Patent Application
20220259332
Increasing evidence that the gut microbiota impacts multiple features of human biology has catalyzed efforts to develop microbiota-directed interventions that improve health status. For similar reasons, there is also a heightened interest in how drugs and other over-the-counter remedies alter the gut microbial community and vice-versa. Better methods are needed, however, to understand how gut microbial community members dynamically interact with microbiota-directed interventions, drugs, and over-the-counter remedies.
In an aspect, the present disclosure provides a composition comprising a plurality particles of one type or a plurality of particles of more than one type, each type comprising (a) a core comprising a tag, (b) a unique compound of interest or a combination of compounds of interest (“the particle-bound compound(s) of interest”) and (b) a unique label, wherein the particle-bound compound(s) of interest are stably attached to the core. Typically, the particle-bound compound(s) of interest remain substantially unaltered during transit through an intestinal tract of a subject that lacks a gut microbiota. In some embodiments, the tag for each particle type is a paramagnetic metal oxide and the core further comprises a coating, wherein the coating comprises an organosilane. A compound of interest may be a drug or a biomolecule.
In another aspect, the present disclosure provides a method for measuring a gut microbiota's functional activity, the method comprising: (a) orally administering to a subject a composition comprising a plurality of particles comprising (i) a core comprising a tag, (ii) a compound of interest or a combination of compounds of interest (“the particle-bound compound(s) of interest”), and (iii) an optional label, wherein the particle-bound compound(s) of interest are stably attached to the core; and wherein structural information and/or amount of the particle-bound compound(s) of interest is known (the “input data”); (b) recovering particles from biological material obtained from the subject; and (c) identifying structural changes to the recovered particle-bound compound(s) of interest and/or measuring the amount of the recovered particle-bound compound(s) of interest (the “recovered data”) and determining the difference between the recovered data and the input data.
In another aspect, the present disclosure provides a method for measuring a gut microbiota's functional activity, the method comprising: (a) orally administering to a subject a composition comprising a plurality of retrievable particles of more than one type, each type of retrievable particle comprising (i) a core comprising a tag, (ii) a compound of interest or a combination of compounds of interest (“the particle-bound compound(s) of interest”), and (iii) a unique label, wherein the particle-bound compound(s) of interest are stably attached to the core, and wherein structural information and/or amount of the particle-bound compound(s) of interest is known (the “input data”); (b) recovering particles from biological material obtained from the subject and then separating the recovered particles by type; and (c) for each type of particle, identifying structural changes to the recovered particle-bound compound(s) of interest and/or measuring the amount of the recovered particle-bound compound(s) of interest (the “recovered data”) and determining the difference between the recovered data and the input data.
In another aspect, the present disclosure encompasses methods to measure modification of a compound of interest in a subject, the methods comprising: (a) orally administering to a subject a composition comprising a plurality of retrievable particles, the retrievable particles comprising a core, a compound of interest or a combination of compounds of interest (“the particle-bound compound(s) of interest”), and an optional label, wherein the particle-bound compound(s) of interest are stably attached to the core, and wherein structural information and/or amount of the particle-bound compound(s) of interest is known (the “input data”), (b) recovering particles from biological material obtained from the subject, and (c) identifying structural changes to the recovered particle-bound compound(s) of interest and/or measuring the amount of the recovered particle-bound compound(s) of interest (the “recovered data”) and determining the difference between the recovered data and the input data.
In another aspect, the present disclosure encompasses methods to measure modification of a compound of interest in a subject, the methods comprising: (a) orally administering to a subject a composition comprising a plurality of retrievable particles of more than one type, each type of retrievable particle comprising a core, a unique compound of interest or a combination of compounds of interest (“the particle-bound compound(s) of interest”), and a unique label, wherein the particle-bound compound(s) of interest are stably attached to the core, and wherein structural information and/or amount of the particle-bound compound(s) of interest is known (the “input data”); (b) recovering particles from biological material obtained from the subject and then separating the recovered particles by type; and (c) for each type of particle, identifying structural changes to the recovered particle-bound compound(s) of interest and/or measuring the amount of the recovered particle-bound compound(s) of interest (the “recovered data”) and determining the difference between the recovered data and the input data.
In another aspect, the present disclosure encompasses methods to measure glycan degradation in a subject, the methods comprising (a) orally administering to a subject a composition comprising a plurality of retrievable particles of more than one type, each type of retrievable particle comprising a core, a unique glycan or a combination of glycans (“the particle-bound glycan(s)”), and a unique label, wherein the particle-bound glycan(s) are stably attached to the core, and wherein the amount of the particle-bound glycan(s) is known (the “input amount”); (b) recovering particles from biological material obtained from the subject and then separating the recovered particles by type; and (c) for each type of particle, measuring the amount of the recovered particle-bound glycan(s) (the “recovered amount”) and determining the difference between the recovered data and the input data.
Other aspects and iterations of the invention are described more thoroughly below.
The application file contains at least one photograph executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.
The present disclosure provides artificial food particles and methods of using the artificial food particles. An “artificial food particle” refers to a retrievable particle that is administered to gut microbiota, the particle comprising a tag, a compound of interest, and optionally a label. Non-limiting examples of suitable compounds of interest include biomolecules and drugs. The tag and optional label provide means to recover food particles and/or to sort recovered food particles into discrete groups. In some embodiments, artificial food particles of the present disclosure are administered to a subject, recovered from the subject, and then analyzed to determine how the artificial food particles changed during transit through the subject's intestinal tract. A variety of changes may occur to the particle including but not limited to degradation of a compound of interest, modification of a compound of interest, attachment or adherence of one or more microbial species, etc. In other embodiments, artificial food particles of the present disclosure are administered to a subject, optionally recovered from the subject, and then the subject's gut microbiota is analyzed to determine how the artificial food particles' transit through the subject's intestinal tract changed the gut mirobiota, gut microbiome, and/or functional outcome(s) of the gut microbiome (e.g., protein expression, enzymatic activities, etc.). In other embodiments, artificial food particles of the present disclosure may be mixed with a biological sample comprising gut microbiota (e.g., a fecal or cecal sample), recovered from the mixture after a suitable amount of time, and then analyzed to determine how the artificial food particle and/or the microbiota and/or microbiome changed. In still further embodiments, artificial food particles of the present disclosure may be mixed with an in vitro culture of one or more gut microbial species (e.g., previously isolated from a biological sample), recovered from the mixture after a suitable amount of time, and then analyzed to determine how the artificial food particle and/or abundance of the microbial species and/or functional activity of the microbial species. Accordingly, artificial food particles of the present disclosure can be used to characterize the composition and/or functional state of a subject's gut microbiota/microbiome, and/or to test the effect of a compound, a drug, a food, a food ingredient, a nutritional supplement, a herbal remedy, a lifestyle modification, or a behavioral modification on the compositional and/or functional state of a subject's gut microbiota/microbiome. In particular, the methods disclosed herein can be used to develop and test microbiota-directed foods.
These and other aspects of the present disclosure are detailed further below. First, several definitions that apply throughout this disclosure are presented.
As used herein, “about” refers to numeric values, including whole numbers, fractions, percentages, etc., whether or not explicitly indicated. The term “about” generally refers to a range of numerical values, for instance, ±0.5-1%, ±1-5% or ±5-10% of the recited value, that one would consider equivalent to the recited value, for example, having the same function or result. In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.
The term “comprising” means “including, but not necessarily limited to”; it specifically indicates open-ended inclusion or membership in a so-described combination, group, series and the like. The terms “comprising” and “including” as used herein are inclusive and/or open-ended and do not exclude additional, unrecited elements or method processes.
As used herein, the term “fiber preparation” refers to a composition comprising dietary fiber that (i) is intended as an ingredient in a food, and (ii) has been prepared from a plant source including, but not limited to, fruits, vegetables, legumes, oilseeds, and cereals, or has been otherwise manufactured to have a composition similar to a fiber preparation prepared from a plant source. “Prepared from a plant source,” as used herein, indicates plant material has undergone one or more treatment step (e.g., grinding, milling, shelling, hulling, extraction, fractionation, etc.). Plant-derived fiber preparations that are economical for use in human foods typically are mixtures of diverse molecular composition comprising not only dietary fiber but also protein, fat, carbohydrate, etc. A skilled artisan will appreciate that fiber preparations prepared by different manufacturing processes may have different compositions, and a proximate analysis may be used to evaluate the suitability of a fiber preparation.
A proximate analysis of a composition (e.g., a fiber preparation, a food item) refers to an analysis of the composition's moisture, protein, fat, dietary fiber, carbohydrate and ash content, which are expressed as the content (wt %) in the composition, respectively. Fiber, protein, fat, ash, and water content can be defined by Association of Official Agricultural Chemists (AOAC) 2009.01, AOAC 920.123, AOAC 933.05, AOAC 935.42, AOAC 926.08, respectively, and carbohydrate can be defined as (100−(Protein+Fat+Ash+Moisture). Analysis of the dietary fiber may provide further information by which to evaluate the suitability of a preparation.
The term “dietary fiber” refers to edible parts of plants, or analogous glycans and carbohydrates, that are resistant to digestion and adsorption in the human small intestine with complete or partial fermentation in the large intestine. The term “dietary fiber” includes glycans, lignin, and associated plant substances. Total dietary fiber, soluble dietary fiber, and insoluble dietary fiber are terms of art defined by the methodology used to measure their relative amount. As used herein, total dietary fiber is defined by AOAC method 2009.01; soluble dietary fiber and insoluble dietary fiber are defined by AOAC method 2011.25.
The term “carbohydrate” refers to an organic compound with the formula Cm(H2O)n, where m and n may be the same or different number, provided the number is greater than 3.
As used herein, the term “glycan” refers to a homo- or heteropolymer of two or more monosaccharides linked glycosidically. As such, the term “glycan” includes disaccharides, oligosaccharides and polysaccharides. The term also encompasses a polymer that has been modified, whether naturally or otherwise; non-limiting examples of such modifications include acetylation, alkylation, esterification, etherification, oxidation, phosphorylation, selenization, sulfonation, or any other manipulation. Glycans may be linear or branched, may be produced synthetically or obtained from a natural source, and may or may not be purified or processed prior to use.
The term “compositional glycan equivalent” refers to a fiber preparation with a substantially similar glycan content as the composition to which it is being compared. A glycan equivalent may be substituted about 1:1 for its comparison composition because the glycan equivalent has a glycan content similar to the composition it is replacing. For instance, if about 30 wt % of pea fiber preparation is to be replaced with a compositional glycan equivalent thereof, one of skill in the art would use about 30 wt % of the pea fiber glycan equivalent. A compositional glycan equivalent may be defined in terms of its monosaccharide content and optionally by an analysis of the glycosidic linkages. Methods for measuring monosaccharide content and analyzing glycosidic linkages are known in the art, and described herein.
The term “functional glycan equivalent” refers to a fiber preparation with substantially similar function as the composition to which it is being compared. The amount of a functional glycan equivalent needed to achieve a substantially similar function may be about the same as the comparison composition, or may be less. For instance, a compositional glycan equivalent will typically have substantially similar function as its comparison composition on a 1:1 (weight) basis. However, a functional glycan equivalent that is an enriched bioactive fraction of a composition may have substantially similar function as the initial composition, but comprise less material, and therefore, less weight than the initial composition. The present disclosure contemplates these and other functional glycan equivalents, as illustrated in Example 12. Substantially similar function may be measured by any method detailed in the Examples herein, in particular the ability to affect total abundance(s) of microbial community members, relative abundance(s) of microbial community members, expression of microbial genes, abundance of microbial gene products (e.g. proteins), activity of microbial proteins, and/or observed biological function of a microbial community.
A “food” is an article to be taken by mouth. The form of the food can vary, and includes but is not limited to a powder form which may be reconstituted or sprinkled on a different food; a bar; a drink; a gel, a gummy, a candy, or the like; a cookie, a cracker, a cake, or the like; and a dairy product (e.g., yogurt, ice cream or the like).
A “microbiota-directed food,” as used herein, refers to a food that selectively promotes the representation and expressed beneficial functions of targeted human gut microbes.
The term “microbiota” refers to microorganisms that are found within a specific environment, and the term “microbiome” refers to a collection of genomes from all the microorganisms found in a particular environment. Accordingly, the term “gut microbiota” refers to microorganisms that are found within a gastrointestinal tract of a subject, and a “gut microbiome” refers to a collection of genomes from all the microorganisms found in the gastrointestinal tract of a subject. The functional outcome of a microbiome refers to measures of gene expression, protein abundance, enzymatic activity and the like, which are encoded by the microbiome.
The “health” of a subject's gut microbiota may be defined by its features, namely its compositional state and/or its functional state. The “compositional state” of a gut microbiota refers to the presence, absence or abundance (relative or absolute) of microbial community members. The community members can be described by different methods of classification typically based on 16S rRNA sequences, including but not limited to operational taxonomic units (OTUs) and amplicon sequence variants (ASVs). The “functional state” of a gut microbiota refers to expression of microbial genes, observed biological functions, and/or phenotypic states of the community. A subject with an unhealthy gut microbiota has a measure of at least one feature of the gut microbiota or microbiome that deviates by 1.5 standard deviation or more (e.g., 2 std. deviation, 2.5 std. deviation, 3 std. deviation, etc.) from that of healthy subjects with similar environmental exposures, such as geography, diet, and age. To “promote a healthy gut microbiota in a subject” means to change the feature of the microbiota or microbiome of the subject with the unhealthy gut microbiota in a manner towards the healthy subjects, and encompasses complete repair (i.e., the measure of gut microbiota health does not deviate by 1.5 standard deviation or more) and levels of repair that are less than complete. Promoting a healthy gut microbiota in a subject also includes preventing the development of an unhealthy gut microbiota in a subject.
The “fiber degrading capacity” of a subject's gut microbiota is defined by its compositional state and its functional state, specifically the absence, presence and abundance of primary and secondary consumers of dietary fiber. An increase in the fiber degrading capacity of a subject may be effected by increasing the abundance of microorganisms with genomic loci for import and metabolism of glycans, as exemplified by polysaccharide utilization loci (PULs) and/or loci encoding CAZymes; and/or increasing the abundance or expression of one or more proteins encoded by a PUL and/or one or more CAZyme (with or without concomitant changes in microorganism abundance).
As used herein, “statistically significant” is a p-value <0.05, <0.01, <0.001, <0.0001, or <0.00001.
The term “substantially similar” generally refers to a range of numerical values, for instance, ±0.5-1%, ±1-5% or ±5-10% of the recited value, that one would consider equivalent to the recited value, for example, having the same function or result.
The terms “relative abundance” and “fractional abundance” as used herein describe an amount of one or more microorganism. Relative abundance means the percent composition of a microorganism of a particular kind relative to the total number of microorganisms in the area. Fractional abundance is the relative abundance divided by 100. For example, the “relative abundance of Bacteroides in a subject's gut microbiota” is the percent of all Bacteroides species relative to the total number of bacteria constituting the subject's gut microbiota, as measured in a suitable sample. “Total abundance” refers to the total number of microorganisms. Suitable samples for quantifying gut microbiota include a fecal sample, a cecal sample or other sample of the lumen. A variety of methods are known in the art for quantifying gut microbiota. For example, a fecal sample, a cecal sample or other sample of the lumenal contents of the large intestine may be collected, processed, plated on appropriate growth media, cultured under suitable conditions (i.e., temperature, presence or absence of oxygen and carbon dioxide, agitation, etc.), and colony forming units may be determined. Alternatively, sequencing methods or arrays may be used to determine abundance. The Examples detail one method, COPRO-Seq, where relative abundance is defined by the number of sequencing reads that can be unambiguously assigned to the species' genome after adjusting for genome uniqueness. 16S rRNA gene sequencing methods can also be used and are well known in the art.
These and other aspects of the present disclosure are detailed further below.
One aspect of the present disclosure is an artificial food particle. As used herein, the terms “artificial food particle,” “particle” and “microbiota functional activity biosensor” are interchangeable. Particles of the present disclosure comprise a compound of interest. In some embodiments, a compound of interest is a compound that is altered, degraded and/or removed from the particle by gut microorganisms during the particles' transit through a subject's gut. In other embodiments, a compound of interest is a compound that binds to gut microorganisms or that gut microorganisms bind to, such that the particle-bound microorganisms may be recovered from biological material. Non-limiting examples of suitable compounds of interest include biomolecules and drugs. Particles may be comprised of only one compound of interest (e.g., a specific glycan, lipid, nucleic acid sequence, protein, etc.). Alternatively, a particle may have multiple compounds of interest of the same type (e.g., multiple glycans, multiple lipids, multiple nucleic acid sequences, multiple proteins, etc.) or multiple compounds of interest of different types (e.g., one or more glycan and one or more lipid, etc.). Compounds of interest can be processed into a particle or attached to a core to make a particle by a variety of methods known in the art.
Particles of the present disclosure are also retrievable, meaning particles can be recovered from biological material obtained from a subject, following administration of the particles to the subject, mixing of the particles with a biological sample obtained from the subject, or mixing of the particles with an in vitro culture of gut microbial species. Recovery of particles is facilitated by the use of a tag. Particles of the present disclosure may optionally comprise a label to facilitate further separation of recovered particles for downstream analyses. In addition, particles of the present disclosure are preferably designed such that they remain substantially unaltered during transit through an intestinal tract of a subject that lacks a gut microbiota (e.g., a germ-free animal). These and other details of an artificial food particle of the present disclosure are further described below.
a) Compound of Interest
Particles of the present disclosure comprise one or more compound of interest. Non-limiting examples of suitable compounds of interest include biomolecules and drugs. The term “compound of interest” encompasses derivatives of a given compound. As used herein, a “derivative” refers to a compound that has been modified by a chemical reaction to include one or more new functional groups. For instance, non-limiting examples of a polysaccharide derivative include a cyano-ester, a cyano-ether, an isocyanide, an isonitrile, a carbylamines, a nitrile, and a carbonitrile of the polysaccharide.
In some embodiments, a particle comprises a drug or a combination of drugs. In other embodiments, a particle comprises a drug or a combination of drugs, and at least one other compound of interest. As used herein, the term “drug” refers to a compound intended for use in the diagnosis, cure, mitigation, treatment of disease, or prevention of disease. In certain embodiments, a drug may also be a type of biomolecule. Although studies on the mechanisms of action and off-target spectra of various drugs aim to improve their efficacy and reduce their side effects, the role of gut microorganisms in these processes and/or the effect of the drug on the composition of the gut microbiome is rarely considered. Particles of the present disclosure can be used to systematically test the effect of a given drug on the composition of the gut microbiota and/or microbiome, and/or identify and optionally quantify gut microbiota-dependent changes to a drug (including changes to structure and/or activity). Classes of drugs that affect the gut microbiota/microbiome composition are known in the art. For example, see, Maier et al. Nature, 2018, 555:623-628. Drugs that are affected by gut microbiota are also known in the art. For example, see, Wallace et al. Science, 2010, 330(6005): 831-835, or Zimmermann et al., Science, 2019, 363(6427). Non-limiting examples of drugs classes that may be of interest include antibiotics, antidiabetics, antihistamines, anti-inflammatories, antimetabolites, antineoplastic agents, antipsychotics, calcium-channel blockers, chemotherapeutics, hormones, proton-pump inhibitors, pscyholeptics. However, the present disclosure is not limited to any one particular drug class.
In some embodiments, a particle comprises a biomolecule or a combination of biomolecules. In other embodiments, a particle comprises a biomolecule or a combination of biomolecules, and at least one other compound of interest. In certain embodiments, a particle comprises a first biomolecule and at least one other biomolecule. The term “biomolecule” refers to carbohydrates, lipids, nucleic acids, and proteins, whether produced synthetically or by a cell or living organism. In some examples, artificial food particles may be produced using a food ingredient. Many food ingredients that are economical for use in human foods are mixtures of diverse molecular composition; they contain active and inactive fractions (from the perspective of the gut microbiota) with different structural features and biophysical availability. Without wishing to be bound by theory, it is hypothesized that the source of food ingredient (e.g., the cultivar of a food staple and/or the waste stream from food manufacturing, etc.), as well food-processing technologies may affect the molecular composition of a food ingredient. Although the use of fiber preparations and individual glycans are described in detail below and also in the Examples, these descriptions are not limiting.
In some embodiments, a particle comprises a carbohydrate. In other embodiments, a particle comprises a carbohydrate and at least one other compound of interest. In certain embodiments, a particle comprises a carbohydrate and at least one other biomolecule. A “carbohydrate,” as used herein, refers to a monosaccharide, disaccharide, oligosaccharide or a polysaccharide.
In some embodiments, a particle comprises a lipid or combination of lipids. In other embodiments, a particle comprises a lipid and at least one other compound of interest. In certain embodiments, a particle comprises a lipid or combination of lipids, and at least one other biomolecule. A “lipid,” as used herein, refers to a compound that is soluble in nonpolar solvents, and includes fatty acids, fatty acid derivatives (e.g., monoglycerides, diglycerides, triglycerides, phospholipids, etc.), sterols, and fat-soluble vitamins (e.g. vitamins, A, D, E, K, etc.). The term “lipid” includes glycolipids.
In some embodiments, a particle comprises a nucleic acid or a combination of nucleic acids. In other embodiments, a particle comprises a nucleic acid and at least one other compound of interest. In certain embodiments, a particle comprises a nucleic acid or a combination of nucleic acids, and at least one other biomolecule. The terms “polynucleotide”, “polynucleotide sequence”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
In some embodiments, a particle comprises a protein or a combination of proteins. In other embodiments, a particle comprises a protein or a combination of proteins, and at least one other compound of interest. In certain embodiments, a particle comprises a protein and at least one other biomolecule. The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non amino acids. The terms also encompass an amino acid polymer that has been modified; non-limiting examples of such modifications include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
In some embodiments, a particle comprises a glycan or a combination of glycans. In other embodiments, a particle comprises a glycan and at least one other compound of interest. In certain embodiments, a particle comprises a glycan and at least one other biomolecule. In still other embodiments, a particle comprises a first glycan and at least one other glycan. A “glycan,” as used herein, refers to a homo- or heteropolymer of two or more monosaccharides linked glycosidically. As such, the term “glycan” includes disaccharides, oligosaccharides and polysaccharides. The term also encompasses a polymer that has been modified, whether naturally or otherwise; non-limiting examples of such modifications include acetylation, alkylation, esterification, etherification, oxidation, phosphorylation, selenization, sulfonation, or any other manipulation, such as conjugation with a labeling component. Glycans may be linear or branched, may be produced synthetically or obtained from a natural source, and may or may not be purified or processed prior to use.
A glycan may be defined, in part, in terms of its monosaccharide content and its glycosyl linkages. For example, plant arabinans are composed of 1,5-α-linked L-arabinofuranosyl residues, and these can be branched at 0-2 or 0-3 by single arabinosyl residues or short side chains (Beldman et al., 1997; Ridley et al., 2001; Mohnen, 2008). 1,5-Linked arabinan structures may exist as free polymers unattached to pectic domains or attached to pectic domains (Beldman et al., 1997; Ridley et al., 2001).
As is understood in the art, due to the mechanism of side chain synthesis, a plant glycan is not a single chemical entity but is rather a mixture of glycans that have a defined backbone and variable amounts of substituents/branching. It is routine in the art to indicate the presence of variable amounts of a substituent by indicating its fractional abundance. For instance, when R1 and R2 are each H, the glycan depicted below is an arabinan—specifically, a polymer consisting of 1,5-α-linked L-arabinofuranosyl residues:
The formula indicates that (1) the polymer backbone consists of 1,5-α-linked L-arabinofuranosyl residues, and (2) there are 4 types of arabinose components—namely, component a—2,3,5-arabinofuranose, component b—5-arabinofuranose, component c—2,5-arabinofuranose, and component d—3,5-arabinofuranose. The fractional abundance of each component is indicated by the values assigned to a, b, c, and d, respectively. The sum of all the values is about 1 (allowing for a small amount of error in the measurements). A value of zero (0) indicates the component is never present in the polymer. A value of one (1) indicates the component accounts for 100% of the polymer. A value of 0.5 indicates that the component accounts for 50% of the polymer. The arrangement of the components within the polymer can vary, as is understood in the art, and is not defined by the order depicted.
Artificial food particles may be produced using a composition comprising a single glycan, or a composition comprising 2, 3, 4, 5, or more glycans (e.g., “a glycan composition”). Glycan compositions may be prepared by using commercially available preparations of a glycan, by first purifying (partially or completely) a desired glycan from a natural source, or by biological or chemical synthesis of a desired glycan. The number and specific structures of glycans to include may be informed by the intended use of the particle and/or by compositional or functional knowledge of the intended subject's gut microbiome, including but not limited to the presence/absence of certain bacterial species, the absolute or relative abundance of certain bacterial species, the level of expression of bacterial genes in polysaccharide utilization loci (PULs), and/or the abundance of bacterial PUL protein products. Additional non-glycan components may also be present in the glycan composition.
In some examples, artificial food particles may be produced using one or more glycans obtained from a fiber preparation. The glycans obtained from a fiber preparation may be partially or completely purified from a fiber preparation prior to use, or a fiber preparation may be used “as is”. Non-limiting examples of fiber preparations include citrus pectin preparations, pea fiber preparations, citrus peel preparations, yellow mustard bran preparations, soy cotyledon preparations, orange fiber preparations, orange peel preparations, tomato peel preparations, inulin preparations, potato fiber preparations, apple pectin preparations, sugar beet fiber preparations, oat hull fiber preparations, acacia extract preparations, barley beta-glucan preparations, barley bran preparations, oat beta-glucan preparations, apple fiber preparations, rye bran preparations, barley malted preparations, wheat bran preparations, wheat aleurone preparations, maltodextrin preparations (including but not limited to resistant maltodextrin preparations), psyllium preparations, cocoa preparations, citrus fiber preparations, tomato pomace preparations, rice bran preparations, chia seed preparations, corn bran preparations, soy fiber preparations, sugar cane fiber preparations, resistant starch 4 preparations. Exemplary fiber preparations are provided in Table A and the paragraphs that follow. Suitable fiber preparations also include those that are substantially similar to the exemplary fiber preparations provided in Table A and the paragraphs that follow. As demonstrated herein, a fiber preparation contains active and inactive fractions with different structural features and biophysical availability, from the perspective of the gut microbiota. Accordingly, preferred fiber preparations may also have substantially similar monosaccharide content and/or glycosyl linkages. Fiber preparations may be prepared from plant material by methods known in the art. Methods for measuring monosaccharide content and performing a glycosyl linkage analysis are known in the art, and described herein.
(i) Barley Fiber Preparations
In some embodiments, an artificial food particle may be produced using one or more glycan obtained from a barley fiber preparation. Barley fiber preparations may be prepared according to methods known in the art, and evaluated as described herein. Commercial sources may also be used.
In some embodiments, a composition comprises one or more barley fiber preparation in an amount that does not exceed 45 wt % of the composition. The amount may also be expressed as individual values or a range. For instance, the barley fiber preparation(s) in these embodiments may be about 1 wt %, 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, 8 wt %, 9 wt %, 10 wt %, 11 wt %, 12 wt %, 13 wt %, 14 wt %, 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, or 45 wt %. In some examples, the barley fiber preparation(s) may be about 1 wt % to about 45 wt %, about 10 wt % to about 45 wt %, or about 20 wt % to about 45 wt % of the composition. In some examples, the barley fiber preparation(s) may be about 1 wt % to about 25 wt % or about 10 wt % to about 25 wt % of the composition, or about 1 wt % to about 20 wt % or about 10 wt % to about 20 wt % of the composition.
In an exemplary embodiment of a suitable barley fiber preparation, the total dietary fiber is comprised of about 5 wt % to about 15 wt %, or about 10 wt % to about 15% of insoluble dietary fiber and/or about 40 wt % to about 50 wt %, or about 42 wt % to about 47 wt % of high molecular weight dietary fiber. In some embodiments, the total dietary fiber is about 35 wt % to about 55 wt %, about 40 wt % to about 55 wt %, or about 45 wt % to about 55 wt % of the preparation. In other embodiments, the total dietary fiber is about 35 wt % to about 50 wt % or about 30 wt % to about 45 wt % of the preparation. In still further embodiments, the barley fiber preparation comprises about 15 wt % to about 20 wt % protein, about 2 wt % to about 5 wt % fat, about 65 wt % to about 75 wt % carbohydrate, about 2 wt % to about 7 wt % moisture, and about 1 wt % to about 3 wt % ash.
In another exemplary embodiment of a suitable barley fiber preparation, the total dietary fiber is comprised of about 5 wt % to about 15 wt %, or about 10 wt % to about 15% of insoluble dietary fiber and about 40 wt % to about 50 wt %, or about 42 wt % to about 47 wt % of high molecular weight dietary fiber; the total dietary fiber is about 35 wt % to about 55 wt %, about 40 wt % to about 55 wt %, or about 45 wt % to about 55 wt % of the preparation; and the barley fiber preparation comprises about 15 wt % to about 20 wt % protein, about 2 wt % to about 5 wt % fat, about 65 wt % to about 75 wt % carbohydrate, about 2 wt % to about 7 wt % moisture, and about 1 wt % to about 3 wt % ash.
In another exemplary embodiment, a suitable barley fiber preparation is substantially similar to the preparation described in Table B.
In each of the embodiments, a suitable barley fiber preparation may also have a monosaccharide content that is substantially similar to the preparation exemplified in Table C; glycosyl linkages substantially similar to the preparation exemplified in Table F, or both.
In another exemplary embodiment, a suitable barley fiber preparation has a monosaccharide content that is substantially similar to the preparation exemplified in Table B and glycosyl linkages that are substantially similar to the preparation exemplified in Table E
(ii) Citrus Fiber Preparations
In some embodiments, an artificial food particle may be produced using one or more glycan obtained from a citrus fiber preparation.
Citrus fiber preparations may be prepared according to methods known in the art from citrus fruits including, but not limited to, clementine, citron, grapefruit, kumquat, lemon, lime, orange, tangelo, tangerine, and yuzu, and evaluated as described herein. Commercial sources may also be used.
In some embodiments, a composition comprises one or more citrus fiber preparation in an amount that does not exceed 25 wt % of the composition. The amount may also be expressed as individual values or a range. For instance, the citrus fiber preparation(s) in these embodiments may be about 1 wt %, 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, 8 wt %, 9 wt %, 10 wt %, 11 wt %, 12 wt %, 13 wt %, 14 wt %, 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, or 25 wt %. In some examples, the citrus fiber preparation(s) may be about 1 wt % to about 25 wt %, about 1 wt % to about 20 wt %, or about 1 wt % to about 15 wt % of the composition. In some examples, the citrus fiber preparation(s) may be about 5 wt % to about 25 wt %, about 5 wt % to about 20 wt %, or about 5 wt % to about 15 wt % of the composition. In some examples, the citrus fiber preparation(s) may be about 10 wt % to about 25 wt %, about 10 wt % to about 20 wt %, or about 10 wt % to about 15 wt % of the composition.
In an exemplary embodiment of a suitable citrus fiber preparation, the total dietary fiber is comprised of about 30 wt % to about 40 wt %, or about 30 wt % to about 35% of insoluble dietary fiber and/or about 65 wt % to about 75 wt %, or about 65 wt % to about 70 wt % of high molecular weight dietary fiber. In some embodiments, the total dietary fiber is about 60 wt % to about 80 wt %, about 60 wt % to about 75 wt %, or about 60 wt % to about 70 wt % of the preparation. In other embodiments, the total dietary fiber is about 65 wt % to about 80 wt %, about 65 wt % to about 75 wt %, or about 65 wt % to about 70 wt % of the preparation. In still further embodiments, the citrus fiber preparation comprises about 5 wt % to about 10 wt % protein, about 1 wt % to about 3 wt % fat, about 75 wt % to about 85 wt % carbohydrate, about 5 wt % to about 10 wt % moisture, and about 1 wt % to about 4 wt % ash.
In another exemplary embodiment of a suitable citrus fiber preparation, the total dietary fiber is comprised of about 30 wt % to about 40 wt %, or about 30 wt % to about 35% of insoluble dietary fiber and/or about 65 wt % to about 75 wt %, or about 65 wt % to about 70 wt % of high molecular weight dietary fiber; the total dietary fiber is about 65 wt % to about 80 wt %, about 65 wt % to about 75 wt %, or about 65 wt % to about 70 wt % of the preparation; and the citrus fiber preparation comprises about 5 wt % to about 10 wt % protein, about 1 wt % to about 3 wt % fat, about 75 wt % to about 85 wt % carbohydrate, about 5 wt % to about 10 wt % moisture, and about 1 wt % to about 4 wt % ash.
In another exemplary embodiment, a suitable citrus fiber preparation is substantially similar to the preparation described in Table B.
In each of the above embodiments, a suitable citrus fiber preparation may also have a monosaccharide content that is substantially similar to the preparation described in Table C; glycosyl linkages substantially similar to the preparation exemplified in Table G; or both.
In another exemplary embodiment, a suitable citrus fiber preparation has a monosaccharide content that is substantially similar to the preparation exemplified in Table C and glycosyl linkages that are substantially similar to the preparation exemplified in Table G.
(iii) Citrus Pectin Preparations
In some embodiments, an artificial food particle may be produced using one or more glycan obtained from a citrus pectin preparation. Citrus pectin preparations may be prepared according to methods known in the art from citrus fruits including, but not limited to, clementine, citron, grapefruit, kumquat, lemon, lime, orange, tangelo, tangerine, and yuzu, and evaluated as described herein. Commercial sources may also be used.
In some embodiments, a composition comprises one or more citrus pectin preparation in an amount that does not exceed 10 wt % of the composition. The amount may also be expressed as individual values or a range. For instance, the amount of citrus pectin in these embodiments may be about 1 wt %, 2 wt %, 3 wt %, 4 wt %, 5 wt %, 6 wt %, 7 wt %, 8 wt %, 9 wt %, or 10 wt %. In some examples, the citrus pectin preparation(s) may be about 1 wt % to about 10 wt %, about 1 wt % to about 8 wt %, or about 1 wt % to about 6 wt % of the composition. In some examples, the citrus pectin preparation(s) may be about 1 wt % to about 4 wt %, or about 1 wt % to about 2 wt % of the composition.
In an exemplary embodiment of a suitable citrus pectin preparation, the total dietary fiber is comprised of about 1 wt % to about 10 wt %, or about 1 wt % to about 5% of insoluble dietary fiber and/or about 85 wt % to about 95 wt %, or about 90 wt % to about 95 wt % of high molecular weight dietary fiber. In some embodiments, the total dietary fiber is about 75 wt % to about 95 wt %, about 80 wt % to about 95 wt %, or about 85 wt % to about 95 wt % of the preparation. In other embodiments, the total dietary fiber is about 85 wt % to about 90 wt % or about 90 wt % to about 95 wt % of the preparation. In still further embodiments, the citrus pectin preparation comprises about 2 wt % or less of protein, about 1 wt % to about 2 wt % fat, about 85 wt % to about 95 wt % carbohydrate, about 1 wt % to about 6 wt % moisture, and about 3 wt % to about 6 wt % ash.
In another exemplary embodiment of a suitable citrus pectin preparation, the total dietary fiber is comprised of about 1 wt % to about 10 wt %, or about 1 wt % to about 5% of insoluble dietary fiber and about 85 wt % to about 95 wt %, or about 90 wt % to about 95 wt % of high molecular weight dietary fiber; the total dietary fiber is about 85 wt % to about 95 wt %, about 85 wt % to about 90 wt %, or about 90 wt % to about 95 wt % of the preparation; and the citrus pectin preparation comprises about 2 wt % or less of protein, about 1 wt % to about 2 wt % fat, about 85 wt % to about 95 wt % carbohydrate, about 1 wt % to about 6 wt % moisture, and about 3 wt % to about 6 wt % ash.
In another exemplary embodiment, a suitable citrus pectin preparation is substantially similar to the preparation described in Table B.
In each of the above embodiments, a suitable citrus fiber preparation may also have a monosaccharide content substantially similar to the preparation exemplified in Table C; glycosyl linkages substantially similar to the preparation exemplified in Table E; or both.
In another exemplary embodiment, a suitable citrus pectin preparation has a monosaccharide content that is substantially similar to the preparation exemplified in Table C and glycosyl linkages that are substantially similar to the preparation exemplified in Table G.
(iv) High Molecular Weight Inulin Preparations
In some embodiments, an artificial food particle may be produced using one or more glycan obtained from a high molecular weight inulin preparation. Inulin is defined by AOAC method 999.03.
High molecular weight inulin is comprised of fructose units linked together by β-(2,1)-linkages, which are typically terminated by a glucose unit. High molecular weight inulin preparations may be prepared according to methods known in the art, and evaluated as described herein. Commercial sources may also be used.
In some embodiments, a composition comprises one or more high molecular weight inulin preparation in an amount that is at least 28 wt % of the composition. The amount may also be expressed as individual values or a range. For instance, the high molecular weight inulin preparation(s) in these embodiments may be about 29 wt %, 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47 wt %, 48 wt %, 49 wt %, 50 wt %, or more. In some examples, the high molecular weight inulin preparation(s) may be about 30 wt % to about 50 wt %, about 30 wt % to about 45 wt %, or about 30 wt % to about 40 wt % of the composition. In some examples, the high molecular weight inulin preparation(s) may be about 35 wt % to about 50 wt %, about 35 wt % to about 45 wt %, or about 35 wt % to about 40 wt % of the composition. Inulin is defined by AOAC method 999.03.
In an exemplary embodiment of a suitable high molecular weight inulin preparation, the total dietary fiber is comprised of about 0.5 wt % or less of insoluble dietary fiber and/or about 55 wt % to about 65 wt %, or about 57 wt % to about 62 wt % of high molecular weight dietary fiber. In some embodiments, the total dietary fiber is about 75 wt % to about 95 wt %, about 80 wt % to about 95 wt %, or about 85 wt % to about 95 wt % of the preparation. In other embodiments, the total dietary fiber is about 85 wt % to about 99 wt %, 90 wt % to about 99 wt %, or about 95 wt % to about 99 wt % of the preparation. In still further embodiments, the high molecular weight inulin preparation comprises no more than 1 wt % of protein, about 2 wt % to about 5 wt % fat, about 85 wt % to about 95 wt % carbohydrate, about 2 wt % to about 7 wt % moisture, and no more than 2 wt % ash.
In an exemplary embodiment of a suitable high molecular weight inulin preparation, the total dietary fiber is comprised of about 0.5 wt % insoluble dietary fiber and about 55 wt % to about 65 wt %, or about 57 wt % to about 62 wt % of high molecular weight dietary fiber; the total dietary fiber is about 85 wt % to about 99 wt %, 90 wt % to about 99 wt %, or about 95 wt % to about 99 wt % of the preparation; and the high molecular weight inulin preparation comprises no more than 1 wt % of protein, about 2 wt % to about 5 wt % fat, about 85 wt % to about 95 wt % carbohydrate, about 2 wt % to about 7 wt % moisture, and no more than 2 wt % ash.
In another exemplary embodiment, a suitable high molecular weight inulin preparation is substantially similar to the preparation described in Table B.
In each of the above embodiments, about 99% of the inulin in a suitable high molecular weight inulin preparation may have a degree of polymerization (DP) that is greater than or equal to 5. In some example, the DP for the inulin in a suitable preparation may range from 5 to 60. Alternatively or in addition, the average DP may be less than or equal to 23.
(v) Pea Fiber Preparations
In some embodiments, an artificial food particle may be produced using one or more glycan obtained from a pea fiber preparation. Pea fiber preparations may be prepared according to methods known in the art, and evaluated as described herein. Commercial sources may also be used.
In some embodiments, a composition comprises one or more pea fiber preparation in an amount that is at least 15 wt % of the composition. The amount may also be expressed as individual values or a range. For instance, the pea fiber preparation(s) in these embodiments may be about 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47 wt %, 48 wt %, 49 wt %, 50 wt %, 51 wt %, 52 wt %, 53 wt %, 54 wt %, 55 wt %, 56 wt %, 57 wt %, 58 wt %, 59 wt %, 60 wt %, 61 wt %, 62 wt %, 63 wt %, 64 wt %, 65 wt %, or more. In some examples, the pea fiber preparation(s) may be about 15 wt % to about 75 wt %, about 25 wt % to about 75 wt %, or about 35 wt % to about 75 wt % of the composition. In some examples, the pea fiber preparation(s) may be about 15 wt % to about 65 wt %, about 25 wt % to about 65 wt %, or about 35 wt % to about 65 wt % of the composition. In some examples, the pea fiber preparation(s) may be about 30 wt % to about 85 wt %, about 40 wt % to about 85 wt %, or about 50 wt % to about 85 wt % of the composition.
In an exemplary embodiment of a suitable pea fiber preparation, the total dietary fiber is comprised of about 55 wt % to about 65 wt %, or about 60 wt % to about 65% of insoluble dietary fiber and/or about 60 wt % to about 70 wt %, or about 65 wt % to about 70 wt % of high molecular weight dietary fiber. In some embodiments, the total dietary fiber is about 60 wt % to about 80 wt %, about 60 wt % to about 75 wt %, or about 60 wt % to about 70 wt % of the preparation. In other embodiments, the total dietary fiber is about 65 wt % to about 80 wt %, about 65 wt % to about 75 wt %, or about 65 wt % to about 70 wt % of the preparation. In still further embodiments, the pea fiber preparation comprises about 7 wt % to about 12 wt % protein, no more than 2 wt % fat, about 75 wt % to about 85 wt % carbohydrate, about 5 wt % to about 10 wt % moisture, and about 1 wt % to about 4 wt % ash.
In an exemplary embodiment of a suitable pea fiber preparation, the total dietary fiber is comprised of about 55 wt % to about 65 wt %, or about 60 wt % to about 65% of insoluble dietary fiber and about 60 wt % to about 70 wt %, or about 65 wt % to about 70 wt % of high molecular weight dietary fiber; the total dietary fiber is about 65 wt % to about 80 wt %, about 65 wt % to about 75 wt %, or about 65 wt % to about 70 wt % of the preparation; and the pea fiber preparation comprises about 7 wt % to about 12 wt % protein, no more than 2 wt % fat, about 75 wt % to about 85 wt % carbohydrate, about 5 wt % to about 10 wt % moisture, and about 1 wt % to about 4 wt % ash.
In another exemplary embodiment, a suitable pea fiber preparation is substantially similar to the preparation described in Table B.
In each of the above embodiments, a suitable pea fiber preparation may also have a monosaccharide content that is substantially similar to the preparation exemplified in Table C; glycosyl linkages substantially similar to the preparation exemplified in Table D, Table 13, Table 14, Table 16, or Table 17; or both.
In another exemplary embodiment, a suitable pea fiber preparation has a monosaccharide content that is substantially similar to the preparation exemplified in Table B and glycosyl linkages that are substantially similar to the preparation exemplified in Table C, Table 13, Table 14, Table 16, or Table 17.
In another exemplary embodiment a suitable pea fiber preparation has a monosaccharide content that has about 10 wt % to about 90 wt % arabinose, and arabinose linkages that are substantially similar to the preparation exemplified in Table C, Table 13, Table 14, Table 16, or Table 17. In some examples, arabinose may be about 10 wt % to 20 wt %, or about 15 wt % to about 20 wt %. In some examples, arabinose may be about 20 wt % to 30 wt %, about 20 wt % to about 25 wt %, or about 25 wt % to about 30 wt %. In some examples, arabinose may be about 50 wt % to 90 wt %, about 60 wt % to about 90 wt %, or about 70 wt % to about 90 wt %. In some examples, arabinose may be about 50 wt % to 80 wt %, about 60 wt % to about 80 wt %, or about 70 wt % to about 80 wt %.
In another exemplary embodiment, a suitable pea fiber preparation has a monosaccharide content that has a substantially similar arabinose content as the preparation exemplified in Table B and arabinose glycosyl linkages that are substantially similar to the preparation exemplified in Table C, Table 13, Table 14, Table 16, or Table 17.
In another exemplary embodiment, a suitable pea fiber preparation is substantially similar to the Fiber 8 fraction or the enzymatically destarched Fiber 8 fraction described in Example 10.
In all the aforementioned, a suitable pea fiber preparation may also comprise arabinan of formula (I):
wherein a is about 0.1 to about 0.3, b is about 0.4 to about 0.6, c is about 0.1 to about 0.4, d is about 0.04 to about 0.06 (calculated from the fractional abundance of arabinose linkages where the arabinose contained a 5-linkage, as determined by partially methylated alditol acetate GC-MS analysis); and wherein R1 and R2 are each independently selected from H, a glycosyl, a sugar moiety (modified or not), an oligosaccharide (branched or not), or a polysaccharide (branched or not), and a polysaccharide containing galacturonic acid, galactose, and rhamnose.
Alternatively, in all the aforementioned embodiments, a suitable pea fiber preparation may also comprise arabinan of formula (I):
wherein a is about 0.2 to about 0.3, b is about 0.5 to about 0.6, c is about 0.2 to about 0.4, d is about 0.04 to about 0.06 (calculated from the fractional abundance of arabinose linkages where the arabinose contained a 5-linkage, as determined by partially methylated alditol acetate GC-MS analysis); and wherein R1 and R2 are each independently selected from H, a glycosyl, a sugar moiety (modified or not), an oligosaccharide (branched or not), or a polysaccharide (branched or not), and a polysaccharide containing galacturonic acid, galactose, and rhamnose.
Alternatively, in all the aforementioned embodiments, a suitable pea fiber preparation may also comprise arabinan of formula (I):
wherein a is about 0.1 to about 0.2, b is about 0.4 to about 0.5, c is about 0.2 to about 0.4, d is about 0.04 to about 0.06 (calculated from the fractional abundance of arabinose linkages where the arabinose contained a 5-linkage, as determined by partially methylated alditol acetate GC-MS analysis); and wherein R1 and R2 are each independently selected from H, a glycosyl, a sugar moiety (modified or not), an oligosaccharide (branched or not), or a polysaccharide (branched or not), and a polysaccharide containing galacturonic acid, galactose, and rhamnose.
Alternatively, in all the aforementioned embodiments, a suitable pea fiber preparation may also comprise arabinan of formula (I):
wherein a is about 0.2 to about 0.3, b is about 0.4 to about 0.5, c is about 0.3 to about 0.4, d is about 0.04 to about 0.06 (calculated from the fractional abundance of arabinose linkages where the arabinose contained a 5-linkage, as determined by partially methylated alditol acetate GC-MS analysis); wherein R1 and R2 are each independently selected from H, a glycosyl, a sugar moiety (modified or not), an oligosaccharide (branched or not), or a polysaccharide (branched or not), and a polysaccharide containing galacturonic acid, galactose, and rhamnose.
Alternatively, in all the aforementioned embodiments, a suitable pea fiber preparation may also comprise arabinan of formula (I):
wherein a is about 0.20, b is about 0.47, c is about 0.28, d is about 0.05 (calculated from the fractional abundance of arabinose linkages where the arabinose contained a 5-linkage, as determined by partially methylated alditol acetate GC-MS analysis); wherein R1 and R2 are each independently selected from H, a glycosyl, a sugar moiety (modified or not), an oligosaccharide (branched or not), or a polysaccharide (branched or not), and a polysaccharide containing galacturonic acid, galactose, and rhamnose.
The molecular weight of the arabinan may be about 2 kDa to about 500,000 kDa, or more. In one example, the molecular weight of the arabinan may be about 1000 kDa to about 500,000 kDa. In one example, the molecular weight of the arabinan may be about 1000 kDa to about 200,000 kDa. In one example, the molecular weight of the arabinan may be about 1000 kDa to about 100,000 kDa. In one example, the molecular weight of the arabinan may be about 1000 kDa to about 10,000 kDa. In one example, the molecular weight of the arabinan may be about 10,000 kDa to about 500,000 kDa. In one example, the molecular weight of the arabinan may be about 10,000 kDa to about 200,000 kDa. In one example, the molecular weight of the arabinan may be about 100,000 kDa to about 500,000 kDa.
The total amount of all arabinans of formula (I) in a suitable pea fiber preparation may vary. In some embodiments, the total amount may be at least 10 wt %. For example, the total amount may be about 10 wt %, about 15 wt %, about 20 wt %, about 25 wt %, about 30 wt %, about 35 wt %, about 40 wt %, about 45 wt %, about 50 wt %, about 55 wt %, about 60 wt %, about 65 wt %, about 70 wt %, about 75 wt %, about 80 wt %, about 85 wt %, about 90 wt %, about 95 wt %. In some embodiments, the total amount may be at least 20 wt %, at least 30 wt %, at least 40 wt %, at least 50 wt %, at least, 60 wt %, at least, 70 wt %, at least, 80 wt %, at least 90 wt %. In some embodiments, the total amount may be about 10 wt % to about 50 wt %, about 20 wt % to about 50 wt %, about 30 wt % to about 50 wt %, about 40 wt % to about 50 wt %. In some embodiments, the total amount may be about 30 wt % to about 70 wt %, about 40 wt % to about 70 wt %, about 50 wt % to about 70 wt %, about 60 wt % to about 70 wt %. In some embodiments, the total amount may be about 50 wt % to about 90 wt %, about 60 wt % to about 90 wt %, about 70 wt % to about 90 wt %, about 80 wt % to about 90 wt %.
(vi) Sugar Beet Fiber Preparations:
In some embodiments, an artificial food particle may be produced using one or more glycan obtained from a sugar beet fiber preparation. Sugar beet fiber preparations may be prepared according to methods known in the art, and evaluated as described herein. Commercial sources may also be used.
In some embodiments, a composition comprises one or more sugar beet fiber preparation in an amount that is at least 15 wt % of the composition. The amount may also be expressed as individual values or a range. For instance, the pea fiber preparation(s) in these embodiments may be about 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %, 20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27 wt %, 28 wt %, 29 wt %, 30 wt %, 31 wt %, 32 wt %, 33 wt %, 34 wt %, 35 wt %, 36 wt %, 37 wt %, 38 wt %, 39 wt %, 40 wt %, 41 wt %, 42 wt %, 43 wt %, 44 wt %, 45 wt %, 46 wt %, 47 wt %, 48 wt %, 49 wt %, 50 wt %, 51 wt %, 52 wt %, 53 wt %, 54 wt %, 55 wt %, 56 wt %, 57 wt %, 58 wt %, 59 wt %, 60 wt %, 61 wt %, 62 wt %, 63 wt %, 64 wt %, 65 wt %, or more. In some examples, the sugar beet fiber preparation(s) may be about 15 wt % to about 65 wt %, about 25 wt % to about 65 wt %, or about 35 wt % to about 65 wt % of the composition. In some examples, the sugar beet fiber preparation(s) may be about 15 wt % to about 55 wt %, about 25 wt % to about 55 wt %, or about 35 wt % to about 55 wt % of the composition. In some examples, the sugar beet fiber preparation(s) may be about 15 wt % to about 45 wt %, about 25 wt % to about 45 wt %, or about 35 wt % to about 45 wt % of the composition.
In an exemplary embodiment of a suitable sugar beet fiber preparation, the total dietary fiber is comprised of about 55 wt % to about 65 wt %, or about 60 wt % to about 65% of insoluble dietary fiber and/or about 75 wt % to about 85 wt %, or about 80 wt % to about 85 wt % of high molecular weight dietary fiber. In some embodiments, the total dietary fiber is about 70 wt % to about 90 wt %, about 70 wt % to about 85 wt %, or about 70 wt % to about 80 wt % of the preparation. In other embodiments, the total dietary fiber is about 75 wt % to about 90 wt %, about 80 wt % to about 90 wt %, or about 80 wt % to about 85 wt % of the preparation. In still further embodiments, the sugar beet fiber preparation comprises about 7 wt % to about 12 wt % protein, about 1 wt % to about 3 wt % fat, about 75 wt % to about 85 wt % carbohydrate, about 5 wt % to about 10 wt % moisture, and about 3 wt % to about 6 wt % ash.
In an exemplary embodiment of a suitable sugar beet fiber preparation, the total dietary fiber is comprised of about 55 wt % to about 65 wt %, or about 60 wt % to about 65% of insoluble dietary fiber and about 75 wt % to about 85 wt %, or about 80 wt % to about 85 wt % of high molecular weight dietary fiber, the total dietary fiber is about 75 wt % to about 90 wt %, about 80 wt % to about 90 wt %, or about 80 wt % to about 85 wt % of the preparation; and the sugar beet fiber preparation comprises about 7 wt % to about 12 wt % protein, about 1 wt % to about 3 wt % fat, about 75 wt % to about 85 wt % carbohydrate, about 5 wt % to about 10 wt % moisture, and about 3 wt % to about 6 wt % ash.
In another exemplary embodiment, a suitable sugar beet preparation is substantially similar to the preparation described in Table B.
(vii) Glycan Equivalents
In each of the above embodiments, a compositional glycan equivalent thereof and/or a functional glycan equivalent thereof may be used as an alternative for a barley fiber preparation, a citrus fiber preparation, a citrus pectin preparation, a high molecular weight inulin preparation, a pea fiber preparation, and/or a sugar beet fiber preparation.
In some embodiments, a suitable functional glycan equivalent of a barley fiber preparation, a citrus fiber preparation, a citrus pectin preparation, a high molecular weight inulin preparation, a pea fiber preparation, or a sugar beet fiber preparation has a substantially similar function as a respective preparation identified in Table 2A. Substantially similar function may be measured by any one or more method detailed in the Examples herein, in particular the ability to affect relative or total abundances of microbial community members, in particular primary and secondary fiber degrading microbes, more particularly Bacteroides species; and/or expression of one or more microbial genes or gene product, in particular one or more gene or gene product encoded by polysaccharide utilization loci (PULs) and/or one or more CAZyme. In an exemplary embodiment, a suitable functional glycan equivalent is a fiber preparation that is enriched for one or more bioactive glycan, as compared to a barley fiber preparation, a citrus fiber preparation, a citrus pectin preparation, a high molecular weight inulin preparation, a pea fiber preparation, or a sugar beet fiber preparation used in the Examples.
For instance, a suitable functional glycan equivalent of a fiber preparation may have a similar effect on the relative abundance of Bacteroides species in a subject's gut microbiota. In another example, a suitable functional glycan equivalent of a fiber preparation may have a similar effect on the total abundance of Bacteroides species in a subject's gut microbiota. In another example, a suitable functional glycan equivalent of a fiber preparation may have a similar effect on the relative abundance of a subset of Bacteroides species. In another example, a suitable functional glycan equivalent of a fiber preparation may have a similar effect on the total abundance of a subset of Bacteroides species. In one example, the subset of Bacteroides species may include one or more species chosen from B. caccae, B. cellulosilyticus, B. finegoldfi, B. massiliensis, B. ovatus, B. thetaiotaomicron, and B. vulgatus. In another example, a suitable functional glycan equivalent may have a similar effect on the relative abundance of one or more species chosen from Bacteroides ovatus, Bacteroides cellulosilyticus, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides caccae, Bacteroides finegoldfi, Bacteroides massiliensis, Collinsella aerofaciens, Escherichia coli, Odoribacter splanchnicus, Parabacteroides distasonis, a Ruminococcaceae sp., and Subdoligranulum variabile.
Alternatively or in addition, a suitable functional glycan equivalent may have a similar effect on the abundance or activity of one or more protein encoded by one or more polysaccharide utilization locus (PUL) and/or one or more CAZyme. In some examples, the PULs are chosen from PUL5, PULE, PUL7, PUL27, PUL31, PUL34, PUL35, PUL38, PUL42, PUL43, PUL73, PUL75, PUL83, and PUL97.
Although the Examples utilize a gnotobiotic mouse model where the mouse is colonized with a defined gut microbiota, the methods detailed in the Examples may also be used to measure effects in a gnotobiotic mouse model where the mouse is colonized with intact uncultured gut microbiota obtained from human(s), as well as to measure effects directly in humans.
(viii) Bioactive Glycans
Applicants have identified fiber preparations that promote a healthy gut microbiota in a subject, and further discovered that each fiber preparation has a number of bioactive glycans responsible for the observed beneficial effect(s). Thus, in another aspect, the present disclosure provides a composition comprising an enriched amount of a bioactive glycan, wherein “an enriched amount” refers to an amount of the bioactive glycan that is more than is found in a naturally occurring plant or plant part, and more than is found in commercially available fiber preparations. A composition comprising an enriched amount of a bioactive glycan may be a purified (partially or completely) fraction from a commercially available fiber preparation. Alternatively, a composition comprising an enriched amount of a bioactive glycan may comprise a chemically synthesized version of the bioactive glycan. The bioactive glycan may be enriched by about 10 wt % wt to about 50 wt %, about 50 wt % to about 100 wt % or more. For instance, the bioactive glycan may be enriched by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold or more. In another example, the bioactive glycan may be enriched by about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold or more. In another example, the bioactive glycan may be enriched by about 500-fold, 1000-fold, or more.
Bioactive glycans of barley fiber, citrus fiber, citrus pectin, high molecular weight inulin, pea fiber, and sugar beet can be identified as detailed herein. For instance, a bioactive glycan of pea fiber includes a compound of formula (I), wherein m is 0.14; n is 1; p is >0.1; and R1 is a pectic fragment:
Example 12 describes methods for obtaining a composition that is enriched for this bioactive glycan. Alternative purification methods may be used to obtain a composition that is enriched for this bioactive glycan. Alternatively, a chemically synthesized version of the bioactive glycan may be used.
b) Particle Design
Compounds of interest can be processed into a particle or attached to a core to make a particle (each instance “particle-bound compound”) by a variety of methods known in the art. The particles may be spherical or irregularly shaped. The particles may have a diameter across the widest portion of about 1 μm and about 100 μm, about 1 μm and about 50 μm, about 1 μm and about 25 μm, about 1 μm and about 15 μm, about 1 μm and about 10 μm, or about 1 μm and about 5 μm.
In some embodiments, a compound of interest or a plurality of compounds of interest may be incorporated into a core or layered over a core as a coating. Generally speaking, these cores or coatings may also comprise binders, lubricants, and/or other excipients that aid in compression, spheronization, granulation, extrusion or other methods known in the art for forming a particle. Without wishing to be bound by theory, incorporation of a compound of interest into a particle may affect the availability of the compound for members of the gut microbiota. Physical partitioning of a compound of interest to different locations within a particle may be a strategy to affect microbial access to and/or utilization of the compound.
In some embodiments, a compound of interest or a combination of compounds of interest are attached to a core. The core may be spherical or irregularly shaped, and typically comprises an inert polymer. Non-limiting examples of suitable cores include nonpareil spheres, latex beads, microcrystalline cellulose beads, silica beads, agarose beads, polystyrene beads or beads made from other polymers, quantum dots (including but not limited to quantum dots of small inorganic dye doped beads, such as those described at www.bangslabs.com/products/fluorescent-microspheres).
A suitable core may also be paramagnetic metal oxide particle comprising a paramagnetic core and an optional coating. The paramagnetic core may be a paramagnetic crystalline core composed of magnetically active metal oxide crystals which range from about 10 to about 500 angstroms in diameter. The cores may be uncoated or, alternatively, coated associated with a polysaccharide, a protein, a polypeptide, an organosilane or any composite thereof. By way of illustration, the polysaccharide coating may comprise dextran of varying molecular weights, the protein coating may comprise bovine or human serum albumin, and the organosilane coating may comprise an alkoxysilane or a halosilane. With coatings, the overall particle diameter may range from about 10 upward to about 5,000 angstroms. In the case of coated particles, the coatings can serve as a base to which a compound of interest or combination of compounds can be attached. In an exemplary embodiment, the core may be a paramagnetic particle comprising ferric oxide and a coating comprising an organosilane. Suitable paramagnetic particles are known in the art. See, for example, U.S. Pat. Nos. 4,695,392, 4,695,393, 4,770,183, 4,827,945, 4,951,675, 5,055,288, 5,069,216, and 5,219,554.
In certain embodiments, a core has a zwitterionic surface. For instance, if the surface of a core is modified by the addition of functional groups with a positive charge (e.g., a reactive amine), it may be desirable to further modify the surface with functional groups that carry a negative charge (e.g., a phosphonate), thereby creating a zwitterionic surface. Without wishing to be bound by theory, creating a zwitterionic surface as described above may reduce non-specific binding to the core's surface. A core's zeta potential can be used to monitor addition of functional groups, such that the zeta potential following derivatization is approximately the same as the zeta potential prior to any derivatization. In some embodiments, suitable cores may have a zeta potential of about −15 mV to about −35 mV, in some embodiments about −20 mV to about −35 mV, in some embodiments about −20 mV to about −30 mV, in some embodiments about −22 mV to about −30 mV, in some embodiments about −25 mV to about −30 mV.
The attachment of a compound of interest, or multiple compounds of interest, to a core is achieved by reaction of functional groups that are present on the exterior surface of the core (each a “surface functional group”) with a functional group on a compound of interest (or derivative thereof). As a result of such a reaction, a stable attachment is formed. As used herein, the terms “stable attachment” or “stably attached” refer to an attachment that remains substantially unaltered during transit through an intestinal tract of subject that lacks a gut microbiota (e.g., a germ-free animal) and/or can resist washing with 1% SDS/6M Urea/HNTB for 10 minutes at room temperature. Compounds of interest may be attached to a core through existing functional groups on the core and compound. Alternatively, the compound of interest and/or core may be derivatized with one or more functional group to produce more desirable properties—for instance, to generate a different reactive group for attachment and/or to add a spacer. A non-limiting example of a suitable spacer is an n PEG spacer, where n is an integer from 1 to 50 (inclusive), preferably 1 to 25 (inclusive), more preferably 1 to 10 (inclusive). Other spacers known in the art may also be used, including but not limited to peptide spacers. Numerous chemistries are known in the art that are suitable for the above purpose.
For instance, a compound of interest may be stably attached to a core via a biotin-avidin interaction. In some embodiments, a compound of interest may be derivatized with streptavidin and a core may be derivatized with biotin. In other embodiments, a compound of interest may be derivatized with biotin and a core may be derivatized with streptavidin. In various embodiments, the avidin protein may be a tetrameric avidin (e.g., chicken egg white avidin or a modified form thereof), a dimeric avidin from bacteria (e.g. streptavidin or a modified form thereof), or a monomeric avidin. In further embodiments, a spacer is present between the functional group (i.e. streptavidin or biotin) and the surface of the core or compound of interest.
In another example, a compound of interest may be stably attached to a core that is derivatized with one or more reactive nucleophile. Suitable nucleophiles include but are not limited to amines, hydroxyl amine, hydrazine, hydrazide, cysteine. In further embodiments, a zwitterionic surface may be generated after derivatization with one or more type of reactive nucleophile. Cores may be functionalized with reactive nucleophiles, and subsequent zwitterionic surfaces created, by methods known in the art or detailed in the examples. If a compound of interest does not have a functional group that is reactive with the nucleophile, the compound of interest can be derivatized with appropriate functional groups.
In another example, a compound of interest may be stably attached to a core that is derivatized with one or more type of reactive amine. In further embodiments, a zwitterionic surface may be generated after derivatization with one or more type of reactive amine. Cores may be functionalized with reactive amines, and subsequent zwitterionic surfaces created, by methods known in the art or detailed in the examples. If a compound of interest does not have a functional group that is reactive with an amine, the compound of interest can be derivatized with appropriate functional groups.
In an exemplary embodiment, a compound of interest with an eletrophilic functional group (e.g., aldehyde, ketone, cyano-ester, etc.) may be stably attached to a core functionalized with one or more reactive nucleophile (e.g., an amine, a hydroxyl amine, a hydrazine, a hydrazide, a cysteine, etc.). The electrophile may be naturally occurring in the compound of interest (e.g. the reducing end chemistry of a polysaccharide) or may be created by derivatization (e.g., creating aldehydes from vicinal hydroxyls by sodium periodate oxidation, creating cyano-esters from the hydroxyls naturally present, etc.). The reaction between the electrophile and the nucleophile will form a bond that may or may not need further chemistry applied to it. For instance, reaction of an amine with the reducing end of a polysaccharide yields an imine that needs to be reduced with a hydride donor to create a stable bond, a reaction termed reductive amination. Reaction of an amine with a cyano-ester yields an isourea that also can be reduced with a hydride donor to form a stable bond. Reaction with a stronger nucleophile (e.g., hydroxyl amine, hydrazide, etc.) forms other intermediates (i.e., hydrazide reaction forms a hydrazone) that may or may not require reduction.
In an exemplary embodiment, the core is a silica particle or a particle comprising a silica coating (e.g., a paramagnetic particle comprising a silica coating, etc.). Surface modification of silica particles is commonly achieved by reaction with an alkoxysilane or halosilane. Alkoxysilanes will bind forming 1-3 Si—O—Si links to the surface in a condensation reaction with the surface silanol groups. The halosilanes will typically hydrolyze substituting the halide for alcohol group which can similarly undergo condensation forming 1-3 Si—O—Si links with surface silanol groups. In anhydrous conditions, halosilanes will react directly with surface silanol groups. A wide variety of alkoxysilanes/halosilanes are commercially available. Suitable alkoxysilanes/halosilanes include but are not limited to 3-aminopropyl triethoxysilane (APTS) and 3-mercaptopropyl trimethoxysilane (MPTS). APTS and MPTS allow for facile linker chemistry with other frequently used linking moieties such as n-hydroxysuccinide (NHS) functionalized molecules, isothiocynates, cyano-esters, malemides, etc. These linking moieties may be present on a compound of interest. For instance, cores functionalized with APTS may be reacted with CDAP-activated polysaccharides. Alternatively, these linking moieties may be used to attach further functional groups to the core. For instance, cores functionalized with APTS may be reacted with amine-reactive biotin conjugates or amine-reactive streptavidin conjugates to create a core derivative with biotin or streptavidin, respectfully.
In further embodiments, one or more compounds of interest may be stably attached to a core using the any of the chemistries described herein in a manner that creates multiple layers. For instance, a core functionalized with a reactive amine may be reacted with a compound of interest with a reducing chemistry to create an initial bond that is then reduced to form a stable bond, thereby creating a core with a first layer of a compound of interest (“the layered core”). A second layer comprising the same or different compound of interest may be produced by either using existing reactive groups present in the first layer or creating new reactive groups in the first layer, and then reacting a compound of interest with the appropriate chemistry to from a core layered with a first and then a second compound of interest. Alternative designs are also encompassed by the present disclosure. For instance, each layer may or may not differ in terms of the compounds of interest, the absolute amount of each compound, the ratio of compounds in a given layer, etc.
Those having ordinary skill in the art, in light of this specification, will realize that depending on the nature of the functional groups that are present on the surface of the beads and the nature of the functional groups that are present on the compound of interest (or derivative thereof), other types of interactions may occur via which compounds of interest can be stably attached to a core. Multiple types of chemistries may also be used. Choice of a suitable chemistry may also be influenced, in part, by a physical property of the compound of interest. For instance, certain chemistries are more amendable to compounds that are water soluble, or partially water soluble, whereas other chemistries are more amendable to compounds that are typically insoluble.
The amount of a compound of interest attached to a core can vary. For instance, the conjugation chemistry and the type of compound may affect the amount of compound attached. Generally, at least about 0.5 pg of a compound of interest is attached to a core. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 5 μg. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 1 μg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1 μg. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 0.5 μg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 0.5 μg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 0.1 μg. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 50 ng. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 50 ng. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 10 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1 ng. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 500 pg. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 100 pg. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 50 pg.
In each of the above embodiments, the core further comprises a tag that facilitates recovery of particles from biological material obtained from a subject, following administration of the particles to the subject. Said tag may be incorporated into the core itself, attached to the exterior surface of the core, layered over the core as a coating, or any combination thereof. When attached to the exterior surface of the core, attachment may occur using the same or a different chemistry than used to attach compounds of interest. Suitable tags include metals, fluorescent compounds, quantum dots, biotin, peptides, and nucleic acids, among others. In some examples, the tag is a purification or affinity tags (e.g., CBP, FLAG-tag, GST, HA-tag, HBH, MBP, Myc, E-tag, NE-tag, S-tag, TAP, V5, AviTag, SBP, Strep-tag, polyhistidine, polyarginine, polyglutamine, thioredoxin-tag, etc.). In other examples, the tag is a metal oxide or other magnetic or paramagnetic material, typically incorporated into the core. As is known in the art, magnetic and paramagnetic particles may have a variety of different structures. For instance, magnetic particles may be distributed in a volume of a polymer matrix, magnetic particles may form a shell around a polymer core, magnetic particles may form a core that is surrounded by a polymer shell, or combinations thereof. See, for instance, Philippova et al., European Polymer Journal, 2011, 47: 542-559. Non-limiting examples of magnetic cores that may be used include Dynabeads® (Dynal AS, Oslo, Norway), MagMax™ beads (Applied Biosystems, Foster City, Calif.), BioMag® beads (Polysciences, Inc., Warrington, Pa.) BcMag™ beads (BioClone Inc., San Diego, Calif.), PureProteome™ magnetic beads (Millipore Corporation), or the like.
c) Optional Labels
Particles of the present disclosure may further comprise a label. One or more labels may be incorporated into a particle, attached to a particle, or attached to the compound of interest by methods known in the art. Preferably, addition of a label does not substantially alter the transit time of a particle through a subject's intestinal tract. Non-limiting examples of suitable labels include fluorescent compounds, quantum dots, biotin, polynucleotide sequences, radioisotopes and purification or affinity tags (e.g., CBP, FLAG-tag, GST, HA-tag, HBH, MBP, Myc, E-tag, NE-tag, S-tag, TAP, V5, AviTag, SBP, Strep-tag, polyhistidine, polyarginine, polyglutamine, thioredoxin-tag, etc.). Use of a label facilitates further separation of recovered particles for downstream analyses or imaging. As such, the label should be different than the tag described in Section I(b). For instance, if the tag is a first fluorochrome, the label should be a second fluorochrome. The method used to attach a label to a particle may be the same or different than the method used to attach a compound of interest to the particle.
d) Exemplary Embodiments
In one example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 2 or more glycans, a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, pea fiber, citrus peel, yellow mustard, soy cotyledon, orange fiber (coarse), orange fiber (fine), orange peel, tomato peel, inulin (low molecular weight), potato fiber, apple pectin, sugar beet fiber, oat hull fiber, acacia extract, inulin (high molecular weight), barley beta-glucan, barley bran, oat beta-glucan, apple fiber, rye bran, barley malted, wheat bran, wheat aleurone, maltodextrin (including but not limited to resistant maltodextrin), psyllium, cocoa, citrus fiber, tomato pomace, rice bran, chia seed, corn bran, soy fiber, sugar cane fiber, resistant starch 4. In each of the above embodiments, the glycan(s) are attached to the core either directly or indirectly, preferably by an irreversible interaction. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or directly or indirectly attached to the core or the glycan via the same method used with the glycan(s) or a different method.
In another example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, pea fiber, citrus peel, yellow mustard, soy cotyledon, orange fiber (coarse), orange fiber (fine), orange peel, tomato peel, inulin (low molecular weight), potato fiber, apple pectin, sugar beet fiber, oat hull fiber, acacia extract, inulin (high molecular weight), barley beta-glucan, barley bran, oat beta-glucan, apple fiber, rye bran, barley malted, wheat bran, wheat aleurone, maltodextrin (including but not limited to resistant maltodextrin), psyllium, cocoa, citrus fiber, tomato pomace, rice bran, chia seed, corn bran, soy fiber, sugar cane fiber, resistant starch 4. In each of the above embodiments, the glycan(s) are attached to the core via an avidin-biotin interaction, preferably a streptavidin-biotin interaction. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or attached to the core or the glycan via an avidin-biotin interaction (the same or different than used with the glycan(s)) or by other methods known in the art.
In another example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, pea fiber, citrus peel, yellow mustard, soy cotyledon, orange fiber (coarse), orange fiber (fine), orange peel, tomato peel, inulin (low molecular weight), potato fiber, apple pectin, sugar beet fiber, oat hull fiber, acacia extract, inulin (high molecular weight), barley beta-glucan, barley bran, oat beta-glucan, apple fiber, rye bran, barley malted, wheat bran, wheat aleurone, maltodextrin (including but not limited to resistant maltodextrin), psyllium, cocoa, citrus fiber, tomato pomace, rice bran, chia seed, corn bran, soy fiber, sugar cane fiber, resistant starch 4. In each of the above embodiments, the glycan(s) are derivatized to generate cyano-esters from the hydroxyls naturally present and the derivatized glycan(s) are attached to cores comprising amine functional groups on the surface. In still further embodiments, the cores are also functionalized with phosphonates. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or attached to the core or the glycan via the amine functional groups on the core's surface (using the same or different chemistry than used with the glycan(s)) or by other methods known in the art.
In another example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, pea fiber, citrus peel, yellow mustard, soy cotyledon, orange fiber (coarse), orange fiber (fine), orange peel, tomato peel, inulin (low molecular weight), potato fiber, apple pectin, sugar beet fiber, oat hull fiber, acacia extract, inulin (high molecular weight), barley beta-glucan, barley bran, oat beta-glucan, apple fiber, rye bran, barley malted, wheat bran, wheat aleurone, maltodextrin (including but not limited to resistant maltodextrin), psyllium, cocoa, citrus fiber, tomato pomace, rice bran, chia seed, corn bran, soy fiber, sugar cane fiber, resistant starch 4. In each of the above embodiments, the core is functionalized with APTS and the glycan(s) are CDAP-activated. In still further embodiments, the cores are also functionalized with phosphonates. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or attached to the core or the glycan via the amine functional groups on the core's surface (using the same or different chemistry than used with the glycan(s)) or by other methods known in the art.
In another example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, orange fiber (coarse), orange (fine), inulin, pea fiber, sugar beet fiber, soy cotyledon, yellow mustard bran, and barley bran. In each of the above embodiments, the glycan(s) are attached to the core either directly or indirectly, preferably by an irreversible interaction. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or directly or indirectly attached to the core or the glycan via the same method used with the glycan(s) or a different method.
In another example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, orange fiber (coarse), orange (fine), inulin, pea fiber, sugar beet fiber, soy cotyledon, yellow mustard bran, and barley bran. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or attached to the core or glycan via an avidin-biotin interaction (the same or different than used with the glycan(s)) or by other methods known in the art.
In another example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, orange fiber (coarse), orange (fine), inulin, pea fiber, sugar beet fiber, soy cotyledon, yellow mustard bran, and barley bran. In each of the above embodiments, In each of the above embodiments, the core is functionalized with APTS and the glycan(s) are CDAP-activated. In still further embodiments, the cores are also functionalized with phosphonates. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or attached to the core or the glycan via the amine functional groups on the core's surface (using the same or different chemistry than used with the glycan(s)) or by other methods known in the art.
In another example, an artificial food particle comprises a core comprising a tag, one or more glycans, and an optional label. In some embodiments, a particle has a single glycan. In other embodiments, a particle has a combination of 5 or more glycans, a combination of 10 or more glycan, or a combination of 20 or more glycans. In other embodiments, a particle has a combination of two to twenty glycans. In other embodiments, a particle has a combination of two to ten glycans. In any of the aforementioned embodiments, the glycan may be a polymer that is a homo- or heteropolymer consisting of two or more monosaccharides linked glycosidically. As such, the glycan is understood to not contain any modifications (e.g., the glycan is not a glycoconjugate of any kind). In still other embodiments, a particle has a combination of glycans obtained from a fiber preparation. In certain embodiments, the fiber preparation is selected from citrus pectin, orange fiber (coarse), orange (fine), inulin, pea fiber, sugar beet fiber, soy cotyledon, yellow mustard bran, and barley bran. In each of the above embodiments, the glycan(s) are derivatized to generate cyano-esters from the hydroxyls naturally present and the derivatized glycan(s) are attached to cores comprising amine functional groups on the surface. In still further embodiments, the cores are also functionalized with phosphonates. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to a core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to a core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or attached to the core or the glycan via the amine functional groups on the core's surface (using the same or different chemistry than used with the glycan(s)) or by other methods known in the art.
In any of the aforementioned embodiments, the glycan polymer(s) may be a polymer that has been modified, whether naturally or otherwise; non-limiting examples of such modifications include acetylation, alkylation, esterification, etherification, oxidation, phosphorylation, selenization, sulfonation, or any other manipulation.
In any of the aforementioned embodiments, the particle may comprise one layer of glycans or more than one layer glycans. As described above, the glycans can be arranged in a variety of different patterns when multiple layers are present.
In another example, an artificial food particle comprises a core, at least one compound of interest, and a label, wherein the core is a paramagnetic particle comprising a silica coating. In some embodiments, a particle comprises one compound of interest. In other embodiments, a particle comprises a combination of 5 or more compounds of interest, a combination of 10 or more compounds of interest, or a combination of 20 or more compounds of interest. In other embodiments, a particle comprises a combination of two to twenty compounds of interest. In other embodiments, a particle comprises a combination of two to ten compounds of interest. In certain embodiments, one or more of the compounds of interest are a biomolecule. In some examples, each compound of interest is a glycan. In still further examples, the core is functionalized with an organosilane reagent, which is optionally APTS, and the glycan(s) are CDAP-activated, and the cores are optionally functionalized with phosphonates. In some embodiments, the amount of the compound of interest attached to the core is about 0.5 pg to about 500 ng, or about 0.5 pg to about 50 ng, or about 0.5 pg to about 5 ng. In some embodiments, the amount of the compound of interest attached to the core is about 0.5 pg to about 500 pg, or about 0.5 pg to about 50 pg. In some embodiments, the amount of the compound of interest attached to the core is about 1 pg to about 1000 pg, or about 1 pg to about 100 pg, or about 1 pg to about 50 pg. When present, the label can be incorporated into the core, or attached to the core or the glycan via the amine functional groups on the core's surface (using the same or different chemistry than used with the glycan(s)) or by other methods known in the art.
In an additional aspect, the present disclosure provides compositions comprising a plurality of artificial food particles. Suitable artificial food particles are described in Section I, the disclosures of which are incorporated into this section by reference. Compositions may comprise a plurality of particles that are compositionally identical or may comprise a plurality of particles of different types. Particles of different types differ in one more aspects including but not limited to the compounds of interest, particle design (e.g., compounds incorporated into a core, coating a core, or attached to a core), the type of core, the label (if present), and the chemistry used to stably attach a compound of interest and/or a label to a core.
In certain embodiments, the present disclosure provides a composition comprising a plurality of particles of more than one type, each type of particle comprising a unique compound of interest or combination of compounds of interest, and a unique label. In exemplary embodiments, all the particles have the same general design, meaning all the particles have the compound(s) of interest either incorporated into a core, or coating a core, or attached to a core. However, in embodiments where the compound(s) of interest are attached to a core, the type of core and the chemistry used to stably attach the compound of interest and/or the label to the core may vary between particle types.
In further embodiments, the present disclosure provides a composition comprising a plurality of particles of more than one type, each type of particle comprising a core, a compound of interest or combination of compounds of interest, and a unique label, wherein the compound(s) of interest and label are stably attached to the core. In various embodiments, the core may be the same between types of particles, may differ between particles, or a combination thereof. In each of the aforementioned embodiments, the chemistry used to stably attach the compound of interest and/or the label to the core may vary or be the same between particle types.
In still further embodiments, the present disclosure provides a composition comprising a plurality of particles of more than one type, each type of particle comprising a core, a glycan or combination of glycans, and a unique label, wherein the glycan(s) and label are stably attached to the core. In various embodiments, the core may be the same between types of particles, may differ between particles, or a combination thereof. In each of the aforementioned embodiments, the chemistry used to stably attach the glycan(s) and/or the label to the core may vary or be the same between particle types.
The number of particle types in a composition is not limited. For instance, compositions of the present disclosure may comprise 5 or fewer particle types, 10 or fewer particle types, 15 or fewer particle types, 20 or fewer particle types, 30 or fewer particle types 40 or fewer particle types 50 or fewer particle types, or more than 50 particle types.
The number of particles in each composition can vary. In embodiments comprising a plurality of particles of more than on type, compositions may contain an equal number of particles for each particle type. Alternatively, compositions may contain different numbers of particles for each particle type. In another alternative, compositions may contain a number of particles for each particle type such that the compounds of interest are provided in approximately the same amount.
Compositions of the present disclosure may be formulated for oral administration, and may further comprise inert excipients. Oral preparations may be enclosed in gelatin capsules or compressed into tablets. Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the composition may further comprise various sweetening, flavoring, coloring, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof. Oral preparations may also be formulated to provide immediate release, time-released, pH-dependent release or enteric release of the particles.
Compositions of the present disclosure may be formulated as a liquid. Liquid preparations are formulated for oral administration, and may be aqueous or oily suspensions, emulsions, syrups, or elixirs. Such liquid formulations may further comprise various sweetening, flavoring, coloring, emulsifying, suspending agents, and/or preservatives, as well as diluents or nonaqueous vehicles. Suspending agent include, but are not limited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Diluents include, but are not limited to, water, ethanol, glycerin, and combinations thereof. Nonaqueous vehicles include, but are not limited to, edible oils, almond oil, fractionated coconut oil, oily esters, propylene glycol, and ethyl alcohol.
Compositions of the present disclosure may also be formulated as a solid by methods known in the art. Solid formulations may be a tablet; a caplet; a pill; a powder such as a sterile packaged powder, a dispensable powder, and an effervescent powder; a capsule including both soft or hard gelatin capsules; a lozenge; a sachet; a sprinkle; a reconstitutable powder or shake; a troche; a pellet; a granule; a semisolid or a gel. Compositions formulated as a solid may be fast disintegrating. Compositions formulated as a solid may provide immediate release, sustained release, enteric release, time-delayed release, or combinations thereof.
In an additional aspect, the present disclosure provides a method to measure modifications that occur to a compound of interest after oral administration to a subject. In one embodiment, the method comprises: (a) orally administering to a subject a composition of Section II, wherein structural information and/or amount of the particle-bound compound(s) of interest is known (the “input data”), (b) recovering particles from biological material obtained from the subject, and (c) identifying structural changes to the recovered particle-bound compound(s) of interest and/or measuring the amount of the recovered particle-bound compound(s) of interest (the “recovered data”) and determining the difference between the recovered data and the input data. In another embodiment, the method comprises: (a) admixing, ex vivo, a composition of Section II and a sample of the subject's gut microbiota, wherein structural information and/or amount of the particle-bound compound(s) of interest is known (the “input data”), (b) recovering particles from the admixture after a suitable amount of time (e.g., hours or days), and (c) identifying structural changes to the recovered particle-bound compound(s) of interest and/or measuring the amount of the recovered particle-bound compound(s) of interest (the “recovered data”) and determining the difference between the recovered data and the input data. In another embodiment, the method comprises: (a) admixing a composition of Section II to an in vitro culture of one or more gut microbial strains, wherein structural information and/or amount of the particle-bound compound(s) of interest is known (the “input data”), (b) recovering particles from the admixture after a suitable amount of time (e.g., hours or days), and (c) identifying structural changes to the recovered particle-bound compound(s) of interest and/or measuring the amount of the recovered particle-bound compound(s) of interest (the “recovered data”) and determining the difference between the recovered data and the input data. The modification may be cleavage, degradation (partial or complete), acetylation, alkylation, deamidation, deglycosylation, delipidation, esterification, etherification, glucuronidation, glycosylation, hydrolysis, lipidation, methylation, methylesterification, oxidation, phosphorylcholination, phosphorylation, proteolysis, reduction, ring opening, selenization, sulfation, sulfonation, or any other manipulation. Compositions may be orally administered by methods known in the art, which for the avoidance of doubt, includes but is not limited to buccal administration, sublabial administration, sublingual administration, and by gavage.
In certain embodiments, a composition of Section II is a composition comprising a plurality of particles of more than one type, each type of particle comprising a unique compound of interest or combination of compounds of interest, and a unique label. After administering said composition to a subject, the method comprises recovering the particles from biological material obtained from the subject and then separating the recovered particles by type; and for each type of particle, measuring the amount of the compound(s) of interest on the recovered particles (the “recovered amount”) and calculating the difference between the recovered amount and the input amount.
Preferred subjects are humans or nonhuman animals. In some embodiments, a subject is a human. In other embodiments, the subject is a non-human mammal, a bird, a fish, a reptile, or an amphibian. In various embodiments, the nonhuman animal may be a companion animal (e.g., dog, cat, etc.), a livestock animal (e.g., cow, pig, horse, sheep, goat, etc.), a zoological animal, or a research animal (e.g., a non-human primate, a rodent, etc.). In one example, the subject is a germ-free mouse. In another example, the subject is a germ-free mouse that was colonized with a consortium of bacterial strains. In a further example, the subject is a germ-free mouse that was colonized with intact uncultured microbiota from a human donor. In still a further example, the subject is a germ-free mouse that was colonized with intact uncultured microbiota from a human donor in need of a dietary intervention. Human subjects in need of a dietary intervention may be a subject that consumes a diet high in saturated fat and/or low in fruits and vegetables, a subject that is overweight or obese, a subject diagnosed with a disease including but not limited to type I diabetes, type II diabetes, cardiovascular disease, a neurological disease, a neurodegenerative disease, or an inflammatory disease.
When the subject has a gut microbiota, the modification(s) to the compound of interest are typically mediated, at least in part if not completely, by the subject's gut microbiota. As such, in embodiments where the subject has a gut microbiota, the present disclosure provides a method to measure gut microbiota-mediated modifications that occur to a compound of interest after oral administration to a subject. When a modification is solely dependent upon gut microorganisms (i.e., due to the functional activity of a gut microbiota), then the difference between the input data and the recovered data is the gut microbiota-dependent modification, which is a measure of the gut microbiota's functional activity. Germ-free animals can be used to evaluate the contribution of any microbiota-independent modifications, and this contribution (if present) can be removed from the final measurement.
The results from the aforementioned methods may be used to characterize the functional state of a subject's gut microbiota/microbiome, which may then be compared to an earlier measurement for the same subject or an average measurement for a suitable comparator (e.g., healthy subjects, subjects with a similar health/disease status, etc.). In this way, the methods may provide a personalized measure of in vivo microbiome activity and health characteristics that may aide in diagnosis of a disease, influence prognosis and/or guide medical treatment, enable personalized food design or nutrition guidance, or allow for other actions to improve the subject's health. For example, the aforementioned methods may be used to measure disease state biomarkers comprising microbiota activity and/or structural information regarding the microbiota/microbiome. As another example, the aforementioned methods may be used to measure the effect of a drug or other therapeutic intervention on microbiota function in order to improve dosing, efficacy and/or adherence. As another example, the aforementioned methods may be used to measure microbiota functional activity restoration following acute surgery or antibiotic administration in order to enable early identification and prevention of adverse events that often require readmission. The above uses are non-limiting, and are intended to only illustrate the scope uses encompassed by the present disclosure.
In various embodiments, the aforementioned methods may further comprise quantifying at least one additional aspect of the subject's gut microbiota and/or the subject's health before, after, or before and after administering a composition of Section II. Non-limiting examples of an additional aspect of the subject's gut microbiota that may be quantified include changes in the representation of bacterial taxa, genes encoding carbohydrate-active enzymes (CAZymes) and/or polysaccharide utilization loci (PULs), and/or genes encoding proteins and enzymes in various metabolic pathways, as well as changes in the abundance of proteins encoded by one or more bacterial PUL, abundance of CAZYmes, abundance of all Firmicutes, abundance of a subset of Firmicutes species, proportional representation of all Firmicutes, proportional representation of a subset Firmicutes species, abundance of all Bacteroides species, abundance of a subset of Bacteroides species, proportional representation of all Bacteroides species, proportional representation of a subset Bacteroides species, and microbial metabolites.
Biological material obtained from a subject administered the composition may be a blood sample or, more preferably, cecal or fecal matter. Biological material may be used immediately or may be frozen and stored indefinitely. A skilled artisan will appreciate that the amount of biological material needed may vary depending upon a variety of factors, including the amount of the composition administered, the type of tag and/or the type of label, as well as the amount of compound, label or tag per particle.
In one example of the aforementioned embodiments, a method to measure glycan degradation comprises (a) orally administering to a subject a composition comprising a plurality of particles of one type, the particles comprising a core, a glycan or combination of glycans, and a label, wherein the glycan(s) and label are stably attached to the core, and wherein the amount of particle-bound glycan is known (the “input data”); (b) recovering particles from biological material obtained from the subject; and (c) measuring the amount of particle-bound glycan for the recovered particles (the “recovered data”) and calculating the amount of glycan degraded, which is the difference between the input data and recovered data.
In another example, a method for measuring glycan degradation comprises (a) orally administering to a subject a composition comprising a plurality of particles of more than one type, each type of particle comprising a core, a glycan or combination of glycans, and a unique label, wherein the glycan(s) and label are stably attached to the core, and wherein the amount of bead-bound glycan per particle type is known (the “input data”); (b) recovering particles from biological material obtained from the subject and then separating the recovered particles by type; and (c) for each recovered particle type, measuring the amount of glycan per particle type (the “recovered data”) and calculating the amount of glycan degraded, which is the difference between the input data and recovered data. In various embodiments, the core may be the same between types of particles, may differ between types of particles, or a combination thereof. In each of the aforementioned embodiments, the chemistry used to stably attach the glycan(s) and/or the label to the core may vary or be the same between particle types. In certain examples of each of the aforementioned embodiments, one or more type of particle comprises a combination of glycans obtained from a fiber preparation.
In each of the above embodiments, particle-bound glycan may be measured by GC-MS after the glycans are release from the cores, as described in the Examples. Briefly, particle-bound glycans are released from the core (e.g., by acid hydrolysis) and the mass of each monosaccharide detected in a sample of each type of bead can be determined by GC-MS and this mass then divided by the final count of beads in each sample to produce a measurement of mass of recoverable monosaccharide per bead. Through routine experimentation, the types of monosaccharaides detected can be optimized. Other methods known in the art may also be used. For instance, other instrumentations such as LC-MS, HPLC, or HPAE-PAD may be used. Alternatively or in addition, any analytical method that quantifies monosaccharides may be used.
In each of the above embodiments, the input data may include structural information about the glycans, in addition to or as an alternative to the amount of particle-bound glycan per particle type. The Examples describe, for instance, methods to analyze carbohydrate linkage analysis. Without wishing to be bound by theory, potentially important information about the ability of an individual's gut microbiota to process specific linkages within a glycan may be missed by a monosaccharide analysis of particle-bound glycan. Methods are also known in the art to analyze other types of glycan modifications, including but not limited to amino-modification, acetylation, alkylation, esterification, etherification, methylation, methylesterification, oxidation, phosphorylcholination, phosphorylation, ring-opening, selenization, sulfation and sulfonation.
A skilled artisan will appreciate that degradation and/or modification of other compounds of interest (e.g., other biomolecules or drugs) may be also measured in view of the disclosures in Section II, the Examples, and methods known in the art to measure degradation or other structural changes to drugs, proteins, lipids, nucleic acids, etc.
In an additional aspect, the present disclosure provides a method to recruit gut microorganisms in vivo, and optionally isolate them. The method comprises: orally administering to a subject a composition of Section II, and optionally recovering particles from biological material obtained from the subject and isolating DNA from the recovered beads and then sequencing the DNA to identify the particular species of microbes that were bound to the recovered beads.
In some embodiments, recruiting gut microorganisms in vivo to a food particle may be used to create novel microenvironments in vivo. For instance, a food particle may comprise two or more types of glycans in order to recruit particular bacterial taxa with complementary functional activities. In another example, a food particle may comprise a biomolecule that a particular bacterial species metabolizes and a drug toxic to the bacterial species, in order to recruit the bacterial species to be in physical proximity to the drug.
In embodiments where the particles are recovered in order to isolate gut microorganisms, isolating gut microorganisms may be used to better understand or define the fiber degrading capacity a subject's gut microbiota. The “fiber degrading capacity” of a subject's gut microbiota is defined by its compositional state, specifically the absence, presence and abundance of primary and secondary consumers of dietary fiber. Microbes that are primary consumers initiate degradation of dietary fibers, while secondary consumers utilize glycans that are released by primary consumers. Without wishing to be bound by theory, stratification of particle-associated microbial communities may be seen with recovered particles. For instance, the most closely adherent microorganisms may include primary consumers, while more loosely adherent microorganisms may include secondary consumers. Alternatively, stratification may not be observed. By using different particles that have different compounds of interest, or compounds of interest arranged within the particle in varying manners, it is possible to evaluate how the availability of a compound (or access to a compound) affects the relationship between primary consumers, secondary consumers, or primary consumers and secondary consumers.
In certain embodiments, the method may further comprise an additional sorting step to enrich for microbe-bound beads. For instance, in step (b), the biological material (or a fraction thereof) may be treated with a DNA or protein stain prior to recovering the particles, and the recovered particles may be further sorted to select those recovered particles labeled with the stain. In an alternative approach, after recovering particles from biological material obtained from the subject, the recovered particles may be treated with a DNA or protein stain and the treated particles may be further sorted to select those recovered particles labeled with the stain. Non-limiting examples of suitable DNA and protein stains include Propidium iodide, DAPI, 7AAD, Syto DNA dyes (Invitrogen), LIVE/DEAD (Invitrogen). Alternatives to DNA stains may also be used. For instance, antibodies, aptamers, or other reagents may be used to specifically label microbial specific proteins, RNA, lipids, and/or carbohydrates.
In an additional aspect, the present disclosure provides methods to measure one or more changes in a subject's gut microbiota. The change measured may be a change in the functional state and/or compositional state of the gut microbiota/microbiome. In one embodiment, the method comprises measuring at least one microbe-mediated modification at a first time and at a second time, and calculating the difference between the obtained values to measure the change in the subject's gut microbiota. Methods to measure microbe-mediated modification(s) are detailed in Section III and incorporated into this section by reference. In another embodiment, the method comprises isolating gut microorganisms at a first time and at a second time, and calculating the difference (either absolute or relative) between the isolated organisms to measure the change in the subject's gut microbiota and/or microbiome. Methods to isolate gut microorganisms are detailed in Section IV and incorporated into this section by reference. In each embodiment, the amount of time that elapses between the first and second measurement may vary. For instance, the amount of time may be hours, days, weeks, or even months.
In various embodiments, the aforementioned methods may be used to test the effect of a compound, a drug, a food, a food ingredient, a nutritional supplement (e.g., a fiber preparation, a prebiotic, a probiotic, a vitamin supplement, a mineral supplement, combinations thereof, etc.), an herbal remedy, a lifestyle modification, or a behavioral modification on the compositional and/or functional state of a subject's gut microbiota. For instance, the aforementioned methods may further comprise a step between the first and second measurement, or between isolation of gut microorganisms the first and second time, wherein the subject is administered a compound, a drug, a food, a food ingredient, a nutritional supplement, or an herbal remedy. Alternatively, or in addition, the method may further comprise a step between the first and second measurement, or between isolation of gut microorganisms the first and second time, wherein the subject engages in a lifestyle or behavioral modification. Non-limiting examples of lifestyle or behavior modifications include increased or decreased exercise, increased or decreased amounts of relaxation, increased or decreased caloric intake, increased or decreased fiber intake, increased or decreased fruit and/or vegetable consumption, increased or decreased fat consumption, increased or decreased alcohol consumption, or the like.
The first measurement or isolation is typically used to establish a baseline or starting condition. This may occur immediately prior to the lifestyle or behavioral modification, or administering the item to be tested, or at a reasonable time before as determined by one of skill in the art through routine experimentation. Similarly, the second measurement or isolation may occur immediately after the lifestyle or behavioral modification, or administering the item to be tested, or at a reasonable time before as determined by one of skill in the art through routine experimentation (e.g., hours, days, or weeks). In various embodiments, the lifestyle or behavioral modification or administration of the item to be tested may occur once or more than once between the first and second measurement/first and second isolation.
In one example, the present disclosure provides a method to test the effect of a food, a food ingredient, or a nutritional supplement on the functional state of a subject's gut microbiota, the method comprising (a) at a first time, measuring degradation of at least one biomolecule of interest according to the method of Section III, (b) administering an amount of a food, a food ingredient, or a nutritional supplement to the subject, (c) at a second time, after the administration of the food, repeating the measurement of step (a), and (d) calculating the difference between the values obtained from step (c) and step (a). In some embodiments, the food, food ingredient, or nutritional supplement is administered daily, and the second measurement occurs within 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the food, food ingredient, or nutritional supplement is administered daily, and the second measurement occurs within 6, 7, 8, 9, 10, or 11 hours. In some embodiments, the food, food ingredient, or nutritional supplement is administered daily, and the second measurement occurs in about 1 to 12 hours or 12 to 24 hours. In some embodiments, the food, food ingredient, or nutritional supplement is administered daily, and the second measurement occurs about 1, 2, 3, 4, 5, or 6 days later. In some embodiments, the food, food ingredient, or nutritional supplement is administered daily, and the second measurement occurs about a week later. In each of the above embodiments, the food, food ingredient, or nutritional supplement may be administered multiple times a day, rather than once a day. Alternatively, the food, food ingredient, or nutritional supplement may be administered less frequently (e.g., every other day, once a week, etc.).
In one example, the present disclosure provides a method to test the effect of a lifestyle or behavioral modification on the functional state of a subject's gut microbiota, the method comprising (a) at a first time, measuring degradation of at least one biomolecule of interest according to the method of Section III, (b) performing a lifestyle or behavioral modification, (c) at a second time, after the lifestyle or behavioral modification, repeating the measurement of step (a), and (d) calculating the difference between the values obtained from step (c) and step (a). In some embodiments, the lifestyle or behavioral modification occurs daily, and the second measurement occurs within 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the lifestyle or behavioral modification occurs daily, and the second measurement occurs within 6, 7, 8, 9, 10, or 11 hours. In some embodiments, the lifestyle or behavioral modification occurs daily, and the second measurement occurs in about 1 to 12 hours or 12 to 24 hours. In some embodiments, the lifestyle or behavioral modification occurs daily, and the second measurement occurs about 1, 2, 3, 4, 5, or 6 days later. In some embodiments, the lifestyle or behavioral modification occurs daily, and the second measurement occurs about a week later. In each of the above embodiments, the lifestyle or behavioral modification may occur multiple times a day, rather than once a day. Alternatively, the lifestyle or behavioral modification may occur less frequently (e.g., every other day, once a week, etc.).
In another example, the present disclosure provides a method to test the effect of the functional state of a subject's gut microbiota on a drug, the method comprising (a) at a first time, measuring degradation of the drug according to the method of Section III, wherein the drug is the compound of interest, (b) administering a pharmaceutical composition comprising the drug to the subject, (c) at a second time, after the administration of the pharmaceutical composition, repeating the measurement of step (a), and (d) calculating the difference between the values obtained from step (c) and step (a). In some embodiments, the pharmaceutical composition is administered daily, and the second measurement occurs within 1, 2, 3, 4, 5, or 6 hours. In some embodiments, the pharmaceutical composition is administered daily, and the second measurement occurs within 6, 7, 8, 9, 10, or 11 hours. In some embodiments, the pharmaceutical composition is administered daily, and the second measurement occurs in about 1 to 12 hours or 12 to 24 hours. In some embodiments, the pharmaceutical composition is administered daily, and the second measurement occurs about 1, 2, 3, 4, 5, or 6 days later. In some embodiments, the pharmaceutical composition is administered daily, and the second measurement occurs about a week later. In each of the above embodiments, the pharmaceutical composition may be administered multiple times a day, rather than once a day. Alternatively, the pharmaceutical composition may be administered less frequently (e.g., every other day, once a week, etc.).
In another aspect, the present disclosure provides methods to develop and test microbiota-directed foods. A “microbiota-directed food,” as used herein, refers to a food that selectively promotes the representation and expressed beneficial functions of targeted human gut microbes.
For instance, the methods of Section III, Section IV, or Section V may be used to directly characterize how gut microorganisms with distinct, as well as overlapping, nutrient harvesting capacities respond to different food ingredients, or combinations of food ingredients, and use this information to develop a microbiota-directed food. As a non-limiting example, the methods of Section III, Section IV, or Section V may be used to test a plurality of biomolecules of the same type (e.g., arabinan) that have different molecular structures to identify bioactive component(s) to include in a microbiota-directed food (i.e., the structure(s) that are preferentially utilized by targeted gut microbiota). As another non-limiting example, the methods of Section III, Section IV, or Section V may be used to screen a food ingredient (e.g., pea fiber, fish oil, hydrolyzed whey protein isolate, etc.) provided by different suppliers to identify a source that maximizes the representation and/or expressed beneficial functions of targeted human gut microbes.
The methods of Section III, Section IV, or Section V may also be used to directly characterize how gut microorganisms with distinct, as well as overlapping, nutrient harvesting capacities respond to a potential microbiota-directed food and use this information to modify the composition of the microbiota-directed food to maximize the desired effect (e.g. maximizes the representation and/or expressed beneficial function(s) of targeted human gut microbes). As a non-limiting example, the methods of Section III, Section IV, or Section V may be used iteratively to test, refine/modify, retest, refine/modify, retest etc. a microbiota-directed food.
The methods of Section III, Section IV, or Section V may also be used to create a personalized microbiota-directed food for a given subject. As a non-limiting example, the methods of Section III, Section IV, or Section V may be used to directly characterize the compositional and/or functional state of a subject's gut microbiota and use this information to develop or select an appropriate microbiota-directed food to promotes the representation and expressed beneficial functions of targeted human gut microbes that will improve the health or well-being of that subject.
The following examples illustrate various iterations of the invention and in some instances demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Therefore, all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
The references listed below are cited in the Examples that follow.
A food-grade, pea fiber preparation was purchased from a commercial supplier. The compositional analysis of the pea fiber preparation is found in Table B. Wheat Arabinoxylan and Icelandic Moss Lichenan were purchased from Megazyme (P-WAXYL, P-LICHN) and yeast alpha-mannan was purchased from Sigma-Aldrich (M7504). Polysaccharides were solubilized in water (at a concentration of 5 mg/mL for pea fiber and 20 mg/mL for arabinoxylan and lichenan), sonicated and heated to 100° C. for 1 minute, then centrifuged at 24,000×g for 10 minutes to remove debris. TFPA-PEG3-biotin (Thermo Scientific), dissolved in DMSO (10 mg/mL) was added to the polysaccharide solution at a ratio of 1:5 (v/v). The sample was subjected to UV irradiation for 10 minutes (UV-B 306 nm, 7844 mJ total), and then diluted 1:4 to facilitate desalting on 7 kD Zeba spin columns (Thermo Scientific).
Biotinylated polysaccharide was mixed with one of several biotinylated fluorophores (PF-505, PF-510LSS, PF-633, PF-415; all at a concentration of 50 ng/mL; all obtained from Promokine). A 500 μL aliquot of this preparation was incubated with 107 paramagnetic streptavidin-coated silica beads (LSKMAGT, Millipore Sigma) for 24 hours at room temperature. Beads were washed by centrifugation three times with 1 mL HNTB buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, 0.1% BSA) followed by addition of 5 μg/mL streptavidin (Jackson Immunoresearch) in HNTB (30 min incubation at room temperature). Beads were washed as before and then incubated with 250 μL of the biotinylated polysaccharide preparation. The washing, streptavidin, and polysaccharide incubation steps were repeated three times.
Bead preparations were assessed using an Aria III cell sorter (BD Biosciences) to confirm adequate labeling. Beads were incubated with 70% ethanol for 1 minute in a biosafety cabinet, then washed three times with 1 mL sterile HNTB using a magnetic stand. The different bead types were combined, diluted, and aliquoted to 107 beads per 650 μL HNTB in sterile Eppendorf microcentrifuge tubes. The number of beads in each aliquot was counted using an Aria III cell sorter and CountBright fluorescent microspheres (BD Bioscience).
Bead preparations were analyzed by GC-MS to quantify the amount of carbohydrate bound. Beads were sorted back into their polysaccharide types based on fluorescence using an Aria III sorter (average sort purity, 96%). Sorted samples were centrifuged (500×g for 5 minutes) to pellet beads and the beads were transferred to a 96-well plate. All bead samples were incubated with 1% SDS/6M Urea/HNTB for 10 minutes at room temperature to remove exogenous components, washed three times with 200 μL HNTB using a magnetic plate rack, and then stored overnight at 4° C. prior to monosaccharide analysis. The number and purity of beads in each sorted sample was determined by taking an aliquot for analysis on the Aria III cell sorter. Equal numbers of beads from each sample were transferred to a new 96-well plate and the supernatant was removed with a magnetic plate rack. For acid hydrolysis, 200 μL of 2M trifluoroacetic acid and 250 ng/mL myo-inositol-D6 (CDN Isotopes; spike-in control) were added to each well, and the entire volume was transferred to 300 μL glass vials (ThermoFisher; catalog number C4008-632C). Another aliquot was taken to verify the final number of beads in each sample. Monosaccharide standards were included in separate wells and subjected to the hydrolysis protocol in parallel with the other samples. Vials were crimped with Teflon-lined silicone caps (ThermoFisher) and incubated at 100° C. with rocking for 2 h. Vials were then cooled, spun to pellet beads, and their caps were removed. A 180 μL aliquot of the supernatant was collected and transferred to new 300 μL glass vials. Samples were dried in a SpeedVac for 4 hours, methoximated in 20 μL O-methoxyamine (15 mg/mL pyridine) for 15 h at 37° C., followed by trimethylsilylation in 20 μL MSTFA/TMCS [N-Methyl-N-trimethylsilyltrifluoroacetamide/2,2,2-trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, chlorotrimethylsilane] (ThermoFisher) for 1 h at 70° C. One half volume of heptane (20 μL) was added before loading the samples for injection onto a 7890B gas chromatography system coupled to a 5977B MS detector (Agilent). The mass of each monosaccharide detected in each sample of sorted beads was determined using monosaccharide standard curves. This mass was then divided by the final count of beads in each sample to produce a measurement of mass of recoverable monosaccharide per bead.
This example describes an alternative method used to attach polysaccharides to paramagnetic glass beads. To covalently immobilize polysaccharides onto paramagnetic glass beads for use as biosensors of gut microbiota biochemical function, a bead with unique chemical functionality was developed. Amine functional groups were added to the bead surface as a chemical handle because of their nucleophilic nature at neutral pH and their utility in multiple bioconjugation reactions (Koniev et al., 2015). It was hypothesized that the amine functional group could be used for two critical functions: 1) addition of a fluorophore for the multiplexed analysis of multiple bead types within a single animal or subject, and 2) the covalent immobilization of an activated polysaccharide (
To install amines on the bead surface, the activated amine-silyl reagent (3-aminopropyl)triethoxysilane (APTS) was reacted with bead in the presence of water. Under the same reaction conditions, a zwitterionic surface could be generated with 3-(trihydroxysilyl)propyl methylphosphonate (THPMP) to an APTS containing reaction. The additional phosphonate functionality was important to reduce nonspecific binding to the bead surface (Bagwe et al., 2006). The zeta potential of surface modified paramagnetic silica beads was used to monitor the addition of both amine and phosphonate functional groups onto the bead surface (
N-Hydroxysuccinim ide ester (NHS)-activated fluorophores were covalently bound to the bead surface to facilitate the multiplexed analysis of multiple bead types within a single animal. With fluorescent amine-phosphonate paramagnetic glass beads in hand, we next sought to covalently immobilize polysaccharides of interest of the bead surface. Strategies for bioconjugation with polysaccharides are lacking compared to proteins, peptide, and nucleic acids due to the limited chemical functionality naturally occurring within polysaccharides. We chose to activate polysaccharides using a cyano (CN—) donor to generate a cyano-ester. Suitable cyano-donors include, but are not limited to, cyanogen bromide (CNBr) (Glabe et al., 1983) and the organic nitrile donor 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Lees et al., 1996). Both donors have been used for the generation of affinity matrixes on agarose beads and the synthesis of polysaccharide-conjugate vaccines; specifically, CDAP activation and conjugation was used for the development of the pneumococcal-conjugate vaccines (Lees et al., 1996). We chose CDAP because of its solubility in DMSO and the fact that it is less pH sensitive and less toxic than CNBr. CDAP was dissolved in DMSO and added to a solution of polysaccharide in the presence of catalytic triethylamine. CDAP nonspecifically generates cyano-ester electrophiles from the hydroxyls naturally present within a polysaccharide (
Polysaccharide immobilization on the bead surface was quantified via acid hydrolysis of surface-immobilized polysaccharide and quantification of the liberated monosaccharides using gas chromatography mass spectrometry (GC-MS). Polysaccharide was hydrolyzed using 2 M trifluoroacetic acid and liberated monosaccharides were quantified as silylated methoxyamine-reduced monosaccharides using free monosaccharides as standards. Beads were enumerated with flow cytometry and an equal number of each bead type were assayed in parallel. Beads lacking surface amines, or beads reacted with polysaccharides not activated with CDAP, lacked surface-immobilized polysaccharide (
Multiple types of polysaccharide-coated beads labeled with distinct fluorophores were pooled and gavaged into gnotobiotic mouse models as biosensors of gut community biochemical function. Polysaccharide degradation was measured as a function of 1) community composition, and 2) diet. Pooled beads were gavaged into germ-free mice 4 hours prior to animals were euthanized; beads were subsequently isolated from the mouse cecum based on their density and magnetic properties. Polysaccharide degradation was quantified as the amount of polysaccharide remaining covalently bound to the bead after passage through the gut and recovery from the cecum (
The ability of a microbiota to degrade a commercially available preparation of sugar beet arabinan (Megazyme; cat. no.: P-ARAB) was determined by comparing amine phosphonate beads coated with the carbohydrate to control beads whose surface amines were acetylated. Sugar beet arabinan is a polymer containing the monosaccharides arabinose, galactose, rhamnose, and galacturonic acid. Neutral monosaccharides were quantified after hydrolysis of bead-bound polysaccharide. Arabinose liberated during acid hydrolysis of sugar beet arabinan-coated beads was used as a marker of arabinan degradation. Comparison of input beads to beads passed through germ-free animals demonstrates that sugar beet arabinan is not digested by host enzymes during passage through a mouse (
Bacteroides ovatus
Bacteroides cellulosilyticus
Bacteroides thetaiotaomicron
Bacteroides thetaiotaomicron
Bacteroides vulgatus
Bacteroides caccae
Bacteroides finegoldii
Bacteroides massiliensis
Collinsella aerofaciens
Escherichia coli
Odoribacter splanchnicus
Parabacteroides distasonis
Ruminococcaceae sp.
Subdoligranulum variabile
Further details are provided below for the materials and methods used in the above experiments.
Synthesis of amine phosphonate beads: To a solution of microscopic (10 μm) paramagnetic silica beads (Millipore Sigma; Cat no: LSKMAGN01) in water, equal molar amounts of (3-aminopropyl)triethoxysilane (APTS) (Sigma Aldrich) and 3-(trihydroxysilyl)propyl methylphosphonate (THPMP) (Sigma Aldrich) were added (Bagwe et al., 2006; Soto-Cantu et al., 2012). The reaction was allowed to proceed for 5 hours at 50° C. with shaking. The reaction was terminated with repeated washing of beads with water using a magnet.
Zeta potential measurement: Zeta potential was measured to track modification of the bead surface. Zeta potential measurements were obtained on a Malvern ZEN3600 using disposable Malvern zeta potential cuvettes. Measurements were obtained with the default settings of the instrument, using the refractive index of SiO2 as the material, and water as the dispersant. Beads were resuspended to a concentration of 5×105/mL in 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES; pH 7.2) and analyzed in triplicate. Zeta potential of starting beads and beads monofunctionalized with APTS or THPMP were used as standards.
Fluorophore labeling of amine phosphonate beads: Fluorophores were covalently bound to the bead surface to facilitate the multiplexed analysis of multiple bead types within a single animal. N-Hydroxysuccinimide ester (NHS)-activated fluorophores were dissolved in dimethyl sulfoxide (DMSO) at 1 mM. Resuspended fluorophore was diluted into a solution of 20 mM HEPES (pH 7.2) and 50 mM NaCl to a final concentration of 100 nM and incubated with amine phosphonate beads for 50 minutes at 22° C. Beads were washed repeatedly with water to terminate the reaction. The extent of fluorophore labeling was assessed on each bead type using flow cytometry. The concentration of fluorophore used was the lowest at which the bead populations could be reliably and easily distinguished via flow cytometry. Fluorophores and their sources: Alexa Fluor 488 NHS ester (Life Technologies; cat. no.: A20000), Promofluor 415 NHS ester (PromoKine; cat. no.: PK-PF415-1-01), Promofluor 633P NHS ester (PromoKine; cat. no.: PK-PF633P-1-01), and Promofluor 510-LSS NHS ester (PromoKine; cat. no.: PK-PF510LSS-1-01).
Amine phosphonate bead acetylation: Acetylation of bead surface amines was used to confirm the specific linkage of both fluorophore and polysaccharides to the bead surface. Acetylated beads were also used as an empty bead control when gavaged into mice. Bead surface amines were acetylated using acetic anhydride under anhydrous conditions. Amine phosphonate beads were washed repeatedly with multiple solvents with the goal of resuspending the beads in anhydrous methanol; beads were washed in water, then methanol, then anhydrous methanol. Pyridine (0.5 volume equivalents) was then added as a base followed by acetic anhydride (0.5 volume equivalents). The reaction was allowed to proceed for 3 hours at 22° C. and then quenched with repeated washing with water. The described acetylation conditions had no effect on the fluorescence of any of the four fluorophores tested.
Polysaccharide conjugation to amine phosphonate beads: Polysaccharides were dissolved at 3-10 mg/mL in 50 mM HEPES (pH 8) with heat and sonication. To a solution of polysaccharide (5 mg/mL) containing trimethylamine (0.5 equivalent), 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP; Sigma Aldrich; 1 eq.) dissolved in DMSO was added. The optimal concentration of CDAP was found to be 0.2 mg of CDAP per mg of polysaccharide. The polysaccharide/CDAP solution was mixed for 5 minutes at 22° C. to allow for polysaccharide activation. Amine phosphonate beads resuspended in 50 mM HEPES (pH 8) were added to the activated polysaccharide solution and the reaction was allowed to proceed for 15 hours at 22° C. Any aggregated beads were resuspended with light sonication. The resulting isourea linkage between the bead and polysaccharide was reduced by addition of 2-picoline borane dissolved in DMSO (10% wt:wt) and incubation for 40 minutes at 40° C. The reaction was terminated with repeated washing in water and then 20 mM HEPES (pH 7.2) 50 mM NaCl. The described reaction conditions for polysaccharide conjugation or reduction had little or no effect on the fluorescence of any of the four fluorophores tested.
Bead counting: The absolute number of beads in a solution was determined with flow cytometry using CountBright Absolute Counting Beads (ThermoFisher Scientific; cat. no.: C36950) according to the manufacturer's suggested protocol.
Bead pooling and gavage into gnotobiotic mice: Pools of equal number of each bead type were prepared from fluorophore-labeled polysaccharide-coated amine phosphonate beads. The required number of a given bead type was sterilized with 70% ethanol for 10 minutes before washing with sterile water and 20 mM HEPES (pH 7.2), 50 mM NaCl, 0.01% bovine serum albumin, and 0.01% Tween-20. The different bead types were then pooled into a single mixture.
Pooled bead mixtures (10-15×106 beads) were gavaged into gnotobiotic mice 4-6 hours prior to sacrifice. Beads were harvested from cecal contents using bead density and magnetism. Beads were sorted back into the original bead type using fluorescence-activated cell sorting (FACS; BD FACSAria III).
Quantitation of polysaccharide degradation: Polysaccharide degradation was determined by quantifying the amount of monosaccharide hydrolyzed from bead-bound polysaccharide after bead passage through a mouse. To do so, an equal number of beads were placed in crimp-top glass vials and hydrolyzed using 2 M trifluoroacetic acid for 2 hours at 95° C. The solution was reduced to dryness under reduced pressure. Liberated monosaccharides were reduced with methoxyamine (15 mg/mL in pyridine) for 15 hours at 37° C. Hydroxyl groups were silylated using N-Methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) 1% 2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, chlorotrimethylsilane (TCMS) (ThermoFisher Scientific; Cat. no.: TS-48915) for 1 hour at 60° C. Samples were diluted with heptane and analyzed by GC-MS on Agilent 7890A gas chromatography system, coupled with a 5975C mass spectrometer detector (Agilent). Monosaccharide composition and quantitation were determined using chemical standards simultaneously derivatized.
As described in Example 1, several layers of a single glycan were applied to a bead by serial incubation of the beads (obtained from the manufacturer with streptavidin covalently bound) with biotin-glycan, then streptavidin, then biotin-glycan, then streptavidin, then biotin-glycan. This is possible since streptavidin has four biotin binding sites, allowing it to link the previous layer of biotin-glycan to a new layer of biotin-glycan. This method may can be modified to create a bead with layers of different glycans, or alternating layers of glycans, by using multiple types of biotin-glycan (e.g., biotin-glycan 1, biotin-glycan 2, biotin, etc.).
As described in Example 2, glycans were conjugated to amine phosphonate beads by first activing the glycans with CDAP. Multi-layered beads can also be prepared by the CDAP method because a diamine can serve the same linking function between previous and new layers, since it has two amine groups.
The procedures above have been performed with alternating arabinoxylan and mannan layers. Other chemistries may also be used.
In the present study, we describe an in vivo approach for identifying fibers and their bioactive components that selectively increase the fitness of a group of human gut Bacteroides, and the different mechanisms these organisms deploy when encountering these nutrient resources and one another. The bacterial targets for fiber-based manipulation originated from our previous study of twins stably discordant for obesity (Ridaura et al., 2013). Fecal microbiota from these twin pairs transmitted discordant adiposity and metabolic dysfunction phenotypes to recipient germ-free mice. Co-housing mice shortly after they received microbial communities from lean (Ln) or obese (Ob) co-twins prevented recipients of the Ob donor microbiota from developing obesity and associated metabolic abnormalities. Analysis of their gut communities revealed that invasion of Bacteroides species from Ln into Ob microbiota, notably B. thetaiotaomicron, B. vulgatus, B. caccae, and B. cellulosilyticus, correlated with protection from the increased adiposity and metabolic phenotypes that developed in co-housed Ob-Ob controls. Invasion was diet-dependent, occurring when animals consumed a human diet designed to represent the lower tertile of consumption of saturated fats and upper tertile of consumption of fruits and vegetables (high in fiber) in the USA, but not when they consumed a diet representing the upper tertile of saturated fat and lower tertile of fruit and vegetable consumption (Ridaura et al., 2013). Here we identify dietary fiber preparations and constituent bioactive components that increase the fitness of these targeted Bacteroides (B. thetaiotaomicron, B. vulgatus, B. caccae, and/or B. cellulosilyticus) in vivo in the high saturated fatty acid-low fruits and vegetables (HiSF-LoFV) diet context. To do so, we first colonized germ-free mice with a defined consortium of sequenced bacterial strains cultured from a Ln donor in an obesity-discordant twin pair. Mice were fed 144 different diets generated by supplementing the HiSF-LoFV formulation with 34 different food-grade fiber preparations in different combinations at different concentrations. Armed with a consortium that contained targeted Bacteroides species, each in the form of a library of tens of thousands of transposon (Tn) mutant strains, and employing high resolution mass spectrometry, we subsequently characterized the effects of monotonous feeding of selected fiber preparations on the community's expressed proteome and on the fitness of Tn mutants. By identifying polysaccharide processing genes whose expression was increased and that functioned as key fitness determinants, we inferred which components of the fiber preparations were bioactive. Time series proteomic analyses of the complete community and derivatives lacking one or more Bacteroides, revealed nutrient harvesting strategies resulting in, as well as alleviating interspecies competition for fiber components. Finally, administering artificial food particles coated with dietary polysaccharides to gnotobiotic mice with deliberately varied community membership further established the contributions of individual Bacteroides species to glycan processing in vivo.
A schematic of the experimental design for screening 34 food grade fibers is shown in
We analyzed the relative abundance of each member of the defined community at two time points at the end of each diet treatment by collecting fecal samples and performing 16S rRNA gene sequencing. Binning the data according to the fiber preparation present at 8% concentration revealed potent and specific effects on distinct taxa (
Bacteroides
thetaiotaomicron
Bacteroides
cellulosilyticus
Bacteroides
vulgatus
Bacteroides caccae
Bacteroides ovatus
Parabacteroides
distasonis
Escherichia coli
Ruminococcaceae
Subdoligranulum
variabile
Collinsella
aerofaciens
Bacteroides
massiliensis
Odoribacter
splanchnicus
Bacteroides
finegoldii
Peptococcus niger
Dorea longicatena
Bacteroides
thetaiotaomicron
Bacteroides
cellulosilyticus
Bacteroides
vulgatus
Bacteroides caccae
Bacteroides ovatus
Parabacteroides
distasonis
Escherichia coli
Ruminococcaceae
Subdoligranulum
variabile
Collinsella
aerofaciens
Bacteroides
massiliensis
Odoribacter
splanchnicus
Bacteroides
finegoldii
Peptococcus niger
Dorea longicatena
Bacteroides
thetaiotaomicron
Bacteroides
cellulosilyticus
Bacteroides
vulgatus
Bacteroides
caccae
Bacteroides
ovatus
Parabacteroides
distasonis
Escherichia coli
Ruminococcaceae
Subdoligranulum
variabile
Collinsella
aerofaciens
Bacteroides
massiliensis
Odoribacter
splanchnicus
Bacteroides
finegoldii
Peptococcus
niger
Bacteroides
thetaiotaomicron
Bacteroides
cellulosilyticus
Bacteroides
vulgatus
Bacteroides
caccae
Bacteroides
ovatus
Parabacteroides
distasonis
Escherichia coli
Ruminococcaceae
Subdoligranulum
variabile
Collinsella
aerofaciens
Bacteroides
massiliensis
Odoribacter
splanchnicus
Bacteroides
finegoldii
Peptococcus
niger
Bacteroides
thetaiotaomicron
Bacteroides
cellulosilyticus
Bacteroides
vulgatus
Bacteroides
caccae
Bacteroides
ovatus
Parabacteroides
distasonis
Escherichia coli
Ruminococcaceae
Subdoligranulum
variabile
Collinsella
aerofaciens
Bacteroides
massiliensis
Odoribacter
splanchnicus
Bacteroides
finegoldii
Peptococcus niger
Bacteroides
thetaiotaomicron
Bacteroides
cellulosilyticus
Bacteroides
vulgatus
Bacteroides
caccae
Bacteroides
ovatus
Parabacteroides
distasonis
Escherichia coli
Ruminococcaceae
Subdoligranulum
variabile
Collinsella
aerofaciens
Bacteroides
massiliensis
Odoribacter
splanchnicus
Bacteroides
finegoldii
Peptococcus niger
Several possible mechanisms could account for the increase of a target Bacteroides in response to fiber administration, including indirect effects involving other species. Therefore, we sought to determine which polysaccharides in the fiber preparations caused the target species to expand and whether they acted directly on those species by serving as nutrient sources for their growth. To do so, we simultaneously quantified community-wide protein expression and assessed the contributions of proteins to bacterial fitness using a forward genetic screen. The screen was based on genome-wide transposon (Tn) mutagenesis and a method known as multi-taxon INsertion Sequencing (INSeq), which allows simultaneous analysis of Tn mutant libraries generated from different Bacteroides species in the same recipient gnotobiotic mouse. We employed five INSeq libraries constructed using type strains corresponding to four Bacteroides species present in the Ln co-twin donor culture collection. The quality and performance of these libraries had been characterized previously in vitro and in vivo (30,300-167,000 isogenic Tn mutants/library; single site of Tn insertion/strain; 11-26 Tn insertions/gene; 71-92% genes covered/genome; (Hibberd et al., 2017; Wu et al., 2015)). Additionally, we simplified the community used in these experiments by omitting six strains from the original 20 member consortium that were not robust colonizers in the HiSF-LoFV diet context (Faith et al., 2014; Ridaura et al., 2013). All mice were colonized with the resulting 15-member community while consuming the base (unsupplemented) HiSF-LoFV diet. Animals were divided into five groups (n=6 animals/group) and were either continued on the base HiSF-LoFV diet or, two days after gavage, switched to the HiSF-LoFV diet supplemented with one of the fibers identified in the screen. We tested pea fiber, citrus pectin, orange peel, and tomato peel, each at a concentration of 10% (w/w), based on their ability to increase the representation of one or more of the targeted Bacteroides (
Consistent with results obtained from seven days of fiber administration in the screening experiments, we observed a statistically significant expansion of B. thetaiotaomicron VPI-5482 in mice consuming pea fiber (ANOVA, P<0.05;
Structural analyses of lead fibers—We used permethylation and gas-chromatography-mass spectrometry to analyze the monosaccharide composition and glycosidic linkages of polysaccharides present in pea fiber and citrus pectin. After accounting for starch (typically degraded and absorbed by the host) and cellulose (not metabolized by the target Bacteroides; (McNulty et al., 2013)), the most abundant polysaccharide in pea fiber was arabinan, consisting of a linear 1,5-linked arabinose backbone with arabinose residues as side chains at position 2 or 3 (
High-resolution proteomic analysis of community gene expression—The results of these biochemical analyses raised the possibility that metabolism of arabinan in pea fiber and methylated homogalacturonan in citrus pectin were involved in the responses of target Bacteroides. To test this hypothesis, we turned to high-resolution shotgun proteomic analysis, focusing on fecal samples obtained on day 6 of the monotonous feeding experiment. After considering only peptides that uniquely mapped to a single seed protein, 11,493 proteins were advanced to quantitative analysis (summed abundances; 59% from community members, 36% from mouse and 2% from diet; see Methods). We calculated a z-score for each expressed protein from each bacterial species using the abundances of all proteins assigned to that individual species in a given sample. This allowed us to determine changes in the abundance of each protein irrespective of changes in the abundance of that species in the community. In the case of the Bacteroides species represented by INSeq libraries, we considered the measured abundance of a given protein to reflect the summed contributions of all the mutant strains of that species (thus representing the level of expression we would expect from a corresponding wild-type strain). Linear models were constructed using limma (Smyth, 2004; Ting et al., 2009) and significant effects were identified between bacterial protein abundances and supplementation of the control diet with pea fiber and citrus pectin (245 and 450 proteins, respectively; |fold-change|>log 2(1.2), P<0.05, FDR corrected). Bacteroides contain multiple polysaccharide utilization loci (PULs) in their genomes. PULs provide a fitness advantage by endowing a species with the ability to sense, import, and process complex glycans using their encoded carbohydrate-responsive transcription factors, SusC/SusD-like transporters, and carbohydrate active enzymes (CAZymes) (Glenwright et al., 2017; Kotarski and Salyers, 1984; Martens et al., 2011; McNulty et al., 2013; Shepherd et al., 2018). Eighty-five of the proteins whose levels were significantly altered by pea fiber and 134 that were significantly affected by citrus pectin were encoded by PULs (Terrapon et al., 2018).
Ranking proteins by the pea-fiber induced increase in their abundance disclosed that in B. thetaiotaomicron, 6 of the top 10 were encoded by PULs 7, 73, and 75. PUL7 is known to be involved in arabinan metabolism (Lynch and Sonnenburg, 2012; Schwalm et al., 2016), and encodes characterized and predicted arabinofuranosidases in glycoside hydrolase (GH) family 43, GH51, and GH146. PUL75 carries out the degradation of rhamnogalacturonan I (RGI) (Luis et al., 2018), but its expression is also triggered by exposure to purified arabinan in vitro (Martens et al., 2011). PUL73 processes homogalacturonan (Luis et al., 2018) and encodes CAZymes that cleave linked galacturonic acid residues and remove methyl and acetyl esters from galacturonic acid [polysaccharide lyase (PL)1, GH105, GH28, CE8, CE12 family members]. B. ovatus proteins encoded by predicted RGI-processing PULs (PUL97) (Luis et al., 2018) were among the most increased by pea fiber administration. Supplementation of the HiSF-LoFV diet with citrus pectin resulted in increased abundance of proteins encoded by a B. cellulosilyticus PUL that is induced by homogalacturonan in vitro (PUL83). In addition, citrus pectin induced expression of proteins in several B. finegoldii PULs (PUL34, 35, 42, and 43) that encode galacturonan-processing enzymes (GH28, GH105, GH106, PL11 subfamily 1, CE8 and CE12). This latter finding correlates with the organism's citrus pectin-driven expansion.
Combining proteomic and INSeq analyses—As noted above, we colonized mice with INSeq libraries and then fed them the base HiSF-LoFV diet for two days before switching the experimental groups to fiber-supplemented diets. We measured the abundances of Tn mutant strains, and calculated log ratios between fecal samples collected on experimental day 6 (posttreatment) and day 2 (pre-treatment); results were compared to the reference HiSF-LoFV treatment arm to focus on genes that had significant fitness effects in the context of these fibers (P<0.05, FDR corrected; see Methods; 223 genes, 24% in PULs. Genes exhibiting a significant positive fold-change in protein abundance and negative effect on fitness when mutated appear in the bottom right quadrant of the orthogonal protein-fitness plots shown in
Genes in PULs were ranked by the magnitude of pea-fiber-dependent increases in the abundances their protein products and decreases in strain fitness when they were disrupted by a Tn insertion. The results revealed genes in three PULs (PUL7 in B. thetaiotaomicron, PUL5 in B. cellulosilyticus, and PUL27 in B. vulgatus;
The increased fitness cost of mutations in B. ovatus RGI-processing PUL97, but not the B. thetaiotaomicron RGI-processing PUL75, indicated that these species utilize different carbohydrates in the pea fiber-supplemented diet (RGI and arabinan, respectively;
A parallel analysis of mice monotonously fed citrus pectin revealed that five genes encoded by galacturonan-processing PUL83 in B. cellulosilyticus were among the most abundantly expressed and most important for fitness compared to the base diet condition (
Together, our proteomic and INSeq datasets revealed the microbial genes required during fiber-driven expansion, highlighted the polysaccharides that contributed to the fitness effects of these fibers and provided evidence for functional overlap in the nutrient harvesting strategies of B. cellulosilyticus and B. vulgatus, in two distinct fiber conditions. The dominance of B. cellulosilyticus in diverse diet contexts led us to ask whether (and how) this species directly competes with other community members for polysaccharides.
We performed a direct test for interactions between B. cellulosilyticus and other species by comparing the defined 15-member community, to the derivative 14-member community lacking B. cellulosilyticus. Using an experimental design that mimicked the monotonous feeding study described above, groups of germ-free mice were colonized with these two communities and fed the HiSF-LoFV diet with or without 10% (w/w) pea fiber or citrus pectin. COPRO-Seq analysis was used to determine the abundance of each strain as a proportion of all strains other than B. cellulosilyticus, thereby controlling for the compositional effect of removing this species. Defined this way, the abundance of B. thetaiotaomicron did not increase upon omission of B. cellulosilyticus in the presence of pea fiber, suggesting minimal competition between these two species for arabinan (
To directly test the capacity of competing Bacteroides to process the same nutrient substrate in vivo, a bead-based glycan degradation assay was developed (
Germ-free mice were colonized with either B. cellulosilyticus or B. vulgatus alone and fed a HiSF-LoFV diet supplemented with 10% (w/w) pea fiber. Seven days after colonization, all mice were gavaged with an equal mixture of the three bead types (5×106 of each type/animal, n=5-6 animals). Mice were euthanized 4 h later, beads were recovered from their cecum and colon, and the mass of monosaccharides on the different purified bead types was quantified. The fluorescent signal present on all bead types persisted after intestinal transit, confirming that the biotin-streptavidin interactions were stable under these conditions (
A follow-up experiment of identical design was performed except that animals fed HiSF-LoFV supplemented with pea fiber were gavaged 12 days rather than seven days after colonization with a collection of four rather than three types of beads. These beads were either empty (no glycan bound) or coated with (i) the soluble, starch-depleted fraction of pea fiber, or wheat arabinoxylan, or lichenan from Icelandic moss, a control glycan low in arabinose (81% glucose/8% mannose/6% galactose/2% arabinose). Beads were recovered, purified by flow cytometry and analyzed using GC-MS. The degradation of bead-bound pea fiber and arabinoxylan was similar to that observed on day 7.
To control for microbe-independent polysaccharide degradation, germ-free mice were given a gavage of arabinoxylan-coated, pea-fiber coated, lichenan-coated, and empty beads (n=13 animals). We collected all fecal samples produced during an 8 h period (from 4 to 12 hours after gavage). Assays of the arabinoxylan-, pea fiber-, and lichenan-coated beads purified from fecal samples obtained from each germ-free animal revealed no significant degradation of these polysaccharides after passage through their intestines (
Given the observation that several species can metabolize pea fiber arabinan in vivo, whether the absence of B. cellulosilyticus would compromise the efficiency with which the community carried out this function was assessed. Mice consuming the unsupplemented HiSF-LoFV diet were given pea fiber-coated, arabinoxylan-coated, lichenan-coated, and empty beads 12 days after colonization with (i) the 15-member consortium or (ii) the derivative 14-member community lacking B. cellulosilyticus. Analysis of beads recovered from the cecal and colonic contents of these mice disclosed that the level of pea fiber degradation was not affected by the absence of B. cellulosilyticus (
B. vulgatus
B. cellulosilyticus
The in vivo bead-based glycan degradation assays revealed that in contrast to arabinan, the capacity of the community to process arabinoxylan was not rescued by other species in the absence of B. cellulosilyticus (
As discussed above, the abundances of B. vulgatus proteins involved in pea fiber or citrus pectin degradation were unchanged upon removal of its competitor B. cellulosilyticus. In contrast, B. ovatus exhibited metabolic flexibility, with proteins encoded by two arabinoxylan-processing PULs (PUL26 and PUL81) predominating among those whose abundances were increased when B. cellulosilyticus was absent versus present (
Monosaccharide and linkage analysis verified that arabinoxylan was present in the HiSF-LoFV diet; this conclusion was based on finding abundant 4-linked xylose with branching 4,3-linked xylose, and terminal arabinose (Tables 4-5). We also detected small amounts of 3-linked glucose (indicative of hemicellulose beta-glucans), galacturonic acid and rhamnose. The presence of these structures in the base HiSF-LoFV diet are consistent with the observed increase in abundance of proteins in B. ovatus PULs shown or predicted to process beta-glucan, rhamnogalacturonan, and host glycan when B. cellulosilyticus is present (
Based on these results, we reasoned that metabolic flexibility allows B. ovatus to acclimate to the presence of B. cellulosilyticus by shifting its nutrient harvesting strategies, de-emphasizing arabinoxylan degradation, thus mitigating competition between the two species. To test this notion further, we performed an experiment omitting B. cellulosilyticus, B. ovatus, or both species from the 15-member consortium introduced into mice. Animals were fed the base HiSF-LoFV diet for 12 days and fecal samples were collected as in previous experiments. Confirming our earlier results, COPRO-Seq revealed that the abundance of B. ovatus was increased in the absence of B. cellulosilyticus (
We sought to directly measure the functional outcome of metabolic flexibility in B. ovatus and establish that this species degraded arabinoxylan in the community lacking B. cellulosilyticus. Therefore, arabinoxylan-beads, as well as empty and yeast alpha-mannan coated control beads, were administered to the four groups of mice described above, with all mice consuming the base HiSF-LoFV diet. In the absence of B. cellulosilyticus, significant degradation of arabinoxylan was still detected (
Together, these experiments show that, in contrast to the persistent competition for arabinan and homogalacturonan exhibited by B. vulgatus, B. ovatus avoids competition for arabinoxylan via acclimation to the presence of its potential competitor, B. cellulosilyticus. This conclusion is based on several observations; (i) the HiSF-LoFV diet contains arabinoxylan polysaccharides, which can be metabolized by both species in question, (ii) omission of B. ovatus did not cause detectable expansion of B. cellulosilyticus, (iii) proteins encoded by B. ovatus arabinoxylan PULs were significantly increased when B. cellulosilyticus was absent, (iv) genes in B. ovatus arabinoxylan PULs were more important for fitness when B. cellulosilyticus was absent, and (v) B. ovatus was responsible for the residual arabinoxylan degradation that took place in the absence of B. cellulosilyticus.
Together, Examples 4-8 show that, in contrast to the persistent competition for arabinan and homogalacturonan exhibited by B. vulgatus, B. ovatus avoids competition via acclimation to the presence of its potential competitor, B. cellulosilyticus. This conclusion is based on the observations that (i) omission of B. ovatus did not cause detectable expansion of B. cellulosilyticus, (ii) proteins encoded by B. ovatus arabinoxylan PULs were significantly increased when B. cellulosilyticus was absent, (iii) genes in B. ovatus arabinoxylan PULs were significantly more important for fitness when B. cellulosilyticus was absent, and (iv) B. ovatus was responsible for the residual arabinoxylan degradation that took place in the absence of B. cellulosilyticus.
Combining (i) high resolution proteomics, (ii) forward genetic screens for fitness determinants, (iii) a collection of glycan-coated artificial food particles, and (iv) deliberate manipulations of community membership in gnotobiotic mice fed ‘representative’ high-fat, low-fiber USA diet led to the direct characterization of how human gut Bacteroides with distinct, as well as overlapping, nutrient harvesting capacities respond to different food-grade fibers. Our approach allowed us to identify bioactive components in compositionally complex fibers that impact specific members of the microbiota. Obtaining this type of information can inform food manufacturing practices by directing efforts to seek sources of and enrich for these active components; e.g., through judicious selection of cultivars of a given food staple, food processing methods or an existing waste stream from food manufacturing to mine for these components.
Deliberately manipulating membership of a consortium of cultured, sequenced human-donor derived microbes prior to their introduction into gnotobiotic mice fed a human diet, with or without fiber supplementation, provides an opportunity to determine whether and how organisms compete and what mechanisms they use to avoid competition. Simultaneous harvest of a particular dietary resource by two species is theoretically possible whenever they both contain a genetic apparatus sufficient for metabolism of that resource. We provide evidence that competition for particular glycans in fiber preparations is realized in such a model community, since glycan-degrading genes were expressed and required for fitness in both species, and negative interactions were observed in strain omission experiments. These omission experiments disclosed distinct relationships between B. vulgatus, B. ovatus and B. cellulosilyticus; namely, the ability of B. ovatus to acclimate to the presence of a competitor (B. cellulosilyticus) as opposed to the persistent competition between B. vulgatus and B. cellulosilyticus for the same resource. A healthy human gut microbiota has great strain-level diversity. Determining which strains representing a given species to select as a lead candidate probiotic agent, or for incorporation into synbiotic (prebiotic plus probiotic) formulations, is a central challenge for those seeking to develop next generation microbiota-directed therapeutics. Identifying organisms with metabolic flexibility, as opposed to those that are more prone to competing with other community members, could contribute to understanding how certain strains are capable of coexisting with the residents of diverse human gut communities.
Particles present in foods prior to consumption, or generated by physical and biochemical/enzymatic processing of foods during their transit through the gut, provide community members with opportunities to attach to their surfaces, and harvest surface-exposed nutrient resources. The ability of organisms to adhere to such particles, the carrying capacity of particles (size relative to nutrient content), and the physical partitioning their component nutrients can be envisioned as affecting competition, conflict avoidance, and cooperation. The ability of a given gut microbial community to degrade different fiber components was quantified in our studies using artificial food particles composed of fluorescently labeled, paramagnetic microscopic beads coated with different polysaccharides. This approach provides an additional dimension for characterizing the functional properties of a microbial community, and has a number of advantages. First, the measurement of polysaccharides coupled to magnetic beads is not confounded by the presence in the gut of structurally similar (or even identical) dietary or microbial polysaccharides. Second, this technology, when applied to gnotobiotic mice, permits simultaneous testing of multiple glycans in the same animal, allowing a direct comparison of the degradative capabilities of different assemblages of human gut microbes in vivo. For example, we were able to demonstrate non-redundant arabinoxylan degradation carried out by B. cellulosilyticus in this community, despite the presence of another arabinoxylan degrader, B. ovatus. Third, applied directly to humans, these diagnostic biosensors' could be used to quantify functional differences between their gut microbiota, and physical associations between carbohydrates and strains of interest, as a function of host health status, nutritional status/interventions, or other perturbations. As such, results obtained with these biosensors could facilitate ongoing efforts to use machine learning algorithms that integrate a variety of parameters, including biomarkers of host physiologic state and features of the microbiota, to develop more personalized nutritional recommendations (Zeevi et al., 2015). Lastly, this technology could be used to advance food science. The bead coating strategy employed was successful with over 30 commercially available polysaccharide preparations and the assay has been extended to measure the degradation of other biomolecules, including proteins. Particles carrying components of food that have been subjected to different processing methods, or particles bearing combinations of nutrients designed to attract different sets of primary (and secondary) microbial consumers could also be employed in preclinical models to develop and test food prototypes optimized for processing by the microbiota representative of different targeted human consumer populations.
Gnotobiotic mice—All experiments involving mice were carried out in accordance with protocols approved by the Animal Studies Committee of Washington University in St. Louis. For screening different fiber preparations, germ-free male C57BL/6J mice (10-16 weeks-old) were singly housed in cages located within flexible plastic isolators. Cages contained paper houses for environmental enrichment. Animals were maintained on a strict light cycle (lights on at 0600 h, off at 1900 h). Mice were fed a LoSF-HiFV diet for five days prior to colonization. After colonization, the community was allowed to stabilize on the LoSF-HiFV diet for an additional five days. One group of control mice remained on this diet for the rest of the experiment and a second control group was switched to the HiSF-LoFV diet for the rest of the experiment.
Mice in the experimental group first received an introductory diet containing equal parts of all fiber preparations employed in a given screen (totaling 10% of the diet by weight), and then received a series of diets containing different fiber preparations as described in
For monotonous feeding experiments, mice were fed the control HiSF-LoFV diet in its pelleted form for two weeks prior to colonization. Two days after colonization, mice were switched to paste diets containing 10% of the powdered fiber preparation mixed into the base diet (or the base diet in paste form without added fiber) for the remainder of the experiment. As noted above, these diets were delivered in freshly hydrated aliquots every two days. Fecal samples, including those obtained prior to colonization, were collected on the days indicated in
Defined microbial communities—The screening experiments used cultured, sequenced bacterial strains obtained from a fecal sample that had been collected from a lean co-twin in an obesity-discordant twin-pair [Twin Pair 1 in (Ridaura et al., 2013); also known as F60T2 in (Faith et al., 2013)]. Isolates were grown to stationary phase in TYGS medium (Goodman et al., 2009) in an anaerobic chamber (atmosphere; 75% N2, 20% CO2, 5% H2). Equivalent numbers of organisms were pooled (based on OD600 measurements). The pool was divided into aliquots that were frozen in TYGS/15% glycerol, and maintained at −80° C. until use. On experimental day 0, aliquots were thawed, the outer surface of their tubes were sterilized with Clidox (Pharmacal) and the tubes were introduced into gnotobiotic isolators. The bacterial consortium was administered through a plastic tipped oral gavage needle (total volume, 400 μL per mouse). Based on inconsistent colonization observed in screening experiment 1, one isolate (Enterococcus fecalis; average relative abundance, 2.1%) was not included in screening experiments 2 and 3.
Model communities containing INSeq libraries—Ten strains selected from the human donor-derived community described above were colony purified, and each frozen in 15% glycerol and TYGS medium. Recoverable CFUs/mL were quantified by plating on brain-heart-infusion (BHI) blood agar. The identity of strains was verified by sequencing full-length 16S rRNA amplicons. On the day of gavage, stocks of these strains were thawed in an anaerobic chamber and mixed together along with each of five multi-taxon INSeq libraries (B. thetaiotaomicron VPI-5482, B. thetaiotaomicron 7330, B. cellulosilyticus WH2, B. vulgatus ATCC-8482, B. ovatus ATCC-8483) whose generation and characterization have been described in earlier publications (Hibberd et al., 2017; Wu et al., 2015). An aliquot of this mixture was administered by oral gavage to germ-free mice housed in gnotobiotic isolators (2×106 CFUs of each donor organism plus an OD600 0.5 of each INSeq library per mouse recipient; total gavage volume, 400 μL). For B. cellulosilyticus, B. vulgatus, B. ovatus, or B. cellulosilyticus and B. ovatus omission experiments, gavage mixtures were prepared in parallel without these organisms. The absence of one or both of these strains was verified by COPRO-Seq analysis of both the gavage mixture and fecal samples collected throughout the experiment from recipient mice.
Fiber-rich food ingredient mixtures—HiSF-LoFV and LoSF-HiFV diets were produced using human foods, selected based on consumption patterns from the National Health and Nutrition Examination Survey (NHANES) database (Ridaura et al., 2013). Diets were milled to powder (D90 particle size, 980 □m), and mixed with pairs of powdered fiber preparations [one preparation at 8% (w/w) and the other preparation at 2% (w/w)]. Fiber content was defined for each preparation [Association of Official Agricultural Chemists (AOAC) 2009.01], as was protein, fat, total carbohydrate, ash, and water content [protein AOAC 920.123; fat AOAC 933.05; ash AOAC 935.42; moisture AOAC 926.08; total carbohydrate (100−(Protein+Fat+Ash+Moisture)]. The powdered mixtures were sealed in containers and sterilized by gamma irradiation (20-50 kilogreys, Steris, Mentor, OH). Sterility was confirmed by culturing the diet under aerobic and anaerobic conditions (atmosphere, 75% N2, 20% CO2, 5% H2) at 37° C. in TYG medium, and by feeding the diets to germ-free mice followed by COPRO-Seq analysis of their fecal DNA.
Monosaccharide and linkage analysis of fiber preparations—Uronic acid (as GalA) was measured using the m-hydroxybiphenyl method (Thibault, 1979). Sodium tetraborate was used to distinguish GlcA and GalA (Filisetti-Cozzi and Carpita, 1991). The degree of methylation of galacturonic acid (pectins) in the sample was estimated as previously described (Levigne et al., 2002). Samples were hydrolyzed with 1M H2SO4 for 2 h at 100° C. and individual neutral sugars were analyzed as their alditol acetate derivatives (Englyst and Cummings, 1988) by gas chromatography. To fully release glucose from cellulose, a pre-hydrolysis step was carried out by incubation in 72% H2SO4 for 30 minutes at 25° C. prior to the hydrolysis step. Linkage analysis was performed after carboxyl reduction of uronic acid with NaBD4/NaBH4 according to a previously published procedure (Pettolino et al., 2012) with minor modifications (this procedure allows galactose, galacturonic acid and methylesterified galacturonic acid to be distinguished). Methylation of carboxyl-reduced samples was performed as described in (Buffetto et al., 2015).
Polysaccharides from the HiSF-LoFV diet were isolated by sequential alkaline extractions (Pattathil et. al., 2012). Briefly, lipids were removed from a sample of powdered HiSF-LoFV by sequential incubation in 80% ethanol, 100% ethanol, and acetone. The dried precipitate was suspended in 1M KOH containing 0.5% (w/w) NaBH4 and stirred overnight. The solution was neutralized and the supernatant was collected by centrifugation (this material is referred to as fraction 1 (F1)). The insoluble material was suspended in 1M KOH/0.5% (w/w) NaBH4 overnight, and the supernatant was collected (referred to as F2). The insoluble material was suspended in 4M KOH/0.5% (w/w) NaBH4 overnight and the supernatant was collected (referred to as F3). Each fraction was dialyzed (SnakeSkin 3.5K MWCO, Thermo Scientific) in water, lyophilized, and then treated for 4 hours at 37° C. with amyloglucosidase (36 units/mg) and alpha-amylase (100 units/mg; both enzymes from Megazyme). Enzymes were inactivated by boiling and samples were dialyzed and lyophilized. Measurement of the dry mass of each fraction before and digestion revealed that the total starch content of the base HiSF-LoFV diet was 22% (w/w) (note a comparable analysis the pea fiber yielded a value of 3.6%, meaning that HiSF-LoFV diet supplemented with 10% pea fiber contains a total starch content of 20% by weight).
HiSF-LoFV diet polysaccharides were analyzed by the Center for Complex Carbohydrate Research at the University of Georgia in Athens. Glycosyl composition analysis was performed by combined GC-MS of the per-O-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis (Santander et al., 2013). Briefly, samples (300-500 μg) were heated with methanolic HCl in a sealed screw-top glass test tube for 17 h at 80° C. After cooling and removal of the solvent under a stream of nitrogen, samples were derivatized with Tri-Sil® (Pierce) at 80° C. for 30 min. GC-MS analysis of the TMS methyl glycosides was performed on an Agilent 7890A GC interfaced to a 5975C mass selective detector (MSD), using a Supelco Equity-1 fused silica capillary column (30 m×0.25 mm ID).
Glycosyl linkage analysis of HiSF-LoFV diet polysaccharides was performed as previously described with slight modification (Heiss et. al., 2009). Samples were permethylated, depolymerized, reduced and acetylated, and the resulting partially methylated alditol acetates (PMAAs) were analyzed by GC-MS. About 1 mg of the sample was used for linkage analysis. The sample was suspended in 200 μL of dimethyl sulfoxide and left to stir for 1 day. Permethylation of the sample was affected by two rounds of treatment with sodium hydroxide (15 minutes) and methyl iodide (45 minutes). The permethylated material was hydrolyzed using 2 M TFA (2 hours in sealed tube at 121° C.), reduced with NaBD4, and acetylated using acetic anhydride/TFA. The resulting PMAAs were analyzed on an Agilent 7890A GC interfaced to a 5975C MSD (electron impact ionization mode); separation was performed on a 30 m Supelco SP-2331 bonded phase fused silica capillary column.
V4-16S rRNA gene sequencing—DNA was isolated from fecal samples by first bead-beating the sample with 0.15 mm-diameter zirconium oxide beads and a 5 mm-diameter steel ball in 2× buffer A (200 mM NaCl, 200 mM Tris, 20 mM EDTA), followed by extraction in phenol:chloroform:isoamyl alcohol, and further purification (QiaQuick 96 purification kit; Qiagen, Valencia, Calif.). PCR amplification of the V4 region of bacterial 16S rRNA genes was performed as described (Bokulich et al., 2013). Amplicons with sample-specific barcodes were pooled for multiplex sequencing using an Illumina MiSeq instrument. Reads were demultiplexed and rarefied to 5000 reads per sample. Reads sharing 99% nucleotide sequence identity [99% ID operational taxonomic units (OTUs)], that mapped to a reference OTU in the GreenGenes 16S rRNA gene database (McDonald et al., 2012) were assigned to that OTU. The 16S rRNA gene could not be amplified in multiple fecal DNA samples from mice fed 8% cocoa fiber. A small subset of reads (<5%) representing additional V4-16S rDNA amplicon sequences produced from colony-purified stocks of Bacteroides ovatus, Parabacteroides distasonis, Dorea longicatena, and Collinsella aerofaciens were omitted from our analyses of fecal DNA samples. Streptococcus thermophilus, an organism heavily used in cheese processing, was also omitted based on its detection in DNA isolated from samples of the sterile HiSF-LoFV diet.
COPRO-Seq analyses of bacterial species abundances—Libraries were prepared from fecal DNA using sonication and addition of paired-end barcoded adaptors (McNulty et al., 2013) or by tagmentation using the Nextera DNA Library Prep Kit (Illumina) and combinations of custom barcoded primers (Adey et al., 2010). Libraries were sequenced using an Illumina NextSeq instrument [1,011,017±314,473 reads/sample (mean±SD) across experiments]. Reads were mapped to bacterial genomes with previously published custom Perl scripts (see below) adapted to use Bowtie II for genome alignments (Hibberd et al., 2017); samples represented by less than 150,000 uniquely mapped reads were omitted from the analysis.
Community-wide quantitative proteomics—Lysates were prepared from fecal samples by bead beating in SDS buffer (4% SDS, 100 mM Tris-HCl, 10 mM dithiothreitol, pH 8.0) using 0.15 mm diameter zirconium oxide beads, followed by centrifugation at 21,000×g for 10 minutes. Pre-cleared protein lysates were further denatured by incubation at 85° C. for 10 minutes, and adjusted to 30 mM iodoacetamide to alkylate reduced cysteines. After incubation in the dark for 20 minutes at room temperature, protein was isolated by chloroform-methanol extraction. Protein pellets were then washed with methanol, air dried, and re-solubilized in 4% sodium deoxycholate (SDC) in 100 mM ammonium bicarbonate (ABC) buffer, pH 8.0. Protein concentrations were measured using the BCA (bicinchoninic acid) assay (Pierce). Protein samples (250 □g) were then transferred to a 10 kDa MWCO spin filter (Vivaspin 500, Sartorius), concentrated, rinsed with ABC buffer, and digested in situ with sequencing-grade trypsin (Clarkson et al., 2017). The tryptic peptide solution was then passed through the spin-filter membrane, adjusted to 1% formic acid to precipitate the remaining SDC, and the precipitate removed from the peptide solution with water-saturated ethyl acetate. Peptide samples were concentrated using a SpeedVac, measured by BCA assay and analyzed by automated 2D LC-MS/MS using a Vanquish UHPLC with autosampler plumbed directly in-line with a Q Exactive Plus mass spectrometer (Thermo Scientific) outfitted with a 100 μm ID triphasic back column [RP—SCX-RP; reversed-phase (5 μm Kinetex C18) and strong-cation exchange (5 μm Luna SCX) chromatographic resins; Phenomenex] coupled to an in-house pulled, 75 μm ID nanospray emitter packed with 30 cm Kinetex C18 resin. For each sample, 12 μg of peptides were autoloaded, desalted, separated and analyzed across four successive salt cuts of ammonium acetate (35, 50, 100 and 500 mM), each followed by a 105-minute organic gradient. Eluting peptides were measured and sequenced by data-dependent acquisition on the Q Exactive Plus (Clarkson et al., 2017).
MS/MS spectra were searched with MyriMatch v.2.2 (Tabb et al., 2007) against a proteome database derived from the genomes of the strains in the defined model community concatenated with major dietary protein sequences, common protein contaminants, and reversed entries to estimate false-discovery rates (FDR). Since the relative abundance of B. thetaiotaomicron 7330 was low on day 6 [0.05%±0.041% (mean±SD) for all groups], we chose to analyze all peptides that mapped to the B. thetaiotaomicron VPI-5482 proteome, regardless of whether they also mapped to B. thetaiotaomicron 7330. Peptide spectrum matches (PSM) were required to be fully tryptic with any number of missed cleavages, and contain a static modification of 57.0214 Da on cysteine and a dynamic modification of 15.9949 Da on methionine. PSMs were filtered using IDPicker v.3.0 (Ma et al., 2009) with an experiment-wide FDR <1% at the peptide-level. Peptide intensities were assessed by chromatographic area-under-the-curve (label-free quantification option in IDPicker). To remove cases of extreme sequence redundancy, the community meta-proteome was clustered at 100% sequence identity post-database search [UCLUST; (Edgar, 2010)] and peptide intensities were summed to their respective protein groups/seeds to estimate overall protein abundance. Proteins were included in the analysis only if they were detected in more than 3 biological replicates in at least one experimental group. Missing values were imputed to simulate the limit of detection of the mass spectrometer, using mean minus 2.2× standard deviation with a width of 0.3× standard deviation. Four additional imputed distributions produced results that were in general agreement with this approach in terms of fold-abundance change induced by fiber treatment and statistical significance.
Multi-taxon INSeq—Multi-taxon INSeq allows simultaneous analysis of multiple mutant libraries in the same recipient gnotobiotic mouse owing to the fact that the mariner Tn vector contains Mmel sites at each end plus taxon-specific barcodes. Mmel digestion cleaves genomic DNA at a site 20-21 bp distal to the restriction enzyme's recognition site so that the site of Tn insertion and the relative abundance of each Tn mutant can be defined in given diet/community contexts by sequencing the flanking genomic sequence and taxon-specific barcode (Wu et al., 2015). Purified fecal DNA was processed as described previously (Wu et al., 2015). DNA was digested with Mmel and the products were ligated to sample-specific barcoded adaptors. Sequencing was performed on an Illumina HiSeq 2500 instrument, with a custom indexing primer providing the strain-specific barcode for the insertion. Analysis of mutant strain frequencies was carried out using custom software. Log ratios of the abundances of Tn mutant strains on experimental days 6 and 2 (corresponding to the period of fiber treatment compared to just prior to fiber exposure) were calculated for each mouse.
PUL nomenclature and homology—All PUL assignments were made based on “new assembly” genomes present in the CAZy PUL database_(www.cazy.org/PULDB) (Terrapon et al., 2018). All boundaries of PULs were algorithmically defined (listed as ‘predicted PUL’ in PULDB). The algorithmically defined boundaries of B. thetaiotaomicron PUL7 were extended to include the adjacent arabinose operon based on previously published experimental datasets (Schwalm et al., 2016). A cluster of three or more adjacent CAZymes was defined as a ‘polysaccharide utilization complement’. Homology between genes in PULs was determined using a reciprocal BLASTp approach with an E-value threshold of 1×10−9, querying each protein product contained within a CAZy-annotated PUL against reference genomes from other species in the community.
Generation of glycan-coated magnetic beads—Wheat Arabinoxylan and Icelandic Moss Lichenan were purchased from Megazyme (P-WAXYL, P-LICHN) and yeast alpha-mannan was purchased from Sigma-Aldrich (M7504). Polysaccharides were solubilized in water (at a concentration of 5 mg/mL for pea fiber and 20 mg/mL for arabinoxylan and lichenan), sonicated and heated to 100° C. for 1 minute, then centrifuged at 24,000×g for 10 minutes to remove debris. TFPA-PEG3-biotin (Thermo Scientific), dissolved in DMSO (10 mg/mL) was added to the polysaccharide solution at a ratio of 1:5 (v/v). The sample was subjected to UV irradiation for 10 minutes (UV-B 306 nm, 7844 mJ total), and then diluted 1:4 to facilitate desalting on 7 kD Zeba spin columns (Thermo Scientific).
Biotinylated polysaccharide was mixed with one of several biotinylated fluorophores (PF-505, PF-510LSS, PF-633, PF-415; all at a concentration of 50 ng/mL; all obtained from Promokine). A 500 μL aliquot of this preparation was incubated with 107 paramagnetic streptavidin-coated silica beads (LSKMAGT, Millipore Sigma) for 24 hours at room temperature. Beads were washed by centrifugation three times with 1 mL HNTB buffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, 0.1% BSA) followed by addition of 5 μg/mL streptavidin (Jackson Immunoresearch) in HNTB (30 min incubation at room temperature). Beads were washed as before and then incubated with 250 μL of the biotinylated polysaccharide preparation. The washing, streptavidin, and polysaccharide incubation steps were repeated three times. Bead preparations were assessed using an Aria III cell sorter (BD Biosciences) to confirm adequate labeling, and then analyzed by GC-MS (see below) to quantify the amount of carbohydrate bound.
Administration and recovery of beads—Beads were incubated with 70% ethanol for 1 minute in a biosafety cabinet, then washed three times with 1 mL sterile HNTB using a magnetic stand. The different bead types were combined, diluted, and aliquoted to 107 beads per 650 μL HNTB insterile Eppendorf microcentrifuge tubes. The number of beads in each aliquot was counted using an Aria III cell sorter and CountBright fluorescent microspheres (BD Bioscience). Tubes containing beads were introduced into gnotobiotic isolators and the beads were administered by oral gavage (600 μL per mouse). Separate aliquots of control beads, used to establish input carbohydrate content were stored in the dark at 37° C. until collection of experimental beads from mouse fecal or cecal samples had been completed.
For germ-free mouse experiments, animals were fed the HiSF-LoFV diet for two weeks and then gavaged with beads; all fecal pellets were collected during the 4- to 12-hour interval that followed gavage. During this time period, bedding was removed and mice were placed on grated cage bottoms (with access to food and water); cage bottoms were placed just above a 0.5 cm deep layer of sterile water on the floor of the cage, to prevent pellets from drying. For colonized animals, cecal and colonic contents were collected four hours after administration of beads at the time of euthanasia. Recovered samples were immediately placed in sterile water on ice.
Fecal, cecal, and input samples were vortexed and filtered through nylon mesh (100 μm pore-diameter). The resulting suspension of luminal contents was layered over sterile Percoll Plus (GE Health Care) and centrifuged for 5 minutes at 500×g. Beads were collected from underneath the Percoll layer and washed four times using a magnetic stand, each time with 1 mL fresh HNTB. Recovered beads were counted by flow cytometry as before, filtered through nylon mesh (40 μm pore diameter, BD Biosciences) and stored at 4° C. overnight. Beads were sorted back into their polysaccharide types based on fluorescence using an Aria III sorter (average sort purity, 96%). Sorted samples were centrifuged (500×g for 5 minutes) to pellet beads and the beads were transferred to a 96-well plate. All bead samples were incubated with 1% SDS/6M Urea/HNTB for 10 minutes at room temperature to remove exogenous components, washed three times with 200 μL HNTB using a magnetic plate rack, and then stored overnight at 4° C. prior to monosaccharide analysis.
Analysis of bead-bound glycan by GC-MS—The number and purity of beads in each sorted sample was determined by taking an aliquot for analysis on the Aria III cell sorter. Equal numbers of beads from each sample were transferred to a new 96-well plate and the supernatant was removed with a magnetic plate rack. For acid hydrolysis, 200 μL of 2M trifluoroacetic acid and 250 ng/mL myo-inositol-D6 (CDN Isotopes; spike-in control) were added to each well, and the entire volume was transferred to 300 μL glass vials (ThermoFisher; catalog number C4008-632C). Another aliquot was taken to verify the final number of beads in each sample. Monosaccharide standards were included in separate wells and subjected to the hydrolysis protocol in parallel with the other samples. Vials were crimped with Teflon-lined silicone caps (ThermoFisher) and incubated at 100° C. with rocking for 2 h. Vials were then cooled, spun to pellet beads, and their caps were removed. A 180 μL aliquot of the supernatant was collected and transferred to new 300 μL glass vials. Samples were dried in a SpeedVac for 4 hours, methoximated in 20 μL O-methoxyamine (15 mg/mL pyridine) for 15 h at 37° C., followed by trimethylsilylation in 20 μL MSTFA/TMCS [N-Methyl-N-trimethylsilyltrifluoroacetamide/2,2,2-trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, chlorotrimethylsilane] (ThermoFisher) for 1 h at 70° C. One half volume of heptane (20 μL) was added before loading the samples for injection onto a 7890B gas chromatography system coupled to a 5977B MS detector (Agilent). The mass of each monosaccharide detected in each sample of sorted beads was determined using monosaccharide standard curves. This mass was then divided by the final count of beads in each sample to produce a measurement of mass of recoverable monosaccharide per bead.
Quantification and Statistical Analysis—Using data from days 6 and 7 of each diet treatment, a mixed effects model was generated in the R programming environment for each species in each of three fiber screening experiments. The relative abundance of that species in feces (or the relative abundance scaled by fecal DNA yield) was used as the dependent variable, and the concentration of administered fiber (10 to 13 fibers tested per experiment), as well as experimental day were used as independent variables. Mixed effects models incorporated terms to describe repeated measures of individual mice. In rare cases where B. cellulosilyticus failed to colonize (5 of 60 mice), the animals were not considered biological replicates since they harbored a distinct microbiota; they were omitted from the models. ANOVA (with Satterthwaite approximation for degrees of freedom) was performed to evaluate the significance of individual terms in models (FDR corrected P value cutoff of 0.01). Models were evaluated based on conditional R2 values (incorporating random factors) and plots of the residuals and Cook's distance (no samples were excluded based on these assessments).
For COPRO-Seq analyses, differences between groups were assessed using mixed-effect models with time as a categorical variable, including day 2 as a pre-treatment time point. For omission experiments, the abundance of each strain as a proportion of all other strains except the omitted strain or strains was used for statistical tests. Significant terms in models were identified using ANOVA (FDR corrected P value cutoff of 0.05). Mann-Whitney U test was used for analyses of individual time-points of interest.
For quantitative proteomics, significant differences in protein abundance were determined using limma (Ting et al., 2009). For multi-taxon INSeq analyses, mutant strain abundances were analyzed using limma-voom (Law et al., 2014) after quantile normalization. The general linear model framework in limma-voom allowed us to perform moderated t-tests to determine the statistical significance (P<0.05, FDR corrected) of differences in fitness in the context of the control versus fiber-supplemented diets. A Mann-Whitney U test was used to calculate significant differences in monosaccharide abundance between bead samples. All tests were two-tailed.
Data and Software Availability—Datasets of V4-16S rRNA sequences in raw format prior to post-processing and data analysis, plus COPRO-Seq and INSeq datasets have been deposited at the European Nucleotide Archive under study accession PRJEB26564. All LC-MS/MS proteomic data have been deposited into the MassIVE data repository under accession numbers MSV000082287 (MassIVE) and PXD009535 (ProteomeXchange). INSeq software: github.com/mengwu1002/Multi-taxon_analysis_pipeline. COPRO-Seq software: github.com/nmcnulty/COPRO-Seq.
Beads were coated with one of 14 different glycans, as described in Example 1. The glycans are shown along the x-axis of
These results demonstrated that microbes from the mouse cecum bind to particular plant polysaccharides (Arabinan from Ghatti Gum). Three technical replicates are shown with standard deviation.
This approach can be extended to fecal samples obtained from humans. It could also be extended to encompass the oral administration of beads to mice, humans, or other animals, with the addition of DNA sequencing of recovered beads to identify the particular species of microbes that bind to the beads in vivo.
This example describes experiments to determine if there was a bioactive component of the pea fiber preparation used in Examples 2-6 that was responsible for increasing the representation of targeted Bacteroides represented in a model human gut community installed in gnotobiotic mice. The pea fiber preparation was subjected to extraction under increasingly harsh conditions with aqueous solutions to differentially solubilize constituents (Pattathil et al.) (
Fraction 8, obtained using the harshest conditions (4 M KOH for 24 hours at 22° C.) and containing high relative content of arabinose and galactose, was selected for further evaluation. Based on its monosaccharide composition and the results obtained from PMAA linkage analysis (Tables 13, 14), it appears that (i) fraction 8 is largely composed of arabinan that is predominately branched at the 2—, or doubly branched at the 2- and 3-positions of a linear α1-5 L-arabinofuranose backbone (
The method for pea fiber arabinan isolation was scaled up using a procedure similar to what was employed in the initial fractionation to supply sufficient quantities for studies in gnotobiotic mice (yield 22%±2% wt:wt) (
Next Fraction 8 (150 mg) was solubilized in 50 mM sodium malate (pH 6)+2 mM calcium chloride (30 mL) via incubation in a 95° C. water bath and sonication to yield a 5 mg/mL solution. To this, 3.5 mg of amyloglucoside (Megazyme; cat. no.: E-AMGFR) and 1.25 mg of alpha-amylase (Megazyme, cat. no.:E-PANAA) were added as 3 mg/mL stock solutions in 50 mM sodium malate (pH 6)+2 mM calcium chloride. Starch was digested via incubation at 37° C. for 4 hours. The digestion was terminated via enzyme denaturation by incubation at 90° C. for 30 min. The glucose product resulting from starch digestion was removed with extensive dialysis against ddH20 using 3.5 kDa molecular weight cut off Snakeskin dialysis tubing (ThermoFisher, cat. no,: 88244). The sample was dried via lyophilization to yield enzymatically destarched Fraction 8. Monosaccharide analysis and glycosyl linkage analysis was performed as described above (Table 16 and Table 17). The enzymatically destarched Fraction 8 was then used in the following animal experiment.
Four groups of adult C57BL/6J male mice fed the HiSF-LoFV diet were colonized with a defined community comprising 14 cultured, sequenced human gut bacterial strains (Ridaura et al.) (n=5 mice/arm; Table 15,
Mice were given ad libitum access to the diets for 10 days at which point all animals were gavaged with polysaccharide-coated paramagnetic fluorescent beads. Animals were sacrificed 4 hours after gavage of the beads. Bacterial community composition was assessed via short read shotgun sequencing (COPRO-Seq) of DNA purified from serially-collected fecal samples and from cecal contents harvested at the conclusion of the experiment (McNulty et al.).
Principal components analysis of the relative abundances of community members in fecal samples collected on day 11 post-colonization revealed that all 3 experimental diets produced microbial community configurations that were distinct from those in mice consuming the control unsupplemented HiSF-LoFV diet (
A time series analysis of the effects of the different glycans on the representation of community members in the fecal microbiota of mice belonging to the four treatment groups is presented in
We took advantage of the fact that the gene content of the community was known and performed mass spectrometry-based fecal meta-proteomic analysis to define the responses of community members to the different glycan preparations. Bacteroides sp. possess multiple polysaccharide utilization loci (PULs); a shared feature of PULs is an adjacent pair of susC and susD homologs responsible for binding extracellular polysaccharide fragments and importing them into the periplasm. PUL genes adjacent to these susC/susD homologs encode various carbohydrate active enzymes (CAZymes) involved in polysaccharide depolymerization (Anderson and Salyers, 1989; Terrapon et al. 2018). Expression of PUL genes is regulated in ways that allow the bacteria to acquire nutrients within the highly competitive environment of the gut.
Additionally, multi-taxon insertion site sequencing (INSeq) of the five strains represented as Tn mutant libraries was used to identify genes with significant contributions to bacterial fitness in each diet context (Wu and Gordon et al., 2015). Fitness was calculated as (i) the log 2 ratio of the number of sequencing reads originating from the site of insertion of the Tn in the organism in fecal communities sampled on dpg 6 versus dpg2, relative to (ii) the same ratio calculated in mice monotonously fed the unsupplemented HiSF-LoFV diet. A negative score indicates that a gene is important for fitness. The score of each gene was parametrized using linear models generated with limma (Richie and Smythe, 2015) to identify those whose effects on fitness were significantly different compared to when the unsupplemented HiSF-LoFV diet was being consumed. The results disclosed that the fitness scores of 332, 195, and 75 genes were significantly altered during diet supplementation with pea fiber, PFABN, or SBABN, respectively (adjusted p value <0.05, FDR-corrected).
Plots of fitness score versus change in protein abundance were subsequently generated for all genes in these Bacteroides (
B. vulgatus ATCC 8482 provided another example of PULs that target arabinan but function as supplement source-specific fitness determinants. PUL27 and PUL12 contain genes belonging to GH43 GH51 and GH146 families that have specificity for L-arabinofuranosyl structures found in arabinan (Luis et al, 2018). Expression of PUL27 is responsive to all three supplements (
We next sought to quantify how the in vivo degradative capacity of each individual mouse's microbiota changed with dietary fiber supplementation. To do so, we employed microscopic paramagnetic silica beads (average diameter=10 μm) with covalently bound glycans from enzymatically destarched Fraction 8 or purified sugar beet arabinan. Each bead type could be distinguished based on its distinct covalently linked fluorophore. Empty control beads contained no bound glycan. Beads were pooled and gavaged into mice colonized with the defined community and fed either the unsupplemented HiSF-LoFV, or the HiSF-LoFV supplemented with the pea fiber preparation, enzymatically destarched Fraction 8, or the purified sugar beet arabinan. A separate group of animals that were maintained as germ-free fed enzymatically destarched Fraction 8 supplemented HiSF-LoFV served as controls (n=5 mice/treatment group)
Animals from all groups were euthanized 4 hours after gavage of the bead mixture. Beads were then separated from cecal contents based on their density and magnetism, and each bead type was purified using fluorescence activated cell sorting (FACS) (
Comparison of germ-free controls to animals containing the defined consortium of human gut bacteria established that removal of arabinan from the different bead types was colonization-dependent. Moreover, no arabinose was detected in the empty beads that were administered to germ-free or colonized animals (
aThese 2 peaks overlapped; percentages were estimated based on MS fragmentation
aPeak overlapped with another peak; percentage estimated based on MS fragmentation
Bacteroides ovatus
Bacteroides cellulosilyticus
Bacteroides thetaiotaomicron
Bacteroides thetaiotaomicron
Bacteroides vulgatus
Bacteroides caccae
Bacteroides finegoldii
Bacteroides massiliensis
Collinsella aerofaciens
Escherichia coli
Odoribacter splanchnicus
Parabacteroides distasonis
Ruminococcaceae sp.
Subdoligranulum variabile
Introduction: Increasing effort is being directed to deciphering how components of diets consumed by various human populations impact the composition and expressed functional features of their gut microbial communities (e.g., Johnson et al., 2019; Ghosh et al., 2020). A hoped-for benefit from obtaining this knowledge is to gain new insights about how food ingredients, and their biotransformation by the microbiota, are linked to various aspects of human physiology, and new ways to both define and improve nutritional status. However, there are many formidable challenges. The gut microbiota is complex, dynamic and exhibits considerable intra- and interpersonal variation in its configurations (Lloyd-Price et al., 2017). The chemical compositions of food staples are being catalogued at ever deepening levels of detail using higher through-put analytical methods, such as mass spectrometry. Even as this knowledge is being acquired, the nature of the ‘bioactive’ components recognized by members of the microbiota, and the pathways through which these chemical entities are metabolized by community members to influence their functions and those of the host remain poorly defined. Furthermore, much needs to be learned about the effects of current methods of food processing on the representation of these bioactives (Wolf et al., 2019; Carmody et al., 2019), and the mechanisms that determine whether and how microbes compete and/or cooperate for these food components (Patnode et al., 2019).
Dietary plant fibers epitomize these challenges and opportunities. Fibers are complex mixtures of biomolecules whose composition varies depending upon their source, their method of initial recovery, and the food processing technologies used to incorporate them into food products that have satisfactory organoleptic properties (texture, taste, smell) (Caffall and Mohnen, 2009). The vast majority of studies testing the biological effects of fibers have been performed with preparations whose biochemical features are largely uncharacterized. Fiber components include but are not limited to polysaccharides, proteins, fatty acids, polyphenols and other plant-derived small molecules (Nicolson et al., 2012; Scalbert et al., 2014). Separating and/or purifying component glycans from crude fiber mixtures can be very challenging; even if separation is achieved, painstaking analysis of features such as glycosidic linkages is required to define their structures (Pettolino et al., 2012). Knowing that a given microbiota member has a suitable complement of genes for acquiring and processing a given glycan structure does not necessarily predict whether that organism will be a consumer in vivo. Other factors need to be considered. For example, an individual's microbiota may harbor a number of organisms with the capacity to compete or cooperate with one another for utilization of a given type of glycan. A given dietary fiber typically contains a multiplicity of glycans. The physical-chemical structure of a fiber (e.g., its size, surface properties/nutrient composition) in a given region of the gut could influence which set of microbes attach to its surface, how its associated microbes prioritize consumption of its component glycans and how/whether particle-associated microbes can share products of glycan metabolism with one another.
The examples illustrate an approach for addressing some of these questions using pea fiber as an example. Pea fiber was selected based on results obtained from a recently published screen we conducted of 34 types of food-grade plant fibers obtained from various sources, including the waste streams of food manufacturing (Patnode et al., 2019). The screen was conducted in gnotobiotic mice colonized with a defined consortium of cultured sequenced human gut bacterial strains, including several saccharolytic Bacteroides species. Mice were fed a low fiber diet formulated to represent the upper tertile of saturated fat consumption and lower tertile of fruits and vegetable consumption by individuals living in the USA, as reported in the NHANES database. Supplementation of this diet with fiber from the seed coat of the pea, Pisum sativum, produced a significant increase in the abundance of Bacteroides thetaiotaomicron (Patnode et al., 2019). An arabinan-enriched fraction from raw pea fiber was purified and its structure defined (Example 12—Fraction 8, referred to in this example as PFABN). Forward genetic and proteomic analyses were used to compare its biological effects, versus those of unfractionated pea fiber and an arabanin from sugar beet with distinct glycosidic linkages, on members of a defined bacterial consortium containing human gut Bacteroides that was established in gnotobiotic mice (Example 12). A generalizable method for covalently attaching different glycans to microscopic paramagnetic glass beads with different covalently bound fluorophores was described (Example 12). Introduction of these ‘Microbiota Functional Activity Biosensors’ (MFABs) into gnotobiotic mice fed the HiSF-LoFV diet with or without glycan supplementation followed by their recovery from the gut allowed us to directly compare the capacity of these glycans to be metabolized by this community (Example 12 and this example). Co-localizing pea fiber arabinan with another type of polysaccharide not found in the diet (glucomannan) on an MFAB surface enhanced the efficiency of microbial community metabolism of bead-associated glucomannan when animals were given pea-fiber supplemented HiSF-LoFV diet (this example). Collectively, these findings illustrate how knowledge of the bioactive components of fibers, and the capacity to directly measure microbiota function with MFABs, could provide new approaches for designing ‘next generation’ prebiotics and foods that are more accessible to, and have a greater impact on, the gut microbiota (and by extension, the host).
Covalent linkage of various fluorescent labels and glycans to paramagnetic MFABs—To quantify PFABN and SBABN utilization as a function of diet, a versatile way to covalently link polysaccharides to recoverable, paramagnetic, microscopic glass beads that could function as biosensors of their degradation was sought. For covalent polysaccharide immobilization on a bead surface, a cyano-transfer reaction employed in the synthesis of polysaccharide-conjugate vaccines was adapted (Lees et al., 1996; Shafer et al., 2000).
Using SBABN as a test case, we found that a 1:7 mol ratio of CDAP to its calculated moles of hexose (assuming for the purpose of a generalizable calculation, that the polysaccharide is only composed of hexose), resulted in consistent and specific SBABN immobilization without ligand over-activation (manifested by aggregation and carbamoylation of hydroxyl groups) (
Quantifying polysaccharide degradation with MFABs in gnotobiotic mice—PFABN and SBABN were immobilized onto amine plus phosphonate-derivatized beads. Beads acetylated with acetic anhydride after fluorophore labeling were used as controls (
The quantities of neutral monosaccharides liberated by acid hydrolysis from the surfaces of beads recovered from the cecums of germ-free mice were not significantly different from the amounts liberated from the input bead preparations with one exception—a slight, albeit statistically significant, increase in galactose (
In contrast to germ-free controls, the masses of arabinan was significantly decreased when PFABN- or SBABN-coated beads were recovered from colonized mice fed the unsupplemented HiSF-LoFV diet (
Beads coated in PFABN revealed that xylan (xylose monosaccharide remaining on PFABN beads) was more efficiently processed by the microbiota in all three supplemented diet contexts (
Co-localization of distinct glycans on the same bead: As noted in the Introduction, plant-derived dietary fibers have complex physical-chemical properties manifest in part by their mixtures of different glycan structures and by their varying shapes and surface properties. Fiber particles are impacted by (i) methods, such as extrusion, that are commonly used to incorporate fibers into food products so that these products have acceptable organoleptic properties (Gualberto et al., 1997; Shahidi et al., 1998), and (ii) the mechanical forces and digestive enzymes (both host and microbial) that are encountered as food passes through the gastrointestinal tract. We reasoned that the MFAB platform could provide a way of testing whether deliberately co-localizing distinct polysaccharides would result in their synergistic utilization by microbial community members.
To explore this notion, we turned to glucomannan, a hemicellulosic linear 13(1-4) polysaccharide composed of D-mannose and D-glucose. We found that among the pea fiber-responsive Bacteroides identified above, only B. ovatus and B. cellulosilyticus were able to grow in minimal medium containing glucomannan as the sole carbon source (
Based on these considerations, we hypothesized that supplementing the diet with pea fiber would induce expression of PULs in community members so that they could readily utilize bead-associated PFABN; moreover, those community members that could utilize PFABN and express β-mannosidases would be able to more efficiently access/metabolize glucomannan positioned on the same bead. To test this hypothesis, we synthesized beads coated with PFABN alone, glucomannan alone, or both glycans together, as well as control acetylated beads that lack a bound polysaccharide (
Discussion—The bead-based Microbiota Functional Activity Biosensors (MFAB) described in this report represent a platform technology for measuring biochemical activities expressed by a microbial community. Installing specific functional groups on the surfaces of microscopic paramagnetic glass beads using commercially available organosilane reagents creates a biorthogonal ‘handle’ for covalent attachment of ligands. This approach represents an alternative to a procedure we described recently, where bifunctional biotinylated ligands are generated prior to immobilization on glass beads coated with streptavidin (Patnode et al., 2019). By immobilizing ligand directly on the bead surface, MFABs possess considerably more sites for ligand attachment than do streptavidin beads. Higher ligand attachment density enables higher levels of ligand loading, which increases the dynamic range of a functional activity readout.
Crude dietary fibers contain various polysaccharides intercalated within a dense cellulose-lignin matrix. The chemistry for covalent attachment employed with MFABs not only allows for dense ligand presentation, but also enables multiple ligands to be simultaneously immobilized to create ‘hybrid’ beads that can be used to model the effects of physical co-localization of different fiber components on microbial utilization. In principle, a wide range of different glycan combinations with varying stoichiometries can be explored owing to the fact that different hybrid bead types, each with its own fluorophore, can be created and tested simultaneously in vitro and in vivo (the latter using defined communities or intact uncultured microbial communities).
The identification of bioactive components of fibers and their combination with other prebiotic glycans offers an approach for creating formulations with enhanced capacity to alter the expressed properties of targeted members of a microbial community. Extrapolating, producing such combinations could provide a way of realizing the health benefits of fiber-containing foods but at lower amounts of total fiber. This last feature would help food scientists surmount the challenge of dealing with the unsatisfactory organoleptic properties commonly encountered with high fiber content food formulations.
The approach we describe in this report for ligand immobilization does not require the synthesis of bifunctional ligands (or fluorophores); instead, custom functional groups can be incorporated into the probe through modification of the organosilane donor molecule. As such, the MFAB platform provides an opportunity to develop chemistries for nondestructively releasing ligands for analysis (Bielski et al., 2013). For example, characterization of microbial utilization of polysaccharides needs to move beyond relatively ‘simple’ GC-MS measurements of monosaccharides released from the surface of recovered beads to readouts of glycan structures recovered from the bead surface (prior to and after exposure to microbes). This information would provide a more informed view of functional properties (saccharolytic activities) expressed by a microbial community as a function of the donor and diet, as well as greater insights about structure/activity relationships of existing or new candidate prebiotic and synbiotic formulations.
Purification of pea fiber arabinan (PFABN): Fractionation of pea fiber—Raw pea fiber was fractionated using serial extractions with aqueous buffers of increasing harshness (Pattathil et al., 2012). Pea fiber (Rattenmaier; Cat. No.: Pea Fiber EF 100) (5 g) was defatted by stirring at 23° C. for two hours in 60 mL of 80% (vol:vol) ethanol. Fiber was pelleted by centrifugation (3,500×g, 5 minutes) and the supernatant was removed. Neat ethanol was added to the pelleted fiber and the solution was mixed for two minutes. Fiber was centrifuged (3,500×g, 5 minutes) and the supernatant was removed. Neat acetone was added to the pelleted fiber, the solution was mixed for two minutes, centrifuged (3,500×g, 10 minutes), and the supernatant was removed. The resulting ‘defatted’ pea fiber was dried in a chemical hood overnight. Defatted pea fiber was subsequently resuspended in 200 mL of 50 mM ammonium oxalate (pH=5.7) and stirred at 23° C. for 20 hours. The suspension was centrifuged (7,000×g, 15 minutes), the supernatant was collected, concentrated [Amicon Stirred Cell concentrator (Millipore Sigma; Cat. No.: UFSC20001) with a 3 kDa molecular weight cut-off ultrafiltration disk (Millipore Sigma; Cat. No.: PLBC06210)] and then dialyzed extensively against water [3.5 KDa molecular weight cut-off dialysis tubing (Thermo Scientific; Cat. No.; 88244) or 3.5 KDa molecular weight cut-off Slide-A-Lyzer dialysis cassettes (Thermo Scientific)]. The precipitate from the dialysis was recovered by centrifugation (15,000×g, 15 minutes). The precipitate and soluble material from the dialysis, representing fractions one and two, respectively, were dried with lyophilization.
The pellet from the ammonium oxalate extraction was washed with 200 mL of water, centrifuged (4,000×g, 15 minutes), and the supernatant was discarded. The pellet was resuspended in 200 mL of 50 mM sodium carbonate (pH=10) containing 0.5% (wt:wt) sodium borohydride and stirred at 23° C. for 20 hours. The suspension was centrifuged (6,000×g, 15 minutes) and the supernatant was collected. Borohydride was quenched by slowly adding glacial acetic acid. A stringy precipitate began to form as the pH decreased. The suspension was concentrated (as above); the insoluble and soluble portions of the resulting concentrated carbonate suspension were separated with centrifugation (15,000×g, 15 minutes), yielding fractions three and four, respectively. Fractions were dialyzed and dried with lyophilization.
The pellet from the carbonate extraction was washed with water before resuspension in 200 mL of 1 M potassium hydroxide containing 1% wt:wt sodium borohydride and stirring for 20 hours at 23° C. The suspension was centrifuged (6,000×g, 15 min) and the supernatant was removed. Five drops of 1-octanol were added to prevent foaming during borohydride quenching. A light precipitate began to form in the solution as the pH decreased. The suspension was concentrated; the insoluble and soluble portions of the concentrated 1 M hydroxide extract were separated with centrifugation (15,000×g, 15 minutes), yielding fractions five and six, respectively. Fractions were dialyzed and dried with lyophilization.
The pellet from the 1 M hydroxide extraction was washed with water before resuspension in 200 mL of 4 M potassium hydroxide containing 1% wt:wt sodium borohydride. The mixture was stirred at 23° C. for 20 hours. The suspension was then centrifuged (6,000×g, 15 min) and the supernatant was removed. 1-Octanol were added to prevent foaming during borohydride quenching; during this process, a precipitate formed, then dissolved, then reformed as the pH was lowered to 6.0. The resulting suspension was concentrated; the insoluble and soluble portions of the concentrated 4 M hydroxide extract were separated with centrifugation (15,000×g, 15 min), yielding fractions seven and eight, respectively. Fractions were dialyzed and dried with lyophilization. Note that after each extraction, sodium azide was added to a final concentration of 0.05% prior to concentration and dialysis.
Purification of pea fiber arabinan (PFABN): Characterization of pea fiber fractions—Each of the eight fractions was resuspended in water (1 mg/mL) by heating to 90° C. and sonication (Branson Sonifer). Insoluble material was removed by centrifugation (18,000×g, 5 minutes). The soluble material was assayed for protein content (bicinchoninic acid assay; Thermo Scientific; Cat. No.: 23227) using bovine serum albumin as a standard, DNA content (UV-visible absorbance spectroscopy, Denovix DS-11 spectrophotometer) and total carbohydrate content (phenol-sulfuric acid assay, Masuko et al., 2005) using D-glucose as a standard (Table 18). The molecular size of each fraction was measured using an Agilent 1260 high performance liquid chromatography (HPLC) system equipped with an evaporative light scattering detector. An Agilent Bio Sec-5 column (Cat. No.: 5190-2526) and guard were used with water as the mobile phase. Unbranched pullulan was used as length standards (Shodex; Cat. No.: Standard P-82). The monosaccharide composition of each fraction was measured using polysaccharide methanolysis followed by GC-MS (Doco et al., 2001). [1,2,3,4,5,6-2H]-Myo-inositol (CDN Isotopes; Cat. No.: D3019) was used as an internal standard. Two-fold dilutions of free monosaccharide standards (L-arabinose, D-galactose, D-galacturonic acid, D-glucose, D-glucuronic acid, D-mannose, D-rhamnose, D-xylose) were simultaneously derivatized and used to quantify the absolute abundance of each monosaccharide in each fraction. GC-MS peaks were quantified using metaMS (Wehrens et al., 2014). Glycosyl linkage analysis was performed on fractions five, seven, and eight at the Complex Carbohydrate Research Center (University of Georgia) employing previously described methods (Anumula and Taylor, 1992). Fraction eight was enriched in arabinan and designated PFABN.
Purification of pea fiber arabinan (PFABN): Procedure for scaled up isolation of PFABN—The isolation procedure described above was slightly modified to recover gram quantities of PFABN. Raw pea fiber was resuspended at 50 mg/mL in 1 M potassium hydroxide containing 0.5% (wt:wt) sodium borohydride and stirred at room temperature for 24 hours. The suspension was centrifuged (3,900×g, 20 minutes) and the supernatant was discarded. The pellet from the 1M potassium hydroxide extraction was resuspended in 4 M potassium hydroxide containing 0.5% (wt:wt) sodium borohydride (50 mg/mL), and stirred at room temperature for 24 hours. The suspension was centrifuged and the supernatant was collected and neutralized with 4 M acetic acid. Neat ethanol was added [7.5:1 (vol:vol)] and polysaccharide was precipitated at −20° C. Precipitated polysaccharide was isolated by centrifugation (3,900×g, 20 minutes), and rinsed with 250 mL of 80% ethanol (4° C.) three times. The pellet was dried overnight under a dry nitrogen stream. The entire procedure was repeated five times to isolate 51 grams of PFABN (overall yield 22%). Isolated PFABN was pulverized (Spex SamplePrep Freezer/Mill; Metuchen, N.J.; Model 6870) and total carbohydrate content was defined (phenol-sulfuric acid assay).
Gas chromatography-mass spectrometry of neutral monosaccharide composition—Purified PFABN was suspended in water at a concentration of 1 mg/mL and transferred to 8 mm crimp top glass vials (Fisher Scientific; Cat. No.: C4008-632C). 1754 of 2 M trifluoroacetic acid containing 15 ng of D6-myo-inositol was added and the vials were capped with Teflon-coated aluminum caps (Fisher Scientific; Cat. No.: C4008-2A). PFABN was hydrolyzed for 2 hours at 95° C. Samples were then centrifuged (3,200×g, 5 minutes), the supernatant was transferred to a new glass vial and the material was dried under reduced pressure. Samples were subsequently oximated by adding 20 μL of methoxyamine (15 mg/mL pyridine) and incubating the solution overnight at 37° C. 20 μL of MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide plus 1% TCMS (2,2,2-trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, chlorotrimethylsilane) (Thermo Scientific; Cat. No.: TS-48915) were added and the solution was incubated at 70° C. for one hour. The material was subsequently diluted with 20 heptane before analysis using an Agilent 7890A gas chromatography system coupled with an Agilent 5975C mass spectrometer detector. Employing L-arabinose, D-galactose, D-glucose, D-mannose, D-rhamnose, D-xylose standards, peaks were identified and quantified using metaMS (Wehrens et al., 2014); peak areas were corrected using a D6-myo-inositol standard and quantified using linear fits of two-fold diluted standards.
PFABN linkage analysis—PFABN was enzymatically de-starched using amyloglucosidase and □-amylase (Megazyme; Cat. No. E-AMGFR and E-PANAA, respectively). To do so, PFABN was first resuspended by heating at 95° C. in a solution containing 50 mM sodium malate (pH=6) and 2 mM calcium chloride (5 mg/mL). Based on the manufacturer's measurement of the specific activities of these two enzymes, we added an amount that should be sufficient to degrade all starch within the PFABN fraction within one minute; nonetheless, we allowed degradation to proceed for 4 hours at 37° C. before terminating the reaction by incubation at 95° C. for 20 minutes. Polysaccharide was dialyzed extensively against water and dried by lyophilization. Complete digestion of starch was confirmed with GC-MS analysis of neutral monosaccharides.
Glycosyl-linkage analysis was performed on the de-starched PFABN at the Complex Carbohydrate Research Center (University of Georgia) using previously described methods (Anumula and Taylor, 1992). Briefly, polysaccharide (1 mg) was taken up in dimethyl sulfoxide, permethylated in the presence of NaOH base, hydrolyzed for 2 hours in 2 M trifluoroacetic acid at 121° C., reduced overnight with sodium borohydride and acetylated with acetic anhydride and pyridine. Inositol was used as an internal standard. The resulting partially methylated alditol acetates were analyzed by GC-MS [HP-5890 instrument interfaced with a 5970 mass selective detector using a SP2330 capillary column (30×0.25 mm ID, Supelco) and a temperature program of 60° C. for 1 min, increasing to 170° C. at 27.5° C./minute, and to 235° C. at 4° C./minute with a 2-minute hold, and finally to 240° C. at 3° C./minute with 12-minute hold]. Sugar beet arabinan (Megazyme; Cat. No. P-ARAB) was analyzed simultaneously. The resulting linkage data are presented in Table 16.
Generation of microbiota functional activity biosensors: Synthesis of amine phosphonate beads—Paramagnetic, 10 μm-diameter glass beads (Millipore Sigma; Cat. No.: LSKMAGN01) were incubated at 23° C. overnight in a solution of 20 mM HEPES (pH 7.4) and 100 mM NaCl. Equal molar amounts of (3-aminopropyl)triethoxysilane (ATPS; Sigma Aldrich, Cat. No. 440140) and 3-(trihydroxysilyl)propyl methylphosphonate (THPMP; Sigma Aldrich, Cat. No. 435716) were subsequently added to a suspension of hydrolyzed NHS-ester-activated beads in deionized water (Bagwe et al, 2006; Soto-Cantu and Russo et al, 2012). Beads were derivatized at a density of 5×106/mL and the organosilane reagents were included at 1000-fold excess of what would be required to coat the bead surface (based on 4 silane molecules per nm2; Soto-Cantu and Russo, 2012). The reaction was allowed to proceed for 5 hours at 50° C. with shaking and then terminated with three cycles of washing in water (using a magnet to recover the beads after each wash cycle). Beads were stored at 4° C. in a sterile solution of 20 mM HEPES (pH 7.2) and 100 mM NaCl.
Bead Zeta potential was measured to characterize the extent of modification of the bead surface; Zeta potential was determined for beads reacted with organosilane reagents and beads subjected to surface amine acetylation. Zeta potential measurements were made on a Malvern ZEN3600 instrument using disposable zeta potential cuvettes (Malvern). Beads were resuspended to a concentration of 5×105/mL in 10 mM HEPES (pH 7.2) passed through a 0.22 μm filter (Millipore) and analyzed in triplicate. Measurements were obtained with the default settings of the instrument, using the refractive index of SiO2 as the material and water as the dispersant.
Generation of microbiota functional activity biosensors: Amine phosphonate bead acetylation—Beads were washed repeatedly with multiple solvents with the goal of resuspending the beads in anhydrous methanol; to do so, beads were washed in water, then methanol, then anhydrous methanol (1 volume equivalent; 5×106 beads/mL). Pyridine (0.5 volume equivalents) was then added as a base followed by acetic anhydride (0.5 volume equivalents). The reaction was allowed to proceed for 3 hours at 22° C. and then terminated by repeated washing in water. Beads were stored in 20 mM HEPES (pH 7.2) and 100 mM NaCl at 4° C.
Generation of microbiota functional activity biosensors: Fluorophore labeling of amine plus phosphonate beads—Beads were labeled with the following N-hydroxysuccinimide ester (NHS)-activated fluorophores: (i) Alexa Fluor 488 NHS ester (Life Technologies; Cat. No.: A20000); (ii) Promofluor 415 NHS ester (PromoKine; Cat. No.: PK-PF415-1-01); (iii) Promofluor 633P NHS ester (PromoKine; Cat. No.: PK-PF633P-1-01) and (iv) Promofluor 510-LSS NHS ester (PromoKine; Cat. No.: PK-PF510LSS-1-01). NHS-activated fluorophores were dissolved in dimethyl sulfoxide (DMSO) at 1 mM. The stock solution of each fluorophore was diluted in DMSO to 10 μM. The fluorophore was conjugated to amine plus phosphonate beads in 20 mM HEPES (pH 7.2) and 100 mM NaCl (3×106 beads/mL reaction; final concentration of fluorophore in the reaction, 100 nM). The reaction was allowed to proceed for 50 minutes at 22° C. and then terminated by repeated washing with water. Beads were stored in 20 mM HEPES (pH 7.2) and 100 mM NaCl at 4° C.
Generation of microbiota functional activity biosensors: Polysaccharide conjugation to fluorophore-labeled amine plus phosphonate beads—Polysaccharides were resuspended at a concentration of 5 mg/mL in 50 mM HEPES (pH 7.8) using heat and sonication. Trimethylamine (TEA, 0.5 equivalent), and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP; 1 eq; Sigma Aldrich; Cat. No.: RES1458C) dissolved in DMSO (50 mg/mL) were added to the polysaccharide solution. The optimal concentration of CDAP for polysaccharide activation, without overactivation and aggregation was found to be 0.2 mg/mg of polysaccharide. The polysaccharide/TEA/CDAP solution was mixed for 2 minutes at 22° C. to allow for polysaccharide activation. Fluorophore-labeled amine plus phosphonate beads resuspended in 50 mM HEPES (pH 7.8) were added to the activated polysaccharide solution and the reaction was allowed to proceed for 15 hours at 22° C. (final polysaccharide concentration typically 3.5 mg/mL). Any aggregated beads were disrupted by gentle sonication. Polysaccharide-conjugated beads were reduced by adding 2-picoline borane (1 eq; Sigma Aldrich; Cat. No.: 654213) dissolved in DMSO (10% wt:wt) and incubating the mixture for 40 minutes at 40° C. The reaction was terminated with repeated washing with water. Beads were stored in 20 mM HEPES (pH 7.2) and 100 mM NaCl at 4° C.
Beads were counted using flow cytometry. Typically, 5 μL of a polysaccharide-coated bead solution were added to 200 μL of HNTB [20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH=7.4), 100 mM NaCl, 0.01% bovine serum albumin (wt:wt), and 0.01% Tween-20 (wt:wt)] containing CountBright Absolute Counting Beads (Thermo Scientific; Cat. No. C36950). Beads were analyzed using flow cytometry on a FACSAriaIII instrument (BD Biosciences).
Generation of microbiota functional activity biosensors: Quantification of bead-bound polysaccharide—Polysaccharide-degradation from beads was quantified by GC-MS as described above with the following modifications. Polysaccharide-coated beads were counted using flow cytometry. Beads for hydrolysis were transferred to a 96-well skirted PCR plate (Multimax; Cat. No.: 2668; 3-7×104 beads/well) and washed three times in water using a magnet. Beads were resuspended in 175 μL of 2M trifluoroacetic acid containing 15 ng of D6-myo-inositol as an internal standard, and then transferred into 8 mm crimp top glass vials. An aliquot was removed from the vial and flow cytometry was used to determine the number of beads that had been transferred to that vial. The quantity of monosaccharide released from a bead was determined from the linear fit of standards divided by the number of beads transferred into the hydrolysis vial. For quantifying relative polysaccharide degradation, the absolute amount of monosaccharide released from the bead surface was divided by the mass of that monosaccharide quantified on input beads (with results expressed as a percentage).
In vitro growth assays—Bacterial stocks, previously stored at −80° C., were struck onto Brain-heart infusion (BHI; Becton Dickinson) agar plates supplemented with 10% (vol:vol) horse blood. Plates were incubated in an anaerobic growth chamber (Coy Laboratory Products; atmosphere 3% hydrogen, 20% CO2, and 77% N2). Single colonies were picked and grown overnight on a defined Bacteroides minimal medium (McNulty and Gordon, 2013) containing 5 mg/mL D-glucose. Bacteria were then diluted 1:500 (vol:vol) into Bacteroides minimal medium supplemented with a carbon source at a final concentration of 0.5% (wt:wt), and distributed into the wells of a 96-well half-area plate (Costar; Cat. No.; 3696). Plates were sealed with an optically clear membrane (Axygen; Cat. No.; UC500) and growth at 37° C. was monitored by measuring optical density at 600 nm every 15 minutes (Biotek Eon instrument with a BioStack 4). Carbon sources tested include D-glucose, PFABN, SBABN and glucomannan (Megazyme; Cat. No.; P-GLCML). All conditions were tested in quadruplicate. Readings obtained from control wells inoculated with bacteria but lacking a carbon source were averaged, and subtracted from data obtained from carbon-supplemented cultures to generate background subtracted OD600 growth curves.
Gnotobiotic mouse experiments: Colonization—Germ-free male C57BL/6J mice were maintained within flexible plastic isolators under a strict 12 h light cycle (lights on a 0600) and fed an autoclavable mouse chow (Envigo; Cat. No.: 2018S). Animals were colonized with a 14-member microbial community of cultured, sequenced bacterial strains composed of a mixture of type strains [or their Tn mutant library equivalent (Wu et al, 2015; Hibberd et al, 2017)] and strains isolated from the lean co-twin of an obesity discordant twin pair [Twin Pair 1 in Ridaura et al., 2013)]. Bacterial strains were grown to early stationary phase in gut microbiota medium (GMM) or LYBHI medium (Goodman et al., 2011). Monocultures were stored at −80° C. after addition of an equal volume of PBS (pH 7.4) supplemented with 30% glycerol (vol:vol). Gavage pools were prepared (2×106 CFUs per strain; equal volumes of each INSeq library) and introduced into mice using a plastic tipped oral gavage needle. Animals receiving communities with Tn mutant libraries were individually housed in cages containing cardboard shelters (for environmental enrichment).
Five days prior to colonization, mice were switched to a HiSF-LoFV diet. This diet was produced using human foods as described (Ridaura et al., 2013), freeze-dried and milled (D90 particle size 980 μm). The milled diet and each of the three diet supplements, were weighed, and transferred (separately) into sterile screw top containers (Fisher Scientific; Cat. No.; 22-150-244). Diets were sterilized by gamma irradiation (20-50 kilogreys, Steris, Mentor, OH). Sterility was confirmed by culturing material in TYG medium under aerobic and anaerobic conditions. The HiSF-LoFV diet and supplement were combined after transfer into gnotobiotic isolators [raw pea fiber at 10% (wt:wt); PFABN at 2% (wt:wt) and SBABN at 2% (wt:wt)]. Diets were mixed into a paste after adding sterile water (15 mL/30 g of diet). The paste was pressed into a small plastic tray and placed on the floor of the cage. Fresh diet was introduced every two days and in sufficient quantity to allow access ad libitum. Autoclaved bedding (Aspen wood chips; Northeastern Products) was changed at least weekly and immediately following a diet switch.
Gnotobiotic mouse experiments: Gavage and recovery of polysaccharide-coated beads from mice—Each bead type was individually sterilized by washing in 70% ethanol (vol:vol) twice on a magnetic tube stand before resuspension in HNTB. A pool of 10-15×106 beads (2.5-3.75×106 per bead type) in 400 μL of HNTB was prepared for each mouse; 350 μL of the pool were introduced by oral gavage; the remaining 50 □L was analyzed as the input beads (see above). Beads were isolated from the cecums of mice four hours after gavage or from all fecal pellets that had been collected from a given animal during the 3- to 6-hour period following gavage.
Recovered beads were resuspended in 10 mL of HNTB by pipetting and subsequently by vortexing. The resulting slurry was passed through a 100 μm nylon filter (Corning; Cat. No.: 352360). Beads were isolated from the suspension by centrifugation (500×g, 5 minutes) through Percoll Plus (GE Healthcare; Cat. No.: 17544502) in a 50 mL conical tube. Beads were recovered from the bottom of the tube; recovered beads from each animal were distributed into four 1.5 mL sterile tubes and washed at least three times with HNTB on a magnetic tube stand until macroscopic particulate debris from intestinal contents were no longer observed. The material from four tubes were subsequently recombined and beads were stored in HNTB containing 0.01% (wt:wt) sodium azide at 4° C.
Bead types were purified by fluorescence-activated sorting (FACSAriaIII; BD Biosciences). Aliquots of input beads were sorted throughout the procedure to quantify and monitor sort yield and purity. Bead purity typically exceeded 98%. Sorted beads were centrifuged (1,500×g, 5 minutes), the supernatant was aspirated, and beads were transferred into a 0.2 mL 96-well skirted PCR plate. Beads were washed with HNTB using a magnetic plate holder and stored at 4° C. in HNTB plus 0.01% (wt:wt) sodium azide until analysis. Beads were subjected to acid hydrolysis of the bound polysaccharide and the amount of liberated neutral monosaccharides was determined by GC-MS. All samples of a given bead type were analyzed in the same GC-MS run; however, the order of analysis of a given bead type recovered from animals representing different treatment groups was randomized. If sufficient beads were available, each bead type from each animal was analyzed up to three times.
Gnotobiotic mouse experiments: COmmunity PROfiling by sequencing (COPRO-Seq)—DNA was isolated from fecal samples by bead beading with 250 μL 0.1 mm zirconia/silica beads and one 3.97 mm steel ball in 500 μL of 2× buffer A (200 mM Tris, 200 mM NaCl, 20 mM EDTA), 210 μL 20% (wt:wt) sodium dodecyl sulfate, and 500 μL of phenol:chloroform:amyl alcohol (pH 7.9; 25:24:1) for four minutes. 420 μL of the aqueous phase was removed; DNA was purified (QIAquick 96 PCR purification kit; Qiagen) according to the manufacture's protocol and eluted into 10 mM Tris-HCl (pH 8.5). Sequencing libraries were prepared from purified DNA by tagmentation with the Nextera DNA Library Prep Kit (Illumina; Cat. No.: 15028211) and custom barcoded primers (Adey et al., 2010). Libraries were sequenced (Illumina Nextseq instrument, 75-nt unidirectional reads) to a depth 1×106 reads per sample. Reads were demultiplexed and mapped to community member bacterial genomes, 2 ‘spiked-in’ bacterial genomes for absolute abundance calculation, and 2 ‘distractor’ genomes [Faecalibacterium prausnitzii; GenBank assembly accession: GCA_902167865.1; Bifidobacterium longum subsp. infantis; GenBank assembly accession: GCA 902167615.1; Raman et al., 2019], using custom Perl scripts adapted to use Bowtie 2 (Langmead and Salzberg, 2012) (https://gitlab.com/hibberdm/COPRO-Seq).
To calculate bacterial absolute abundance, an aliquot containing a known number of two bacteria strains not encountered in mammalian gut communities or in the diet was ‘spiked-in’ to each fecal sample prior to DNA extraction (Stammler et al., 2016) [30 μL of a 2.22×108 cells/mL suspension of Alicyclobacillus acidiphilus DSM 14558 (GenBank assembly accession: GCA_001544355.1) and 30 μL of a 9.93×108 cells/mL suspension of Agrobacterium radiobacter DSM 30147 (GenBank assembly accession: GCA_000421945.1); Wolf et al., 2019]. COPRO-Seq provides an output counts table that is normalized to the informative genome size of each bacterial genome; this is used to generate a normalized relative abundance table. The calculated relative abundances of the spike-in genomes were 0.40±0.19% and 0.29%±0.16 (mean±s.d.), respectively. For a given taxa i, in sample j, the absolute abundance in genome equivalents per gram of feces was calculated using the normalized relative abundance and the A. acidophilus spike-in (A.a):
To identify bacterial taxa that respond to each diet treatment, absolute abundance data from fecal samples collected after diet supplementation were fit using a linear mixed effects model (Ime4 package; Bates et al., 2015). The dependence of bacterial abundance on ‘diet by day’ was tested. ‘Animal’ was included as a random variable. Tukey HSD p-values from the linear models were corrected for multiple hypotheses (Benjamini and Hochberg, 1995). Estimated marginal means were calculated from linear models (emmeans package) of absolute abundances for each diet group. To simplify visualization of the effects of each diet supplement, estimated marginal mean values were expressed as a ratio of the marginal mean of all mice prior to the diet switch on dpg2. Diet-responsive bacterial strains were defined as those whose absolute abundance was significantly different [p<0.01, linear mixed-effects model (Gaussian); two-way ANOVA with Tukey's HSD, FDR-corrected] in 3 of the 6 total diet comparisons [i.e., (i) HiSF-LoFV vs pea fiber, (ii) HiSF-LoFV vs PFABN, (iii) HiSF-LoFV vs SBABN, (iv) pea fiber vs PFABN, (v) pea fiber vs SBABN, or (vi) PFABN vs SBABN], and the estimated marginal mean of the diet effect was greater than 1.5 for at least one diet-supplemented group.
Tn insertion site sequencing (INSeq)—Multi-taxon INSeq (Wu et al., 2015) was used to simultaneously measure genetic fitness determinants in five Bacteroides sp. (four of which were identified as fiber responsive). Briefly, Mmel digestion cleaves genomic DNA at a site 20-21 bp distal to the restriction enzyme's recognition sequence in the mariner transposon vector. This flanking genomic DNA, and a taxon-specific barcode inserted into the transposon, allow quantitation of each unique insertion mutant member of a given Bacteroides INSeq library.
Purified fecal DNA was processed as previously described (Wu et al., 2015). Genomic DNA was digested with Mmel, size selected, ligated to sample-specific adapter primers, size selected, amplified by PCR, and a specific 131 bp final product isolated from a 4% (wt:wt) MetaPhore (Lonza) DNA gel. Purified DNA was sequenced, unidirectionally, on an Illumina HiSeq 2500 platform (50-nt reads) using a custom primer that captures the species-specific barcode. Quantitation of each insertion mutant's abundance (read counts) was determined using custom software (https://github.com/mengwu1002/Multi-taxon_analysis_pipeline; Wu et al., 2015). Count data were normalized for library depth (within the same species), a pseudo count of 8 was added, and the data were log2 transformed. Transformed count data from dpg 2 and dpg 6 were used to build linear models (limma package; Ritchie et al., 2015) to identify diet supplement-specific genes that significantly altered bacterial abundance (relative to unsupplemented HiSF-LoFV diet). P-values from the linear models were corrected for multiple hypotheses with the Benjamini-Hochberg method.
Meta-proteomic analysis—The protocol for meta-proteomic analysis of fecal samples has been described in detail in our previous publications (Patnode et al., 2019). Only data from peptides that uniquely map to a single protein were considered for analysis. Summed peptide abundance data for each protein was log 2 transformed. Missing data was imputed to simulate ‘instrument limit of detection’ by calculating the mean and standard deviation of each protein in samples where a protein was detected in more than three mice within a given treatment group. Missing values were imputed as mean minus 2.2 times the standard deviation with a width equal to 0.3 times the standard deviation. For species where greater than 100 proteins were quantified, data were normalized with cyclic loess normalization (limma package). Loess-normalized protein abundance data were then used to build linear models (limma package) to identify diet-supplement-responsive proteins (relative to levels in control mice receiving the unsupplemented HiSF-LoFV diet) at dpg 6. P-values from the linear models were corrected for multiple hypotheses (Benjamini and Hochberg, 1995).
PULs that were upregulated during diet supplementation were identified using geneset enrichment analysis with GAGE (Luo and Woolf, 2009). PUL gene annotations were identical to those employed in Patnode et al. (2019). All genes within a PUL were annotated as a gene set. We required that more than five quantified proteins change in abundance unidirectionally upon diet supplementation in a given PUL for that PUL to be considered. Significantly enriched PULs were identified using a one-sample Z-test; p-values were corrected for multiple hypotheses with the Benjamini-Hochberg method.
This application claims priority to U.S. Provisional Application No. 62/876,379, filed Jul. 19, 2019, the disclosures of which are incorporated herein by reference.
This invention was made with government support under DK070977, DK078669, and DK107158 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/42678 | 7/17/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62876379 | Jul 2019 | US |
