Foundation guild transplantation

Abstract

The disclosure is directed to methods for culturing, transferring, and maintaining a microbiota containing a predominance of a select group of beneficial microorganisms to alleviate and / or treat gut dysbiosis, including gastrointestinal symptoms thereof, in a subject suffering from dysbiosis.

Description

Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033FOUNDATION GUILD TRANSPLANTATIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application no. 63 / 715,940, filed November 4, 2024, entitled “FOUNDATION GUILD TRANSPLANTATION,” the disclosure of which is incorporated by reference herein in its entirety.FIELD OF DISCLOSURE

[0002] The present disclosure is related to the field of microbiology. More specifically, the disclosure is directed to culturing or fermentation processes that enrich beneficial microbiota for transplantation.BACKGROUND

[0003] There are about 100 trillion diverse types of microorganisms in the gut microbiota, including, for example, bacteria, archaea, eukaryotes, and viruses. The gut is in a state of dysbiosis when the gut has a higher diversity and abundance of pathogenic bacteria than beneficial bacteria. Gut dysbiosis has been associated with chronic diseases such as irritable bowel disease (IBS), chronic kidney disease (CKD), and Type 2 Diabetes among others. Although fecal microbiota transplantation (“FMT”) is a conventional medical procedure for transferring healthy bacteria from a donor’s stool sample to a recipient’s colon, direct transfer of such fecal microbiota has been shown to have limited efficacy in improving the suffering recipients’ health condition. See, e.g., Yang et al. Cell Metabolism. 35: 1548-1562, 2023. Another issue with FMT is a potential side effect where the recipient is infected with another bacterium or virus from the donor, or complications such as thrombotic cutaneous gangrene (“TCG”) arise in inflammatory bowel disease (“IBD”) patients. Also, the microbiomes of individuals differ, such that the combination of microorganisms found to be beneficial for one individual may not be beneficial for another. Therefore, there is a need to effectively restore and maintain beneficial bacteria of the microbiota of an individual suffering from dysbiosis, thereby treating the individual.SUMMARY

[0004] The disclosure provides methods of in vitro fermentation or culturing a healthy donor sample for use in Foundation Guild Transplantation (“FGT”) into a recipient subject suffering1ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 from dysbiosis. As described here, the healthy donor sample is modified and enriched for beneficial acetate- and butyrate-producing bacteria. Other features and advantages of the disclosure will be apparent from the detailed description and claims.

[0005] It is therefore an object of this disclosure to provide a method, comprising (a) mixing a stool sample from a healthy individual, where the sample from the healthy individual comprises one or more bacteria of TABLE 1, and a preservation buffer; (b) incubating the mixed sample (a) and an enrichment buffer under reducing conditions, forming SAMPLE A; (c) adding the enrichment buffer to SAMPLE A, forming enriched SAMPLE A; and (c) incubating enriched SAMPLE A under reducing conditions, forming SAMPLE B, where SAMPLE B comprises an enriched foundation guild of one or more bacteria of TABLE 1. Such method can further comprise repeating steps (b)-(d) (forming SAMPLE A' and SAMPLE B '). In some aspects of the disclosure, the preservation buffer comprises polysaccharides (e.g., prebiotics and fibers: agave, artichokes, fructooligosaccaraides (FOS), galactooligosaccharides (GOS), inulin, oat and lentil fibers, psyllium, resistant starch, wheat dextrin; digestion-resistant maltodextrin (e.g., Fibersol®, Fibersol®-2), high fiber formulation (e.g., NBT-NM108); and any combination of oligosaccharides or polysaccharides that can preserve and encourage the growth of one or more bacteria listed in TABLE 1) and an isotonic solvent (e.g., bicarbonate buffer, citrate buffer, PBS buffer (or capsules), saline, Ringer’s solution, lactated Ringer’s solution, dextrose in water). Additional aspects provide for the polysaccharides of the preservation buffer in an amount of 1% of the total volume of the preservation buffer to 15% of the total volume of the preservation buffer. In further aspects of the disclosure, at least one of the polysaccharides is selected from the group consisting of: inulin and digestion-resistant maltodextrin; or any combination of oligosaccharides or polysaccharides that can preserve and encourage the growth of one or more bacteria listed in TABLE 1. Additional aspects provide for the enrichment buffer to comprise polysaccharides (prebiotics and fibers: agave, artichokes, fructooligosaccaraides (FOS), galactooligosaccharides (GOS), inulin, oat and lentil fibers, psyllium, resistant starch, wheat dextrin; digestion-resistant maltodextrin (e.g., Fibersol®, Fibersol®-2), high fiber formulation (e.g., NBT-NM108); and any combination of oligosaccharides or polysaccharides that can preserve and encourage the growth of one or more bacteria listed in TABLE 1) and isotonic solvent (e.g., bicarbonate buffer, citrate buffer, PBS buffer (or capsules), saline, Ringer’s solution, lactated Ringer’s solution, dextrose in water). In some aspects of the disclosure, the polysaccharides of the enrichment buffer are in an amount of 3% of the total volume of the enrichment buffer to 50% of the total volume of the enrichment2ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 buffer. Further aspects provide for at least one of the polysaccharides of the enrichment buffer comprises a high fiber botanical -based agent that can preserve and encourage the growth of one or more bacteria listed in TABLE 1. In additional aspects of the method described here, SAMPLE B (or SAMPLE B') comprises an enriched foundation guild of one or more bacteria of TABLE 1, where the foundation guild is the predominant bacteria in the sample.

[0006] Another object of the disclosure is to provide a method, comprising: (a) screening a healthy individual interested in being a potential microbiome donor; (b) culturing a stool sample from the healthy individual as described here, wherein the stool sample contains one or more bacteria of TABLE 1; (c) transferring SAMPLE B (or SAMPLE B') into a recipient subject in need thereof; and (d) administering a fiber mix formulation to the recipient subject.BRIEF DESCRIPTION OF FIGURES

[0007] FIGs. 1A-1B demonstrate the Foundation Guild Transplantation (FGT) pipeline. In FIG. 1 A, the left side displays the pipeline from “Interested potential donors” up to “Screening, analysis, + review” (solid arrows). Once accepted, the approved screened donor’s pipeline can be seen with green arrows starting with their original GUILD: SAVE PLUS sample (right side). “PC” refers to polycarbonate. FIG. IB shows the upper panel of screening interested participants and lower panel of in vitro fermentation or culturing the sample from approved donor for use in FGT.

[0008] FIG. 2 shows an exemplary flowchart of the in vitro fermentation or culturing method described here (Project 1). The left side displays incubation time mark of each of the 7 timepoints (baseline, T2, T3, T4, T5, T6, and T7), while the right side displays name of the 4 different collection tube types, with corresponding buffer, as well as a brief explanation of activity and purpose at each timepoint. In Project 1, Donor A is female, and Donor B is male.

[0009] FIG. 3 illustrates a series of six panels representing Bray-Curtis Principal Coordinates Analysis (PCoA) plots to compare microbial community compositions for two donors (Project 1). Each donor has three distinct sets of samples, with each set encompassing seven consecutive time points. Panels in the top row depict the results for Donor B (male), with each panel dedicated to one of the three sample sets. Panels in the bottom row show the data for Donor A (female), similarly organized by sample set.

[0010] FIG. 4 presents TABLE 3, which demonstrates consistency of top amplicon sequence variants (AS Vs) in Repeat samples for Donor A and Donor B at T3 (48hr + 48hr).

[0011] FIG. 5 presents TABLE 4, which demonstrates consistency of Top ASVs in Repeat samples for Donor A at T6 (48 hr + 48 hr +24 hr + 24 hr + 24 hr).3ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0012] FIG. 6 presents TABLE 5, which demonstrates consistency of Top ASVs in Repeat samples for Donor B at T6 (48 hr + 48 hr +24 hr + 24 h r+ 24 hr).

[0013] FIGs. 7A-7B present TABLE 6, which demonstrates relative abundance levels of top ASVs in the last sample of Donor A at TABLE 6 and the relative abundance levels of these ASVs at baseline.

[0014] FIGs. 8A-8B present TABLE 7, which demonstrates relative abundance levels of top ASVs in the last sample of Donor B at T7 and the relative abundance levels of these ASVs at baseline.

[0015] FIG. 9 shows an exemplary flowchart of the in vitro fermentation or culturing method described here (Project 2). The left side displays incubation time mark of each of the 6 timepoints (baseline, T2, T3, T4, T5, and T6), while the right side displays name of the 4 different collection tube types, with corresponding buffer, as well as a brief explanation of activity and purpose at each timepoint. In Project 2, Donor A is male, and Donor B is female.

[0016] FIG. 10 illustrates a series of six panels representing Bray-Curtis Principal Coordinates Analysis (PCoA) plots to compare microbial community compositions for Donor A (male) in Project 2. This donor has six distinct sets of samples, with each set encompassing seven consecutive time points. Panels in the top row depict the results for Donor A transfer samples, with each panel dedicated to one of the three sample sets. Panels in the bottom row show the data for Donor A predict samples, similarly organized by sample set. In each panel, points represent individual samples, plotted according to their principal coordinates to reflect differences in microbial community composition. Samples from the same set are connected by a trajectory line, highlighting the temporal development of the microbiome over the seven time points.

[0017] FIG. 11 illustrates a series of six panels representing Bray-Curtis Principal Coordinates Analysis (PCoA) plots to compare microbial community compositions for Donor B (female) in Project 2. This donor has six distinct sets of samples, with each set encompassing seven consecutive time points. Panels in the top row depict the results for Donor B transfer samples, with each panel dedicated to one of the three sample sets. Panels in the bottom row show the data for Donor B predict samples, similarly organized by sample set. In each panel, points represent individual samples, plotted according to their principal coordinates to reflect differences in microbial community composition. Samples from the same set are connected by a trajectory line, highlighting the temporal development of the microbiome over the seven time points.4ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0018] FIG. 12 presents TABLE 7, which shows the relative abundance of top ASVs across timepoints in Donor A (Project 2).

[0019] FIG. 13 presents TABLE 8, which shows relative abundance of top ASVs across timepoints in Donor B (Project 2).

[0020] FIG. 14 shows an exemplary flowchart of the in vitro fermentation or culturing method described here (Project 3). The left side of the timeline displays the incubation time mark of each of the 4 timepoints (baseline, T2, T3, and T4), while the right side of the timeline displays a brief explanation of activity and purpose at each timepoint with corresponding collection tube type. In Project 3, Donor A is male, and Donor B is female.

[0021] FIGs. 15A-15D show beta diversity (Project 3). Beta Diversity Principal Coordinates Analysis (PCoA) Plot Comparing Microbial Communities of Two Donors Across Four Timepoints. FIGs. 15A-15D comprise four panels, each illustrating a PCoA plot based on different measures of beta diversity to compare microbial communities from Donor A and Donor B across four timepoints. The panels are labeled as follows: (FIG. 15 A) Bray-Curtis distance, (FIG. 15B) Jaccard distance, (FIG. 15C) Weighted UniFrac, and (FIG. 15D) Unweighted UniFrac. Each panel displays samples from Donor A as circle dots and samples from Donor B as triangular dots, facilitating clear visual differentiation between the two donors. The samples as presented in the legend represent different sampling timepoints for baseline, T2 samples, T3 samples, and T4 samples.

[0022] FIGs. 16A-16D show alpha diversity metrics across timepoints in Donor A. FIGs. 16A-16D comprise four panels, each representing a different alpha diversity metric for samples collected from Donor A at four distinct time points. The panels are organized as follows: (FIG. 16A) Shannon Index, (FIG. 16B) ASV (Amplicon Sequence Variant) number, (FIG. 16C) Faith's Phylogenetic Diversity, and (FIG. 16D) Evenness. Each panel features four boxplots corresponding to the timepoints: baseline, T2, T3, and T4.

[0023] FIGs. 17A-17D show alpha diversity metrics across timepoints in Donor B (Project 3). FIGs. 17A-17D comprise four panels, each representing a different alpha diversity metric for samples collected from Donor B at four distinct time points. The panels are organized as follows: (FIG. 17A) Shannon Index, (FIG. 17B) ASV (Amplicon Sequence Variant) number, (FIG. 17C) Faith's Phylogenetic Diversity, and (FIG. 17D) Evenness. Each panel features four boxplots corresponding to the timepoints: baseline, T2, T3, and T4.

[0024] FIG. 18 presents TABLE 9, which shows the dynamics of group-level relative abundance in Donor A (Project 3).5ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0025] FIG. 19 presents a graphical representation of TABLE 9, which describes the dynamic shifts in the relative abundance of two key ASV groups within Donor A across four timepoints: baseline, T2, T3, and T4. The x-axis is marked by these timepoints, while the y-axis denotes the relative abundance of each group, expressed in percentage format.

[0026] FIG. 20 presents TABLE 10, which shows the dynamics of group-level relative abundance in Donor B (Project 3).

[0027] FIG. 21 presents a graphical representation of TABLE 10, which describes the dynamic shifts in the relative abundance of two key ASV groups within Donor B across four timepoints: baseline, T2, T3, and T4. The x-axis is marked by these time points, while the y- axis denotes the relative abundance of each group, expressed in percentage format.

[0028] FIG. 22 presents TABLE 11, which shows consistency of top ASVs in repeat samples for Donors A and B at T4 (48hr + 24hr + 24hr) (Project 3).

[0029] FIG. 23 shows an exemplary workflow diagram (Project 4).

[0030] FIG. 24 demonstrates pH change over time of donor samples. Upper line - Donor A; Lower line Donor B.

[0031] FIG. 25 illustrates relative abundance of Faecalibacterium ASVs in Donor A (upper graph) and Donor B (lower graph). The upper line in both donors (the upper and lower graphs) is ASV001E g_Faecalibacterium, while the lower line in both is ASV000Y g_F aecalib acterium .

[0032] FIG. 26 presents an in vitro fermentation workflow (S-00182601 (T3)).

[0033] FIGs. 27A-27B demonstrates Pioneer-Guild Composition stability.

[0034] FIG. 28 shows a table of live colony counts.

[0035] FIG. 29 presents a Quality Assurance (QA) / Quality Control (QC) Testing matrix Summary.

[0036] FIG. 30 presents a stool and transplant material pathogen-screening checklist.DETAILED DESCRIPTION

[0037] The disclosure features methods of preparing donor microbiota samples for transplanting into a recipient during Foundation Guild Transplantation (“FGT”), which involves re-seeding the recipient’s gut microbiome with beneficial acetate- and butyrate- producing bacteria referred to as the “foundation guild”. The disclosure is based, at least in part, on the discovery that there is a need for genome-specificity, database-independence, and interaction-focused aggregation to identify, prepare, restore, and maintain beneficial bacteria (e.g., foundation guild) in a subject in need of treatment for dysbiosis. Technologies that restore6ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 and maintain the ecological dominance of the foundational guild are described here, which are useful in promoting and preventing dysbiosis, diet-related diseases (DRDs), and symptoms thereof, for personalized treatments or treatment for the masses. FGT is a next-generation transplantation therapeutic. FGT transfers a healthy donor microbiota to an appropriate recipient via colonoscopy. The process in which donor samples are prepared for FGT is unique and novel.

[0038] The gut microbiota has been found to be health-supporting when one or more bacteria from a defined group of beneficial bacteria, i.e., foundation guild (“FG”) (TABLE 1), is present in ecologically dominant position over another group of pathogenic or detrimental bacteria, named pathobiont guild (“PG”). When the gut is in a state of dysbiosis, the pathobiont guild is present in greater abundance than the beneficial foundation guild. In order to improve human health and treat gut dysbiosis, the beneficial FG bacteria should be dominant in a healthy gut and those pathogenic PG bacteria should be kept at bay. See, e.g., Wu et al. Cell. S0092- 8674(24)01038-9, 2024. doi: 10.1016 / j.cell.2024.09.019.

[0039] However, in some patients, the diversity of the FG bacteria has been lost due to antibiotic medications, infections, or other medical conditions to an extent that the FG is no longer able to dominate the PG in subjects or patients suffering from gut dysbiosis. In order to assist or treat these patients by restoring a health-supporting gut microbiota dominated by FG bacteria, a healthy donor needs to be identified to provide “seeds” of FG (by virtue of, for example, a stool / fecal sample containing one or more bacteria of TABLE 1) to be transplanted into the gut of the subjects or patients in need of treatment in order to obtain a health-supporting gut microbiota dominated by FG.

[0040] However, the fecal sample from a healthy donor contains both FG and PG bacteria and many FG bacteria may be low in abundance to a non-detectable level.

[0041] Definitions

[0042] As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. All terms used herein are intended to have their ordinary meaning in the art unless otherwise provided. All concentrations are in terms of percentage by weight of the specified component relative to the entire weight of the composition, unless otherwise defined.

[0043] As used herein, “a” or “an” shall mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” mean one or more than one. As used herein “another” means at least a second or more.7ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0044] As used herein, all ranges of numeric values include the endpoints and all possible values disclosed between the disclosed values. The exact values of all half-integral numeric values are also contemplated as specifically disclosed and as limits for all subsets of the disclosed range. For example, a range of from 0.1% to 3% specifically discloses a percentage of 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%. Additionally, a range of 0.1 to 3% includes subsets of the original range including from 0.5% to 2.5%, from 1% to 3%, from 0.1% to 2.5%, etc. It will be understood that the sum of all weight % of individual components will not exceed 100%.

[0045] By “consist essentially” it is meant that the ingredients include only the listed components along with the normal impurities present in commercial materials and with any other additives present at levels which do not affect the operation of the embodiments disclosed herein, for example at levels less than 5% by weight or less than 1% or even 0.5% by weight.

[0046] By “alteration” is meant a change (increase or decrease) in, for example, the expression levels, structure, or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, a 25% change, a 40% change, or a 50% or greater change in expression levels.

[0047] By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or condition.

[0048] In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0049] By “ decreases” is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages.

[0050] As used herein, the term “disease” refers to any condition or disorder that interferes with or damages normal functions of a cell, tissue, or organ. For example, gut dysbiosis can lead to several dysbiosis-related diseases including but not limited to cancer (e.g., breast cancer,8ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 colon / colorectal cancer, hepatocellular carcinoma, esophageal cancer, gallbladder carcinomas, gastric cancer, laryngeal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, stomach cancer); gastrointestinal diseases (e.g., H. pylori and C. difficile infections, inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis); gastrointestinal issues (e.g., diarrhea, constipation, gas, acid reflux); small intestinal bacterial overgrowth (“SIBO”); metabolic diseases (e.g., Type 1 diabetes (i.e., insulin-dependent diabetes mellitus) and Type 2 diabetes (i.e., non-insulin-dependent diabetes mellitus)); inflammatory diseases (e.g., rheumatoid arthritis, asthma, non-alcoholic fatty liver disease (“NAFLD”)); obesity; autism spectrum disorders; mental health diseases (e.g., Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy, dementia, anxiety, attention deficit hyperactivity disorder (“ADHD”), depression, bipolar disorder, schizophrenia, obsessive-compulsive disorder (“OCD”), post-traumatic stress disorder (“PTSD”)). Various embodiments described here are directed to methods of alleviating, reducing, or treating gut dysbiosis, which in turn alleviates, treats, or reduces the symptoms of any of the dysbiosis-related diseases described here.

[0051] The term, “dysbiosis” including “gut dysbiosis” as used herein, is meant an imbalance in a microorganism population of the gut in a subject, which negatively impacts the subject by the presence of an increase in unfavorable or damaging microorganisms or decrease in beneficial bacteria. This imbalance leads to a variety of health issues and diseases. Dysbiosis can be further characterized as L- and D-type dysbiosis. Symptoms of dysbiosis may manifest and include without limitation, aching joints, acne, acid reflux or heartburn, ADHD or issues with concentration, anxiety, bloating, chronic fatigue, depression, digestive problems, food intolerance, gas, inflammation, psoriasis, skin rashes, trouble urinating, and vaginal or rectal infections or itching, and the like.

[0052] As used herein, the term “Fibersol®” or “Fibersol®-2”, used interchangeably here, is a concentrated dietary fiber. It is also labeled as a soluble vegetable (e.g., corn) fiber, a maltodextrin or digestion-resistant maltodextrin. Fibersol® can be used as a polysaccharide in the preparation of FGT compositions described here.

[0053] By the term “foundation guild” as used herein is meant beneficial microorganisms, including acetate- and butyrate-producing bacteria. Such exemplary bacteria include species 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,9ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033Paraprevotella, Phascolarctobacterium, Roseburia, Ruminiclostridium, or Subdoligranulum, and 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, and Veillonellaceae. The foundation guild bacteria are described as Guild 1 in PCT / US23 / 66191 (see, e.g., Tables 1-2, FIGs. 42A-4XX) and PCT / US24 / 26282 (see, e.g., Tables 1-2), both of which are incorporated by reference in its entirety and for their teaching regarding Guild 1 (aka Foundation Guild). Non-limiting examples of bacteria of the foundation guild are found in TABLE 1 and include:

[0054] TABLE 110ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03311ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03312ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03313ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03314ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03315ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03316ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03317ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0055] As used herein, “Foundation Guild Nutrition” or “FGN” or “fiber mix formulation” used interchangeably here is meant to be a formulation comprising a mixture of fiber useful for increasing proliferation of or maintaining the foundation guild bacteria. In some embodiments, the FGN is administered to the recipient subject after foundation guild transplantation to maintain the presence and / or dominance of beneficial foundation guild bacteria in the gut of the recipient subject. The FGN or fiber mix formulation useful after FGT is described in PCT / US2021 / 026258 and incorporated by reference for such teachings, dosages, administration, and the like, and also in its entirety. Briefly, the FGN or fiber mix formulation comprises a mixture of bran (e.g., corn bran, wheat bran, sorghum bran, oat bran), inulin, and digestion resistant maltodextrin, where the mixture has a ratio of dietary fibers from bran: dietary fibers from inulin: dietary fibers from digestion resistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage or support growth of one or more bacteria of TABLE 1, ranging from about 1 : 1 : 1 to about 4: 1 : 1, where the inulin is present in a range of 5-10% of total weight of the mixture; and where the mixture has a uniform particle size between 150 pm and 300 pm. The FGN can be administered as a supplement for consumption daily, every other day, every two days, every three days, every four days, every five days, every six days, or weekly, monthly, multiple times a day, with or without food, in between meals, or as needed, or as frequently as needed to promote and maintain a healthy gut microbiome comprising the beneficial foundation guild bacteria.

[0056] The term “Foundation Guild Transplantation” or “Foundation Guild Transfer” or “FGT”, all used interchangeably here, is meant a procedure that transfers beneficial bacteria, such as foundation guild bacteria from a healthy donor to a recipient patient to treat microbiome imbalances. The foundation guild bacteria can be transferred directly or indirectly to a recipient patient’s gastrointestinal tract, such as but not limited to, the colon. The foundation guild bacteria can be enriched and formulated into a composition from stool or feces of an appropriate donor.

[0057] As used herein, the term “inulin” is a dietary fiber classified as fructans, and is a type of plant-based polysaccharide, typically extracted from chicory among other plants. Inulin can be used as a polysaccharide in the preparation of FGT compositions described here.18ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0058] By the term “NBT-NM108” is meant a high fiber botanical -based agent that modulates the gut microbiota, restores unhealthy, altered gut microbiome, enhances microbial diversity and function in the gastrointestinal (GI) tract, and re-establishes a healthy microbiome in the GI tract. As a result, recipients of NBT-NM108 acquire increased immunological activity against various opportunistic pathogens and decreased pathogen-induced inflammation. NBT- NM108 can be used as a polysaccharide in the preparation of FGT compositions described here.

[0059] As used herein, the term “normal gut microbiota” refers to a population of microorganisms present in, or substantially similar to, the gut of a healthy individual, such as but not limited to a healthy donor.

[0060] The term “pathobiont” or “pathobiont guild” (“PG”) as used here refers to a microorganism that is opportunistic as a result of changes in a healthy microbiome, such that the microorganism becomes pathogenic allowing for certain diseases to result.

[0061] As used here, the term “polysaccharides” of the preservation buffer and enrichment buffer described here includes oligosaccharides. Such “polysaccharides” include any one or more oligosaccharides or polysaccharides that preserve and encourage the growth of one or more bacteria listed in TABLE 1. Non-limiting examples of “polysaccharides” of the disclosure include: prebiotics and fibers: agave, artichokes, fructooligosaccaraides (FOS), galactooligosaccharides (GOS), inulin, oat and lentil fibers, psyllium, resistant starch, wheat dextrin; digestion-resistant maltodextrin (e.g., Fibersol®, Fibersol®-2), high fiber formulation (e.g., NBT-NM108); and any combination of oligosaccharides or polysaccharides that can preserve and encourage the growth of one or more bacteria listed in TABLE 1.

[0062] As used herein, the term “subject” refers to any living or non-living organism including, but not limited to, a human, a non-human mammal, or a non-human animal. Any human or non-human animal can serve as a subject, including but not limited to mammal, reptile, avian, amphibian, fish, ungulate, ruminant, bovine (e.g., cattle), equine (e.g., horse), caprine and ovine (e.g., sheep, goat), swine (e.g., pig), cam elid (e.g., camel, llama, alpaca), monkey, ape (e.g., gorilla, chimpanzee), ursid (e.g., bear), poultry, dog, cat, mouse, rat, fish, dolphin, whale and shark. In some embodiments, a subject is a male or female of any age (e.g., a man, a woman, or a child).19ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0063] As used herein, the term “undesirable gut microbiome” is meant a community of microorganisms comprising a pathogen or having a biological activity associated with a pathogenic process.

[0064] “Unit dosage forms”, also referred to as “unitary dosage forms”, as used here, often denote those forms of therapeutics supplied in a manner that does not require further weighing or measuring to provide the dosage (e.g., tablet, capsule, caplet, suppository, etc.). For example, a unit dosage form refers to a physically discrete unit suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material (e.g., enriched donor sample, SAMPLE B, SAMPLE B') calculated to produce the desired therapeutic effect, in association with any suitable pharmaceutical vehicle (e.g., carrier, excipient, diluent). Exemplary, non-limiting unit dosage forms include a tablet (e.g., a chewable tablet), caplet, capsule (e.g., a hard capsule or a soft capsule), lozenge, gel cap, suppository, suspensions.

[0065] Foundation Guild Transplantation

[0066] Foundation guild transplantation (“FGT”) is a next-generation transplantation therapeutic. FGT transfers an enhanced healthy donor microbiota to an appropriate recipient subject(s) via, for example, colonoscopy. FGT can be used to treat recipients suffering from dysbiosis, including those with a severe depletion or absence of the beneficial bacteria of the foundation guild. The process in which donor stool samples are prepared for FGT is unique and novel. In one embodiment, the FGT process involves screening individuals interested in being potential donors; once approved, the process involves culturing a donor sample (e.g., fecal or microbiota sample), thereby enriching for beneficial microorganisms, such as acetate- and butyrate-producing bacteria (i.e., foundation guild) forming an enriched sample; analyzing the enriched sample; transplanting the enriched sample; and maintaining the dominance of the foundation guild in the recipient’s microbiota.

[0067] Another embodiment of the disclosure provides for enriching a donor sample after being screened, analyzed, and approved. For example, the donor sample’s foundation guild bacteria are enriched through a series of advanced in vitro fermentation or culturing steps (see, e.g., FIGs. 1A-1B). Refining the donor’s sample before transplantation allows for dominance of a select group of acetate- and butyrate-producing bacteria, such as but not limited to, one or more bacteria listed in the foundation guild of TABLE 1, or gut organisms disclosed in Guild 1 of PCT / US23 / 66191 (see, e.g., Tables 1-2, FIGs. 42A-4XX) and PCT / US24 / 26282 (see, e.g., Tables 1-2), both of which are incorporated by reference in its entirety and for their teaching20ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 regarding Guild 1 (aka Foundation Guild). This enrichment process also reduces populations of opportunistic pathogens rendering the FGT process safer than existing fecal matter transplantation (“FMT”) protocols.

[0068] Screening Potential Donors

[0069] Some embodiments are directed to the screening process of individuals interested in being a donor for FGT. In a preliminary screening, interested individuals take and complete a preliminary screening questionnaire (e.g., online (e.g., on a computer, a mobile device), on paper). Ideally, once an interested individual is identified, the interested individual can undergo the preliminary screening questionnaire initially before the other screenings described here. However, in some instances, any of the screenings described here can occur simultaneously or essentially simultaneously. The questionnaire acts to select an appropriate donor who is interested and fits the inclusion and exclusion criteria such as, but not limited to: INCLUSION: Age of at least 18 years old; EXCLUSION: Risk factors for transmittable diseases (e.g., new sexual contact in last six months, recent needle stick accident, recent transfusion, i.v. drug use, risk for variant Creutzfeldt-Jakob disease, sex for drugs or money, homosexuality, or tattoos); Travel to tropical area in last 3 months; Known history of tropical infection; Use of antibiotics in the 3 months prior to donation; Diarrhea (>3 loose or watery stools per day for at least 2 consecutive days or >8 loose stools in 48 hours; Household contacts with active gastrointestinal infection; Abnormal blood or stool test result suggestive of active / current disease; Any gastrointestinal illness (inflammatory bowel disease, irritable bowel syndrome, gastrointestinal malignancies, or major gastrointestinal surgery) or complaints; History of autoimmune or atopic illness or ongoing immune modulating therapy; History of chronic pain syndromes (fibromyalgia, chronic fatigue) or neurologic or neurodevelopmental disorders; Metabolic syndrome, obesity (BMI of >30), moderate to severe undernutrition; and History of malignancy / receipt of chemotherapy.

[0070] Medical Screening

[0071] In a further embodiment, an interested individual will submit to a medical screening questionnaire in order to determine if the individual is healthy enough to be considered a potential donor. Ideally, once an interested individual qualifies after the preliminary screening questionnaire, the individual will undergo the medical screening. However, in some instances, any of the screenings can occur simultaneously or essentially simultaneously. The medical screening questionnaire can take place over the phone or in person.21ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0072] Blood Screening

[0073] Another embodiment is directed to the blood screening of an interested individual. Blood will be drawn and screened from interested individuals who are deemed healthy enough after the medical screening. Their blood sample will be screened for certain health blood parameters as well as the presence of any disease / infection markers. Pursuant to potential donor study participant protocols, interested individuals will provide the necessary informed consent in line with the laws, policies, and regulations governing such procedures prior to giving blood. For research purposes, the individual’s blood sample will be given a deidentified study participant ID #. Ideally, once an interested individual qualifies after the preliminary screening and medical screening, the individual will undergo the blood screening. However, in some instances, any of the screenings can occur simultaneously or essentially simultaneously. The blood sample will be screened for, for example, any of the following: Bacterial Blood Screening (Treponema pallidum serology, Helicobacter pulori EIA); Viral Blood Screening (e.g., Cytomegalovirus (CMV), Espsteil Barr virus serology, Hepatitis A virus IgM, Hepatitis B virus surface, Hepatitis C virus antibody, HIV Ab / Ag including p24, HTLV 1 and 2 antibodies, JC virus serology, Syphilis); Protozoal Blood Screening (e.g., Entamoeba histolytica latex agglutination and dipstick, Strongyloids stercoralis serology, Schistosoma spp.); Other Blood Screening (e.g., Complete blood count, Complete metabolic panel (Alanine transaminase (ALT), Albumin, Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Bilirubin, blood urea nitrogen (BUN), Calcium, carbon dioxide (bicarbonate), Chloride, Creatine, Glucose, Potassium, Sodium), Erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP)).

[0074] Stool and Foundation Guild Screening

[0075] In some embodiments, once the interested individual’s blood screening result or blood panel report has been reviewed and approved, a full stool sample will be taken from the individual for analysis. The collection of the stool sample serves two purposes: (1) to screen for any potential gut pathogens and (2) to see if the Foundation Guild bacteria within the sample can be optimized through in vitro fermentation or culturing. Ideally, once an interested individual qualifies after the preliminary, medical, and blood screenings, the individual will undergo the stool and foundation guild bacteria screening. However, in some instances, any of the screenings can occur simultaneously or essentially simultaneously. Non-limiting stool screening tests include: Clostridioides difficile (e.g., Off label toxin PCR, Enzyme immunoassay (EIA)); Bacterial (e.g., Enteric pathogen culture (e.g., Salmonella, Shigella,22ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033Camplyobacter), Shiga toxin (STEC) or E. coli 0157 culture (including EPEC) (testing can occur via nucleic acid amplification test (NAAT)), Listeria spp., H. pylori EIA, Vibrio spp., Multi-drug-resistant organisms (MDROs) (e.g., Methicillin-resistant staphylococcus aureus (MRSA), Vancomyocin-resistant enterococcus (VRE) culture, carbapenem-resistant Enterob acteriaceae (CRE), Extended Spectrum beta-lactamase (ESBL)- producing Enterob acteriaceae); Viral (e.g., Adenovirus EIA, Norovirus EIA or real-time PCR, Rotavirus EIA, SARS-CoV2, Monkeypox (e.g., MPV, MPVX, or hMPXV)); and Protozoal (e.g., Ovum and parasite microscopic examination, Microporidia microscopic examination, Giardia fecal antigen / EIA, Cryptosporidium EIA, Isospora and Cyclospora microscopic examination).

[0076] In vitro Fermentation or Culturing

[0077] In order to prepare and optimize foundation guild bacteria for Foundation Guild Transplantation (FGT), a series of in vitro fermentation transfer steps are performed. In some embodiments, immediately prior to stool sample collection from the interested individual who passed or was approved based on prior screenings described here (“donor”), the donor will complete a questionnaire regarding the individual’s current health (e.g., symptoms the day of stool collection, bacterial or viral infection exposures (e.g., COVID-19), etc.). By modifying a sample from a potential donor to allow for the dominance of a select group of acetate- and butyrate-producing bacteria (e.g., foundation guild), i.e., one or more bacteria of TABLE 1, improves the success of FGT by reducing opportunistic pathogenic bacteria and populations thereof, which also provides for a safe and effective transplantation. One embodiment provides for an in vitro fermentation method comprising: collecting a stool sample from the donor (e.g., 1g stool / 5ml isotonic solvent / buffer); preparing the stool sample into (1) a baseline sample (in an ethanol-based buffer (e.g., 70-95% ethanol; “GUILD: QUEST”) and (2) a sample for further processing (in a buffer that preserves (i.e., preservation buffer) the beneficial bacteria, i.e., the foundation guild bacteria; “GUILD-SAVE PLUS”), where the buffer comprises an isotonic solvent (e.g., bicarbonate buffer, citrate buffer, PBS buffer (or capsules), saline, Ringer’s solution, lactated Ringer’s solution, dextrose in water); polysaccharides (or oligosaccharides) (e.g., prebiotics and fibers: agave, artichokes, fructooligosaccaraides (FOS), galactooligosaccharides (GOS), inulin, oat and lentil fibers, psyllium, resistant starch, wheat dextrin; digestion-resistant maltodextrin (e.g., Fibersol®, Fibersol®-2), high fiber formulation (e.g., NBT-NM108)); and reducing conditions (e.g., anaerobic chamber, reducing agents (e.g., formic acid, hydrogen gas, iron, lithium, lithium aluminum hydride, L-Cysteine, magnesium, oxalic acid, sodium, sodium 2-mercaptoethanesulfonic acid, sodium borohydride, sulfite23ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 compounds, zinc); incubating the sample for further processing (2) in a buffer that enriches the beneficial bacteria (i.e., foundation guild bacteria), where the buffer comprises: an isotonic solvent (e.g., saline, PBS buffer, Ringer’s solution, lactated Ringer’s solution, dextrose in water); polysaccharides (e.g., prebiotics and fibers: agave, artichokes, fructooligosaccaraides (FOS), galactooligosaccharides (GOS), inulin, oat and lentil fibers, psyllium, resistant starch, wheat dextrin; digestion-resistant maltodextrin (e.g., Fibersol®, Fibersol®-2), high fiber formulation (e.g., NBT-NM108); and reducing conditions (e.g., anaerobic chamber, reducing agents (e.g., formic acid, hydrogen gas, iron, lithium, lithium aluminum hydride, L-Cysteine, magnesium, oxalic acid, sodium, sodium 2-mercaptoethanesulfonic acid, sodium borohydride, sulfite compounds, zinc), at a temperature of 4°C or greater (e.g., 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42); 40°C or less (e.g., 39, 37, 35, 33, 31, 29, 27, 25, 23, 21, 19, 17, 15, 13, 11, 9, 7, 5, 3); or 4°C - 40°C (e.g., 5°C - 39°C; 6°C - 38°C; 7°C - 37°C; 8°C - 36°C; 9°C - 35°C; 10°C - 34°C; 11°C - 33°C; 12°C - 32°C; 13°C - 31°C; 14°C - 30°C; 15°C - 29°C; 16°C - 28°C; 17°C - 27°C; 18°C - 26°C; 19°C - 25°C; 20°C - 24°C; 21°C - 23°C)), and under agitation (e.g. centrifuge, orbital shaker, rocking shaker, rotating shaker, shaker, stirrer, vortex) at 5 rpm or greater (e.g., 15, 25, 35, 45, 55, 65, 75, 85, 95, 105), 100 rpm or less (e.g., 90, 80, 70, 60, 50, 40, 30, 20, 10), or 5 rpm - 100 rpm (e.g., 10 rpm - 95 rpm, 15 rpm - 90 rpm, 20 rpm - 85 rpm, 25 rpm - 80 rpm, 30 rpm - 75 rpm, 35 rpm - 70 rpm, 40 rpm - 65 rpm, 45 rpm - 60 rpm, 50 rpm - 55 rpm), for 1 hour or longer (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51); 50 hours or shorter (e.g., 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22 ,20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 0.5); 1 hour - 50 hours (e.g., 2 hours - 49 hours; 3 hours - 48 hours; 4 hours - 47 hours; 5 hours - 46 hours; 6 hours - 45 hours; 7 hours - 44 hours; 8 hours - 43 hours; 9 hours - 42 hours; 10 hours - 41 hours; 11 hours - 40 hours; 12 hours - 39 hours; 13 hours - 38 hours; 14 hours - 37 hours; 15 hours - 36 hours; 16 hours - 35 hours; 17 hours - 34 hours; 18 hours - 33 hours; 19 hours - 32 hours; 20 hours - 31 hours; 21 hours - 30 hours; 22 hours - 29 hours; 23 hours - 28 hours; 24 hours - 27 hours; 25 hours - 26 hours), forming SAMPLE A (“GUILD: PREDICT1”); incubating SAMPLE A in a buffer that enriches the beneficial bacteria (i.e., foundation guild bacteria), where the buffer comprises: an isotonic solvent (e.g., saline, PBS buffer, Ringer’s solution, lactated Ringer’s solution, dextrose in water); polysaccharides (e.g., prebiotics and fibers: agave, artichokes, fructooligosaccaraides (FOS), galactooligosaccharides (GOS), inulin, oat and lentil fibers, psyllium, resistant starch, wheat dextrin; digestion-resistant maltodextrin (e.g., Fibersol®, Fibersol®-2), high fiber formulation (e.g., NBT-NM108); and reducing conditions (e.g., anaerobic chamber, reducing agents e.g.,24ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 formic acid, hydrogen gas, iron, lithium, lithium aluminum hydride, L-Cysteine, magnesium, oxalic acid, sodium, sodium 2-mercaptoethanesulfonic acid, sodium borohydride, sulfite compounds, zinc), at a temperature of 4°C or greater (e.g., 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42); 40°C or less (e.g., 39, 37, 35, 33, 31, 29, 27, 25, 23, 21, 19, 17, 15, 13, 11, 9, 7, 5, 3); or 4°C - 40°C (e.g., 5°C - 39°C; 6°C - 38°C; 7°C - 37°C; 8°C - 36°C; 9°C - 35°C; 10°C - 34°C; 11°C - 33°C; 12°C - 32°C; 13°C - 31°C; 14°C - 30°C; 15°C- 29°C; 16°C - 28°C; 17°C - 27°C; 18°C - 26°C; 19°C - 25°C; 20°C - 24°C; 21°C - 23°C)), and under agitation (e.g. rocking shaker, orbital shaker, rotating shaker) at 5 rpm or greater (e.g., 15, 25, 35, 45, 55, 65, 75, 85, 95, 105), 100 rpm or less (e.g., 90, 80, 70, 60, 50, 40, 30, 20, 10), or 5 rpm - 100 rpm (e.g., 10 rpm - 95 rpm, 15 rpm - 90 rpm, 20 rpm - 85 rpm, 25 rpm- 80 rpm, 30 rpm - 75 rpm, 35 rpm - 70 rpm, 40 rpm - 65 rpm, 45 rpm - 60 rpm, 50 rpm - 55 rpm), for 1 hour or longer (e.g., 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51); 50 hours or shorter (e.g., 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22 ,20, 18, 16, 14, 12, 10, 8, 6, 4, 2, 0.5); 1 hour - 50 hours (e.g., 2 hours - 49 hours; 3 hours - 48 hours; 4 hours - 47 hours; 5 hours - 46 hours; 6 hours - 45 hours; 7 hours - 44 hours; 8 hours - 43 hours; 9 hours - 42 hours; 10 hours - 41 hours; 11 hours - 40 hours; 12 hours - 39 hours; 13 hours - 38 hours; 14 hours - 37 hours; 15 hours - 36 hours; 16 hours - 35 hours; 17 hours - 34 hours; 18 hours - 33 hours; 19 hours - 32 hours; 20 hours - 31 hours; 21 hours - 30 hours; 22 hours - 29 hours; 23 hours - 28 hours; 24 hours - 27 hours; 25 hours- 26 hours), forming SAMPLE B (“Guild-Predict2”). In various embodiments, these steps are repeated as needed. Further embodiments provide for methods of mixing and incubating a stool sample from a healthy individual (e.g., a selected FGT donor), a preservation buffer, and an enrichment buffer under reducing conditions (e.g., anaerobic chamber, reducing agents (e.g., formic acid, hydrogen gas, iron, lithium, lithium aluminum hydride, L-Cysteine, magnesium, oxalic acid, sodium, sodium 2-mercaptoethanesulfonic acid, sodium borohydride, sulfite compounds, zinc), or alternatively mixing and incubating a stool sample from a healthy individual (e.g., a selected FGT donor) and either a preservation buffer or an enrichment buffer, under reducing conditions (e.g., anaerobic chamber, reducing agents (e.g., formic acid, hydrogen gas, iron, lithium, lithium aluminum hydride, L-Cysteine, magnesium, oxalic acid, sodium, sodium 2-mercaptoethanesulfonic acid, sodium borohydride, sulfite compounds, zinc) to enrich foundation guild bacteria of TABLE 1, where the mixing and incubating steps can occur simultaneously or mixing occurs prior to incubating. Some embodiments are directed to a preservation buffer comprising polysaccharides (2% of total volume of preservation buffer; 0.5% or greater (e.g., 0.7, 0.9, 1.1, 1.3, 1.5, 1.7, 1.9, 2.1, 2.3, 2.5, 2.7, 2.9, 3.1, 3.3, 3.5, 3.7,25ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-0333.9, 4.1, 4.3, 4.5, 4.7, 4.9, 5.1, 5.3, 5.5, 5.7, 5.9, 6.1, 6.3, 6.5); 15% or less (e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6.8, 6.6, 6.4, 6.2, 6, 5.8, 5.6, 5.4, 5.2, 5, 4.8, 4.6, 4.4, 4.2, 4, 3.8, 3.6, 3.4, 3.2, 3, 2.8,2.6, 2.4, 2.2, 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.6, 0.4, 0.2); 0.5%-l 5% (e.g., 0.6%-14%; 0.7%-13%; 0.8%-12%; 0.9%-l 1%; l%-10.5%; 1.5%-10%; 2%-9.5%; 2.5%-9%; 3%-8.5%; 3.5%-7%; 4%-6.5%; 4.5%-6%; 5%— 5.5%) in an isotonic solvent, such as but not limited to, saline. One embodiment of the disclosure provides for a preservation buffer comprising two different polysaccharides, where they are in a 1 : 1 ratio. In additional embodiments, an enrichment buffer comprises polysaccharides (6% of total volume of enrichment buffer; 1% or greater (e.g., 1.1,I.3, 1.5, 1.7, 1.9, 2.1, 2.3, 2.5, 2.7, 2.9, 3.1, 3.3, 3.5, 3.7, 3.9, 4.1, 4.3, 4.5, 4.7, 4.9, 5.1, 5.3, 5.5, 5.7, 5.9, 6.1, 6.3, 6.5, 6.7, 6.9, 7.1, 7.3, 7.5, 7.7, 7.9, 8.1, 8.3, 8.5, 8.7, 8.9, 9.1, 9.3, 9.5,9.7, 9.9, 10.1, 20, 30, 40, 50, 55); 50% or less (e.g., 45, 35, 25, 19.8, 19.6, 19.4, 19.2, 19, 18.8, 18.6, 18.4, 18.2, 18, 17.8, 17.6, 17.4, 17.2, 17, 16.8, 16.6, 16.4, 16.2, 16, 15.8, 15.6, 15.4, 15.2, 15, 14.8, 14.6, 14.4, 14.2, 14, 13.8, 13.6, 13.4, 13.2, 13, 12.8, 12.6, 12.4, 12.2, 12, 11.8, 11.6,I I.4, 11.2, 11, 10.8, 10.6, 10.4, 10.2, 10, 9.8, 9.6, 9.4, 9.2, 9, 8.8, 8.6, 8.4, 8.2, 8, 7.8, 7.6, 7.4, 7.2, 7, 6.8, 6.6, 6.4, 6.2, 6, 5.8, 5.6, 5.4, 5.2, 5, 4.8, 4.6, 4.4, 4.2, 4, 3.8, 3.6, 3.4, 3.2, 3, 2.8, 2.6, 2.4, 2.2, 2, 1.8, 1.6, 1.4, 1.2, 1, 0.8, 0.6, 0.4); l%-50% (e.g., 1.5%-49.5%; 2%-49%; 2.5%- 48.5%; 3%-48%; 3.5%-47.5%; 4%-47%; 4.5% 46.5%; 5%-46%; 5.5%-45.5%; 6%-45%; 6.5%-44.5%; 7%-44%; 7.5%-43.5%; 8%-43%; 8.5%-42.5%; 9%-42%; 9.5%-41.5%; 10%- 41%; 10.5%-40.5%; l l%-40%; 11.5%-35.5%; 12%-35%; 12.5%-34.5%; 13%-34%; 13.5%— 33.5%) in an isotonic solvent, such as but not limited to, saline. Another embodiment of the disclosure provides for an enrichment buffer comprising three different polysaccharides, where they are in a 2: 1 : 1 ratio, or any ratio of soluble and insoluble fibers that can preserve and encourage the growth of one or more bacteria of TABLE 1. Further embodiments are directed to preservation buffers and enrichment buffers comprising at least one polysaccharide selected from the group consisting of: inulin, digestion-resistant maltodextrin (Fibersol®), and a high fiber botanical -based agent (NBT-NM108), or combinations thereof. In additional embodiments, the preservation buffer comprises inulin and digestion-resistant maltodextrin in a 1 : 1 ratio. Some embodiments are directed to enrichment buffers comprising a high fiber botanical-based agent:inulin:digestion-resistant maltodextrin in a 2: 1 : 1 ratio. In some embodiments it is understood that the polysaccharides may not solubilize completely or become homogenous as the polysaccharides include both soluble and insoluble polysaccharides. However, the buffers containing polysaccharides are mixed sufficiently to remove any aggregates or clumps. Moreover, all samples, solutions, buffers, equipment, and materials are sterilized and sterile for FGT. Non-limiting sterilization methods include26ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 autoclaving, dry heat sterilization, chemical sterilization using ethylene oxide, hydrogen peroxide, peracetic acid, etc., radiation, steam sterilization. In some embodiments, for long term storage, the enriched donor sample for transfer or SAMPLE B or SAMPLE B' can be stored in a sterile cryoprotectant at -80°C. Commonly used techniques for safely storing bacteria are used. Examples of useful cryoprotectants include, but are not limited to, DMSO, ethylene glycol, glycerin, glycerol, methanol, propylene glycol, serum, and serum albumin. For example, glycerol can be used in an amount sufficient to protect the bacteria, such as in a 1 part bacteria to 1 part cryoprotectant. Some aspects provide for glycerol to be used at 5%-50% (e.g., 10%-45%; 15%-40%; 20%-35%; 25%-30%).

[0078] Sample Screening Analysis and Review

[0079] Additional embodiments are directed to methods of analyzing the samples collected from the interested individual and processed during the in vitro fermentation or culturing steps to determine if the interested individual is approved or rejected as a potential FGT donor. This analysis serves to confirm that the interested individual’ s sample is sufficiently healthy for FGT purposes and that the foundation guild bacteria in the sample has been enriched. The samples for analysis include: the baseline sample (1) (“GUILD: QUEST”), the sample for further processing (2) (“GUILD: SAVE PLUS”), SAMPLE A (“GUILDTREDICT1”), and SAMPLE B (“GUILD PREDICT2”) and / or SAMPLE B' (“GUILD:TRANSFER”). Analyzing each of the samples from different timepoints involves DNA extraction, polymerase chain reaction (PCR) amplification, PCR purification, and nucleic acid sequencing. See, PCT / US23 / 66191 and PCT / US24 / 26282 for their teachings regarding the foundation guild and analysis, both of these publications are incorporated herein by reference in their entirety. Analysis of metagenomic datasets identified two competing guilds, the foundation guild specialized in fiber fermentation and acetate- / butyrate- production and another guild characterized by virulence and antibiotic resistance. This genome-specific, database independent, and interaction focused, identified a core microbiome signature as biomarkers for health maintenance and disease management applicable in the disclosure. See, Wu et al. Cell. 80092-8674(24)01038-9. doi: 10.1016 / j.cell.2024.09.019, incorporated by reference in its entirety and for teachings of the genomic analysis, microbiome signature as a health biomarker, and foundation guild. In some embodiments, the nucleic acid sequencing is selected from the group consisting of: 16s rRNA sequencing, internal transcribed spacer (ITS) rRNA sequencing, shotgun metagenomic sequencing, and shallow shotgun sequencing, or combinations thereof. Additional embodiments are directed to repeating the in vitro fermentation or culturing steps in order to27ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 result in sufficient levels of foundation guild and / or stable levels of foundation guild (i.e., for example, when at least one of the bacteria of TABLE 1 reaches a level of at least 1% of the total abundance of all bacteria in the sample), and an interested individual being approved as a FGT donor, or combinations thereof, where the presence of beneficial bacteria of the FG are the majority of bacteria in the sample and / or gut microbiome. Once the interested individual’s stool sample has been screened and analyzed, a determination of which interested individuals are approved or rejected as a potential FGT donor is made.

[0080] Donor Registration and Official Stool Donation for FGT

[0081] In some embodiments, a new sample from the approved FGT donor or a previously collected sample for further processing (i.e., GUILD: SAVE PLUS) sample from the approved FGT donor undergoes further in vitro fermentation or culturing using the previously mentioned in vitro fermentation or culturing steps. Briefly, immediately prior to sample collection, the approved donor is asked to complete a health questionnaire (e.g., symptoms the day of stool collection, bacterial or viral infection exposures (e.g., COVID-19), etc.). Some embodiments provide for methods of the disclosure comprising collecting a new stool sample from the approved FGT donor or using the previously collected sample for further processing (i.e., GUILD: SAVE PLUS). If collected, the method comprises preparing the stool sample into (1) a baseline sample (“GUILD: QUEST”) and (2) a sample for further processing in a preservation buffer (“GUILD: SAVE PLUS”); incubating the sample for further processing (2) in a buffer to enrich the beneficial bacteria (i.e., foundation guild bacteria) under reducing conditions, at a temperature of 4°C - 40°C, under agitation at 5 rpm-100 rpm, for 1 hour - 50 hours, forming SAMPLE A' (“GUILD :PREDICT”); incubating SAMPLE A' in an enrichment buffer to enrich the beneficial bacteria (i.e., foundation guild bacteria) under reducing conditions, at a temperature of 4°C - 40°C, under agitation at 5 rpm-100 rpm, for 1 hour - 50 hours, forming SAMPLE B' (“GUILD: TRANSFER”), wherein SAMPLE B' (“GUILD: TRANSFER”) comprises a sugar alcohol (e.g., glycerol, mannitol, sorbitol, xylitol); screening the samples for analysis: baseline sample (1) (“GUILD: QUEST”), the sample for further processing (2) (“GUILD: SAVE PLUS”), SAMPLE A' (“GUILD PREDICT”), SAMPLE B' (“GUILD: TRANSFER”) by DNA extraction, PCR, PCR purification, nucleic acid sequencing (e.g., 16s rRNA sequencing, internal transcribed spacer (ITS) rRNA sequencing, shotgun metagenomic sequencing, and shallow shotgun sequencing, or combinations thereof). The SAMPLE B' (“GUILD: TRANSFER”) can undergo additional analyses prior to FGT, including but not limited to, testing for Clostridoides difficde infection, enteropathogenic E. coli (EPEC)28ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 infection, SARS-CoV-2 infection, Shiga toxin-producing E. coli (STEC) infection. See, e.g., FIG. 2. In some embodiments, SAMPLE B or SAMPLE B' can be further analyzed to ensure that the foundation guild has been enriched or is the predominant bacteria in the sample for transplantation.

[0082] Methods for Transplantation

[0083] In some embodiments, a composition comprises the enriched donor sample for transfer or FGT (e.g., SAMPLE B, SAMPLE B') described here, as prepared by or formulated using the disclosed methods. Additional aspects provide for such compositions that are pharmaceutical compositions, further comprising a pharmaceutically acceptable vehicle (e.g., carriers, excipients, diluents). The pharmaceutically acceptable vehicle does not destroy the pharmacological activity of the disclosed sample and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the sample. The enriched donor sample and / or composition can be administered to an individual suffering from dysbiosis or in need of beneficial bacteria (e.g., foundation guild). Administration is either directly or indirectly to the gut or gastrointestinal (GI) tract, including colon, of an individual in need thereof, such as the individual suffering from dysbiosis. In another embodiment, routes of administering such compositions include, but are not limited to: colonoscopy, endoscopy, enema, enteroscopy, esophagogastroduodenoscopy, nasoduodenal catheter, nasoenteric tube, oral, sigmoidoscopy, and the like. One embodiment of the disclosure provides for a composition for FGT comprising the enriched donor sample for transfer (i.e., SAMPLE B or SAMPLE B'), where the donor sample comprises one or more bacteria of TABLE 1 that is predominant in the sample, where the composition is a solid composition or a liquid composition. In another aspect, the solid composition is selected from the group consisting of: tablet, pill, powder, granule, capsule, lozenge, semisolids, and the like. Additional aspects of the disclosure are directed to liquid compositions selected from the group consisting of: solutions, suspensions, emulsions, elixirs, syrups, and the like. In other aspects, the compositions for FGT are formulated as rectal dosage forms in the form of, but not limited to, suppositories, rectal capsules, semisolids, and liquid preparations.

[0084] In some embodiments, the transfer or transplantation of a donor microbiota modified to predominantly containing a select group of acetate and butyrate producers, known as the “Foundation Guild”, i.e., one or more bacteria of TABLE 1, supported by additional fiber as an energy source in the gut microbiota and can alleviate GI symptoms found in recipients with severe dysbiosis such as IBD patients who are resistant to any available form of therapies. In29ACTIVE 715868553v1Customer Number: 67395 Docket No. : 117465-022601 / PCT | T2025-033 additional embodiments, the enriched sample for transfer described herein, including liquid forms, suspensions, solid forms, and the like, is present in a unit dosage form. Some embodiments are directed to such compositions or unit dosage forms described here comprising the enriched donor sample for transfer, SAMPLE B or SAMPLE B' containing one or more bacteria of TABLE 1 predominant in the sample, in an amount sufficient to alleviate or treat dysbiosis, diseases associated with dysbiosis, or symptoms thereof, where the amount is 5 g or greater (e.g., 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105); 100 g or less (e.g., 98, 96, 94, 92, 90, 88, 86, 84, 82, 80, 78, 76, 74, 72, 70, 68, 66, 64, 62, 60, 58, 56, 54, 52, 50, 48, 46, 44, 42, 40, 38, 36, 34, 32, 30, 28, 26, 24, 22, 20, 18,16, 14, 12, 10, 8, 6, 4, 2); or 5g - 100g (e.g., 6g - 99g; 7g - 98g; 8g-97g; 9g-96g; 10g -95g; l lg-94g; 12g-93g; 13g-92g; 14g-91g; 15g-90g; 16g-89g; 17g-88g; 18g-87g; 19g-86g;20g-85g; 21g-84g; 22g-83g; 23g-82g; 24g-81g; 25g-80g; 26g-79g; 27g-78g; 28g-77g;29g-76g; 30g-75g; 31g-74g; 32g-73g; 33g-72g; 34g-71g; 35g-70g; 36g-69g; 37g-68g;38g-67g; 39g-66g; 40g-65g; 41g-64g; 42g-63g; 43g-62g; 44g-61g; 45g-60g; 46g-59g;47g-58g; 48g-57g; 49g-56g; 50g-55g; 51g-54g; 52g-53g).

[0085] In some embodiments, an effective amount of the FGT compositions or unit dosage forms described here can comprise 105colony forming units (CFU) or greater (e.g., 107, 109, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031); 1042or less (e.g., IO40, 1038, 1036, 1034, 1034, 1032, IO30, 1028, 1026, 1024, 1022, IO20, 1018, 1016, 1014, 1012, 1010, 108, 106, 104, 103); 105- 1042(e.g., 106- 1041; 107- IO40; 108- 1039; 109- 1038; IO10- 1037; 1011- 1036; 1012- 1035; 1013- 1034; 1014- 1033; 1015- 1032; 1016- 1031; 1017- IO30; 1018- 1029; 1019- 1028; IO20- 1027; 1021- 1026; 1022- 1025; 1023- 1024). In some embodiments, the FGT composition or unit dosage form can be administered daily, every other day, every two days, every three days, every four days, every five days, every six days, weekly, every other week, every two weeks, every three weeks, monthly, or as needed, where the FGT composition or unit dosage form can be administered once, twice, or three or more times, or as needed.

[0086] Further embodiments are directed to FGT maintenance using foundation guild nutrition (“FGN”), where after FGT or administration of the enriched donor sample, SAMPLE B, SAMPLE B', or composition or unit dosage form comprising any of the samples thereof, to a subject in need thereof, the method comprises further administering to the subject a supplement. FGN comprises beneficial nutrients that maintain dominance of the foundation guild in the gut of the recipient subject while preventing pathogenic bacteria from overtaking30ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 the beneficial foundation guild. Accordingly, the FGN provides optimal substrates and environmental conditions for the transplanted foundation guild to flourish. In some embodiments, the FGN can be personalized to the recipient subject’s gut microbiome. Exemplary FGN or fiber mix formulation is found in PCT / US2021 / 026258, which is incorporated herein by reference in its entirety and for its teachings regarding the FGN or fiber mix formulation that promotes the growth of the acetate- and butyrate-producing bacteria and inhibits the growth of pathogens and detrimental bacteria. Various embodiments of the disclosure include the administration of the fiber mix formulation a mixture comprising bran, soluble polysaccharides, and insoluble polysaccharides. In some embodiments, the fiber mix formulation comprises dietary fibers from bran, dietary fibers from inulin, and dietary fibers from digestion resistant maltodextrin. In some embodiments, after FGT described here, the method further comprises administering to the subject in need thereof, an effective amount of the FGN or fiber mix formulation comprising a mixture of bran, inulin, and digestion resistant maltodextrin, to promote a healthy gut microbiome in the subject, such that the beneficial acetate-producing and butyrate-producing gut bacteria sufficiently proliferate to be predominant in the gastrointestinal tract of the subject, thereby maintaining and promoting a healthy gut microbiome. In some embodiments, the FGN or fiber mix formulation comprises a mixture of bran (e.g., corn bran, wheat bran, sorghum bran, oat bran), inulin, and digestionresistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria of TABLE 1. Additional aspects provide for the mixture of the FGN described here that 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. In some aspects of the FGN, the inulin is present in a range of 5-10% of total weight of the mixture. Further aspects of the FGN described relate to the mixture having a uniform particle size between 150 pm and 300 pm. Additional aspects provide for a combination therapy utilizing the methods described here in the preparation of a donor sample for FGT and FGT into a subject suffering from dysbiosis, followed by administration of the FGN described here (see also PCT / US2021 / 026258, which is incorporated by reference). In some aspects, the FGN comprises a high fiber botanical -based agent (NBT-NM108), where the subject having received the FGT is administered (e.g., orally, by colonoscopy, by enema) the FGN as frequently as needed to maintain the beneficial FG gut microbiome.

[0087] Some embodiments of the disclosure are directed to a method, comprising: (a) screening a healthy individual interested in being a potential microbiome donor; (b) culturing31ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 a stool sample from the healthy individual in accordance with in vitro fermentation or culturing described here, where the stool sample comprises one or more bacteria of TABLE 1; (c) transferring the enriched donor sample, SAMPLE B or SAMPLE B', into a recipient subject in need thereof (for example, the recipient subject suffers from dysbiosis and / or a dysbiosis- related disease or symptoms thereof); and (d) administering a fiber mix formulation to the recipient subject. Such method treats the recipient subject suffering from dysbiosis and / or a dysbiosis-related disease, or alleviates or reduces symptoms of the dysbiosis-related disease.

[0088] Data and results from EXAMPLES 1-3 described the methods for conducting Foundation Guild Transplantation as described here, which is a novel method for enriching the key beneficial Foundation Guild bacteria through a series of in vitro fermentation transfer steps (see, e.g., FIG. 26). The end products can be delivered, for example, via colonoscopy to transfer those enriched bacteria to chronically diseased patients with gut dysbiosis, or L-Type Dysbiosis.

[0089] In various embodiments described throughout the disclosure, compared to conventional fecal microbiota transplantation (FMT), Foundation Guild Transplantation (FGT) utilizes a fiber-mix growth media (see, e.g., U.S. Pub. No. 2023 / 0142707, incorporated by reference here) to selectively enrich the Foundation Guild bacteria in the donor sample. The methods described here lower the number of opportunistic pathogens in the transplant material, improving the overall efficacy of the transplantation while reducing risks from potential infections.

[0090] The ability of the GS205 buffer in the Guild: Save sample preservation kit to collect, preserve and enrich the key butyric acid-producing Foundation Guild bacteria, such as bacteria from the Faecalibacterium genus was tested (see, e.g., EXAMPLES 4-6). For many donors, key Foundation Guild bacteria strains, especially butyric acid-producing gut bacteria from the Faecalibacterium genus, may be present in the donor stool samples, but at a very low level. These bacteria serve critical ecological functions in the gut microbiota by producing butyric acid and other short-chain fatty acids that can inhibit the growth or directly kill the pathobiont guild members. However, their growth requires the blend of dietary fibers in large quantities. See, e.g., U.S. Pub. No. 2023 / 0142707, which is incorporated by reference in its entirety. These nutrients are severely lacking in the modern-day western-style diet. Therefore, many stool samples from the healthy donors may only contain small amounts of these beneficial Foundation Guild bacteria. When beneficial Foundation Guild bacteria grow, they produce short-chain fatty acids (“SCFAs”) like acetic and butyric acid as byproducts of the digestion32ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 and fermentation of complex carbohydrates. These SCFAs will reduce the pH of the surrounding environment, inhibiting the growth or directly killing opportunistic pathogens in the pathobiont guild. By measuring pH levels in donor stool samples enriched with GS205, fermentation occurring within the sample can be determined, which indicates growth of the Foundation Guild bacteria. Example 4 and FIG. 24 demonstrate that pH data from the donor samples showed a reduction of pH levels after incubation in the Guild: Save GS205 buffer.

[0091] In patients or subjects with severe L-type dysbiosis, such as but not limited to those with severe-stage inflammatory bowel disease (IBD), the gut environment is hostile to healthy, beneficial gut bacteria, specifically obligatory anaerobes including members from the genus Faecalibacterium, due to the high levels of free radicals and oxidative stress. This type of environment would significantly reduce the Foundation Guild bacteria’s transplant efficiency to a patient’s gut, as the newly introduced obligatory anaerobic Foundation Guild members would be destroyed immediately. In some embodiments as described, Foundation Guild Transplantation was developed and it selects and enriches beneficial Foundation Guild members that are not as sensitive to oxidative stress, such as, bacteria from genera like Bifidobacterium or Lactobacillus. These beneficial bacteria serve as the “pioneer species” for colonizing the gut of a patient with severe L-type dysbiosis (e.g., severe-IBD) via transplantation to pave the way for subsequent Foundation Guild transplants with bacteria that are more sensitive to oxidative stress. By first transplanting the pioneer species, they will tolerate the intense initial oxidative stress inside the patient’s gut and modulate the gut environment so that it becomes more suitable for the other, more oxygen-sensitive Foundation Guild bacteria to survive in the second transplantation. See, e.g., EXAMPLE 5.

[0092] Demonstrated Advantages over Conventional FMT

[0093] Compared to the conventional Fecal Microbiota Transplantation (FMT), Foundation Guild Transplantation (FGT) offers several advantages. For example, the serial culturing and enrichment of the Foundation Guild bacteria using proprietary buffers, fiber-mix substrates, and culturing techniques removes the dangerous pathogens that can cause infections in patients, especially patients with compromised immune systems. Additionally, by enriching beneficial Foundation Guild bacteria, it eliminates the randomness of the gut microbiome membership from the original donor’s fecal sample through selective enrichment of a key group of shortchain fatty acid (SCFA)-producing gut bacteria. This process makes FGT more effective at inhibiting and removing the pathogens inside the patient’s gut, thus making the clinical improvements more reproducible, and enables the production of more transplant material from33ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 the same amount of donor fecal samples, thereby reducing manufacturing cost significantly. See, e.g., Example 6, FIGs. 29-30. The FGT protocol also has a high translational readiness. For example, the protocol described here is ready for clinical and commercial purposes to produce FGT transplant materials that are ready for patients.

[0094] The data presented in Examples 4-6 support the embodiments of the methods described here. Specifically, the embodiments of the described methods: (a) enable selective preservation and amplification of Foundation Guild organisms from donor sources; (b) establish a two-stage ecological restoration process through pioneer species followed by strict- anaerobic colonization; and (c) produce standardized, pathogen-free, functionally potent transplant compositions.

[0095] In some embodiments, the present disclosure provides Foundation Guild Transplantation (FGT), a selective-enrichment and transplantation process for restoring beneficial gut microbial consortia. FGT employs a fiber-based growth medium to selectively amplify Foundation Guild (FG) bacteria that produce short-chain fatty acids such as butyrate, thereby suppressing opportunistic pathogens. Accordingly, the Foundation Guild Transplantation (FGT) technology of the disclosure provides a reproducible and mechanistically defined means for restoring gut-ecosystem balance between Foundation Guild and Pathobiont Guild microorganisms, representing a significant advance over conventional fecal microbiota transplantation (FMT).

[0096] 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 in connection with the various embodiments of the disclosure is intended to be illustrative, and not restrictive.EXAMPLES

[0097] The following examples illustrate specific aspects of the instant description. The examples should not be construed as limiting, as the example merely provides specific understanding and practice of the embodiments and its various aspects.

[0098] Example 1 : Exemplary Project 1

[0099] In accordance to FIG. 2, samples were collected from two donors, Donor A and Donor B, as exemplified in TABLE 2, which shows the sample collection schedule, the number of samples collected at each timepoint, as well as the type of tube it was collected in (Project 1). In Project 1, Donor A is female, and Donor B is male.34ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0100] TABLE 2

[0101] Individual sample Bray-Curtis Principal Coordinates Analysis (“PCoA”) plots were used to compare microbial communities for the two donors of Project 1. See, FIG. 3; FIG. 4 (TABLE 3). TABLE 3 demonstrated the consistency of top amplicon sequence variants (ASVs) in repeat samples for Donors A and B at T3 (48hr + 48hr). TABLE 3 is divided into two panels, one for each donor, which demonstrated the consistency of the top amplicon sequence variants (ASVs) across three repeat samples taken at timepoint T3. Each panel corresponds to a donor (A and B) and includes three columns, each representing a different repeat sample (e.g., Donor A Project 1 T3 Sample Repeat 1, Repeat 2, and Repeat 3). Within each column, the ASVs are listed by their relative abundance in descending order, showcasing which ASVs dominated the samples. The purpose of this arrangement was to highlight the reproducibility of microbial community assessments, as evidenced by the similarity in the top ASVs identified in each repeat. By comparing these lists side by side, the table effectively illustrated that despite the inherent variability in biological sampling and sequencing, the top ASVs remained remarkably consistent across the repeat samples for each donor at the T3 timepoint, reinforcing the reliability of the microbial community data obtained.

[0102] FIG. 5 (TABLE 4) showed the consistency of the top amplicon sequence variants (ASVs) across three repeat samples taken at timepoint T6 during Project 1. The three columns each represented a different repeat sample (e.g., Donor A Project 1 T6 Sample Repeat 1, Repeat 2, and Repeat 3, left to right). Within each column, the ASVs are listed by their relative35ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 abundance in descending order, showcasing which AS Vs dominated the samples. FIG. 6 (TABLE 5) demonstrates the consistency of the top amplicon sequence variants (ASVs) across three repeat samples taken at timepoint T6 during Project 1. The three columns each represented a different repeat sample (e.g., Donor B Project 1 T6 Sample Repeat 1, Repeat 2, and Repeat 3). Within each column, the ASVs are listed by their relative abundance in descending order, showcasing which ASVs dominate the samples. FIGs. 7A-7B (TABLE 6) present a comparative analysis of the relative abundance of top amplicon sequence variants (ASVs) in the microbiome sample of Donor A at the last timepoint, T7, in Project 1. The table is organized with two main columns: the first showing the relative abundance of each ASV at baseline, and the second displaying its abundance at T7. The ASVs are listed in descending order based on their abundance at T7, highlighting the significant shifts in microbial presence over the sampling period. The top ASVs, such as Lactobacillus ASV03YU, Lactobacillus fermentum ASV005K, Weissella ASV05PN, Lactobacillus ASV0W23, Megasphaera ASVODGN, Bifidobacterium ASV0004, Blautia ASV000H, Bifidobacterium ASV000J, Leuconostoc ASV07UY, and Lactobacillus ASV0W22, are notable for their marked increase from very low levels at baseline to higher relative abundance at T7. Similarly, FIGs. 8A-8B (TABLE 7) present a comparative analysis of the relative abundance of top amplicon sequence variants (ASVs) in the microbiome sample of Donor B at the last timepoint, T7, in Project 1. The table is organized with two main columns: the first showing the relative abundance of each ASV at baseline, and the second displaying its abundance at T7. The ASVs are listed in descending order based on their abundance at T7, highlighting the significant shifts in microbial presence over the sampling period. The top ASVs, such as Lactobacillus ASV030T, Lactobacillus brevis ASV0FD6, Lactobacillus ASV0W2E, Lactobacillus ASV0W2L, Lactobacillus casei ASV0W24, Lactobacillus ASV0W1X, Lactobacillus delbrueckii ASV005Z, Lactobacillus ASV03YU, are notable for their marked increase from very low levels at baseline to higher relative abundance at T7.

[0103] Example 2: Exemplary Project 2

[0104] In accordance to the flowchart of FIG. 9, samples were collected from two donors, Donor A (male) and Donor B (female). The sample collection schedule, the number of samples collected at each timepoint, as well as the type of tube it was collected in Project 2 were described.

[0105] Individual sample Bray-Curtis PCoA plots were used to compare microbial community compositions for different samples from Donor A, i.e., Transfer and Predict, of36ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033Project 2. See, FIG. 10. Individual sample Bray-Curtis PCoA plots were used to compare microbial community compositions for Donor B, i.e., Transfer and Predict, of Project 2. See, FIG. 11. TABLE 7 (see, FIG. 12) details the relative abundance of the top amplicon sequence variants (AS Vs) in samples from Donor A. The table was organized to reflect changes across multiple timepoints. The AS Vs are ranked based on their average relative abundance in the T6 transfer sample, listed in descending order. The table features several columns, each representing a different timepoint: baseline, T2, T3, T4 predict, T4 transfer, T5 predict, T5 transfer, T6 predict, and finally, T6 transfer as presented in FIG. 9 (Project 2). Each column shows the average relative abundance of the AS Vs at the respective timepoint, allowing for a direct comparison of microbial dynamics from the beginning of the sampling period through to T6. TABLE 8 (see, FIG. 13) details the relative abundance of the top amplicon sequence variants (AS Vs) in samples from Donor B. The table was organized to reflect changes across multiple timepoints. The AS Vs are ranked based on their average relative abundance in the T6 transfer sample, listed in descending order. The table features several columns, each representing a different timepoint: baseline, T2, T3, T4 predict, T4 transfer, T5 predict, T5 transfer, T6 predict, and finally, T6 transfer as presented in FIG. 9 (Project 2). Each column shows the average relative abundance of the AS Vs at the respective timepoint, allowing for a direct comparison of microbial dynamics from the beginning of the sampling period through to T6.

[0106] Example 3: Exemplary Project 3

[0107] In accordance to the flowchart of FIG. 14, samples were collected from two donors, Donor A (male) and Donor B (female). The sample collection schedule, the number of samples collected at each timepoint, as well as the type of tube it was collected in Project 3 were described.

[0108] Beta diversity PCoA plots comparing microbial communities of two donors across four timepoints are presented in FIGs. 15A-15D. The timepoint scheme allowed for an immediate visual assessment of changes and similarities in the microbial communities over time and between the different computational approaches to measuring diversity. FIGs. 15A- 15D provided insights into the temporal dynamics and diversity patterns shaped by the selected distance metrics. Alpha diversity metrics from Donor A at four distinct timepoints (i.e., baseline, T2, T3, and T4) are shown in FIGs. 16A-16D. Notably, the boxplots for T3 and T4 showed markedly lower diversity scores across all metrics compared to those observed at baseline and T2. This pattern suggested a significant reduction in microbial diversity in the37ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 later stages of sampling. Alpha diversity metrics from Donor B at four distinct timepoints (i.e., baseline, T2, T3, and T4) are shown in FIGs. 17A-17D. Notably, the boxplots for T4 showed markedly lower diversity scores across all metrics compared to those observed at baseline, T2, and T3. This pattern suggested a significant reduction in microbial diversity in the later stages of sampling. FIG. 18 presents TABLE 9, which details the shifts in relative abundance of two distinct groups of amplicon sequence variants (ASVs) within Donor A over the course of the study, presented across four time points: baseline, T2, T3, and T4. The first row highlighted a group that has been enriched during the study period, showing a dramatic increase from a mere 0.2% at baseline to an overwhelming 99.4% by T4 (Enriched Guild). Conversely, the second row tracked a group of ASVs that had experienced a substantial reduction, decreasing from 63.2% at baseline to just 0.6% at T4 (Reduced ASV). TABLE 9 effectively captured the significant transformations within the microbial community of Donor A in Project 3, illustrating a clear shift from one dominant group to another over time. FIG. 19 presents a graphical representation of TABLE 9. FIG. 20 presents TABLE 10, which details the shifts in relative abundance of two distinct groups of ASVs within Donor B over the course of the study, presented across four time points: baseline, T2, T3, and T4. The first row highlights a group that has been enriched during the study period, showing a dramatic increase from a mere 0.0% at baseline to an overwhelming 98.6% by T4 (Enriched Guild). Conversely, the second row tracks a group of ASVs that experienced a substantial reduction, decreasing from 86.5% at baseline to just 1.4% at T4 (Reduced ASV). This table effectively captures the significant transformations within the microbial community of Donor B. FIG. 21 presents a graphical representation of TABLE 10. FIG. 22 presents TABLE 11, which shows the consistency of top ASVs in repeat samples for Donor A (left) and Donor B (right) in Project 3. TABLE 11 is divided into two panels, one for each donor, to demonstrate the consistency of the top ASVs across three repeat samples taken at timepoint T4 during Project 3. Each panel corresponded to a donor (A and B) and included three columns, each representing a different repeat sample (e.g., Donor A Project 3 T4 Sample Repeat 1, Repeat 2, and Repeat 3). Within each column, the ASVs are listed by their relative abundance in descending order, showcasing which ASVs dominated the samples. The purpose of this arrangement was to highlight the reproducibility of microbial community assessments, as evidenced by the similarity in the top ASVs identified in each repeat. By comparing these lists side by side, the table effectively illustrated that despite the inherent variability in biological sampling and sequencing, the top ASVs remained remarkably consistent across the repeat samples for each donor at the T4 timepoint, reinforcing the reliability of the microbial community data obtained.38ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0109] Example 4: Exemplary Project 4

[0110] Enrichment of Foundation Guild Bacteria in Donor Fecal Material[OHl] A kit containing buffers for sample preservation (e.g., Guild: Save sample preservation kit, GS205 buffer) was developed to preserve and enrich beneficial Foundation Guild bacteria. The dietary fibers (inulin and fibersol-2) in the GS205 buffer provided a source of complex carbohydrates for the Foundation Guild bacteria to grow.

[0112] Fecal samples from two donors that had a low-abundance of butyric-acid-producing Foundation Guild microorganisms were tested. Fecal samples from the two donors were collected in the fiber-containing preservation medium and tested. From each donor, three fecal samples were collected using the Guild:Quest fixation buffer kits to establish the baseline for both donors (timepoint 1), and six fecal samples from each donor were also collected using the Guild: Save preservation kits. The pH and 16S results were compared to see if the GS205 buffer in the Guild: Save kit could indeed preserve and enrich key butyric-producing Foundation Guild bacteria.

[0113] Materials and Methods

[0114] Each Guild:Quest kit contained about 1 g of fecal sample suspended in 7 ml of ethanol, and each Guild: Save kit contained about 1 g of fecal sample suspended in 8ml of GS205 buffer. The samples were left in the Guild: Save buffer tubes to incubate for 96 hours total (timepoints 2-5). The first 48 hours were incubated at room temperature, at around 20°C to mimic shipping times in the real world. The second 48 hours were incubated at 37°C to test the viability of the buffer under accelerated growth conditions. Two 1 ml aliquots were taken from each sample every 24 hours to be tested for pH and DNA sequencing. See, FIG. 23 of a graph of the overall design of Project 4; FIG. 24 pH change over time of Donor A and Donor B tested samples.

[0115] Briefly, the sample mixture was incubated for approximately 96 hours (48 h at 20 °C followed by 48 h at 37 °C). Sequential sampling demonstrated acidification and enrichment of Faecalibacterium ASVs 000Y and 001E, that increased from 0.27 % and 2.37 % to 2.8 % and 8.8 %. The GS205 formulation therefore preserved and selectively amplified Foundation Guild bacteria within donor material prior to further fermentation.

[0116] Results (pH / 16S / SCFA Data)39ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0117] The 16S sequencing data (FIG. 25) show the relative abundance of the top 10 amplicon sequence variants (AS Vs) in each time point. The results demonstrated that the GS205 buffer in the Guild: Save kit could preserve and enrich important beneficial Foundation Guild bacteria. For example, for Donor-A at baseline time point 1, Faecalibacterium ASV000Y and ASV001E only had an average abundance of 0.27% and 2.37%, respectively, inside the samples. After 24 hours of incubating at room temperature, their average abundance levels increased to 2.8% and 8.81%, respectively. The average abundance levels for these bacteria remained stable through time points 2-5, indicating the buffer’s ability to preserve these beneficial Foundation Guild bacteria, in addition to enrich their abundance levels inside the stool sample. Results from donor B also showed similar preservation and enrichment of the key Foundation Guild bacteria.

[0118] In summary, Example 4 demonstrated that the Guild: Save GS205 fiber medium preserved and enriched butyrate-producing Foundation Guild (FG) organisms (e.g., Faecalibacterium ASVs 000Y, 001E). A pH decrease (from -pH 6.8 to ~ pH 5.5) confirmed active short-chain fatty acid fermentation during incubation. Example 4 also demonstrated that the relative abundance of FG bacteria increased 5- to 10-fold compared to ethanol control samples.

[0119] Example 5: Exemplary Project 5

[0120] Oxidative-Tolerant Pioneer Species for Severe L-Type Dysbiosis

[0121] Experimental Design

[0122] Previously frozen samples from previously made FGT samples, which had already gone through 48 hours of incubation at room temperature and another 48 hours of incubation at 37°C in the Guild:Save buffer (S-00182601), or had already gone through the Guild:Save incubation steps and 24 hours incubation at 37°C in the Guild:Predict GS108 buffer (S- 00182597) were used. These samples were used and processed through the entire FGT protocol as described (FIG. 26; overall workflow chart below using the S-00182601 sample). DNA of aliquoted samples at every step of the FGT serial enrichment protocol were obtained. To analyze the microbial makeup of the Foundation Guild bacteria 16S sequencing was performed (FIGs. 27A-27B) as well as anaerobic and aerobic colony plate count using GAM and LB agar plates. See, FIG. 28.

[0123] Specifically, oxidative-tolerant Foundation Guild members (Bifidobacterium, Lactobacillus') were enriched from previously fermented samples (S-00182601 and S-40ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03300182597) using GS205 and GS108 media followed by Guild:Transfer cultivation. Metagenomic profiling revealed dominance of these pioneer species, whose abundances remained stable during serial transfer. Anaerobic plating yielded viable counts of approximately 107CFU / mL, demonstrating metabolic activity and stability of the pioneer consortium. This phase preconditioned the intestinal environment for subsequent colonization by strict-anaerobic FG organisms.

[0124] Microbial Community Dynamics

[0125] In the 16S sequencing data (FIGs. 27A-27B), Bars 1-4 represent samples enriched from S-00182601 after 24 hours in Guild:Predict buffer (bar 1), and after another 24 hours in Guild:Transfer buffer (bars 2-4). Bars 5-11 show the results from samples enriched from S- 00182597. This data demonstrated that after in vitro fermentation using the Foundation Guild Transplantation protocol, the samples were predominately dominated by beneficial Foundation Guild bacteria members from the Lactobacillus and Bifidobacterium genera. The membership of the bacteria also remained stable throughout the fermentation process.

[0126] Viability and Stability Data

[0127] The bacterial colony plate counts of FIG. 28 demonstrated that the bacteria remaining in the transplant materials remained viable and were able to grow after the FGT’s serial enrichment protocol. The samples were spread on GAM plates that either contained or did not contain additional soluble fibers (inulin and fibersol-2 at 1% w / v). After 72 hours of incubation in the anaerobic chambers, the colonies that formed on the GAM plates were counted and the concentration was calculated. The Foundation Guild Transplant material contained 107CFU / ml cells in the transplant materials produced from the FGT protocol, indicating adequate viability of the beneficial Foundation Guild (FG) bacteria.

[0128] In summary, Example 5 identified Bifidobacterium and Lactobacillus as oxidativeresistant FG members capable of serving as pioneer species in severely dysbiotic (L-type) environments. Also, serial enrichment maintained stable community structure and viability 107CFU / mL. Example 5 also established a two-phase ecological restoration: pioneer inoculation strict-anaerobe recolonization.

[0129] Example 6: Quality Assurance / Quality Control (QA / QC) and Safety Validation

[0130] Analytical Tests and Acceptance Criteria41ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033

[0131] For QA / QC of the Foundation Guild Transplant material, aliquots at every time point of the FGT protocol were collected to ensure each transplant material contained a high count of viable live Foundation Guild bacterial cells that: (1) Could not induce disease or did not contain any antibiotic resistance. Specifically, the Foundation Guild bacteria remaining in the transplant material did not contain any antibiotic resistance genes (ARG) and virulence factor genes (VF) except genes related to Immune Modulation (see, e.g., Zhao et al. Cell 187: 1-16, 2024, which is incorporated by reference in its entirety, where Figure S7A highlights the number of VF genes in the Foundation Guild bacteria group CIA from the QD-TCG dataset) below highlighting the Foundation Guild bacteria group CIA). This additional requirement further ensured that the Foundation Guild bacteria remaining in the transplant material was not capable of inducing any diseases inside the patient’s gut. (2) They were highly effective at producing short-chain fatty acids, such as acetic or butyric acid, which lowered the pH of the environment. The transplant material did not have any pathogens that could cause severe diseases, according to the FDA.

[0132] Quality-Control and Safety Testing of FGT Compositions

[0133] Each batch of FGT composition was subjected to the following analyses:

[0134] Viability: > 106CFU / mL on GAM agar ± 1 % fiber after 72 h anaerobic culture.

[0135] pH: 5.0-6.5.

[0136] Short-Chain Fatty Acids: acetate and butyrate production confirmed by gas chromatography.

[0137] Genomic Verification: Presence of target FG genomes as defined in Cell 2024.

[0138] Pathogen Exclusion: Negative for C. difficile, enteric bacteria (Salmonella, Shigella, Campylobacter, Vibrio, Listeria, E. coli 0157 / STEC / EPEC), multi-drug-resistant organisms (MRSA, VRE, CRE, ESBL), viral agents (Adenovirus, Norovirus, Rotavirus, SARS-CoV-2, Monkeypox), and protozoa (Giardia, Cryptosporidium, Isospora, Cyclospora, Microsporidia).

[0139] Genetic Safety: Absence of antibiotic-resistance and virulence-factor genes.

[0140] Only those compositions that met all criteria were released for clinical administration. See, FIG. 29

[0141] In summary, Example 6 defined standardized analytical specifications: (i) Viability106CFU / mL; (ii) pH 5.0-6.5; (iii) Negative for all FDA-listed stool pathogens; (iv) No antibiotic-resistance or virulence genes. Example 6 also confirmed the readiness for Good42ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 manufacturing practice (GMP) manufacturing and investigational new drug (IND)-level documentation.

[0142] Overall Findings and Significance

[0143] In the summary depicted in FIG. 29, it was demonstrated by 16S sequencing, live colony counts, and pH reading data as described here (Examples 4-6) that the Foundation Guild Transplantation protocol and buffers: (1) preserved and enriched the butyric-producing Foundation Guild bacteria from healthy donors’ fecal samples, and (2) enriched the abundance of Foundation Guild bacteria that belonged to the more oxygen-tolerating genera, including Bifidobacterium and Lactobacillus.

[0144] The data described throughout the disclosure demonstrated that Foundation Guild Transplantation (FGT) provided targeted, fiber-guided enrichment of beneficial FG consortia; implemented a two-stage ecological restoration overcoming oxidative barriers in severe dysbiosis; and produced standardized, pathogen-free, functionally potent live biotherapeutic compositions. Together, these results substantiated the novelty, reproducibility, and translational applicability of FGT beyond conventional fecal microbiota transplantation (FMT).

[0145] Experimental data demonstrated that donor materials preserved in Guild: Save GS205 buffer exhibited a five- to ten-fold increase in Faecalibacterium abundance and reduced pH due to active fermentation. A complementary pioneer-species phase using oxidative-tolerant FG members (Bifidobacterium, Lactobacillus') enabled colonization of severely dysbiotic intestines prior to strict-anaerobic FG engraftment. Quality-control analysis confirmed viability, 20 mM, and absence of pathogens or antibiotic-resistance genes. FGT as described here provided a reproducible, safe, and mechanistically defined method for ecological restoration of the gut microbiome, which is an advancement over conventional fecal microbiota transplantation (FMT).SPECIFIC EMBODIMENTS

[0146] Non-limiting specific embodiments are described below each of which is considered to be within the present disclosure.

[0147] Embodiment 1. A method, comprising:(a) mixing a stool sample from a healthy individual and a preservation buffer;43ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 wherein the preservation buffer comprises polysaccharides and an isotonic solvent (e.g., bicarbonate buffer, citrate buffer, PBS buffer (or capsules), saline, Ringer’s solution, lactated Ringer’s solution, dextrose in water), wherein the polysaccharides of the preservation buffer are in an amount of 1% of the total volume of the preservation buffer to 15% of the total volume of the preservation buffer, wherein at least one of the polysaccharides is selected from the group consisting of: inulin and digestionresistant maltodextrin, or any combination of oligosaccharides or polysaccharides that can preserve and encourage the growth of one or more bacteria of TABLE 1;(b) incubating the mixed sample (a) and an enrichment buffer under reducing conditions (e.g., anaerobic chamber, reducing agents (e.g., formic acid, hydrogen gas, iron, lithium, lithium aluminum hydride, L-Cysteine, magnesium, oxalic acid, sodium, sodium 2-mercaptoethanesulfonic acid, sodium borohydride, sulfite compounds, zinc), forming SAMPLE A; wherein the enrichment buffer comprises polysaccharides and an isotonic solvent (e.g., bicarbonate buffer, citrate buffer, PBS buffer (or capsules), saline, Ringer’s solution, lactated Ringer’s solution, dextrose in water), wherein the polysaccharides of the enrichment buffer are in an amount of 3% of the total volume of the enrichment buffer - 50% of the total volume enrichment buffer, wherein at least one of the polysaccharides of the enrichment buffer comprises high fiber botanical -based agent that can preserve and encourage growth of one or more bacteria of TABLE 1,(c) adding the enrichment buffer to SAMPLE A, forming enriched SAMPLE A; and44ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033(d) incubating enriched SAMPLE A under reducing conditions (e.g., anaerobic chamber, reducing agents (e.g., formic acid, hydrogen gas, iron, lithium, lithium aluminum hydride, L-Cysteine, magnesium, oxalic acid, sodium, sodium 2- mercaptoethanesulfonic acid, sodium borohydride, sulfite compounds, zinc), forming SAMPLE B, wherein SAMPLE B comprises an enriched foundation guild of one or more bacteria of TABLE 1.

[0148] Embodiment 2. The method of embodiment 1, further comprising repeating steps (b)-(d) (forming SAMPLE A' and SAMPLE B').

[0149] Embodiment 3. The method of embodiment 1, wherein the preservation buffer comprises polysaccharides in an amount of 2% of the total volume of the preservation buffer.

[0150] Embodiment 4. The method of embodiment 3, wherein the polysaccharides comprise inulin and digestion-resistant maltodextrin, or any combination of polysaccharides that can preserve and encourage growth of one or more bacteria of TABLE 1.

[0151] Embodiment 5. The method of embodiment 4, wherein inulin and digestionresistant maltodextrin are in a 1 : 1 ratio.

[0152] Embodiment 6. The method of embodiment 1, wherein the enrichment buffer comprises polysaccharides in an amount of 6% of the total volume of the enrichment buffer.

[0153] Embodiment 7. The method of embodiment 6, wherein the polysaccharides comprise a high fiber botanical -based agent (NBT-NM108), inulin, and digestion-resistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria of TABLE 1.

[0154] Embodiment 8. The method of embodiment 7, wherein the high fiber botanicalbased agent, inulin, and digestion-resistant maltodextrin are in a 2: 1 : 1 ratio, or any ratio of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria of TABLE 1.

[0155] Embodiment 9. The method of embodiment 1, further comprising administering SAMPLE B to a subject suffering from dysbiosis.

[0156] Embodiment 10. The method of embodiment 9, wherein administering is through a route selected from the group consisting of: colonoscopy, endoscopy, enema, enteroscopy,45ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 esophagogastroduodenoscopy, nasoduodenal catheter, nasoenteric tube, oral, and sigmoidoscopy.

[0157] Embodiment 11. The method of embodiment 9, further administering a fiber mix formulation to the subject suffering from dysbiosis.

[0158] Embodiment 12. The method of embodiment 11, wherein the fiber mix formulation comprises a mixture of bran, inulin, and digestion-resistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria of TABLE 1.

[0159] Embodiment 13. A method, comprising:(a) screening a healthy individual interested in being a potential microbiome donor;(b) culturing a stool sample from the healthy individual in accordance with embodiment 1, wherein the stool sample comprises one or more bacteria of TABLE 1;(c) transferring SAMPLE B (or SAMPLE B') into a recipient subject in need thereof; and(d) administering a fiber mix formulation to the recipient subject.

[0160] Embodiment 14. The method of embodiment 13, wherein the screening comprises: health screening, medical screening, blood screening, and stool screening.

[0161] Embodiment 15. The method of embodiment 13, wherein the transferring occurs through a route selected from the group consisting of: colonoscopy, endoscopy, enema, enteroscopy, esophagogastroduodenoscopy, nasoduodenal catheter, nasoenteric tube, oral, and sigmoidoscopy.

[0162] Embodiment 16. The method of embodiment 13, wherein the recipient subject in need thereof suffers from dysbiosis.

[0163] Embodiment 17. The method of embodiment 13, wherein the recipient subject suffers from a dysbiosis-related disease.

[0164] Embodiment 18. The method of embodiment 17, wherein the dysbiosis-related disease is selected from the group consisting of: cancer (e.g., breast cancer, colon / colorectal cancer, hepatocellular carcinoma, esophageal cancer, gallbladder carcinomas, gastric cancer, laryngeal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, stomach cancer); gastrointestinal diseases (e.g., H. pylori and C. difficile infections, inflammatory bowel disease46ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033(IBD) (e.g., Crohn’s disease, ulcerative colitis); gastrointestinal issues (e.g., diarrhea, constipation, gas, acid reflux); small intestinal bacterial overgrowth (“SIBO”); metabolic diseases (e.g., Type 1 diabetes (i.e., insulin-dependent diabetes mellitus) and Type 2 diabetes (i.e., non-insulin-dependent diabetes mellitus)); inflammatory diseases (e.g., rheumatoid arthritis, asthma, non-alcoholic fatty liver disease (“NAFLD”)); obesity; autism spectrum disorders; mental health diseases (e.g., Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy, dementia, anxiety, attention deficit hyperactivity disorder (“ADHD”), depression, bipolar disorder, schizophrenia, obsessive-compulsive disorder (“OCD”), post- traumatic stress disorder (“PTSD”)).

[0165] Embodiment 19. The method of embodiment 13, wherein the fiber mix formulation comprises a mixture of bran, inulin, and digestion-resistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria listed in TABLE 1.

[0166] Embodiment 20. The method of embodiment 13, wherein the administering occurs at least once daily.

[0167] Embodiment 21. The method of embodiment 17, wherein the method treats the dysbiosis-related disease of the recipient subject.

[0168] Embodiment 22. The method of embodiment 13, wherein the method treats the recipient subject suffering from a dysbiosis-related disease, or alleviates or reduces symptoms of the dysbiosis-related disease.

[0169] As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present disclosure, it is intended that all subject matter contained in the above description, or defined in the appended embodiments, be interpreted as descriptive and illustrative of the present disclosure. Many modifications and variations of the present disclosure are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications and variances which fall within the scope of the appended embodiments.

[0170] All documents cited or referenced herein and all documents cited or referenced in the herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document47ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033 incorporated by reference herein, are hereby incorporated by reference, and may be employed in the practice of the disclosure.48ACTIVE 715868553v1

Claims

Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-033CLAIMS1. A method, comprising:(a) mixing a stool sample from a healthy individual and a preservation buffer; wherein the preservation buffer comprises polysaccharides and an isotonic solvent, wherein the polysaccharides of the preservation buffer are in an amount of 1% of the total volume - 15% of the total volume, wherein at least one of the polysaccharides is selected from the group consisting of: inulin and digestionresistant maltodextrin, or any combination of oligosaccharides or polysaccharides that can preserve and encourage the growth of one or more bacteria listed in TABLE 1;(b) incubating the mixed sample (a) and an enrichment buffer under reducing conditions, forming SAMPLE A; wherein the enrichment buffer comprises polysaccharides and an isotonic solvent, wherein the polysaccharides of the enrichment buffer are in an amount of 3% of the total volume - 50% of the total volume, wherein at least one of the polysaccharides of the enrichment buffer comprises high fiber botanical -based agent that can preserve and encourage the growth of one or more bacteria listed in TABLE 1.(c) adding the enrichment buffer to SAMPLE A, forming enriched SAMPLE A; and(d) incubating enriched SAMPLE A under reducing conditions, forming SAMPLE B, wherein SAMPLE B comprises an enriched foundation guild of one or more bacteria of TABLE 1.

2. The method of claim 1, further comprising repeating steps (b)-(d).

3. The method of claim 1, wherein the preservation buffer comprises polysaccharides in an amount of 2% of the total volume.49ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-0334. The method of claim 3, wherein the polysaccharides comprise inulin and digestionresistant maltodextrin, or any combination of polysaccharides that can preserve and encourage growth of one or more bacteria of TABLE 1.

5. The method of claim 4, wherein inulin and digestion-resistant maltodextrin are in a 1 : 1 ratio.

6. The method of claim 1, wherein the enrichment buffer comprises polysaccharides in an amount of 6% of the total volume.

7. The method of claim 6, wherein the polysaccharides comprise a high fiber botanicalbased agent (NBT-NM108), inulin, and digestion-resistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria of TABLE 1.

8. The method of claim 7, wherein the high fiber botanical -based agent, inulin, and digestion-resistant maltodextrin are in a 2: 1 : 1 ratio, or any ratio of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria of TABLE 1.

9. The method of claim 1, further comprising administering SAMPLE B to a subject suffering from dysbiosis.

10. The method of claim 9, wherein administering is through a route selected from the group consisting of: colonoscopy, endoscopy, enema, enteroscopy, esophagogastroduodenoscopy, nasoduodenal catheter, nasoenteric tube, oral, and sigmoidoscopy.

11. The method of claim 9, further administering a fiber mix formulation to the subject suffering from dysbiosis.

12. The method of claim 11, wherein the fiber mix formulation comprises a mixture of bran, inulin, and digestion-resistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria of TABLE 1.

13. A method, comprising:(a) screening a healthy individual interested in being a potential microbiome donor;(b) culturing a stool sample from the healthy individual in accordance with claim1, wherein the stool sample comprises one or more bacteria of TABLE 1;(c) transferring SAMPLE B into a recipient subject in need thereof; and(d) administering a fiber mix formulation to the recipient subject.

14. The method of claim 13, wherein the screening comprises: health screening, medical screening, blood screening, and stool screening.50ACTIVE 715868553v1Customer Number: 67395 Docket No.: 117465-022601 / PCT | T2025-03315. The method of claim 13, wherein the transferring occurs through a route selected from the group consisting of: colonoscopy, endoscopy, enema, enteroscopy, esophagogastroduodenoscopy, nasoduodenal catheter, nasoenteric tube, oral, and sigmoidoscopy.

16. The method of claim 13, wherein the recipient subject in need thereof suffers from dysbiosis.

17. The method of claim 13, wherein the recipient subject suffers from a dysbiosis-related disease.

18. The method of claim 17, wherein the dysbiosis-related disease is selected from the group consisting of: cancer (e.g., breast cancer, colon / colorectal cancer, hepatocellular carcinoma, esophageal cancer, gallbladder carcinomas, gastric cancer, laryngeal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, stomach cancer); gastrointestinal diseases (e.g., H. pylori and C. difficile infections, inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis); gastrointestinal issues (e.g., diarrhea, constipation, gas, acid reflux); small intestinal bacterial overgrowth (“SIBO”); metabolic diseases (e.g., Type 1 diabetes (i.e., insulin-dependent diabetes mellitus) and Type 2 diabetes (i.e., non-insulin- dependent diabetes mellitus)); inflammatory diseases (e.g., rheumatoid arthritis, asthma, nonalcoholic fatty liver disease (“NAFLD”)); obesity; autism spectrum disorders; mental health diseases (e.g., Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, epilepsy, dementia, anxiety, attention deficit hyperactivity disorder (“ADHD”), depression, bipolar disorder, schizophrenia, obsessive-compulsive disorder (“OCD”), post-traumatic stress disorder (“PTSD”)).

19. The method of claim 13, wherein the fiber mix formulation comprises a mixture of bran, inulin, and digestion-resistant maltodextrin, or any combination of soluble and insoluble fibers that can preserve and encourage growth of one or more bacteria listed in TABLE 1.

20. The method of claim 13, wherein the administering occurs at least once daily.

21. The method of claim 17, wherein the method treats the dysbiosis-related disease of the recipient subject.51ACTIVE 715868553v1

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