Synergistic prebiotic fiber blends, compositions, and methods of use

Abstract

Synergistic prebiotic fiber blend compositions, ingestible formulations comprising same, and methods of use to promote synergistically beneficial microbes in the gut microbiome.

Description

SYNERGISTIC PREBIOTIC FIBER BLENDS, COMPOSITIONS, AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims prior to U.S. provisional patent application no. 63 / 640,540, which was filed April 30, 2024, and which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to synergistic prebiotic fiber blends, compositions, ingestible formulations comprising same, and methods of use to promote beneficial microbes in the gut microbiome.BACKGROUND

[0003] The human gut microbiota is a complex ecosystem composed of trillions of microorganisms that inhabit and interact with each other and the host throughout the gastrointestinal tract.1This microbial community plays an important role in maintaining overall health and well-being by participating in a range of physiological processes, such as nutrient metabolism, immune modulation, and defense against pathogens.1 3The presence and quantity of different beneficial microbes, each with specific functions, are key to maintaining a balanced ecosystem within the gut.4 6In this sense, dietary fibers, as the main energy source for gut microbes, play a pivotal role in shaping the composition and function of the gut microbiota, ultimately influencing human health outcomes.7,8Notably, microbes have different fiber degradation abilities and preferences,9 11and even small differences in dietary fiber structures can lead to marked shifts in bacterial outcomes.12 13

[0004] “Dietary fiber” refers mainly to a diverse group of plant-derived carbohydrates that resists digestion in the upper gastrointestinal tract and reaches the large intestine intact.14Although humans lack the enzymes necessary to break down fiber, it serves as a valuable energy source for gut microbes, which possess the enzymatic machinery to ferment most of these complex carbohydrates.15Through fermentation, gut microbes convert fiber into various metabolites, including short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, which are relevant due to their health-promoting effects locally and systemically.16

[0005] It is important to note that the structures of dietary fibers align with bacterial utilization capabilities. Dietary fibers differ in structural features, such as the degree of branching, glycosidic linkages, and solubility, which influence their fermentability by gut bacteria.11While previous studies have explored the impact of individual dietary fiber types on gut microbiota modulation and SCFA production, few studies have focused on the collective effects of fiber mixtures. In synthetic communities, we have demonstrated that bacterial taxa employ various prioritization strategies for fiber utilization when present in a mixture, allowing them to coexist even in highly competitive environments, such as the gut microbiome.17Moreover, given that individuals respond differently to different fiber types,18,19it is likely that offering a broad range of fibers, as opposed to a single type of fiber, would be a more effective strategy to elicit consistent responses across different individuals. Still most dietary interventions and investigations on gut microbial communities have focused on isolated fibers or specific fiber sources,0 23overlooking the intricate interactions and possible synergistic effects that arise when multiple fiber types are consumed together. Thus, there is a critical knowledge gap regarding how the gut microbiota responds to dietary fiber mixtures and the subsequent implications for SCFA production.

[0006] N.V. Nutricia, USPN 5,792,754, discloses a composition for enteral administration. The composition contains 5-120 g dietary fiber per daily dose. The fiber consists of (i) soluble nonstarch polysaccharides, (ii) insoluble non-starch polysaccharides, (iii) oligosaccharides, resistant starch or a mixture thereof, and (iv) lignin (see, e.g., claim 1).

[0007] N.V. Nutricia, USPAPN 2011 / 0091445, discloses dietary fiber compositions for the normalization of the intestinal flora of HIV-infected subjects. The dietary fibers are described as typically resistant to digestion and absorption in the human small intestine with preferably a complete or partial fermentation in the large intestine (see, e.g., para.

[0016] ). Preferably, at least one dietary fiber can stimulate the growth of bifidobacteria in the gut (see, e.g., para.

[0016] ). In an embodiment, the composition comprises at least two different dietary fibers selected from a group, including degradation products thereof, and an acid oligosaccharide dietary fiber selected from a group, including degradation products thereof (see, e.g., para.

[0034] ).

[0008] N.V. Nutricia, USPAPN 2015 / 0182579, discloses the use of cereal dietary fiber to delay or otherwise reduce a sating effect of a medical nutrition or medicament. In an embodiment, the dietary fiber is composed of at least 15 wt.% non-acetogenic saccharide units (see, e.g., claim 5).

[0009] Nestec S.A., USPAPN 2016 / 0100617, discloses a nutritional composition comprising a fructo-oligosaccharide, a polysaccharide that is not partially hydrolyzed guar gum, and inulin (see, e.g., claim 1).

[0010] Optibiotix Ltd., USPAPN 2023 / 0292806, discloses a composition comprising prebiotic, chromium, and soluble fiber, wherein the composition is formed by agglomeration into granules.

[0011] Previously, we designed a fiber mixture that promoted different microbial groups and showed localized and systemic benefits to subjects with Parkinson’s disease related to improvements in the gut community structure.24Our hypothesis continues to be that systematically designed fiber mixtures provide a plethora of structures that can accommodate the metabolic requirements and preferences of a wide range of gut bacteria, thereby supporting a more balanced and diverse gut microbiota than any single fiber. We developed novel fiber mixtures that render more consistent responses across people than single fibers. Here, we elucidated the effects of systematically designed fiber mixtures compared to their single fiber components on human gut microbiota composition and SCFA production in vitro.

[0012] In view of the above, it is an object of the present disclosure to provide fiber blends that have a synergistic and unexpected effect to support a wide range of beneficial bacteria, produce consistent inter-individual responses, and are effective in suppressing potential pathogens. It is a further object of the present disclosure to provide a method of systematically formulating fiber blends, through synergism, to promote a diverse range of health-associated bacterial groups and high fermentation metabolites in the gut. These and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein.SUMMARY

[0013] Provided is a composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as com resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides). In embodiments, the composition promotes Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and Faecalibacterium prausnitzii. In embodiments, the composition further promotes Lachnospira, Subdoligranulum, Blautia,Roseburia, Agathobacter, and Eubacterium eligens. The composition has a synergistic effect. In embodiments, the composition suppresses Proteobacteria, which include potentially pro- inflammatory bacteria. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as corn resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0014] By way of example, a composition is provided comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides. In embodiments, the varying amount by weight of each can be, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%). The varying amount by weight in examples can be an equal amount for each of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides. In other examples, the varying amount by weight can be 0% for up to two of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides.

[0015] Further provided is composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides). In embodiments, the composition promotes Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and other Clostridium cluster XlVa bacteria. In embodiments, the composition further promotes Lachnospira, Subdoligranulum, Blautia, Roseburia, Agathobacter, and Eubacterium eligens. The composition has a synergistic effect. In embodiments, the composition suppresses Proteobacteria, which include potentially pro-inflammatory bacteria. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each ofat least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0016] By way of example, a composition comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides. In embodiments, the varying amount by weight of each can be, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).17. The varying amount by weight in examples can be an equal amount for each of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides. In other examples, the varying amount by weight can be 0% for up to two of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides.

[0017] Still further provided is a composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, the above two compositions. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of the two compositions, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0018] Ingestible formulations comprising an above-described composition are also provided. The ingestible formulation can be a supplement, a powder sachet, a powder for a shake, a liquid shake, a prebiotic shot, a snack, or a meal replacement. The ingestible formulation can comprise from about 2.5 grams to about 25 grams of the composition.

[0019] In view of the above, a method of synergistically promoting Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and Faecalibacterium prausnitzii in the gut microbiome of a subject is further provided. The method comprises administering to the subject a composition comprising (or consisting essentially of or consisting of), such as a compositioncomprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as corn resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides) in an amount effective to promote synergistically said bacteria or an ingestible formulation comprising same. In embodiments, the method further synergistically promotes Lachnospira, Siibdoligramihim , Blautia, Roseburia Agathobacter and Eubacterium eligens. In embodiments, the method suppresses Probacteria, which include potentially pro-inflammatory bacteria. In embodiments of the method, the composition comprises (or consists essentially of or consists of) an amount by weight of each of each of at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as corn resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments of the method, the composition comprises (or consists essentially of or consists of) both compositions described above, such as equal amounts of both compositions. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of the two compositions, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0020] By way of example, a method is provided comprising administering to the subject a composition comprising (or consisting essentially of or consisting of), a composition is provided comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides. In embodiments, the varying amount by weight of each can be, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%). The varying amount by weight in examples can be an equal amount for each of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides. In other examples, the varying amount byweight can be 0% for up to two of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides.

[0021] Also in view of the above, a method of synergistically promoting Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and other Clostridium cluster XlVa bacteria in the gut microbiome of a subject is provided. The method comprises administering to the subject a composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides) in an amount effective to promote synergistically said bacteria or an ingestible formulation comprising same. In embodiments, the method further synergistically promotes Lachnospira, Subdoligranulum , Blautia, Roseburia, Agathobacter, and Eubacterium eligens. In embodiments, the method suppresses Probacteria, which include potentially pro-inflammatory bacteria. In embodiments of the method, the composition comprises (or consists essentially of or consists of) an amount by weight of each of at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments of the method, the composition comprises (or consists essentially of or consists of) both compositions described above, such as equal amounts of both compositions. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of the two compositions, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0022] By way of example, a method is provided comprising administering to the subject a composition comprising (or consisting essentially of or consisting of), a composition comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, andfructooligosaccharides. In embodiments, the varying amount by weight of each can be, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).17. The varying amount by weight in examples can be an equal amount for each of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides. In other examples, the varying amount by weight can be 0% for up to two of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides.FIGURES

[0023] Fig. 1A is a heatmap of relative abundances (presented as Z-scores) showing clusters of bacterial species exhibiting similar patterns of abundance depending on the type of substrate present. Hierarchical clustering of taxa was performed using Euclidean distances. Four main clusters of taxa (red, green, purple, and blue) were observed and associated with fiber types.

[0024] Fig. IB shows the short-chain fatty acid (SCFA) profiles (percentage of acetate (blue), propionate (red), and butyrate (green) of total SCFAs) in relation to fiber type. No added fibers (blank), arabinoxylan (AX), arabinoxylan-oligosaccharides (AXOS), pectic galactan (PG), wheat bran (WB), arabinan (A), green banana resistant starch (BRS), high-amylose com resistant starch (CRS), wheat resistant starch (WRS), resistant a-glucan from enzymatic modification of isomaltodextrin (RAG), Chitin-glucan (CG), beta-glucan (BG), konjac glucomannan (KG) pectin (PEC), galactooligosaccharides (GOS), xylooligosaccharides (FOS) and fructooligosaccharides (FOS).

[0025] Fig. 2A is a heatmap of relative abundances (presented as Z-scores) showing clusters of bacterial species exhibiting similar patterns of abundance depending on the fibers selected for two mixtures, namely mixture 1 and mixture 2. Hierarchical clustering of taxa was performed using Euclidean distances. Four main clusters of taxa (red, green, purple, and blue) were observed and associated with fiber types.

[0026] Fig. 2B shows the SCFA profile (mM) per 50 mg carbohydrate in relation to fiber type. Results presented refer to the initial in vitro fecal fermentation experiment using pooled fecal samples for no added fibers (blank), arabinoxylan (AX), arabinoxylan-oligosaccharides (AXOS),pectic galactan (PG), wheat bran (WB), arabinan (A), green banana resistant starch (BRS), high- amylose corn resistant starch (CRS), wheat resistant starch (WRS), resistant a-glucan from enzymatic modification of isomaltodextrin (RAG), Chitin-glucan (CG), beta-glucan (BG), konjac glucomannan (KG) pectin (PEC), galactooligosaccharides (GOS), xylooligosaccharides (FOS) and fructooligosaccharides (FOS).

[0027] Fig. 2C shows the total SCFA (mM) per 50 mg carbohydrate in relation to fiber type. Results presented refer to the initial in vitro fecal fermentation experiment using pooled fecal samples for no added fibers (blank), arabinoxylan (AX), arabinoxylan-oligosaccharides (AXOS), pectic galactan (PG), wheat bran (WB), arabinan (A), green banana resistant starch (BRS), high- amylose corn resistant starch (CRS), wheat resistant starch (WRS), resistant a-glucan from enzymatic modification of isomaltodextrin (RAG), Chitin-glucan (CG), beta-glucan (BG), konjac glucomannan (KG) pectin (PEC), galactooligosaccharides (GOS), xylooligosaccharides (FOS) and fructooligosaccharides (FOS).

[0028] Fig. 3A is a heatmap of relative abundances (presented as Z-scores) showing clusters of bacterial species exhibiting similar patterns of abundance depending on individual fibers and mixture E

[0029] Fig. 3B is a heatmap of relative abundances (presented as Z-scores) showing clusters of bacterial species exhibiting similar patterns of abundance depending on individual fibers and mixture 2.

[0030] Fig. 3C is a heatmap of relative abundances (presented as Z-scores) showing responses of bacterial species to mixtures 1, 2 and 3.

[0031] Fig. 3D shows alpha diversity analysis, as measured by the Shannon index, of single fibers versus their corresponding mixtures. The asterisks denote significance (*, P < 0.05; **, P < 0.01). Results are presented for no added fibers (blank), arabinoxylan (AX), pectin (PEC), high-amylose com resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides (AXOS), green banana resistant starch (BRS), chitin- glucan (CG), and galactooligosaccharides (GOS).

[0032] Fig. 4A shows acetate production in response to individual fibers and mixtures 1, 2 and 3. The asterisks denote significance (*, P < 0.05; **, P < 0.01). Results are presented for no added fibers (blank), arabinoxylan (AX), pectin (PEC), high-amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides(AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0033] Fig. 4B shows propionate production in response to individual fibers and mixtures 1, 2 and 3. The asterisks denote significance (*, P < 0.05; **, P < 0.01). Results are presented for no added fibers (blank), arabinoxylan (AX), pectin (PEC), high-amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides (AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0034] Fig. 4C shows butyrate production in response to individual fibers and fiber mixtures 1, 2 and 3. The asterisks denote significance (*, P < 0.05; **, P < 0.01). Results are presented for no added fibers (blank), arabinoxylan (AX), pectin (PEC), high-amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides (AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0035] Fig. 4D shows total SCFA production (mM) per 50 mg carbohydrate in relation to individual fibers and mixtures 1, 2 and 3. The asterisks denote significance (*, P < 0.05; **, P < 0.01). Results are presented for no added fibers (blank), arabinoxylan (AX), pectin (PEC), high- amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides (AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0036] Fig. 5A is a heat tree of relative abundances of bacterial taxa (represented by colored nodes) promoted by mixture 1 over single fiber components. Red-colored nodes indicate a significantly higher (p < 0.05 after "fdr" correction) proportion of a taxon in fiber treated samples. Node sizes are proportional to the number of ASVs within each taxon. Names of taxon discussed in the text were added and its representative nodes marked with gray squares. Results presented refer to the second in vitro fecal fermentation experiments using individual fecal samples from 10 people. Arabinoxylan (AX), pectin (PEC), high-amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides (AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0037] Fig. 5B is a heat tree of relative abundances of bacterial taxa (represented by colored nodes) promoted by mixture 2 over single fiber components. Red-colored nodes indicate a significantly higher (p < 0.05 after "fdr" correction) proportion of a taxon in fiber treated samples. Node sizes are proportional to the number of ASVs within each taxon. Names of taxon discussed in the text were added and its representative nodes marked with gray squares. Results presented refer to the second in vitro fecal fermentation experiments using individual fecal samples from 10 people. Arabinoxylan (AX), pectin (PEC), high-amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides (AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0038] Fig. 6 is a set of heat trees of relative abundances of bacterial taxa (represented by colored nodes) promoted by mixtures 1, 2 and 3, showing that mixture 3 had a similar, and perhaps intermediate, effect to mixtures 1 and 2. Red-colored nodes indicate a significantly higher (p < 0.05 after "fdr" correction) proportion of a taxon in fiber mixture treated samples. Node sizes are proportional to the number of species within each taxon. Names of taxon discussed in the text were added and its representative nodes marked with gray squares.

[0039] Fig. 7 is a PCoA plot of weighted UniFrac measure of beta diversity showing the direction in community changes, averaged by donor, for single fibers compared to mixtures 1, 2 and 3. Results presented refer to the second in vitro fecal fermentation experiments using individual fecal samples from 10 people. Arabinoxylan (AX), pectin (PEC), high-amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan- oligosaccharides (AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0040] Fig. 8 shows violin plots displaying the centered-log-ratio transformed relative abundances of genera identified as differentially abundant by ANCOM II across various fiber types. Results presented refer to the second in vitro fecal fermentation experiments using individual fecal samples from 10 people. The treatments are as follow: no added fibers (blank), arabinoxylan (AX), pectin (PEC), high-amylose corn resistant starch (CRS), konjac glucomannan (KG), fructooligosaccharides (FOS), arabinoxylan-oligosaccharides (AXOS), green banana resistant starch (BRS), chitin-glucan (CG), and galactooligosaccharides (GOS).

[0041] Fig. 9 shows Violin plots displaying the percent of variance of bacterial taxa explained after partitioning variance by fiber types and donors across samples. Single fiber group was designated for fermentations with single fibers (arabinoxylan, pectin, high-amylose corn resistant starch, konjac glucomannan, fructooligosaccharides, arabinoxylan-oligosaccharides, green banana resistant starch, chitin-glucan, and galactooligosaccharides) and Fiber mixture group was designated for fermentations with the fiber mixtures (Mixtures 1, 2 and 3).DESCRIPTION

[0042] The present disclosure is predicated on the systematic formulation of dietary fiber blends, which have been surprisingly and unexpectedly discovered to have synergistic effects in the promotion of a diverse range of health-associated bacterial groups and high fermentation metabolites in the gut. Distinct outcomes for these blends, which weren’t observed for the individual fibers of the blends, included (i) unique promotion of beneficial bacteria (e.g., some taxa were exclusively promoted by the fiber blends, a broader number of beneficial microbes were promoted by the fiber blends, and the intensity of promotion for several taxa was higher than the promotion by any individual fiber of the blend), (ii) higher production of short-chain fatty acids (SCFAs) compared to the sum of the proportional SCFA part of the individual fiber components, (iii) unmatched suppression of potentially pathogenic bacteria taxa (i.e., proteobacteria phylum), and (iv) greater consistency in the microbial response across different individuals by the fiber blends compared to the individual fibers of the blends. Furthermore, combining fibers with overlapping bacterial targets failed to provide further benefits, with an “averaged,” as opposed to “synergistic,” response benefit. The response achieved with the fiber blends was more consistent than that which can be achieved by individual fibers.

[0043] In view of the above, provided is a composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as com resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides). In embodiments, the composition promotes Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and Faecalibacterium prausnitzii. In embodiments, the composition further promotes Lachnospira, Subdoligranulum, Blautia,Roseburia, Agathobacter, and Eubacterium eligens. The composition has a synergistic effect. In embodiments, the composition suppresses Proteobacteria, which include potentially pro- inflammatory bacteria. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of each of at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as corn resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%). Based on the data obtained for donors in the experiments described herein, below, one having ordinary skill in the art would understand how to vary the relative amounts of each fiber type to promote or suppress different gut microbial communities based on, e.g., geographic regional differences in diet, etc.

[0044] In embodiments of this composition, the composition does not contain chromium. In embodiments of this composition, the composition does not contain non-acetogenic saccharide units. In embodiments of this composition, the composition does not contain cereal non-resistant starch. In embodiments of this composition, the composition is not administered to an HIV- infected patient. In embodiments of this composition, the composition does not further comprise (or further consist essentially of or further consist of) colostrum, such as bovine colostrum, and / or antibodies. In embodiments of this composition, the composition does not further comprise (or further consist essentially of or further consist of) probiotic bacteria.

[0045] By way of example, a composition is provided comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides. In embodiments, the varying amount by weight of each may be, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%). The varying amount by weight in examples may be an equal amount for each of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides. Inother examples, the varying amount by weight may be 0% for up to two of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides.

[0046] In embodiments, the composition comprises (or consists essentially of or consists of) glucomannan, fructan, and pectin, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of glucomannan, fructan, and pectin, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0047] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of glucomannan, fructan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of an amount by weight of each of glucomannan, fructan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0048] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of glucomannan, fructan, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of glucomannan, fructan, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0049] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of glucomannan, pectin, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of glucomannan, pectin, and resistant starch, wherein the amount by weight of each is, independently of eachother, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0050] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of glucomannan, pectin, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of glucomannan, pectin, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0051] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of glucomannan, resistant starch, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of glucomannan, resistant starch, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0052] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of fructan, pectin, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of fructan, pectin, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0053] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of fructan, pectin, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of fructan, pectin, andarabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0054] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of fructan, resistant starch, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of fructan, resistant starch, and arabinoxylan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0055] Also in view of the above, provided is a composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides). In embodiments, the composition promotes Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and other Clostridium cluster XlVa bacteria. In embodiments, the composition further promotes Lachnospira, Subdoligranulum, Blautia, Roseburia, Agathobacter, and Eubacterium eligens. The composition has a synergistic effect. In embodiments, the composition suppresses Proteobacteria, which include potentially pro-inflammatory bacteria. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%). Based on the data obtained for donors in the experiments described herein, below, one having ordinary skill in the art would understand how to vary the relative amounts of each fibertype to promote or suppress different gut microbial communities based on, e.g., geographic regional differences in diet, etc.

[0056] By way of example, a composition comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides. In embodiments, the varying amount by weight of each may be, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).17. The varying amount by weight in examples may be an equal amount for each of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides. In other examples, the varying amount by weight may be 0% for up to two of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides.

[0057] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of arabinoxylanoligosaccharides, glucomannan, and fructan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of arabinoxylanoligosaccharides, glucomannan, and fructan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0058] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of arabinoxylanoligosaccharides, glucomannan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of arabinoxylanoligosaccharides, glucomannan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0059] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of arabinoxylanoligosaccharides, glucomannan, and glucan, whereinthe amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of arabinoxylanoligosaccharides, glucomannan, and glucan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0060] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of arabinoxylanoligosaccharides, fructan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of arabinoxylanoligosaccharides, fructan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0061] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of arabinoxylanoligosaccharides, fructan, and glucan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of arabinoxylanoligosaccharides, fructan, and glucan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0062] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of glucomannan, fructan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of glucomannan, fructan, and resistant starch, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0063] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of glucomannan, fructan, and glucan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of glucomannan, fructan, and glucan, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0064] In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of fructan, resistant starch, and glucan wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%). In embodiments, the composition comprises (or consists essentially of or consists of) equal amounts by weight of fructan, resistant starch, and glucan wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

[0065] A composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, the above two compositions is also provided. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of the two compositions, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).

[0066] An ingestible formulation comprising one or more of the foregoing compositions is also provided. For example, an ingestible formulation comprising the composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, arabinoxylan, pectin, com resistant starch, konjac glucomannan, and fructooligosaccharides is also provided. The ingestible formulation can be a supplement, a powder sachet, a powder for a shake, a liquid shake, a prebiotic shot, a snack, or a meal replacement. The ingestible formulation can comprise from about 2.5 grams to about 25 grams of the composition, such as from about 2.5 to 25 grams, 2.5 toabout 25 grams, 2.5 to 20 grams, 2.5 to 15 grams, 2.5 to 10 grams, 2.5 to 5 grams, 5 grams to 25 grams, 10 grams to 20 grams, 15 grams to 25 grams, or 20 to 25 grams.

[0067] An ingestible formulation comprising the composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides is also provided. The ingestible formulation can be a supplement, a powder sachet, a powder for a shake, a liquid shake, a prebiotic shot, a snack, or a meal replacement. The ingestible formulation can comprise from about 2.5 grams to about 25 grams of the composition, such as from about 2.5 to 25 grams, 2.5 to about 25 grams, 2.5 to 20 grams, 2.5 to 15 grams, 2.5 to 10 grams, 2.5 to 5 grams, 5 grams to 25 grams, 10 grams to 20 grams, 15 grams to 25 grams, or 20 to 25 grams.

[0068] An ingestible formulation comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, the above two compositions is also provided. The ingestible formulation can comprise from about 2.5 grams to about 25 grams of the compositions, such as from about 2.5 to 25 grams, 2.5 to about 25 grams, 2.5 to 20 grams, 2.5 to 15 grams, 2.5 to 10 grams, 2.5 to 5 grams, 5 grams to 25 grams, 10 grams to 20 grams, 15 grams to 25 grams, or 20 to 25 grams.

[0069] The preparation of prebiotic fibers and composition comprising same is within the ordinary skill in the art. Likewise, the preparation of ingestible formulations comprising a composition comprising prebiotic fibers is also within the ordinary skill in the art.

[0070] In view of the above, also provided is a method of synergistically promoting Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and Faecalibacterium prausnitzii in the gut microbiome of a subject. To that end, a method is provided that comprises administering to a subject one or more of the foregoing compositions. For example, a the method comprises administering to the subject a composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as corn resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides) in an amount effective to promote synergistically said bacteria or an ingestible formulation comprising same. The ingestible formulation can be a supplement, apowder sachet, a powder for a shake, a liquid shake, a prebiotic shot, a snack, or a meal replacement. The ingestible formulation can comprise from about 2.5 grams to about 25 grams of the composition, such as from about 2.5 to 25 grams, 2.5 to about 25 grams, 2.5 to 20 grams, 2.5 to 15 grams, 2.5 to 10 grams, 2.5 to 5 grams, 5 grams to 25 grams, 10 grams to 20 grams, 15 grams to 25 grams, or 20 to 25 grams. The method can further promote I.achnospira.Subdoligranulum, laulia, Roseburia, Agathobacter, and Eubacterium eligens. In embodiments, the method suppresses Probacteria, which include potentially pro-inflammatory bacteria. In embodiments of the method, the composition comprises (or consists essentially of or consists of) an amount by weight of each of each of at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as corn resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%). In embodiments of the method, the composition comprises (or consists essentially of or consists of) both compositions described above, such as equal amounts of both compositions. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of the two compositions, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).

[0071] Also in view of the above, provided is a method of synergistically promoting Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and other Clostridium cluster XlVa bacteria in the gut microbiome of a subject. The method comprises administering to the subject a composition comprising (or consisting essentially of or consisting of), such as a composition, comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides) in an amount effective to promotesynergistically said bacteria or an ingestible formulation comprising same. The ingestible formulation can be a supplement, a powder sachet, a powder for a shake, a liquid shake, a prebiotic shot, a snack, or a meal replacement. The ingestible formulation can comprise from about 2.5 grams to about 25 grams of the composition, such as from about 2.5 to 25 grams, 2.5 to about 25 grams, 2.5 to 20 grams, 2.5 to 15 grams, 2.5 to 10 grams, 2.5 to 5 grams, 5 grams to 25 grams, 10 grams to 20 grams, 15 grams to 25 grams, or 20 to 25 grams. The method can further promote Lachnospirci, Stibdoligranulum, Blautia, Roseburia, Agathobacter, and Eubacterium eligens. In embodiments, the method suppresses Probacteria, which include potentially pro- inflammatory bacteria. In embodiments of the method , the composition comprises (or consists essentially of or consists of) an amount by weight of each of at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides), wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%). In embodiments of the method, the composition comprises (or consists essentially of or consists of) both compositions described above, such as equal amounts of both compositions. In embodiments, the composition comprises (or consists essentially of or consists of) an amount by weight of each of the two compositions, wherein the amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).EXPERIMENTAL SECTION

[0072] The following experimental section is intended to illustrate the present disclosure. The section is not intended to limit the scope of the appended claims in any way.Materials

[0073] Individual fibers were tentatively selected to promote bacterial groups previously related to health benefits such as Bacteroides, Ruminococcus, Clostridium cluster XlVa, and Bifidobacteria, as well as to support production of different SCFAs, with special emphasis on propionate and butyrate. In total, 16 different fibers were screened for their ability to promote different bacterial groups and metabolites related to health (Table 1).Table 1. Fibers selected for tentative preferential support of health-related bacterial taxa and metabolites.Source / Brand AcronymBacteroides and propionateArabinoxylan Corn / Agrifiber AXArabinoxylan-oligosaccharides Corn / Agrifiber AXOSPectic galactan Potato / Megazyme PGWheat Bran Wheat / Bob's Red Mill WBArabinan Sugar beet / Megazyme ARumi Clostridium cluster XlVa and butyrateType Green banana / Natural Evolution BRSType High-amylose corn (Hylon VII) / Ingredion CRSTypeWheat (Fibersym) / MGP WRSEnzymatic modified Isomaltodextrin / Resistant a-glucan Hayashibara RAGClostridium Cluster XlVa and butyrateChitin-glucan Aspergillus niger (KiOtransine) / Kitozyme CGBeta-glucan Barely / GrainFrac BBGluco manan Konjac root / Bulksupplements KGClostridium cluster IPectin Citrus Fiber (Citri-Fi 100) / Fiberstar PECGalactooligosaccharides Plant source / Health Aid GOSXylooligosaccharides Corncob / Nutrasumma XOSFructooligosaccharides Chicory root (Orafit) / Beneo FOS

[0074] Based on studies from our laboratory and others, Bacteroides and propionate were proposed to be promoted by arabinoxylan (AX), arabinoxylan-oligosaccharides (AXOS), pectic galactan (PG), wheat bran (WB), and arabinan (A)25 2X(Table 1). Ruminococcus is known for starch degradation, with several butyrate producers from the Clostridium cluster XlVa benefitingfrom cross-feeding in starch.29 31Thus, type II resistant starches from green banana (BRS) and high-amylose corn (CRS), type IV resistant starch from wheat (WRS), and a resistant a-glucan (isomaltodextrin) (RAG) were tested to support Ruminococcus + Clostridium cluster XI Va and butyrate production (Table 1). Chitin-glucan (CG), beta-glucan (BG), and konjac glucomannan (KG) were also tested for support of Clostridium cluster XlVa and butyrate production3233(Table 1). Pectin (PEC) was specifically tested for promotion of Faecalibacterium prausnilzii, which is also a member of the Clostridium cluster XlVa with special relevance to intestinal health.34Finally, galactooligosaccharides (GOS), xylooligosaccharides (XOS), and fructooligosaccharides (FOS) were tested for Bifidobacteria support.35All material sources are summarized in Table 1.First set of in vitro fecal fermentations (pooled fecal sample)

[0075] In vitro fermentations of the 16 dietary fibers (described above and in Table 1) and the blank (no fiber added) were performed in triplicate according to the methodology described by Cantu-Jungles et al.36using a pooled fecal inocula from three healthy donors (two males and one female; ages between 24 and 40; average age = 33). Briefly, carbonate-phosphate buffer was prepared and sterilized by autoclaving at 121°C for 20 min. The buffer was then cooled to room temperature, oxygen was removed by bubbling with carbon dioxide, and cysteine hydrochloride (0.25 g / liter of buffer) was added as a reducing agent. The prepared buffer was then placed into the anaerobic chamber the day before experimentation to complete buffer reduction. On the day of the experiment, freshly collected fecal samples from the three donors were pooled (in equal amounts) and homogenized with carbonate-phosphate buffer (1 :3 [wt / vol]), followed by filtration through four layers of cheesecloth. Then, one ml of this fecal inoculum was added to Balch tubes (Chemglass Life Sciences, Vineland, NJ) containing 50 mg of the dietary fiber substrate and 4 ml of the carbonate-phosphate buffer. Tubes were closed with butyl rubber stoppers (Chemglass Life Sciences), sealed with aluminum seals (Chemglass Life Sciences), and incubated at 37°C in a shaker incubator (150 rpm; MaxQ 6000; Thermo Fisher, Waltham, MA) for 24 hrs. Aliquots of the baseline sample and samples after 24-hr fermentation were prepared and stored at -80°C until further use for SCFA analysis (0.5 ml) and DNA sequencing (1 ml). All sample manipulation was conducted under an anaerobic atmosphere (85% N2, 5% CO2, and 10% H2).Human stool collection and use were approved by the Institutional Review Board at Purdue University (IRB protocol no. 1510016635).

[0076] SCFA analysis (pooled fecal sample). Samples for SCFA analyses were prepared as previously described (10) and analyzed using a gas chromatograph (GC-FID 7890 A; Agilent Technologies Inc.) on a fused silica capillary column (Nukon Supelco no. 40369-03 A; Bellefonte, PA) under the following conditions: injector temperature at 230°C, initial oven temperature at 100°C, and temperature increase of 8°C / min to 200°C with a hold for 3 min at final temperature. Helium was used as a carrier gas at 0.75 ml / min. Quantification was performed based on relative peak areas using external standards of acetate (A38S), propionate (A258), and butyrate (AC108111000) and an internal standard of 4-methylvaleric acid (AAAI 540506) from Fisher Scientific (Hampton, NH).

[0077] DNA extraction and 16S rRNA gene amplicon sequencing (pooled fecal sample).Stored samples for DNA extraction were thawed and centrifuged (13,000 rpm for 15 min), and supernatants were discarded. Automated DNA extraction of the precipitates was performed using the QIAcube Connect instrument (Qiagen, Germantown, MD) with the QIAamp PowerFecal Pro DNA kit (Qiagen) per manufacturer’s instructions. The V4 region of the 16S rRNA gene was amplified using primers 515F and 806R. The primers contained 5' common sequence tags (known as common sequence 1 and 2 [CS1 and CS2]). First-stage PCR amplifications were performed in 10-pl reaction mixtures in 96-well plates, using MyTaq HS 2x master mix (Bioline, Memphis, TN). PCR conditions were 95°C for 5 min, followed by 28 cycles of 95°C for 30 sec, 55°C for 45 sec, and 72°C for 60 sec. Amplicons were generated using a two-stage PCR amplification protocol as described previously (37). The primers contained 5' common sequence tags (known as common sequence 1 and 2 [CS1 and CS2]) as described previously (38). Subsequently, a second PCR amplification was performed in 10-pl reaction mixtures in 96-well plates. A master mix for the entire plate was made using MyTaq HS 2* master mix. Each well received a separate primer pair with a unique 10-base barcode, obtained from the Access Array Barcode Library for Illumina (Fluidigm, South San Francisco, CA; catalog no. 100-4876). These Access Array primers contained the CS1 and CS2 linkers at the 3' ends of the oligonucleotides. Cycling conditions were 95°C for 5 min, followed by 8 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec. Samples were then pooled in equal volume using an EpMotion5075 liquid handling robot (Eppendorf, Hamburg, Germany). The pooled library was purified using anAMPure XP cleanup protocol (0.6x, vol / vol; Agencourt, Beckman-Coulter, Indianapolis, IN) to remove fragments smaller than 300 bp. The pooled libraries, with a 20% phiX spike-in, were loaded onto an Illumina MiniSeq mid-output flow cell (2 x 153 paired-end reads). Based on the distribution of reads per barcode, the amplicons (before purification) were re-pooled to generate a more balanced distribution of reads. The re-pooled library was purified using AMPure XP cleanup, as described above. The re-pooled libraries, with a 20% phiX spike-in, were loaded onto a MiniSeq flow cell and sequenced (2 x 153 paired-end reads). Fluidigm sequencing primers, targeting the CS1 and CS2 linker regions, were used to initiate sequencing. Demultiplexing of reads was performed on instrument. Library preparation, pooling, and sequencing were performed at the University of Illinois at Chicago Genome Research Core (GRC) within the Research Resources Center (RRC).Second set of in vitro fecal fermentations (individual fecal samples)

[0078] Fecal material for the experiment was collected from 10 donors with good intestinal health (5 males and 5 females; age between 17 and 64; average age = 46). The fecal samples were collected only once and anonymously and are, therefore, considered not subjected to the WMO in The Netherlands. To collect the sample, the subjects used the FecesCatcher, a specimen collection device consisting of biodegradable paper to be placed under the toilet seat (fecesvanger.nl). Fecal material was collected with a sterile plastic spoon and placed in a tube that was, in turn, placed in an anaerobic jar (materials provided by TNO) with an AnaeroGen Sachet (Thermo Fisher Diagnostics GMBH). The jar was kept at 4 °C until delivery at the laboratory (within 24 hrs of collection). There, the material was introduced into an anaerobic chamber, diluted 1 :3 with phosphate-buffered saline, and homogenized. Finally, 20% glycerol was added before storing the material at -80 °C. Individual fermentations were performed with fecal material incubated anaerobically in the i-screen (intestinal screening) system.37First, the fecal samples were pre-cultured overnight in modified standard ileal efflux medium (SIEM) in anaerobic conditions at 37 °C with shaking at 300 rpm.38The microbiota were then transferred to microtiter plates, and the fibers were added at a concentration of 4 mg / mL. The i-screen incubation started with a fecal bacterial load of approximately 109CFU / mL. The microbiota were cultured in SIEM with pH adjusted to 5.8. All compounds were tested in triplicate. After 24hrs of anaerobic fermentation, the incubation material was sampled for DNA isolation and metabolite analysis.

[0079] SCFA analysis (individual fecal samples). Supernatant samples were diluted 20x with 75% methanol. Internal standard solution (50 pL of d3-acetic acid, d3-propionic acid, d3-butyric acid, and d9-valeric acid) was added to 50 pL of diluted fecal material. This was followed by 50 pL of 50 mM 3 -nitrophenylhydrazine solution (75% methanol in water), 50 pL of 50 mM 1- ethyl-3 -(3 -dimethylamino-propyl) carbodiimide solution (75% methanol in water), and 50 pL of pyridine (7.5% in 75% methanol). Samples were incubated for 30 min at 600 rpm at room temperature. Then 250 pL of 2% of formic acid were added, and the samples were mixed. The samples were stored at -80°C until analysis. The derivatized SCFA were analyzed by LC-MS using a high-resolution mass spectrometer (Q-Exactive, Thermo, USA) equipped with an electrospray source (HESI). The mass spectrometer was operated in positive ion mode at a resolution of 17,500. Data were acquired by scanning from m / z 100 to 700. Separation of the derivatized SCFA was done with an Acquity H-Class UPLC system (Waters) fitted with an Acquity BEH-C18 column (Waters, 150 x 2.1 mm, 1.7 pm). Mobile phase A was 0.1% formic acid in water, and mobile phase B was 100% acetonitrile. The gradient used was 16% B (0 min), 25% B (min), 40% B (9 min), followed by column wash-out at 95% B and equilibration at 16% B, at a flow rate of 0.35 mL / min and a column temperature of 40 °C. The injection volume was 2.0 pL.

[0080] SCFA concentrations were obtained by the analysis of calibration standards in 75% methanol in water (in total, seven concentrations for each SCFA). Concentration ranges were 0 to 100 pM (acetic acid), 0 to 50 pM (propionic and butyric acid), and 0 to 10 pM (iso-butyric, valeric, iso-valeric, and 2-methylbutyric acid). The calibration standards were lOOx diluted prior to adding internal standard solution and derivatization.

[0081] DNA extraction and 16S rRNA gene amplicon sequencing (individual fecal samples). Automated DNA extraction of the precipitates was performed using the QIAcube Connect instrument (Qiagen, Germantown, MD) with the Dneasy 96 Powersoil Pro QIAcube HT kit (Qiagen) per manufacturer’s instructions. Changes in the microbiota composition were analyzed by using 16S rDNA amplicon sequencing. The V4 hypervariable region was targeted. A total of 100 pg of DNA was amplified as described by Kozich et al.,39with the exception that 30 cycles were used instead of 35, applying F515 / R806 primers.40Primers included Illumina adapters anda unique 8 nt sample index sequence key.39The amplicon libraries were pooled in equimolar amounts and purified using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). Amplicon quality and size were analyzed on a Fragment Analyzer (Advanced Analytical Technologies, Inc., Heidelberg, Germany). Paired-end sequencing of amplicons (approximately 400 base pairs) was conducted on the Illumina MiSeq platform (Illumina, Eindhoven, The Netherlands).Bioinformatics

[0082] Samples from both the first set (pooled fecal fermentations) and the second set (individual fecal fermentations) of in vitro fecal fermentations were processed in a similar manner initially. Sequence reads were supplied as paired-end FASTQ sequence files and imported into QIIME 2 (q2) version 2-2021.11 for analysis.41Amplicon sequence variants (ASVs) were generated using DADA242with sequences trimmed at 153 bp, and taxonomic assignment was carried out using the q2-feature-classifier plugin against the Silva reference database classifier with 99% similarity, specific for the V4 16S region (v. 138). Sequence alignment and construction of a phylogeny tree were obtained using the Qiime2 pipeline align- to-tree-mafft-fasttree. Non-rarefied ASVs were collapsed at the species level, and relative abundances were used for downstream analysis and visualization. Heatmaps of relative abundances were created using R Stats software version 3.6.3 (R Core Team, Vienna, Austria), and SCFA data were visualized using GraphPad Prism (V. 9.5.1).

[0083] Additional analyses were performed for the second set of in vitro fecal fermentations to evaluate differences in community structure of single fibers versus the designed fiber mixtures. To minimize the effects of sequencing depth on diversity measurements, the number of reads from each sample was rarefied to 7,600 and alpha and beta diversities were calculated using the q2-diversity plugin, which included Shannon Index for alpha diversity and weighted UniFrac for beta diversity. Statistical differences in alpha diversity were calculated using the q2-alpha-group- significance plugin. Detection of taxa significantly different from the blank at all taxonomic levels was achieved using the Wilcoxon Rank Sum test corrected for multiple comparisons and visualized through differential phylogenetic trees using METACODE. Differentially abundant genera of each fiber substrate versus the blank were also evaluated using a stricter linear mixed- effects model implemented in ANCOM-II (Analysis of Composition of Microbiomes-II, R-codehttps: / / github.com / FrederickHuangLin / ANCOM), accounting for the donor as the random effect and adjusting for time (before versus after substrate fermentation). Each fiber treatment was individually compared to the control group (blank) in ANCOM-II, and a genus was determined to be significant if it surpassed the 0.9 threshold for statistical significance. The variancePartition method was also used to quantify the variation in gut microbial taxa attributable to donor and fiber type, when fibers were used individually or as a mixture.43PCoA plots, differential phylogenetic trees, and violin plots of differentially abundant genera detected through ANCOM in response to fiber treatments and from variancePartition analysis were created using R Stats software version 3.6.3 (R Core Team, Vienna, Austria).Fiber mixture design

[0084] A range of 16 dietary fibers was selected for their ability to promote different groups of microbes related to health, namely Bacteroides Ruminococcus (from both Clostridium Clusters IV and XlVa), other Clostridium cluster XlVa members, Faecalibacterium prausnitzii (Clostridium cluster IV), and Bifidobacteria. After 24 hrs of in vitro fecal fermentation using a pooled fecal sample from an American cohort of healthy adults, most abundant community members were clustered using Euclidean distances (Fig. 1A). Four main bacterial clusters were identified representing groups of bacterial species that exhibit similar patterns of abundance that are dependent on the type of substrate present (labeled and colored from 1 to 4 on the left side of the heatmap, Fig. 1A). RAG, CG, BB, and KG were most promotive of Cluster 1 (in red), resistant starches BRS, CRS and WRS were most promotive of Cluster 2 (in green), oligosaccharides GOS, XOS and FOS were most promotive of Cluster 3 (in purple), and AX, AXOS, PG, WB, A and PEC were most promotive of Cluster 4 (in blue) (Fig. 1A). Moreover, specific SCFA profiles were related to the fiber type, with several fibers promotive of Cluster 1 being also related to propionate production, and fibers related to promotion of Cluster 2 related to butyrate production (Fig. IB).

[0085] Thus, individual fibers were selected to make mixtures different in composition that would promote all four complementary bacterial clusters as well as different SCFAs to support community diversity. Fiber mixture 1 was composed of AX, PEC, CRS, KG and FOS in equal amounts (Fig. 2A). Fiber mixture 2 was composed of AXOS, BRS, CG, KG, and GOS in equalamounts (Fig. 2A). Finally, fiber mixture 3 was composed of equal amounts of fiber mixtures 1 and 2.Designed mixtures better support microbial diversity and SCFA production than single fibers

[0086] In the second set of experiments, in vitro fecal fermentations were conducted with each fiber mixture and the individual fibers comprising each mixture separately. This was performed using fecal inocula from 10 distinct donors from a cohort from healthy adults from the Netherlands. Promotion of the clusters (defined above from the pooled fecal fermentation set of experiments) was then evaluated in each donor separately, and a high interindividual response variability was observed (Figs. 3A, 3B and 3C). Also, no Butyrivibrio or Catenibacterium taxa were observed (Figs. 3A and 3B) due to differences in microbiota composition from the two experimental sets. Fiber responses were not always consistent with the first experimental set as noted for several taxa like Ruminococcus torques group (not promoted by any of the fibers tested), Subdoligranulum (better supported by PEC than FOS in mixture 1), and Bacteroides (better supported by KG than AX in mixture 1), among others. These observations, in alignment with prior reports, indicate that fiber responses can vary across donors19,44,45and even more so between donors from different regions of the world, which will likely have different gut microbial communities,46as was the case in this study. Still, several fiber responses observed were congruent with expectations based on the initial set of experiments. For instance, Parabacteroides, Lachnospiraceae, and Roseburia in cluster 1 (outlined in red) were well promoted by KG, a component of both mixtures (Figs. 3A and 3B). In cluster 2 (in green), CRS most effectively promoted Ruminococcaceae (C AG-352) in mixture 1, and BRS was the most effective promoter of Agathobacter in mixture 2 (Figs. 3A and 3B). For cluster 3 (in pink), FOS and GOS from mixture 1 and 2, respectively, were the most effective at promoting Anaerostipes and Bifidobacterium (Figs. 3A and 3B). In cluster 4 (in blue), PEC most effectively promoted Faecalibacterium in mixture 1, and AXOS was the most effective promoter of Bacteroides in mixture 2 (Figs. 3A and 3B).

[0087] In both experimental sets, no single individual fiber was particularly effective at supporting all clusters of taxa (Figs. 1A, 3A and 3B). However, when mixtures of the single fibers were composed, a broader support of different taxa was achieved (Figs. 3A and 3B).Beyond that, for some taxa, like Fusicatenibacter , Agathohacter , Roseburia, Blautia and Dorea the mixtures performed better than any of the single fibers tested (Figs. 3A and 3B). Thus, the mixture of fibers targeted toward different bacterial taxa was overall better for broader community support. When comparing the three mixtures (Fig. 3C), mixture 1 seemed to offer support to the broadest range of taxa. Importantly, blending mixtures 1 and 2 to produce mixture 3 did not provide additional support for new microbes, but rather an intermediate effect to those observed for fiber mixtures 1 and 2 was achieved (Fig. 3C).

[0088] These findings were further substantiated by alpha diversity analysis, as measured by the Shannon index, of single fibers versus their corresponding mixtures (Fig. 3D). Mixture 1 outperformed all its individual fiber components in supporting alpha diversity, and mixture 2 was superior to most of its single fiber components (with the exception of AXOS) in supporting alpha diversity (Fig. 3D). Additionally, combining mixtures 1 and 2 (both of which were designed for the support of similar clusters of taxa) into mixture 3 did not further enhance alpha diversity (Fig. 3D). No significant differences were detected among the three mixtures tested (Fig. 3D). Hence, the composition of mixtures with fibers of overlapping bacterial targets does not confer additional benefits to diversity support and highlights the importance of mixtures composed of different target-specific single fibers.

[0089] Regarding SCFA production, mixtures 1, 2, and 3 were among the most promotive of acetate, butyrate, and propionate production, and higher than some of their single fiber components (Figs. 4A, 4B, and 4C). Also, the three mixtures performed similarly in increasing the production of the three SCFAs (Figs. 4A, 4B, and 4C). Accordingly, the production of total SCFAs was more pronounced for the mixtures than for several of their individual fiber components (Fig. 4D). Forty percent of the single fibers composing mixture 1 and 80% of the single fibers composing mixture 2 had a lower total SCFA production than the mixtures. SCFA production of the mixtures was not merely a summation of SCFA production of each single fiber component but was significantly higher in amount suggesting a synergistic effect when fibers are combined in mixtures, which is more advantageous for SCFA production.

[0090] Overall, systematically designed mixtures that support complementary groups of bacteria were better than single fibers at supporting a diverse range of gut microbes as well as the production of health-related metabolites, namely butyrate, propionate, and acetate.Synergistic effect of fibers in a mixture result in unique promotion of bacterial taxa

[0091] Differential phylogenetic trees were used for the visualization of significant changes in microbial taxa abundance at multiple taxonomic levels for each fiber type compared to the blank, using data from the second set of in vitro fecal fermentations. In agreement with heatmaps and alpha diversity results discussed above, mixtures 1 and 2 promoted more bacterial taxa (represented by colored nodes in the heat tree) than any of their single fiber components (Figs. 5A and 5B). This is clearly observed for several taxa, including those belonging to the Lachnospiraceae family (at the left side of the heat tree). Lachnospiraceae is an important family of bacteria related to health, which includes a variety of butyrate producers. Mixtures 1 and 2 promoted, respectively, 13 and 12 different tree tip nodes (representing different species) of Lachnospiraceae, while their summed single fiber components ranged from promoting only 1 in mixture 1 (FOS and CRS) and 0 in mixture 2 (CG), to a maximum of 5 (KG) Lachnospiraceae nodes in both mixtures. Surprisingly, not only were more taxa promoted by the mixtures, but several new taxa were found which were not promoted by their individual component fibers fermented separately. For instance, some Blautia spp were uniquely promoted by the mixtures, but not by their individual fibers. Also, at the class level, Bacilli were only promoted by the mixtures, and not by their individual fibers. At the phylum level, Actinobacteria was significantly promoted in mixture 1 (and phylogenetically at the genus level, Bifidobacterium was also significantly promoted), but not by any of mixture l's single fiber components. Similarly, the Firmi cutes phylum was only promoted by mixture 2, but not by any of its single fiber components, compared to the blank. Interestingly, Proteobacteria, a phylum known to have several pathogens and usually related to a proinflammatory phenotype, presented increases in relative abundances when several of the single fibers were tested in comparison to the blank, but this was not observed when fibers were blended together. Mixture 3 had a similar, and perhaps intermediate effect, to fibers 1 and 2 (Fig. 6), corroborating results previously presented.

[0092] To understand how the different fibers tested changed the overall community structure, shifts in beta diversity before and after fermentation were evaluated using weighted UniFrac samples. Interestingly, the direction of changes in PCoA plots, averaged by donor, were markedly different for single fibers compared to those obtained from fiber mixtures (Fig. 7). While all single fibers trended in a direction towards the left side of the PCoA plot, all three fiber mixtures shifted towards the right side of the graph. Taken together, these results show thatchanges in community structure promoted by fiber mixtures are not merely a summation or average of the changes promoted by the individual fibers of which they are composed. Rather, a synergistic effect is observed when blending fibers together to mixtures, which culminates in unique changes in specific taxa as well as in the overall community structure that are not observed in the mixture components separately.Mixtures are more consistent in changing the gut microbial community

[0093] The specific genera enhanced by single fibers and fiber mixtures, as compared to the blank, were identified using ANCOM II. Ten bacterial genera were identified by ANCOM as being promoted by one or more types of fiber. These included Enterococcus from the Bacilli phylum, promoted by all three mixtures, and several members from the Lachnospiraceae family, such as Anaerostipes (promoted by FOS and mixture 1), Lachnospira (promoted by mixtures 1 and 3), Subdoligranulum (promoted by mixture 1), Blautia (promoted by mixtures 1 and 3), Roseburia (promoted by mixture 1), Agathobacter (promoted by mixture 3), and the Eubacterium eligens group (promoted by mixture 2). Parabacteroides were supported by BRS and CG (Table 2).Table 2. W values of differentially abundant genera identified through ANCOM-II from fiber treatment comparisons versus the blankDifferentially abundant taxa* Wg Parabacteroides 28 g Roseburia 27g _ Parabacteroides 25 g _ Lachnospira 40 g _ Faecalibacterium 27* Taxa were detected at the 0.6 threshold level across 10 donors. Results are presented for green banana resistant starch (BRS). chitin-glucan (CG), pectin (PEC), fructooligosaccharides (FOS), and mixtures 1, 2 and 3 (MIX 1, MIX 2 and MIX 3, respectively). No taxa were detected to be differentially promoted for arabinoxylan (AX), arabinoxylan-oligosaccharides (AXOS), galactooligosaccharides (GOS), high-amylose com resistant starch (CRS), and konjac glucomannan (KG) compared to tire blank.

[0094] Centered log ratios of genera detected by ANCOM were plotted for all fiber types in violin plots to evaluate the distribution of donors' responses (Fig. 8). Notably, genera such as Enterococcus, Anaeroslipes, and Lachnospira were promoted to some extent in all individuals by the three fiber mixtures. While some single fibers could promote these genera, no single fiber was effective in all donors (except for BRS in promoting Enterococcus)' . The fiber mixtures also generally promoted Subdoligranulum and Blautia in all subjects to some degree, with few individual fibers displaying such consistent effects across subjects. Another observation was that no single fiber or fiber mixture was universally successful in promoting Roseburia, although the mixtures did support this genus in a larger number of subjects compared to any individual fiber. In the case of genera such as Agathobacter and the Eubacterium eligens group, variations in response were observed across subjects for all tested fibers. Yet intriguingly, the responses to the mixtures closely mirrored those to the most promotive individual fibers, even though they comprised only a fraction of the mixture and other individual fibers in the mix showed little or no response. The only taxa detected through ANCOM to be better supported by single fibers than the mixtures was Parabacteroides. In several instances, the mixtures were more promotive oftaxa than any of their individual fiber components (i.e., Enterococcus in all mixtures, Blautia in mixture 1 and Anaerostipes and Lachnospira in mixture 2). It is also noteworthy that in several instances donors with taxa that were not responsive to individual fibers became responsive when fibers were blended together. For instance, in the case of donor 5, neither Enterococcus nor Anaerostipes was promoted when exposed to the individual fibers that make up mixture 1. Yet, surprisingly, these bacteria were promoted when fermented with mixture 1. This was also observed with the same donor for Anaerostipes and mixture 2, with Lachnospira in donor 3 with mixture 2, among others.

[0095] The observed improved consistency in response of bacterial taxa promoted across donors could infer some degree of predictability of gut microbiota responses related to fiber. To compare the overall predictability of responses across people when using individual fibers versus fiber mixtures we have performed a variance partitioning analysis, which determined how much variation in the gut microbiota response was attributed to the donor, fiber treatment (single fibers versus fiber mixtures), or residuals (unexplained variation that cannot be accounted for by the fibers or donor of fecal sample). Results showed that more variance is explained by the treatment when fibers are presented as a mixture compared to individual fibers (Fig. 9). The multimodal distribution for variance explained by donor, fiber and residuals with single fibers also indicates that the response to single fibers tested here is not as straightforward and may depend on more precise or individual factors specific to each donor's microbiota. Similarly, the lack of explained variability for individual fibers was significantly higher than for mixtures, as shown by greater residuals (Fig. 9). These results corroborate the above analysis and suggest that better consistency in responses across different donors is achieved by using mixtures rather than individual fibers.Discussion

[0096] Several groups of microbes in the gut have been related to human health and also have been proposed to have preferential utilization of some dietary fiber types.2947 ,1In this study, three fiber mixtures composed of single fibers that together could stimulate all four distinct bacterial clusters observed in a first set of pooled in vitro fecal fermentation experiments was systematically designed, aiming to support different microbial groups related to health and produce a variety of different SCFAs. The following dietary fibers were selected: AX, CRS, KGand FOS (to compose mixture 1) and AXOS, BRS, KG, and GOS (to compose mixture 2) - due their abilities to promote similar targets including Bacteroides, Ruminococcus + Clostridium cluster XlVa, other Clostridium cluster XlVa, and Bifidobacteria, respectively. Moreover, PEC was included in mixture 1 for promotion of Faecalibacterium prausnitzii from Clostridium Cluster IV, and CG was included in mixture 2 for promotion of other Clostridium cluster XlVa bacteria. To understand if increasing the number of fiber types in a mixture that have similar / overlapping bacterial targets further improve the response, we have also designed mixture 3 composed of equal amounts of mixtures 1 and 2.

[0097] The three designed mixtures were tested against their individual components in a second set of in vitro fecal experiments performed individually for 10 different donors. Despite the differences observed compared to the first set of experiments, single fibers were still overall promotive of specific bacterial taxa, whereas the fiber mixtures, as expected, gave better support to a broader number of bacteria than any of the individual fibers. Unexpectedly, not only more of the targeted groups were promoted by the mixtures, but also, for several taxa, the support was as good or better than that of the summed individual fibers, despite they each only represented 20% of the mixtures 1 and 2. Surprisingly, different and new taxa were promoted by the mixtures, but not by any of their single fiber components indicating a synergistic effect of fibers when present together as a mixture. Similarly, the SCFA production from mixture fermented samples was not an average of its composed fibers, but instead was produced in higher levels for all mixtures, despite several of its components leading to low-SCFA production individually.

[0098] These results indicate that there is a heretofore unreported synergistic advantage in using a range of substrates to support different bacterial taxa simultaneously. This notion resonates with the principles of resource hierarchical utilization, as observed in other microbial studies, where species reciprocally prioritize certain resources over others, ensuring that they do not directly compete for the exact same resource at the same time.17Such differentiation in fiber preferences can allow for stable coexistence of different species in complex microbial communities when using fiber mixtures, aligned with the higher diversity we observed compared to single fibers. Furthermore, the support of diverse bacterial species can lead to beneficial interactions among them that can include the production of growth-promoting substances, alterations to the local environment that are beneficial for growth, or the suppression of potential pathogens.52 24Metabolic cooperation can also be beneficial where some bacteria producemetabolic byproducts that other bacteria can utilize as substrates (i.e., cross-feeding), which can support their growth even when they are not directly fed.54Whereas deciphering specific mechanisms is out of the scope of this study, probably several of these factors act together to produce the observed synergistic effects of fibers toward gut microbial promotion and diversity, where each bacterial species can better thrive, despite individual members not receiving a large amount of direct substrate.

[0099] Several genera were detected to be promoted by the mixtures through ANCOM II, all belonging to Firmicutes, and mostly to the Lachnospiraceae family from Clostridium cluster XlVa, such as, Lachnospira, Subdoligranulum, Blautia, Roseburia, Agathobacter , and the Eubacterium eligens group. Whereas other groups were initially targeted by the mixtures, such as Bacteroides and bifidobacteria, taxa from Clostridium cluster XlVa was notably the main target for 40% of the fibers included in mixture 1 (CRS and KG), 60% of the components included in mixture 2 (BRS, KG and CG), and 50% of the components of mixture 3 (CRS, BRS, KG and CG). Notably, each component from mixtures 1 and 2 was tailored towards specific taxa in the Clostridium cluster XlVa group, so that there was little overlap in their fiber components regarding preferential support of taxa. The Clostridium cluster XlVa is a key group of gut bacteria that plays an essential role in maintaining gut health and homeostasis.47These mucosal bacteria are crucial in the production of short-chain fatty acids, particularly butyrate, which provides numerous health benefits including nourishing the gut lining, supporting the immune system, and potentially protecting against certain types of colorectal cancer.47Moreover, we envision that other fiber mixtures could be systematically put together to improve the support of other bacterial groups related to health that were not the main target of this study.

[0100] Another observation is that blending fibers 1 and 2 together to make mixture 3 did not provide additional advantages regarding increasing the number of bacteria supported as observed from alpha diversity, heatmaps, differential phylogenetic trees, intensity and similarity of responses across donors as observed in violin plots, and SCFA production. Thus, mixtures with fibers of overlapping bacterial targets do not seem to provide further benefits and these results highlight the importance of systematic selection of fibers to compose mixtures that are targetspecific.

[0101] Interestingly, mixtures were more consistent in promotion of specific genera across different donors than single fibers. In general, bacterial genera respond distinctively to fibertypes, and blending fibers could increase chances that the appropriate fiber type for a specific genus is present for more donors. However, that does not seem to explain fully the observed results. Upon further evaluation of some specific donor responses, it was noted that for some taxa only the mixture, and not any of the single fibers making up the mixture, led to bacterial enhancement compared to the control group. It is possible that the factors previously discussed (i.e., changes in competitive pressures for substrate utilization, and beneficial interactions among supported bacteria) could also contribute to more homogeneous responses across people for several selected taxa. In agreement, when variability predicted by the fiber treatment was evaluated, higher residuals were observed for individual fibers, highlighting the challenges in predicting and achieving consistent responses in different people with individual fibers. On the other hand, the greater amount of variance explained by the fiber treatment, when presented as mixtures, indicates a greater level of predictability and consistency in their impact on gut microbiota of different people.

[0102] Previously, we proposed and showed that single fibers of high specificity regarding their physicochemical features promote homogeneous in vitro fecal fermentation taxa responses across people, whereas those of low specificity do not.44,55Here, we show that another strategy to achieve some consistency in response of fibers of lower specificity could be designed fiber mixtures. Overall, systematically designed fiber mixtures show promise synergistically increasing amount and diversity of beneficial gut microbes, supporting short chain fatty acid production beyond the summation of single fibers in a mixture, and achieving more consistency in fiber responses across people. Also, support of some bacterial taxa was only attained through fiber mixtures and not any of their individual fiber components. Thus, these findings underscore the importance of viewing prebiotic fiber responses as products of the gut microbiota’s intricate ecological interactions, rather than merely the cumulative response of its single fiber constituents.References1. Mohajeri MH, Brummer RJM. Rastall RA, Weersma RK, Harmsen HJM, Faas M, EggersdorferM. The role of the microbiome for human health: from basic science to clinical applications. European Journal of Nutrition 2018 57:1 [Internet] 2018 [cited 2023 Oct 9]: 57:1-14. Available from: https: / / link[dot]springer[dot]com / article / 10[dot] 1007 / s00394-018-1703-42. Clemente JC, Ursell LK, Parfrey LW, Knight R. The impact of the gut microbiota on human health: An integrative view. Cell 2012; 148: 1258-70.3. Greenhalgh K, Meyer KM, Aagaard KM. Wilmes P. The human gut microbiome in health: establishment and resilience of microbiota over a lifetime. Environ Microbiol [Internet] 2016 [cited 2022 Jul 21]; 18:2103-16. 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Available from: http: / / www[dot]ncbi[dot]nlm[dot]nih[dot]gov / pubmed / 2394165748. El Aidy S, Van den Abbeele P, Van de Wiele T, Louis P, Kleerebezem M. Intestinal colonization: How key microbial players become established in this dynamic process. BioEssays [Internet] 2013 [cited 2019 Jan 27]; 35:n / a-n / a. Available from: http: / / www[dot]ncbi[dot]nlm[dot]nih[dot]gov / pubmed / 2394608849. Watts AM, West NP, Zhang P, Smith PK, Cripps AW, Cox AJ. The Gut Microbiome of Adults with Allergic Rhinitis Is Characterised by Reduced Diversity and an Altered Abundance of Key Microbial Taxa Compared to Controls. Int Arch Allergy Immunol [Internet] 2021 [cited 2022 Aug 8]; 182:94-105. Available from: https: / / www[dot]karger[dot]com / Article / FullText / 51053650. Miquel S, Martin R, Rossi O, Bermudez-Humaran LG, Chatel JM, Sokol H, Thomas M, Wells JM, Langella P. Faecalibacterium prausnitzii and human intestinal health. Curr Opin Microbiol 2013; 16:255-61.51. Zhao Q, Yu J, Hao Y, Zhou H, Hu Y, Zhang C, Zheng H, Wang X, Zeng F, Hu J, et al. Akkermansia muciniphila plays critical roles in host health. https: / / doi[dot]org / 101080 / 1040841X20222037506 [Internet] 2022 [cited 2022 Jun 28]; : 1— 19. Available from: https: / / www[dot]tandfonline[dot]com / doi / abs / 10.1080 / 104084 lX[dot]2022[dot]203750652. Coyte KZ, Rakoff-Nahoum S. Understanding Competition and Cooperation within the Mammalian Gut Microbiome. Curr Biol [Internet] 2019 [cited 2023 Oct 9]; 29:R538. Available from: / pmc / articles / PMC6935 13 / 53. Figueiredo ART, Kramer J. Cooperation and Conflict Within the Microbiota and Their Effects On Animal Hosts. Front Ecol Evol 2020; 8:507161.54. Rakoff-Nahoum S, Foster KR, Comstock LE. The evolution of cooperation within the gut microbiota. Nature 2015 533:7602 [Internet] 2016 [cited 2023 Oct 9]; 533:255-9. Available from: https : / / www [dot] nature [dot] co m / artic le s / nature 1762655. Cantu-Jungles T, Hamaker B. New view on dietary fiber selection for predictable shifts in gut microbiota. mBio [Internet] 2020 [cited 2020 Feb 23]; ll:e02179-19. Available from: http: / / mbio[dot]asm[dot]org / lookup / doi / l O[dot] 1128 / mBio.02179-19The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.Any use of section headings and subheadings is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one section heading or subheading is intended to constitute a disclosure under each and every other section heading or subheading.Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined and are described or discussed elsewhere, the definitions and all descriptions and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof.Further, all publications and patents mentioned herein are incorporated by reference in their entireties for all purposes. In the event of inconsistent usages between this document andthose documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylan, pectin, com resistant starch, konjac glucomannan, and fructooligosaccharides.

2. The composition of claim 1, which promotes Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and Faeccilibacterium prausnitzii .

3. The composition of claim 2, which further promotes Lachnospira, Subdoligranulum, Blautia, Roseburia, Agathobacter, and Eubacterium eligens.

4. The composition of claim 2 or 3, which has a synergistic effect.

5. The composition of any one of claims 2-4, which suppresses Proteobacteria.

6. The composition of any one of claims 1-5, wherein the varying amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).

7. The composition of claim 6, wherein the varying amount by weight is an equal amount for each of arabinoxylan, pectin, com resistant starch, konjac glucomannan, and fructooligosaccharides.

8. The composition of any one of claims claim 1-6, wherein the varying amount by weight is 0% for up to two of arabinoxylan, pectin, corn resistant starch, konjac glucomannan, and fructooligosaccharides.

9. A composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weightof, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylan, pectin, resistant starch (such as com resistant starch, resistant maltodextrin, or resistant dextrin), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides).

10. A composition comprising (or consisting essentially of or consisting of) varying amounts by weight of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides.

11. The composition of claim 10, which promotes Bacteroides, Ruminococcus, Clostridium cluster XlVa, Bifidobacteria, and other Clostridium cluster XlVa bacteria.

12. The composition of claim 11, which further promotes Lachnospira, Subdoligranulum, Blautia, Roseburia, Agathobacter, and Eubacterium eligens.

13. The composition of claim 11 or 12, which has a synergistic effect.

14. The composition of any one of claims 10-13, which suppresses Proteobacteria.

15. The composition of any one of claims 10-14, wherein the varying amount by weight of each is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%; e.g., 5% to 35%, 10% to 35%, 15% to 35%, 20% to 35%, 25% to 35%, 30% to 35%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, or 10% to 15%).

16. The composition of claim 15, wherein the varying amount by weight is an equal amount for each of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides.

17. The composition of any one of claims 10-15, wherein the varying amount by weight is 0% for up to two of arabinoxylooligosaccharides, banana resistant starch, chitin glucan, konjac glucomannan, and fructooligosaccharides.

18. A composition comprising (or consisting essentially of or consisting of), such as a composition comprising (or consisting essentially of or consisting of) equal amounts by weight of, at least three (such as any combination of three, any combination of four, or all five) of arabinoxylooligosaccharides, resistant starch (such as banana resistant starch, resistant maltodextrin, or resistant dextrin), glucan (such as chitin glucan), glucomannan (such as konjac glucomannan), and fructan (such as fructooligosaccharides).

19. The composition of either of claims 6 or 15, wherein the composition comprises (or consists essentially of or consists of) the varying amount by weight of each of the two compositions is, independently of each other, an amount between about 5% and about 40% (such as about 5% to 40%, 5% to about 40%, or 5% to 40%).

20. The composition of any one of claims 1-19, wherein the composition is an ingestible formulation, wherein the ingestible formulation is a supplement, a powder sachet, a powder for a shake, a liquid shake, a prebiotic shot, a snack, or a meal replacement, and wherein the ingestible formulation comprises from about 2.5 grams to about 25 grams of the composition.

21. A method of synergistically promoting Bacteroides, Ruminococcus, Clostridium cluster XfVa, Bifidobacteria, and Faecalibact rium prausnitzii in the gut microbiome of a subject, which method comprises administering to the subject a composition of any one of claims 1-9 in an amount effective to promote synergistically said bacteria or an ingestible formulation comprising same.

22. A method of synergistically promoting Bacteroides, Ruminococcus, Clostridium cluster XfVa, Bifidobacteria, and other Clostridium cluster XTVa bacteria in the gut microbiome of a subject, which method comprises administering to the subject a composition of any one ofclaims 10-19 in an amount effective to promote synergistically said bacteria or an ingestible formulation comprising same.

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