RELATED APPLICATIONS

[1] This patent application claims the benefit of and the priority to U.S. Provisional Patent Application Serial No. 63/006,896, filed April 8, 2020, the entirety of which is incorporated by reference in its entirety.

SEQUENCE LISTING

[2] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 7, 2021, is named 117465-016701PCT_SL.txt and is 49,521 bytes in size.

FIELD

[3] The present disclosure relates to a fiber mix formulation useful for in vitro and in vivo indications.

BACKGROUND

[4] The gut ecosystem is regarded as foundational for human health. Developing strategies to promote and maintain a healthy gut microbiota is one of the key priorities in recent medical research. Dysbiosis (an imbalance of beneficial and detrimental intestinal microbes) is associated with metabolic diseases, including obesity and type 2 diabetes.

SUMMARY

[5] Covered embodiments are defined by the claims, not this summary. This summary is a high-level overview of various aspects and introduces some of the concepts that are further described in the Detailed Description section below. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all drawings, and each claim.

[6] Aspects of the present disclosure relate to a formulation comprising a mixture of bran, inulin, and digestion resistant maltodextrin, wherein the bran consists essentially of com bran, wheat bran, sorghum bran, and oat bran, wherein the mixture has a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 1:1:1 to about 4: 1:1, wherein the inulin is present in a range of 5-10% of total weight of the mixture, and wherein the mixture has a uniform particle size between 150 pm and 300 pm.

[7] In some embodiments, the ratio of fiber contributed by each of the com bran, wheat bran, sorghum bran, and oat bran is about 1 : 1 : 1 : 1 by weight.

[8] In some embodiments, the formulation comprises about 7-8% inulin by weight. In some embodiments, the comprises about 7.8% inulin by weight. In some embodiments, the total fiber content is about 47% (w/w). In some embodiments, the digestion resistant maltodextrin is Fibersol-2. In some embodiments, the Fibersol-2 is present at a final percentage of about 7.8% (w/w).

[9] In some embodiments, the formulation further comprises xanthan gum. In some embodiments, the xanthan gum is present at a final percentage of about 4% (w/w).

[10] Other aspects of the present disclosure relate to methods for promoting a healthy gut microbiome in a subject in need thereof, the method comprising enterally administering to the subject an effective amount of the formulation comprising a mixture of bran, inulin, and digestion resistant maltodextrin, wherein the bran consists essentially of com bran, wheat bran, sorghum bran, and oat bran, wherein the mixture has a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 1:1:1 to about 4:1:1, wherein the inulin is present in a range of 5-10% of total weight of the mixture, and wherein the mixture has a uniform particle size between 150 pm and 300 pm, wherein the effective amount is sufficient to increase proliferation of acetate-producing and butyrate- producing gut bacteria in the subject’s gut, thereby promoting the healthy gut microbiome in the subject.

[11] In some embodiments, the effective amount of the formulation comprises 30-90 grams of fiber per day. In some embodiments, the effective amount of the formulation comprises 30- 60 grams of fiber per day. In some embodiments, the effective amount of the formulation comprises 30-120 grams of fiber per day.

[12] In some embodiments, the acetate-producing and butyrate-producing gut bacteria species are classified in a bacterial genus comprising at least one of Bifidobacterium, Ruminococcus, Eubacterium, Clostridium, Alistipes, Bacteroides, Blautia, Butyricicoccus, Butyricimonas, Collinsella, Coprococcus, Dorea, Eggerthella, Faecalibacterium, Fusicatenibacter, Intestinimonas, Lachnoclostridium, Lachnospiraceae, Megasphaera, Parabacteroides, Paraprevotella, Phascolarctobacterium, Roseburia, Ruminiclostridium, or Subdoligranulum, wherein the acetate-producing and butyrate-producing gut bacteria species are classified in a bacterial family comprising at least one of Acidaminococcaceae , Bacteroidaceae , Bifidobacteriaceae , Clostridiaceae, Clostridiales , Coriobacteriaceae , Eggerthellaceae , Enter obacteriaceae , Erysipelotrichaceae , Lachnospiraceae , Marinifilaceae , Prevotellaceae , Rikenellaceae , Ruminococcaceae , Tannerellaceae , or Veillonellaceae .

[13] In some embodiments, the acetate-producing and butyrate-producing gut bacteria species are detected by 16S rRNA amplicon sequencing targeting one or a few of the variable regions such as V4, V3-V4 or the full-length gene. In some embodiments, the 16S rRNA V4 amplicon sequencing identifies at least one of an amplicon sequence variant (ASV) characteristic of the acetate-producing and butyrate-producing gut bacteria. In some embodiments, each of the ASVs represents a unique bacterial strain or a group of highly similar strains of the acetate-producing and butyrate-producing gut bacteria.

[14] In some embodiments, the subject in need thereof has an imbalance of gut microbiota. In some embodiments, the imbalance of gut microbiota is characterized by low levels of acetate-producing and butyrate-producing gut bacteria. In some embodiments, the low levels of acetate-producing and butyrate-producing gut bacteria are less than 5% of total gut microbiota in the subject in need thereof. For example, the acetate-producing and butyrate- producing gut bacteria species are classified in a bacterial genus comprising at least one of Bifidobacterium , Ruminococcus or Eubacterium. In some embodiments, the acetate-producing and butyrate-producing gut bacteria species are detected by 16S rRNA V4 amplicon sequencing. In some embodiments, the 16S rRNA V4 amplicon sequencing identifies at least one of an amplicon sequence variant (ASV) characteristic of the acetate-producing and butyrate-producing gut bacteria. In some embodiments, each of the ASVs represents a unique bacterial strain or a group of highly similar strains of the acetate-producing and butyrate- producing gut bacteria.

[15] In some embodiments, the administering increases the acetate-producing and butyrate- producing gut bacteria.

[16] Aspects of present disclosure relate to a fiber mix formulation comprising a mixture of bran, inulin, and digestion resistant maltodextrin useful. The mixture of soluble dietary fiber (SDF) and insoluble dietary fiber (ISDF) with a ratio of ISDF:SDF ranging from about 0.5:1 to about 1:0.5 has synergistic effect on gut microbiota composition, promoting the growth of the acetate- and butyrate-producing bacteria and inhibiting the growth of pathogens and detrimental bacteria. In some embodiments, the fiber mix formulation restores a healthier gut microbiota. In some embodiments, the fiber mix formulation restores a healthier gut microbiota and alleviate symptoms in patients with various diseases or indications which are known to have a dysbiotic gut microbiota, such as obesity, prediabetes, type 2 diabetes, multiple sclerosis Parkinson disease, Prader-Willi syndrome, alleviation of side effects (diarrhea, nausea, vomiting, bloody stool etc.) of cancer chemotherapy.

[17] In some embodiments, the subject in need thereof is obese. In some embodiments, the subject in need thereof has type 2 diabetes. In some embodiments, the subject in need thereof has diabetic kidney disease. In some embodiments, the subject in need thereof is obese with respiratory viral infection such as COVID-19. In some embodiments, the subject in need thereof has type 2 diabetes with respiratory viral infection such as COVID-19. In some embodiments, the subject in need thereof is prediabetic with respiratory viral infection such as COVID-19. In some embodiments, the subject in need thereof has multiple sclerosis. In some embodiments, the subject in need thereof has Parkinson disease. In some embodiments, the subject in need thereof has Prader-Willi syndrome. In some embodiments, the subject in need thereof has cancer and administration of the formulation alleviates side effects associated with cancer chemotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[18] Some embodiments of the disclosure are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the embodiments shown are by way of example and for purposes of illustrative discussion of embodiments of the disclosure. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the disclosure may be practiced.

[19] Figure 1A and IB depict postprandial glycemic response in healthy controls (Figure 1A) and individuals with prediabetes/type 2 diabetes (Figure IB). *P<0.05 vs white bread at the same time point.

[20] Figure 2 shows Principal Coordinates Analysis plot of beta diversity at baseline and after 48h fermentation based on the Bray-Curtis distance. N=3-5/group. Blank is the baseline of fermentation. Other symbols indicate the position of all 15 fiber groups after 48 hours fermentation.

[21] Figure 3 shows comparison of responders between 1% Bran Mix and 2% Bran Mix in in vitro fermentation.

[22] Figure 4 shows comparison of responders between 1% Inulin and 2% Inulin in in vitro fermentation.

[23] Figure 5 shows comparison of responders between 1% Fibersol-2 and 2% Fibersol-2 in in vitro fermentation.

[24] Figure 6 shows comparison of responders between 0.5% Fibersol-2:0.5% Inulin and 1% Fibersol-2: 1% Inulin in in vitro fermentation.

[25] Figure 7 shows comparison of positive responders among 0.5% Fibersol-2:0.5% Inulin, 1% Fibersol-2: 1% Inulin, 1% Fibersol-2, 2% Fibersol-2, 1% Inulin and 2% Inulin groups in in vitro fermentation.

[26] Figure 8 shows positive responder comparison among 1% Fibersol-2: 1% Bran Mix, 1% Fibersol-2, 1% Bran Mix, 2% Fibersol-2, and 2% Bran Mix in in vitro fermentation.

[27] Figure 9 shows positive responder comparison among 1% Inulin: 1% Bran Mix, 1% Inulin, 1% Bran Mix, 2% Inulin, and 2% Bran Mix in in vitro fermentation.

[28] Figure 10 shows positive responder comparison among 0.5% Fibersol-2:0.5% Inulin, 0.5% Fibersol-2:0.5% Inulin:0.1% Bran Mix, 0.5% Fibersol-2:0.5% Inulin:0.5% Bran Mix, and 0.5% Fibersol-2:0.5% Inulin: 1% Bran Mix in in vitro fermentation.

[29] Figure 11 shows negative responder comparison among 0.5% Fibersol-2:0.5% Inulin, 0.5% Fibersol-2:0.5% Inulin:0.1% Bran Mix, 0.5% Fibersol-2:0.5% Inulin:0.5% Bran Mix, and 0.5% Fibersol-2:0.5% Inulin: 1% Bran Mix in in vitro fermentation.

[30] Figure 12 shows positive responder comparison among 1% Bran Mix, 0.5% Fibersol- 2:0.5% Inulin: 1% Bran Mix, 0.25% Fibersol-2:0.25% Inulin: 1% Bran Mix, and 0.05% Fibersol-2:0.05% Inulin: 1% Bran Mix in in vitro fermentation.

[31] Figure 13 shows negative responder comparison among 1% Bran Mix, 0.5% Fibersol- 2:0.5% Inulin: 1% Bran Mix, 0.25% Fibersol-2:0.25% Inulin: 1% Bran Mix, and 0.05% Fibersol-2:0.05% Inulin: 1% Bran Mix in in vitro fermentation. [32] Figure 14 is a Table showing positive and negative responders to different combination of fibers and 16S rRNA V4 region sequences (SEQ ID NOs: 1-117)

DETAILED DESCRIPTION

[33] Among those benefits and improvements that have been disclosed, other objects and advantages of this disclosure will become apparent from the following description taken in conjunction with the accompanying figures. Detailed embodiments of the present disclosure are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the disclosure that may be embodied in various forms. In addition, each of the examples given regarding the various embodiments of the disclosure which are intended to be illustrative, and not restrictive.

[34] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases "in one embodiment," “in an embodiment,” and "in some embodiments" as used herein do not necessarily refer to the same embodiment(s), though it may. Furthermore, the phrases "in another embodiment" and "in some other embodiments" as used herein do not necessarily refer to a different embodiment, although it may. All embodiments of the disclosure are intended to be combinable without departing from the scope or spirit of the disclosure.

[35] As used herein, the term "based on" is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of "a," "an," and "the" include plural references. The meaning of "in" includes "in" and "on."

[36] As used herein, terms such as “comprising” “including,” and “having” do not limit the scope of a specific claim to the materials or steps recited by the claim.

[37] As used herein, the term “consisting essentially of’ limits the scope of a specific claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic or characteristics of the specific claim, e.g., fiber mix formulations and use thereof for restoring and/or maintaining a gut microbiota that supports human health. For instance, by using “consisting essentially of’ the fiber mix formulations described herein do not contain any unspecified ingredients that have a direct beneficial or adverse therapeutic effect on treatment of a disorder associated with dysbiosis of gut microbiota. Also, by using the term “consisting essentially of’ the composition may comprise substances that do not have therapeutic effects on the treatment of a disorder associated with dysbiosis of gut microbiota; such ingredients include carriers, excipients, adjuvants, flavoring agents, etc. that do not affect gut microbiota.

[38] As used herein, terms such as “consisting of’ and “composed of’ limit the scope of a specific claim to the materials and steps recited by the claim.

[39] The term “treatment” or any grammatical variation thereof ( e.g ., treat, treating, and treatment etc.), as used herein, includes but is not limited to, alleviating a symptom of a disease or condition; and/or reducing, suppressing, inhibiting, lessening, or affecting the progression, severity, and/or scope of a disease or condition.

[40] The term “amelioration” or any grammatical variation thereof (e.g., ameliorate, ameliorating, and amelioration etc.), as used herein, includes, but is not limited to, delaying the onset, or reducing the severity of a disease or condition (e.g., diarrhea, bacteremia and/or endotoxemia). Amelioration, as used herein, does not require the complete absence of symptoms.

[41] The term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.

[42] As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refer to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. In general, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least or equal to ±1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%.

Fiber Mix

[43] In some embodiments, the subject invention provides a method for restoring and/or maintaining a gut microbiota that supports human health, wherein said method comprises: administering to a subject in need thereof, a fiber mix formulation comprising oat bran, com bran, sorghum bran, wheat bran, inulin, and digestion resistant maltodextrin; and optionally, therapeutically acceptable carriers, electrolytes, vitamins, buffering agents, and flavoring agents. In some embodiments, the digestion resistant maltodextrin is Fibersol-2. In a particular embodiment, the administering is via enteral route. In a particular embodiment, the fiber mix formulation further comprises water.

[44] The present inventors have gathered comprehensive evidence relating to the role of dietary fibers in establishing and maintaining a gut microbiota that supports human health. By incorporating foods that are rich in whole grains, traditional Chinese medicinal foods, and prebiotics, the present inventors demonstrated that a diet high in dietary fibers having diverse physicochemical structures restores a healthier gut microbiota, which, in turn, confers clinical benefits including weight loss, reduction of inflammation and improved glucose homeostasis. As shown herein, among hundreds of gut bacteria that possess the genetic capacity to utilize dietary fibers, the fiber mix formulation described herein selectively promotes a small group of acetate- and butyrate-producing bacteria. Acetate and butyrate from bacterial carbohydrate fermentation provide energy substrates locally for colonocytes and function as signaling molecules in many metabolic pathways including those that regulate immune response, appetite and insulin secretion. The present inventors refer to this group of bacteria as the “foundation guild” of a healthy gut microbiota structure. The foundation guild comprises bacteria from different groups. When the foundation guild becomes predominant in a gut microbial community, the guild produces acetate and butyrate to create a mildly acidic gut environment, which is inhibitory to many detrimental bacteria or pathogens and therefore shapes the gut microbiota towards a healthier structure.

[45] Adopting a high-fiber dietary regimen in everyday life, however, is challenging if at all possible. The fiber mix formulation described herein was carefully formulated to include a large amount of diverse fibers with different physicochemical structures to maximize short- chain fatty acid (SCFA) production by gut bacteria that differ with respect to their preferred fermentation substrates.

[46] In the present inventors’ clinical trials using the fiber mix formulation described herein, the average fiber intake was 33-44 g/day, which greatly exceeds the typical intake of fiber at 15 g/day among adults in the United States. Such a significant discrepancy between the amount fiber that may be needed for health benefits and that which is routinely consumed, underscores the potential benefits of the fiber mix formulation described herein to the general population.

[47] The fiber mix formulation described herein provides soluble and insoluble dietary fibers as fermentation substrates that restore and/or maintain a gut microbiota that supports human health. The fiber mix primarily consists of ingredients that are rich in soluble and insoluble fibers of diverse physicochemical structures. In some embodiments, the fiber mix formulation comprises one or more soluble fibers. In some embodiments, the soluble fiber can be fructo- oligosaccharide (FOS), galacto-oligosaccharides (GOS), trans-galacto-oligosaccharides (TOS), resistant starch (RS), pectic oligosaccharide (POS), raffinose family oligosaccharides (RFO), polydextrose, inulin, fibersol-2 or any combination of the foregoing.

[48] In some embodiments, the fiber mix formulation comprises a mixture of bran (insoluble dietary fibers), inulin (soluble dietary fibers) and/or digestion resistant maltodextrin (soluble dietary fibers). In some embodiments, the fiber mix formulation further comprises xanthan gum.

[49] In some embodiments, the bran mix comprises oat bran, corn bran, sorghum bran, wheat bran or combinations thereof. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 1 : 1 : 1 to about 4: 1 : 1. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 2:1:1 to about 4:1:1. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 3:1:1 to about 4:1:1. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 1:1:1 to about 2: 1 : 1. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 1:1:1 to about 3:1:1. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 2:1:1 to about 3:1:1. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 3:1:1 to about 4:1:1. For example, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin (e.g. Fibersol-2) can be 1:1 :1, 3:1:1, 4:1:1. In some embodiments, the fiber mix comprises a ratio of insoluble dietary fibers to soluble dietary fibers ranging from about 0.5:1 to about 1 :0.5. In some embodiments, the fiber mix comprises a ratio of insoluble dietary fibers to soluble dietary fibers ranging from about 0.5:1 to about 1:1. In some embodiments, the fiber mix comprises a ratio of insoluble dietary fibers to soluble dietary fibers ranging from about 1 : 1 to about 1:0.5. For example, the fiber mix comprises a ratio of insoluble dietary fibers to soluble dietary fibers of about 0.5:1, about 0.6:1, about 0.7:1, about 0.8:1, about 0.9:1, about 1:1, about 1:0.9, about 1:0.8, about 1:0.7, about 1:0.6, or about 1:0.5.

[50] Oat bran, corn bran, sorghum bran, wheat bran, inulin and Fibersol-2 are mixed such that the total dietary fibers from the brans and those from inulin and Fibersol-2 are in the ratio of between about 1:1:1 and about 4:1:1, for example about 1:1:1, about 2: 1:1, about 3:1:1, or about 4:1:1.

[51] It should be noted that each bran has a different content of dietary fiber. In some embodiments, each bran contributes equal amount of dietary fiber in the fiber mix composition. In some embodiments, the composition of the fiber mix 1 comprises between about 30% to 50% (w/w) of total dietary fiber content. The total dietary fiber content includes insoluble and soluble fibers. In some embodiments, the composition of the fiber mix 1 comprises between about 36% to 47% (w/w) of total dietary fiber content. In some embodiments, the composition of the fiber mix as shown in Table 1 comprises 36.8% (w/w) of total dietary fiber content.

Table 1

[52] In some embodiments, a thickener, such as xanthan gum, can be then added to this mix to improve texture and palatability. (see Table 2)

Table 2 [53] In some embodiments, the amount of soluble fiber such as inulin ranges from 1% to 30% (w/w) relative to the total weight of the fiber mix. In some embodiments, the amount of inulin ranges from 5% to 25% (w/w) relative to the total weight of the fiber mix. In some embodiments, the amount of inulin ranges from 10% to 20% (w/w) relative to the total weight of the fiber mix. In some embodiments, the amount of inulin ranges from 7% to 8% (w/w) relative to the total weight of the fiber mix. According to certain exemplary embodiments, the amount of inulin ranges from 5% to 15% (w/w) relative to the total weight of the fiber mix. In some embodiments, the amount of inulin ranges from 7% to 8% (w/w) relative to the total weight of the fiber mix. In some embodiments, the amount of inulin ranges from 7.5% (w/w) relative to the total weight of the fiber mix. In some embodiments, the amount of inulin ranges from 5% to 15%, 6% to 15%, 7% to 15%, 8% to 15%, 9% to 15%, 10% to 15%, 11% to 15%, 12% to 15%, 13% to 15%, and 14% to 15% (w/w) relative to the total weight of the fiber mix. Each possibility represents a separate embodiment of the formulations, methods and uses described herein.

[54] In some embodiments, the mixture of bran, inulin, and digestion resistant maltodextrin comprises has a uniform particle size between 150 pm and 300 pm. In some embodiments, the mixture has a uniform particle size higher than 150 pm and lower than 300 pm. For example, the mean particle size can be 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 pm.

[55] All ingredients are sourced from commercial food suppliers. To prepare the fiber mix, the bran ingredients are first mixed at the specified ratios, the mixture is then milled to yield a uniform particle size between 150 and 300 pm and then roasted at 135°C for 5 minutes for sterilization. The insoluble bran fiber mix may be further mixed with soluble fiber(s). A sample of the finished project is tested for nutrient profiling, microbial load check, and stability and shelf life by an ISO 17025 accredited food nutritional laboratory (Eurofms Nutrition Analysis Center). The remaining fiber mix is packaged into sachets and kept at 4°C and 60-70% humidity for long-term storage. In some embodiments, the fiber mix is stable for at least 1 year at room temperature.

[56] The fiber mix described herein comprises fibers of a diverse range of physicochemical structures that effectively promote members of the foundation guild to increase SCFA production. The diverse range of physicochemical structures confers to the fiber the ability to promote the foundation guild of microbes despite a myriad of expected differences in guild members across individuals. The foundation guild of gut bacteria is able to utilize nondigestible but fermentable carbohydrates and become predominant in the gut ecosystem as the foundation of a healthy gut microbiota. The foundation guild of gut bacteria provides SCFAs, including but not limited to acetic acid, butyric acid and propionate, that supports human health. In some embodiments, the SCFA producers play a structural role in the gut ecosystem by producing SCFAs that acidify the gut and create a gut environment unfavorable to pathogenic and detrimental bacteria, producing antimicrobials and occupying available ecological niches (competitive exclusion effect) etc.

[57] Many gut bacteria possess the genetic capacity to ferment carbohydrates but not all of them are equally efficient in generating SCFAs. Gut bacteria also differ in their tolerance to an acidified gut environment. Without being bound to the theory among many gut bacteria that possess the genetic capacity to produce SCFAs, only a select group are active SCFA producers that can form an interactive group (e.g. via cross-feeding or other mechanisms) that collectively function as a “guild” to ferment carbohydrates and generate SCFAs. This guild of SCFA producers can be regarded as the foundation for a healthy gut microbiota. In some embodiments, the microbiota can be profiled using 16S rRNA amplicon sequencing targeting one or a few of the variable regions such as V4, V3-V4 or the full-length gene. The microbiota can also be profiled based on shotgun metagenomics.

[58] The fiber mix is primarily intended to be consumed as a supplement (e.g., mixed with water and consumed as a drink) or it may be incorporated into daily food (e.g., as a substitute for conventional flour) to restore and/or maintain a gut microbiota that supports human health. For example, the fiber mix can be consumed as smoothies, baking products, as substitution of part of flours

[59] In each and every embodiment of administering a fiber mix formulation described herein, the fiber mix formulation may be administered or consumed as a single combined unit comprising all of the components or as multiple units, wherein each of the units comprises at least one of the components of the fiber mix formulation, such that administration/consumption of the multiple units in total provides all of the components of the single combined unit of the fiber mix formulation. For example, the fiber mix formulation may be consumed or administered in multiple units: one unit comprising the bran, one unit comprising the inulin, and one unit comprising the digestion resistant maltodextrin. In another embodiment, the fiber mix formulation may be consumed or administered in multiple units: one unit comprising the bran and the inulin, and one unit comprising the digestion resistant maltodextrin. Additional multiple unit administrations are encompassed herein.

[60] Fiber mix formulations described herein are suitable for the general population. In an embodiment, fiber mix formulations described herein may be administered to healthy individuals who want to maintain healthy gut microbiota. In another embodiment, fiber mix formulations described herein are administered to individuals in need thereof who have dysbiosis of gut microbiota or who are at risk for dysbiosis of gut microbiota.

[61] Other aspects of the present disclosure relate to methods for promoting a healthy gut microbiome in a subject in need thereof, the method comprising enterally administering to the subject an effective amount of the formulation comprising a mixture of bran, inulin, and digestion resistant maltodextrin, wherein the bran consists essentially of com bran, wheat bran, sorghum bran, and oat bran, wherein the mixture has a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 1:1:1 to about 4:1:1, wherein the inulin is present in a range of 5-10% of total weight of the mixture, and wherein the mixture has a uniform particle size between 150 pm and 300 pm, wherein the effective amount is sufficient to increase proliferation of acetate-producing and butyrate- producing gut bacteria in the subject’s gut, thereby promoting the healthy gut microbiome in the subject. In some embodiments, the fiber mix comprises a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin ranging from about 1:1:1 to about 4:1:1, wherein the inulin is present in a range of 7-8% of total weight of the mixture, and wherein the mixture has a uniform particle size between 150 pm and 300 pm, wherein the effective amount is sufficient to increase proliferation of acetate-producing and butyrate-producing gut bacteria in the subject’s gut, thereby promoting the healthy gut microbiome in the subject.

[62] In some embodiments, the acetate-producing and butyrate-producing gut bacteria species are classified in a bacterial genus comprising at least one of Bifidobacterium, Ruminococcus, Eubacterium, Clostridium, Alistipes, Bacteroides, Blautia, Butyricicoccus, Butyricimonas, Collinsella, Coprococcus, Dorea, Eggerthella, Faecalibacterium, Fusicatenibacter, Intestinimonas, Lachnoclostridium, Lachnospiraceae, Megasphaera, Parabacteroides, Paraprevotella, Phascolarctobacterium, Roseburia, Ruminiclostridium, or Subdoligranulum, wherein the acetate-producing and butyrate-producing gut bacteria species are classified in a bacterial family comprising at least one of Acidaminococcaceae , Bacteroidaceae , Bifidobacteriaceae , Clostridiaceae, Clostridiales , Coriobacteriaceae , Eggerthellaceae , Enter obacteriaceae , Erysipelotrichaceae , Lachnospiraceae , Marinifilaceae , Prevotellaceae , Rikenellaceae , Ruminococcaceae , Tannerellaceae , or Veillonellaceae .

[63] In some embodiments, the acetate-producing and butyrate-producing gut bacteria species are detected by 16S rRNA amplicon sequencing targeting one or a few of the variable regions such as V4, V3-V4 or the full-length gene. In some embodiments, the 16S rRNA V4 amplicon sequencing identifies at least one of an amplicon sequence variant (ASV) characteristic of the acetate-producing and butyrate-producing gut bacteria. In some embodiments, each of the ASVs represents a unique bacterial strain or a group of highly similar strains of the acetate-producing and butyrate-producing gut bacteria.

[64] In some embodiments, the subject in need thereof has an imbalance of gut microbiota. In some embodiments, the imbalance of gut microbiota is characterized by low levels of acetate-producing and butyrate-producing gut bacteria. In some embodiments, the low levels of acetate-producing and butyrate-producing gut bacteria are less than 5% of total gut microbiota in the subject in need thereof. In some embodiments, the administering increases the acetate-producing and butyrate-producing gut bacteria.

[65] In some embodiments, the fiber mix formulation comprising a mixture of soluble dietary fiber (SDF) and insoluble dietary fiber (ISDF) with a ratio of ISDF:SDF ranging from about 0.5:1 to about 1:0.5 has synergistic effect on gut microbiota composition, promoting the growth of the acetate- and butyrate-producing bacteria and inhibiting the growth of pathogens and detrimental bacteria. In some embodiments, the fiber mix formulation restores a healthier gut microbiota. In some embodiments, the fiber mix formulation restores a healthier gut microbiota and alleviate symptoms in subjects with various diseases or indications which are known to have a dysbiotic gut microbiota. In some embodiments, the fiber mix formulation alleviate symptoms in patients having obesity, prediabetes, type 2 diabetes, multiple sclerosis, Parkinson disease, Prader-Willi syndrome, or alleviate of side effects of cancer chemotherapy.

[66] In some embodiments, the subject in need thereof has prediabetes. In some embodiments, the subject in need thereof is obese. In some embodiments, the subject in need thereof has type 2 diabetes. In some embodiments, the subject in need thereof has diabetic kidney disease. In some embodiments, the subject in need thereof is obese with respiratory viral infection such as COVID-19. In some embodiments, the subject in need thereof has prediabetes with respiratory viral infection such as COVID-19. In some embodiments, the subject in need thereof is diabetic with respiratory viral infection such as COVID-19.

[67] Overgrowth of pathogens in the gut of diabetic or prediabetic patients may drive the more severe form of COVID-19. Such pathogens can contribute to the cytokine storm and sepsis, which are the major causes of mortality in these high-risk patients.

[68] In some embodiments, the subject in need thereof has multiple sclerosis. In some embodiments, the subject in need thereof has multiple sclerosis. In some embodiments, the subject in need thereof has Parkinson disease. In some embodiments, the subject in need thereof has Prader-Willi syndrome.

[69] The fiber mix may also be used as a standardized reagent (a supply of fermentable carbohydrates having diverse physicochemical structures at known concentrations) in an in vitro system, e.g. in vitro fermentation in Figure 2, to directly compare the bacterial fermentation capacity of gut microbiota of different individuals. Such fermentation assays may serve as diagnostic test assays for gut microbiota functions.

Dosage Forms

[70] A dosage unit of a fiber mix formulation described herein may be mixed together or combined such that ionic or covalent bonds are formed between and/or among components of the formulation. The fiber mix formulation described herein may be administered enterally (e.g., via oral or rectal route), using dosage forms known to those of ordinary skill in the pharmaceutical arts. Depending on the particular location or method of delivery, different dosage forms, e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the fiber mix formulation described herein to a patient in need of thereof. The fiber mix formulation described herein may also be administered as a salt form of any one of the components described herein.

[71] A fiber mix formulation described herein can be administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and/or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and conventional pharmaceutical practice. Depending on the mode of administration, the fiber mix formulation described herein may be formulated to provide, e.g., maximum and/or consistent dosing for oral or rectal administration. While the fiber mix formulation described herein may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier. The carrier may be solid or liquid, depending on the type and/or location of administration selected.

[72] Techniques and compositions for making useful dosage forms a fiber mix formulation described herein are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington's Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); each of which is incorporated herein by reference in its entirety.

[73] For example, the fiber mix formulation described herein may be included in a tablet. Tablets may contain, e.g., suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents and/or melting agents. For example, oral administration may be in a dosage unit form of a tablet, gel cap, caplet or capsule, wherein the active drug component is combined with a non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, mixtures thereof, and the like. Suitable binders for use with the fiber mix formulation described herein include: starch, gelatin, natural sugars (e.g., glucose or beta-lactose), corn sweeteners, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants for use with the fiber mix formulation described herein may include: sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, mixtures thereof, and the like. Disintegrators may include: starch, methylcellulose, agar, bentonite, mixtures thereof, and the like.

[74] The fiber mix formulation described herein may be administered in the form of liposome delivery systems, e.g., small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles, whether charged or uncharged. Liposomes may include one or more: phospholipids (e.g., cholesterol), stearylamine and/or phosphatidylcholines, mixtures thereof, and the like. [75] The fiber mix formulation described herein may also be coupled to one or more soluble, biodegradable, bioacceptable polymers as drug carriers or as a prodrug. Such polymers may include: polyvinylpyrrolidone, pyran copolymer, Poly(N-(2-hydroxypropyl) methacrylamide)- phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, mixtures thereof, and the like. Furthermore, the fiber mix formulation described herein may be coupled one or more biodegradable polymers to achieve controlled release of the fiber mix formulation described herein. Biodegradable polymers for use with the fiber mix formulation described herein include: polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels, mixtures thereof, and the like.

[76] In one embodiment, gelatin capsules (gel caps) may include the fiber mix formulation described herein and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Like diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as immediate-release, mixed-release or sustained-release formulations to provide for a range of release of the fiber mix formulation described herein over a period of minutes to hours. Compressed tablets may be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere. An enteric coating may be used to provide selective disintegration in, e.g., the gastrointestinal tract.

[77] For oral administration in a liquid dosage form, components of the fiber mix formulation described herein may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents, mixtures thereof, and the like.

[78] Liquid dosage forms of the fiber mix formulation described herein for oral administration may also include coloring and flavoring agents that increase patient tolerance of the formulation and therefore, promote compliance with a dosing regimen. In general, water, a suitable oil, saline, aqueous dextrose (e.g., glucose, lactose and related sugar solutions) and glycols (e.g., propylene glycol or polyethylene glycols) may be used as suitable carriers for parenteral solutions. Solutions for parenteral administration include generally, a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering salts. Antioxidizing agents such as sodium bisulfite, sodium sulfite and/or ascorbic acid, either alone or in combination, are suitable stabilizing agents. Citric acid and its salts and sodium EDTA may also be included to increase stability. In addition, parenteral solutions may include pharmaceutically acceptable preservatives, e.g., benzalkonium chloride, methyl- or propyl paraben, and/or chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, relevant portions incorporated herein by reference.

[79] Capsules. Capsules may, for example, be prepared by filling standard two-piece hard gelatin capsules each with the fiber mix formulation described herein, 10 to 500 milligrams of powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams of cellulose and 6 milligrams magnesium stearate.

[80] Soft Gelatin Capsules. A mixture of active ingredients (the fiber mix formulation described herein) is dissolved in a digestible oil such as soybean oil, cottonseed oil or olive oil. The active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 10-500 milligrams of the active ingredient. The capsules are washed and dried.

[81] Tablets. Tablets are prepared in bulk by conventional procedures so that the dosage unit may, for example, be 100-1,500 milligrams of the fiber mix formulation described herein, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 50-275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.

[82] To provide an effervescent tablet with the fiber mix formulation described herein, appropriate amounts of, e.g., monosodium citrate and sodium bicarbonate, are blended together and then roller compacted, in the absence of water, to form flakes that are then crushed to give granulates. The granulates are then combined with the active ingredient, drug and/or salt thereof, conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants. [83] Suspension. An aqueous suspension is prepared for oral administration so that each 5 ml contain 100 mg of finely divided active ingredients (fiber mix formulation described herein), 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 ml of vanillin.

[84] For mini-tablets, the fiber mix formulation described herein is compressed into a hardness in the range 6 to 12 Kp. The hardness of the final tablets is influenced by the linear roller compaction strength used in preparing the granules or granulates, which are influenced by the particle size of, e.g., the monosodium hydrogen carbonate and sodium hydrogen carbonate. For smaller particle sizes, a linear roller compaction strength of about 15 to 20 KN/cm may be used.

[85] Examples of suitable oral dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral forms may also include minerals and other materials to make them compatible with the delivery system chosen.

[86] The composition of the fiber mix formulation described herein may be formulated for release that is immediate, rapid, extended, bi-phasic, etc. By “immediate release” is meant a release of an active agent to an environment over a period of seconds to no more than about 30 minutes once release has begun and release begins within no more than about 2 minutes after administration. An immediate release does not exhibit a significant delay in the release of drug. By “rapid release” is meant a release of an active agent to an environment over a period of 1- 59 minutes or 0.1 minute to three hours once release has begun and release can begin within a few minutes after administration or after expiration of a delay period (lag time) after administration. As used herein, the term “extended release” profile assumes the definition as widely recognized in the art of pharmaceutical sciences. An extended release dosage form will release drug at substantially constant rate over an extended period of time or a substantially constant amount of drug will be released incrementally over an extended period of time. An extended release tablet generally effects at least a two-fold reduction in dosing frequency as compared to the drug presented in a conventional dosage form (e.g., a solution or rapid releasing conventional solid dosage forms). By “controlled release” is meant a release of an active agent to an environment over a period of about eight hours up to about 12 hours, 16 hours, 18 hours, 20 hours, a day, or more than a day. By “sustained release” is meant an extended release of an active agent to maintain a constant drug level in the blood or target tissue of a subject to which the device is administered. The term “controlled release”, as regards to drug release, includes the terms “extended release”, “prolonged release”, “sustained release”, or “slow release”, as these terms are used in the pharmaceutical sciences. A controlled release can begin within a few minutes after administration or after expiration of a delay period (lag time) after administration.

[87] A slow release dosage form is one that provides a slow rate of release of drug so that drug is released slowly and approximately continuously over a period of 3 hours, 6 hours, 12 hours, 18 hours, a day, 2 or more days, a week, or 2 or more weeks, for example.

[88] A timed-release dosage form is one that begins to release drug after a predetermined period of time as measured from the moment of initial exposure to the environment of use.

[89] A targeted release dosage form generally refers to an oral dosage form that designed to deliver the fiber mix formulation described herein to a particular portion of the gastrointestinal tract of a subject. An exemplary targeted dosage form is an enteric dosage form that delivers a drug into the middle to lower intestinal tract but not into the stomach or mouth of the subject. Other targeted dosage forms can deliver to other sections of the gastrointestinal tract such as the stomach, jejunum, ileum, duodenum, cecum, large intestine, small intestine, colon, or rectum.

[90] In some embodiments, the fiber mix formulation is mixed with aqueous solution. In some embodiments, an effective amount of the fiber mix formulation is mixed with an aqueous solution. For example, the fiber mix formulation can be prepared by mixing 30 g of fiber mix in 500 ml of water. In some embodiments, the 30g of fiber mix comprises about 1 lg of dietary fiber.

[91] In some embodiments, the formulation described herein can be taken at about 60 grams/day with no adverse gut reaction. In some embodiments, the formulation can be administered to a subject in need thereof in a dosage that ranges from about 1 g to about 120 g/day without adverse gut reaction. In some embodiments, the formulation described herein can be taken at about 30-120 grams, about 30-90 grams, about 30-60 grams, about 30-50 grams or about 30 to 40 grams/day with no adverse gut reaction.

[92] In some embodiments, the effective amount of the formulation comprises about 10 to 50 grams of dietary fiber per day. In some embodiments, the effective amount of the formulation comprises about 10 to 120 grams of dietary fiber per day. In some embodiments, the effective amount of the formulation comprises 11 to 44 grams of dietary fiber per day. For example, the effective amount of the formulation comprises 11 grams of dietary fiber per day, 22 grams of dietary fiber per day, 33 grams of dietary fiber per day or 44 grams of dietary fiber per day. In some embodiments, the effective amount of the formulation comprises 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more grams of fiber per day.

[93] In some embodiments, the method comprises administering orally to a subject in need thereof an effective amount of the formulation once a day, twice a day, three times a day, four times a day. In some embodiments, the method comprises administering the formulation orally to a subject in need thereof four times a day. For example, the method comprises administering the formulation orally to a subject in need thereof before each main meal and 2 hours after dinner.

[94] In some embodiments, administration results in the reduction of pathogens in the gut. For example, administration results in the reduction of pathogens by at least 10%, 20%, 30%. 40%, 50%, 60%, 70%, 80%, 90% or more. In some embodiments, administration results in the reduction of pathogens in the gut to non-detectable levels. In some embodiments, administration results in the reduction of levels of inflammation. In some embodiments, administration results in the reduction of risk of bacterial sepsis.

EXAMPLES

[95] Variations, modifications and alterations to embodiments of the present disclosure described above will make themselves apparent to those skilled in the art. All such variations, modifications, alterations and the like are intended to fall within the spirit and scope of the present disclosure, limited solely by the appended claims.

Example 1- Process of making fiber mix

[96] The components include wheat bran, oat bran, com bran, sorghum bran, inulin, Fiber sol -2 and xanthan gum. [97] An exemplary batch of the insoluble component of the fiber mix (the bran mix) comprising oat bran, corn bran, sorghum bran and wheat bran at the ratios listed in Table 3 was made.

Table 3: Bran mix composition.

[98] The bran mix was sent to Safe Sterilization USA for mixing, milling and roasting as described above. In some embodiments, the wheat bran, oat bran, com bran and sorghum bran are in the form of powder or flakes. In some embodiments, the wheat bran, oat bran, com bran and sorghum bran sieved to remove brans of particle size lower than 150 pm or greater than 300pm.

[99] Samples of the wheat bran, oat bran, corn bran and sorghum bran can be processed and sieved to confirm that the particular size is from 150-300 pm.

[100] Thereafter, inulin, fibersol-2 and xanthan gum were added to the bran mix at a final percentage of 7.5, 7.5 and 4% (w/w).

[101] In some embodiments, inulin, fibersol-2 and xanthan gum is in the form of powder.

[102] The wheat bran, oat bran, com bran and sorghum bran are mixed so that each bran contributes to the same amount of dietary fibers. This bran mix is then combined with inulin and Fibersol-2 so that the ratio of dietary fiber from bran mix to that of inulin and that of Fibersol-2 is 4: 1 : 1. Finally, xanthan gum is added to achieve a total percentage of 4% (w/w).

[103] The percentage (w/w) of each component of the fiber mix is: oat bran 42.0%, sorghum bran 15.8%, wheat bran 12.5%, com bran 10.8%, inulin 7.5%, Fibersol-2 7.5% and xanthan gum 4.0% as shown in Table 2.

[104] Oat bran, sorghum bran, what bran and com bran are first mixed at the specified ratios. This bran mixture is milled to yield a uniform particle size between 150 and 300 pm, mixed with inulin and Fibersol-2 at specified ratios, xanthan gum is added at a percentage of 4% (w/w), and finally roasted at 135°C for 5 minutes for sterilization. NBT-NM108 is then packaged into heat-sealed foil sachets and kept at 4°C and 60-70% humidity for long-term storage.

[105] Nutrient profile (including total dietary fiber content) of each ingredient and the final product can be determined by an ISO 17025 accredited food nutritional laboratory (Eurofms Nutrition Analysis Center).

[106] Microbial load, stability and shelf life of each ingredient can be determined by an ISO 17025 accredited food nutritional laboratory.

Example 2 Fermentation characteristics

[107] To explore the fermentation characteristics of different fiber combinations and their interactions with intestinal microbiota, 15 different fiber formulas were fermented in vitro with human fecal samples from a healthy donor for 48 hours anaerobically. These fibers formulas are soluble dietary fibers (SDFs) and insoluble dietary fibers (ISDF)s combined at different ratios (Table 4). SDFs were exemplified by Inulin and fibersol-2; and ISDFs by a bran mix (fiber content ratio of corn bran: sorghum bran: oat bran: wheat bran=l : 1 : 1 : 1).

[108] In vitro culture setup

[109] The direct effect of the fiber mix composition on the human gut microbiota was determined using in vitro fermentation. Human fecal material (within 2 h of collection) was suspended in phosphate-buffered saline (PBS)-based medium (Table 5) in a 1:3 ratio (w/v). The suspension was then mixed in a 1:4 ratio with a PBS-based fiber solution with each fiber mixture (Table 4) at 37°C for 6, 12, 24, and 48 hours under anaerobic conditions. The final concentration of bran mixture (w/v) is shown in Table 4. This system was set up as triplicates or quintuplicate.

[110] Prior to culture, the bran mix was pre-treated with digestive enzymes (pepsin, amyloglucosidase and pancreatin) and dialyzed to mimic the digestion and absorption of macronutrients in the human gastrointestinal system.

[111] Table 4. Composition of the 15 Fiber formulas.

*The weight percentage is the weight of these fibers in the fermentation system [w/v].

Table 5. PBS medium (1000 ml).

[112] Fermenta samples, gas production, and pH data were collected or measured at the baseline and different timepoints of fermentation. These fermenta samples were sequenced for 16S rRNA gene V4 region with Ion torrent platform. All samples were downsized to 20,000 reads. 1499 ASVs were retained for analysis. Each ASV represent a unique type of gut bacteria. Taxonomic position of each ASV was assigned at different levels depending on the novelty of these sequences.

[113] Fermentation with the mix of ISDFs with SDFs had higher gas production than those with same concentration of SDF

[114] Gas production of all 15 different fiber formulas were recorded after 48 hours fermentation (Table 6). Gas production may reflect the fermentability of fibers and their formula. Inulin fermentation produced more gas than fibersol-2, indicating that inulin may be a fast fermentable SDF while fibersol-2 a slow fermentable SDF. Both SDFs produce more gas than the ISDF, bran mix, indicating that SDF are more fermentable than ISDFs. 1%F1%I and 2% Inulin produce less gas than 1% Inulin, which is probably due to the low pH induced by these two fibers formula that inhibit the survival or growth of gas-producing bacteria (Table

7)·

[115] Table 6 shows the gas production after 48h fermentation with different fibers. Tukey test were used for gas production comparison. Significance difference were shown in letters. Blank is fermentation with only fecal sample, no fiber is added in the Blank group.

Table 6

* P<0.05. If groups have same letters, it means there is no significant difference between these two groups. E.g. l%Inulin and 0.5%F0.5%I1%B groups both have f, which means there is no significant difference in their gas production results

[116] The combination of ISDFs with SDFs had higher gas production than the same concentration of SDF, which shows a synergistic fermentation effect between SDFs and ISDFs. For example, produced gas volume: 1%I1%B > 2% Inulin; 1%F1%B ~ 2%Fibersol2; 0.5%F0.5%I1%B > 1 %F 1 %I. But fiber with low ISDF/SDF ratio did not show such promotion. For example, 0.5%F0.5%I0.1%B group produced similar gas with 0.5%F0.5%I.

[117] Fermentation with SDF produce more acids than those with same concentration of ISDF

[118] pH was reported to be able to regulate the fermentation mode (Mohd-Zaki, Z., et ah, Influence of pH Regulation Mode in Glucose Fermentation on Product Selection and Process Stability. Microorganisms, 2016. 4(1): p. 2.). At high pH, propionate type fermentation is the main process, producing propionate and acetate without any substantial gas production. When the pH decrease, the fermentation type may shift to butyrate-type or ethanol-type fermentation, producing butyrate or ethanol and acetate with hydrogen and carbon-dioxide as by-products. pH data at baseline and the endpoint of the fermentation are reported in Table 7. SDFs decreased pH more significantly than ISDFs. Regarding SDF, inulin decreased pH more significantly than fibersol-2. The gas production and pH data combined, 2% Inulin produced less gas than 1% Inulin, which may be because the lower pH of 2% Inulin group, 3.78 ± 0.02, is inhibitory to many gas-producing bacteria. The growth and survival of some gas-producing bacteria may be prevented or reduced in such low pH and couldn’t produce more gas as 1% Inulin group. Fibersol-2 produced less acid and gas compared to inulin, which is also consistent with previous study (lickinger, E.A., et al., Glucose-Based Oligosaccharides Exhibit Different In Vitro Fermentation Patterns and Affect In Vivo Apparent Nutrient Digestibility and Microbial Populations in Dogs. The Journal of Nutrition, 2000. 130(5): p. 1267-1273). The formula which combined ISDF with SDF optimized pH so that more fermentation can take place as indicated by high amount of gas production.

[119] Table 7 shows the pH at baseline and the endpoint of fermentation with different fibers. Wilcoxon test were used to compare the pH before and after fermentation. Asterisk indicate the significant difference between before (Time Oh) and after fermentation (Time 48h). **P<0.01. Tukey test were used for pH comparison among all treatments at baseline or after 48 hours fermentation. There is no significance difference among pH of 16 groups before fermentation. Significance difference among pH of 16 groups after fermentation were shown in letters.

Table 7

[120] The generation of gas and the change of pH are two primary indicators reflecting the degree of fermentation. Unexpectedly, the inventors found SDF could increase the fermentability of ISDF as indicated by synergistic gas production. Without being bound by the theory, when bacteria are cultured with the mix of SDF and ISDF, some gas producing bacteria grow faster than when they were only cultured with either SDF or ISDF alone. This shows that synergistic effects of the combination of SDFs and ISDFs on fermentation.

Example 3: Fecal microbiota composition change induced by fiber formula

[121] Changes in the microbiota composition were assessed after the fecal microbiota were fermented with different combinations of fibers. In total, 1499 ASVs were identified, which included all the unique bacteria that could be detected in all treatment groups and time points.

[122] The gut microbiota composition showed a significant shift after 48 hours fermentation with all 15 fiber formulas as indicated by principal coordinates analysis (PCoA, multivariate analysis of variance (MANOVA) test) based on Bray-Curtis distance b-diversity based on the Bray-Curtis distance of ASVs shows that 48h fermentation with fiber formula induced a shift of microbiota along PCI compared to baseline samples (Figure 2). All baseline samples were clustered together with blank group at 48h. There was a significant clustering by treatment groups for samples from 48h fermenta along PCI and these groups are separated based on their fermentability. The slowest fermentable ISDF groups, 1% Bran Mix and 2% Bran Mix, were clustered together and located at the right of the PCoA plot. The microbiota from groups with the faster fermentable SDF and higher amount of SDF shifted toward left along PCI. Groups with inulin located toward the left with 2% fibersol-2 group at the left of the PCoA plot.

Example 4: Positive or negative responders to fiber mixtures [123] Positive and negative responders to different combination of fibers were identified (see Table at Figure 14).

[124] Principal coordinate analysis (PCoA) was performed to compare and visualize the changing patterns of the pre- and post-fermentation samples based on Bray-Curtis distance. The principal coordinate, along which the pre- and post-fermentation samples can be segregated, was selected for further analysis. Pearson correlation was performed between the abundance of each ASV and the scores of the selected principal coordinate (de Goffau et al. Diabetes, 2013, 62(4): 1238-44). P value was adjusted by Benjamini-Hochberg procedure to control for the false positives that arise from multiple statistical comparisons. The ASVs, which have significant correlations (adjusted P < 0.05) with the selected principal coordinate, were identified as responders.

[125] The fermentation of ISDFs: when cultured with 1% bran mix, 53 ASVs were positive responders and 43 negative responders. In 2% Bran Mix, 83 were positive responders and 44 negative responders. 42 new ASVs responded positively when the bran mix dose was doubled from 1% (Figure 3). One ASV, Coprococcus ASV004E, is positive responders in 2% Bran Mix but negative responder in 1% Bran Mix. 1% or 2% Bran Mix shared 33 negative responders but both have around 10 unique negative responders (Figure 3). The data suggests that the relationship between substrate dose and microbiota composition is dynamic rather than a simple linear relationship.

[126] The fermentation of SDFs: increased concentration of inulin reduced the number of positive responding ASVs (43 in 1% vs 34 in 2% inulin) but increase the number of negative responding ASVs (50 in 1% vs 75 in 2% inulin). Among the 43 positive responders in 1% inulin, 15 no longer responded when the inulin dose was doubled (Figure 4), which is probably because these 15 ASVs couldn’t grow well in the lower pH induced by 2% Inulin.

[127] Increased concentration of another soluble fiber, fibersol-2, also reduced the number of positive responding ASVs (73 in 1% vs 60 in 2% fibersol-2; Figure 5) but increased the number of negative responding ASVs (38 in 1% vs 48 in 2% fibersol-2; Figure 5).

[128] Different SDFs, inulin and fibersol-2, were combined and fermented with fecal microbiota. Increased concentration of SDFs increased the number of positive responding ASVs (26 in 0.5%F0.5%I vs 34 in 1 %F 1 %I; Figure 6) and the number of negative responding ASVs (24 in 0.5%F0.5%I vs 55 in 1%F1%I; Figure 6). [129] The positive responders from 0.5% fiber sol -2: 0.5% inulin, 1% fibersol-2: 1% inulin, 1% fibersol-2, 2% fibersol-2, 1% inulin and 2% inulin groups were compared (Figure 7). The unique 10 positive responders from 1% fibersol-2: 1% inulin contain 4 potential butyrate producing bacteria, including Blautia ASV005H, Blautia massiliensis ASV002F, Blautia ASV006C, Blautia massiliensis ASV04LB, and one beneficial bacterium, Bifidobacterium ASV06TU. The unique 2 positive responders from 0.5% fibersol-2:0.5% inulin contain one beneficial bacterium, Bifidobacterium ASV0004.

[130] The data show that the combination the two different SDFs promote more beneficial bacteria compared to single SDF groups. The data show that relationship between substrate dose and microbiota composition is dynamic rather than a simple linear relationship.

[131] The fermentation of single SDF combined with ISDFs: The combination of one single SDF, inulin or fibersol-2, with ISDFs introduced new negative and positive responders compared to SDF alone or ISDF alone groups. The combination of fibersol-2 and bran mix have 8 unique positive responders compared to either 1% or 2% Fibersol-2 and 1% or 2% BranMix groups (Figure 8). These 8 unique positive responders included three potential butyrate producing bacteria, such as Clostridium butyricum ASV07RL, Faecalibacterium ASV002D, and Blautia ASV006C.

[132] The combination of inulin and bran mix have 9 unique positive responders compared to either 1% or 2% inulin and 1% or 2% Bran Mix groups (Figure 9). These 9 unique positive responders contain a beneficial bacterium, Bifidobacterium ASV0004, and three potential butyrate producing bacteria, including Anaerostipes ASV001P, Anaerostipes ASV002J, and Clostridium butyricum ASV07RL.

[133] These data show that synergistic effect was present in the combination of single SDF with ISDFs. The combination of single SDFs with ISDFs promoted unique beneficial bacteria compared with single SDF or ISDFs alone.

[134] The fermentation of SDFs combined with ISDFs: The combination of two SDFs, inulin and fibersol-2, with ISDFs was fermented with fecal microbiota. Their positive and negative responders were identified. Different ratios between SDFs and ISDFs were compared.

[135] First, SDF: ISDF=1 : 1 (0.5%F0.5%I1%B), SDF: ISDF=1 :0.5 (0.5%F0.5%I0.5%B) and SDF: ISDF=1:0.1 (0.5%F0.5%I0.1%B) were compared with SDF formula (0.5%F0.5%I) group. [136] 1 of the 15 positive responders in SDF: ISDF=1:0.1 (0.5%F0.5%I0.1%B) group was not promoted by 0.5%F0.5%I group. The only one new ASV promoted by SDF: ISDF=1:0.1 (0.5%F0.5%I0.1%B) group is a potential butyrate-producing bacterium, Parabacteroides ASV000Q (Figure 10).

[137] 18 of the 38 positive responders in SDF: ISDF=1:0.5 (0.5%F0.5%I0.5%B) group were not promoted by 0.5%F0.5%I group (Figure 10). 10 unique positive responders of SDF: ISDF=1:0.5 (0.5%F0.5%I0.5%B) group also contain three potential butyrate producing bacteria, Parabacteroides ASV000L, Parabacteroides ASV001J, and Butyricicoccus ASV0067.

[138] 10 of the 18 positive responders in SDF: ISDF=1 : 1 (0.5%F0.5%I1%B) group were not promoted by 0.5%F0.5%I group (Figure 10). 3 unique positive responders of SDF: ISDF=1:1 (0.5%F0.5%I1%B) group contain one potential butyrate producing bacteria, Blautia ASV005H. 7 ASVs were promoted by both SDF: ISDF=1:1 (0.5%F0.5%I1%B) and SDF: ISDF=1:0.5 (0.5%F0.5%I0.5%B) groups (Figure 10), which includes a beneficial bacterium Bifidobacterium ASV000J, and three potential butyrate producing bacteria, Blautia ASV001 A, Clostridium butyricum ASV07IF, and Parabacteroides distasonis ASV000V.

[139] SDF: ISDF=1:1 (0.5%F0.5%I1%B) and SDF: ISDF=1:0.5 (0.5%F0.5%I0.5%B) groups share 14 negative responders which were not inhibited by SDF: ISDF=1:0.1 (0.5%F0.5%I0.1%B) and 0.5%F0.5%I groups (Figure 11). Among these 14 unique bacteria were Akkermansia ASV0007 and Bilophila ASV0022. Some Akkermansia species, which adhere tightly to intestinal epithelium, was reported to be correlated to the dextran sodium sulfate-induced recurring colitis (Berry, D., et al., Intestinal Microbiota Signatures Associated with Inflammation History in Mice Experiencing Recurring Colitis. Frontiers in microbiology, 2015, 6: p. 1408-1408).

[140] Members of Bilophila genus has been shown to be pathogenic and detrimental by producing toxic metabolites such as hydrogen sulfide and proinflammatory antigens such as endotoxin (Devkota, S., et al., Dietary-fat-induced taurocholic acid promotes pathobiont expansion and colitis in 1110-/- mice. Nature, 2012. 487(7405): p. 104-108).

[141] Promotion of the butyrate-producing bacteria and inhibition of the potential detrimental bacteria indicated that SDF: ISDF=1:1 (0.5%F0.5%I1%B) and SDF: ISDF=1:0.5 (0.5%F0.5%I0.5%B) groups could exert more benefits to host. Again, these data suggests that the synergistic effect present in the combination of SDF and ISDF groups that could bring more benefits compared to single SDF or ISDF groups.

[142] Second, SDF: ISDF=1:1 (0.5%F0.5%I1%B), SDF: ISDF=0.5:1 (0.25%F0.25%I1%B) and SDF: ISDF=0.1:1 (0.05%F0.05%I1%B) groups were compared with 1% BranMix group.

[143] 11 of the 18 positive responders in SDF: ISDF=1 : 1 (0.5%F0.5%I1%B) group were not promoted by 1% BranMix group (Figure 12). Among these 11 ASVs, 5 ASVs were also promoted by SDF: ISDF=0.5:1 (0.25%F0.25%I1%B) group, including two potential beneficial bacteria, Faecalibacterium ASV0014 and Blautia ASV001A. 1 ASV was promoted by SDF: ISDF=1 : 1 (0.5%F0.5%I1%B) group, which is Bifidobacterium ASV000J. The unique 5 ASVs only promoted by SDF: ISDF=1:1 (0.5%F0.5%I1%B) contain one beneficial bacterium, Bifidobacterium ASV0004, and one potential butyrate producing bacterium, Blautia ASV005H.

[144] 24 of the 44 positive responders in SDF: ISDF=0.5:1 (0.25%F0.25%I1%B) group were not promoted by 1% BranMix group (Figure 12). Among these 24 ASVs, 8 ASVs were also promoted by SDF: ISDF=1:1 (0.5%F0.5%I1%B) group, including three potential butyrate producing bacteria, Butyricimonas ASV0036, Eubacterium hallii group ASV003U, and Blautia ASV001A. The unique 12 ASVs only promoted by SDF: ISDF=0.5:1 (0.25%F0.25%I1%B) contain one potential butyrate producing bacteria, Clostridium butyricum ASV07RL.

[145] Although only 1/10 SDF was added in ISDF in the SDF: ISDF=0.1:1 (0.05%F0.05%I1%B) group, 16 out of 50 positive responders in this group were not promoted by l%Bran Mix group (Figure 12), which includes three potential butyrate-producing bacteria mentioned above. This suggests that 10 folders lower of soluble fiber (i.e., fibersol-2 and inulin) still changed the response of specific ASVs to l%Bran Mix group and that combination of SDF and ISDF promote more beneficial bacteria than ISDF alone group.

[146] One potential detrimental bacterium was found in the 7 unique negative responders of SDF: ISDF=1:1 (0.5%F0.5%I1%B) group, which is Akkermansia ASV0007 (Figure 13).

[147] In summary, SDF: ISDF=1:1 (0.5%F0.5%I1%B), SDF: ISDF=0.5:1 (0.25%F0.25%I1%B) and SDF: ISDF=0.1:1 (0.05%F0.05%I1%B) groups promote more beneficial bacteria compared to the l%Bran Mix group.

[148] Conclusion [149] Promotion of the butyrate-producing bacteria and inhibition of the potential detrimental bacteria indicated that the combination of SDF with ISDF at the ratio from SDF: ISDF=1:0.5 (Fibersol:Inulin:BranMix=l:l:l) and SDF: ISDF=1:1 (Fibersol:Inulin:BranMix=l:l:2), and SDF: ISDF=0.5: 1 (Fibersol:Iinulin:BranMix= 1:1:4) groups promoted more beneficial bacteria and suppressed pathogenic bacteria.

Example 5

[150] In some embodiments, a batch of the insoluble component of the fiber mix (the bran mix) comprising oat bran, corn bran, sorghum bran and wheat bran is sent to Safe Sterilization USA for mixing, milling and roasting as described in Example 1. Thereafter, one, two, three or more soluble fibers are added. In some embodiments, the one or more soluble fiber comprises fructo-oligosaccharide (FOS), galacto-oligosaccharides (GOS), trans-galacto- oligosaccharides (TOS), resistant starch (RS), pectic oligosaccharide (POS), raffmose family oligosaccharides (RFO), polydextrose, inulin, and/or fibersol-2. In some embodiments, the one or more soluble fiber comprises fructo-oligosaccharide (FOS), galacto-oligosaccharides (GOS), trans-galacto-oligosaccharides (TOS), resistant starch (RS), pectic oligosaccharide (POS), raffmose family oligosaccharides (RFO), and/or polydextrose.

[151] The resulting formulation changes the dynamics of the fermentation so that a unique combination of SCFA-producing bacteria is selectively promoted as described in Example 2.

[152] When soluble fibers are used alone, the dosage cannot be too high as they are readily fermentable and produce a large amount of gas that can induce bloating, diarrhea as adverse gut effects. Humans cannot tolerate a high dosage of such soluble fibers (e.g. more than 20 grams/day).

[153] When combined with bran fiber mix, the fermentation process slows and produces less gas so that adverse effect minimized.

[154] In some embodiments, the formulation described herein can be taken at 60 grams/day with no adverse gut reaction.

Example 6-Type 2 Diabetes

[155] Since patients with type 2 diabetes is one of the key populations that may benefit from gut microbiota-targeted interventions, the present inventors tested the effect of the bran mix of Table 1 on postprandial glycemic response. The present inventors showed that the bran mix induced minimal changes in blood glucose in individuals who have normal glycemic control, prediabetes or type 2 diabetes (Figures 1A-1B), suggesting that this fiber mix does not negatively impact glucose homeostasis.

Example 7- COVID-19

[156] All enrolled participants are between 18 and 69 years of age having the following: (1) prediabetes or type 2 diabetes and (2) mild to moderate COVID- 19-like symptoms based on the symptom list from the CDC. Mild COVID-19 symptoms include fever or chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea or vomiting, and diarrhea, no clinical signs indicative of moderate, severe, or critical illness severity. Moderate Covid-19 symptoms include any symptom of mild illness or shortness of breath with exertion, clinical signs suggestive of moderate illness with COVID-19, such as respiratory rate ^ 20 breaths/min, oxygen saturation level > 93% on room air at sea level, heart rate ^ 90 beats/min, no clinical signs indicative of severe or critical illness severity.

[157] Individuals having the following are excluded from the study:

Receiving vancomycin monotherapy or oral broad-spectrum antibiotics Inability to receive oral fluids

Self-reported allergy or intolerance to any ingredients in the formulation Surgery involving the intestinal lumen within the last 30 days

Documented diagnosis of celiac disease, inflammatory bowel disease or irritable bowel syndrome

Pregnancy or breastfeeding Bariatric surgery

Chronic lung disease or moderate/severe asthma

Heart failure, coronary artery disease, congenital heart disease, cardiomyopathies or pulmonary hypertension

Chronic kidney disease on dialysis Chronic liver disease (cirrhosis)

Immunocompromised, e.g. cancer treatment, bone marrow/organ transplant, immune deficiency, poorly controlled HIV/AIDS, prolonged use of steroids or other immunosuppressant medications

[158] All enrolled participants receive self-supportive care for the treatment of COVID-19 and they have access to a series of videos specific to their assigned group that provide them with an overview of the study schedule and what is required of them during the study. The research team provide participants with a digital oral thermometer (Medtus, MO) to monitor body temperature, a pulse oximeter (IMDK, Shenzhen, China) to monitor oxygen saturation level and pulse rate, and a glucometer (Curo Fit, Yorba Linda, CA) to monitor blood glucose with step-by-step instructions. The treatment group take formulation described at Table 2, that provides fermentable dietary fibers as perturbation to improve the gut microbiota. The formulation is taken in the form of drinks up to four times a day (before each main meal and 2 h after dinner) from Day 1 to Day 28. Each drink is prepared by mixing a sachet of formulation mix (30 g) with 500 ml of water. The control group follow the same schedule except they drink the same volume of water without formulation. After 28 days of intervention, all participants remain in the study for follow-up until Day 56. If participants require hospitalization during the study, they discontinue all study procedures during hospitalization but remain in the study for follow-up until Day 56. Participants have access to an interactive online personal dashboard to review their study progress and the data that they have reported.

[159] All prior patents, publications, and test methods referenced herein are incorporated by reference in their entireties.

[160] While several embodiments of the present disclosure have been described, it is understood that these embodiments are illustrative only, and not restrictive, and that many modifications may become apparent to those of ordinary skill in the art. For example, all dimensions discussed herein are provided as examples only, and are intended to be illustrative and not restrictive.

[161] Any feature or element that is positively identified in this description may also be specifically excluded as a feature or element of an embodiment of the present as defined in the claims.