 48h treatment, the bacterial taxa responsible for the segregation were similar for the various treatments (almost fully explained by LD1). but the extent of the effect differed amongst treatments. In the mucosal environment, a less distinct segregation of the treatments and negative control were observed along the LD1 axis, suggesting a less pronounced impact of the treatment on the mucosal microbial community composition (FIG.38, Panels A-B). Nevertheless, the conditions with organic psyllium displayed a shift in the opposite direction compared to Natureknit - Organic and organic inulin, with the negative control positioned intermediate to these groups along the LD1 axis.

[0218] Differential abundance analysis: LEfSe and treeclimbR are statistical analysis tools used to detect which gut bacteria are affected by treatment. In this analysis, the negative control is used as a reference. Both analysis tools are used in parallel, as they apply different statistical methods, and thus their combined use provides more exhaustive insights into treatment- induced community shifts than either method alone. By looking at consistencies across donors (in this analysis, each donor is considered a replicate measurement), detected bacterial enrichments account for interindividual variation. An overview of the affected taxa is provided in FIG.39; relative abundances of affected taxa are displayed in FIG.40-FIG.42, Three different color codes are applied in FIG.39, depending on (1) consistency across donors and (2) effect size. Most significant are bacterial enrichments in red, as those passed the thresholds of statistical and biological significance, meaning that the effect was (1) consistent across donors and (2) with meaningful effect size, thus likely to impact metabolite production. Bacterial enrichments indicated in green pass the threshold for biological significance, meaning that the size of the effect is likely to be reflected at metabolic level, but the effect was not consistent across donors due to interindividual variation (statistical significance was not reached). Finally, bacterial enrichments indicated in blue indicate that the enrichment was observed in a majority' of donors (statistical significance was reached), but the extent of the enrichment was mild and not necessarily reflected at the metabolic level.

[0219] FIG.40 depicts box plots displaying relative abundances (%) of genera Bifidobacterium, Bacteroides, Eisenbergiella, Lachnospiraceae ND3007 group, Ruminococcus, [Ruminococcus] torques group, and Monoglobus in the various conditions, 24h after start of incubation in the luminal environment. indicates statistically significant enrichment in treatment versus blank, as identified by LEfSe and / or treeclimbR. FIG.41 depicts box plots displaying relative abundances (%) of genera Bacteroides, Anaerostipes, Eisenbergiella, [Eubacterium] halli group, Lachnospiraceae ND3007 group, Lachnospiraceae NK4A136 group, Marvinbiyantia, and Monoglobus in the various conditions, 48h after start of incubation in the luminal environment. indicates statistically significant enrichment in treatment versus blank, as identified by LEfSe and / or treeclimbR. FIG.42 depicts box plots displaying relative abundances (%) of genera Bifidobacterium, Eisenbergiella, and Lachnospiraceae ND3007 group in the various conditions, 48h after start of incubation in the mucosal environment. indicates statistically significant enrichment in treatment versus blank, as identified by LEfSe and / or treeclimbR.

[0220] Overall, treatment-induced bacterial enrichments were most pronounced after 24 hours of incubation in the luminal environment. Specifically, supplementation with NatureKnit - Organic and organic inulin stimulated the growth of species of the Actinobacteriaceae phylum, particularly Bifidobacterium, with approximately 2% and 7.5% across donors, respectively. Depending on the species, Bifidobacterium acts either as a primary7substrate degrader or a secondary consumer, producing beneficial metabolites like acetate and lactate. These metabolites drive a series of trophic interactions with other bacteria, ultimately leading to the production of downstream metabolites such as propionate and butyrate. The effect of organic psyllium on microbial community7composition was associated with a significant enrichment of Bacteroides, which was increased by approximately 11% across donors. The Bacteroides genus primarily participates in substrate degradation, producing metabolites such as acetate, propionate, or succinate. Further, shifts in Firmicutes genera were observed in response to all three treatments. Supplementation of NatureKnit - Organic statistically and biologically enriched Ruminococcus species and organic inulin the Ruminococcus torques group. Ruminococcus species are key cellulose degraders, capable of producing acetate and succinate. To a lesser extent. NatureKnit - Organic and organic psyllium treatment stimulated the growth of Monoglobus, a member of the Oscillospiraceae family. The enrichment of the Monoglobus genus could be linked with Monoglobus pectinilyticus, the only representative of this bacterial genus. This species is specialized in the fermentation of pectin in the human colon, suggesting that the Monoglobus genus was (at least partially) involved in the degradation of the fiber fraction of the NatureKnit - Organic test product. Furthermore, organic psyllium supplementation enriched Eisenbergiella, and Lachnospiraceae ND3007 group, belonging to the Lachnospiraceae family, known for their pectin-degrading properties and producing SCFA.

[0221] After 48h, supplementation of organic psyllium still exhibited particularly strong Bacteroidogenic effects, increasing Bacteroides abundance by approximately 12% across donors in the luminal environment. In contrast, upon NatureKnit - Organic and organic inulin treatment, enrichment of Bifidobacterium was diminished after 48h of incubation. Moreover, during the incubation period, NatureKnit - Organic treatment promoted the grow th of various genera within the Firmicutes and Bacteroidota phyla, with the most pronounced effects observed for Monoglobus. Lachnospiraceae ND3007 group, and Lachnospiraceae NK4A136 group, followed by Eisenbergiella. Anaerostipes, and Marvinbryantia. Lachnospiraceae as Lachnospiraceae ND3007 group and NK4A214 group have demonstrated a positive impact ongut health, amongst others through the production of SCFA. Anaerostipes is a bacterial genus known for its versatile metabolic capabilities, but mostly for the production of butyrate. This genus also produces acetate and lactate, and interestingly, can convert acetate into butyrate. Marvinbryantia, member of the Lachnospiraceae family, is capable of producing succinate, lactate, and butyrate. Enrichment of species belonging to the Eisenbergiella, Marvinbryantia, and Monoglobus genera was also observed upon administration of organic psyllium, while none of these species were enriched upon administration of organic inulin. On the other hand. Eubacterium halli group was statistically enriched upon organic inulin supplementation. Eubacterium hallii is considered an important microbe due to its ability to utilize glucose, acetate and lactate, to produce butyrate.

[0222] Finally, in the mucosal colonic environment, no significant NatureKnit - Organic treatment-induced bacterial enrichments were observed. In contrast, organic inulin statistically and biologically enriched Bifidobacterium species, while organic psyllium enriched various species within the Firmi cutes phylum, i.e., it statistically and biologically enriched Lachnospiraceae ND3007 group and statically enriched Eisenbergiella.

[0223] In view of the above data, the metabolic activity analysis indicated that the gut microbiota of all nine donors were able to ferment the substrates which became available upon administration of NatureKnit - Organic, organic inulin, and organic psyllium. More specifically, the overall pH decrease and gas pressure increase (significantly) enhanced as compared to the negative control. The observed changes in metabolic activity' were confirmed by metagenomic sequencing, i.e., the generated diversity plots revealed that the different test products induced shifts in microbial community composition in both the luminal and mucosal environment, with strongest shifts being observed upon administration of NatureKnit - Organic and organic psyllium.

[0224] The saccharolytic activity of the gut microbiota also resulted in significantly enhanced total SCFA and acetate levels following administration of the different test products. Indeed, several primary substrate degraders were stimulated following product supplementation. NatureKnit - Organic supplementation stimulated Bifidobacterium species (24h only), but also members of the Lachnospira and Monoglobus genera were stimulated. Stimulation of the Bifidobacteriaceae was also observed upon administration of organic inulin, while a stimulation of mainly Bacteroides was observed following administration of the organic psyllium test product. Furthermore, lactate concentrations initially accumulated andwere subsequently converted to secondary metabolites such as propionate and buty rate. Efficient cross-feeding interactions were thus established within the gut microbial communities of the different donors, with enhanced levels of lactate accumulation (and conversion) being observed upon administration of NatureKnit - Organic and organic inulin as compared to the negative control. Regarding the NatureKnit - Organic test product, mainly the Lachnospiraceae family and Monoglobus species were further stimulated, while supplementation of organic psyllium still exhibited particularly strong Bacteroidogenic effects. Finally, administration of NatureKnit - Organic and organic psyllium, on the one hand, had no significant effect on bSCFA levels and a limited reducing effect on NH4-N levels. Administration of organic inulin, on the other hand, had a mild reducing effect on both bSCFA and NH4-N levels.

[0225] Upon comparison of the different test products, it was overall concluded that administration of NatureKnit - Organic resulted in a significantly increased total SCFA production by the gut microbial communities of the different adult human donors, with enhanced propiogenic and butyrogenic properties being observed, which might result in positive health outcomes, mainly related to stimulation of butyrate-producing groups such as Lachnospiraceae ND3007 group, Lachnospiraceae NK4A 136 group, and Anaerostipes species.

[0226] Finally, comparison between Example 2 (using an organic form of NatureKnit) and Example 1 (using a non-organic form of NatureKnit) indicated that similar trends were observed for both formulations when focusing on the microbial fermentation activity7across donors. The propiogenic and butyrogenic properties even tended to be slightly higher upon administration of the organic form of NatureKnit. Most of the stimulated bacterial taxa observed upon administration of the organic form were also identified upon administration of the non-organic test products. For example, FIG.43 depicts data showing observed Taxa (by count of species) in the lumen at 48 hours. Here, the left graph (P1516) shows the results from the composition of Example 1, while the right graph (P1716) shows corresponding data using the composition of Example 2. As can be readily seen, the results for the complex compositions of Example 1 (FiberKnit) and Example 2 (FiberKnit - Organic) reach statistical significance against the comparative composition.

[0227] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by' the term “about.” As used herein, the terms "about" and "approximately", when referring to aspecified, measurable value (such as a parameter, an amount, a temporal duration, and the like), is meant to encompass the specified value and variations of and from the specified value, such as variations of + / -10% or less, alternatively + / -5% or less, alternatively + / -!% or less, alternatively + / -0. 1% or less of and from the specified value, insofar as such variations are appropriate to perform in the disclosed embodiments. Thus, the value to which the modifier "about" or "approximately" refers is itself also specifically disclosed. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0228] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0229] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. As also used herein, and unless the context dictates otherwise, the term "coupled to" is intended to include both direct coupling (in which two elements that are coupled to each other contact each other) and indirect coupling (in which at least one additional element is located between the two elements). Therefore, the terms "coupled to" and "coupled with" are used synonymously.

[0230] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements,components, or steps that are not expressly referenced. Where the specification or claims refer to at least one of something selected from the group consisting of A. B, C . . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMSWhat is claimed is:

1. A dietary fiber composition, comprising: a plurality of distinct dietary fiber materials, wherein each of the dietary fiber materials is derived from distinct plant materials, and wherein each of the dietary fiber materials compnses fiber-bound polyphenols, optionally in combination with a nutritionally acceptable carrier; and wherein the distinct dietary' fibers and fiber-bound polyphenols have a composition that, upon oral administration to a subject at an effective dosage, increase short chain fatty acid concentration, microbial biodiversity, and / or microbial abundance in a gut of the subject as compared to administration of a weight-equivalent dosage of inulin and / or psyllium.

2. The dietary’ fiber composition of claim 1, wherein the distinct plant materials comprise edible fruit materials and / or edible vegetable materials.

3. The dietary fiber composition of claim 1, wherein the distinct plant materials are processed plant materials.

4. The dietary’ fiber composition of claim 3, wherein the processed plant materials are comminuted plant materials, expressed plant materials, and / or extracted plant materials.

5. The dietary’ fiber composition of claim 1, wherein the distinct dietary fiber materials are dehydrated fiber materials.

6. The dietary fiber composition of claim 1, wherein the fiber-bound polyphenols in the dietary fiber materials naturally occur in the plant materials from which the dietary fiber materials are derived.

7. The dietary fiber composition of claim 1, wherein at least one of the distinct dietary fiber materials further comprises a soluble saccharide and / or a non-polyphenol flavor component.

8. The dietary fiber composition of claim 1, wherein the fiber-bound polyphenols are present in the dietary fiber materials in an amount of at least 0. 1 wt%.

9. The dietary fiber composition of claim 1, wherein each of the distinct dietary' fibers are comminuted.

10. The dietary fiber composition of claim 9, wherein the comminuted dietary fibers have an average particle size of equal or less than 500 pm.

11. The dietary fiber composition of claim 1, yvherein the effective dosage is equal or less than 10,000 mg, preferably betyveen 1,000 and 5,000 mg.

12. The dietary fiber composition of claim 1, wherein the effective dosage is between 250 mg and 2,500 mg.

13. The dietary fiber composition of claim 1 , wherein the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the short chain fatty' acid concentration in the gut of the subject.

14. The dietary fiber composition of claim 1 , wherein the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the microbial biodiversity' in the gut of the subject.

15. The dietary fiber composition of claim 1 , wherein the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the microbial abundance in the gut of the subject.

16. The dietaty fiber composition of claim 1, wherein the composition is formulated as a powder.17 A dietary- fiber composition, comprising: a plurality- of distinct dietary' fiber materials, wherein each of the dietary' fiber materials is derived from distinct plant materials, and wherein each of the dietary fiber materials comprises fiber-bound polyphenols; and wherein the distinct dietary fibers and fiber-bound polyphenols are dehydrated and ground to have an average particle size of equal or less than 500 pm, thereby enhancing, upon oral administration to a subject, short chain fatty acid concentration, microbial biodiversity’, and / or microbial abundance in a gut of the subject as compared to corresponding unground dietary fiber materials on a weight-equivalent basis.

18. The dietary fiber composition of claim 17, wherein the distinct plant materials comprise edible fruit materials and / or edible vegetable materials.

19. The dietary fiber composition of claim 17, wherein the distinct plant materials are processed plant materials.

20. The dietary fiber composition of claim 17, wherein the processed plant materials are comminuted plant materials, expressed plant materials, and / or extracted plant materials.

21. The dietary fiber composition of claim 17, wherein the dehydrated fiber materials are airdried materials, heat-dried materials, vacuum dried materials, pressure-dried materials, infrared dried materials, microwave-dried materials, or freeze-dried materials.

22. The dietary fiber composition of claim 17, wherein the dehydrated fiber materials have a residual water content of equal or less than 10 wt%.

23. The dietary fiber composition of claim 17, wherein at least one of the distinct dietary fiber materials further comprises a soluble saccharide and / or a non-polyphenol flavor component.

24. The dietary fiber composition of claim 17, wherein the fiber-bound polyphenols are present in the dietary fiber materials in an amount of at least 0. 1 wt%.

25. The dietary fiber composition of claim 17, wherein the distinct dietary fibers and fiberbound polyphenols have an average particle size of between 100 pm and 500 pm.

26. The dietary fiber composition of claim 17, wherein the comminuted dietary fibers have an average particle size of equal or less than 500 pm.

27. The dietary fiber composition of claim 17, wherein the effective dosage is equal or less than 10,000 mg, preferably between 500 mg and 5,000 mg.

28. The dietary fiber composition of claim 17, wherein the effective dosage is between 250 mg and 2,500 mg.

29. The dietary fiber composition of claim 17, wherein the distinct dietary fibers and fiberbound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the short chain fatty acid concentration in the gut of the subject.

30. The dietary fiber composition of claim 17, wherein the distinct dietary fibers and fiberbound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the microbial biodiversity' in the gut of the subject.

31. The dietary fiber composition of claim 17, wherein the distinct dietary fibers and fiberbound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase the microbial abundance in the gut of the subject.

32. The dietary' fiber composition of claim 17, wherein the composition enhances the short chain fatty acid concentration, the microbial biodiversity, and / or the microbial abundance by at least 10% as compared to corresponding unground dietary fiber materials on a weightequivalent basis. A dietary fiber composition, comprising: a plurality of distinct dietary fiber materials, wherein each of the dietary fiber materials is derived from distinct plant materials, and wherein at least one of the dietary fiber materials is impregnated with one or more polyphenols.

34. The dietary fiber composition of claim 33, wherein the distinct plant materials comprise edible fruit materials and / or edible vegetable materials.

35. The dietary fiber composition of claim 33, wherein the distinct plant materials are processed plant materials.

36. The dietary' fiber composition of claim 33, wherein the at least one fiber material is impregnated with a single type of polyphenols.

37. The dietary fiber composition of claim 33, wherein the at least one fiber material is impregnated with multiple and distinct types of polyphenols.

38. The dietary fiber composition of claim 33, wherein the one or more polyphenols are from a plant from which at least one of the distinct plant materials are derived from.

39. The dietary' fiber composition of claim 33, wherein the at least one of the dietary fiber materials is thermally or chemically impregnated with the at least one or more polyphenols.40 A dietary' fiber composition, comprising a dietary' fiber material that is impregnated with one or more polyphenols.

41. The dietary fiber composition of claim 40, wherein the one or more polyphenols are from a single plant type.

42. The dietary7fiber composition of claim 40, wherein the one or more polyphenols are from multiple and distinct plant types.

43. The dietary fiber composition of claim 40, wherein the one or more polyphenols comprise a synthetic polyphenol.

44. The dietary fiber composition of claim 40, wherein the dietary fiber material is derived from an edible fruit material and / or an edible vegetable material.

45. The dietary fiber composition of claim 40, wherein the dietary fiber material comprises a synthetic fiber material.

46. The dietary7fiber composition of claim 40, wherein the dietary fiber material comprises a fiber material that contains equal or less than 0.01wt% fiber-bound polyphenols before impregnation with the one or more polyphenols.

47. The dietary7fiber composition of claim 40, wherein the dietary fiber material comprises at least 0.1 wt% of fiber-bound polyphenols after impregnation with the one or more polyphenols.

48. The dietary7fiber composition of claim 40, wherein the dietary fiber material is thermally or chemically impregnated with the at least one or more polyphenols.

49. The dietary' fiber composition of claim 40, wherein the composition further comprises a soluble saccharide and / or a non-polyphenol flavor component.50 A method of preparing a dietary fiber composition, comprising: providing a plurality of distinct dietary fiber materials, wherein each of the dietary fiber materials is derived from distinct plant materials; rapidly dry ing, or freezing and subsequently dry ing, the distinct dietary7fiber materials to so produce respective dehydrated dietary7fiber materials, wherein each of the dehydrated dietary fiber materials comprises fiber-bound polyphenols; and comminuting and combining the dehydrated dietary fiber materials to thereby form the dietary fiber composition.

51. The method of claim 50, wherein the distinct plant materials comprise edible fruit materials and / or edible vegetable materials.

52. The method of claim 50, wherein the distinct plant materials are processed plant materials.

53. The method of claim 50, wherein the processed plant materials are comminuted plant materials, expressed plant materials, and / or extracted plant materials.

54. The method of claim 50, wherein the step of rapidly drying is performed under a protocol that prevents microbial spoilage.

55. The method of claim 50, wherein the step of rapidly drying is performed to achieve a residual water content of equal or less than 10 wt%.

56. The method of claim 50, wherein the step of rapidly drying comprises air-drying, heatdrying, vacuum drying, infrared drying, microwave drying, or freeze- dry ing.

57. The method of claim 50. wherein the step of rapidly drying or drying comprises a step of thermal or chemical setting to produce the fiber-bound polyphenols.

58. The method of claim 50, wherein the step of comminuting is performed to achieve an average particle size of equal or less than 500 pm.

59. The method of claim 50, wherein the step of comminuting comprises milling, grinding, or shearing.60 A method of preparing a dietary fiber composition, comprising: providing a dietary fiber material; rapidly dehydrating, or freezing and subsequently drying, the distinct dietary fiber materials in the presence of one or more exogenously added polyphenols to so produce a dehydrated dietary fiber material with fiber-bound polyphenols; and optionally comminuting the dehydrated dietary fiber material with fiber-bound polyphenols.

61. The method of claim 60. wherein the fiber material is derived from a plant material.

62. The method of claim 61, wherein the plant material comprises an edible fruit material and / or an edible vegetable material.

63. The method of claim 61, wherein the plant material comprises a processed plant material.

64. The method of claim 61, wherein the processed plant material is a comminuted plant material, an expressed plant material, and / or an extracted plant material.

65. The method of claim 60. wherein the step of rapidly dehydrating or drying is performed to reduce residual water in the dehydrated dietary fiber material to equal or less than 10 wt%.

66. The method of claim 60, wherein the one or more exogenously added polyphenols are from a single plant material.

67. The method of claim 60, wherein the one or more exogenously added polyphenols are from multiple and distinct plant materials.

68. The method of claim 60, wherein the one or more exogenously added polyphenols are present in the dehydrated dietary fiber material with fiber-bound polyphenols in an amount of at least 0.1 wt%.

69. The method of claim 60, wherein the dehydrated dietary fiber material with fiber-bound polyphenols is comminuted to an average particle size of equal or less than 500 pm.

70. A method of preparing a dietary supplement and / or functional food, comprising formulating the dietary fiber composition of any one of claims 1-49 into liquid or solid to so form an article suitable for oral ingestion.

71. The method of claim 70, wherein the article is solid article.

72. The method of claim 71, wherein the solid article is a capsule, a ready -to-mix powder, a snack bar, an energy bar, a chew, a gummy, a confectionary' article, a baked good, or a cereal product.

73. The method of claim 70, wherein the article is liquid article.

74. The method of claim 70. wherein the liquid article is an energy’ drink, a juice beverage, a coffee, a fermented beverage, a tea, a smoothie, or a soda.

75. A method of increasing short chain fatty acid concentration, microbial biodiversity, and / or microbial abundance in a gut of a subject, comprising: administering to the subject a plurality of distinct dietary fiber materials;wherein each of the dietary fiber materials is derived from distinct plant materials, and wherein each of the dietary fiber materials comprises fiber-bound polyphenols; and wherein the distinct dietary fibers and fiber-bound polyphenols have a composition that, upon oral administration to the subject at an effective dosage, increase short chain fatty acid concentration, microbial biodiversity’, and / or microbial abundance in the gut of the subject.

76. The method of claim 75, wherein the plurality' of distinct dietary' fiber materials are administered at a dosage of between 250 mg and 2,500 mg or between 500 mg and 5,000 mg.

77. The method of claim 75 or claim 76, wherein the plurality of distinct dietary' fiber materials are administered over a period of at least 2 weeks.

78. The method of claim 75, wherein administration increases the short chain fatty’ acid concentration in the gut of the subject.

79. The method of claim 75. wherein administration increases the microbial biodiversity in the gut of the subject.

80. The method of claim 75. wherein administration increases the microbial abundance in the gut of the subject.

81. The method of claim 75, wherein the plurality' of distinct dietary' fiber materials are provided in a composition of any one of claims 1-49.

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