Glycan polymers and related methods thereof

Abstract

To provide glycan polymer preparations and methods for treating subjects with a disease or disorder using the same.SOLUTION: The disclosure provides a method for treating a subject having a disease or disorder associated with an unwanted level of a metabolite (e.g., a short chain fatty acid (SCFA) such as propionate or butylate, ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), a uremic solute (e.g., p-cresol or indole), a lipopolysaccharide (LPS), or bile acid (e.g., secondary bile acid), comprising: optionally, selecting a glycan polymer preparation based on that it modulates the production or level of the metabolite; and administering an amount of the glycan polymer preparation effective to modulate the level of the metabolite, thereby treating the disease or disorder.SELECTED DRAWING: None

Description

[Technical Field]

[0001] RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 430895, filed December 6, 2016, U.S. Provisional Patent Application No. 62 / 446316, filed January 13, 2017, and U.S. Provisional Patent Application No. 62 / 430849, filed December 6, 2016, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] The human microbiota is complex and varies from individual to individual depending on genetics, age, sex, stress, nutrients, and diet. The microbiota performs many activities and can affect the physiology of the host. Modulation of the intestinal microbiota can change community function and interactions with the host. A limited number of probiotic bacteria are known in the art, and some associations with health benefits have been documented when probiotic bacteria are ingested by humans. Some foods are considered "prebiotic" foods, containing substances that can promote the growth of specific bacteria that are thought to be beneficial to the human host. Clinical trials using these substances have shown conflicting results regarding their effectiveness, and their impact on human health has generally been described as minimal. Therefore, there is a need for novel solutions that can modulate the microbiota and improve human health. Summary of the Invention [Means for solving the problem]

[0003] Described herein are methods of treating a subject with a disease or disorder using glycan polymer preparations and compositions thereof.

[0004] Thus, in one aspect, the present invention relates to a method of treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., a short-chain fatty acid (SCFA) (e.g., propionate or butyrate), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes (e.g., p-cresol or indole), lipopolysaccharide (LPS), or bile acid (e.g., secondary bile acid)), optionally comprising selecting a glycan polymer preparation based on its ability to regulate the production or level of the metabolite, and administering an amount of the glycan polymer preparation effective to regulate the level of the metabolite, thereby treating the disease or disorder.

[0005] In another aspect, the present invention is directed to a method of treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., a short-chain fatty acid (SCFA) (e.g., propionate or butyrate), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes (e.g., p-cresol or indole), lipopolysaccharide (LPS), or bile acid (e.g., secondary bile acid)), optionally comprising determining that a glycan polymer preparation regulates the production or level of the metabolite, and administering an amount of the glycan polymer preparation effective to regulate the level of the metabolite, thereby treating the disease or disorder.

[0006] In another aspect, the present invention relates to a method for regulating the production or level of a product (e.g., short-chain fatty acids (SCFAs), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, or bile acids) in a subject's body (e.g., gut (colon, intestine), blood, urine, organs (e.g., liver, kidney), brain), comprising administering to the subject (e.g., orally or rectally) an effective amount of a glycan polymer preparation sufficient to regulate the production or level of the product, wherein the glycan polymer is optionally a substrate for microbial components of the colon or intestine.

[0007] In another aspect, the present invention relates to a method for selecting a glycan polymer preparation for use as a substrate for a glycosidase enzyme (e.g., a CAZy family) of a preselected human gut microorganism (e.g., selected for its glycosidase profile), comprising: a) obtaining a value for the glycosidase (e.g., CAZy family) profile of the microorganism; b) identifying, designing, or selecting a glycan polymer that can serve as a substrate for the microorganism based on the glycosidase (e.g., CAZy family) profile; c) optionally, i. Assembling a panel of human gut microorganisms (e.g., a single strain, a group of designed strains, or an ex vivo group (e.g., from a fecal sample) containing the microorganism of interest; ii. Contacting the panel of microorganisms with a test glycan preparation; iii. Evaluating the growth of the human gut microorganism (of interest); d) selecting a glycan polymer preparation.

[0008] In another aspect, the present invention is directed to a glycan preparation produced or selected by the methods described herein.

[0009] In another aspect, the invention provides a glycan polymer preparation comprising a glycan polymer, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising i) glucose, mannose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond, or ii) glucose, mannose, or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond, and wherein the glycan polymer is selected from the group consisting of i) GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, GH13 subfamily 36, GH113, or GH112. CAZy family, ii) GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13, GH13 subfamily 9, GH13 subfamily 31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77 or GH94 CAZy family, iii) GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8 or GH13 subfamily 14 or iv) the GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy families.

[0010] In another aspect, the invention provides a glycan polymer preparation, e.g., the preparation comprises at least about 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, and the glycan polymer comprises i) xylose, arabinose, fucose, or rhamnose subunits, or combinations thereof, and at least one alpha-glycosidic bond, or ii) xylose, arabinose, fucose, or rhamnose subunits, or combinations thereof, and at least one beta-glycosidic bond, and the glycan polymer comprises i) GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, or GH13 subfamily 14. The present invention relates to glycan polymer preparations that are substrates for one or more, e.g., two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family, or ii) the GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy families.

[0011] In another aspect, the invention provides a glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, and the glycan polymer comprises i) glucose or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond, or ii) glucose or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond, and the glycan polymer is selected from the group consisting of i) GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, GH13 CAZy family, or ii) GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT 10, GH77, GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, Gh28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, GH24 CAZy families.

[0012] In another aspect, the invention provides a glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising glycan polymers comprising arabinose, galactose, xylose, or glucose subunits, or combinations thereof, and at least one alpha-glycosidic linkage, and comprising a glycan polymer selected from: i) GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family; or ii) GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92 are substrates for one or more, e.g., two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family. , directed to glycan polymer preparations.

[0013] In another aspect, the invention relates to a glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, and the glycan polymer comprises glucose and at least one alpha-glycosidic bond, and is a substrate for one or more, e.g., two, three, four, or more, human gut microbial glycosidase enzymes selected from i) GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family, or ii) GH23, GH24, or GH33 CAZy family.

[0014] In another aspect, the invention provides a glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising glycan polymers comprising i) glucose or xylose subunits, or a combination thereof, and at least one alpha-glycosidic bond, or ii) glucose or xylose subunits, or a combination thereof, and at least one beta-glycosidic bond, wherein the glycan polymers are selected from the group consisting of i) GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family, or ii) GH2, GH31, GH23, GH13, or GH24 Glycan polymer preparations are directed to glycan polymer preparations that are substrates for one or more, eg, two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family.

[0015] In another aspect, the invention provides a glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising glycan polymers comprising glucose, xylose, arabinose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic linkage, and the glycan polymers are selected from the group consisting of i) GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family, or ii) GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106. Glycan polymer preparations are directed to glycan polymer preparations that are substrates for one or more, eg, two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family.

[0016] In another aspect, the present invention is directed to a unit dosage form comprising the glycan preparation described herein.

[0017] In another aspect, the present invention is directed to a pharmaceutical composition comprising the glycan preparation described herein.

[0018] In another aspect, the present invention is directed to a set of pharmaceutical compositions each comprising a glycan polymer preparation described herein or a portion thereof, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

[0019] In another aspect, the present invention is directed to a medical food comprising the glycan preparation described herein.

[0020] In another aspect, the present invention is directed to a set of medical foods each comprising a glycan polymer preparation or portion thereof described herein, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

[0021] In another aspect, the present invention is directed to a dietary supplement comprising the glycan preparation described herein.

[0022] In another aspect, the present invention is directed to a set of dietary supplements each comprising a glycan polymer preparation described herein or a portion thereof, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

[0023] In another aspect, the present invention is directed to a food ingredient comprising the glycan preparation described herein.

[0024] In another aspect, the present invention is directed to a set of food ingredients each comprising a glycan polymer preparation described herein or a portion thereof, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

[0025] In another aspect, the present invention is directed to a method for producing a co-preparation, comprising providing a preparation of human gut microbes, providing a glycan polymer preparation as described herein, wherein the glycan polymer is a substrate for the human gut microbes, and combining the human gut microbes with the glycan polymer.

[0026] In another aspect, the present invention is directed to a synbiotic co-preparation comprising a preparation of human gut microbes and a preparation of glycan polymers described herein.

[0027] In another aspect, the present invention is directed to a method of transplanting human gut microbes into the colon or large intestine of a human subject in need thereof, comprising administering to the subject a synbiotic co-formulation described herein in an amount and for a time effective to transplant the human gut microbes.

[0028] In another aspect, the present invention is directed to a method of treating a subject having dysbiosis, comprising administering a composition comprising a glycan polymer preparation and a microbial preparation described herein in an amount effective to treat the dysbiosis.

[0029] In another aspect, the present invention is directed to a glycan polymer preparation described herein, comprising a glycan polymer that is a substrate for a human gut microbial glycosidase enzyme of a spore-forming microorganism (e.g., a spore-forming bacterial taxon).

[0030] In another aspect, the invention provides a glycan polymer preparation, optionally, e.g., the preparation comprises at least about 0.5, 1, 2, 5, 10, 50, or 100 kg, and / or further optionally, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising: a. xylose or arabinose subunits, or a combination thereof, and at least one alpha-glycosidic bond; b. xylose or arabinose subunits, or a combination thereof, and at least one beta-glycosidic bond; c. galactose, xylose, or arabinose subunits, or a combination thereof. a. a galactose, xylose, or arabinose subunit, or a combination thereof, and at least one beta-glycosidic bond; b. a galactose, xylose, or arabinose subunit, or a combination thereof, and at least one alpha-glycosidic bond; c. a glucose, xylose, or arabinose subunit, or a combination thereof, and at least one alpha-glycosidic bond; e. a glucose, xylose, or arabinose subunit, or a combination thereof, and at least one alpha-glycosidic bond; f. a glucose, xylose, or arabinose subunit, or a combination thereof, and at least one beta-glycosidic bond; g. a xylose, arabinose, glucose, or galactose subunit, or a combination thereof, and at least one alpha-glycosidic bond; h.The glycan polymer comprises xylose, arabinose, glucose, or galactose subunits, or a combination thereof and at least one beta-glycosidic linkage, or a combination thereof and at least one beta-glycosidic linkage, and is selected from the group consisting of GT5, GT35, GT3, GH97, GH95, GH92, GH89, GH88, GH78, GH77, GH57, GH51, GH43 subfamily 34, GH43 subfamily 24, GH43 subfamily 10, GH42, GH36, GH35, GH33, GH32, GH31, GH3, GH29, GH28, GH27, GH24, GH20, GH2, GH16, GH133, GH130, GH13 subfamily 8, GH13 subfamily 38, GH13 subfamily 14, GH13, GH123, GH115, GH109, ​​and GH105. The present invention targets glycan polymer preparations that are substrates for human gut microbial glycosidase enzymes, one of the CAZy family.

[0031] In another aspect, the present invention is directed to a method for producing a co-preparation, comprising providing a preparation of spore-forming microorganisms (e.g., spore-forming human gut microorganisms), providing a glycan polymer preparation (described herein) in which the glycan polymer is a substrate for the spore-forming microorganisms, and combining the preparation of spore-forming microorganisms with the glycan polymer preparation.

[0032] In another aspect, the present invention provides a method for producing a preparation of a glycan polymer, e.g., a glycan polymer that is a substrate for a glycosidase enzyme present in a human gut microorganism, comprising the steps of: providing a plurality of glycan subunits, e.g., sugar monomers or sugar dimers, suitable for producing the glycan polymer; and contacting the plurality of glycan subunits with a glycosidase enzyme molecule, e.g., derived from a human gut microorganism, under conditions that result in incorporation of the glycan subunits into the glycan polymer (e.g., by a condensation reaction), thereby The present invention relates to a method comprising the steps of: producing a glycan polymer preparation that is a substrate for human gut microorganisms; and optionally, i) the glycan polymer preparation comprises at least about 0.25, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400, or 500 kilograms of glycan polymer; and / or ii) the glycan polymer preparation is produced at a yield (determined on a weight / weight basis as a percentage of the input glycan subunits) of at least about 15%, 30%, 45%, 60%, or about 75%.

[0033] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, mannose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymer of the preparation is a substrate for human gut microorganisms comprising a glycosidase enzyme of the GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, GH13 subfamily 36, GH113, or GH112 CAZy family; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from the GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, GH13 subfamily 36, GH113, or GH112 CAZy family.

[0034] In another aspect, the present invention relates to a method for preparing a glycan polymer comprising administering to a subject a glycan polymer selected from the group consisting of GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13.0, GH13.9, GH13.31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77, GH94 providing a plurality of glucose-, mannose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the creation of a glycan polymer preparation that is a substrate for human gut microorganisms comprising a glycosidase enzyme of the CAZy family; and coupling the plurality of glycan subunits to a glycosidase enzyme selected from one of the GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13.0, GH13.9, GH13.31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77, or GH94 CAZy family. The present invention relates to a method for producing a glycan polymer preparation, comprising the step of contacting a glycan polymer preparation with a glycan polymer.

[0035] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of xylose, arabinose, galactose, and / or glucose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymer of the preparation is a substrate for human gut microorganisms comprising a glycosidase enzyme of the GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family.

[0036] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of xylose, arabinose, galactose, and / or glucose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms comprising glycosidase enzymes of the GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92 CAZy family; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92 CAZy family.

[0037] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose- and / or sialic acid-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family.

[0038] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose- and / or sialic acid-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms comprising glycosidase enzymes of the GH23, GH24, or GH33 CAZy family; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH23, GH24, or GH33 CAZy families.

[0039] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, xylose-, mannose-, arabinose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymer of the preparation is a substrate for human gut microorganisms comprising a glycosidase enzyme of the GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family.

[0040] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, xylose-, mannose-, arabinose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GH2, GH31, GH23, GH13, or GH24 CAZy families; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH2, GH31, GH23, GH13, or GH24 CAZy families.

[0041] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, xylose-, arabinose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymer of the preparation is a substrate for human gut microorganisms comprising a glycosidase enzyme of the GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family.

[0042] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, xylose-, arabinose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are a substrate for human gut microorganisms comprising glycosidase enzymes of the GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106 CAZy family; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106 CAZy family.

[0043] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, mannose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymer of the preparation is a substrate for human gut microorganisms containing glycosidase enzymes of the GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, GH13 subfamily 14 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from the group consisting of GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, and GH13 subfamily 14 CAZy family.

[0044] In another aspect, the present invention provides a method for preparing a glycan polymer comprising administering to a subject a glycan polymer selected from the group consisting of GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77 The present invention relates to a method for producing a glycan polymer preparation, the method comprising: providing a plurality of glucose-, mannose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, the glycan subunits being a substrate for human gut microorganisms comprising a CAZy family glycosidase enzyme; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the following CAZy families: GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77.

[0045] In another aspect, the present invention relates to a method for producing a glycan polymer preparation, comprising: providing a plurality of xylose, arabinose, fucose, and / or rhamnose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymer of the preparation is a substrate for human gut microorganisms containing glycosidase enzymes of the GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, GH13 subfamily 14 CAZy family; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from the group consisting of GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, and GH13 subfamily 14 CAZy family.

[0046] In another aspect, the present invention is directed to a method for producing a glycan polymer preparation, comprising: providing a plurality of glycan subunits, e.g., monomers or dimers, of a substrate in column E of Table 23 under conditions that result in the production of a glycan polymer preparation, e.g., conditions in columns F, G, H, I, J, K, and / or L in the same row as the substrate and glycosidase enzyme; Contacting the plurality of glycan subunits of the substrate with a glycosidase enzyme in column A in the same row as the substrate.

[0047] In another aspect, the present invention provides a method for preparing a glycan polymer comprising administering to a subject a glycan polymer selected from the group consisting of GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77 The present invention relates to a method for producing a glycan polymer preparation, the method comprising: providing a plurality of xylose-, arabinose-, fucose-, and / or rhamnose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, the glycan subunits being a substrate for human gut microorganisms comprising a CAZy family glycosidase enzyme; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the following CAZy families: GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77.

[0048] In another aspect, the present invention provides a method for preparing a glycan polymer comprising the steps of: GT3, GH97, GH43 subfamily 2, The present invention relates to a method for producing a glycan polymer preparation, the method comprising: providing a plurality of glucose- and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, the glycan subunits being a substrate for human gut microorganisms containing glycosidase enzymes of the 4, GH27, GH133, GH13 subfamily 8, and GH13 CAZy family; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the following: GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, and GH13 CAZy family.

[0049] In another aspect, the present invention provides a method for preparing a glycan polymer comprising the steps of: GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH 51, GT10, GH77, GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, Gh28, GT35, G T28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, GH24 providing a plurality of glucose- and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation that is a substrate for human gut microorganisms containing a CAZy family glycosidase enzyme; , GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77, GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, GH28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, GH24 CAZy family.

[0050] In another aspect, the present invention is directed to a glycan polymer preparation produced, producible, or preparable by the methods disclosed herein, e.g., by the methods described herein.

[0051] In another aspect, the invention is directed to a glycan polymer preparation selected or selectable by the methods disclosed herein, eg, by the methods described herein.

[0052] In another aspect, the present invention is directed to a therapeutic nutritional product comprising the glycan polymer preparation described herein.

[0053] In another aspect, the invention is directed to a reaction mixture described herein, e.g., produced by any one of the methods described herein, comprising a plurality of glycan subunits, e.g., sugar monomers or sugar dimers, suitable for producing a glycan polymer, in an amount suitable to produce a glycan polymer preparation comprising at least 0.25, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400, or 500 kilograms of glycan polymer, and / or under conditions suitable for obtaining a yield of at least about 15%, 30%, 45%, 60%, or about 75% (as determined on a weight / weight basis as a % of the input glycan subunits); and a glycosidase enzyme molecule (e.g., one or more glycosidase enzymes associated with glycotaxa class 1, class 2, class 3, class 4, class 5, class 6, or class 7).

[0054] In another aspect, the present invention is directed to a method of making a pharmaceutical composition, medical food, dietary supplement, food ingredient, or therapeutic nutritional product comprising formulating a preparation described herein into the pharmaceutical composition, medical food, dietary supplement, food ingredient, or therapeutic nutritional product.

[0055] In another aspect, the present invention is directed to fractions, eg, molecular weight fractions, of the glycan polymer preparations described herein.

[0056] In another aspect, the invention relates to a method for producing, evaluating, selecting, classifying, or providing a preparation of glycan polymers produced or preparable by the methods described herein, comprising obtaining a candidate preparation; obtaining a value for a parameter related to the preparation, such as a physical parameter, e.g., molecular weight, e.g., average molecular weight or molecular weight distribution, glycan subunit composition or purity, or a parameter related to a biological property, e.g., the ability to regulate the growth of human gut microorganisms, the ability to regulate microbial metabolites produced by microorganisms, e.g., in an ex vivo assay, or the ability to regulate a biomarker, e.g., an inflammatory or immune biomarker, a toxic or waste compound, a bacterial compound, e.g., in a human subject; and comparing the value with a reference value, thereby producing, evaluating, selecting, classifying, or providing a preparation of glycan polymers.

[0057] In another aspect, the present invention is directed to a method for producing a pharmaceutical composition for modulating a target human gut microorganism, comprising the steps of providing a plurality of glycan subunits; contacting the plurality of glycan subunits with a glycosidase enzyme composition having glycosidase activity present in the target gut microorganism under conditions that result in incorporation of the glycan subunits into a glycan polymer; optionally purifying the glycan polymer; and formulating the glycan polymer as a pharmaceutical composition for administration to the intestine and modulation of gut microorganisms, thereby producing a pharmaceutical composition for modulating the target human gut microorganism.

[0058] In another aspect, the present invention is directed to a purified preparation of glycosidase enzyme molecules comprising a glycosidase enzyme encoded by a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124, wherein the glycosidase enzyme is present in a human gut microorganism.

[0059] In another aspect, the present invention is directed to a vector comprising a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124, wherein the nucleic acid encodes a glycosidase enzyme present in human gut microbes, and the vector can be used to express the glycosidase enzyme.

[0060] In another aspect, the present invention is directed to a reaction mixture comprising a glycosidase enzyme encoded by a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124, and a substrate for the glycosidase enzyme, e.g., a glycan subunit, e.g., a monomer or dimer, wherein the substrate is present in an amount sufficient to form a glycan polymer, e.g., by condensation. [Brief explanation of the drawings]

[0061] [Figure 1] Representative SEC curve from 16 minutes to 20.5 minutes for a glu100 sample showing the average molecular weight at both the leading and trailing edges of the curve and the molecular weight at 10% of maximum absorption. [Figure 2] Representative anomeric region of the 1H-13C HSQC spectrum of a glu100 sample, including signal distribution of alpha- and beta-glycosidic linkages. [Figure 3A] Figures 3A-3C show representative anomeric regions of the 1H-13C HSQC spectra of the glu50gal50 (Figure 3A), glu100 (Figure 3B), and gal100 (Figure 3C) samples, illustrating the additive effect of the fingerprint peaks. [Figure 3B] Figures 3A-3C show representative anomeric regions of the 1H-13C HSQC spectra of the glu50gal50 (Figure 3A), glu100 (Figure 3B), and gal100 (Figure 3C) samples, illustrating the additive effect of the fingerprint peaks. [Figure 3C]Figures 3A-3C show representative anomeric regions of the 1H-13C HSQC spectra of the glu50gal50 (Figure 3A), glu100 (Figure 3B), and gal100 (Figure 3C) samples, illustrating the additive effect of the fingerprint peaks. [Figure 4] Representative GC chromatograms of three representative permethylated and hydrolyzed glycans, glu50gal50 (Figure 4A), man52glu29gal19 (Figure 4B), and glu100 (Figure 4C), showing the distribution of regiochemistry assigned by comparison to known standards. [Figure 5] 1 is a graph showing processed SEC traces comparing lactose (gray, β-galacto-1,4-glucose) with glycans (black) generated by treatment of lactose with β-galactosidase as described in Example 2. [Figure 6] 1 is a graph showing processed SEC traces comparing cellobiose (gray, β-gluco-1,4-glucose) with glycans (black) produced by treatment of cellobiose with β-glucosidase as described in Example 4. The shift in maximum peak intensity of the DP2 material is caused by the formation of allocellobiose (e.g., β-gluco-1,6-glucose), which slightly shifts the average apparent Mw of the DP2 material. [Figure 7] FIG. 7A shows processed SEC traces comparing (FIG. 7A) maltobiose (gray, α-gluco-1,4-glucose) with glycans (black) produced by treatment of maltobiose with α-glucosidase as described in Example 5, and (FIG. 7B) maltobiose (gray) with glycans (black) from Example 18 purified by yeast fermentation as described in Example 9. While maltose can be digested by yeast, some DP2 material persists through transglycosylation, in which maltose (α-gluco-1,4-glucose) is converted to allomaltose (e.g., α-gluco-1,6-glucose; α-gluco-1,3-glucose), which is digested less efficiently by yeast. [Figure 8]1 is a graph showing processed SEC traces comparing melibiose (gray, α-galacto-1,6-glucose) with glycans (black) generated by treatment of melibiose with α-galactosidase as described in Example 3. The shift in maximum peak intensity of the DP2 material is caused by the formation of allomellibiose (e.g., α-gluco-1,4-glucose), which slightly shifts the average apparent Mw of the DP2 material. [Figure 9] 1 is an image showing fluorescent carbohydrate electrophoresis (FACE) analysis of reaction mixtures from the reverse hydrolysis of glucose by β-glucosidase. Lane 1 is pure protein, and lanes 2-4 are reactions in trimethyl phosphate, diethylene glycol dimethyl ether, and tetraethylene glycol dimethyl ether, respectively, as described in Example 7. [Figure 10] 1 is a graph showing a raw data SEC comparison of glycans produced by treating lactose with β-galactosidase after 300 minutes with glycans produced by treating lactose with β-galactosidase in the presence of d-galactose after 1200 minutes (i.e., conversion up to DP≧3), as described in Example 8. The trace shows that the addition of d-galactose significantly slows the reaction, but also shifts the product distribution toward increased amounts of DP≧3 oligosaccharides. [Figure 11] Graph showing processed SEC data for the results of charcoal fractionation of glycans aimed at removing monomers from the sample without further fractionation. The three curves represent the parent glycan, the monomer fraction (apparent peak molecular weight approximately 200) removed from the parent by elution with 1% EtOH, and the remaining fraction isolated by elution with 50% EtOH. [Figure 12]Schematic diagram of oligosaccharide synthesis via substrate-selective transglycosylation, as described in Example 6. In each reaction, enzyme selectivity for transglycosylation of the non-reducing end monomer results in a distinct mixture of products. In this diagram, "A" and "B" can represent different monomers, different stereochemistry of the glycosidic bond, different regiochemistry of the glycosidic bond, or any combination thereof. [Figure 13] 1 is a graph showing the SEC curves of glycans generated by treating lactose with β-galactosidase after 300 minutes, as described in Examples 11-18. [Figure 14] 1 is a graph showing the SEC curves of glycans generated by treating lactose with β-glucosidase after 300 minutes, as described in Examples 11-18. [Figure 15] FIG. 1 is a chart showing all genomes annotated and used in genome analyses from the Human Microbiome Project, as well as the percentage of genomes by genus that encode each indicated metabolite. [Figure 16] 16A-16B are charts showing the percentage of genomes encoding CAZy families significantly enriched in butyrate producers (P<0.001, Wilcox rank sum, FDR-corrected and identified in >10% of butyrate producers). (FIG. 16A) Percentage of butyrate and non-butyrate producers encoding at least one enzyme from the indicated family. (FIG. 16B) Percentage of non-butyrate producers encoding any CAZyme that is individually significantly enriched in butyrate producers. [Figure 17] 1 is a chart showing the most abundant families in butyrate producers, ordered by average gene number. The chart represents the mean ± standard deviation. [Figure 18]Charts showing the percentage of genomes encoding CAZy families that were significantly depleted in TMA-lyase-positive genomes (P<0.05, Wilcox rank sum, FDR corrected). (Figure 18A) Percentage of TMA-lyase-positive and -negative genomes encoding at least one enzyme from the indicated family. (Figure 18B) Percentage of TMA-lyase-positive and -negative genomes encoding any CAZyme that was significantly depleted in TMA-lyase-positive genomes. [Figure 19] 1 is a chart showing the most abundant families in TMA-lyase negative genomes, ordered by average gene number. Chart represents mean ± standard deviation. [Figure 20] 20A-20B are charts showing the percentage of genomes encoding CAZy families that were significantly depleted in urease-positive genomes (P<0.05, Wilcox rank sum, FDR corrected). (FIG. 20A) Percentage of urease-positive and -negative genomes encoding at least one enzyme from the indicated family. (FIG. 20B) Percentage of urease-positive and -negative genomes encoding any CAZyme that was significantly depleted in urease-positive genomes. [Figure 21] 1 is a chart showing the most abundant families in urease-negative genomes, ordered by average gene number. Chart represents mean ± standard deviation. [Figure 22] 22A-22B are graphs showing the results of a LASSO linear regression model of SCFA production as a function of glycan composition, allowing for all quadratic interaction terms. (FIG. 22A) SCFA production from the ex vivo model and (FIG. 22B) defined groups. [Figure 23] Graphs showing the relative abundance of Bacteroides cellulolyticus strains in a defined group of 15 strains grown for 48 hours in the presence of carbohydrates (black circles in Figures 23A and 23B) or in the presence of the indicated carbohydrates with the addition of a glycan polymer preparation (e.g., Glu100) at 18 hours (gray triangles in Figure 23B). Mean relative abundance ± st.dev. is shown. [Figure 24] Graphs showing the relative abundance of Bacteroides cellulolyticus strains in defined groups of 14 strains. Figure 24A shows the relative abundance of B. cellulolyticus grown in the presence of various carbohydrates for 48 hours (filled circles) or in the presence of the indicated carbohydrates with B. cellulolyticus added for 18 hours (gray triangles). Figure 24B shows the relative abundance of B. cellulolyticus grown in the same defined groups of 14 strains in the presence of various carbohydrates and B. cellulolyticus added for 18 hours (filled circles) or in the presence of the indicated carbohydrates with B. cellulolyticus added for 18 hours and with a glycan polymer preparation (Glu, gray triangles). Mean relative abundance ± st.dev. is shown. [Figure 25] 25A-25D are graphs showing the 16S rRNA sequencing analysis results and correlation with butyrate production for the bacterial panel screened in Example 23. As shown, several taxa are highly correlated with butyrate levels: (FIG. 25A) Clostridiaceae (ρ=0.406 p-value 0.003), (FIG. 25B) Lachnospiraceae Roseburia (ρ=0.333 p-value 0.018), (FIG. 25C) Bacteroides fragilis (ρ=0.483 p-value 0), (FIG. 25D) Turicibacteraceae Turicibacter (ρ=0.554 p-value 0). [Figure 26-1]Figures 26-1 and 26-2 show graphs showing the correlation between 16S rRNA sequencing analysis results and acetate production for the bacterial panel screened in Example 23. In the ex vivo assay, some taxa are highly correlated with acetate levels. As shown, some taxa are highly correlated with acetate levels. (Figure 26A) Clostridiaceae (ρ=0.428 p-value=0.002), (Figure 26B) Bacteroides uniformis (ρ=0.525 p-value=0), (Figure 26C) Ruminococcaceae Oscillospira (ρ=-0.791 p-value=0), (Figure 26D) Bacteroides ovatus (ρ=0.405 p-value=0.004), (Figure 26E) Bacteroidales Rikenellaceae (ρ=-0.739 p-value=0), (Figure 26F) Clostridiales Ruminococcaceae (ρ=-0.83 p-value=0). [Figure 26-2]Figures 26-1 and 26-2 show graphs showing the correlation between 16S rRNA sequencing analysis results and acetate production for the bacterial panel screened in Example 23. In the ex vivo assay, some taxa are highly correlated with acetate levels. As shown, some taxa are highly correlated with acetate levels. (Figure 26A) Clostridiaceae (ρ=0.428 p-value=0.002), (Figure 26B) Bacteroides uniformis (ρ=0.525 p-value=0), (Figure 26C) Ruminococcaceae Oscillospira (ρ=-0.791 p-value=0), (Figure 26D) Bacteroides ovatus (ρ=0.405 p-value=0.004), (Figure 26E) Bacteroidales Rikenellaceae (ρ=-0.739 p-value=0), (Figure 26F) Clostridiales Ruminococcaceae (ρ=-0.83 p-value=0). [Figure 27] 27A-27D are graphs showing the 16S rRNA sequencing analysis results and correlation with propionate production for the bacterial panel screened in Example 23. As shown, several taxa are highly correlated with propionate levels: (FIG. 27A) Bacteroides ovatus (ρ=0.678 p-value=0), (FIG. 27B) Bifidobacterium (ρ=-0.781 p-value=0), (FIG. 27C) Ruminococcus bromii (ρ=-0.72 p-value=0), and (FIG. 27D) Bacteroides uniformis (ρ=0.559 p-value=0). [Figure 28]The mean number of CAZyme genes detected in spore-forming and non-spore-forming bacteria for each CAZyme family and subfamily. Only families in which genes were significantly enriched in spore-forming bacteria and detected in more than 10% of spore-forming bacterial genomes are shown (P<0.05, Wilcox rank sum, FDR corrected). [Figure 29] Percentage of genomes encoding CAZy families significantly enriched in genomes of sporulating versus non-sporulating bacteria (P<0.001, Wilcox rank sum, FDR-corrected and identified in >10% of sporulating bacteria). (A) Percentage of sporulating and non-sporulating bacteria encoding at least one enzyme from the indicated family. (B) Percentage of sporulating and non-sporulating bacteria encoding any CAzyme family or subfamily that is individually significantly enriched in sporulating bacteria. [Figure 30-1] Figures 30-1 through 30-4 show graphs (Figures 30A, 30C, 30E, and 30G) showing the percentage of genomes encoding CAZy families significantly enriched in metabolite-converting genomes, and charts (Figures 30B, 30D, 30F, and 30H) showing the most abundant families in metabolite-converting genomes, sorted by average gene number. The percentages of secondary bile acid-converting and non-converting genomes (Figure 30A), genomes encoding CAZy families exclusively encoded in non-indole-producing bacteria (Figure 30C), genomes encoding CAZy families significantly depleted in p-cresol-producing genomes (Figure 30E), and genomes encoding CAZy families significantly depleted in propionate-producing genomes (Figure 30G) are also shown. Charts showing the most abundant families in the secondary bile acid conversion genome (Figure 30B), the indole-negative genome (Figure 30D), the p-cresol-negative genome (Figure 30F), and the propionate-negative genome (Figure 30H) are shown. Charts represent the mean ± standard error. [Figure 30-2]Figures 30-1 through 30-4 show graphs (Figures 30A, 30C, 30E, and 30G) showing the percentage of genomes encoding CAZy families significantly enriched in metabolite-converting genomes, and charts (Figures 30B, 30D, 30F, and 30H) showing the most abundant families in metabolite-converting genomes, sorted by average gene number. The percentages of secondary bile acid-converting and non-converting genomes (Figure 30A), genomes encoding CAZy families exclusively encoded in non-indole-producing bacteria (Figure 30C), genomes encoding CAZy families significantly depleted in p-cresol-producing genomes (Figure 30E), and genomes encoding CAZy families significantly depleted in propionate-producing genomes (Figure 30G) are also shown. Charts showing the most abundant families in the secondary bile acid conversion genome (Figure 30B), the indole-negative genome (Figure 30D), the p-cresol-negative genome (Figure 30F), and the propionate-negative genome (Figure 30H) are shown. Charts represent the mean ± standard error. [Figure 30-3] Figures 30-1 through 30-4 show graphs (Figures 30A, 30C, 30E, and 30G) showing the percentage of genomes encoding CAZy families significantly enriched in metabolite-converting genomes, and charts (Figures 30B, 30D, 30F, and 30H) showing the most abundant families in metabolite-converting genomes, sorted by average gene number. The percentages of secondary bile acid-converting and non-converting genomes (Figure 30A), genomes encoding CAZy families exclusively encoded in non-indole-producing bacteria (Figure 30C), genomes encoding CAZy families significantly depleted in p-cresol-producing genomes (Figure 30E), and genomes encoding CAZy families significantly depleted in propionate-producing genomes (Figure 30G) are also shown. Charts showing the most abundant families in the secondary bile acid conversion genome (Figure 30B), the indole-negative genome (Figure 30D), the p-cresol-negative genome (Figure 30F), and the propionate-negative genome (Figure 30H) are shown. Charts represent the mean ± standard error. [Figure 30-4]Figures 30-1 through 30-4 show graphs (Figures 30A, 30C, 30E, and 30G) showing the percentage of genomes encoding CAZy families significantly enriched in metabolite-converting genomes, and charts (Figures 30B, 30D, 30F, and 30H) showing the most abundant families in metabolite-converting genomes, sorted by average gene number. The percentages of secondary bile acid-converting and non-converting genomes (Figure 30A), genomes encoding CAZy families exclusively encoded in non-indole-producing bacteria (Figure 30C), genomes encoding CAZy families significantly depleted in p-cresol-producing genomes (Figure 30E), and genomes encoding CAZy families significantly depleted in propionate-producing genomes (Figure 30G) are also shown. Charts showing the most abundant families in the secondary bile acid conversion genome (Figure 30B), the indole-negative genome (Figure 30D), the p-cresol-negative genome (Figure 30F), and the propionate-negative genome (Figure 30H) are shown. Charts represent the mean ± standard error. [Figure 31]Graphs showing the increase in relative abundance of Lachnospiraceae bacteria in ex vivo populations when grown in the presence of melibiose (e.g., melibiose-1) (FIG. 31A) or raffinose (e.g., raffinose-1) (FIG. 31B) along with alpha-galactosidase and alpha-galactooligosaccharides synthesized via either melibiose or raffinose. Figure 31A shows that enzymes 19 and 20 are alpha-galactosidases encoded in bacterial genomes from Lachnospiraceae, demonstrating specific enrichment of these taxa (melibiose-enz19-1 and melibiose-enz20-1) compared to alpha-galactosidases from other species (melibiose-enz16-1 and melibiose-enz17-1), which did not show similar specific enrichment for Lachnospiraceae bacteria. Figure 31B shows that enzyme 19 is an alpha-galactosidase encoded in bacterial genomes from the Lachnospiraceae family, showing specific enrichment in that taxon (raffinose-enz19-1) compared to an alpha-galactosidase from a different species (raffinose-enz16-1), which did not show similar specific enrichment for Lachnospiraceae bacteria. [Figure 32]32A-32C are graphs showing the increase in relative abundance in ex vivo populations of Bifidobacterium (FIG. 32A), Bacteroides (FIG. 32B), and Roseburia (FIG. 32C) bacteria when grown in the presence of lactulose (lactulose-1) and beta-galactooligosaccharides synthesized via GH42 beta-galactosidase (enz23) and lactulose (lactulose-enz23-1). Enzyme 23 is a beta-galactosidase encoded by bacterial genomes from Bifidobacteria species, and beta-galactooligosaccharides synthesized using this enzyme (lactulose-enz23-1) showed enrichment in Bifidobacterium, Roseburia, and Bacteroides compared with lactulose alone. GH42 beta-galactooligosaccharides showed enrichment in GH42 glycosidases from Bacteroides and Firmicutes genomes, common gut microbiome commensals (Figure 32D). DETAILED DESCRIPTION OF THE INVENTION

[0062] The present invention features at least one method of treating a subject with a disease or disorder (e.g., those described herein) using a glycan polymer preparation. In embodiments, the glycan polymer preparation is selected based on its ability to regulate the production or level (e.g., undesirable level) of a metabolite (e.g., short-chain fatty acids (SFCA) (e.g., propionate or butyrate), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes (e.g., p-cresol or indole), lipopolysaccharide (LPS), or bile acid (e.g., secondary bile acid)). An undesirable level of a metabolite may be too high or too low. In some embodiments, the metabolite is associated with a desired (e.g., beneficial) effect on the subject's health. In other embodiments, the metabolite is associated with an undesirable (e.g., harmful) effect on the subject's health. In some embodiments, the methods described herein include increasing a metabolite. In other embodiments, the method includes decreasing a metabolite. In some embodiments, the metabolite is a microbial (e.g., bacterial) metabolite. In some embodiments, a first metabolite (e.g., one produced by taxon A) is modulated to modulate a second metabolite (e.g., one produced by taxon B). In some embodiments, the second metabolite is associated with a disease or disorder. Undesirable levels of a metabolite can occur anywhere in a subject's body (e.g., the gastrointestinal tract, including the colon and intestines, organs, including the liver and kidneys, feces, blood, brain, nervous system, heart, urine, and other locations). In some embodiments, microbiota production of a metabolite (e.g., in the gut) is modulated to locally affect metabolite levels (e.g., locally increase or decrease a metabolite). In some embodiments, microbiota production of a metabolite (e.g., in the gut) is modulated to systemically affect metabolite levels (e.g., systemically increase or decrease a metabolite). In some embodiments, modulating a first metabolite (e.g., metabolite A, e.g., in the gut) results in modulating a second metabolite (e.g., metabolite B, e.g., in a site of the body other than the gut).In some embodiments, a glycan polymer preparation is administered to a subject in need thereof, wherein the glycan polymer is a substrate (e.g., a preferred substrate) for a specific glycosidase mechanism of a class of microbial metabolite producers. In some embodiments, a glycan polymer preparation is administered to a subject in need thereof, wherein the glycan polymer is a substrate (e.g., a preferred substrate) for a specific glycosidase mechanism of a class of microbial metabolite non-producers. In some embodiments, the balance between metabolite producers and metabolite non-producers (e.g., the relative abundance of microbial taxa in a body site, such as the intestine) is adjusted to regulate the level of metabolites produced by the site. In some embodiments, adjusting the balance between producers and non-producers to regulate metabolite levels treats diseases or disorders associated with metabolic dysregulation. In some embodiments, the subject has dysbiosis in a site, such as the intestine. Further provided herein are glycan polymer preparations that are substrates (e.g., preferred substrates) for microbial metabolite producers or non-producers. In some embodiments, the glycan polymer preparation is tailored to the glycosidase enzyme profile of the microbial taxon or metabolite producer or non-producer, respectively. That is, glycan polymers are substrates (e.g., preferred substrates) for glycosidases present in the genomes of producers or non-producers. In some embodiments, glycosidases are enriched or exclusive to one class (e.g., metabolite producers) relative to another class (e.g., non-producers). Further provided herein are microbial taxa with tailored co-formulations of glycan polymers (e.g., synbiotics) and glycosidase repertoires (glycosidase profiles) that can (preferentially) use glycan polymers as substrates. In some embodiments, the co-formulations are used to increase the population of microbial taxa in microbial sites, such as the intestine.

[0063] The glycan polymers described herein can be tailored to target specific gut microorganisms, e.g., human gut microorganisms. In some embodiments, glycoside hydrolase (glycosidase) enzymes are selected to tailor the glycan polymer to a specific microorganism. In some embodiments, the glycoside hydrolase (glycosidase) profile of the microorganism is determined, and the glycan polymer is tailored to that profile using (e.g., in vitro) one or more identified glycoside hydrolases (glycosidases), e.g., to produce a glycan polymer preparation under conditions suitable for producing the glycan polymer. The glycoside hydrolase can be isolated (and optionally immobilized, e.g., on a suitable substrate). In some embodiments, the glycoside hydrolase can be extracted from a microorganism (e.g., a microbial extract containing the glycoside hydrolase). In some embodiments, microbial cells (e.g., bacteria) containing glycoside hydrolase on their surface and / or intracellularly can be used. In some embodiments, a supernatant containing the glycoside hydrolase (e.g., from a microbial culture) can be used. In some embodiments, the glycoside hydrolase (glycosidase) profile of a particular microorganism is not known or determined, but enzymes derived from the microorganism are used to produce glycan polymers in the methods described herein (e.g., in isolated, extracted, whole-cell, or supernatant form). In some embodiments, the glycan polymer preparation produced as described herein is a specific substrate for a particular microorganism (or group of microorganisms, e.g., a group of microorganisms with the same or similar glycosidase profile) and its glycosidase machinery. In some embodiments, the glycan polymer preparation is specifically fermented by a microorganism or group of microorganisms, e.g., in the gastrointestinal tract of a human subject (e.g., the glycan polymer is fermented at a faster rate or to a greater extent than another microorganism (or group of microorganisms), e.g., with a different glycosidase profile). ... Glycan polymers confer a growth advantage to certain microorganisms. In some embodiments, glycan polymers can be used to regulate the production of microbial metabolites, such as metabolites produced by specific microorganisms or microbial metabolites not produced by specific microorganisms. In the latter case, a specific microorganism may compete with another microorganism, a microorganism that produces an undesirable microbial metabolite, and successful competition by a specific microorganism may result in lower levels of the microbial metabolite. In some embodiments, glycan polymers can be used to promote the transplantation of specific microorganisms administered to a subject in need of transplantation into the subject's microbiota (e.g., the intestinal microbiota, e.g., the colonic microbiota). In some embodiments, glycan polymers confer a growth advantage to certain microorganisms, allowing them to successfully compete for, for example, space and nutrients, to more successfully transplant into the existing microbiota at the transplant site (e.g., the intestine).

[0064] DEFINITIONS The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Terms used hereinafter should be understood according to their general common sense unless otherwise indicated.

[0065] "Abundance" of a microbial taxon, as used herein, is a relative term and refers to the presence of the microbial taxon relative to other taxa in a defined microbial ecological niche, such as the community within the gastrointestinal tract, or in the entire host organism (e.g., a human or experimental animal model of disease).

[0066] As the terms are used herein, "obtain" or "obtaining" refers to obtaining a value, e.g., a numerical value, or an image, or a physical entity (e.g., a sample), for example, by "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" means performing a process (e.g., performing a synthetic or analytical method or protocol) to obtain a value or physical entity. "Indirectly obtaining" refers to receiving a value or physical entity from another entity or source (e.g., a third party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes performing a process that involves a physical change of a physical substance or the use of a machine or device. An example of directly obtaining a value includes obtaining a sample from a human subject. Directly obtaining a value includes performing a process that uses a machine or device, e.g., an NMR spectrometer, to obtain an NMR spectrum.

[0067] For example, when used herein in relation to a type of glycan polymer, "distinct" means that it is chemically and / or structurally distinct from others. For example, two sugars are "distinct" if they are chemically different (e.g., fucose and xylose) or structurally different (e.g., cyclic vs. acyclic, L vs. D). Two dimers are distinct if they consist of the same two monomers, but one pair contains alpha-1,4 linkages and the other pair contains beta-1,6 linkages. Distinct entities may have any other suitable distinct characteristics or properties known in the art and / or detectable by the methods described herein.

[0068] As used herein, a "dosing regimen," "administration regimen," or "treatment regimen" is a modality of drug administration that achieves a therapeutic goal. A dosing regimen includes one, two, three, or four of the following definitions: route of administration, unit dose, dosing frequency, or length of treatment.

[0069] "Dysbiosis" refers to an imbalanced state of the microbiota, e.g., in the gastrointestinal tract, in which the normal diversity, ratio of a first bacterial taxon to a second bacterial taxon, and / or function of the ecological network (e.g., metabolic product production) is disrupted or unstable. This undesirable, e.g., unhealthy, state can be due to a number of factors, including, but not limited to, a decrease or increase in the diversity of the microbiota (e.g., bacterial taxa), an overgrowth of one or more pathogens or pests, or a shift in the biological microbial community that no longer provides an essential function to the host subject and therefore, in one embodiment, no longer promotes health in the subject or is associated with undesired symptoms. In one embodiment, metabolic product production is modulated to contribute to the development of a disease or disorder.

[0070] The terms "effective amount" and "therapeutically effective amount" of a composition (e.g., pharmaceutical composition, etc.) or agent refer to an amount sufficient to provide the desired effect of the composition or agent. In some embodiments, a physician or other medical professional will determine the appropriate amount and administration regimen. An effective amount also refers to the amount of a composition (e.g., pharmaceutical composition, etc.) or agent that prevents the onset or recurrence of a medical condition.

[0071] "Microbial transplantation" or simply "transplantation" refers to the colonization (e.g., proliferation) of a target niche (e.g., the human gut, such as the colon or intestine) with a microbial taxon that is underrepresented (e.g., relative to a healthy reference subject) or absent (e.g., undetectable) in a human subject prior to transplantation (e.g., by administering the microbial taxon to the control, e.g., in the form of a synbiotic described herein). The transplanted microbial taxon may colonize for a transient period or may exhibit long-term stability in the microbiota occupying the subject following transplantation of the microbial taxon. In some embodiments, the transplanted microbial taxon may induce an environmental shift in the target niche that represents a shift from dysbiosis to a healthy state.

[0072] "Fructooligosaccharides" or "FOS," as these terms are used herein, refer to fructose polymers consisting of the following sequence, optionally including a terminal glucose: (Fru)n-Glc, consisting of one or more of beta-2,1, beta-2,6, alpha-1,2, and beta-1,2 glycosidic linkages (where n is typically 3-10). Variants include inulin-type beta-1,2 and levan-type beta-2,6 linkages between fructosyl units in the backbone. In one embodiment, the FOS are produced by the methods described in any of references 8, 24, 25, 61, 67, 69, 72, 170, or 176-186, or 21, 29, 170, 176, or 222 of Meyer, Biotechnological Production of Oligosaccharides—Applications in the Food Industry, Chapter two, Food technology and Industry, 2015, (Meyer 2015), which together with the respective references thereto are incorporated by reference herein. In one embodiment, the FOS are those described in or made by the methods described in Sangeetha et al. 2005, 2014 found in Diez-Municio et al., 2014, Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases, 2014, Microbial Biotechnology, 7:315-313 (Diez-Municio et al. 2014), which are incorporated herein by reference in their entirety.In one embodiment, the FOS is selected from the group consisting of B. macerans, Z. mobilis, L. reutri, A. niger, A. japonicas, A. foetidus, A. sydowi,b, A. pulllans, C. purpurea, F. oxysporum, and the like. In some embodiments, the FOS are produced by enzymes derived from P. rum, P. citrinum, P. frequentans, P. spinulosum, P. rigulosum, P. parasitica, S. brevicaulis, S. cerevisiae, or K. marxianus. In other embodiments, the FOS are produced by the enzymatic action of a fructosyltransferase, β-fructofuranosidase (EC 3.2.1.26), inulosucrase (EC 2.4.1.9), levansucrase (EC 2.4.1.10), or endo-inulinase.

[0073] As used herein, "galactooligosaccharides" or "GOS" refers to a mixture of substances produced from lactose and having 2 to 8 saccharide units, one of which is a terminal glucose, the remaining units are galactose, and a disaccharide containing two units of galactose. In one embodiment, GOS is a mixture of galactopyranosyl oligomers (DP = 3 to 8) that are predominantly linked by β-(1,4) or β-(1,6) linkages, although minor percentages of β-(1,2) or β-(1,3) linkages may also be present. The terminal glucosyl residue is linked to the galactosyl unit by a β-(1,4) linkage.

[0074] GOS is synthesized by the reverse action of β-galactosidase (EC 3.2.1.23) on lactose at relatively high concentrations of lactose. In one embodiment, the GOS is synthesized by the enzymatic action of β-galactosidase from a Bifidobacterium, e.g., Bifidobacterium longum, Kluyveromyces sp., Kluyveromyces marxianus, an Aspergillus sp., e.g., Aspergillus oryzae, Escherichia coli K-12, Bacillus circulans, Lactobacillus bulgaricus, S. singularis, S. thermophiles, or C. laurentii. In one embodiment, the GOS is a GOS disclosed in or made by the methods described in any of references 8, 105, or 196-206, or 105, 120, 198, 202-205, or 223-227 of Meyer 2015, which are incorporated herein by reference in their entirety. In one embodiment, the GOS is a GOS described in or made by the methods described in Panesar et al. 2006 or Torres et al. 2010, 2014, which are incorporated herein by reference in their entirety.

[0075] "Glucooligosaccharides" or "GLOS," as the term is used herein, refer to polymers of glucose subunits. The primary linkages in GLOS are (Glc)n[α(1→2), α(1→3), α(1→4), and α(1→6)]. In one embodiment, GLOS is made using dextransucrase (EC 2.4.1.5). In one embodiment, GLOS can be produced using enzymes derived from bacteria (L. mesenteroides; L. citreum). In one embodiment, GLOS is prepared using Diez-M GLOS is described in or produced by the methods described in any of Remaud et al., 1992, Chung and Day, 2002, or Kim et al., 2014, as found in Unicio et al., 2014, Synthesis of novel bioactive lactose-derived oligosaccharides by microbial glycoside hydrolases, 2014, Microbial Biotechnology, 7:315-313, which documents, together with their respective references, are incorporated herein by reference.

[0076] As used herein, a "glycan polymer preparation" (also referred to as a "glycan polymer preparation," "glycan preparation," or "glycan polymer") is a preparation containing glycan polymers that exhibit a desired effect (e.g., a therapeutic effect). In some embodiments, the glycan polymer preparation is one or more naturally occurring oligosaccharides, such as glucooligosaccharides, mannanoligosaccharides, inulin, lycunose, maltotetraose, nigerotetraose, nystose, sesemose, stachyose, isomaltotriose, nigerotriose, maltotriose, melezitose, maltotriose, raffinose, kestose, fructooligosaccharides, and the like. , 2'-fucosyllactose, galactooligosaccharide, glycosyl, idraparinux, isomaltooligosaccharide, maltodextrin, xylooligosaccharide, agar, agarose, alginic acid, alguronic acid, alpha-glucan, amylopectin, amylose, arabioxiran, beta-glucan, callose, capsulan, carrageenan, cellodextrin, cellulin, cellulose, chitin, chitin nanofibril, chitin-glucan complex , Chitosan, Chrysolaminarin, Curdlan, Cyclodextrin, Alpha-Cyclodextrin, Dextran, Dextrin, Dialdehyde Starch, Ficoll, Fructan, Fucoidan, Galactoglucomannan, Galactomannan, Galactosaminogalactan, Gellan Gum, Glucan, Glucomannan, Glucuronoxylan, Glycocalyx, Glycogen, Hemicellulose, Hypromellose, Icodextrin, Kefiran, Laminarin , lentinan, levan polysaccharide, lichenin, mannan, mucus, natural gum, paramylon, pectic acid, pectin, pentastarch, plant glycogen, pullulan, poligeenan, polydextrose, porphyran, pullulan, schizophyllan, sepharose, sinistrin, sizophyllan, sugammadex, welan gum, xanthan gum, xylan, xyloglucan, zymosan, etc. In some embodiments, the glycan polymer is present as a salt, e.g., a pharmaceutically acceptable salt.

[0077] As used herein, "glycan subunit" refers to an individual unit of a glycan species disclosed herein, e.g., a component that makes up a glycan species. In one embodiment, the glycan subunit is a monomer. In one embodiment, the glycan subunit is a dimer. In one embodiment, the glycan subunit is a monosaccharide. In one embodiment, the glycan subunit is a disaccharide. In some embodiments, the glycan subunit is a carbohydrate and may be selected from sugar alcohols, short-chain fatty acids, sugar acids, imino sugars, deoxy sugars, and amino sugars. In some embodiments, the glycan subunit is erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, fucose, fuculose, rhamnose, mannoheptulose, sedoheptulose, etc. In some embodiments, the glycan subunit is glucose, galactose, arabinose, mannose, fructose, xylose, fucose, or rhamnose. In embodiments, the glycan comprises distinct glycan subunits, such as a first and a second monosaccharide, or a first and a second disaccharide. In embodiments, the glycan comprises distinct glycan subunits, such as a first, a second, a third, a fourth, and / or a fifth distinct glycan subunit.

[0078] As used herein, a "glycosidase enzyme molecule" includes a polypeptide that retains or has the activity of a glycosidase enzyme, for example, it retains or has at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 99.9% of the turnover rate of the glycosidase enzyme, or it retains or has at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 99.9% of the specificity of the glycosidase enzyme, or it retains or has at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 99.9% of the affinity of the glycosidase enzyme for the glycan subunit. In some embodiments, the glycosidase enzyme molecule comprises a polypeptide having an activity of the glycosidase enzyme that is at least about 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, or 500% of the turnover rate of the glycosidase enzyme, or at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, or 500% of the affinity of the glycosidase enzyme for a glycan subunit. In some embodiments, the glycosidase enzyme molecule is a fragment (e.g., an active fragment) of the glycosidase enzyme. In some embodiments, the glycosidase enzyme molecule differs at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100 or more amino acid residues compared to the glycosidase enzyme. In some embodiments, the glycosidase enzyme molecule includes at least 1, 2, 3, 4, 5, 10, 25, 50, 75, 100 amino acid mutations (e.g., deletions, additions, or substitutions) compared to the glycosidase enzyme.

[0079] As used herein, "glycosidase enzymes" includes glycosidases (also called "glycoside hydrolases" (GHs)), glycosyltransferases (GTs) and lyases.

[0080] As used herein, "glycotaxon" refers to bacterial microorganisms (e.g., human gut microorganisms) classified according to the presence (e.g., absence) of metabolic (e.g., enzymatic) function. In some embodiments, taxa may be classified according to CAZy glycosidase / glycohydrolase (GH) or CAZy glycosyltransferase (GT) enzyme function. In some embodiments, bacterial taxa may be classified into any one of glycotaxon class 1, glycotaxon class 2, glycotaxon class 3, glycotaxon class 4, glycotaxon class 5, glycotaxon class 6, or glycotaxon class 7. In some embodiments, glycotaxon class 1 contains but and / or buk gene-containing bacterial taxa. In some embodiments, glycotaxon class 2 contains cutC gene-negative bacterial taxa. In some embodiments, glycotaxon class 3 contains urease gene-negative bacterial taxa. In some embodiments, glycotaxon class 4 excludes one or more propionate production-related enzymes selected from propionate kinase, propionate-CoA transferase, propionate-CoA ligase, propionyl-CoA carboxylase, methylmalonyl-CoA carboxytransferase, (S)-methylmalonyl-CoA decarboxylase, methylmalonate semialdehyde dehydrogenase, and propanal dehydrogenase. In some embodiments, glycotaxon class 5 comprises an enzyme associated with bile acid production (e.g., secondary bile acid production) selected from 7alpha-hydroxysteroid dehydrogenase, 12alpha-hydroxysteroid dehydrogenase, 7beta-hydroxysteroid dehydrogenase (NADP+), 2beta-hydroxysteroid dehydrogenase, 3beta-hydroxycholanic acid 3-dehydrogenase (NAD+), 3alpha-hydroxycholanic acid dehydrogenase (NADP+), 3beta-hydroxycholanic acid 3-dehydrogenase (NADP+), 3alpha-hydroxybile acid-CoA-ester 3-dehydrogenase, 3alpha-hydroxycholanic acid dehydrogenase (NAD+), bile acid CoA-transferase, bile acid 7alpha-dehydratase, and bile acid CoA ligase.In some embodiments, glycotaxon class 6 excludes one or more indole production-related enzymes (e.g., tryptophanase). In some embodiments, glycotaxon class 7 excludes one or more p-cresol production-related enzymes selected from 4-hydroxyphenylacetate decarboxylase and aldehyde ferredoxin oxidoreductase.

[0081] As used herein, "isomaltooligosaccharides" or "IMOS" refers to a mixture of oligosaccharides with primarily α-(1,6)-linked glucose residues, with a degree of polymerization (DP) ranging from 2 to 6, and oligosaccharides with a mixture of α-(1,6) and sometimes α-(1,4) glycosidic linkages, such as panose. In one embodiment, IMOS contain glucosyl residues linked to maltose or isomaltose by α-(1,6) glycosidic linkages. In one embodiment, IMOS are produced using starch as a feedstock. In one embodiment, they are produced from corn starch and consist of isomaltose, isomaltotriose, and panose. In one embodiment, IMOS are the product of an enzymatic transglycosidase reaction using an immobilized enzyme combination in which starch is liquefied using α-amylase (EC 3.2.1.1) and pullulanase (EC 3.2.1.41). In a second step, the intermediate product is treated with both β-amylase (EC 3.2.1.2) and α-glucosidase (EC 3.2.1.20). The beta-amylase first hydrolyzes the liquefied starch to maltose. The transglucosidase activity of the α-glucosidase then produces an isomaltooligosaccharide mixture containing oligosaccharides with both α-(1,6)- and α-(1,4)-linked glucose residues. In one embodiment, the IMOS are those described in or made by the methods described in any of references 2, 217-219, 12, 152, 159, or 236 of Meyer 2015, which are incorporated herein by reference in their entirety. In one embodiment, the IMOS are those described in or made by the methods described in Panesar et al. 2006 or Torres et al. 2010, 2014, which are found in Diez-Municio et al. 2014, which are incorporated herein by reference in their entirety. In one embodiment, the IMOS are synthesized by enzymatic hydrolysis of starch with α-amylase or pullulanase, or with β-amylase and α-glucosidase, in that order.In one embodiment, the IMOS is selected from A. niger, Bacillus spp., B. subtilis, B. stearothermoff. It is synthesized by enzymes derived from B. stearothermophilus, T. maritime, A. carbonarious, or L. mesenteroides.

[0082] As used herein, an "isolated" or "purified" glycan polymer preparation is substantially pure and free of contaminants, such as pathogens, enzymes, or other unwanted biological substances, or toxic or other unwanted organic or inorganic compounds. In some embodiments, a pure or isolated compound, composition, or preparation may contain trace amounts of solvents and / or salts (e.g., less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, less than 0.5%, or 0.1% by w / w, w / v, v / v, or mole %). A purified compound or preparation contains at least about 60% (w / w, w / v, v / v, or mole %), at least about 75%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the compound of interest. For example, a purified (substantially pure) or isolated glycan polymer preparation is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% glycan polymer (i.e., free of any solvent, e.g., water, in which the glycan polymer preparation can be dissolved), for example, during production, extraction / purification, and / or processing, separated from co-occurring components (e.g., the glycan polymer is substantially free of undesired compounds). Purity can be measured by any appropriate standard method, for example, by column chromatography (e.g., size exclusion chromatography (SEC)), thin layer chromatography (TLC), gas chromatography (GC), high performance liquid chromatography (HPLC), or nuclear magnetic resonance (NMR) spectroscopy. Purified or purity may also define the degree of sterility, safe for administration to a human subject, eg, lacking viable infectious or toxic agents.

[0083] As used herein, "microbiome" refers to the genetic content of the microbial community ("microbiota") that inhabits both persistently and transiently in and on a subject (e.g., a human subject), including eukaryotes, archaea, bacteria, and viruses (including bacterial viruses (e.g., phages)), where "genetic content" encompasses all genomic DNA, RNA such as ribosomal RNA and messenger RNA, epigenomes, plasmids, and other types of genomic information. In some embodiments, microbiome specifically refers to the genetic content of the microbial community in an ecological niche.

[0084] "Microbiota," as used herein, refers to the community of microorganisms (chronically and transiently) occurring in and on a subject (e.g., a human subject), and includes eukaryotes, archaea, bacteria, and viruses (including bacterial viruses, e.g., phages). In some embodiments, microbiota specifically refers to the community of microorganisms in an ecological niche.

[0085] "Pathobiont" or "(opportunistic) pathogen," as used herein, refers to a commensal organism that is capable of causing disease only when certain genetic and / or environmental conditions are present in a subject (e.g., a human subject).

[0086] As used herein, the term "pathogenic" (e.g., "pathogenic bacteria") refers to a substance, microorganism, or condition that has the ability to cause disease. In certain contexts, pathogens also include microorganisms (e.g., bacteria) that are associated with a disease or condition, but for which a (direct) causative relationship has not been or has not yet been established. As used herein, the term "pathogen" refers to viruses, parasites, and bacteria, or other pathogens that can cause infection in a subject, e.g., a human.

[0087] As used herein, a "pharmaceutical composition" refers to a composition or preparation having pharmacological activity or other direct effect in the alleviation, treatment, or prevention of disease, and / or a finished dosage form or formulation thereof, and intended for human use. Pharmaceutical compositions are typically produced under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be sterile or non-sterile. If non-sterile, such pharmaceutical compositions typically meet the microbiological standards and standards for non-sterile pharmaceutical products set forth in the United States Pharmacopoeia (USP) or the European Pharmacopoeia (EP). Pharmaceutical compositions can further comprise or be co-administered with additional active agents, such as additional therapeutic agents.

[0088] The pharmaceutical composition may also include, for example, additional therapeutic agents, polyphenols, prebiotic substances, probiotic bacteria, pharmaceutically acceptable excipients, solvents, carriers, or any combination thereof. Any of the glycan polymer preparations described herein can be formulated as a pharmaceutical composition.

[0089] The term "subject" (or "patient"), as used herein, refers to any human subject. The term does not denote a particular age or sex. A subject may include a pregnant woman. A subject may include newborns (preterm, full-term), infants up to 1 year of age, young children (e.g., 1-12 years old), teenagers (e.g., 13-19 years old), adults (e.g., 20-64 years old), and seniors (65 years old and older). A subject does not include agricultural animals, e.g., farm animals or livestock, e.g., cows, horses, sheep, pigs, chickens, etc.

[0090] A "substantial reduction," as used herein, is a reduction of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.9%, or 100% (e.g., for a biomarker or metabolite).

[0091] A "substantial increase," as used herein (e.g., with respect to a biomarker or metabolite), is an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, or greater than 1000%.

[0092] As used herein with respect to glycosidase enzymes and / or glycosidase enzyme molecules, the term "substrate" refers to a glycan polymer that is the product of a glycosidase enzyme molecule or has the structure of a glycan polymer created by a glycosidase enzyme molecule, which is a substrate for a glycosidase enzyme, for example, a glycosidase expressed in human intestinal microorganisms. In embodiments, the glycosidase enzyme molecule catalyzes the polymerization of glycan subunits to form a substrate under appropriate reaction conditions, and the glycosidase enzyme cleaves the bond between the glycan subunits of the substrate (in embodiments, the same bond formed by the glycosidase enzyme molecule) under appropriate reaction conditions. In one embodiment, the glycosidase enzyme molecule and the glycosidase enzyme have the same primary amino acid sequence, for example, are the same enzyme. In embodiments, the substrate has one or more of the following properties: i) the turnover rate of the substrate and the glycosidase enzyme is similar to that of a naturally occurring substrate of the glycosidase enzyme to the extent that it is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% of that of at least one naturally occurring substrate of the glycosidase enzyme.The turnover rate can be expressed, for example, in terms of glycosidic bonds cleaved per unit time, e.g., per minute or hour, or the rate of depolymerization of glycan polymers per unit time, e.g., per hour or minute; ii) a binding constant for the glycosidase enzyme is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% of at least one naturally occurring substrate of the glycosidase enzyme, and in embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 95, or 99% of at least one naturally occurring substrate of the glycosidase enzyme. and iii) the binding motif for the glycosidase enzyme, the binding motif for the glycosidase enzyme molecule, and at least one naturally occurring substrate of the glycosidase enzyme share one or more particular glycan subunits, e.g., a particular sugar dimer, a particular sugar branch point, a particular alpha- or beta-configuration, a particular regiochemistry, e.g., 1,2-, 1,3-, 1,4-, 1,5-, or 1,6-linkages; and iv) the substrate promotes the growth or metabolism of human gut microorganisms expressing the enzyme molecule.

[0093] As used herein, "synthetic" refers to an artificial compound or preparation, such as a glycan polymer preparation, that does not occur in nature. In one embodiment, the non-enzymatic polymer catalyst described herein is used to synthesize the glycans of the preparation under appropriate reaction conditions, for example, by a polymerization reaction that creates oligomers from individual glycan subunits added to the reaction. In some embodiments, the non-enzymatic polymer catalyst can act as a hydrolytic agent, breaking glycosidic bonds. In other embodiments, the non-enzymatic polymer catalyst can form glycosidic bonds. In one embodiment, the glucosidase enzyme molecule described herein is used to synthesize the glycans of the preparation under appropriate reaction conditions, for example, by a polymerization reaction that creates oligomers from individual glycan subunits added to the reaction. In some embodiments, the glycosidase enzyme molecule can act as a hydrolytic agent, breaking glycosidic bonds. In other embodiments, the glycosidase enzyme molecule can form glycosidic bonds. In one embodiment, solid-phase oligosaccharide synthesis is used to synthesize the glycans of the preparation under appropriate reaction conditions, for example, by polymerization reactions that create oligomers from individual glycan subunits added to the reaction mixture. Synthetic glycan polymer preparations can also include glycan polymers that are not isolated from natural oligosaccharide or polysaccharide sources. While glycan polymer preparations are not isolated from natural oligosaccharide or polysaccharide sources, it is understood that the glycan subunits that make up the glycan polymer can be, and often are isolated from, natural oligosaccharide or polysaccharide sources, including those listed herein, or are synthesized de novo.

[0094] As used herein, the terms "treating" and "treatment" refer to the administration of an agent or composition to a subject (e.g., a symptomatic subject suffering from an adverse condition, disorder, or disease) to affect a reduction in the severity and / or frequency of symptoms, eliminate the symptoms and / or their underlying cause, and / or promote the amelioration or repair of damage, and / or prevent an adverse condition, disorder, or disease in an asymptomatic subject (e.g., a human subject) predisposed to, or at risk of developing, the particular adverse condition, disorder, or disease.

[0095] A "therapeutic nutritional product" is a food product that provides a therapeutic effect when administered alone or in combination with a second therapy (e.g., a drug therapy), and when combined with the second therapy, it provides an additive or synergistic therapeutic effect, or alleviates or reduces a negative effect of the second therapy (e.g., reduced side effects). A therapeutic nutritional product may be a food product that is administered in conjunction with dietary recommendations (e.g., by a physician or nutritionist, or other professional in nutrition, human nutrition), and dietary regulations (e.g., based on the subject's medical condition and individual needs). (based on

[0096] "Xylooligosaccharides" or "XOS," as the term is used herein, refer to sugar oligomers of xylose units linked by β-(1,4) bonds. The number of xylose residues varies from 2 to 10, but is primarily composed of xylobiose, xylotriose, and xylotetraose. Arabinofuranosyl, glucopyranosyluronic acid, or their 4-O-methyl derivatives (2- or 3-acetyl or phenolic substituents) may also be present, resulting in branched XOS. In one embodiment, XOS are primarily linear β-(1,4)-linked XOS (primarily xylobiose, xylotriose, and xylotetraose) and some oligosaccharides with branched arabinose residues. In one embodiment, XOS are produced using β-xylanase from lignocellulosic materials. In one embodiment, xylan is enzymatically hydrolyzed to xylooligosaccharides by endo-β-1,4-xylanase (EC 3.2.1.8) or beta-xylosidase (EC 3.2.1.9). In one embodiment, XOS is produced by enzymatic degradation of xylan, for example, by endo-β-1,4-xylanase, exo-β-1,4-xylosidase, α-glucuronosidase, α-L-arabinofuranosidase, acetyl xylan esterase, ferulic acid esterase, or p-coumaric acid esterase. In one embodiment, the XOS is XOS produced by the methods disclosed in or described in any of references 152, 159, 162, 179, 214-216, or 232 of Meyer 2015, which are incorporated herein by reference. In one embodiment, the XOS is XOS described in or produced by the methods described in Casci and Rostal, 2006, found in Diez-Municio et al., 2014, which are incorporated herein by reference along with their respective references.In one embodiment, the XOS is synthesized by a xylanase from any of T. reesei, T. harzianum, T. viride, T. koningii, T. longibrachiatum, P. chyrosporium, G. trabeum, or A. oryzae.

[0097] Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising of". Hereinafter, when a group is defined as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of these embodiments.

[0098] Where an indefinite or definite article is used when referring to a singular noun e.g. "a", "an" or "the", this includes a plural of that noun unless specifically stated otherwise.

[0099] Claims and disclosures relating to methods of treatment or diagnosis are considered to be equivalent embodiment and claim disclosures to "a compound, composition, product, etc. for use in ..." or "the use of a compound, composition, product, etc. in the manufacture of a medicament, pharmaceutical composition, diagnostic composition, etc. for ...," indicating that such compound, composition, product, etc. is to be used in a diagnostic or treatment method that may be performed on the human or animal body. Thus, if an embodiment or claim refers to a "method of treatment by administering a compound to a human or animal suspected of having a disease," this is also considered to be a disclosure of "the use of a compound in the manufacture of a medicament for treating a human or animal suspected of having a disease" or "a compound for use in treating a human or animal suspected of having a disease." By way of example, a reference to a method of treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., short chain fatty acids (SCFAs), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, lipopolysaccharide (LPS), or bile acids) by administering an amount of a glycan polymer preparation means (i) treating an undesirable level of a metabolite (e.g., short chain fatty acids (SCFAs), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO) (ii) a glycan polymer preparation for use in treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., short-chain fatty acid (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, lipopolysaccharide (LPS), or bile acid); or (iii) a glycan composition for use in the manufacture of a medicament for treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., short-chain fatty acid (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, lipopolysaccharide (LPS), or bile acid).

[0100] Whenever reference is made to a method of treating an individual or a population of individuals by administration (e.g., of a glycan composition), such reference, in a preferred embodiment, contemplates analytical, diagnostic steps, etc. in the course of such treatment that may be useful in determining, for example, whether an individual or population is susceptible to a particular treatment due to its microbiome composition, whether it is successful, etc. For example, a reference to a method of treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., short-chain fatty acid (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, lipopolysaccharide (LPS)), or bile acid) by administering an amount of a glycan polymer preparation is also considered to be a disclosure of such a method, wherein, in a preferred embodiment, the subject (i) is first tested for the nature and level of the metabolite before initiating treatment, (ii) is tested for the composition of its microbiome to match the administration of the glycan composition to the microbial glycosidase enzyme composition in the gut, (iii) is tested during the course of treatment to monitor the effect of administration of the glycan composition on the level of the metabolite, etc.

[0101] The terms "obtainable by," "producible by," and the like are used to indicate that the claim or embodiment refers to the compound, composition, product, etc. itself, i.e., that the compound, composition, product, etc. can be obtained or produced by a method described for producing the compound, composition, product, etc., but that the compound, composition, product, etc. can also be obtained or produced by methods other than those described. The terms "obtainable by," "produced by," and the like indicate that the compound, composition, product is obtained or produced by the specific method recited. The terms "obtainable by," "producible by," and the like also disclose terms such as "obtained by," "produced by," and the like, as preferred embodiments of "obtainable by," "producible by," and the like.

[0102] It should be further understood that the present disclosure as a preferred embodiment also discloses how the individual aspects and embodiments described herein can be combined. For example, Table 3 discloses the relationship between metabolites and phyla and strains, while Table 5 discloses the relationship between metabolites and diseases. Therefore, a person skilled in the art will consider this information together and understand which microorganisms must be affected to reduce the level of a metabolite, for example, to treat a specific disease.

[0103] As used herein, "homology" and "sequence identity" (used interchangeably herein) are a measure of how similar a sequence (e.g., an amino acid sequence or a nucleic acid sequence) is to another sequence. Calculation of "homology" or "sequence identity" (these terms are used interchangeably herein) between two sequences is performed as follows: For optimal comparison, the sequences are aligned (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Optimal alignment is determined as the best score using the GAP program in the GCG software package, using a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

[0104] Methods of Making Glycan Polymers Glycan polymer preparations can be produced using any method known in the art.

[0105] The glycan polymer composition may comprise the glycans described herein, dietary fiber, e.g., FOS (fructooligosaccharides), other sugars (e.g., monomers, dimers, e.g., lactulose), and sugar alcohols, and optionally other components, such as polyphenols, fatty acids, peptides, micronutrients, such as those described in WO 2016 / 172658, "MICROBIOME REGULATORS AND RELATED USES THEREOF," and microorganisms such as bacteria.

[0106] The glycan preparations described in WO 2016 / 122889, "GLYCAN THERAPEUTICS AND RELATED METHODS THEREOF" and WO 2016 / 172657, "GLYCAN THERAPEUTICS AND METHODS OF TREATMENT" (which are incorporated by reference in their entireties) are suitable for the methods and compositions described herein.

[0107] Preparations containing glycan polymers can be produced by non-enzymatic catalysts, such as the polymer catalysts described in WO 2012 / 118767, "POLYMERIC ACID CATALYSTS AND USES THEREOF," or other suitable methods. Other acid catalysts (e.g., solid catalysts) may also be used. Methods for preparing the polymer catalysts and solid-supported catalysts described herein can be found in WO 2014 / 031956, "POLYMERIC AND SOLID-SUPPORTED CATALYSTS, AND METHODS OF DIGESTING CELLULOSIC MATERIALS USING SUCH CATALYSTS." For example, glycans produced using catalysts such as those described in WO 2016 / 007778, "OLIGOSACCHARIDE COMPOSITIONS AND METHODS FOR PRODUCING THEREOF" are suitable for the methods and compositions described herein. All patent applications are incorporated herein by reference in their entirety.

[0108] How many In some embodiments, glycan polymers are produced by solid-phase oligosaccharide synthesis, e.g., using various protecting groups to achieve glycan synthesis. Exemplary methods are described in "Solid-Phase Oligosaccharide Synthesis and Combinatorial Carbohydrate Libraries," Peter H. Seeberger and Wilm-Christian Haase, American Chemical Society, 2000; and "Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research," Thomas J. Boltje et al., Nat. Chem. 2009 November 1;1(8):611-622.

[0109] In some embodiments, glycan polymers can be synthesized using enzymatic catalysts (e.g., isolated or bacterially expressed glycosidases or glycosyltransferases) as described herein to synthesize glycans by polymerization reactions that generate oligomers from individual glycan subunits added to the reaction. Exemplary methods are described in "Synthesis and Purification of Galacto-Oligosaccharides: State of the Art", Carlos Vera et al., World J. Microbiol Biotechnol. 2016;32:197; "Synthesis of Novel Bioactive Lactose-Derived Oligosaccharides by Microbial Glycoside Hydrolases", Marina Diez-Municio et al., Microbial Biotechnol. 2014;7(4),315-331; and "Methods of Improving Enzymatic Trans-Glycosylation for Synthesis of Human Milk Oligosaccharide Biomimetics", Birgitte Zeuner et al., J. Agric. Food Chem. 2014,62,9615-9631, WO 2005 / 003329, "NOVEL GALACTOOLIGOSACCHARIDE COMPOSITION AND THE PREPARATION THEREOF,” all of which are incorporated herein by reference.

[0110] In some embodiments, glycan preparations can be prepared using glycan polymers such as starch and other fibers, such as dietary fiber (as described herein), and one or more glycan (or fiber) properties, such as the degree of polymerization (e.g., depolymerization), the degree of branching (e.g., debranching), or glycosidic bond distribution (e.g., by adding new types of glycosidic bonds or removing existing bonds), can be prepared by subjecting them to a catalyst (e.g., an acid catalyst, a solid or polymer catalyst, an enzyme catalyst) to change the properties. An exemplary method for corn syrup is described in Example 101 of U.S. Patent Application Publication No. 2016 / 0007642, which is incorporated by reference. Other methods, such as those used to prepare digestion-resistant starch (e.g., as described in MGSajilata et al., "Resistant Starch - A Review," Comprehensive Reviews in Food Science and Food Safety - Vol. 5, 2006, and U.S. Patent Application Publication No. 2006 / 0257977, "Slowly digestible starch"), such as heat treatment, enzymatic treatment, chemical treatment, or a combination thereof, can be used to produce the glycan preparations described herein.

[0111] The present invention features a method for producing a glycan polymer or preparation that is a substrate for gut microorganisms (e.g., human gut bacteria). The starting material for the method is a glycan subunit that includes a sugar monomer (e.g., a monosaccharide), a sugar dimer (e.g., a disaccharide), a sugar trimer (e.g., a trisaccharide), or a combination thereof.

[0112] The starting material may comprise a furanose or pyranose sugar. In some embodiments, the starting material comprises a tetrose, pentose, hexose, or heptose. In some embodiments, the starting material comprises glucose, galactose, arabinose, mannose, fructose, xylose, fucose, and rhamnose. The glycan subunit starting material may be in their L- or D-form, alpha- or beta-configuration, and / or deoxygenated form, if applicable, and any combination thereof.

[0113] The glycan subunits used in the methods described herein can include a monosaccharide, such as a C5 monosaccharide or a C6 monosaccharide. In some embodiments, the monosaccharide is a C5 monosaccharide. In some embodiments, the monosaccharide is a C6 monosaccharide. The glycan subunit can include a disaccharide, such as a disaccharide comprising a C5 monosaccharide or a C6 monosaccharide. In some embodiments, the disaccharide comprises a C5 monosaccharide. In some embodiments, the disaccharide comprises two C5 monosaccharides. In some embodiments, the disaccharide comprises a C6 monosaccharide. In some embodiments, the disaccharide comprises two C6 monosaccharides. In some embodiments, the disaccharide comprises one C5 monosaccharide and one C6 monosaccharide.

[0114] The glycan subunit starting material used herein may be glycolaldehyde, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, fucose, fuculose, rhamnose, mannoheptulose, sedoheptulose, neuraminic acid, N-acetylneuraminic acid, N-acetylgalactosamine, N-acetylglucosamine, fructosamine, galactosamine, glucosamine, sorbitol, glycerol, erythritol, threitol, arabitol, xylitol, mannitol, sorbitol, galactitol, fucitol, and lactic acid.

[0115] The glycan subunit starting material used herein may be a disaccharide or larger subunit selected from acarbiosin, N-acetyllactosamine, allolactose, cellobiose, chitobiose, galactose-alpha-1,3-galactose, gentiobiose, isomalt, isomaltose, isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose, laminaribiose, maltitol, maltose, mannobiose, melibiose, melibiulose, neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose, sucralose, sucrose, sucrose acetate isobutyrate, octaacetylsucrose, trehalose, turanose, vicianose, and xylobiose.

[0116] In some embodiments, the glycan subunit is an unactivated glycan subunit. In some embodiments, the glycan subunit is an activated glycan subunit, for example, activated with a nucleoside, a nucleotide (e.g., UTP, UDP, UMP, GTP, GDP, GMP, ATP, ADP, AMP, CTP, CDP, CMP), or a phosphate group. In some embodiments, the glycan subunit is a UDP sugar or a UMP sugar.

[0117] In some embodiments, the glycan subunits are substituted or derivatized with an acetyl group, acetate ester, sulfate half ester, phosphate ester, or pyruvyl cyclic acetal group, or are otherwise derivatized, for example, with one or more hydroxyl or amine groups.

[0118] In some embodiments, the glycan subunit comprises an amino sugar, a deoxy sugar, an imino sugar, a sugar acid, or a sugar alcohol. Exemplary amino sugars include acarbose, N-acetylemannosamine, N-acetylmuramic acid, N-acetylneuraminic acid, N-acetyletalosaminuronic acid, arabinopyranosyl-N-methyl-N-nitrosourea, D-fructose-L-histidine, N-glycolylneuraminic acid, ketosamine, kidamycin, mannosamine, 1B-methylseleno-N-acetyl-D-galactosamine, muramic acid, muramyl dipeptide, phosphoribosylamine, PUGNAc, sialyl-Lewis A, sialyl-Lewis X, validamycin, voglibose, N-acetylgalactosamine, N-acetylglucosamine, aspartylglucosamine, bacilitiol, daunosamine, desosamine, fructosamine, galactosamine, glucosamine, meglumine, and perosamine. Exemplary deoxysugars include 1-5-ahydroglucitol, cladinose, colitose, 2-deoxy-D-glucose, 3-deoxyglucasone, deoxyribose, dideoxynucleotides, digitalose, fludeoxyglucose, sarmentose, and sulfoquinovose. Exemplary iminosugars include castanospermine, 1-deoxynojirimycin, iminosugars, miglitol, miglustat, and swainsonine. Exemplary sugar acids include N-acetylneuraminic acid, N-acetyltalosamnuronic acid, and the like. acid), aldaric acid, aldonic acid, 3-deoxy-D-manno-oct-2-urosonic acid, glucuronic acid, glucosamineuronic acid, glyceric acid, N-glycolylneuraminic acid, iduronic acid, isosaccharinic acid, pangamic acid, sialic acid, threonic acid, urosonic acid, uronic acid, xylonic acid, gluconic acid, ascorbic acid, ketodeoxyoctursonic acid, galacturonic acid, galactosaminuronic acid, mannuronic acid, mannosaminuronic acid, tartaric acid, mucic acid, saccharic acid, lactic acid, oxalic acid, succinic acid, hexanoic acid, fumaric acid, maleic acid, butyric acid, citric acid, glucosaminic acid, malic acid, succinamic acid, sebacic acid, and capric acid.Exemplary sugar alcohols include methanol, ethylene glycol, glycerol, erythritol, threitol, arabitol, ribitol, xylitol, mannitol, sorbitol, galactitol, iditol, volemitol, fucitol, inositol, maltotriitol, maltotetriitol, and polyglycitol.

[0119] In some embodiments, the glycan subunit starting material is in the form of a salt (e.g., a pharmaceutically acceptable salt), such as, for example, hydrochloride, hydroiodate, hydrobromide, phosphate, sulfate, methanesulfate, acetate, formate, tartrate, malate, citrate, succinate, lactate, gluconate, pyruvate, fumarate, propionate, aspartate, glutamate, benzoate, ascorbate, etc.

[0120] The glycan subunits used in the methods described herein can be obtained from any commercially available, known source or produced according to any method known in the art. In some embodiments, hydrolysis can be used to produce the constituent monosaccharides or oligosaccharides suitable for producing the glycans described herein. Glycan units, such as monosaccharides, can be in many different forms, for example, conformations. It may exist in isomeric, cyclic, acyclic, stereoisomer, tautomer, anomer, and isomeric forms.

[0121] Reaction Conditions for Producing Glycan Polymers Using Non-Enzymatic Polymeric Catalysts In some embodiments, the glycan units and catalyst (e.g., polymeric catalyst or solid-supported catalyst) can be reacted for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 16 hours, at least 24 hours, at least 36 hours, or at least 48 hours, or 1 to 24 hours, 2 to 12 hours, 3 to 6 hours, 1 to 96 hours, 12 to 72 hours, or 12 to 48 hours.

[0122] In some embodiments, the degree of polymerization of one or more oligosaccharides produced according to the methods described herein can be controlled by the reaction time. For example, in some embodiments, the degree of polymerization of one or more oligosaccharides is increased by increasing the reaction time, while in other embodiments, the degree of polymerization of one or more oligosaccharides is decreased by decreasing the reaction time.

[0123] Reaction Temperature In some embodiments, the reaction temperature is maintained in the range of about 25° C. to about 150° C. In particular embodiments, the temperature is about 30° C. to about 125° C., about 60° C. to about 120° C., about 80° C. to about 115° C., about 90° C. to about 110° C., about 95° C. to about 105° C., or about 100° C. to 110° C.

[0124] Amount of Glycan Units The amount of glycan units used in the methods described herein relative to the amount of solvent used can affect reaction rate and yield. The amount of glycan units used can be characterized by dry solids content. In certain embodiments, dry solids content refers to the total solids content of the slurry as a percentage of dry weight. In some embodiments, the dry solids content of glycan units is about 5% to about 95% by weight, about 10% to about 80% by weight, about 15% to about 75% by weight, or about 15% to about 50% by weight.

[0125] The amount of catalyst used in the methods described herein can vary depending on several factors, including, for example, the type of glycan unit selected, the concentration of the glycan unit, and the reaction conditions (e.g., temperature, time, and pH). In some embodiments, the weight ratio of catalyst to glycan unit is about 0.01 g / g to about 50 g / g, about 0.01 g / g to about 5 g / g, about 0.05 g / g to about 1.0 g / g, about 0.05 g / g to about 0.5 g / g, about 0.05 g / g to about 0.2 g / g, or about 0.1 g / g to about 0.2 g / g.

[0126] Solvent In certain embodiments, the method using the catalyst is carried out in an aqueous environment. One suitable aqueous solvent is water, which can be obtained from a variety of sources. Generally, water sources containing low concentrations of ionic species (e.g., sodium, phosphorus, ammonium, or magnesium salts) are preferred, as low concentrations of such ionic species can reduce the effectiveness of the catalyst. In some embodiments, where the aqueous solvent is water, the water has a resistivity of at least 0.1 megaohm-centimeters, at least 1 megaohm-centimeters, at least 2 megaohm-centimeters, at least 5 megaohm-centimeters, or at least 10 megaohm-centimeters.

[0127] Water content Furthermore, as the dehydration reaction of the present method progresses, water is produced with each coupling of one or more glycan units. In certain embodiments, the methods described herein may further comprise monitoring the amount of water present in the reaction mixture and / or the ratio of water to monomer or catalyst over a period of time. In some embodiments, the method further comprises removing at least a portion of the water produced in the reaction mixture (e.g., by removing at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100%, for example, by vacuum filtration). However, it should be understood that the amount of water relative to monomer can be adjusted based on the reaction conditions and specific catalyst used.

[0128] Any method known in the art can be used to remove water in the reaction mixture, for example, by vacuum filtration, vacuum distillation, heating, and / or evaporation. In some embodiments, the method includes including water in the reaction mixture.

[0129] In some embodiments, the present disclosure provides a method for producing an oligosaccharide composition by mixing a glycan unit having an acidic and ionic moiety and a catalyst to form a reaction mixture, producing water in the reaction mixture, and removing at least a portion of the water produced in the reaction mixture. In some variations, at least a portion of the water is removed to maintain a water content in the reaction mixture of less than 99% by weight, less than 90% by weight, less than 80% by weight, less than 70% by weight, less than 60% by weight, less than 50% by weight, less than 40% by weight, less than 30% by weight, less than 20% by weight, less than 10% by weight, less than 5% by weight, or less than 1% by weight.

[0130] In some embodiments, the degree of polymerization of one or more oligosaccharides produced according to the methods described herein can be adjusted by adjusting or controlling the concentration of water present in the reaction mixture. For example, in some embodiments, the degree of polymerization of one or more oligosaccharides is increased by decreasing the water concentration, while in other embodiments, the degree of polymerization of one or more oligosaccharides is decreased by increasing the water concentration. In some embodiments, the water content of the reaction is adjusted during the reaction to adjust the degree of polymerization of one or more oligosaccharides produced.

[0131] In one example, one or more monomers, dimers, trimers, or other oligosaccharides can be added to a round-bottom flask equipped with an overhead stirrer and a jacketed short-path condenser along with 1-50% (1-10%, 1-20%, 1-30%, 1-40%, 1-60%, 1-70%) of one or more of the catalysts described herein by dry weight. Water or another compatible solvent (0.1-5 equivalents, 1-5 equivalents, 1-4 equivalents, 0.1-4 equivalents) can be added to the dry mixture, and the slurry can be mixed at a low speed (e.g., 10-100 rpm, 50-200 rpm, 100-200 rpm) using a paddle sized to fit the contours of the selected round-bottom flask as closely as possible. The mixture is heated to 70-180°C (70-160°C, 75-165°C, 80-160°C) under a vacuum pressure of 10-1000 mbar. The reaction may be stirred for 30 minutes to 6 hours, with water constantly being removed from the reaction. Reaction progress may be monitored by HPLC. The solid mass resulting from the process may be dissolved in a volume of water sufficient to create a solution of approximately 50 Brix (grams of sugar per 100 g of solution). Once dissolution is complete, the solid catalyst may be removed by filtration, and the oligomer solution may be concentrated to approximately 50-75 Brix, for example, by rotary evaporation. Optionally, an organic solvent may be used; water-immiscible solvents may be removed by biphasic extraction, and water-miscible solvents may be removed, for example, by rotary evaporation simultaneously with the concentration step.

[0132] Reaction Conditions for Producing Glycan Polymers Using Glycosidase Enzyme Molecules Glycan polymers produced using the methods described herein can be produced by condensation (e.g., reverse hydrolysis) and / or transglycosylation of glycosidic bonds catalyzed by glycosidase enzyme molecules (e.g., hydrolases, transferases, or lyases). In some embodiments, the properties of glycan polymers produced according to the methods described herein can be adjusted by reaction conditions, such as reaction time, reaction temperature, concentration or amount of glycan subunits, concentration or amount of glycosidase enzyme molecules, solvent, or further processing steps, for example, as described herein. In some embodiments, the reaction conditions of the methods described herein reflect physiological conditions, e.g., a pH of 5-7.5 and a temperature of 35°C-60°C. In some embodiments, the reaction conditions of the methods described herein differ from physiological conditions.

[0133] Reaction Time: In some embodiments, the glycosidase enzyme molecule and the starting material (e.g., glycan subunit) are reacted for at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 16 hours, at least 24 hours, at least 36 hours, or at least 48 hours. In some embodiments, the glycosidase enzyme molecule and the starting material (e.g., glycan subunit) are reacted for 1 to 24 hours, 2 to 12 hours, 3 to 6 hours, 1 to 96 hours, 12 to 72 hours, or 12 to 48 hours. In some embodiments, the degree of polymerization (DP) of the glycan polymers produced according to the methods described herein can be controlled by the reaction time. For example, in some embodiments, the DP of the glycan polymers is increased by increasing the reaction time, while in other embodiments, the DP of the glycan polymers is decreased by decreasing the reaction time.

[0134] Reaction Temperature In some embodiments, the reaction temperature is maintained in the range of about 4°C to about 150°C. In certain embodiments, the temperature is about 4°C to about 30°C, about 4°C to about 125°C, about 30°C to about 125°C, about 60°C to about 120°C, about 80°C to about 115°C, about 90°C to about 110°C, about 95°C to about 105°C, or about 100°C to 110°C. In some embodiments, the reaction temperature is room temperature (e.g., about 25°C). In some embodiments, the reaction temperature is physiological temperature (e.g., about 30°C). In some embodiments, the reaction temperature is about 60°C.

[0135] In some embodiments, the reaction is slowed or substantially stopped after a period of time by increasing the temperature, for example through denaturation of the enzyme, hi some embodiments, the reaction is slowed or substantially stopped by increasing the temperature to above about 45°C, above about 50°C, above about 60°C, above about 70°C, above about 80°C, above about 90°C, above about 100°C, above about 110°C, or higher.

[0136] Concentration or amount of glycan subunit The concentration or amount of glycan subunit used in the methods described herein relative to the amount of solvent used can affect reaction rate and yield. In some embodiments, the concentration or amount of glycan subunit is about 10 mg / mL, about 25 mg / mL, about 50 mg / mL, about 75 mg / mL, about 100 mg / mL, about 200 mg / mL, 300 mg / mL, about 400 mg / mL, about 500 mg / mL, about 750 mg / mL, about 1 g / mL or more.

[0137] The amount of glycan subunits used can be characterized by their dry solids content. In certain embodiments, the dry solids content refers to the total solids content of the slurry as a percentage of dry weight. In some embodiments, the dry solids content of the glycan subunits is about 5% to about 95% by weight, about 10% to about 80% by weight, about 15% to about 75% by weight, or about 15% to about 50% by weight.

[0138] Concentration or Amount of Glycosidase Enzyme Molecules The concentration or amount of glycan enzyme molecules used in the methods described herein can vary depending on several factors, including, for example, the selection of the type of glycan subunit, the concentration of the glycan subunit, and the reaction conditions (e.g., temperature, time, and pH). In some embodiments, the concentration or amount of glycosidase enzyme molecules is about 0.1 U / mL, about 0.5 U / mL, about 1 U / mL, about 5 U / mL, about 10 U / mL, about 25 U / mL, about 50 U / mL, or more. In some embodiments, the concentration or amount of glycosidase enzyme molecules is 0.1-5 U / mL, 1-25 U / mL, or 1-50 U / mL. In some embodiments, the weight ratio of glycosidase enzyme molecules to glycan subunits is about 0.01 g / g to about 50 g / g, about 0.01 g / g to about 5 g / g, about 0.05 g / g to about 1.0 g / g, about 0.05 g / g to about 0.5 g / g, about 0.05 g / g to about 0.2 g / g, or about 0.1 g / g to about 0.2 g / g.

[0139] Solvent In some embodiments, the reaction solvent is a biocompatible solvent. In certain embodiments, the reaction solvent is an aqueous solvent, e.g., water or a water mixture. In some embodiments, the reactant solvent is water or a mixture of water and a miscible solvent such as acetone, ethanol, isopropanol, polyethylene glycol, t-butanol, or other solvent. In some embodiments, the reaction solvent is an organic solvent (e.g., a pure organic solvent).

[0140] Solvents may be added to the reaction mixture to increase the reaction rate or overall reaction yield, for example, by increasing the accessibility of the glycosidase enzyme molecule to the glycan subunits. Exemplary solvents include organic solvents such as DMSO and toluene.

[0141] Additional Reaction Components The reaction mixture may include additional components such as salts, detergents, metals, chelators, acids, bases, cofactors, coenzymes, vitamins, amino acids, prosthetic groups, nucleosides, nucleotides, or any combination thereof. In some embodiments, the reaction mixture may include NAD + , NADH, NADP + , NADPH, FAD, FADH, coenzyme A, biotin, pyridoxal phosphate, or methylcobalamin. In some embodiments, the inclusion of the additional component improves reaction yield, enzyme turnover rate, enzyme stability, glycan subunit stability, glycan polymer stability, or any combination thereof.

[0142] Glycosidase Enzymes Described herein are methods for preparing glycan polymers that are substrates for glycosidase enzymes, such as those present in human intestinal microorganisms. In their natural environment, such as those expressed by enterobacteria in the intestines of a subject, glycosidases use glycan polymers as substrates, for example, they recognize specific glycan polymers and hydrolyze the glycosidic bonds in the glycan polymer. This hydrolysis can result in the release of monomers or dimers from the glycan polymer, shortening the glycan polymer, and / or debranching (e.g., removing the glycosidic branch point of the glycan polymer). Glycosidase action provides microorganisms with glycan degradation products that can be converted into energy. This process is called glycan fermentation. Many glycosidases are specific, e.g., they have recognition motifs at the end (e.g., exoglycosidases) or within (e.g., endoglycosidases) of the glycan chain, they can recognize specific sugars or sugar combinations (e.g., glu-glu or glu-gal), and can be even more selective in stereochemistry and / or regiochemistry (e.g., recognizing alpha versus beta glycosidic linkages, and / or 1→2 versus 1→3 versus 1→6 linkages). Some glycosidase enzymes are more promiscuous, with a greater variety of glycan polymer substrates.

[0143] In an artificial environment and under appropriate conditions, glycosidase enzymes can produce glycan polymers, for example, through condensation and / or transglycosylation reactions. The produced glycan polymers can have a higher degree of polymerization than the input and can exhibit branching and steric and / or regiochemical diversity (with respect to alpha-beta glycosidic bonds and linkages). Exemplary glycosidase enzymes include hydrolases, transferases, or lyases. Glycosidase enzymes can be characterized in various ways, such as by their sequence, size, or function. In some embodiments, glycosidase enzymes are associated with bacteria from specific taxonomic groups. In some embodiments, glycosidase enzymes have CAZy family names (i.e., family names provided by the Carbohydrate Active enZYme database (http: / / www.cazy.org / )) based on analysis of genomic, structural, and biochemical information, such as the glycosyl hydrolase (GH) family or the glycosyltransferase (GT) family. In some embodiments, the glycosidase enzyme (e.g., a naturally occurring glycosidase enzyme, e.g., one expressed by an intestinal microorganism) is a glycosidase enzyme molecule (e.g., a glycosidase used in a method of making a glycan polymer described herein). In some embodiments, the glycosidase enzyme molecule is 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the glycosidase enzyme (e.g., by DNA sequence, RNA sequence, or amino acid sequence). In other embodiments, the glycosidase enzyme molecule contains deletions, additional sequence, point mutations, conservative or non-conservative amino acid changes, codon optimization, purification tags, folding / stability-enhancing mutations, etc. compared to the glycosidase enzyme. In some embodiments, the glycosidase enzyme is a member of the GH CAZY family. In some embodiments, the glycosidase enzyme is a member of the GT CAZY family.In some embodiments, the glycosidase enzyme molecule is related to (or derived from) a glycosidase enzyme having one or more sequence (e.g., DNA, RNA, or amino acid sequence) modifications, such as those described herein.

[0144] In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is present in a human intestinal microorganism. The glycosidase enzyme can be isolated from the microorganism. In some embodiments, the glycosidase is present in a microbial supernatant, a microbial extract, a microbial cell mass, or isolated to essential purity (e.g., an essentially pure enzyme fraction). In some embodiments, the glycosidase enzyme is sourced from a human intestinal microorganism. In some embodiments, the glycosidase enzyme is sourced from a yeast, fungus, or bacterium. In one embodiment, the glycosidase enzyme is sourced from a bacterium, such as a human intestinal bacterium. In some embodiments, the bacterial taxon is one of Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Spirochaetes, Synergistetes, Tenericutes, Proteobacteria, Verrucomicrobia, Euroarchaeota, e.g., a bacterial taxon listed in Table 2. In some embodiments, the human gut microbes are a species having the bacterial taxon Actinobacteria. In some embodiments, the human gut microbes are a species having the bacterial taxon Bacteroidetes. In some embodiments, the human gut microbes are a species having the bacterial taxon Firmicutes. In some embodiments, the human gut microbes are species having the bacterial taxon Fusobacteria. In some embodiments, the human gut microbes are species having the bacterial taxon Spirochaetes. In some embodiments, the human gut microbes are species having the bacterial taxon Synergistetes. In some embodiments, the human gut microbes are species having the bacterial taxon Tenericutes.In some embodiments, the human gut microbes are species having the bacterial taxon Proteobacteria. In some embodiments, the human gut microbes are species having the bacterial taxon Verrucomicrobia. In some embodiments, the human gut microbes are species having the bacterial taxon Euroarchaeota. In some embodiments, the human gut microbes are other than Bifidobacterium or Lactobacillus.

[0145] In some embodiments, glycan polymers (e.g., those produced by the methods described herein) are substrates for human gut microbial glycosidase enzymes from a particular CAZy family (e.g., the glycosyl hydrolase (GH) family or the glycosyltransferase (GT) family). In some embodiments, glycan polymers are substrates for human gut microbial glycosidase enzymes from a particular glycosyl hydrolase (GH) family (e.g., one of GH1-GH135) or glycosyltransferase (GT) family (e.g., one of GT1-GT101). In some embodiments, the glycan polymer preparation is selected to be a substrate for a human gut microbe with a particular glycosidase profile (e.g., that expresses (or possesses in its genome) one or more glycosidase genes, e.g., from one or more CAZy families). In some embodiments, glycosidase enzyme molecules are used in the methods described herein to produce glycan polymers that contain one or more functions of glycosidase enzymes present in a particular microorganism (or group of microorganisms). In some embodiments, the glycosidase enzyme molecule comprises the same function as a glucosidase enzyme (e.g., an enzyme present in intestinal microorganisms). In some embodiments, the glycosidase enzyme molecule has structural similarity or a degree of sequence similarity to the glycosidase enzyme. The glycosidase enzyme molecule can be produced by any method known in the art, for example, using standard cloning, genetics, protein expression, protein purification, or protein processing techniques.

[0146] Glycosidase enzyme molecules suitable for the methods of producing glycan polymers described herein can be selected based on their glycosidase enzyme counterparts present in the microorganism, and thus the glycan polymer or its preparation can be tailored to the glycosidase enzymes of the microorganism as a tailored substrate (glycosidase enzyme profile).

[0147] In some embodiments, the glycan polymer (e.g., produced by the methods described herein) is a substrate for a human gut microbial glycosidase enzyme selected from the GT5, GH94, GH13.9, GH13.39, GH13.36, GH113.0, and GH112 CAZy families. In some embodiments, the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from the GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13, GH13 subfamily 9, GH13 subfamily 31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77, and GH94 CAZy families. In some embodiments, the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, and GH13 subfamily 14 CAZy family. In some embodiments, the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28 In some embodiments, the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from the GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, and GH13 CAZy family. In some embodiments, the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from the GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, GH28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GT25, GH51, GH77, GH88, GH24 CAZy family.

[0148] In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is other than one of the GH1, GH2, GH3, GH4, GH5, GH8, GH9, GH10, GH11, GH12, GH13, GH14, GH16, GH26, GH28, GH30, GH31, GH32, GH35, GH42, GH43, GH44, GH50, GH51, GH57, GH62, GH63, GH68, GH70, GH97, GH100, GH116, GH119, or GH122 CAZy families.

[0149] In some embodiments, the methods described herein further comprise identifying in silico the glycosidase profile of a particular microorganism (e.g., of a CAZy family (e.g., the GT family or the GH family)). In some embodiments, identifying the glycosidase profile is performed according to the methods of Examples 11-15. For example, sequenced genomes from commensal bacterial species isolated from healthy human gut microbiomes, e.g., as part of the Human Microbiome Project, can be predicted for their ability to regulate metabolites, e.g., to produce butyrate, convert urea to ammonia via urease, or convert choline to TMA.

[0150] In some embodiments, the glycan polymer is a substrate for a glycosidase enzyme present in a microorganism (e.g., a human gut microorganism) that regulates (e.g., produces) the level of a microbial metabolite. Exemplary metabolites include formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, ascorbic acid, lactic acid, tryptophan, serotonin, indole, succinic acid, trimethylamine (TMA), TMAO (trimethylamine N-oxide), deoxycholic acid, ethylphenyl sulfate, acetylaldehyde, hydrogen peroxide, ammonia, bile acids, lipopolysaccharide (LPS), and / or butanedione. In some embodiments, the metabolite is butyric acid (e.g., butyrate), trimethylamine (TMA), or ammonia. In some embodiments, the metabolite is butyric acid (e.g., butyrate). In some embodiments, the metabolite is acetic acid (e.g., acetate). In some embodiments, the metabolite is propionic acid (e.g., propionate). In some embodiments, the metabolite is trimethylamine (TMA). In some embodiments, the metabolite is ammonia. In some embodiments, the metabolite is lipopolysaccharide (LPS). In some embodiments, the metabolite is a bile acid (e.g., a secondary bile acid). In some embodiments, a substantial increase or decrease in the metabolite can be detected. In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is other than alpha- or beta-galactosidase, alpha- or beta-glucosidase, alpha- or beta-xylosidase, alpha- or beta-mannosidase, or alpha- or beta-fructofuranosidase. In some embodiments, the glycosidase enzyme or glycosidase enzyme molecule is other than alpha- or beta-galactosidase.

[0151] Methods of Producing Glycosidase Enzyme Molecules Glycosidase enzyme molecules can be produced by expression in recombinant host cells, but can also be produced by other methods, such as in vitro transcription and translation and chemical synthesis.

[0152] For cellular expression, one or more nucleic acids (e.g., cDNA or genomic DNA) encoding a glycosidase enzyme molecule can be inserted into a replicable vector for cloning or expression. The vector can be, for example, a plasmid, cosmid, virus genome, phagemid, phage genome, or other autonomously replicating sequence. An appropriate coding nucleic acid sequence can be inserted into the vector by a variety of procedures. For example, an appropriate restriction endonuclease site can be engineered (e.g., using PCR). The coding nucleic acid sequence can then be inserted into the appropriate location using restriction digestion and ligation. Vector components generally include one or more origins of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0153] Glycosidase enzyme molecules may optionally be produced recombinantly by fusion to one or more other components, such as a signal sequence, an epitope or purification moiety, or a tag.

[0154] For bacterial expression, the glycosidase enzyme molecule can be produced with or without a signal sequence. For example, it can be produced intracellularly so that it accumulates in inclusion bodies or a soluble fraction. It can also be secreted, for example, by adding a prokaryotic signal sequence, such as an appropriate leader sequence. Exemplary bacterial host cells for expression include any transformable E. coli K-12 strain (E. coli BL21, C600, ATCC 23724; E. coli HB101 NRRLB-11371, ATCC-33694; E. coli MM294 ATCC-33625; E. coli W3110 ATCC-27325), B. subtilis, Pseudomonas, and other Bacilli. In some embodiments, the bacterial host cell is selected from a proteolytic taxon, for example, a taxon that expresses few or no endogenous glycosidase enzymes.

[0155] Glycosidase enzyme molecules can be expressed in yeast host cells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hansenula, or Pichia pastoris. For yeast expression, glycosidase enzyme molecules can also be produced intracellularly or by secretion using, for example, the yeast invertase leader or alpha factor leader (including Saccharomyces and Kluyveromyces forms), or the acid phosphatase leader, or the C. albicans glucoamylase leader (European Patent No. 362,179, published April 4, 1990).

[0156] Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The replication origin from the plasmid pBR322 is suitable for most Gram-negative bacteria, and the 2-plasmid origin is suitable for yeast.

[0157] Expression and cloning vectors typically contain a selection gene or marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency (such as the URA3 marker in Saccharomyces), or (c) supply critical nutrients unavailable from complex media, such as the gene encoding D-alanine racemase for Bacilli.

[0158] Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the glycosidase enzyme molecule to direct mRNA synthesis. Exemplary promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkaline phosphatase, tryptophan (trp) promoter systems (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)). Promoters for use in bacterial systems may also contain an appropriately positioned Shine-Dalgarno sequence. The T7 polymerase system can also be used to drive expression of nucleic acid coding sequences placed under the control of a T7 promoter.

[0159] Further methods, vectors, and host cells suitable for the synthesis of glycosidase enzyme molecules in recombinant cells are described in Molecular Cloning: A Laboratory Manual, Third Ed., Sambrook et al. (eds.), Cold Spring Harbor Press, (2001) (ISBN: 0879695773).

[0160] Once expressed intracellularly, glycosidase enzyme molecules can be recovered from the culture medium, inclusion bodies, or cell lysates. Cells can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents (e.g., detergents).

[0161] Glycosidase enzyme molecules can be purified from other cellular proteins or polypeptides that can be found in cell lysates or cell culture media.Various methods of protein purification can be used, and such methods are known in the art, for example, as described in Deutscher, Methods in Enzymology, 182 (1990); and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (2010) (ISBN: 1441928332). Exemplary purification procedures include fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica or on a cation exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration, e.g., using Sephadex G-75, Protein A Sepharose columns to remove contaminants such as IgG, affinity columns (e.g., metal chelate columns to bind epitope-tagged forms of the protein, and columns with various ligands to bind any purification moieties associated with the glycosidase enzyme). Purification methods can include the combination of two different ion exchange chromatography steps, e.g., cation exchange chromatography followed by anion exchange chromatography, or vice versa. Glycosidase enzyme molecules can be eluted from ion exchange resins by a variety of methods or processes, including salt and / or pH gradients. In some embodiments, glycosidase enzyme molecules contain purification moieties (such as epitope tags and affinity handles). Such moieties can be used for affinity chromatography and, optionally, can be removed by proteolytic cleavage.

[0162] Anionic or cationic substituents can be attached to the matrix to form anionic or cationic supports for chromatography. Anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Cellulose ion exchange resins such as DE23, DE32, DE52, CM-23, CM-32, and CM-52 are available from Whatman Ltd. (Maidstone, Kent, UK). SEPHADEX™ and other crosslinked ion exchangers are also known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX™, as well as DEAE-, Q-, CM-, and S-SEPHAROSE™ and SEPHAROSE™ Fast Flow are available from Pharmacia AB. DEAE and CM derivatized ethylene glycol-methacrylate copolymers, such as TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or M, are available from Toso Haas Co. (Philadelphia, PA, USA).

[0163] A cation exchange surface is an ion exchange surface that has covalently bound negatively charged ligands and therefore has free cations to exchange with cations in a solution that contacts the surface. Exemplary surfaces include cation exchange resins, such as those in which the covalently bound groups are carboxylate or sulfonate. Commercially available cation exchange resins include CMC-cellulose, SP-Sephadex™, and Fast S-Sepharose™ (Pharmacia).

[0164] Anion exchange surfaces are ion exchange surfaces having covalently attached positively charged groups, such as quaternary amino groups. Exemplary anion exchange surfaces are anion exchange resins such as DEAE cellulose, TMAE, QAE Sephadex™, and Fast Q Sepharose™ (Pharmacia).

[0165] An exemplary purification scheme for glycosidase enzyme molecules involves lysing Escherichia coli (E. coli) cells in a lysis buffer by depth filtration. The material is then subjected to cation exchange chromatography (CEX). The CEX eluate is then run on an anion exchange medium in an anion exchange chromatography (AEX) step. The AEX FT can be subjected to a polishing step. The material can then be processed by UF / DF to, for example, concentrate or desalt the material. UF / DF membranes can be selected based on their nominal molecular weight cut-off ("NMWCO") to retain proteins in the retentate while allowing low molecular weight substances, such as salts, to pass through to the filtrate. For example, any buffer solution or sterile water can be used during the final buffer exchange step, depending on the desired final pH and product conductivity.

[0166] Glycosidase enzyme molecules may contain one or more conservative sequence modifications. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a glycosidase enzyme can be replaced with other amino acid residues from the same side chain family, and the modified glycosidase enzyme molecules can be tested using the functional assays described herein.

[0167] Further Processing Steps Optionally, the preparation may undergo further processing steps. Further processing steps may include, for example, purification steps. Purification steps may include, for example, separation, dilution, concentration, filtration, desalting, or ion exchange, chromatographic separation, or decolorization, or any combination thereof.

[0168] In some embodiments, the methods described herein further comprise a decolorization step. The glycan polymers produced may be decolorized using any method known in the art, including, for example, treatment with absorbents, activated carbon, chromatography (e.g., ion exchange resins), hydrogenation, and / or filtration (e.g., microfiltration).

[0169] In certain embodiments, the produced glycan polymer is contacted with a color-absorbing material at a specific temperature, at a specific concentration, and / or for a specific period of time. In some embodiments, the mass of the color-absorbing species contacted with the glycan polymer is less than 50% of the mass of the glycan polymer, less than 35% of the mass of the glycan polymer, less than 20% of the mass of the glycan polymer, less than 10% of the mass of the glycan polymer, less than 5% of the mass of the glycan polymer, less than 2% of the mass of the glycan polymer, or less than 1% of the mass of the glycan polymer.

[0170] In some embodiments, the glycan polymer is contacted with the color-absorbing material. In certain embodiments, the glycan polymer is contacted with the color-absorbing material for less than 10 hours, less than 5 hours, less than 1 hour, or less than 30 minutes. In certain embodiments, the glycan polymer is contacted with the color-absorbing material for 1 hour.

[0171] In certain embodiments, the glycan polymer is contacted with the color absorbing material at a temperature of 20-100 degrees Celsius, 30-80 degrees Celsius, 40-80 degrees Celsius, or 40-65 degrees Celsius. In certain embodiments, the glycan polymer is contacted with the color absorbing material at a temperature of 50 degrees Celsius.

[0172] In certain embodiments, the color absorbing material is activated carbon. In one embodiment, the color absorbing material is powdered activated carbon. In other embodiments, the color absorbing material is an ion exchange resin. In one embodiment, the color absorbing material is a strong base cation exchange resin in the chloride form. In another embodiment, the color absorbing material is cross-linked polystyrene. In yet another embodiment, the color absorbing material is cross-linked polyacrylate. In certain embodiments, the color absorbing material is Amberlite FPA91, Amberlite FPA98, Dowex 22, Dowex Marathon MSA, or Dowex Optipore SD-2.

[0173] Ion Exchange / Demineralization (Demineralization) In some embodiments, the produced glycan polymers are contacted with a material to remove salts, minerals, and / or other ionic species. In certain embodiments, the glycan polymers are passed through a pair of anion / cation exchange columns. In one embodiment, the anion exchange column contains a weakly basic exchange resin in hydroxide form, and the cation exchange column contains a strongly acidic exchange resin in protonated form.

[0174] Separation and Concentration In some embodiments, the methods described herein further comprise isolating the glycan polymer produced. In certain variations, isolating the glycan polymer comprises separating at least a portion of the glycan polymer from at least a portion of the glycosidase enzyme molecules using any method known in the art, including, for example, centrifugation, filtration (e.g., vacuum filtration, membrane filtration), and gravity sedimentation. In some embodiments, isolating the glycan polymer comprises separating at least a portion of the glycan polymer from at least a portion of any unreacted sugars using any method known in the art, including, for example, filtration (e.g., membrane filtration), chromatography (e.g., chromatographic fractionation), differential solubility, and centrifugation (e.g., differential centrifugation).

[0175] In some embodiments, the methods described herein further comprise a concentration step. For example, in some embodiments, the isolated glycan polymers are subjected to evaporation (e.g., vacuum evaporation) to produce a concentrated glycan polymer preparation. In other embodiments, the isolated glycan polymers are subjected to a spray-drying process to produce an oligosaccharide powder. In certain embodiments, the isolated glycan polymers are subjected to both an evaporation step and a spray-drying step.

[0176] In some embodiments, the methods described herein further comprise a fractionation step. The prepared and purified glycan polymers can then be separated by molecular weight using any method known in the art, including, for example, high performance liquid chromatography, adsorption / desorption (e.g., low-pressure activated carbon chromatography), or filtration (e.g., ultrafiltration or diafiltration).

[0177] In certain embodiments, the produced glycan polymers are fractionated by adsorption onto a carbonaceous material, followed by desorption of the fraction by washing the material with a mixture of organic solvent in water at a concentration of 1%, 5%, 10%, 20%, 50%, or 100%. In one embodiment, the adsorbent material is activated carbon. In another embodiment, the adsorbent material is a mixture of activated carbon and a bulking agent such as diatomaceous earth or Celite 545 in a volume or weight ratio of 5%, 10%, 20%, 30%, 40%, or 50%.

[0178] In further embodiments, the produced glycan polymers are separated by passing them through a high performance liquid chromatography system. In certain variations, the produced glycan polymers are separated by ion affinity chromatography, hydrophilic interaction chromatography, or size exclusion chromatography, including gel permeation and gel filtration.

[0179] In other embodiments, low molecular weight substances are removed by filtration methods. In certain variations, low molecular weight substances may be removed by dialysis, ultrafiltration, diafiltration, or tangential flow filtration. In certain embodiments, filtration is performed in a static dialysis tubing device. In other embodiments, filtration is performed in a dynamic flow filtration system. In other embodiments, filtration is performed in a centrifugal force-driven filtration centrifuge.

[0180] Other processing steps may include any one of those described in the Examples herein. In some embodiments, yeast fermentation is used to remove unreacted components, such as sugar monomers or dimers, or reaction by-products such as sugar monomers.

[0181] Glycan Preparation Characteristics The glycans may have one or more of the characteristics and properties described in WO 2016 / 122889, WO 2016 / 172657, WO 2016 / 007778 and WO 2016 / 172658 (each of which is incorporated by reference in its entirety), as well as the characteristics and properties described herein.

[0182] The glycans produced by the methods described herein can comprise oligosaccharides. In some embodiments, the glycans comprise homooligosaccharides (or homoglycans), where all of the saccharides in the polymer All monosaccharides are of the same type.

[0183] In some embodiments, the glycan comprises a heterooligosaccharide (or heteroglycan), in which multiple types of monosaccharides are present in the polymer. In some embodiments, the glycan has one or more of the properties described herein. In some embodiments, the glycan preparation has one or more of the bulk properties described herein.

[0184] Degree of Polymerization (DP) In some embodiments, glycan polymer preparations, such as those produced using the methods described herein, are polydisperse and exhibit a range of degrees of polymerization.

[0185] Optionally, the preparation may be fractionated to represent, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or greater than 98% short (DP about 1-2), medium (DP about 3-10), long (DP about 11-18), or very long (DP >18) species. In one embodiment, a polydisperse fractionated glycan polymer preparation is provided that comprises at least 85%, 90%, or at least 95% medium length species having a DP of about 3-10. In one embodiment, a polydisperse fractionated glycan polymer preparation is provided that comprises at least 85%, 90%, or at least 95% long species having a DP of about 11-18. In one embodiment, a polydisperse fractionated glycan polymer preparation is provided that comprises at least 85%, 90%, or at least 95% very long species having a DP of about 18-30.

[0186] Optionally, the preparation may be fractionated to represent, for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or greater than 98% of the short (DP about 1-2) or medium (DP about 3-10) glycans in the preparation. Alternatively, or in addition to fractionation, the small DP fraction (e.g., monomers and dimers) is subjected to enzymatic fermentation, e.g., using yeast suitable for degrading these sugars. In one embodiment, a polydisperse fractionated glycan polymer preparation comprising at least 85%, 90%, or at least 95% glycans having a DP of about 3-10 is prepared using the methods described herein.

[0187] In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymers of the glycan preparation have a DP of at least DP3, DP4, DP5, DP6, or DP7. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymers of the glycan preparation have a DP of about DP3 to about DP10, about DP3 to about DP8, about DP3 to about DP6, about DP3 to about DP5, about DP3 to about DP4, about DP2 to about DP4, about DP2 to about DP5, about DP2 to about DP6, about DP2 to about DP8, or about DP2 to about DP10. In some embodiments, less than 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or less than 50% of the glycan polymers of the glycan preparation have a DP of DP2 or less.

[0188] In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of 2 to 25, 3 to 25, 4 to 25, 5 to 25, 6 to 25, 7 to 25, 8 to 25, 9 to 25, 10 to 25, 2 to 30, 3 to 30, 4 to 30, 5 to 30, 6 to 30, 7 to 30, 8 to 30, 9 to 30, or 10 to 30. In one embodiment, the glycan polymer preparation has a degree of polymerization (DP) of at least 3 and less than 30 glycan units.

[0189] In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of 5 or more and less than 30 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of 8 or more and less than 30 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of 10 or more and less than 30 glycan units. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of glycan units between 3, 4, 5, 6, 7, 8 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of glycan units between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of between 3, 4, 5, 6, 7, 8, 9, 10 and 20, 21, 22, 23, 24, 25, 26, 27, 28 glycan units.

[0190] The conversion yield for one or more glycan units (e.g., sugars) in the methods described herein can be determined by any suitable method known in the art, including, for example, high-performance liquid chromatography (HPLC). The average conversion yield can be determined by methods known to those skilled in the art, such as size exclusion, ionic affinity, hydrophilicity, or hydrophobicity chemistry. These methods generally rely on chromatographic separation of materials using an HPLC system equipped with appropriate column chemistry. Chromatographic separation of the starting material from the product then allows for direct comparison of the area under the curve of those materials, which can then be converted into the percent yield of conversion. Example 15 describes specific IAC and SEC approaches that can be used to determine conversion yield. In a preferred embodiment, the conversions referred to herein are determined by the SEC method of Example 15.

[0191] In some embodiments, the yield of conversion of one or more glycan subunits to a glycan polymer preparation having a DP greater than DP1 (DP>1) after combining the glycan subunits with a glycosidase enzyme molecule is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more (determined on a weight / weight basis as a percentage of the input glycan subunits). In some embodiments, the conversion yield of glycan polymers having a DP of at least DP2 to a glycan polymer preparation after combining one or more glycan subunits with a glycosidase enzyme molecule is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more (determined on a weight / weight basis as a percentage of the input glycan subunits).

[0192] In some embodiments, the yield of conversion of one or more glycan subunits to a glycan polymer preparation having a DP of at least DP3 after combining the glycan subunits with a glycosidase enzyme molecule is about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more (determined on a weight / weight basis as a percentage of the input glycan subunits).

[0193] In some embodiments, the conversion yield for a glycan polymer preparation having a DP greater than 1 after mixing one or more glycan units with the catalyst (e.g., 2, 3, 4, 8, 12, 24, or 48 hours after mixing one or more glycan units with the catalyst) is greater than about 50% (e.g., greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%). In some embodiments, the conversion yield for a glycan polymer preparation having a DP greater than 2 after mixing one or more glycan units with the catalyst (e.g., 2, 3, 4, 8, 12, 24, or 48 hours after mixing one or more glycan units with the catalyst) is greater than 30% (e.g., greater than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98%).

[0194] In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of at least 2. In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has a DP of at least 3.

[0195] Average DP In some embodiments, the glycan polymer preparation has an average degree of polymerization (average DP) of about DP2, DP3, DP4, DP5, DP6, DP7, DP8, or DP9. In some embodiments, the glycan polymer preparation has an average degree of polymerization (average DP) of about 2 to about 10, about 2 to about 8, about 2 to about 6, about 2 to about 4, about 3 to about 10, about 3 to about 8, about 3 to about 6, or about 3 to about 4.

[0196] In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymer preparation has an average degree of polymerization (average DP) of about DP5, DP6, DP7, DP8, DP9, DP10, DP11, or DP12. In some embodiments, the average DP of the glycan polymer preparation is about DP5-DP10, about DP6-DP10, about DP6-DP12, about DP6-DP14, about DP8-DP12, about DP8-DP14, about DP8-DP16, about DP10-DP16, about DP10-DP18, about DP4-DP18, about DP6-DP18, or about DP8-DP18.

[0197] The degree of polymerization (DP) distribution (or average) of a glycan polymer preparation can be determined by methods known to those skilled in the art, such as ion affinity chromatography (IAC) or size exclusion chromatography (SEC) measurement of molecular weight (MW) followed by mathematical conversion to an average DP. These methods generally rely on chromatographic separation of materials using an HPLC system equipped with a mass-sensitive column chemistry, such as a size exclusion column or ion affinity column, followed by computational conversion of the distribution to an average MW by comparison with a set of standards with known MW. Once the average molecular weight is determined, the average DP can be calculated by dividing this value by the average weight of the repeating unit of the glycan. Example 15 describes specific IAC and SEC approaches that can be used to determine the average DP as referred to herein. In a preferred embodiment, the average DP referred to herein is determined by the SEC method of Example 15.

[0198] In some embodiments, about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 97% of the glycan polymers of the preparation have an average molecular weight of about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 1000, 1050, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800 g / mol, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 200 and having an average molecular weight of less than 0, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, and 5000 g / mol.

[0199] The average molecular weight (MW) can be determined by methods known to those skilled in the art, such as ion affinity chromatography (IAC) or size exclusion chromatography (SEC). These methods generally rely on chromatographic separation of materials based on HPLC systems equipped with mass-sensitive column chemistry, such as size exclusion or ion affinity columns, followed by computational conversion of their distribution to an average MW by comparison with a set of standards with known MW. Example 15 describes specific IAC and SEC approaches that can be used to determine the average MW as referred to herein. In a preferred embodiment, the average MW referred to herein is determined by the SEC method of Example 15.

[0200] Degree of Branching (DB) In some embodiments, the glycan preparation varies in structure from linear to branched. Branched glycans can contain at least one glycan subunit linked via an alpha or beta glycosidic bond to form a branch. The branching rate or degree of branching (DB) can vary so that the glycan polymers of the preparation contain at least one, at least two, at least three, at least four, at least five, or at least about six branch points in the glycan polymer. In some embodiments, the glycan polymers of the glycan preparation are unbranched (DB=0).

[0201] In some embodiments, glycan preparations (e.g., oligosaccharides or polysaccharides) range from linear to highly branched structures. Unbranched glycans may contain only alpha or only beta linkages. Unbranched glycans may contain at least one alpha linkage and at least one beta linkage. Branched glycans may contain at least one glycan unit linked via an alpha or beta glycosidic bond to form a branch. The branching rate or degree of branching (DB) may vary, such that approximately every 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 15th, 20th, 25th, 30th, 35th, 40th, 45th, 50th, 60th, or 70th unit contains at least one branch point. For example, animal glycogen contains a branch point approximately every 10th unit.

[0202] In some embodiments, a preparation of glycan polymers is provided, the preparation comprising a mixture of branched glycans, the average degree of branching (DB, branch points per residue) is 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.99, 1, or 2. In some embodiments, a preparation of glycan polymers is provided, the average degree of branching is at least 0.01, 0.05, 0.1, 0.2, 0.3, or at least 0.4. In some embodiments, a preparation of glycan polymers is provided, the average degree of branching is about 0.01-0.1, 0.01-0.2, 0.01-0.3, 0.01-0.4, 0.01-0.5, 0.01-0.6, or about 0.01-0.7. In some embodiments, a preparation of glycan polymers is provided, the average degree of branching is about 0.05-0.1, 0.05-0.2, 0.05-0.3, 0.05-0.4, 0.05-0.5, 0.05-0.6, or about 0.05-0.7. In some embodiments, a preparation of glycan polymers is provided, the average degree of branching is not zero. In some embodiments, a preparation of glycan polymers is provided, the average degree of branching is not greater than 0.1 and less than 0.4, or not greater than 0.2 and less than 0.4. In some embodiments, the glycan polymer preparation comprises linear glycans. In some embodiments, the preparation of glycan polymers comprises glycans that exhibit branched or branch-on-branch structures.

[0203] In some embodiments, a preparation of glycan polymers is provided, wherein the average degree of branching (DB) is not 0, is at least 0.01, 0.05, 0.1, or at least 0.2, or is in the range of about 0.01 to about 0.2 or about 0.05 to 0.1.

[0204] The degree of branching (DB) of glycan polymer preparations can be determined by methods known to those skilled in the art, such as permethylation analysis. These methods generally rely on chemical functionalization of the free hydroxyl groups of glycans, followed by total acid hydrolysis and GC-MS analysis of the isolated monomers. Therefore, the fraction of monomers with multiple unfunctionalized hydroxyl groups can be interpreted as being equivalent to the fraction of polymer units that are linked to multiple other units, such as branched fractions. Example 15, as referred to herein, describes a specific permethylation approach that can be used to determine DB. In a preferred embodiment, the DB described herein is determined by permethylation according to Example 15.

[0205] Glycosidic Bonds and Glycosidic Linkages Linkages between individual glycan subunits found in preparations of glycan polymers can include alpha 1->2, alpha 1->3, alpha 1->4, alpha 1->5, alpha 1->6, alpha 2->1, alpha 2->3, alpha 2->4, alpha 2->6, beta 1->2, beta 1->3, beta 1->4, beta 1->5, beta 1->6, beta 2->1, beta 2->3, beta 2->4, and beta 2->6.

[0206] In some embodiments, the glycan polymer preparation comprises only alpha linkages. In some embodiments, the glycan polymer comprises only beta linkages. In some embodiments, the glycan polymer comprises a mixture of alpha and beta linkages.

[0207] In some embodiments, the ratio of alpha-glycosidic linkages to beta-glycosidic linkages in the preparation is about 1:1, 2:1, 3:1, 4:1, or 5:1. In some embodiments, the ratio of beta-glycosidic linkages to alpha-glycosidic linkages in the preparation is about 1:1, 2:1, 3:1, 4:1, or 5:1.

[0208] In some embodiments, the ratio of alpha-glycosidic bonds to beta-glycosidic bonds in the preparation is about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.2:1, 1.5:1, 1.7:1, 2:1, 2.2:1, 2.5:1, 2.7:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or about 10:1.

[0209] In some embodiments, the glycan polymers of the glycan polymer preparation contain both alpha and beta glycosidic linkages selected from the group consisting of 1->2 glycosidic linkages, 1->3 glycosidic linkages, 1->4 glycosidic linkages, 1->5 glycosidic linkages, and 1->6 glycosidic linkages. In some embodiments, the glycan polymer preparation contains at least two, or at least three, alpha and beta 1->2 glycosidic linkages, alpha and beta 1->3 glycosidic linkages, alpha and beta 1->4 glycosidic linkages, alpha and beta 1->5 glycosidic linkages, and / or alpha and beta 1->6 glycosidic linkages.

[0210] In some embodiments, the glycan polymers of the glycan preparation comprise substantially all glycan subunits of the alpha or beta configuration, optionally each comprising about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the other configurations.

[0211] In some embodiments, the preparation of glycan polymers comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, at least 99.9%, or even 100% glycans having alpha glycosidic linkages. In some embodiments, the preparation of glycan polymers comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, at least 99.9%, or even 100% glycans having beta-glycosidic linkages. In some embodiments, a preparation of glycan polymers is provided in which at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85% of the glycans having glycosidic linkages are alpha-glycosidic, and at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85% of the glycans having glycosidic linkages are beta-glycosidic, and the ratio of alpha-glycosidic and beta-glycosidic linkages does not exceed 100%.

[0212] In some embodiments, a preparation of glycan polymers is provided in which at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, at least 99.9%, or even 100% of the glycan glycosidic linkages are one or more of 1->2 glycosidic linkages, 1->3 glycosidic linkages, 1->4 glycosidic linkages, and 1->6 glycosidic linkages. In some embodiments, a preparation of glycan polymers is provided in which at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, at least 20%, or 25% of each of the glycan glycosidic linkages are 1->2, 1->3, 1->4, and 1->6 glycosidic linkages. Optionally, the glycan polymer preparation further comprises at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85% of glycan glycosidic linkages selected from the group consisting of alpha 2->1, alpha 2->3, alpha 2->4, alpha 2->6, beta 2->1, beta 2->3, beta 2->4, and beta 2->6 glycosidic linkages.

[0213] In some embodiments, the glycan polymers of the glycan preparation are alpha 1->2 and alpha 1->3, alpha 1->2 and alpha 1->4, alpha 1->2 and alpha 1->6, alpha 1->2 and beta 1->2, alpha 1->2 and beta 1->3, alpha 1->2 and beta 1->4, alpha 1->2 and beta 1->6, alpha 1->3 and alpha 1->4, alpha 1->3 and alpha 1->6, alpha 1->3 and beta 1->2, alpha 1->3 and beta 1->3, alpha 1->3 and beta 1->4, alpha 1->3 and beta 1->6, alpha 1->4 and alpha 1->6, alpha 1->4 and beta 1->2, alpha 1->4 and beta 1->3, alpha 1->4 and beta 1->6, alpha 1->4 and beta 1->2, alpha 1->4 and beta 1->3, alpha 1-> and at least two glycosidic bonds selected from the group consisting of: 4 and beta 1->4, alpha 1->4 and beta 1->6, alpha 1->6 and beta 1->2, alpha 1->6 and beta 1->3, alpha 1->6 and beta 1->4, alpha 1->6 and beta 1->6, beta 1->2 and beta 1->3, beta 1->2 and beta 1->4, beta 1->2 and beta 1->6, beta 1->3 and beta 1->4, beta 1->3 and beta 1->6, and beta 1->4 and beta 1->6.

[0214] The distribution of glycosidic bonds and linkages can be determined by methods known to those skilled in the art, such as two-dimensional nuclear magnetic resonance spectroscopy (2D NMR). These methods generally rely on quantification of the area under the curve (AUC) of peaks characteristic of a given bond type. Example 15, as referred to herein, describes a specific 2D NMR approach that can be used to determine glycosidic bonds and linkages. In a preferred embodiment, glycosidic bonds and linkages are determined using the 2D NMR method of Example 15.

[0215] L-type and D-type In some embodiments, a preparation of glycan polymers is provided in which at least one glycan subunit is an L-type sugar. In some embodiments, a preparation of glycans is provided in which at least one glycan subunit is a D-type sugar. In some embodiments, a preparation of glycans is provided in which the glycan subunits are naturally occurring or more common (e.g., D-glucose, D-xylose, L-arabinose) L-type or D-type sugars.

[0216] In some embodiments, the glycan polymer preparations (e.g., oligosaccharides and polysaccharides) contain a desired mixture of L- and D-type glycan subunits, for example, a desired ratio of L- to D-type or D- to L-type, such as 1:1, 1:2, 1:3, 1:4, 1:5, etc.

[0217] In some embodiments, the glycan polymer preparation comprises a desired mixture of L- and D-type glycan units, for example, a mixture of L-type to D-type or D-type to L-type in a desired ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:100, 1:150, etc.

[0218] In some embodiments, the glycan polymer preparation comprises glycans having substantially all L- or D-type glycan subunits, optionally containing about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of each of the other types.

[0219] Glycan Unit Content: In some embodiments, glycan polymer preparations are provided in which at least one glycan subunit is a tetrose, pentose, hexose, or heptose. Optionally, the glycan subunits involved in forming the glycans of the glycan polymer preparation are varied. Examples of monosaccharide glycan subunits include hexoses, such as glucose, galactose, and fructose, and pentoses, such as xylose. Monosaccharides generally have the chemical formula: Cx(HO)y, where x is typically ≥ 3. Monosaccharides can be classified by the number of carbon atoms they contain, x, for example, diose (2), triose (3), tetrose (4), pentose (5), hexose (6), and heptose (7). Monosaccharide glycan subunits can exist in acyclic (open-chain) forms. Open-chain monosaccharides with the same molecular graph can exist as two or more stereoisomers. Monosaccharides can also exist in cyclic forms via a nucleophilic addition reaction between a carbonyl group and one of the hydroxyls of the same molecule. The reactants form a ring of carbon atoms closed by a single bridging oxygen atom. In these cyclic forms, the ring usually has five atoms (furanose) or six atoms (pyranose).

[0220] In some embodiments, the glycan polymer preparation comprises a desired mixture of different monosaccharide glycan subunits, such as a mixture of diose (2), triose (3), tetrose (4), pentose (5), hexose (6), or heptose (7). In some embodiments, the glycan polymers of the glycan polymer preparation comprise a desired mixture of pentose (5) and hexose (6).

[0221] In some embodiments, the glycan polymer preparation comprises a desired mixture of two, three, four, or five different glycan subunits, for example, one or more glycan subunits selected from i) monosaccharides selected from glucose, galactose, arabinose, mannose, fructose, xylose, fucose, and rhamnose, ii) acarviosin, n-acetyllactosamine, allolactose, cellobiose, chitobiose, galactose-alpha-1,3-galactose, gentiobiose, isomalt, isomaltose, isomaltulose, kojibiose, lactitol, lactobionic acid, lactose ... ii) one or more glycan subunits selected from disaccharides selected from tulose, laminaribiose, maltitol, maltose, mannobiose, melibiose, melibiulose, neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose, sucralose, sucrose, sucrose acetate isobutyrate, octaacetylsucrose, trehalose, turanose, vicianose, and xylobiose; and iii) acarbose, N-acetylemannosamine, N-acetylmuramic acid, N-acetylneuraminic acid, N-acetyletalosaminuronic acid. acid), arabinopyranosyl-N-methyl-N-nitrosourea, D-fructose-L-histidine, N-glycolylneuraminic acid, ketosamine, kidamycin, mannosamine, 1B-methylseleno-N-acetyl-D-galactosamine, muramic acid, muramyl dipeptide, phosphoribosylamine, PUGNAc, sialyl-Lewis A, sialyl-LewisX), validamycin, voglibose, N-acetylgalactosamine, N-acetylglucosamine, aspartylglucosamine, bacillithiol, daunosamine, desosamine, fructosamine, galactosamine, glucosamine, meglumine, and perosamine; iv) one or more glycan subunits selected from amino sugars selected from 1-5-ahydroglucitol, cladinose, colitose, 2-deoxy-D-glucose, 3-deoxyglucasone, deoxyribose, dideoxy v) one or more glycan subunits selected from deoxysugars selected from nucleotides, digitalose, fludeoxyglucose, sarmentose, and sulfoquinovose; v) one or more glycan subunits selected from iminosugars selected from castanospermine, 1-deoxynojirimycin, miglitol, miglustat, and swainsonine, N-acetylneuraminic acid, N-acetyltalosamnuronic acidacid), aldaric acid, aldonic acid, 3-deoxy-D-manno-octo-2-urosonic acid, glucuronic acid, glucosamineuronic acid, glyceric acid, N-glycolylneuraminic acid, iduronic acid, isosaccharinic acid, pangamic acid, sialic acid, threonic acid, urosonic acid, uronic acid, xylonic acid, gluconic acid, ascorbic acid, ketodeoxyoctulosonic acid, galacturonic acid, galactosaminuronic acid, mannuronic acid, mannosaminuronic acid, tartaric acid, mucic acid, saccharic acid, lactic acid, oxalic acid, succinic acid, hexanoic acid, fumaric acid, maleic acid, butyric acid, citric acid, glucosamine acid, malic acid, succinamic acid, sebacic acid, and capric acid vi) one or more glycan subunits selected from a sugar acid selected from formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid; and vii) one or more glycan subunits selected from a sugar alcohol selected from methanol, ethylene glycol, glycerol, erythritol, threitol, arabitol, ribitol, xylitol, mannitol, sorbitol, galactitol, iditol, volemitol, fucitol, inositol, maltotriitol, maltotetriitol, and polyglycitol.

[0222] Exemplary glycans are described by a three-letter code representing the monomeric sugar components, with the number out of 100 reflecting the proportion of the material the monomer comprises. Thus, "glu100" refers to a glycan produced from an input of 100% D-glucose (glycan unit), and "glu50gal50" refers to a glycan produced from an input of 50% D-glucose and 50% D-galactose (glycan unit), or alternatively, from an input of lactose dimers (glycan unit). As used herein, xyl = D-xylose, ara = L-arabinose, gal = D-galactose, glu = D-glucose, rha = L-rhamnose, fuc = L-fucose, man = D-mannose, sor = D-sorbitol, gly = D-glycerol, and neu = NAc-neuraminic acid.

[0223] In some embodiments, the glycan polymer preparation comprises one glycan unit A selected from i) to vii) above, wherein the glycan unit A comprises 100% of the glycan unit input. For example, in some embodiments, the glycan polymer preparation is selected from homoglycans xyl100, rha100, ara100, gal100, glu100, and man100. In some embodiments, the glycan preparation is selected from homoglycans fuc100 and fru100.

[0224] In some embodiments, the glycan polymer preparation comprises a mixture of two glycan units A and B independently selected from i) to vii) above, wherein A and B can be selected from the same or different groups i) to vii), and A and B can be selected in any desired ratio (e.g., in the range of 1 to 99% A and 99 to 1% B, but not exceeding 100%).

[0225] For example, in some embodiments, the glycan polymer preparation comprises heteroglycans ara50gal50, ara50gal50, xyl75gal25, ara80xyl20, ara60xyl40, ara50xyl50, glu80man20, glu60man40, man80glu20, man60glu40, xyl75ara25, gal75xyl25, Man80gal20, gal75xyl25, Man66gal33, Man75gal25, glu80gal20, glu60gal40, glu40gal60, glu20gal80, gal8 0man20, gal60man40, gal40man60, glu80xyl20, glu60xyl40, glu40xyl60, glu20xyl80, glu80ara20, glu60ara40, glu40ara60, glu20ara80, gal80xyl20, ga l60xyl40, gal40xyl60, gal20xyl80, gal80ara20, gal60ara40, gal40ara60, gal20ara80, man80xyl20, man60xyl40, man40xyl60, man20xyl80, man80ara20, Selected from man60ara40, man40ara60, man20ara80, xyl80ara20, xyl60ara40, glu50gal50, and man62glu38.

[0226] In some embodiments, the glycan polymer preparation comprises a mixture of three glycan units A, B, and C independently selected from i) to vii) above, wherein A, B, and C can be selected from the same or different groups in i) to vii), and A, B, and C can be selected in any desired ratio (e.g., not exceeding 100%, 1 to 99% A, 1 to 99% B, and 1 to 99% C).

[0227] For example, in some embodiments, the glycan polymer preparation comprises heteroglycans xyl75glu12gal12, xyl33glu33gal33, xyl75glu12gal12, glu33gal33fuc33, glu33gal33nman33, glu33gal33xyl33, glu33gal33ara33, gal33man33xyl33, gal33man33ara33, man52glu29gal19, Glu33Man33Xyl33, Glu33Man33Ara33, Glu33Xyl33Ara33, Gal33Man33Xyl33, Gal33Man33Ara33, Gal33Xyl33Ara33, Man33Xy and Glu45Gal45Man10.

[0228] In some embodiments, the glycan polymer preparation comprises a mixture of four glycan units A, B, C, and D independently selected from i) to vii) above, wherein A, B, C, and D can be selected from the same or different groups in i) to vii), and A, B, C, and D can be selected in any desired ratio (e.g., not exceeding 100%, 1 to 99% A, 1 to 99% B, 1 to 99% C, and 1 to 99% D).

[0229] In some embodiments, the glycan polymer preparation comprises a mixture of five glycan units A, B, C, D, and E independently selected from i) to vii) above, wherein A, B, C, D, and E can be selected from the same or different groups in i) to vii), and A, B, C, D, and E can be selected in any desired ratio (e.g., not exceeding 100%, 1 to 99% A, 1 to 99% B, 1 to 99% C, 1 to 99% D, and 1 to 99% E).

[0230] In some embodiments, a preparation of glycan polymers is provided, wherein at least one glycan subunit is selected from the group consisting of glucose, galactose, arabinose, mannose, fructose, xylose, fucose, and rhamnose.

[0231] In some embodiments, the preparation of glycan polymers is selected from a group consisting of, for example, glucose and galactose, glucose and arabinose, glucose and mannose, glucose and fructose, glucose and xylose, glucose and fucose, glucose and rhamnose, galactose and arabinose, galactose and mannose, galactose and fructose, galactose and xylose, galactose and fucose, and galactose and rhamnose, arabinose and mannose, arabinose and fructose, arabinose and xylose, arabinose and fucose, and arabinose and rhamnose, mannose and fructose, mannose and xylose, mannose and fucose, and A desired mixture of two different monosaccharide glycan subunits, such as mixtures of mannose and rhamnose, fructose and xylose, fructose and fucose, and fructose and rhamnose, xylose and fucose, xylose and rhamnose, and fucose and rhamnose, can be prepared in a ratio of, for example, 1:1, 1:2, 1:3, 1:4, or 1:5 or in a mixture of two different monosaccharide glycan subunits. or in the inverse ratios thereof, or 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, or 1:100, or in the inverse ratios thereof.

[0232] In some embodiments, the glycan polymer preparation contains a desired mixture of three different monosaccharide glycan subunits, e.g., for a glucose-containing glycan preparation, glucose, galactose, and arabinose; glucose, galactose, and mannose; glucose, galactose, and fructose; glucose, galactose, and xylose; glucose, galactose, and fucose; glucose, galactose, and rhamnose; glucose, arabinose, and mannose; glucose, arabinose, and fructose; glucose, arabinose, and xylose; glucose, arabinose, and fucose; glucose, arabinose, and rhamnose; glucose, mannose, and fructose; glucose, mannose, and xylose; glucose, mannose, and fucose; glucose, mannose. Rhamnose; glucose, fructose, and xylose; glucose, fructose, and fucose; glucose, fructose, and rhamnose; mixtures of glucose, fucose, and rhamnose, etc., may be used in ratios of, for example, 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:1:2, 1:2:2, 1:3:2, 1:4:2, 1:1:3, 1:2:3, 1:3:3, 1:1:4, 1:2:4, 1:1:5, 1:2:5, etc., or in ratios of 1:1:1, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:9:1, 1:10:1, 1:12:1, 1: 14:1, 1:16:1, 1:18:1, 1:20:1, 1:1:2, 1:2:2, 1:3:2, 1:4:2, 1:5:2, 1:6:2, 1:7:2, 1:8:2, 1:9:2, 1:10:2, 1:1:3, 1:2:3, 1:3:3, 1:4:3, 1:5:3, 1:6:3, 1:7:3, 1:8:3, 1:9 :3, 1:10:3, 1:1:4, 1:2:4, 1:3:4, 1:4:4, 1:5:4, 1:6:4, 1:7:4, 1:8:4, 1:9:4, 1:10:4, 1:1:5, 1:2:5, 1:3:5, 1:4:5, 1:5:5, 1:6:5, 1:7:5, 1:8:5, 1:9:5, 1:10:5, etc.

[0233] In some embodiments, the glycan polymer preparation does not contain N-acetylgalactosamine or N-acetylglucosamine. In some embodiments, the glycan preparation does not contain sialic acid. In some embodiments, the glycan polymer preparation does not contain lipids or fatty acids. In some embodiments, the glycan polymer preparation does not contain amino acids.

[0234] Furanose:pyranose In some embodiments, a preparation of glycan polymers is provided in which at least one glycan subunit is a furanose sugar. In some embodiments, a preparation of glycans is provided in which at least one glycan subunit is a pyranose sugar. In some embodiments, the glycan polymer comprises a mixture of furanose and pyranose sugars. In some embodiments, the ratio of furanose sugars to pyranose sugars in the preparation is about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.2:1, 1.5:1, 1.7:1, 2:1, 2.2:1, 2.5:1, 2.7:1, 3:1, 4:1, 5:1, or about 6:1, or the ratio of furanose sugars to pyranose sugars in the preparation is about 7:1, 8:1, 9:1, or about 10:1.

[0235] In some embodiments, the glycan polymer preparation comprises substantially all furanose or pyranose sugars, optionally containing about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of each of the other sugars.

[0236] In some embodiments, the glycan polymer preparation comprises substantially all pyranose sugars, and the furanose-type glycan units in the preparation are less than about 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, or 5%. In some embodiments, less than 3%, 2%, or 1% of the monomeric glycan units in the preparation are furanose-type.

[0237] Salts In some embodiments, the glycan polymer preparation comprises one or more glycan subunits present in a salt form (e.g., a pharmaceutically acceptable salt form), such as, for example, hydrochloride, hydroiodide, hydrobromide, phosphate, sulfate, methanesulfate, acetate, formate, tartrate, malate, citrate, succinate, lactate, gluconate, pyruvate, fumarate, propionate, aspartate, glutamate, benzoate, etc.

[0238] Derivatization If necessary, the monosaccharide or oligosaccharide glycan subunits of the glycan can be further substituted or derivatized, for example, the hydroxyl group can be etherified or esterified. For example, the glycan (e.g., oligo- or polysaccharide) can contain modified saccharide units, such as 2'-deoxyribose, in which the hydroxyl group is removed, 2'-fluororibose, in which the hydroxyl group is replaced with fluorine, or N-acetylglucosamine, nitrogen-containing forms of glucose (e.g., 2'-fluororibose, deoxyribose, and hexose). The degree of substitution (DS, the average number of hydroxyl groups per glycosyl unit) can be 1, 2, or 3, or another suitable DS. In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the glycan subunits are substituted or derivatized. In some embodiments, the degree of substitution varies between subunits, for example, a certain percentage is not derivatized, exhibits a DS of 1, exhibits a DS of 2, and exhibits a DS of 3. Any desired mixture can be produced, for example, 0-99% of the subunits are underivatized, 0-99% of the subunits exhibit a DS of 1, 0-99% of the subunits exhibit a DS of 2, and 0-99% of the subunits exhibit a DS of 3, totaling 100%. The degree of substitution can be controlled by adjusting the average number of moles of substituents (molar substitution (MS)) added to the glycosyl moiety. The distribution of substituents along the length of the glycan oligosaccharide or polysaccharide chain can be controlled by adjusting the reaction conditions, reagent type, and extent of substitution. In some embodiments, the monomeric subunits are substituted with one or more of acetate ester, sulfate half ester, phosphate ester, or pyruvyl cyclic acetal groups.

[0239] Solubility In some embodiments, the glycan polysaccharide in the preparation The glycan polymer preparation is highly soluble. In some embodiments, the glycan polymer preparation can be concentrated to at least 55 Brix, 65 Brix, 60 Brix, 65 Brix, 70 Brix, 75 Brix, 80 Brix, or at least 85 Brix at 23 ° C. (final solubility limit) without significant solidification or crystallization. In some embodiments, the glycan polymer preparation can be concentrated to at least about 0.5 g / ml, 1 g / ml, 1.5 g / ml, 2 g / ml, 2.5 g / ml, 3 g / ml, 3.5 g / ml, or at least 4 g / ml at 23 ° C. (final solubility limit) without significant solidification or crystallization.

[0240] In some embodiments, the glycan polymer preparation (e.g., oligosaccharides) is branched, e.g., has an average DB of at least 0.01, 0.05, or 0.1, and has a final solubility limit in water at 23°C of at least about 70 Brix, 75 Brix, 80 Brix, or at least about 85 Brix, or at least about 1 g / ml, 2 g / ml, or at least about 3 g / ml.

[0241] In some embodiments, the preparation of glycan polymers has a concentration of at least 0.001 g / L, 0.005 g / L, 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 53 g / L, 54 g / L, 55 g / L, 56 g / L, 57 g / L, 58 g / L, 59 g / L, 60 g / L, 61 g / L, 62 g / L, 63 g / L, 64 g / 400g / L, 500g / L, 600g / L, 700g / L, 800g / L, 900g / L, and 1000g / L. In some embodiments, the glycan polymer preparation is more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or more than 99.5% soluble and has a solubility of 0.001 g / L, 0.005 g / L, 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, 26 g / L, 27 g / L, 28 g / L, 29 g / L, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, 51 g / L, 52 g / L, 5 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 900 g / L, 1000 g / L.

[0242] Sweetness In some embodiments, the glycan polymer preparation has a desired sweetness. For example, sucrose (table sugar) is the prototypical sweet substance. Sucrose in solution has a sweetness perception rating of 1, and other substances are rated relative to this (e.g., fructose is rated 1.7 times the sweetness of sucrose). In some embodiments, the sweetness of the glycan polymer preparation ranges from 0.1 to 500,000 relative to sucrose. In some embodiments, the relative sweetness is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 25000, 50000, 75000, 100000, 150000, 200000, 250000, 300000, 350000, 40000, 450000, 500000, or greater than 500,000 (sucrose is scored as 1). In some embodiments, the glycan polymer preparation is mildly sweet or both sweet and bitter.

[0243] In some embodiments, a preparation of glycan polymers, e.g., a preparation that is substantially DP2+ or DP3+ (e.g., at least 80%, 90%, or at least 95% DP2+ or DP3+, or a fractionated preparation), is substantially insensitive to sweetness, having a relative sweetness of about 0, 0.0001, 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or about 0.8 compared to sucrose (sucrose is scored as 1).

[0244] Fermentability In some embodiments, the glycan polymer preparations disclosed herein are screened to assess their fermentability. The fermentability of a glycan polymer is a function of the number or representation of hydrolyzable glycosidic bonds in the glycan species of the preparation. In some embodiments, fermentability is tested using a glycosidase enzyme or glycosidase enzyme molecule described herein. By using methods described herein, such as utilizing a glycosidase enzyme molecule, the glycan polymer produced is considered to be a substrate for a glycosidase enzyme (e.g., a glycosidase enzyme molecule) that is closely related to the glycosidase enzyme molecule (e.g., shares a high degree of sequence homology, is a derivative of the glycosidase enzyme, shares the same origin (e.g., microbial origin, shares the same glycosidic functional group, is a member of the glycoside hydrolase or glycoside transferase CAZy family, etc.) (e.g., that of human intestinal microorganisms).

[0245] In some embodiments, the fermentation rate of the glycan polymer preparation is 30 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, or 1 minute or less. In some embodiments, the digestibility of the glycan polymer preparation is 30 minutes or more, 45 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, or 10 hours or more, and 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 97%, 99% of the glycans of the preparation are fermented, e.g., degraded, such that the glycan polymers of the preparation exhibit a decrease in average DP (e.g., from DP = 5 to DP = 4) and / or an increase (or decrease) in small molecular weight fractions (e.g., monomers, dimers, trimers) by standard methods (e.g., size exclusion chromatography). In some embodiments, the glycan polymers of the glycan polymer preparation contain less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 30%, 40%, or less than 50% of the linkages that are hydrolyzable by mammalian enzymes (e.g., amylases).

[0246] Suitable assays can be used to assess comparative fermentability (eg, against a benchmark glycan) or to assess absolute digestibility.

[0247] Glycan polymer identification and analysis Glycan polymer preparations can be characterized as needed. For example, the monomer building blocks (e.g., monosaccharide or glycan subunit composition), anomeric configuration of side chains, the presence and position of substituents, degree of polymerization / molecular weight, and linkage pattern can be identified by standard methods known in the art, such as methylation analysis, reductive cleavage, hydrolysis, GC-MS (gas chromatography-mass spectrometry), MALDI-MS (matrix-assisted laser desorption / ionization mass spectrometry), ESI-MS (electrospray ionization mass spectrometry), HPLC (high-performance liquid chromatography with ultraviolet or refractive index detection), HPAEC-PAD (high-performance anion exchange chromatography with pulsed amperometric detection), CE (capillary electrophoresis), IR (infrared) / Raman spectroscopy, and NMR (nuclear magnetic resonance) spectroscopy. For polymers of crystalline consistency, the crystalline structure can be resolved, for example, using solid-state NMR, FT-IR (Fourier transform infrared spectroscopy), and WAXS (wide-angle X-ray scattering). DP, DP distribution, and polydispersity can be determined, for example, by viscosity measurements and SEC (SEC-HPLC, high-performance size-exclusion chromatography). Heterogeneous groups, terminal groups, and substituents can be determined, for example, using SEC with labeling, aqueous analysis, MALDI-MS, FT-IR, and NMR. To identify the monomer components of glycans, methods such as acid-catalyzed hydrolysis, HPLC (high-performance liquid chromatography), or GLC (gas-liquid chromatography) (after conversion to alditol acetates) can be used. To determine the linkages present in glycans, in one example, polysaccharides are methylated with methyl iodide and a strong base in DMSO, hydrolyzed, reduced to partially methylated alditols, acetylated to methylated alditol acetates, and analyzed by GLC / MS (gas-liquid chromatography plus mass spectrometry). In some embodiments, partial depolymerization using acids or enzymes is performed to determine the polysaccharide sequence and structure.The possible structures of the polysaccharide are compared to the structures of hydrolyzable oligomers to determine which of the possible structures can produce the oligomer. To identify the anomeric structure, in one example, intact polysaccharide or oligosaccharide preparations are subjected to enzymatic analysis, e.g., they are contacted with an enzyme that is specific for a particular type of bond, such as β-galactosidase or α-glucosidase, and NMR can be used to analyze the product.

[0248] For example, the distribution (or average) degree of polymerization (DP) of a glycan polymer preparation can be measured, for example, by injecting a sample having a concentration of 10-100 mg / mL into an Agilent 1260 BioPure HPLC (or similar) equipped with a 7.8 x 300 mm BioRad Aminex HPX-42A column (or similar) and an RI detector, as described, for example, in Gomez et al. (Purification, Characterization, and Prebiotic Properties of Pectic Oligosaccharides from Orange Peel Wastes, J Agric Food Chem, 2014, 62:9769). Alternatively, a sample containing the concentration can be injected onto a Dionex ICS5000 HPLC (or similar) equipped with a 4 x 250 mm Dionex CarboPac PA1 column (or similar) and a PAD detector, as described, for example, in Holck et al. (Feruloylated and nonferuloylated arabino-oligosaccharides from sugar beet pectin selectively stimulate the growth of bifidobacterium spp. in human fecal in vitro fermentations, Journal of Agricultural and Food Chemistry, 2011, 59(12), 6511-6519). Integration of the resulting spectrum relative to a standard solution of the oligomer allows the determination of the average DP.

[0249] The molecular weight distribution can be measured, for example, by MALDI mass spectrometry. The oligosaccharide concentration can be measured using a Mettler-Toledo sugar refractometer (or similar), adjusting the final value against a standardization curve to account for the difference in refractive index between monomers and oligomers. .

[0250] The distribution of glycosidic regiochemistry can be characterized by a variety of 2D-NMR techniques, including COSY, HMBC, HSQC, DEPT, and TOCSY analysis, using, for example, standard pulse sequences and a Bruker 500 MHz spectrometer. Peaks can be assigned by correlation with the spectra of naturally occurring polysaccharides with known regiochemistry.

[0251] The monomer composition of an oligomer can be measured, for example, by a complete hydrolysis method in which a known amount of oligomer is dissolved in a strong acid at elevated temperature and allowed sufficient time for total hydrolysis to occur. The concentrations of individual monomers can then be measured by HPLC or GC methods described herein and known in the art to achieve relative abundance measurements as described in Holck et al. Absolute amounts can be measured by spiking the HPLC sample with a known amount of active standard into a detector selected to prevent overlap with any of the critical signals.

[0252] The degree of branching in any given population can be measured, for example, by the methylation analysis method established by Hakomori (J. Biochem. (Tokyo), 1964, 55, 205). From these data, identification of potential repeating units can be established by combining data from total hydrolysis, average DP, and methylation analysis and comparing them to DEPT NMR spectra. Correlation of the anomeric carbon signal counts to these data indicates whether regular repeating units are necessary to satisfy the collected data, as demonstrated, for example, by Harding et al. (Carbohydr. Res. 2005, 340, 1107).

[0253] The molar percentage of species with a degree of polymerization (DP) of n (referred to herein as DP(n)) in a population is determined by high-performance liquid chromatography (HPLC) using, for example, an Agilent 1260 BioInert series instrument equipped with a refractive index (RI) detector and water as the mobile phase on a variety of columns familiar to those skilled in the art. The column is selected from chemistries that best isolate the species of interest, including, but not limited to, HILIC, metal coordination, and aqueous size-exclusion chromatography. The mole percent of DP(n) is determined by the formula: %DP(n) = 100 * AUC[DP(n)] / AUC[DP(total)], where AUC is defined as the area under the curve for the species of interest, determined by calibration to known standards. The mole percentage of glycosidic linkage isomers (% alpha and % beta) is determined by nuclear magnetic resonance (NMR) spectroscopy using a variety of 2D techniques familiar to those skilled in the art. Alpha and beta isomers are distinguished, for example, by their different shifts in the NMR spectrum, and the molar percentage is determined by the following formula: % of glycosidic linkages (glycosidic isomer n) = 100 * AUC [shift (isomer n)] / AUC [shift (isomer alpha + isomer beta)], where AUC is defined as the area under the concentration curve for a particular shift value known to represent the desired isomer n. The molar percentage of regiochemical isomers is determined in an analogous manner using the following formula: % of regioisomers (regioisomer n) = 100 * AUC [shift (regioisomer n)] / AUC [shift (total regioisomers)].

[0254] The relative proportions of monomeric sugars making up an oligomeric population are determined, for example, by the sum of acidic digestion of an oligomeric sample, followed by conversion to alditol acetates, followed by gas chromatographic (GC) analysis of the resulting monomer solution compared to GC of known standards. The molar percentage of monomer (n) (n can be any sugar) is determined by the formula: %(sugar n) = 100 * AUC[sugar n] / AUC[sum of all monomeric sugars].

[0255] In some embodiments, the solubility of a preparation of glycan polymers can be adjusted, for example, by selecting the charge, structure (eg, DP, degree of branching), and / or derivatization of the glycan units.

[0256] Preparations of glycan polymers consisting of certain sugar units uniformly linked in linear chains are usually water-insoluble at 23°C, even when the glycans have low molecular weights with a degree of polymerization (DP) of 20-30. The solubility of glycan polymers can be tuned by modifying the glycosidic linkages in the glycan, for example, by introducing (1->6) linkages. The extra degree of freedom afforded by rotation around the C-5 to C-6 bond results in higher entropy values ​​of solution. Homoglycans with two types of sugar linkages or heteroglycans consisting of two types of sugars are generally more soluble than homogeneous polymers. Ionization of linear homoglycans can add to their solubility (e.g., gel solubility). The viscosity of the solution often depends on the tertiary structure of the glycan.

[0257] Glycan Polymer Formulations and Dosages Also provided herein are methods for producing compositions (e.g., pharmaceutical compositions) containing glycan polymer preparations that meet one or more, two or more, three or more, or four or more of the preparation characteristics described herein (including criteria (i)-(v) above). Specifically, the methods include providing a glycan polymer preparation, obtaining values ​​for one or more, two or more, or three or more characteristics of the preparation, including, for example, i) degree of polymerization (DP), ii) average degree of branching (DB, branch points per residue), iii) ratio of alpha-glycosidic to beta-glycosidic linkages, iv) glycan subunit identity, and v) glycan subunit ratio, and producing a pharmaceutical composition containing the glycan polymer preparation if the desired or predetermined criteria for the preparation are met within a desired range of deviation.

[0258] Methods for formulating glycan polymer preparations into pharmaceutical compositions, medical foods, or dietary supplements are known in the art and may include one or more, two or more, three or more, or four or more of the following steps: (i) formulating the preparation into a drug product; (ii) packaging the preparation; (iii) labeling the packaged preparation; and (iv) selling or offering for sale the packaged and labeled preparation. Formulation of glycan polymer preparations into drug products is known in the art and may include one or more, two or more, three or more, or four or more of the following steps: (i) removing undesirable components from the preparation; (ii) reducing the volume of the preparation; (iii) sterilizing the preparation; (iv) mixing the preparation with a pharmaceutically acceptable excipient or carrier; (v) mixing the preparation with a second drug or pharmaceutical agent; (vi) formulating the preparation to a suitable consistency, such as an aqueous dilution, syrup, or solid; and (vii) formulating the preparation into a suitable dosage form, such as a tablet, pill, or capsule.

[0259] In some embodiments, the glycan polymer preparation undergoes further processing to produce either a glycan polymer syrup or a powder. For example, in one variation, the glycan polymer preparation is concentrated to form a syrup. Any suitable method known in the art for concentrating a solution, such as using a vacuum evaporator, can be used. In another variation, the glycan polymer preparation is spray-dried to form a powder. Any suitable method known in the art for spray-drying a solution to form a powder can be used.

[0260] Pharmaceutical compositions, medical foods, and dietary supplements comprising glycan polymer preparations are provided herein. Optionally, the pharmaceutical compositions, medical foods, and dietary supplements comprising the glycan polymer preparations further comprise a second agent, such as a prebiotic substance and / or probiotic bacteria. In some embodiments, the pharmaceutical compositions, medical foods, and dietary supplements comprising the glycan polymer preparations further comprise micronutrients. In some embodiments, the pharmaceutical compositions, medical foods, and dietary supplements comprising the glycan polymer preparations do not contain prebiotic substances. In some embodiments, the pharmaceutical compositions, medical foods, and dietary supplements comprising the glycan polymer preparations do not contain probiotic bacteria. Optionally, the pharmaceutical compositions, medical foods, and dietary supplements comprising the glycan polymer preparations further comprise one or more excipients or carriers, including diluents, binders, disintegrants, dispersants, lubricants, glidants, stabilizers, surfactants, flavoring agents, and coloring agents.

[0261] In some embodiments, pharmaceutical compositions, medical foods, and dietary supplements (and kits containing same) containing glycan polymer preparations contain one or more micronutrients. In some embodiments, the micronutrient is selected from the group consisting of trace elements, choline, vitamins, and polyphenols. In some embodiments, the micronutrient is a trace metal. Suitable trace elements for use as micronutrients include, but are not limited to, boron, cobalt, chromium, calcium, copper, fluoride, iodine, iron, magnesium, manganese, molybdenum, selenium, and zinc. In some embodiments, the micronutrient is a vitamin. In some embodiments, the micronutrient is a polyphenol.

[0262] Furthermore, if desired, pharmaceutical compositions, medical foods, and dietary supplements containing glycan polymer preparations may contain therapeutic agents, prebiotics, and / or probiotic bacteria. Alternatively or additionally, the therapeutic agents, prebiotics, and / or probiotic bacteria may be administered separately (e.g., before, simultaneously with, or after administration of the glycan polymer) rather than as part of the glycan polymer pharmaceutical composition, medical food, or dietary supplement (e.g., as a co-formulation). In some embodiments, pharmaceutical compositions or medical foods or dietary supplements containing glycan polymer preparations are administered in combination with a recommended or prescribed diet, such as a diet rich in probiotics and / or prebiotic-containing foods, as determined by a physician or other healthcare professional. The therapeutic agents, prebiotics, and / or probiotic bacteria may be administered to regulate the subject's intestinal microbiome. In some embodiments, the combined effect (e.g., in the number or intensity of microbial, genomic, or functional changes) is additive. In other embodiments, the combined effect (eg, in the number or intensity of microbial, genomic, or functional changes) is synergistic.

[0263] In some embodiments, pharmaceutical compositions, medical foods, and dietary supplements comprising the glycan polymer preparations described herein further comprise a prebiotic substance or preparation thereof.

[0264] In some embodiments, prebiotics can be administered to subjects receiving pharmaceutical compositions, medical foods, or dietary supplements containing the glycan polymer preparations described herein. Prebiotics are indigestible substances that, when consumed, can provide beneficial physiological effects to the host by selectively stimulating the growth or activity of a limited number of indigenous bacteria in the digestive tract (Gibson GR, Roberfroid M B. Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. J Nutr. 1995 June; 125(6):1401-12.). Prebiotics, such as dietary fiber or prebiotic oligosaccharides (e.g., crystalline cellulose, wheat bran, oat bran, corn fiber, soybean fiber, beet fiber, etc.), can also provide bacteria with fermentable amounts of carbohydrates, supporting their microbial population (e.g., Increasing the levels of Lactobacillus and Bifidobacteria in the gastrointestinal tract may promote the growth of probiotic and / or commensal bacteria in the intestine.

[0265] Prebiotics include, but are not limited to, various galactan and carbohydrate-based gums, such as psyllium, guar, carrageen, gellan, lactulose, and konjac. In some embodiments, prebiotics include galactooligosaccharides (GOS), lactulose, raffinose, stachyose, lactosucrose, fructo-oligosaccharides (FOS, e.g., oligofructose or oligofructans), inulin, isomalto-oligosaccharides, xylo-oligosaccharides (XOS), palatinose oligosaccharides, isomaltose oligosaccharides (IMOS), transgalactosylated oligosaccharides (e.g., transgalacto-oligosaccharides), transgalactosylated disaccharides, soybean oligosaccharides (e.g., soybean oligosaccharides), chitosan oligosaccharides (thioses), gentio-oligosaccharides, soybean and pectic oligosaccharides. The sugars and / or sugar-soluble materials may include one or more of the following: oligosaccharides, gluco-oligosaccharides, pectin oligosaccharides, palatinose polycondensates, difructose anhydride III, sorbitol, maltitol, lactitol, polyols, polydextrose, linear and branched dextrans, pullalan, hemicellulose, reduced palatinose, cellulose, beta-glucose, beta-galactose, beta-fructose, verbascose, galactinol, xylan, inulin, chitosan, beta-glucan, guar gum, gum arabic, pectin, high sodium alginate, and lambda carrageenan, or mixtures thereof.

[0266] Examples of suitable probiotics include Bacteroides, Blautia, Clostridium, Fusobacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Akkermansia, Faecalibacterium, and Roseburia. Examples of bacteria include, but are not limited to, organisms classified as members of the genera Enterococcus, Prevotella, Bifidobacterium, Lactobacillus, Bacillus, Enterococcus, Escherichia, Streptococcus, Saccharomyces, Streptomyces, and the family Christensenellaceae. Non-exclusive examples of probiotic bacteria that may be used in the methods and compositions described herein include L. acidophilus, L. crispatus, L. casei, L. rhamnosus, L. reuteri, L. fermentum, L. plantarum, L. sporogenes, and Lactobacillus species, such as L. lactis, L. animalis, L. bifidum, L. longum, L. adolescentis, and L. infantis, as well as Lactobacillus species, such as L. lactis, L. animalis, L. bifidum, L. longum, L. adolescentis, and L. infantis.Yeasts such as Saccharomyces boulardii are also suitable as probiotics for administration to the gastrointestinal tract, for example, via oral dosage forms or food. In some embodiments, the probiotic bacterial taxon is not a Bifidobacterium. In some embodiments, the probiotic bacterial taxon is not a Lactobacillus.

[0267] Bacteria beneficial for modulating the gastrointestinal microbiota can include, for example, bacteria that produce organic acids (lactic and acetic acid) or that produce cytotoxic or cytostatic agents (to inhibit pathogen growth), such as hydrogen peroxide (HO) and bacteriocins, which are small antimicrobial peptides that can kill closely related bacteria or exhibit broader spectrum activity (e.g., nisin).

[0268] Beneficial bacteria include Akkermansia, Anaerofilum, Bacteroides, Blautia, Bifidobacterium, Butyrivibrio, Clostridium, Coprococcus, Dialister, Dorea, Fusobacterium, Eubacterium, and Faecalis. one or more species of the genera Lactobacillus, Phascolarctobacterium, Peptococcus, Peptostreptococcus, Prevotella, Roseburia, Ruminococcus and Streptococcus, and / or Akkermansia munisifila The bacterial strains may include one or more of the following species: Clostridium municiphilia, Clostridium minuta, Clostridium coccoides, Clostridium leptum, Clostridium scindens, Dialister invisus, Eubacterium rectal, Eubacterium eligens, Faecalibacterium prausnitzii, Streptococcus salivarius, and Streptococcus thermophilus.In some embodiments, the probiotic or commensal bacteria comprise one or more of the bacteria listed in Table 2.

[0269] The prebiotic substances and probiotic strains that can be combined with the glycan polymers described herein to produce the compositions or kits can be isolated to any purity level by standard methods, and purification can be achieved by conventional means known to those skilled in the art, such as distillation, recrystallization, and chromatography. The cultivated bacteria used in the compositions can be separated from the culture broth by any method, including, but not limited to, centrifugation, filtration, or decantation. The cells separated from the fermentation broth can optionally be washed with water, saline (0.9% NaCl), or any suitable buffer. The resulting wet cell mass can be dried by any suitable method, such as lyophilization.

[0270] In some embodiments, the probiotic bacteria are freeze-dried vegetative cells. In some embodiments, a preparation of spores from spore-forming probiotic bacteria is used.

[0271] In one embodiment, the glycan polymer preparation further comprises a prebiotic and a probiotic. In one embodiment, the pharmaceutical composition comprises a probiotic with a partially attenuated viability (e.g., a mixture containing 10%, 20%, 30%, 40%, 50% or more non-viable bacteria), or a probiotic consisting solely of non-viable microorganisms. The composition may further comprise microbial membranes and / or cell walls isolated and purified from killed microorganisms. Optionally, probiotic organisms can be incorporated into the glycan polymer preparation as a culture in water or other liquid or semi-solid medium in which the probiotics are viable. In another technique, a lyophilized powder containing probiotic organisms can be incorporated into a particulate material or a liquid or semi-solid material by mixing or blending.

[0272] In some embodiments, the pharmaceutical compositions, medical foods, and dietary supplements comprising the glycan polymer preparation further comprise a second therapeutic agent or a preparation thereof. In some embodiments, the therapeutic agent comprises an antibiotic, an antifungal agent, an antiviral agent, or an anti-inflammatory agent (e.g., a cytokine, a hormone, etc.).

[0273] The glycan polymer preparations described herein, other therapeutically active agents, prebiotic substances, micronutrients, and probiotics can be mixed or blended into a single pharmaceutical composition, medical food, or dietary supplement. In other embodiments, they can be contained in separate containers (and / or in various suitable unit dosage forms), but can be packaged together in one or more kits. In some embodiments, the preparations or compositions are not packaged together or placed together. A physician can then administer the preparations or compositions together, for example, before, at the same time, or after each other. In some embodiments, the preparations or compositions act synergistically in regulating the microflora in a subject, for example, in the gastrointestinal tract.

[0274] In some embodiments, the glycan polymer composition comprises 0.1% to 100% of the glycan polymer preparation on a w / w, w / v, v / v, or molar % basis. In another embodiment, the glycan polymer composition is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% by w / w, w / v, v / v, or molar %. , 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% glycan polymer preparation. In one embodiment, the glycan polymer composition is about 1-90%, about 10-90%, about 20-90%, about 30-90%, about 40-90%, about 40-80%, about 40-70%, about 40-60%, about 40-50%, about 50-90%, about 50-80%, about 50-70%, about 50-60%, about 50-60%, about 50-70%, about 50-80%, about 50-60%, about 50-80%, about 50-9 ... %, about 60-90%, about 60-80%, about 60-70%, about 70-90%, about 70-80%, about 70-90%, about 70-80%, about 80-90%, about 90-96%, about 93-96%, about 93-95%, about 94-98%, about 93-99%, or about 90-100% glycan polymer preparation.

[0275] The composition containing the glycan polymer preparation may optionally contain one or more excipients or carriers. The glycan polymer composition may contain from about 1% to about 90% of one or more excipients or carriers on a w / w, w / v, v / v, or molar basis. For example, the glycan polymer composition may be about 1-90%, 1-75%, 1-60%, 1-55%, 1-50%, 1-45%, 1-40%, 1-25%, 1-15%, 1-10%, 10-90%, 10-75%, 10-60%, 10-55%, 10-50%, 10-45%, 10-40%, 10-25%, 10-15%, 15-90%, 15-75%, 15-60%, 15-55%, 15-50%, 15-45%, 15-40%, 15-25% w / w, w / v, v / v, or molar %. , 25-90%, 25-75%, 25-60%, 25-55%, 25-50%, 25-45%, 25-40%, 40-90%, 40-75%, 40-60%, 40-55%, 40-50%, 40-45%, 45-90%, 45-75%, 45-60%, 45-55%, 45-50%, 50-90%, 50-75%, 50-60%, 50-55%, 55-90%, 55-75%, 55-60%, 60-90%, 60-75%, 75-90% of one or more excipients or carriers.

[0276] Medical Foods Also provided herein are glycan polymer preparations formulated as medical foods. Any of the glycan polymer preparations described herein can be formulated as medical foods and pharmaceutical compositions containing the glycan polymer preparation.

[0277] Medical foods are defined in Section 5(b)(3) of the Orphan Drug Act (21 U.S.C. 360ee(b)(3)). Medical foods are formulated to be ingested (oral intake) or administered enterally (e.g., via nutritional supplementation / nasogastric tube) under medical supervision, e.g., by a physician. They are intended for the specific dietary management of a disease or condition, such as dysbiosis or GI tract disease. As used herein, medical foods do not include foods simply recommended by a physician as part of an overall diet to manage symptoms or reduce the risk of a disease or condition. Medical foods containing glycan polymer preparations are synthetic foods (e.g., formulated and / or processed products, such as those prescribed for partial or total nutritional support of patients via oral intake or enteral feeding tubes), and are not naturally occurring foods used in their natural state.

[0278] In some embodiments, the subject has limited or impaired ability to ingest, digest, absorb, or metabolize normal foods or specific nutrients. In other embodiments, the subject has other special medically determined nutritional needs and cannot achieve its nutritional management by changing its normal diet alone. A medical food containing a glycan polymer preparation is administered to a subject in need thereof under medical supervision (which may be ongoing), and the subject will usually receive instructions on the use of the medical food. A medical food may contain one or more food additives, color additives, GRAS excipients, and other drugs or substances suitable for medical foods. A medical food preparation may be a nutritionally complete or incomplete formula.

[0279] Dietary Supplements Any of the glycan polymer preparations described herein can be formulated as dietary supplements, for example, for use in the methods described herein. Dietary supplements are regulated by the Dietary Supplement Health and Education Act of 1994 (DSHEA). Dietary supplements are orally ingested products containing "dietary ingredients" intended to supplement the diet. In addition to the glycan polymer preparations described herein, the "dietary ingredients" in these products can include one or more of the following substances: vitamins, minerals, herbs or other plants, amino acids, and enzymes, organ tissues, glands, and metabolites. Dietary supplements can also be extracts or concentrates, and can be found in many forms, such as tablets, capsules, softgels, gel caps, liquids, or powders. They can also be in other forms, such as bars, but in that case, the information on the label must not represent the product as a conventional food or a separate item of diet. DSHEA requires that all dietary supplements be labeled as dietary supplements, not as general foods.

[0280] Any of the glycan polymer preparations described herein can be formulated as a food ingredient or food additive, for example, for use in the methods described herein. Food ingredients may be generally recognized as safe (GRAS) or require FDA approval. The glycan polymer preparation can be added to any desired food, such as beverages (e.g., fruit juice), dairy products (e.g., milk, yogurt, cheese), cereals (any grain product), bread, spreads, etc.

[0281] The glycan polymer preparations described herein can be formulated for any suitable dosage form, for example, nasal, oral, rectal, or intragastric administration. In some embodiments, the glycan polymer preparations described herein can be formulated for enteral administration. In some embodiments, the glycan polymer preparations described herein can be formulated for tube feeding (nasogastric tube, oral gastric tube, or gastric tube feeding). The dosage forms described herein can be prepared using methods known to those skilled in the art.

[0282] The dosage form may be, for example, a packet containing the glycan polymer preparation in the form of a liquid (wash / rinse), gel, cream, ointment, powder, tablet, pill, capsule, repository, disposable applicator, or medical device (e.g., syringe). Also provided are articles of manufacture, such as a container containing a unit dosage form of the glycan polymer preparation and a label containing instructions for use of the glycan polymer.

[0283] Orally usable composition forms include tablets, push-fit capsules made of gelatin, and soft, sealed capsules made of gelatin and a plasticizer (e.g., glycerol or sorbitol). Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropyl methylcellulose, inert diluents, preservatives, antioxidants, disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), or a lubricant, surfactant, or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets can optionally be coated or scored and can be formulated so as to provide slow or controlled release of the active ingredient therein. The tablets can optionally be provided with an enteric coating, to provide release in a part of the intestine (e.g., colon, lower intestinal tract). All formulations for oral administration can be in a dosage suitable for such administration. Push-fit capsules The capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound and / or other agents (e.g., prebiotics or probiotics) may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. Dragee cores are provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablets or dragee coatings to identify or characterize different combinations of active compound doses.

[0284] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with a water-soluble carrier, such as polyethylene glycol, or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0285] In one embodiment, the provided glycan polymer preparation comprises a softgel formulation. The softgel can comprise a gelatin-based shell surrounding a liquid fill. The shell can be made of gelatin, a plasticizer (e.g., glycerin and / or sorbitol), a modifier, water, a colorant, an antioxidant, or a flavoring. The shell can be made of starch or carrageenan. The outer layer can be enteric coated. In one embodiment, the softgel formulation can comprise a water- or oil-soluble fill solution or a suspension of the composition covered by a gelatin layer.

[0286] Oral solid formulations can include an enteric coating, which can control the location where the glycan polymer preparation is absorbed in the digestive system. For example, the enteric coating can be designed so that the glycan polymer preparation does not dissolve in the stomach, but rather moves to the small intestine and dissolves there. The enteric coating is stable at low pH (e.g., in the stomach) and can dissolve at higher pH (e.g., in the small intestine). Materials that can be used for enteric coating include, for example, alginic acid, cellulose acetate phthalate, plastic, wax, shellac, and fatty acids (e.g., stearic acid, palmitic acid).

[0287] The preparation for oral use can also be provided in liquid dosage form.Liquid preparation can be, for example, in the form of aqueous or oily suspension, solution, emulsion, syrup or elixir, or can be provided as a dry product that is reconstituted with water or other suitable vehicle before use.Such liquid preparation can contain suspending agent, for example, sorbitol, methylcellulose, glucose syrup, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fat; emulsifier, for example, lecithin, sorbitan monooleate, acacia gum; non-aqueous vehicle (can contain edible fat), for example, almond oil, oily ester (such as glycerin, propylene glycol or ethyl alcohol); preservative, for example, methyl p-hydroxybenzoate or propyl p-hydroxybenzoate or sorbic acid, and if necessary, conventional flavoring or coloring agent. In some embodiments, the liquid formulation may contain, for example, a drug in water solution and / or suspension form; and a vehicle containing polyethoxylated castor oil, alcohol, and / or polyoxyethylated sorbitan monooleate, with or without flavoring. Each dosage form may contain an effective amount of glycan polymer, and may optionally contain pharmaceutically inactive agents such as conventional excipients, vehicles, fillers, binders, disintegrants, pH adjusters, buffers, solvents, solubilizers, sweeteners, colorants, and any other inactive agents that can be included in pharmaceutical dosage forms for administration. Examples of such vehicles and excipients can be found in Remington's Pharmaceutical Sciences, 17th edition (1985).

[0288] The pharmaceutical compositions provided herein may be in unit-dosage or multi-dosage form. As used herein, unit-dosage form refers to a physically discrete unit suitable for administration to a human in need thereof. In one embodiment, the unit-dosage form is provided in a package. Each unit dose may contain a predetermined quantity of the active ingredient sufficient to produce the desired therapeutic effect, together with other pharmaceutical carriers or excipients. Examples of unit-dosage forms include, but are not limited to, ampoules, syringes, and individually packaged tablets and capsules. A unit-dosage form may be administered in fractions or multiples thereof. A multi-dosage form is a plurality of identical unit-dosage forms packaged in a single container and may be administered as separate unit-dosage forms. Examples of multi-dosage forms include, but are not limited to, vials, bottles of tablets or capsules, or bottles of pints or gallons. In another embodiment, the multi-dosage form contains different pharmaceutically active agents. For example, a multi-dose dosage form may be provided that includes a first dosage element containing a composition comprising a glycan polymer and a second dosage element containing a prebiotic, a therapeutic agent, and / or a probiotic (which may be in modified release form). In this example, a pair of dosage elements may constitute a single unit dosage. In one embodiment, a kit is provided that includes multiple unit dosages, each unit including a first dosage element containing a composition comprising a glycan polymer preparation and a second dosage element (which may be in modified release form) containing a probiotic, a medicinal agent, a prebiotic, or a combination thereof. In another embodiment, the kit further includes a set of instructions.

[0289] In some embodiments, the unit dosage form contains about 1 mg to about 100 g of a glycan polymer preparation (e.g., a glycan polymer described herein). For example, the unit dosage form may contain about 50 mg to about 50 g, about 500 mg to about 50 g, about 5 g to about 50 g, about 100 mg to about 100 g, about 1 g to about 100 g, about 10 g to about 100 g, about 1 g to about 10 g, about 1 g to about 20 g, about 1 g to about 30 g, about 1 g to about 40 g, about 1 g to about 50 g, about 1 g to about 60 g, about 1 g to about 70 g, about 1 g to about 80 g, about 1 g to about 90 g, about 1 g to about 100 g, about 1 g to about 150 g, or about 1 g to about 200 g of glycan polymer.

[0290] In other embodiments, the unit dosage form contains from about 0.001 mL to about 1000 mL of a glycan polymer (eg, a glycan polymer described herein). For example, the unit dosage form may contain about 0.001 mL to about 950 mL, about 0.005 mL to about 900 mL, about 0.01 mL to about 850 mL, about 0.05 mL to about 800 mL, about 0.075 mL to about 750 mL, about 0.1 mL to about 700 mL, about 0.25 mL to about 650 mL, about 0.5 mL to about 600 mL, about 0.75 mL to about 550 mL, about 1 mL to about 500 mL, about 2.5 mL to about 450 mL, about 5 mL to about 400 mL, about 7.5 mL to about 350 mL, about 10 mL to about 300 mL, about 12.5 mL to about 250 mL, about 15 mL to about 200 mL, about 17.5 mL to about 150 mL, about 20 mL to about 100 mL, or about 25 mL to about 75 mL of glycan polymer.

[0291] In certain embodiments, the unit dosage form contains about 0.001 mL to about 10 mL, about 0.005 mL to about 7.5 mL, about 0.01 mL to about 5 mL, about 0.05 mL to about 2.5 mL, about 0.1 mL to about 1 mL, about 0.25 mL to about 1 mL, or about 0.5 mL to about 1 mL of glycan polymer. In other embodiments, the unit dosage form contains about 0.01 mL to about 10 mL, about 0.025 mL to about 7.5 mL, about 0.05 mL to about 5 mL, or about 0.1 mL to about 2.5 mL of glycan polymer. In other embodiments, the unit dosage form contains about 0.1 mL to about 10 mL, about 0.25 mL to about 7.5 mL, about 0.5 mL to about 5 mL, about 0.5 mL to about 2.5 mL, or about 0.5 mL to about 1 mL of glycan polymer.

[0292] In some embodiments, the unit dosage form, e.g., a tablet, capsule (e.g., a hard capsule, a push-fit capsule, or a soft capsule), or softgel, has a length of about 0.1 inches to about 1.5 inches (e.g., about 0.5 inches and about 1 inch), or about 5 mm to about 50 mm (e.g., about 10 mm to about 25 mm). In some embodiments, the unit dosage form, e.g., a tablet, capsule (e.g., a hard capsule, a push-fit capsule, or a soft capsule), or softgel, has an outer diameter of about 0.05 inches to about 1 inch (e.g., about 0.1 inches to about 0.5 inches), or about 1 mm to about 25 mm (e.g., about 5 mm to about 10 mm).

[0293] Each unit dosage form of the glycan polymer can have a caloric value of about 0.01 kcal to about 1000 kcal. For example, the unit dosage form can have a caloric value of about 0.01 kcal to about 100 kcal, about 0.05 kcal to about 50 kcal, about 0.1 kcal to about 10 kcal, about 0.25 kcal to about 2.5 kcal, about 0.5 kcal to about 5 kcal, about 0.75 kcal to about 7.5 kcal, about 1 kcal to 10 kcal, about 5 kcal to about 50 kcal, or about 10 kcal to about 100 kcal. In certain embodiments, the unit dosage form of the glycan polymer has a caloric value of 10 kcal to about 500 kcal. In certain embodiments, the unit dosage form of the glycan polymer has a caloric value of 1 kcal to about 100 kcal. In certain embodiments, the unit dosage form of the glycan polymer has a caloric value of 0.1 kcal to about 10 kcal.

[0294] In still other embodiments, the unit dosage form may have a caloric value of about 0.001 kcal to about 10 kcal, about 0.005 kcal to about 10 kcal, about 0.01 kcal to about 10 kcal, about 0.025 kcal to about 25 kcal, about 0.05 kcal to about 50 kcal, about 0.075 kcal to about 75 kcal, about 0.1 kcal to 100 kcal, about 0.25 kcal to about 10 kcal, about 0.5 kcal to about 5 kcal, about 0.25 kcal to about 25 kcal, or about 0.1 kcal to about 1 kcal.

[0295] The unit dosage form of the glycan polymer can be formulated to dissolve in an aqueous solution (e.g., water, milk, juice, etc.) and orally administered as a drink, syrup, solution, or suspension. For example, the unit dosage form of the glycan polymer can include cubes, packets, lozenges, pills, tablets, capsules, candies, powders, elixirs, or concentrated syrups formulated to dissolve in an aqueous solution prior to oral administration. In other embodiments, the unit dosage form of the glycan polymer can include cubes, packets, lozenges, pills, tablets, capsules, candies, powders, elixirs, or concentrated syrups formulated to dissolve in vivo, for example, in the mouth, stomach, intestines, or colon of a subject (e.g., a human subject) upon oral administration.

[0296] In some embodiments, the glycan polymer preparation is administered enterally. This preferably includes oral administration or administration by oral or nasogastric tube (including nasojejunal tube, oral gastric tube or oral jejunal tube). In other embodiments, administration includes rectal administration (including enema, suppository, or colonoscopy).

[0297] The dosage forms described herein can be manufactured using methods known to those skilled in the art. For example, in tablet production, an effective amount of prebiotics can be uniformly dispersed in one or more excipients or additives, for example, by high-shear granulation, low-shear granulation, fluidized-bed granulation, or by blending for direct compression. Excipients and additives include diluents, binders, disintegrants, dispersants, lubricants, glidants, stabilizers, surfactants, anti-adherents, adsorbents, sweeteners, and colorants, or combinations thereof. Diluents, also known as fillers, can be used to increase the bulk of the tablet so that it provides a practical size for compression. Non-limiting examples of diluents include lactose, cellulose, microcrystalline cellulose, mannitol, dry starch, hydrolyzed starch, powdered sugar, talc, sodium chloride, silicon dioxide, titanium oxide, dicalcium phosphate dihydrate, calcium sulfate, calcium carbonate, alumina, and kaolin. Binders can impart cohesion to tablet formulations and can be used to help the tablet remain intact after compression. Non-limiting examples of suitable binders include starch (including corn starch and pregelatinized starch), gelatin, sugars (e.g., glucose, dextrose, sucrose, lactose, and sorbitol), cellulose, polyethylene glycol, alginic acid, dextrin, casein, methylcellulose, waxes, natural and synthetic gums, such as acacia, tragacanth, sodium alginate, arabic, xanthan gum, and synthetic polymers such as polymethacrylic acid, polyvinyl alcohol, hydroxypropyl cellulose, and polyvinylpyrrolidone. Lubricants can also facilitate tablet manufacture, non-limiting examples of which include magnesium stearate, calcium stearate, stearic acid, glyceryl behenate, and polyethylene glycol.Disintegrants can facilitate the disintegration of the tablet after administration, non-limiting examples of which include starch, alginic acid, cross-linked polymers such as, for example, cross-linked polyvinylpyrrolidone, e.g., cross-linked polyvinylpyrrolidone, croscarmellose sodium, potassium starch glycolate or sodium starch glycolate, clay, cellulose (e.g., carboxymethylcellulose (e.g., carboxymethylcellulose (CMC), CMC-Na, CMC-Ca)), starch, gums, etc. Non-limiting examples of suitable glidants include silicon dioxide, talc, etc. Stabilizers can inhibit or retard drug degradation reactions, including oxidative reactions. Surfactants can also be included and can be anionic, cationic, amphoteric, or nonionic. Exemplary sweeteners can include stevia extract, aspartame, sucrose, alitame, saccharin, etc. Optionally, the tablets may also contain non-toxic auxiliary substances such as pH buffering agents, preservatives, e.g., antioxidants, wetting agents, or emulsifying agents, solubilizing agents, coating agents, flavoring agents (e.g., mint, cherry, anise, peach, apricot, licorice, raspberry, vanilla), and the like.Additional excipients and additives include aluminum acetate, benzyl alcohol, butylparaben, butylated hydroxytoluene, calcium disodium EDTA, calcium hydrogen phosphate dihydrate, dibasic calcium phosphate, tribasic calcium phosphate, candelilla wax, carnauba wax, hydrogenated castor oil, cetylpyridine chloride, citric acid, colloidal silicon dioxide, copolyvidone, corn starch, cysteine ​​HCl, dimethicone, disodium hydrogen phosphate, sodium erythrosinate, ethylcellulose, gelatin, glycerin, glyceryl monooleate, glyceryl monostearate, glycine, HPMC phthalate (pthalate), hydroxypropyl cellulose, hydroxypropyl methylcellulose, hypromellose, red iron oxide or ferric oxide, yellow iron oxide, iron oxide or ferric oxide, magnesium carbonate, magnesium oxide, magnesium stearate, methionine, methacrylic acid copolymer, methylparaben, silicified microcrystalline cellulose, mineral oil, phosphoric acid, ordinary calcium phosphate, anhydrous calcium phosphate, polaxamer 407, polaxamer 188, ordinary polaxamer, polyethylene oxide, polyoxyethylene 140 stearate, polysorbate 80, potassium bicarbonate, sodium sorbate, potato starch, polysorbate. The following may be included: vidone, propylene glycol, propylene paraben, propyl paraben, retinyl palmitate, sodium saccharin, selenium, silica, silica gel, fumed silica, sodium benzoate, sodium carbonate, sodium citrate dihydrate, crossmellose sodium, sodium laurin sulfate, sodium metabisulfite, sodium propionate, sodium starch, sodium starch glycolate, sodium stearyl fumarate, sorbic acid, sorbitol, sorbitan monooleate, pregelatinized starch, succinic acid, triacetin, triethyl citrate, vegetable stearin, vitamin A, vitamin E, vitamin C, or combinations thereof. The amounts of these excipients and additives may be appropriately selected based on their relationship to the other components and the characteristics of the formulation and production method.

[0298] An immediate release formulation of an effective amount of a glycan polymer preparation may include one or more combinations of excipients that allow for rapid release of the pharmaceutically active agent (e.g., within 1 minute to 1 hour after administration). A controlled release formulation (also referred to as sustained release (SR), extended release (ER, XR, or XL), time-release or timed-release, controlled release (CR), or sustained release) refers to the release of the glycan polymer preparation from the dosage form at a specific, desired time point after the dosage form is administered to a subject (e.g., a human subject).

[0299] In one embodiment, a controlled-release dosage form begins its release and continues its release over an extended period of time. Release can begin almost immediately or can be sustained. Release can be constant, increase or decrease over time, be pulsed, continuous or intermittent, etc. In one embodiment, a controlled-release dosage form refers to the release of a drug from a composition or dosage form in which the drug is released according to a desired profile over an extended period of time. In one aspect, controlled release refers to the delayed release of a drug from a composition or dosage form in which the drug is released according to a desired profile in which release occurs after a period of time.

[0300] Pharmaceutical carriers or vehicles suitable for administering the compounds provided herein include any carrier known to those skilled in the art to be suitable for a particular mode of administration. In addition, the composition may contain one or more components that do not impair the desired action, or components that complement the desired action or have another action.

[0301] In a further embodiment, the dosage form may be an effervescent dosage form. Effervescent means that the dosage form generates gas when mixed with liquids, including water and saliva. Some effervescent agents (or effervescent couples) generate gas through a chemical reaction that occurs upon exposure of the effervescent disintegrant to water or saliva in the oral cavity. This reaction may be the result of a reaction between a soluble acid source and a source of alkaline monocarbonate or alkaline carbonate. The reaction of these two common compounds produces carbon dioxide gas upon contact with water or saliva. The effervescent couple (or individual acids and bases individually) can be coated with a solvent-protecting coating or an enteric coating to prevent premature reaction. Such couples can also be mixed with pre-lyophilized particles (e.g., glycan polymers). The acid source may be any that is safe for human consumption and generally includes food acids, acids, and hydrite antacids, such as citric acid, tartaric acid, amaric acid, humic acid, adipic acid, and succinic acid. Carbonate sources include dry solid carbonates and bicarbonates, such as sodium bicarbonate, sodium carbonate, potassium bicarbonate and potassium carbonate, magnesium carbonate, etc. Also included are reactants that evolve oxygen or other gases and are safe for human consumption. In one embodiment, citric acid and sodium bicarbonate are used.

[0302] In another aspect, the dosage form can be in the form of a candy (e.g., a matrix), such as a lollipop or lozenge. In one embodiment, an effective amount of glycan polymer is dispersed in the candy matrix. In one embodiment, the candy matrix contains one or more sugars (e.g., dextrose or sucrose). In another embodiment, the candy matrix is ​​a sugar-free matrix. The selection of a particular candy matrix can vary widely. Conventional sweeteners (e.g., sucrose), sugar alcohols suitable for use by diabetics (e.g., sorbitol or mannitol), or other sweeteners (e.g., those described herein) can be used. The candy matrix can be very soft and quickly dissolving, or hard and more slowly dissolving. Various forms will have advantages in different situations.

[0303] A candy mass composition containing an effective amount of glycan polymers can be orally administered to a subject in need thereof, including a human adult or child, such that the effective amount of glycan polymers is released into the subject's oral cavity as the candy mass dissolves and is swallowed.

[0304] The dosage forms described herein can also be in the form of pharmaceutical particles produced by a variety of methods, including, but not limited to, high-pressure homogenization, wet or dry ball milling, or small particle precipitation (e.g., nGimat's NanoSpray). Other methods useful for making suitable powder formulations are to prepare a solution of the active ingredient and excipients, followed by precipitation, filtration, and micronization, or by removal of the solution by lyophilization, followed by micronization of the powder to the desired particle size. In one embodiment, the pharmaceutical particles have a final size of 3-1000 microns, e.g., up to 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 microns. In another embodiment, the pharmaceutical particles have a final size of 10-500 microns. In another embodiment, the pharmaceutical particles have a final size of 50-600 microns. In another embodiment, the pharmaceutical particles have a final size of 100-800 microns.

[0305] In another aspect, the present disclosure provides a method for making a unit dosage form described herein, comprising providing a glycan polymer (e.g., a glycan polymer described herein); formulating the glycan polymer into a unit dosage form (e.g., a unit dosage form described herein); packaging the unit dosage form; labeling the packaged, packaged unit dosage form, and / or selling or offering for sale the packaged and labeled unit dosage form.

[0306] The unit dosage forms described herein can also be processed.In one embodiment, this processing includes one or more of the following: processing the dosage form into a pharmaceutical composition, for example, formulating and mixing with a second component, such as an excipient or buffer; dividing into smaller or larger aliquots; disposing of the dosage form into a container, for example, an airtight or liquid-tight container; packaging; integrating a label; shipping or transferring to a different location.In one embodiment, this processing includes one or more of the following: sorting, selecting, accepting or discarding, releasing or withholding, processing into a pharmaceutical composition, shipping or transferring to a different location, formulating, labeling, packaging, releasing into commerce, or selling or offering for sale, depending on whether a predetermined threshold is met.In some embodiments, the processed dosage form comprises the glycan polymer described herein.

[0307] In some embodiments, the processing includes one or more of: processing the dosage form into a pharmaceutical composition, e.g., formulating and mixing with a second component, e.g., an excipient or buffer; dividing into smaller or larger aliquots; processing into a container, e.g., an airtight or liquid-tight container; packaging; associating with a label; shipping or transferring to a different location. In one embodiment, the processing includes one or more of: sorting, selecting, accepting or discarding, releasing or withholding, processing into a pharmaceutical composition, shipping or transferring to a different location, formulating, labeling, packaging, releasing into commerce, or selling or offering for sale, and responding to a decision.

[0308] In another embodiment, an oral dosage form comprising a glycan polymer preparation is provided, wherein the oral dosage form is a syrup. The syrup may contain about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% solids. The syrup may contain about 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% liquid, such as water. The solid may comprise the glycan polymer preparation. The solid may be, for example, about 1-96%, 10-96%, 20-96%, 30-96%, 40-96%, 50-96%, 60-96%, 70-96%, 80-96%, or 90-96% glycan polymer preparation. In another embodiment, the glycan polymer preparation is formulated as a viscous fluid.

[0309] In one embodiment, the composition includes an effervescent component, a neutralizing component, or a water-insoluble dietary fiber. The effervescent component may be at least one member selected from the group consisting of sodium bicarbonate, sodium carbonate, and calcium carbonate. In one embodiment, the neutralizing component may be at least one member selected from the group consisting of citric acid, L-tartaric acid, fumaric acid, L-ascorbic acid, DL-malic acid, acetic acid, lactic acid, and anhydrous citric acid. In one embodiment, the water-insoluble dietary fiber may be at least one member selected from the group consisting of crystalline cellulose, wheat bran, oat bran, corn fiber, soybean fiber, and beet fiber. The formulation may include sucrose fatty acid esters, powdered sugar, fruit juice powder, and / or flavoring materials.

[0310] In some embodiments, the dosage form is formulated to release a pharmaceutical composition comprising a glycan polymer preparation in a specific region of the GI tract, such as the small intestine or large intestine. In some embodiments, the dosage form is formulated to release a pharmaceutical composition comprising a glycan polymer preparation in a specific region of the GI tract, such as the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and / or rectum.

[0311] In some embodiments, the dosage form for the glycan polymer preparation described herein is an enzyme-responsive delivery system. For example, trypsin-responsive polymers can be made using hydrogels cross-linked with peptides that are degraded by trypsin. Trypsin is active in the small intestine. Trypsin-responsive delivery systems can be used to target the delivery of glycan polymer preparations to the small intestine. In another example, an enzyme-digestible hydrogel made of poly(vinylpyrrolidone) cross-linked with albumin is degraded in the presence of pepsin.

[0312] In some embodiments, the dosage form for the glycan polymer preparation described herein is a delivery device that allows for long-term retention at a specific site in the GI tract. For example, a gastroretentive delivery system allows for long-term release of the glycan polymer preparation into the stomach. Gastroretentive delivery can be used for glycan polymer preparations that regulate the bacteria in the stomach or upper small intestine.

[0313] In some embodiments, the dosage form for the glycan polymer preparations described herein is a mucoadhesive delivery system that adheres to the mucosal surface of the stomach. They are typically made of polymers with multiple hydrogen-bonding groups, such as cross-linked polyacrylic acid, sodium carboxymethylcellulose, sodium alginate, carrageenan, Carbopol 934P, or chitosan. It is composed of hydroxylated polycarbophil.

[0314] In some embodiments, the dosage form for the glycan polymer preparations described herein is an expanding delivery system that rapidly increases in size in the stomach, slowing passage through the pylorus. Such systems include systems that expand within the stomach. For example, geometric shapes such as tetrahedrons, rings, and discs can be packed into gelatin capsules. The shape expands as the capsule dissolves. This system can be composed of one or more erodible polymers (e.g., hydroxypropyl cellulose) and one or more non-erodible polymers (e.g., polyolefins, polyamides, polyurethanes). The glycan polymer can then be dispersed within the polymer matrix. Retention time can be fine-tuned by blending polymers. Alternatively, devices made from elastic polymers that are stable at the acidic pH of the stomach but dissolve in the neutral / alkaline conditions along the GI tract can be used. Such polymer formulations can prevent ileus when the device exits the stomach. Supramolecular polymer gels crosslinked by hydrogen bonds between carboxyl groups, such as poly(acryloyl 6-aminocaproic acid) (PA6ACA) and poly(methacrylic acid-co-ethyl acrylate) (EUDRAGIT L 100-55), can also be used. Other systems include swellable excipients such as collagen sponges. For example, hydrogel matrices (e.g., swellable cores: polyvinylpyrrolidone XL, Carbopol 934P, calcium carbonate) swell 2–50 times in the stomach. Superporous hydrogel composites swell to hundreds of times their original volume within minutes. Some systems, such as carbon dioxide-generating inflatable systems surrounded by a hydrophilic membrane, utilize gas generation to achieve expansion.

[0315] In some embodiments, the dosage form for the glycan polymer preparations described herein is a density-controlled delivery system. These systems are designed to float or sink in gastric fluid, which delays their discharge from the stomach. For example, high-density systems cause the device to sink below the pylorus, at the fundus of the stomach, thereby preventing gastric emptying. Other systems are low-density / floating systems. Such devices may contain air trapped in a hollow chamber or low-density materials such as fats, oils, or foam powders. Low density can be achieved, for example, by swelling; for example, hydrocolloid-containing capsules dissolve upon contact with gastric fluid, causing the hydrocolloid to swell and form a viscous liquid. Other polymers include chitosan, sodium alginate, and glycerol monooleate matrices. Low density can also be achieved by gas generation. For example, tablets loaded with carbonate and, optionally, citric acid generate carbon dioxide upon contact with an acidic aqueous medium. The generated carbon dioxide is trapped within the gelling hydrocolloid, causing the system to float. Hydrocolloids include hydroxypropyl methylcellulose and Carbopol 934P.

[0316] In some embodiments, the dosage forms for the glycan polymer preparations described herein are designed to retain the device in the small or large intestine. The location specificity of the device is provided by specific triggering methods, such as pH or enzymes. These include systems designed for mucoadhesion, as well as microneedle pills. Microneedle pills contain a drug reservoir encapsulated in a pH-responsive coating with microneedles. When the pill reaches the desired location in the GI tract and the coating dissolves, the microneedles adhere the pill to the GI tract lining. In other embodiments, the microneedle pill contains a capsule consisting of two chemical compartments, one filled with citric acid and the other with sodium bicarbonate. As the pill dissolves in the digestive system, the barrier between the two substances erodes, causing them to mix and initiate a chemical reaction that pushes the sugar microneedles through the outer layer of the capsule and into the lining of the small intestine. The sugar needles can be filled with a drug that is delivered to nearby blood vessels as the sugar is absorbed.

[0317] In some embodiments, the dosage form for the glycan polymer preparation described herein uses a pH-sensitive polymer coating. For example, a pH-dependent polymer (biphasic or triphasic) may be insoluble at low pH levels (e.g., acid-resistant in the stomach, pH 1-2) and become more soluble as the pH increases, for example, to about 5.5-6.2 in the duodenum, about 5.7 in the ascending colon, about 6.4 in the cecum, about 6.6 in the transverse colon, about 7.0 in the descending colon, about 7.2-7.5 in the ileum, or about 7.5 in the distal small intestine. In one example, TARGIT™ technology can be used for site-specific delivery of glycan polymer preparations in the gastrointestinal (GI) tract. This system uses a pH-sensitive coating on an injection-molded starch capsule to target the terminal ileum and colon.

[0318] In some embodiments, the dosage form for the glycan polymer preparations described herein is a delayed-release or time-controlled release system. Such systems typically use an enteric coating that can be combined with pH sensitivity and time-release functions. For example, an ETP (enteric-coated time-release press-coated) tablet can be used, which consists of three components: a glycan polymer-containing core tablet (rapid-release function), a press-coated swellable hydrophobic polymer layer (e.g., a hydroxypropyl cellulose (HPC) layer), and a time-release function. The duration of the lag phase can be controlled by the weight or composition of the polymer layer and the enteric coating layer (acid-resistant function).

[0319] In some embodiments, the dosage forms for the glycan polymer preparations described herein use Eudragit® enteric coatings for tablets and capsules. Other suitable synthetic polymers include shellac, ethyl cellulose, cellulose acetate phthalate, hydroxypropyl methylcellulose, polyvinyl acetate phthalate, and polyglutamic acid coatings such as poly-γ-glutamic acid (γ-PGA). These coatings combine both mucoadhesive and pH-dependent release strategies. To enhance colon-targeted delivery, Eudragits® are methacrylic copolymers with various side group compositions that change the pH at which they are soluble. For example, the Eudragit® coating system does not result in significant drug release in the stomach (e.g., pH 1.4) and small intestine (e.g., pH 6.3), while significant drug release is observed in the ileocecal junction at pH 7.8.

[0320] In some embodiments, the dosage form for the glycan polymer preparations described herein is a microbially triggered system, such as a polysaccharide-based delivery system. Polysaccharide-based delivery systems include biodegradable, mucoadhesive polymer coatings, such as coatings composed of chitosan and pectin. Other suitable natural polymers include, for example, guar gum, inulin, cyclodextrin, dextran, amylase, chondroitin sulfate, and locust bean gum. These delivery systems can be used to target the delivery of glycan polymer preparations to the small intestine. Coatings made from naturally occurring polysaccharides, such as guar gum, xanthan gum, chitosan, and alginate, are degraded by enzymes in the colonic gut microbiota, such as xylosidase, arabinosidase, and galactosidase. For example, CODES™ technology can be used to deliver glycan polymer preparations. This system combines a polysaccharide coating with a pH-sensitive coating. In some embodiments, this system consists of a core tablet coated with the following three layers of polymer coating: an outer coating composed of Eudragit L. This coating dissolves in the duodenum, exposing the next coating. This next coating is composed of Eudragit E. This layer allows the release of lactulose present in the inner core. Lactulose is metabolized to short-chain fatty acids, which lower the surrounding pH at which the Eudragit E layer dissolves. The dissolution of Eudragit E exposes glycan polymers. Bacteria present in the colon are responsible for the degradation of polysaccharides, which are released from the core tablet. The degradation of polysaccharides can result in the formation of organic acids, which lower the pH of the contents surrounding the tablet.

[0321] In some embodiments, the dosage form for the glycan polymer preparation described herein is a pressure-controlled delivery system. This system takes advantage of the fact that the colon experiences higher pressure than the small intestine. For example, in a water-insoluble ethylcellulose system, the pressure in the lumen of the colon results in the disintegration of the water-insoluble polymer capsule, followed by the release of the glycan polymer. The release profile can be adjusted by changing the thickness of the ethylcellulose, the size of the capsule, and / or the density of the capsule.

[0322] In some embodiments, the dosage form for the glycan polymer preparation described herein is a pulsatile colon-targeted delivery system. For example, this system can be a pulsincap system. The capsule used contains a plug placed within the capsule that controls the release of the glycan polymer. A swellable hydrogel (e.g., hydroxypropylmethylcellulose (HPMC), polymethylmethacrylate, or polyvinyl acetate) seals the drug contents. When the capsule comes into contact with fluid, the plug is extruded from the capsule, releasing the glycan polymer. The release profile can be controlled by varying the length and / or intersection point of the plug with the capsule body. Another system is a port system. The capsule body is surrounded by a semipermeable membrane. The insoluble plug consists of an osmotically active agent and a glycan polymer. When the capsule comes into contact with fluid, the semipermeable membrane allows the fluid to enter, increasing pressure within the capsule body. This extrudes the plug and releases the glycan polymer.

[0323] In some embodiments, the dosage form for the glycan polymer preparations described herein is an osmotically controlled colon-targeted delivery system. An exemplary system, OROS-CT, consists of an osmotic unit (up to 5 or 6 push-pull units) encapsulated in a hard gelatin capsule. The push-pull unit is bilayered with an outer intestinal-impermeable membrane and an inner semipermeable membrane. The inner central portion of the push-pull unit consists of a drug layer and a push layer. The glycan polymer is released through the semipermeable membrane. The capsule body surrounding the push-pull unit dissolves immediately after administration. In the GI tract, the intestinal-impermeable membrane prevents water absorption. The enteric coating dissolves in the small intestine (higher pH, >7), allowing water to enter the unit through the semipermeable membrane, swelling the push layer and forcing the glycan polymer out.

[0324] In some embodiments, the dosage form for the glycan polymer preparations described herein is a "smart pill" that can be used to release the glycan polymer just before it reaches the ileocecal valve.

[0325] In some embodiments, the dosage form for the glycan polymer preparation described herein is a rectal administration formulation. For example, an enema introduces a liquid formulation of the glycan polymer preparation into the rectum. The administered volume is usually less than 10 mL. A suppository introduces the glycan polymer preparation into the rectum. A suppository is a solid dosage form that melts or dissolves when inserted into the rectum and releases the glycan polymer. Common excipients for suppository formulations include cocoa butter, polyethylene glycol, and agar.

[0326] Kits are also contemplated. For example, a kit may include a glycan polymer preparation in unit dosage form and a method for treating a gastrointestinal disorder or condition. The kit may include a package insert containing instructions for use of the glycan polymer in the treatment of a disease state. The kit includes the glycan polymer preparation in suitable packaging for use by a subject (e.g., a human subject) in need thereof. Any of the compositions described herein can be packaged in the form of a kit. The kit may contain a sufficient amount of glycan polymer preparation (optionally, further containing prebiotics, probiotic bacteria, and / or a second therapeutic agent) for a full course of treatment or a portion of a course of treatment. Doses of the glycan polymer preparation may be individually packaged, or the glycan polymer preparation may be provided in bulk, or a combination thereof. Thus, in one embodiment, the kit provides individual doses of the glycan polymer preparation in suitable packaging corresponding to administration points in a treatment regimen, with the doses packaged in one or more packets.

[0327] In one embodiment, the glycan polymer preparation can be provided in bulk in a single container, or in two, three, four, five, or more than five containers. For example, each container can contain enough glycan polymer preparation for a specific week of a one-month treatment program. When multiple bulk containers are provided, the bulk containers can be suitably packaged together to provide enough glycan polymer preparation for all or part of the treatment period. One or more of these containers can be labeled with information useful to the subject in need or the physician administering the treatment protocol, such as the administration schedule.

[0328] The glycan polymer preparation can be packaged with other suitable substances, such as probiotic bacteria, prebiotic substances, or other substances, as described herein. The other substances can be packaged separately from the glycan polymer preparation, mixed with the glycan polymer preparation, or a combination thereof. Thus, in one embodiment, the kit includes all components intended for use in a course of treatment or part of a course of treatment, such as the glycan polymer preparation, and, optionally, a dosage form containing a probiotic, prebiotic, or polymer agent, such as a buffer, excipient, etc. In one embodiment, the glycan polymer preparation is packaged in one package or a set of packages, and additional components, such as probiotic bacteria, prebiotics, and therapeutic agents, are packaged separately from the glycan polymer preparation.

[0329] The kit may further include materials, such as instructions, expected results, testimonials, explanations, warnings, clinical data, information for medical professionals, etc. In one embodiment, the kit includes a label or other information indicating that the kit is to be used only under the direction of a medical professional. The container may further include a spoon, syringe, bottle, cup, applicator, or other measuring or serving device.

[0330] Identification of Bacterial Components In some embodiments, the glycan polymer preparations described herein are administered to a subject (e.g., a human subject) to increase the growth of beneficial bacteria, reduce the growth of pathogens, and / or regulate (microbial) metabolic products (e.g., SCFAs, ammonia, TMA / TMAO, bile acids, LPS, etc.) in the gastrointestinal tract. In some embodiments, the microbial community is shifted to a healthy state by the glycan polymers administered to the microbial community. Microbial changes occurring in the gastrointestinal tract can be analyzed using any number of methods known in the art and described herein.

[0331] As a quantitative method for determining whether glycan polymer preparations cause a shift in the bacterial population in the gastrointestinal tract, quantitative PCR (qPCR) can be performed. Genomic DNA can be extracted from samples using commercially available kits, such as Mo Bio Powersoil®-htp 96-well Soil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA), Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA), or QIAamp DNA Stool Mini Kit (QIAGEN, Valencia, CA), according to the manufacturer's instructions or other standard methods known to those skilled in the art.

[0332] In some embodiments, qPCR can be performed using HotMasterMix (5PRIME, Gaithersburg, MD) and primers specific for a particular (e.g., beneficial or desired) bacterium, and can be performed in a barcoded MicroAmp® Fast Optical 96-well reaction plate (0.1 mL) (Life Technologies, Grand Island, NY) or a BioRad C1000™ Thermal Cycler equipped with a CFX96™ Real-Time System (BioRad, Hercules, CA) that reads fluorescence in the FAM and ROX channels. The Cq value for each well in the FAM channel is determined by CFX Manager™ software version 2.1. The Cq values ​​for a given sample are input into a linear regression model generated from the standard curve to compare the Cq values ​​of the standard curve wells with the known log α of those samples. 10 (cfu / ml) of each experimental sample. 10 (cfu / ml) is calculated. Alternative qPCR modes may be utilized by those skilled in the art.

[0333] In some embodiments, microbial components are identified by characterizing the DNA sequence of the microbial 16S small subunit ribosomal RNA gene (16S rRNA gene). The 16S rRNA gene is approximately 1,500 nucleotides long and is generally highly conserved across organisms, but contains specific variable and hypervariable regions (V1-V9) that harbor sufficient nucleotide diversity to differentiate most species and strain taxa. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294, and 1435-1465, respectively, using numbering based on the E. coli system. (See, e.g., Brosius et al., Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli, PNAS 75(10):4801-4805 (1978)).

[0334] The composition of the microbial community can be estimated by sequencing the entire 16S rRNA gene, or at least one of the V1, V2, V3, V4, V5, V6, V7, V8, and V9 regions of this gene, or by sequencing any combination of variable regions from this gene (e.g., V1-3 or V3-5). In one embodiment, the V1, V2, and V3 regions are used to characterize the microbiota. In another embodiment, the V3, V4, and V5 regions are used to characterize the microbiota. In another embodiment, the V4 region is used to characterize the microbiota.

[0335] Sequences that are at least 97% identical to each other are grouped into operational taxonomic units (OTUs). OTUs containing sequences with 97% similarity correspond approximately to species-level taxa. At least one representative sequence from each OTU is selected and used to obtain a taxonomic assignment for the OTU by comparison with a highly curated reference database of 16S rRNA gene sequences (such as the Greengenes or SILVA database). The relationships between OTUs in a microbial community can be inferred by constructing a phylogenetic tree from representative sequences from each OTU.

[0336] To determine the sequence of the complete 16S sequence or any variable region of the 16S sequence, genomic DNA is extracted from a bacterial sample, 16S rRNA (complete region or specific variable region) is amplified by polymerase chain reaction (PCR), the PCR product is cleaned, and the nucleotide sequence is analyzed to determine the genetic composition of the 16S rRNA gene or the variable region of this gene, using known techniques. If complete 16S sequencing is performed, the sequencing method used can be, but is not limited to, Sanger sequencing. If one or more variable regions, such as the V4 region, are used, sequencing can be performed using the Sanger method or next-generation sequencing methods, such as the Illumina method. Primers designed to anneal to conserved regions of the 16S rRNA gene (e.g., 515F and 805R primers for amplification of the V4 region) can contain unique barcode sequences to enable simultaneous characterization of multiple microbial communities.

[0337] Another method for identifying microbial composition is the characterization of nucleotide markers or genes, specifically highly conserved genes (e.g., "housekeeping" genes), or a combination of these, or whole-genome shotgun sequencing (WGS). Using a defined method, DNA extracted from a bacterial sample is amplified using PCR, with specific genomic regions sequenced to determine the nucleotide sequence of the amplified product. In the WGS method, the extracted DNA is fragmented into pieces of various lengths (300 to approximately 40,000 nucleotides) and sequenced directly without amplification. Sequence data can be generated using any sequencing technology, including, but not limited to, Sanger, Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences, and / or Oxford Nanopore.

[0338] In addition to the 16S rRNA gene, a selected set of genes known to be marker genes in a given species or taxonomic group are analyzed to assess the composition of the microbial community. These genes are alternatively assayed using PCR-based screening strategies. For example, various strains of pathogenic E. coli are differentiated using genes encoding thermolabile (LTI, LTIIa, and LTIIb) and thermostable (STI and STII) toxins, verotoxin types 1, 2, and 2e (VT1, VT2, and VT2e, respectively), cytotoxic necrotizing factors (CNF1 and CNF2), attaching and effacing mechanisms (eaeA), enteroaggregation mechanisms (Eagg), and invasive mechanisms (Einv). The optimal genes used to determine the taxonomic composition of a microbial community through the use of marker genes are well known to those skilled in the art of sequence-based taxonomic identification.

[0339] The sequencing library for microbial whole genome sequencing (WGS) can be prepared from bacterial genomic DNA.For genomic DNA isolated from human or experimental animal samples, DNA can be optionally enriched for bacterial DNA using commercially available kits, such as NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, Ipswich, MA) or other enrichment kits.Sequencing library can be prepared from genomic DNA using commercially available kits, such as Nextera Mate-Pair Sample Preparation Kit, TruSeq DNA PCR-Free or TruSeq Nano DNA, or Nextera XT Sample Preparation Kit (Illumina, San Diego, CA), according to the manufacturer's instructions.

[0340] Alternatively, the library can be sequenced using an Illumina sequencing platform. Other kits compatible with the platform can be used to prepare libraries, such as the NEBNext DNA Library Construction Kit (New England Biolabs, Ipswich, MA). Libraries can then be sequenced using standard sequencing techniques, including, but not limited to, MiSeq, HiSeq, or NextSeq sequencers (Illumina, San Diego, CA).

[0341] Alternatively, whole-genome shotgun fragment libraries were prepared using standard methods in the art. For example, shotgun fragment libraries could be constructed using the GS FLX Titanium Rapid Library Preparation Kit (454 Life Sciences, Branford, CT), amplified using the GS FLX Titanium emPCR Kit (454 Life Sciences, Branford, CT), and sequenced on a 454 sequencer (454 Life Sciences, Branford, CT) according to standard 454 pyrosequencing protocols.

[0342] Bacterial RNA can be isolated from microbial cultures or samples containing bacteria using commercially available kits such as the RiboPure Bacterial RNA Purification Kit (Life Technologies, Carlsbad, CA). Another method for the isolation of bacterial RNA can involve enriching mRNA in a purified sample of bacterial RNA by removing tRNA.

[0343] Alternatively, RNA can be converted to cDNA, which is used to generate a sequencing library using standard methods such as the Nextera XT sample preparation kit (Illumina, San Diego, CA).

[0344] Nucleic acid sequences are analyzed to define taxonomic assignments using sequence homology and phylogenetic substitution methods, or a combination of the two strategies. Similar approaches are used to annotate protein names, protein functions, transcription factor names, and any other taxonomic scheme for nucleic acid sequences. Sequence homology-based methods include BLAST, BLASTx, tBLASTn, tBLASTx, RDP classifier, DNAclust, RapSearch2, DIAMOND, USEARCH, and various implementations of these algorithms, such as QIIME or Mothur. These methods map sequence reads to a reference database and select the best matches. Common databases for taxonomic assignment include KEGG, MetaCyc, NCBI non-redundant database, Greengenes, RDP, and Silva. For functional assignments, reads are mapped to various functional databases, such as COG, KEGG, BioCyc, MetaCyc, and the Carbohydrate-Active Enzyme (CAZy) database. Microbial clades are assigned using software including MetaPhlAn.

[0345] Proteome analysis of microbial populations Glycan polymer preparations can be selected based on their ability to increase the expression of microbial proteins associated with a healthy state or decrease the expression of microbial proteins associated with a pathological state. Proteome analysis of microbial populations can be performed according to protocols known to those skilled in the art (e.g., Cordwell, Exploring and exploiting bacterial proteomes, Methods in Molecular Biology, 2004, 266:115). To identify differentially expressed proteins (e.g., to identify changes in protein expression upon treatment of microbial populations with glycan polymers), proteome analysis can be performed, for example, as described in Juste et al. (Bacterial protein signals are associated with Crohn's disease, Gut, 2014, 63:1566). For example, proteins are isolated from microbial lysates of two samples (e.g., an untreated microbial population and a population treated with glycan polymers). Each protein sample is labeled (e.g., with a fluorescent dye, e.g., Cy3 or Cy5 CyDye DIGE Fluor Minimal Dye, GE Healthcare) and analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). The gel is stained, and protein spots identified as significantly different between the two samples are excised, digested, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). X!TandemPipeline (http: / / pappso.inra.fr / bioinfo / xtandempipeline / ) can be used to identify differentially expressed proteins.

[0346] Glycan polymer preparations can also be selected for administration to human subjects based on their effect in the presence of microbial products. For example, glycan polymer preparations can be selected for their ability to induce or promote the growth of bacteria that produce short-chain fatty acids such as propionate (propionic acid), acetate, and / or butyrate (butyric acid). Similarly, glycan polymer preparations can be selected for their ability to induce or promote the growth of bacteria that produce lactic acid, which can regulate the growth of other bacteria by producing an acidic environment and is also utilized by taxa that produce butyrate. Such analysis can also be used to pair probiotic bacteria with glycan polymers so that the glycan polymers are a substrate for the production of desired fermentation products. In some embodiments, glycan polymers can also be selected for administration to human subjects based on their effect on bacterial taxa that do not produce undesirable metabolic products (e.g., ammonia, uremic solutes, TMA, and the like). In some embodiments, glycan polymers increase the growth of bacterial taxa that do not produce undesirable metabolites, thereby outcompeting (e.g., in terms of space and nutrients) bacterial taxa that do produce undesirable metabolites. By shifting the balance of producers to non-producers in favor of non-producers, the overall level of undesirable metabolites can be reduced. In some embodiments, the balance of SCFA producers to non-producer taxa is shifted toward SCFA producers to increase the level of SCFA production (e.g., butyrate, acetate, propionate). In some embodiments, the balance of ammonia producers to non-producer taxa is shifted toward non-producers (e.g., urease-negative bacterial taxa) to decrease the level of ammonia production. In some embodiments, the balance of TMA producers to non-producer taxa is shifted toward non-producers to decrease the level of TMA production. Metabolites present in unused or spent culture media or biological samples collected from humans can be determined using the methods described herein. Gas or liquid chromatography coupled with mass spectrometry, or1 Unbiased methods known to those skilled in the art can be used to determine the relative concentrations of metabolites in a sample, such as H-NMR. These measurements can be validated by running standard metabolites through the same analytical system.

[0347] For gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) analysis, polar metabolites and fatty acids can be extracted and derivatized using a monophasic or biphasic system of organic solvents and aqueous samples (Fendt et al., "Reductive glutamine metabolism is a function of the α-ketoglutarate to citrate ratio in cells," Nat Commun, 2013, 4:2236; Fendt et al., "Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism," Cancer Res, 2013, 73:4429; Metallo et al., "Reductive glutamine metabolism by IDH1 mediates lipogenesis under hypoxia," Nature, 2011, 481:380). An exemplary protocol for derivatization of polar metabolites involves incubation of metabolites with 2% methoxylamine hydrochloride in pyridine, followed by the formation of the methoxylamine-tBDMS derivative by the addition of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) and 1% tert-butyldimethylchlorosilane (t-BDMCS). The nonpolar fraction, containing triacylglycerides and phospholipids, can be saponified to free fatty acids and esterified to form fatty acid methyl esters, for example, by incubation with 2% H2SO4 in methanol or by using methyl-8 reagent (Thermo Scientific). The derivatized samples can then be analyzed by GC-MS using standard LC-MS methods, for example, a DB-35MS column (30 m x 0.25 mm i.d. x 0.25 μm, Agilent J&W Scientific) installed on a gas chromatograph (GC) compatible with mass spectrometry (MS).Mass isotopomer distributions can be determined by integrating metabolite ion fragments and corrected for natural abundance using standard algorithms, such as those adapted from Fernandez et al. (Fernandez et al., Correction of 13C mass isotopomer distributions for natural stable isotope abundance, J Mass Spectrom, 1996, 31:255). For liquid chromatography-mass spectrometry (LC-MS), polar metabolites can be analyzed using a standard benchtop LC-MS / MS equipped with a column such as a SeQuant ZIC-pHILIC Polymeric column (2.1 x 150 mm; EMD Millipore). Exemplary mobile phases used for separation can include a buffer adjusted to a specific pH value and an organic solvent.

[0348] In combination, or alternatively, the extracted sample may: 1 H-nuclear magnetic resonance ( 1 The sample may be analyzed by H-NMR. The sample may optionally be combined with an isotopically enriched solvent such as DO in the presence of a buffer solution (e.g., NaHPO, NaHPO, pH 7.4 in DO). The sample may also be supplemented with a reference standard (e.g., 5 mM 2,2-dimethyl-2-silapentane-5-sulfonic acid sodium salt (DSS-d, Isotec, USA)) for calibration and chemical shift determination. Prior to analysis, the solution may be filtered or centrifuged to remove any sediment or precipitate, and then transferred to an appropriate NMR tube or container (e.g., a 5 mm NMR tube) for analysis. 1 H-NMR spectra can be acquired on a standard NMR spectrometer, e.g., an Avance II+500 Bruker spectrometer (500 MHz) (Bruker, DE) equipped with a 5 mm QXI-Z C / N / P probe-head and analyzed with spectrum integration software (e.g., Chenomx NMR Suite 7.1; Chenomx Inc., Edmonton, AB). (Duarte et al., 1 H-NMR protocol for exometabolome analysis of cultured mammalian cells, Methods Mol Biol, 2014:237-47).

[0349] or, 1 H-NMR can be performed according to other published protocols known in the art (Chassaing et al., Lack of soluble fiber drives diet-induced adiposity in mice, Am J Physiol Gastrointest Liver Physiol, 2015; Bai et al., Comparison of Storage Conditions for Human Vaginal Microbiome Studies, PLoS ONE, 2012:e36934).

[0350] Administration In some embodiments, the glycan polymer is administered to a subject in need thereof (e.g., a human subject) by enteral administration. In some embodiments, the glycan polymer is administered to a subject in need thereof by oral, nasal, gastric, or rectal administration. In some embodiments, the glycan polymer is administered to a subject in need thereof by tube feeding.

[0351] In some embodiments, the glycan polymer is administered to a subject in need thereof immediately after one or more drug treatments have ended (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after the end of antibiotic treatment). During a course of drug treatment, the glycan polymer preparation may be provided prior to the start of drug treatment (e.g., 1, 2, 3, 4, 5, 6, 7 days before), on the day drug treatment begins, or immediately after antibiotic treatment, e.g., 1, 2, 3, 4, 5, 6, 7 days or more after treatment, and optionally only at the beginning (e.g., for a short period) or throughout the duration of drug treatment, and may be continued for a desired period of time after the end of the drug treatment period (e.g., for 1 to 7 days, 1 to 14 days, or 1 to 21 days thereafter). In some embodiments, administration of the glycan polymer preparation is initiated or continued when one or more side effects occur and / or are diagnosed in conjunction with drug treatment (e.g., digestive disorders or pathogen growth). In some embodiments, the treatment agent causing the dysbiosis is not a drug, but rather radiation treatment or surgery, and the glycan polymer preparation can also be administered as described herein. In some embodiments, the total number and duration of treatment periods are based on the subject's response to treatment. For example, an individual may experience symptom relief 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after treatment with the glycan polymer preparation. In another example, an individual may experience symptom relief 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after treatment with the glycan polymer preparation. Thus, the duration of treatment is determined by the individual subject's response to the glycan polymer preparation and the onset of relief of one or more symptoms. Thus, a subject may experience symptoms at a given dose of glycan polymer preparation, and may need to remain at that dose or a lower dose until symptoms disappear.Therefore, in one embodiment, the duration of treatment is not determined at the beginning, but continues until the maximum dose of glycan polymer preparation is achieved every day, or until the desired level of symptom relief is achieved.In one embodiment, treatment is continuous.

[0352] In one embodiment, a subject (e.g., a human subject) can receive one dose during a first treatment period and a second dose during a second treatment period during a treatment regimen. For example, a subject can receive one dose of glycan polymer preparation over a week period and a second dose over the following week period.

[0353] The subject may self-administer the glycan polymer preparation, which may be provided or recommended (or prescribed) by a medical professional, such as a physician or other qualified medical professional, and optionally test results (e.g., obtained for biomarkers from samples taken from the subject) and / or changes in health and treatment endpoints may be monitored by the medical professional. In some embodiments, the glycan polymer preparation is administered by a medical professional.

[0354] In one embodiment, a subject in need thereof can undergo repeated treatment with a glycan polymer preparation. The treatment can be repeated if symptoms recur or increase to undesirable levels. Alternatively, the treatment can be repeated at regular or predetermined intervals. Thus, treatment can be repeated after approximately 1 month, 2 months, 3 months, 4 months, 6 months, 8 months, 10 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, or more than 5 years, or any combination thereof (e.g., treatment can be repeated after 1 year, and then every 2 to 5 years thereafter). The treatment can be repeated in the same form (e.g., duration, dosage, timing of administration, additional substances, etc.) as used in the initial treatment, or can be modified. For example, the treatment period can be shortened or extended, and the dosage can be increased or decreased.

[0355] In some embodiments, the pharmaceutical composition is administered once, twice, or three times daily. In some embodiments, the pharmaceutical composition is administered twice daily. In some embodiments, the pharmaceutical composition is administered daily for a predetermined number of days (treatment period). In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 35, 42, 49, 56, 63, 70, 100, 200, 300, or 365 days. In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. In some embodiments, the treatment period is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years, or a lifetime.

[0356] In one embodiment, the total duration of treatment for a gastrointestinal disease, disorder, or condition can be about 1 day to 10 years, 1 day to 1 year, 1 day to 6 months, 1 day to 3 months, 1 day to 1 month, 1 day to 1 week, 1 day to 5 days, 1 day to 10 days, 1 week to about 12 weeks, or about 4 weeks to about 10 weeks, or about 4 weeks to about 8 weeks, or about 6 weeks. A subject (e.g., a human subject) may receive an appropriate treatment period, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 treatment periods. During the treatment period, the subject optionally ingests a glycan polymer preparation described herein, along with the ingestion of a food containing prebiotics and / or probiotics. In one embodiment, the glycan polymer preparation can also be administered in combination with another substance (e.g., a probiotic or probiotically beneficial bacteria, a prebiotic substance, or a therapeutic agent) described herein.

[0357] In some embodiments, the glycan polymer preparation can also be combined with an antibiotic that prevents the growth of normal gastrointestinal microflora. Typically, the duration of antibiotic treatment is 1 to 14 days, or 2 to 10 days, or 5 to 7 days. In some embodiments, the glycan polymer is administered to a subject in need thereof immediately after one or more antibiotic treatments have ended (e.g., 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after the end of antibiotic treatment). During a course of antibiotic treatment, the glycan polymer preparation can be administered at the start of antibiotic treatment, immediately after antibiotic treatment, for example, 1, 2, 3, 4, 5, 6, 7 days, or more after treatment, or upon diagnosis of gastric growth of an undesirable pathogen.

[0358] Methods of Treatment Provided herein are methods for treating a subject (e.g., a human subject) with a disease or disorder. In some embodiments, the disease or disorder is associated with levels (e.g., undesirable levels) of metabolites (e.g., short-chain fatty acids (SCFAs), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, lipopolysaccharides (LPS), or bile acids). In some embodiments, the method comprises administering to a human subject a glycan polymer preparation in an amount effective to treat the disease or disorder. In some embodiments, glycan polymer preparations (e.g., those described herein) are useful in treating various diseases, disorders, or conditions. Such diseases, disorders, or conditions may be associated with dysbiosis of the microbiota. The decline of beneficial microbiota can be caused by a variety of factors (e.g., genetic or environmental), including, but not limited to, the use of antibiotics, chemotherapeutic agents, and other dysbiosis-inducing drugs or treatments (e.g., radiation treatment), pathogen infection, pesticide activity, miscalibrated caloric intake (e.g., high fat, high carbohydrate), miscalibrated (non-digestible) fiber intake (e.g., low or no fiber), host factors (e.g., host genetic alterations), etc. In some embodiments, the disease, disorder, or condition is associated with dysbiosis of the gastrointestinal microbiota. In some embodiments, the disease, disorder, or condition is treated by treating the dysbiosis. Symptoms that may be associated with dysbiosis of the gastrointestinal microbiota and / or a gastrointestinal disease, disorder, or condition include, but are not limited to, gas, heartburn, upset stomach, bloating, flatulence, diarrhea, abdominal pain, cramps, nausea, and vomiting. Minor digestive problems related to the stomach and intestines may also include occasional bloating, diarrhea, constipation, gas, or upset stomach.

[0359] In some embodiments, a disease or disorder is associated with metabolite levels (e.g., undesirable levels). Metabolites such as short-chain fatty acids (SCFAs), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, lipopolysaccharides, or bile acids, and the bacteria that produce them, have been associated with a range of diseases. For example, decreased levels of butyrate-producing bacteria have been reported in Crohn's disease (Takahashi et al., (2016)), and decreased levels of butyrate and propionate, and increased levels of acetate, have been reported in fecal samples from patients with Crohn's disease (Galecka et al., (2013)). Butyrate has been reported to reduce the expression of pro-inflammatory cytokines, which may play an important role in inflammatory bowel diseases, including Crohn's disease (Russo I. et al., PLoS One 2012). Other diseases associated with reduced butyrate levels compared to healthy patient populations include ulcerative colitis (Kumari et al., 2013), type 2 diabetes (Qin et al., 2012), atopic dermatitis (Song et al., 2016), colorectal cancer (Wang et al., 2012), and Parkinson's disease (Keshavarzian et al., 2015). Administering glycans that support the growth of microbiota that are directly or indirectly positively correlated with butyrate production to individuals may increase butyrate levels in vivo and ameliorate or prevent Crohn's disease symptoms.

[0360] In some embodiments, it may also be beneficial to administer glycans that reduce the production of one or more short-chain fatty acids to treat some diseases. Butyrate-producing bacteria have been reported to be increased in obese patients compared to healthy individuals (Ross et al., 2015), and butyrate and propionate are increased in the stool of obese patients compared to healthy patients. It has been found that increased levels of acetate are associated with obesity (Payne et al., 2011), and similarly, increased levels of acetate have been associated with obesity (Gao et al., 2014). Therefore, administration of glycans that selectively reduce microbiota directly or indirectly associated with increased butyrate, propionate, and / or acetate may be useful for treating or preventing obesity. Other diseases associated with relatively high acetate levels include malabsorption syndromes (Bala et al., 2006), colorectal cancer (Weir et al., 2013), and Crohn's disease (Galecka et al., 2013). Administration of glycans to a subject to selectively reduce microbiota directly or indirectly associated with increased acetate may be useful for treating or preventing diseases associated with elevated acetate levels, such as obesity, malabsorption syndromes, colorectal cancer, and Crohn's disease.

[0361] In some embodiments, the disease or disorder is associated with levels (e.g., undesirable levels) of short chain fatty acids, such as, for example, acute pouchitis, allergic diseases, AIDS, atherosclerosis, asthma, atopic dermatitis, autism spectrum disorder, chronic functional constipation, celiac disease, chronic atrophi...

Claims

1. A method of treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., a short-chain fatty acid (SCFA) (e.g., propionate or butyrate), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), a uremic solute (e.g., p-cresol or indole), lipopolysaccharide (LPS), or a bile acid (e.g., a secondary bile acid)), optionally comprising selecting a glycan polymer preparation based on its modulation of the production or level of the metabolite, and administering an amount of the glycan polymer preparation effective to effect modulation of the level of the metabolite, thereby treating the disease or disorder.

2. A method for treating a subject having a disease or disorder associated with undesirable levels of a metabolite (e.g., a short-chain fatty acid (SCFA) (e.g., propionate or butyrate), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), a uremic solute (e.g., p-cresol or indole), lipopolysaccharide (LPS), or a bile acid (e.g., a secondary bile acid)), optionally comprising determining that a glycan polymer preparation regulates the production or level of the metabolite, and administering an amount of the glycan polymer preparation effective to regulate the level of the metabolite, thereby treating the disease or disorder.

3. The method of claim 1 or 2, wherein the glycan polymer preparation is administered in response to evidence or knowledge that the glycan polymer preparation regulates the production or level of the metabolite.

3. The glycan polymers of the glycan polymer preparation, or at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% (by weight or number) of the glycan polymers, have one or more (e.g., 2, 3, 4, 5, or 6) of the properties listed in Table 1, and optionally, a. glycan polymers comprising glucose, mannose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic linkage, b. glycan polymers comprising glucose, mannose, or galactose subunits, or a combination thereof, and at least one beta-glycosidic linkage, c. glycan polymers comprising xylose, arabinose, fucose, or rhamnose subunits, or a combination thereof, and at least one alpha-glycosidic linkage, d. glycan polymers comprising xylose, arabinose, fucose, or rhamnose subunits, or a combination thereof, and at least one beta-glycosidic linkage, e.g., glycan polymers comprising xylose, arabinose, fucose, or rhamnose subunits, or a combination thereof, and at least one beta-glycosidic linkage, 4. The method according to any one of claims 1 to 3, wherein the glycan polymer is selected from: f. a glycan polymer comprising glucose or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond; or f. a glycan polymer comprising glucose or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond.

4. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond, optionally the beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a glu-gal preparation); iv. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a glu-man preparation); and v. the glycan polymer preparation further comprises glycan polymers comprising galactose and mannose (e.g., a glu-gal-man preparation).

5. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a glu-gal preparation); iv. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a glu-man preparation); and v. the glycan polymer preparation further comprises glycan polymers comprising galactose and mannose (e.g., a glu-gal-man preparation).

6. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage, optionally the beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a gal-glu preparation); iv. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a gal-man preparation); and v. the glycan polymer preparation further comprises glycan polymers comprising glucose and mannose (e.g., a gal-man-glu preparation).

7. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one beta-glycosidic linkage, optionally the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a gal-glu preparation); iv. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a gal-man preparation); and v. the glycan polymer preparation further comprises glycan polymers comprising glucose and mannose (e.g., a gal-glu-man preparation).

8. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage, optionally the beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); iv. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a man-glu preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising galactose and glucose (e.g., a man-gal-glu preparation).

9. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); iv. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a man-glu preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising galactose and glucose (e.g., a man-gal-glu preparation).

10. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation comprises alpha-1,2 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof; The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises one, two, three, or more, for example, all, of: i. the glycan polymer preparation further comprises glycan polymers comprising a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage, optionally wherein the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. the glycan polymer preparation further comprises glycan polymers comprising fucose (e.g., a gal-fuc preparation); v. the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a gal-man preparation); and vi. the glycan polymer preparation further comprises glycan polymers comprising fucose and mannose (e.g., a gal-fuc-man preparation).

11. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one beta-glycosidic bond, optionally the beta-1,3 glycosidic bond, the beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising fucose (e.g., a gal-fuc preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a gal-man preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising fucose and mannose (e.g., a gal-fuc-man preparation).

12. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises fucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage, optionally a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a fuc-gal preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a fuc-man preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising galactose and mannose (e.g., a fuc-gal-man preparation).

13. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises fucose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-1; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a fuc-gal preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a fuc-man preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising galactose and mannose (e.g., a fuc-gal-man preparation).

14. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage, optionally wherein the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising fucose (e.g., a man-fuc preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising galactose and fucose (e.g., a man-gal-fuc preparation).

15. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising fucose (e.g., a man-fuc preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising galactose and fucose (e.g., a man-gal-fuc preparation).

16. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises one, two, or three of glucose, xylose, and arabinose, and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; The method of any one of claims 1-3A, wherein the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage, optionally the beta-1,3 glycosidic linkage is a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. the glycan polymer preparation comprises glycan polymers comprising glucose; v. the glycan polymer preparation comprises glycan polymers comprising xylose; and vi. the glycan polymer preparation comprises glycan polymers comprising arabinose.

17. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises one, two, or three of glucose, xylose, and arabinose, and at least one beta-glycosidic bond, optionally the beta-1,3 glycosidic bond, beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage being an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. the glycan polymer preparation comprises glycan polymers comprising glucose; v. the glycan polymer preparation comprises glycan polymers comprising xylose; and vi. the glycan polymer preparation comprises glycan polymers comprising arabinose.

18. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer is composed of glucose and at least one alpha and optionally, the alpha-glycosidic linkages are alpha-1,3 glycosidic linkages, and further optionally, the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic linkages, alpha-1,4 glycosidic linkages, alpha-1,6 glycosidic linkages, or combinations thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage; iv. the glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., a glu-gal preparation); v. the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a glu-ara preparation); vi. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a glu-xyl preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising two or three of galactose, arabinose, and xylose.

19. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic linkages, alpha-1,4 glycosidic linkages, alpha-1,6 glycosidic linkages, or combinations thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage; iv. the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., a gal-glu preparation); v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a gal-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a gal-xyl preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising two or three of glucose, arabinose, and xylose.

20. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises one or two of xylose and arabinose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,4 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation comprises glycan polymers comprising xylose; and v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation comprises one, two, three, or more, for example all, of the following: glycan polymers comprising arabinose.

21. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises arabinose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., an ara-gal preparation); iv. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., an ara-xyl preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., an ara-gal preparation). The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising galactose and xylose (e.g., an ara-gal-xyl preparation);

22. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage; iii. the glycan polymer preparation further comprises a glycan polymer comprising arabinose (e.g., a gal-ara preparation); iv. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a gal-xyl preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a gal-xyl preparation). The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises a glycan polymer comprising arabinose and xylose (e.g., a gal-ara-xyl preparation);

23. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises xylose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a xyl-gal preparation); iv. the glycan polymer preparation further comprises a glycan polymer comprising arabinose (e.g., a xyl-ara preparation); and v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers containing galactose and arabinose (e.g., a xyl-ara-gal preparation);

24. The method of any one of claims 1-3A, wherein the glycan polymer and / or glycan polymer preparation comprises one, two, or more, e.g., all, of the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; and iii. the glycan polymer preparation further comprises a glycan polymer comprising one, two, or three of arabinose, galactose, or xylose.

25. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, and optionally, the average degree of polymerization (DP) of the preparation is DP2-4, DP2-6, DP3-10, or DP3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic linkages, alpha-1,3 glycosidic linkages, alpha-1,4 glycosidic linkages, alpha-1,6 glycosidic linkages, or combinations thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage; and iv. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises one, two, three, or more, for example all, of glycan polymers comprising one, two, three, or four of galactose, mannose, arabinose, or sialic acid.

26. The method of any one of claims 1-3A, wherein the glycan polymer and / or glycan polymer preparation comprises one, two, three, or more, e.g., all, of the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage; iii. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a glu-xyl preparation); and iv. the glycan polymer preparation further comprises a glycan polymer comprising one, two, or three of mannose, arabinose, or galactose.

27. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one beta-glycosidic bond, and optionally, the average degree of polymerization (DP) of the preparation is DP2-4, DP2-6, DP3-10, or DP3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, and optionally, the alpha-glycosidic bond is an alpha-1,3 glycosidic bond; iii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a glu-xyl preparation); and v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises one, two, three, or more, for example all, of glycan polymers comprising one, two, or three of mannose, arabinose, or galactose.

28. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises xylose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation has at least one beta- 3. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises one, two, three, or more, for example, all, of: i. the glycan polymer preparation further comprises glycan polymers comprising a -glycosidic bond; iii. the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., a xyl-glu preparation); and iv. the glycan polymer preparation further comprises glycan polymers comprising one, two, or three of mannose, arabinose, or galactose.

29. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises xylose and at least one beta-glycosidic bond, and optionally, the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, and optionally, the alpha-glycosidic bond is an alpha-1,3 glycosidic bond; iii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a xyl-glu preparation); and v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises one, two, three, or more, for example all, of glycan polymers comprising one, two, or three of mannose, arabinose, or galactose.

30. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic bond, alpha-1,4 glycosidic bond, alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a glu-xyl preparation); v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a glu-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., a glu-gal preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising one, two, or three of xylose, arabinose, or galactose, one, two, three, or more, e.g., all of the following.

31. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises xylose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic bond, alpha-1,4 glycosidic bond, alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., a xyl-glu preparation); v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a xyl-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., a xyl-gal preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising one, two, or three of glucose, arabinose, or galactose.

32. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises arabinose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP2-4, DP2-6, DP3-10, or DP3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic bond, alpha-1,4 glycosidic bond, alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., an ara-xyl preparation); v. The method of any one of claims 1 to 3A, wherein the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., an ara-glu preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., an ara-gal preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising one, two, or three of xylose, glucose, or galactose.

33. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic linkages, alpha-1,4 glycosidic linkages, alpha-1,6 glycosidic linkages, or combinations thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage; iv. the glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a gal-xyl preparation); v. the glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a gal-xyl preparation); The method of any one of claims 1-3A, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a gal-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., a gal-glu preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising one, two, or three of xylose, arabinose, or glucose, one, two, or more, e.g., all of the following.

34. The method of any of claims 1 to 33, wherein the glycan polymers, or at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% (by weight or number) of the glycan polymers of the glycan polymer preparation are substrates for a glycosidase enzyme.

35. 35. The method of claim 34, wherein the glycosidase enzyme is present in a human intestinal microorganism.

36. 36. The method of claim 35, wherein the human gut microbe is a member of glycotaxon class 1, a but and / or buk gene-containing bacterial taxon.

37. 36. The method of claim 35, wherein the human gut microbe is a member of glycotaxon class 2, a cutC gene negative bacterial taxon.

38. 36. The method of claim 35, wherein the human gut microorganism is a member of glycotaxon class 3, a urease gene negative bacterial taxon.

39. The human gut microorganism is selected from glycotaxon class 4, propionate kinase, propionate CoA transferase, propionate CoA ligase, propionyl CoA carboxylase, methylmalonyl-CoA carboxytransferase, (S)-methylmalonyl-CoA decarboxylase, methylmalonate semialdehyde dehydrogenase, and propanal dehydrogenase (e.g., Enzyme Commission (EC) number 6.4.

1.

36. The method of claim 35, wherein the bacterial strain is a member of a bacterial taxon that does not contain one or more (e.g., not including 1, 2, 3, 4, or more (e.g., not including all)) propionate production-related enzymes.

40. The human intestinal microorganisms are glycotaxonomic group 5, 7alpha-hydroxysteroid dehydrogenase, 12alpha-hydroxysteroid dehydrogenase, 7beta-hydroxysteroid dehydrogenase (NADP+), 2beta-hydroxysteroid dehydrogenase, 3beta-hydroxycholanic acid 3-dehydrogenase (NAD+), 3alpha-hydroxycholanic acid dehydrogenase (NADP+), 3beta-hydroxycholanic acid 3-dehydrogenase (NADP+), 3alpha-hydroxybile acid-CoA-ester 3-dehydrogenase, 3alpha-hydroxycholanic acid dehydrogenase (NAD+), bile acid 36. The method of claim 35, wherein the bacterial strain is a member of a bacterial taxon that contains one or more (e.g., including 1, 2, 3, 4, or more (e.g., all)) bile acid production (e.g., secondary bile acid production)-related enzymes selected from bile acid CoA-transferase, bile acid 7 alpha-dehydratase, and bile acid CoA ligase (e.g., selected from enzymes corresponding to Enzyme Commission (EC) numbers 1.1.1.159, 1.1.1.176, 1.1.1.201, 1.1.238, 1.1.1.391, 1.1.392, 1.1.393, 1.1.395, 1.1.1.52, 2.8.3.25, 4.2.1.106, and 6.2.1.7).

41. 36. The method of claim 35, wherein the human gut microbe is a member of a bacterial taxon that does not contain one or more (e.g., does not contain one, two, three, four, or more (e.g., does not contain all)) indole production-related enzymes selected from glycotaxon class 6, tryptophanase (e.g., enzymes corresponding to Enzyme Commission (EC) number 4.1.99.1).

42. 36. The method of claim 35, wherein the human gut microbe is a member of a bacterial taxon that does not contain one or more (e.g., does not contain one or both) p-cresol production-related enzymes selected from glycotaxon class 7, 4-hydroxyphenylacetate decarboxylase, and aldehyde ferredoxin oxidoreductase (e.g., selected from enzymes corresponding to Enzyme Commission (EC) numbers 4.1.1.83, 2.6.1.-, 4.1.1.-, and 1.2.7.5).

43. The glycan polymer is selected from the group consisting of GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, and GH13 subfamily 36.

37. The method of claim 34, 35, or 36, wherein the glycosidase is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from the CAZy family of GH113, GH112, or GH113.

44. 37. The method of claim 34, 35, or 36, wherein the glycan polymer is a substrate for a glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the following CAZy family: GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13, GH13 subfamily 9, GH13 subfamily 31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77, or GH94.

45. 38. The method of claim 34, 35, or 37, wherein the glycan polymer is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, or GH13 subfamily 14 CAZy family.

46. 38. The method of claim 34, 35, or 37, wherein the glycan polymer is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from the GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy family.

47. 39. The method of claim 34, 35, or 38, wherein the glycan polymer is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, or GH13 CAZy family.

48. 39. The method of claim 34, 35, or 38, wherein the glycan polymer is a substrate for a glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, GH28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, or GH24 CAZy family.

49. 40. The method of claim 34, 35, or 39, wherein the glycan polymer is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family.

50. 40. The method of claim 34, 35, or 39, wherein the glycan polymer is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from the GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92 CAZy family.

51. 41. The method of claim 34, 35, or 40, wherein the glycan polymer is a substrate for a glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family.

52. 41. The method of claim 34, 35, or 40, wherein the glycan polymer is a substrate for a glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GH23, GH24, or GH33 CAZy family.

53. 42. The method of claim 34, 35, or 41, wherein the glycan polymer is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family.

54. 42. The method of claim 34, 35, or 41, wherein the glycan polymer is a substrate for a glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GH2, GH31, GH23, GH13, or GH24 CAZy family.

55. 43. The method of claim 34, 35, or 42, wherein the glycan polymer is a substrate for one or more, e.g., two, three, four, or more, glycosidase enzymes selected from GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family.

56. 43. The method of claim 34, 35, or 42, wherein the glycan polymer is a substrate for a glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106 CAZy family.

57. The method of claim 1, wherein the step of selecting a glycan polymer comprises selecting the glycan polymer based on the substrate specificity of any one of claims 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, or 56.

58. 58. The method of any one of claims 1 to 57, wherein the metabolite is one of a short chain fatty acid (SCFA) (e.g., butyrate and / or propionate), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), a uremic solute (e.g., p-cresol or indole), or a bile acid (e.g., a secondary bile acid).

59. 59. The method of claim 58, wherein the metabolite is a short chain fatty acid (SCFA).

60. 60. The method of claim 59, wherein the SCFA is acetate, butyrate, and / or propionate.

61. 59. The method of claim 58, wherein the metabolite is TMA and / or TMAO.

62. 59. The method of claim 58, wherein the metabolic product is ammonia.

63. 59. The method of claim 58, wherein the metabolite is a bile acid.

64. 59. The method of claim 58, wherein the metabolite is a uremic solute, for example, p-cresol.

65. 59. The method of claim 58, wherein the metabolite is a uremic solute, e.g., indole.

66. The disease or disorder is diarrhea (e.g., drug toxicity-induced diarrhea, e.g., diarrhea induced by a treatment regimen including the administration of a tyrosine kinase inhibitor or a chemotherapeutic agent (e.g., a FOLFIRI regimen); or radiation-induced diarrhea and radiation-induced acute intestinal symptoms), optionally wherein the SCFA is butyrate, and optionally wherein the level of butyrate is increased (e.g., relative to a subject receiving the same treatment but not administered the glycan polymer preparation, or relative to the level in the subject before administration of the glycan polymer preparation).

67. 61. The method of any of claims 59 or 60, wherein the disease or disorder is selected from Crohn's disease, inflammatory bowel disease, irritable bowel disease, irritable bowel disease-constipation (IBS-C), or ulcerative colitis, and optionally the SCFA is butyrate.

68. 61. The method of claim 59 or 60, wherein the disease or disorder is selected from non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), and optionally the SCFA is butyrate.

69. 61. The method of claim 59 or 60, wherein the disease or disorder is hepatic encephalopathy, and optionally the SCFA is butyrate.

70. 62. The method of claim 61, wherein the disease or disorder is trimethylaminuria (e.g., secondary trimethylaminuria).

71. 62. The method of claim 61, wherein the disease or disorder is a chronic disease (e.g., chronic kidney disease or end-stage renal disease).

72. 62. The method of claim 61, wherein the disease or disorder is a chronic disease (e.g., chronic heart disease, chronic heart failure, chronic vascular disease).

73. 62. The method of claim 61, wherein the disease or disorder is one of nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).

74. 63. The method of claim 62, wherein the disease or disorder is chronic kidney disease.

75. 63. The method of claim 62, wherein the disease or disorder is cirrhosis, optionally accompanied by minimal hepatic encephalopathy (MHE).

76. 63. The method of claim 62, wherein the disease or disorder is hepatic encephalopathy.

77. 63. The method of claim 62, wherein the disease or disorder is a urea cycle disorder.

78. 61. The method of any of claims 59 or 60, wherein the disease or disorder is propionic acidemia.

79. 64. The method of claim 63, wherein the disease or disorder is selected from cirrhosis, alcoholic cirrhosis, primary biliary cirrhosis, or intestinal failure-associated liver disease.

80. 64. The method of claim 63, wherein the disease or disorder is selected from Crohn's disease, inflammatory bowel disease, irritable bowel disease, irritable bowel disease-constipation (IBS-C), or ulcerative colitis.

81. 64. The method of claim 63, wherein the disease or disorder is selected from non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

82. 66. The method of claim 65, wherein the disease or disorder is chronic kidney disease.

83. 66. The method of claim 65, wherein the disease or disorder is hepatic encephalopathy.

84. 66. The method of claim 65, wherein the disease or disorder is hepatic phenylketonuria.

85. 65. The method of claim 64, wherein the disease or disorder is chronic kidney disease.

86. 65. The method of claim 64, wherein the disease or disorder is hepatic encephalopathy.

87. 87. The method of any one of claims 66 to 86, wherein the metabolite level is increased in the subject, or in a suitable sample from said subject having said disease or disorder, e.g., increased compared to a reference, e.g., a predetermined reference value, the level in the subject before treatment, or a healthy control.

88. 87. The method of any one of claims 66 to 86, wherein the metabolite level is decreased in the subject, or a suitable sample from said subject having said disease or disorder, e.g., decreased compared to a reference, e.g., a predetermined reference value, the level in the subject before treatment, or a healthy control.

89. 89. The method of any one of claims 1 to 88, further comprising assessing the level of the metabolite, or symptoms of an undesirable level of the metabolite, e.g., by obtaining the level of the metabolite, optionally before (e.g., as a baseline), during (e.g., to monitor treatment success), and / or after (e.g., to assess recurrence of the disease or disorder) treating the subject.

90. butyrate levels (e.g., systemic levels, e.g., blood or fecal levels) are increased after treatment with, e.g., the glycan polymer preparation.

90. The method of any one of claims 4-9, 36, 43, 44, 59, 60, 66-69, or 87, wherein the rate or level of butyrate production (e.g., by gastrointestinal microorganisms) is increased in a subject treated with the method, compared to a subject not treated with the method.

91. The method of any of claims 10-17, 36, 43, 44, 59, 60, 70, or 88, wherein the level of TMA (e.g., systemic level, e.g., blood or fecal level) is reduced (e.g., the rate or level of conversion of choline to TMA (e.g., by gastrointestinal microorganisms) is reduced), e.g., compared to a subject not treated with the glycan polymer preparation.

92. The method of any of claims 18-20, 37, 45, 46, 61, 70-73, or 88, wherein the level of ammonia (e.g., systemic level, e.g., blood or fecal level) is reduced (e.g., the rate or level of conversion of urea to ammonia (e.g., by gastrointestinal microorganisms) is reduced), e.g., compared to a subject not treated with the glycan polymer preparation.

93. The method of any one of claims 21-24, 39, 49, 50, 59, 60, 78, or 88, wherein the level of propionic acid (e.g., systemic level, e.g., blood or fecal level) is reduced (e.g., the rate or level of propionic acid production (e.g., by gastrointestinal microorganisms) is reduced), e.g., compared to a subject not treated with the glycan polymer preparation.

94. The level of a secondary bile acid (e.g., systemic level, e.g., intestinal or fecal level) is increased (e.g., the rate or level of conversion of bile acids to secondary bile acids (e.g., by gastrointestinal microorganisms) is increased), e.g., compared to a subject not treated with the glycan polymer preparation. The method of any one of claims 25, 40, 51, 52, 63, 79-81, or 87.

95. 89. The method of any one of claims 26-29, 41, 53, 54, 65, 82-84, or 88, wherein the level of indole (e.g., systemic level, e.g., fecal level) is reduced (e.g., the rate or level of indole production (e.g., by gastrointestinal microorganisms) is reduced), e.g., compared to a subject not treated with the glycan polymer preparation.

96. 90. The method of any one of claims 30-33, 42, 55, 56, 64, 85, 86, or 88, wherein the level (e.g., systemic level) of p-cresol is reduced (e.g., the rate or level of tyrosine conversion (e.g., by gastrointestinal microorganisms) to p-cresol is reduced), e.g., compared to a subject not treated with the glycan polymer preparation.

97. 97. The method of any one of claims 1 to 96, further comprising selecting a subject for treatment based on or in response to obtaining one or more of the following findings: a) the subject has an undesirable level of a metabolite (e.g., an undesirable level of a metabolite described in any one of claims 58 to 65); b) the subject has a disease or disorder (e.g., a disease or disorder described in any one of claims 66 to 86); c) the subject has dysbiosis of the gut microbiota (e.g., a miscalibrated level / relative abundance (e.g., of a class 1, class 2, class 3, class 4, class 5, class 6, or class 7 bacterial taxon described in any one of claims 36 to 42); d) the subject has responded to a previous treatment with a glycan polymer (e.g., a glycan polymer described in any one of claims 3 to 33); or e) the subject has undergone a treatment or other circumstances that result in dysbiosis, such as antibiotic treatment or gastric surgery prior to treatment; and optionally, the method further comprising obtaining appropriate values ​​for determining the selection criteria.

98. 98. The method of claim 97, wherein the subject is selected for treatment based on or in response to obtaining findings of any two or more of (a)-(e).

99. 98. The method of claim 97, wherein the subject is selected for treatment based on or in response to obtaining the findings of any three or more of (a)-(e).

100. 98. The method of claim 97, wherein the subject is selected for treatment based on or in response to obtaining findings of any four or more of (a) through (e).

101. 98. The method of claim 97, wherein the subject is selected for treatment based on or in response to obtaining the findings of all of (a) through (e).

102. The method of any of claims 97 to 101, wherein the appropriate value may be obtained by analyzing an appropriate biological sample from the subject.

103. 103. The method of claim 102, wherein the sample is a blood, feces, urine, saliva, or organ tissue sample.

104. 104. The method of any one of claims 1 to 103, wherein the undesirable level of the metabolite is modulated, e.g. reduced (e.g. in the subject, or in an appropriate sample taken from the treated subject), after a period of treatment, by 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% (e.g. when compared to a baseline, e.g. a predetermined baseline value, the level in the subject before treatment, or a healthy control).

105. 105. The method of any one of claims 1 to 104, wherein the undesirable level of the metabolite increases (e.g. in an appropriate sample taken from the treated subject) after a period of treatment by 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% (e.g. when compared to a baseline, e.g. a predetermined baseline value, the level in the subject before treatment, or a healthy control).

106. The method of any one of claims 1 to 105, wherein the treating step further comprises administering a second therapeutic agent (e.g., a therapeutic agent other than the glycan polymer for treating the disease or disorder and / or for regulating the level of the metabolite).

107. 107. The method of any one of claims 1 to 106, wherein the treating step further comprises administering a preparation of gut microbes (e.g., human gut microbes).

108. The intestinal microorganisms (e.g., human intestinal microorganisms) are classified into: i. Class 1 (e.g., but and / or buk gene-containing bacterial taxa), ii. Class 2 (e.g., cutC gene-negative bacterial taxa), iii. Class 3 (e.g., urease gene-negative bacterial taxa), iv. Class 4 (e.g., bacterial taxa lacking one or more propionate production-related enzymes selected from propionate kinase, propionate CoA transferase, propionate CoA ligase, propionyl CoA carboxylase, methylmalonyl-CoA carboxytransferase, (S)-methylmalonyl-CoA decarboxylase, methylmalonate semialdehyde dehydrogenase, and propanal dehydrogenase (e.g., selected from enzymes corresponding to Enzyme Commission (EC) numbers 6.4.1.3, 2.1.3.1, 4.1.1.41, 1.2.1.27, 2.3.3.5, 1.2.1.87, 1.3.1.95, 1.3.8.7, 2.3.1.54, 2.3.1.168, 2.3.1.8, and 2.3.1.222)); v. Class 5 (e.g., 7alpha-hydroxysteroid dehydrogenase, 12alpha-hydroxysteroid dehydrogenase, 7beta-hydroxysteroid dehydrogenase (NADP+), 2beta-hydroxysteroid dehydrogenase, 3beta-hydroxycholanic acid 3-dehydrogenase (NAD+), 3alpha-hydroxycholanic acid dehydrogenase (NADP+), 3beta-hydroxycholanic acid 3-dehydrogenase (NADP+), 3alpha-hydroxybile acid-CoA-ester 3-dehydrogenase, 3alpha vi. Class 6 (e.g., tryptophanase (e.g., 2.8.3.25, 4.2.1.106, and 6.2.1.7)), vi. Class 7 (e.g., tryptophanase (e.g., 2.8.3.25, 4.2.1.106, and 6.2.1.7)), vi. Class 8 (e.g., tryptophanase (e.g., 2.8.3.25, 4.2.1.106, and 6.2.1.7)), vi. Class 9 (e.g., tryptophanase (e.g., 2.8.3.25, 4.2.1.106, and 6.2.1.7)), vi. Class 10 (e.g., tryptophanase (e.g., 2.8.3.25, 4.2.1.106, and 6.2.1.7)),vii. a bacterial taxon lacking one or more indole production-related enzymes selected from class 7 (e.g., 4-hydroxyphenylacetate decarboxylase and aldehyde ferredoxin oxidoreductase (e.g., selected from enzymes corresponding to Enzyme Commission (EC) numbers 4.1.1.83, 2.6.1.-, 4.1.1.-, and 1.2.7.5)), or vii. a bacterial taxon lacking one or more p-cresol production-related enzymes selected from class 7 (e.g., 4-hydroxyphenylacetate decarboxylase and aldehyde ferredoxin oxidoreductase (e.g., selected from enzymes corresponding to Enzyme Commission (EC) numbers 4.1.1.83, 2.6.1.-, 4.1.1.-, and 1.2.7.5).

109. 109. The method of claim 108, wherein the gut microbes are selected based on their relationship with the metabolite (e.g., based on their positive correlation, negative correlation, or lack of correlation with the metabolite).

110. 110. The method of claim 109, wherein the selecting the gut microorganism comprises selecting a gut microorganism from Table 3 based on the gut microorganism's relationship to the metabolite (e.g., based on its positive correlation, negative correlation, or lack of correlation with the metabolite).

111. 111. The method of any of claims 107-110, wherein the glycan polymer is a substrate for the gut microorganisms (e.g., human gut microorganisms).

112. 112. The method of any one of claims 1 to 111, wherein the glycan polymer is a substrate for gut microbial glycosidase enzymes and promotes the growth of the gut microorganisms.

113. The method of any one of claims 1 to 112, wherein the glycan preparation is administered daily.

114. The method of any one of claims 1 to 113, wherein the glycan preparation is administered over a single treatment period.

115. The method of any one of claims 1 to 113, wherein the glycan preparation is administered over multiple treatment periods, for example, wherein the treatment periods are longer than one or both of the adjacent treatment periods, or wherein the treatment periods are shorter than one or both of the adjacent treatment periods.

116. 116. The method of any of claims 1 to 115, wherein the glycan polymer is a substrate for microbial components of the colon or intestine.

117. The method of any of claims 1 to 116, wherein the glycan polymer preparation is administered orally or rectally.

118. A method for regulating the production or level of a product (e.g., short-chain fatty acids (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, or bile acids) in a subject's body (e.g., gut (colon, intestine), blood, urine, organs (e.g., liver, kidney), brain), comprising administering to the subject (e.g., orally or rectally) an effective amount of a glycan polymer preparation sufficient to regulate the production or level of the product, wherein optionally the glycan polymer is a substrate for microbial components of the colon or intestine.

119. The method of claim 118, wherein the microbial component a) produces the product, e.g., thereby increasing the level or production of the product, b) produces a precursor or surrogate product that is converted to the product by the producer taxon, e.g., thereby increasing the level or production of the product, or c) does not produce the product but competes or antagonizes the producer taxon of the product (e.g., competes for space and / or nutrients or produces an antimicrobial substance that is toxic to the producer taxon), e.g., thereby decreasing the relative abundance of the producer taxon and decreasing the level or production of the product.

120. 120. The method of claim 119, wherein the microbial component is selected from the components in Table 2.

121. 120. The method of claim 119, wherein the microbial component is selected from the strains in Table 3.

122. 120. The method of claim 119, wherein the microbial component is selected from components comprising a glycosidase enzyme from the glycosidase family of Table 4.

123. 120. The method of claim 119, wherein the microbial component is selected from components comprising a glycosidase enzyme from the glycosidase family of any one of claims 43 to 55.

124. 122. The method of any of claims 119 or 121, wherein the product is selected from the metabolites of Table 3.

125. 120. The method of claim 119, wherein the product is an SCFA and the subject has a condition selected from the SCFA row of Table 5.

126. 120. The method of claim 119, wherein the product is ammonia and the subject has a condition selected from the ammonia row of Table 5.

127. 120. The method of claim 119, wherein the product is a TMA and the subject has a condition selected from the TMA row of Table 5.

128. 120. The method of claim 119, wherein the product is a bile acid and the subject has a condition selected from the bile acid row of Table 5.

129. 120. The method of claim 119, wherein the product is a uremic solute (e.g., p-cresol or indole) and the subject has a condition selected from the p-cresol or indole row of Table 5.

130. 120. The method of claim 118 or 119, further comprising obtaining the identity of the microorganism (e.g., bacterial taxon) that regulates, e.g., produces, the product.

131. The method of any one of claims 118 to 130, further comprising selecting the glycan preparation based on its ability to regulate the microbial component.

132. The method of any one of claims 118 to 130, wherein the glycan preparation is a substrate for a glycosidase enzyme of the microbial component, for example, the microbial component and the product are from the same row of Table 3.

133. The method of any preceding claim, wherein the subject is a human, such as a human patient.

134. A glycan polymer preparation, such as described herein, for use in a method according to any one of claims 1 to 133.

135. A method for selecting a glycan polymer preparation for use as a substrate for a glycosidase enzyme (e.g., a CAZy family) of a preselected human gut microorganism (e.g., selected for its glycosidase profile), comprising: a) obtaining a value for the glycosidase (e.g., CAZy family) profile of the microorganism; b) identifying, designing, or selecting a glycan polymer that can serve as a substrate for the microorganism based on the glycosidase (e.g., CAZy family) profile; c) optionally, i. Assembling a panel of human gut microorganisms (e.g., a single strain, a group of designed strains, or an ex vivo group (e.g., from a fecal sample) containing the microorganism of interest; ii. Contacting the panel of microorganisms with a test glycan preparation; iii. Evaluating the growth of the human gut microorganism (of interest); d) selecting the glycan polymer preparation.

136. 136. The method of claim 135, wherein (a) comprises finding a glycosidase (e.g., CAZy family) profile value in Table 4.

137. 136. The method of claim 135, wherein (b) comprises identifying, designing, or selecting a glycan polymer found in Table 4.

138. The method of claim 135, wherein (a) comprises finding a value for a glycosidase (e.g., CAZy family) profile in Table 4, and (b) comprises identifying, designing, or selecting a glycan polymer that is a substrate for a glycosidase (e.g., CAZy family) in the same column of Table 4, for example, the glycosidase profile of (a).

139. A glycan preparation produced or selected by the method of any of claims 135 to 138.

140. 1. A glycan polymer preparation comprising a glycan polymer, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising i) glucose, mannose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond, or ii) glucose, mannose, or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond, and wherein i) GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, GH13 subfamily 36, GH113, or GH112. CAZy family, ii) GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13, GH13 subfamily 9, GH13 subfamily 31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77 or GH94 CAZy family, iii) GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8 or GH13 subfamily 14 GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy family, or iv) GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy family. A glycan polymer preparation that is a substrate for one or more, for example, two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family.

141. 1. A glycan polymer preparation, e.g., the preparation comprises at least about 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising glycan polymers comprising i) xylose, arabinose, fucose, or rhamnose subunits, or combinations thereof, and at least one alpha-glycosidic bond, or ii) xylose, arabinose, fucose, or rhamnose subunits, or combinations thereof, and at least one beta-glycosidic bond, wherein i) the preparation comprises GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, or GH13 subfamily 14. GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy family, or ii) GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy family. A glycan polymer preparation that is a substrate for one or more, for example, two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family.

142. 1. A glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, and comprises glycan polymers comprising i) glucose or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond, or ii) glucose or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond, and CAZy family, or ii) GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT 10, GH77, GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, Gh28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, GH24 A glycan polymer preparation that is a substrate for one or more, for example, two, three, four or more, human intestinal microbial glycosidase enzymes selected from the CAZy family.

143. 1. A glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising glycan polymers comprising arabinose, galactose, xylose, or glucose subunits, or a combination thereof, and at least one alpha-glycosidic linkage, and wherein the glycan polymers are selected from the group consisting of i) GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family; or ii) GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92. A glycan polymer preparation that is a substrate for one or more, for example, two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family.

144. A glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, and comprises glycan polymers comprising glucose and at least one alpha-glycosidic bond, and which are substrates for one or more, e.g., two, three, four, or more, human gut microbial glycosidase enzymes selected from i) GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family; or ii) GH23, GH24, or GH33 CAZy family.

145. 1. A glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising glycan polymers comprising i) glucose or xylose subunits, or a combination thereof, and at least one alpha-glycosidic bond, or ii) glucose or xylose subunits, or a combination thereof, and at least one beta-glycosidic bond, wherein i) GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family, or ii) GH2, GH31, GH23, GH13, or GH24. A glycan polymer preparation that is a substrate for one or more, for example, two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family.

146. Glica 1. A glycan polymer preparation, e.g., the preparation comprises at least 0.5, 1, 2, 5, 10, 50, or 100 kg, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising glycan polymers comprising glucose, xylose, arabinose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic linkage, and wherein the glycan polymer preparation is selected from the group consisting of i) GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family, or ii) GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106. A glycan polymer preparation that is a substrate for one or more, for example, two, three, four, or more, human gut microbial glycosidase enzymes selected from the CAZy family.

147. The glycan preparation of any one of claims 140 to 146, wherein the glycan preparation is formulated as a pharmaceutical composition, medical food, dietary supplement, food ingredient, or therapeutic nutritional product, for example, wherein the formulation step comprises dividing the preparation into multiple dosage forms or portions.

148. The glycan preparation of any one of claims 140 to 147, formulated for oral administration as a liquid.

149. The glycan preparation of claim 148, wherein the liquid is a beverage, a syrup, an aqueous solution or an aqueous suspension.

150. The glycan preparation of any one of claims 140 to 147, formulated for oral administration as a solid.

151. 151. The glycan preparation of claim 150, wherein the solid is a tablet, pill, capsule, lozenge, candy, or powder.

152. The glycan preparation of claim 150, wherein the solid is a solid food product.

153. The glycan preparation of claim 151, wherein the powder is formulated for reconstitution in an aqueous solution prior to oral administration.

154. The glycan preparation of any one of claims 140 to 147, formulated for rectal administration as a solid or liquid.

155. The glycan preparation of claim 154, formulated as an enema or suppository.

156. The glycan preparation according to any one of claims 140 to 155, formulated as a delayed release or time-controlled system.

157. The glycan preparation of any one of claims 140 to 156, further comprising a pharmaceutically acceptable carrier or excipient.

158. The glycan preparation of any one of claims 140 to 156, further comprising a food-acceptable carrier or excipient.

159. The glycan preparation of any one of claims 140 to 158, further comprising a second therapeutic agent.

160. The glycan preparation of any one of claims 140 to 159, further comprising a preparation of intestinal microorganisms (e.g., human intestinal microorganisms).

161. The glycan preparation of claim 160, wherein the glycan polymer is a substrate for the intestinal microorganisms.

162. The glycan preparation of claim 161, wherein the glycan polymer is a substrate for a gut microorganism glycosidase enzyme and promotes the growth of the gut microorganism.

163. A unit dosage form comprising the glycan preparation of any one of claims 140 to 162.

164. 164. The unit dosage form of claim 163, formulated for enteral, nasal, oral, or rectal administration, or tube feeding.

165. The unit dosage form, for example, the glycan polymer preparation component of the unit dosage form, has a caloric value of about 0.01 kcal to about 1 kcal, 0.1 kcal to 5 kcal, 0.01 kcal to 10 kcal, or 0.1 kcal to 10 kcal. The unit dosage form of claim 163 or 164.

166. 166. The unit dosage form of any one of claims 163 to 165, formulated for timed and / or targeted release in the colon or large intestine.

167. A pharmaceutical composition comprising a glycan preparation according to any one of claims 140 to 162.

168. A set of pharmaceutical compositions each comprising a glycan polymer preparation or portion thereof according to any one of claims 140 to 162, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

169. A medical food comprising a glycan preparation according to any one of claims 140 to 162.

170. A set of portions of medical foods each comprising a glycan polymer preparation or portion thereof according to any one of claims 140 to 162, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

171. A dietary supplement comprising a glycan preparation according to any one of claims 140 to 162.

172. A set of portions of a dietary supplement each comprising a glycan polymer preparation or portion thereof according to any one of claims 140 to 162, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

173. A food ingredient comprising a glycan preparation according to any one of claims 140 to 162.

174. A set of portions of food ingredients each comprising a glycan polymer preparation or portion thereof according to any one of claims 140 to 162, wherein collectively, the set comprises at least 0.1, 0.5, 1, 2, 5, 10, or 100 kilograms of the preparation.

175. A method for producing a co-preparation, comprising the steps of: providing a preparation of human gut microorganisms; providing a glycan polymer preparation according to any one of claims 140 to 162, wherein the glycan polymer is a substrate for the human gut microorganisms; and combining the human gut microorganisms with the glycan polymer.

176. 176. The method of claim 175, wherein the human gut microbes are selected from the microbes listed in Table 2.

177. 176. The method of claim 175, wherein the human gut microbes are selected from the microbes listed in Table 3.

178. The method of any one of claims 175 to 177, further comprising identifying the CAZy family profile of the human intestinal microorganism, and selecting a substrate glycan polymer preparation based on the identified CAZy family profile of the human intestinal microorganism.

179. 179. The method of any one of claims 175-178, further comprising formulating the co-preparation for oral, nasal or rectal delivery or tube feeding.

180. 180. The method of any one of claims 175-179, further comprising formulating the co-formulation as a time-release formulation.

181. 181. The method of claim 180, wherein release of the preparation occurs in the colon or large intestine.

182. 182. The method of any one of claims 175-181, wherein greater than about 50%, 60%, 70%, 80%, 90%, 95% or 98% of the microorganisms in the preparation are viable after passage through the stomach (e.g., upon reaching the colon or large intestine).

183. 183. The method of any one of claims 175-182, wherein more than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% of the microorganisms of the preparation implant after release in the colon or large intestine.

184. The method of any one of claims 175 to 183, wherein the glycan polymer preparation is produced by glycosidase-directed synthesis, selecting one or more glycosidases from the CAZy family profile identified for the synthesis of the glycan polymer.

185. The method of any one of claims 175 to 183, wherein the glycan polymer preparation is synthesized and designed using a non-enzymatic polymer catalyst based on the identified CAZy family profile.

186. 186. The method of any one of claims 175-185, further comprising formulating said co-preparation into a pharmaceutical composition.

187. 163. A synbiotic co-preparation comprising a preparation of human gut microbes and a preparation of glycan polymers according to any one of claims 140 to 162.

188. 188. The synbiotic co-preparation of claim 187, further comprising a pharmaceutically acceptable excipient or carrier.

189. 189. The synbiotic co-preparation of claim 187 or 188, formulated as a unit dosage form for nasal, oral, gastric or rectal delivery.

190. 190. The synbiotic co-preparation of any one of claims 187 to 189, formulated to protect the human gut microorganisms of the preparation from inactivation by stomach acid.

191. 191. A method of transplanting human gut microbes into the colon or large intestine of a human subject in need thereof, comprising administering to said subject the synbiotic co-preparation of any one of claims 187 to 190 in an amount and for a time effective to transplant said human gut microbes.

192. 192. The method of claim 191, wherein the human subject has a dysbiosis of the gut microbiota, e.g., has been subjected to a treatment or exposure that causes such dysbiosis, e.g., the human subject has been identified as having received said treatment or exposure.

193. 193. The method of claim 191 or 192, wherein the human subject is undergoing antibiotic treatment.

194. 193. The method of claim 191 or 192, wherein the human subject has not received antibiotic treatment.

195. 195. The method of any one of claims 191 to 194, wherein the microbiota of the intestine (e.g., colon or large intestine) is stable (e.g., no significant change in the relative abundance of taxa).

196. 195. The method of any one of claims 191 to 194, wherein the microbiota of the intestine (e.g., colon or large intestine) is unstable (e.g., there is a significant shift in the relative abundance of taxa).

197. 197. The method of any one of claims 191-196, wherein the extent of engraftment is determined by analysis, for example by 16S, quantitative culture, or qPCR, before or after administration of the synbiotic co-formulation.

198. 198. The method of any one of claims 191 to 197, wherein the extent of engraftment is determined through a comparison of the number of organisms administered to the subject in the synbiotic co-formulation with the number of organisms recoverable from the gut of the subject, for example through quantitative culture or qPCR.

199. The human subject is suffering from a disease or disorder listed in Table 5, e.g., acute pouchitis, allergic disease, AIDS, atherosclerosis, asthma, atopic dermatitis, autism spectrum disorder, chronic functional constipation, celiac disease, chronic atrophic gastritis, chronic pouchitis, Clostridium difficile, 200. The method of any one of claims 191-198, wherein the patient has chronic obstructive pulmonary disease (COPD), chronic obstructive pulmonary disease (COPD), celiac disease, colorectal adenoma, colorectal cancer, Crohn's disease, cystic fibrosis, depression, diabetes mellitus (Type I), diabetes mellitus (Type II), diarrhea, eczema, enterostomy, familial Mediterranean fever, food intolerance, graft versus host disease (GvHD), hepatic encephalopathy, hypertension, inflammatory bowel disease, irritable bowel disease, irritable bowel disease-constipation (IBS-C), lung cancer, microscopic colitis, multiple sclerosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), obesity-related asthma, Parkinson's disease (PD), radiation-induced acute bowel symptoms, dysentery, short bowel syndrome, spinal cord injury-associated bowel dysfunction, systemic inflammatory response syndrome, systemic lupus erythematosus, or ulcerative colitis.

200. The human subject has a disease or disorder listed in Table 5, e.g., atherosclerosis, cardiovascular disease, cardiovascular risk in HIV.

199. The method of any one of claims 191 to 198, wherein the patient has: coronary heart disease, carotid atherosclerosis, chronic heart disease, chronic heart failure, chronic kidney disease, chronic vascular disease, colorectal cancer, coronary heart disease, coronary artery disease (CAD), diabetes (type II), end-stage renal disease, HIV, inflammatory bowel disease, ischemic stroke, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), obesity, radiation-induced acute intestinal symptoms (RIAIS), or stroke.

201. 199. The method of any one of claims 191-198, wherein the human subject has a disease or disorder listed in Table 5, e.g., chronic kidney disease, Helicobacter pylori infection, hepatic encephalopathy, or cirrhosis with minimal hepatic encephalopathy (MHE).

202. A method of treating a subject having a dysbiosis, comprising administering a composition comprising a glycan polymer preparation and a microbial preparation described herein in an amount effective to treat the dysbiosis.

203. 203. The method of claim 202, wherein the microorganism is a spore-forming microorganism.

204. 204. The method of claim 202 or 203, wherein the glycan polymer preparation comprises xylose, arabinose, glucose, galactose, or a combination thereof.

205. The glycan polymers of the glycan polymer preparation, or at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% (by weight or number) of the glycan polymers, have one or more (e.g., 2, 3, 4, 5, or 6) of the properties listed in Table 1, and optionally include: a. glycan polymers comprising xylose or arabinose subunits, or a combination thereof, and at least one alpha-glycosidic linkage; b. glycan polymers comprising xylose or arabinose subunits, or a combination thereof, and at least one beta-glycosidic linkage; c. glycan polymers comprising galactose, xylose, or arabinose subunits, or a combination thereof, and at least one alpha-glycosidic linkage; d. glycan polymers comprising galactose, xylose, or arabinose subunits, or a combination thereof, and at least one beta-glycosidic linkage; e.g., glycan polymers comprising galactose, xylose, or arabinose subunits, or a combination thereof, and at least one beta-glycosidic linkage; The method of any one of claims 202 to 204, wherein the glycan polymer is selected from: a. a glycan polymer comprising glucose, xylose, or arabinose subunits, or a combination thereof, and at least one alpha-glycosidic bond; b. a glycan polymer comprising glucose, xylose, or arabinose subunits, or a combination thereof, and at least one beta-glycosidic bond; c. a glycan polymer comprising xylose, arabinose, glucose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond; d. a glycan polymer comprising xylose, arabinose, glucose, or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond; or e.g., a glycan polymer comprising xylose, arabinose, glucose, or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond.

206. The method of any one of claims 202 to 205, wherein the glycan polymers, or at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% (by weight or number) of the glycan polymers of the glycan polymer preparation are substrates for glycosidase enzymes.

207. 207. The method of any one of claims 202 to 206, wherein the glycosidase enzyme is present in spore-forming human intestinal microorganisms.

208. the glycan polymer is GT5, GT35, GT3, GH97, GH95, GH92, GH89, GH88, GH78, GH77, GH57, GH51, GH43 subfamily 34, GH43 subfamily 24, GH43 subfamily 10, GH42, GH36, GH35, GH33, GH32, GH31, GH3, GH29, GH28, GH27, GH24, GH20, GH2, GH16, GH133, GH130, GH13 subfamily 8, GH13 subfamily 38, GH13 subfamily 14, GH13, GH123, GH115, GH109, ​​or GH105 208. The method of any one of claims 202 to 207, which is a substrate for a glycosidase enzyme of one of the CAZy family.

209. 209. The method of any one of claims 202-208, wherein the microorganism is any one of the microorganisms in Table 19, column 1.

210. 209. The method of any one of claims 202-208, wherein the microorganism is any one of the microorganisms in Table 20, column 1.

211. 209. The method of any one of claims 202-208, wherein the microorganism is any one of the microorganisms in Table 21, column 1.

212. The microorganism is any one of the microorganisms in Table 19, column 1, and the glycan preparation is any one of Table 19, column 3, Table 19, column 4, Table 19, column 5, Table 19, column 6, Table 19, column 7, Table 19, column 8, Table 19, column 9, Table 19, column 10. The method of any one of claims 202 to 208.

213. The method of any one of claims 202 to 208, wherein the microorganism is any one of the microorganisms in Table 20, column 1, and the glycan preparation is any one of Table 20, column 2, Table 20, column 3, Table 20, column 4, Table 20, column 5, Table 20, column 6, Table 20, column 7, Table 20, column 8, or Table 20, column 9.

214. The method of any one of claims 202 to 208, wherein the microorganism is any one of the microorganisms in Table 21, column 1, and the glycan preparation is any one of Table 21, column 2, Table 21, column 3, Table 21, column 4, Table 21, column 5, Table 21, column 6, Table 21, column 7, Table 21, column 8, or Table 21, column 9.

215. The glycan polymer preparation described herein comprises a glycan polymer that is a substrate for a human gut microbial glycosidase enzyme of a spore-forming microorganism (e.g., a spore-forming bacterial taxon).

216. A glycan polymer preparation, optionally, e.g., said preparation comprises at least about 0.5, 1, 2, 5, 10, 50, or 100 kg, and / or further optionally, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% pure, comprising: a. xylose or arabinose subunits, or combinations thereof, and at least one alpha-glycosidic bond; b. xylose or arabinose subunits, or combinations thereof, and at least one beta-glycosidic bond; c. galactose, xylose, or arabinose subunits, or combinations thereof, and at least one alpha-glycosidic bond; d. galactose, xylose, or arabinose subunits, or combinations thereof, and at least one beta-glycosidic bond; e.g., glucose, xylose, or arabinose subunits, or combinations thereof, and at least one alpha-glycosidic bond; f. g. glucose, xylose, or arabinose subunits, or combinations thereof, and at least one beta-glycosidic bond, g. xylose, arabinose, glucose, or galactose subunits, or combinations thereof, and at least one alpha-glycosidic bond, h. xylose, arabinose, glucose, or galactose subunits, or combinations thereof, and at least one beta-glycosidic bond, or combinations thereof, and at least one beta-glycosidic bond, and the glycan polymers include GT5, GT35, GT3, GH97, GH95, GH92, GH89, GH88, GH78, GH77, GH57, GH51, GH43 subunits. Family 34, GH43 subfamily 24, GH43 subfamily 10, GH42, GH36, GH35, GH33, GH32, GH31, GH3, GH29, GH28, GH27, GH24, GH20, GH2, GH16, GH133, GH130, GH13 subfamily 8, GH13 subfamily 38, GH13 subfamily 14, GH13, GH123, GH115, GH109, ​​or GH105A glycan polymer preparation that is a substrate for a human gut microbial glycosidase enzyme of the CAZy family.

217. 217. The glycan polymer preparation of claim 215 or 216, wherein the microorganism is any one of the microorganisms in Table 19, column 1.

218. 217. The glycan polymer preparation of claim 215 or 216, wherein the microorganism is any one of the microorganisms in Table 20, column 1.

219. 217. The glycan polymer preparation of claim 215 or 216, wherein the microorganism is any one of the microorganisms in Table 21, column 1.

220. The glycan polymer preparation of any one of claims 215 to 219, wherein the microorganism is any one of the microorganisms in Table 19, column 1, and the glycan preparation is any one of Table 19, column 3, Table 19, column 4, Table 19, column 5, Table 19, column 6, Table 19, column 7, Table 19, column 8, Table 19, column 9, Table 19, column 10.

221. The glycan polymer preparation of any one of claims 215 to 219, wherein the microorganism is any one of the microorganisms in Table 20, column 1, and the glycan preparation is any one of Table 20, column 2, Table 20, column 3, Table 20, column 4, Table 20, column 5, Table 20, column 6, Table 20, column 7, Table 20, column 8, or Table 20, column 9.

222. The glycan polymer preparation of any one of claims 215 to 219, wherein the microorganism is any one of the microorganisms in Table 21, column 1, and the glycan preparation is any one of Table 21, column 2, Table 21, column 3, Table 21, column 4, Table 21, column 5, Table 21, column 6, Table 21, column 7, Table 21, column 8, or Table 21, column 9.

223. A method for producing a co-preparation, comprising: providing a preparation of spore-forming microorganisms (e.g., spore-forming human gut microorganisms); providing a glycan polymer preparation (described herein) in which the glycan polymer is a substrate for the spore-forming microorganisms; and combining the preparation of the spore-forming microorganisms with the glycan polymer preparation.

224. The glycan polymer comprises: a. xylose or arabinose subunits, or a combination thereof, and at least one alpha-glycosidic linkage; b. xylose or arabinose subunits, or a combination thereof, and at least one beta-glycosidic linkage; c. galactose, xylose, or arabinose subunits, or a combination thereof, and at least one alpha-glycosidic linkage; d. galactose, xylose, or arabinose subunits, or a combination thereof, and at least one beta-glycosidic linkage; e. glucose, xylose, or arabinose subunits, or a combination thereof, and at least one alpha-glycosidic linkage; f. glucose, xylose, or arabinose subunits, or a combination thereof, and at least one beta-glycosidic linkage; g. xylose, arabinose, glucose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic linkage; or h.

224. The method of claim 223, comprising one of xylose, arabinose, glucose, or galactose subunits, or a combination thereof and at least one beta-glycosidic bond, or a combination thereof and at least one beta-glycosidic bond.

225. The glycan polymer is selected from the group consisting of GT5, GT35, GT3, GH97, GH95, GH92, GH 89, GH88, GH78, GH77, GH57, GH51, GH43 subfamily 34, GH43 subfamily 24, GH43 subfamily 10, GH42, GH36, GH35, GH33, GH32, GH31, GH3, GH29, GH28, GH27, GH24, GH20, GH2, GH16, GH133, GH130, GH13 subfamily 8, GH13 subfamily 38, GH13 subfamily 14, GH13, GH123, GH115, GH109, ​​or GH105 CAZy family glycosidase enzymes.

226. 226. The method of any one of claims 223-225, wherein the microorganism is any one of the microorganisms in Table 19, column 1.

227. 226. The method of any one of claims 223-225, wherein the microorganism is any one of the microorganisms in Table 20, column 1.

228. 226. The method of any one of claims 223-225, wherein the microorganism is any one of the microorganisms in Table 21, column 1.

229. The method of any one of claims 223 to 228, wherein the microorganism is any one of the microorganisms in Table 19, column 1, and the glycan preparation is any one of Table 19, column 3, Table 19, column 4, Table 19, column 5, Table 19, column 6, Table 19, column 7, Table 19, column 8, Table 19, column 9, Table 19, column 10.

230. The method of any one of claims 223 to 228, wherein the microorganism is any one of the microorganisms in Table 20, column 1, and the glycan preparation is any one of Table 20, column 2, Table 20, column 3, Table 20, column 4, Table 20, column 5, Table 20, column 6, Table 20, column 7, Table 20, column 8, or Table 20, column 9.

231. The method of any one of claims 223 to 228, wherein the microorganism is any one of the microorganisms in Table 21, column 1, and the glycan preparation is any one of Table 21, column 2, Table 21, column 3, Table 21, column 4, Table 21, column 5, Table 21, column 6, Table 21, column 7, Table 21, column 8, or Table 21, column 9.

232. 232. The method of any one of claims 223-231, further comprising formulating the co-preparation for oral, nasal or rectal delivery or tube feeding.

233. 233. The method of any one of claims 223-232, further comprising formulating the co-formulation as a time-release formulation.

234. 234. The method of claim 233, wherein release of the preparation occurs in the colon or large intestine.

235. 235. The method of any one of claims 223-234, wherein greater than about 50%, 60%, 70%, 80%, 90%, 95% or 98% of the microorganisms in the preparation are viable after passage through the stomach (e.g., upon reaching the colon or large intestine).

236. 236. The method of any one of claims 223-235, wherein more than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60% or 75% of the microorganisms in the preparation implant after release in the colon or large intestine.

237. The method of any one of claims 223 to 236, wherein the glycan polymer preparation is produced by glycosidase-directed synthesis, selecting one or more glycosidases from the CAZy family profile identified for the synthesis of the glycan polymer.

238. The method of any one of claims 223 to 237, wherein the glycan polymer preparation is synthesized and designed using a non-enzymatic polymer catalyst based on the identified CAZy family profile.

239. 239. The method of any one of claims 223-238, further comprising formulating said co-preparation into a pharmaceutical composition.

240. 240. A synbiotic co-preparation comprising a preparation of human gut microbes and a preparation of glycan polymers according to any one of claims 223 to 239.

241. 241. The synbiotic co-preparation of claim 240, further comprising a pharmaceutically acceptable excipient or carrier.

242. 242. The synbiotic co-preparation of claim 240 or 241, formulated as a unit dosage form for nasal, oral, gastric or rectal delivery.

243. 243. The synbiotic co-preparation of any one of claims 240 to 242, formulated to protect the human gut microorganisms of the preparation from inactivation by stomach acid.

244. 244. A method of transplanting human gut microbes into the colon or large intestine of a human subject in need thereof, comprising administering to said subject the synbiotic co-formulation of any one of claims 240-243 in an amount and for a time effective to transplant said human gut microbes.

245. The method of claim 244, wherein the human subject has a dysbiosis of the intestinal microbiota, e.g., has undergone a treatment (e.g., an antibacterial treatment, a cancer treatment, etc.) or exposure (e.g., exposure to a pathogen such as a bacterial pathogen (e.g., C. diff.)) that causes such dysbiosis, optionally, e.g., the human subject has been identified as having undergone the treatment or exposure.

246. The method of claim 244 or 245, wherein the human subject is undergoing antibiotic treatment.

247. The method of claim 244 or 245, wherein the human subject has not received antibiotic treatment.

248. 248. The method of any one of claims 244 to 247, wherein the microbiota of the intestine (e.g., colon or large intestine) is stable (e.g., no significant change in the relative abundance of taxa).

249. 248. The method of any one of claims 244 to 247, wherein the microbiota of the intestine (e.g. the colon or large intestine) is unstable (e.g. there is a significant shift in the relative abundance of taxa).

250. 250. The method of any one of claims 244-249, wherein the extent of engraftment is determined by analysis, for example by 16S, quantitative culture, or qPCR, before or after administration of the synbiotic co-preparation.

251. 251. The method of any one of claims 244 to 250, wherein the extent of engraftment is determined through a comparison of the number of organisms administered to the subject in the synbiotic co-formulation with the number of organisms recoverable from the gut of the subject, for example through quantitative culture or qPCR.

252. The method of any embodiment described herein.

253. The composition of any embodiment described herein.

254. A method for producing a preparation of a glycan polymer, e.g., a glycan polymer that is a substrate for a glycosidase enzyme present in a human gut microorganism, comprising the steps of: providing a plurality of glycan subunits, e.g., sugar monomers or sugar dimers, suitable for producing the glycan polymer; and contacting the plurality of glycan subunits with a glycosidase enzyme molecule, e.g., derived from a human gut microorganism, under conditions that result in incorporation of the glycan subunits into the glycan polymer (e.g., by a condensation reaction), thereby , producing a glycan polymer preparation that is a substrate for human gut microorganisms, wherein optionally i) the glycan polymer preparation comprises at least about 0.25, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400, or 500 kilograms of glycan polymers, and / or ii) the glycan polymer preparation is produced at a yield of at least about 15%, 30%, 45%, 60%, or about 75% (determined on a weight / weight basis as a percentage of the input glycan subunits).

255. The method of claim 254, wherein the human intestinal microorganism from which the glycosidase enzyme molecule is derived is of the same taxonomic group, e.g., phylum, order, family, genus or species, as the human intestinal microorganism for which the glycan polymer is a substrate.

256. The method of claim 254, wherein the human intestinal microorganism from which the glycosidase enzyme molecule is derived is of a first taxonomic group, such as a phylum, order, family, genus, or species, and the human intestinal microorganism from which the glycan polymer is a substrate is of a second taxonomic group, such as a phylum, order, family, genus, or species.

257. 257. The method of any of claims 254 to 256, further comprising formulating the glycan polymer preparation into a pharmaceutical composition, a medical food, a dietary supplement, a food ingredient, or a therapeutic nutritional product.

258. 258. The method of any of claims 254 to 257, further comprising dividing the preparation into a plurality of portions, e.g., unit doses or formulations, e.g., for enteral administration, e.g., oral or rectal, or for tube feeding, e.g., nasal, oral, or gastric tube feeding, e.g., dividing the preparation into at least 10, 100, or 1,000 portions.

259. 259. The method of claim 258, wherein the plurality of portions differ by no more than 0.5%, 1%, 2%, 5%, 10%, or 20% by weight with respect to the amount of glycan polymer present in the portions.

260. 260. The method of any one of claims 254 to 259, comprising combining the preparation with an excipient or carrier.

261. 261. The method of claim 260, wherein the excipient or carrier is a pharmaceutically acceptable excipient or carrier.

262. 261. The method of claim 260, wherein the excipient or carrier is a food product.

263. 263. The method of any one of claims 254-262, wherein the glycosidase enzyme and the glycosidase enzyme molecule are independently selected from Table 4 (Column 2), 23 (Column A), 24 (Column A), or 22 (Column 1).

264. 264. The method of any one of claims 254-263, wherein the amino acid sequence encoding the glycosidase enzyme shares at least 95%, 97%, or 99% sequence identity with an amino acid sequence encoded by any one of SEQ ID NOs: 1-124.

265. 265. The method of any one of claims 254-264, wherein the amino acid sequence encoding the glycosidase enzyme shares at least 95%, 97%, or 99% sequence identity with an amino acid sequence encoded by any one of SEQ ID NOs: 12, 18, 31, 38, 39, 48, 56, 57, 64, 68, 72, 83, 84, 92, 93, 99, 104, 110, and 117 of Table 23 or 24.

266. 266. The method of any one of claims 254-265, wherein the amino acid sequence encoding the glycosidase enzyme molecule shares at least 95%, 97%, or 99% sequence identity with an amino acid sequence encoded by any one of SEQ ID NOs: 1-124.

267. 267. The method of any one of claims 254-266, wherein the amino acid sequence encoding the glycosidase enzyme molecule shares at least 95%, 97%, or 99% sequence identity with an amino acid encoded by any one of SEQ ID NOs: 12, 18, 31, 38, 39, 48, 56, 57, 64, 68, 72, 83, 84, 92, 93, 99, 104, 110, and 117 of Table 23 or 24.

268. 268. The method of any one of claims 262 to 267, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is other than from Bifidobacterium.

269. 268. The method of any one of claims 262 to 267, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is other than from Lactobacillus.

270. 270. The method of any one of claims 254 to 269, wherein the glycosidase enzyme and the glycosidase enzyme molecule are of the same human intestinal microbial origin.

271. 271. The method of claim 270, wherein the glycosidase enzyme and the glycosidase enzyme molecule are selected from Table 4 (column 2), 23 (column A), 24 (column A), or 22 (column 1).

272. the amino acid sequences of the glycosidase enzyme and the glycosidase enzyme molecule are at least 95%, 97%, or 272. The method of any one of claims 254 to 271, sharing 99% sequence identity.

273. 273. The method of claim 272, wherein the nucleic acid sequence encoding the amino acid sequence is one of SEQ ID NOs: 1-124.

274. 273. The method of claim 272, wherein the nucleic acid sequence encoding the amino acid sequence is one of SEQ ID NOs: 12, 18, 31, 38, 39, 48, 56, 57, 64, 68, 72, 83, 84, 92, 93, 99, 104, 110, and 117 of Table 23 or 24.

275. 273. The method of claim 272, wherein the glycosidase enzyme and the glycosidase enzyme molecule are selected from Table 4 (column 2), 23 (column A), 24 (column A), or 22 (column 1).

276. 276. The method of any one of claims 272 to 275, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is other than from Bifidobacterium.

277. 276. The method of any one of claims 272 to 275, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is other than from Lactobacillus.

278. 278. The method of any one of claims 254-277, wherein both the glycosidase enzyme and the glycosidase enzyme molecule are from the same CAZy family (e.g., the same GH family (e.g., one or more of GH1-GH135) and / or GT family (e.g., one or more of GT1-GT101), e.g., those listed in Table 4 (column 1), 23 (column C), 24 (column C), or 22 (column 1).

279. 279. The method of any one of claims 254 to 278, comprising obtaining the identity (e.g., taxonomic, 16s) of the human gut microorganism, and optionally its glycosidase profile (e.g., CAZy family profile).

280. 279. The method of any one of claims 254-279, wherein said human gut microbes are selected from the microbial taxa of phylum (column 1), class (column 2) or genus (column 3) listed in Table 2.

281. 280. The method of any one of claims 254-279, wherein the human gut microbes are selected from the microbial taxon of strain (column 1) or phylum (column 2) listed in Table 3.

282. 280. The method of any one of claims 254-279, wherein the human gut microbes are selected from the microbial taxon of genera listed in Table 4, column 3.

283. 280. The method of any one of claims 254-279, wherein the human gut microbes are selected from the microorganisms listed in Table 22, column 1.

284. 280. The method of any one of claims 254-279, wherein the human gut microbes are selected from the microbial taxa listed in Table 19, columns 1 and 2 (Spore Fungi).

285. 280. The method of any one of claims 254-279, wherein the human gut microbes are selected from the microbial taxa listed in Table 20, column 1 (Spore Fungi).

286. 280. The method of any one of claims 254 to 279, wherein the human gut microorganisms are selected from the microorganisms listed in Table 21, column 1 (spore fungi).

287. The method of any one of claims 254 to 286, wherein the human intestinal microorganism is other than Bifidobacterium.

288. The method of any one of claims 254 to 287, wherein the human intestinal microorganism is other than Lactobacillus.

289. The method of claim 279, comprising selecting either or both of a glycosidase enzyme molecule and a glycan subunit in response to the identity of the human gut microorganism and / or its glycosidase gene profile.

290. 290. The method of any one of claims 254 to 289, wherein the glycosidase enzyme molecule (e.g., an isolated glycosidase enzyme molecule or a cell extract containing the glycosidase enzyme molecule) is disposed on, e.g., covalently or non-covalently bound to, a binding substrate (e.g., a solid surface such as the surface of a solid particle, or a matrix material such as a high MW carbon-containing molecule, e.g., agarose, cellulose, etc.).

291. 291. The method of claim 290, wherein the binding substrate is other than a bacterial cell.

292. 292. The method of any one of claims 254 to 291, wherein the contacting step comprises a cell-free process.

293. 293. The method of any one of claims 254 to 292, wherein the human intestinal microorganism is a bacterium.

294. The method of any one of claims 254 to 293, further comprising obtaining a value for a parameter of the preparation, such as a physical parameter, for example, a molecular weight, for example, an average molecular weight or molecular weight distribution, a glycan subunit composition or purity, or a parameter of a biological property, for example, the ability to regulate the growth of the human gut microorganism, the ability to regulate a microbial metabolic product produced by the microorganism, for example, in an ex vivo assay, or the ability to regulate a biomarker, for example, an inflammatory or immune biomarker, a toxic or waste compound, a bacterial compound, for example, in a human subject.

295. 295. The method of claim 294, comprising the step of performing an assay to obtain said value.

296. 295. The method of claim 294, comprising obtaining the value from another party.

297. The method of any one of claims 294 to 296, wherein the value is compared to a reference value to evaluate the glycan preparation, for example, for suitability for use, for example, therapeutic use.

298. 300. The method of any one of claims 254 to 297, wherein the glycosidase enzyme is encoded by a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124.

299. 300. The method of any one of claims 254-298, wherein the glycosidase enzyme is encoded by a nucleic acid sequence selected from one or more of SEQ ID NOs: 12, 18, 31, 38, 39, 48, 56, 57, 64, 68, 72, 83, 84, 92, 93, 99, 104, 110, and 117.

300. 300. The method of any one of claims 254-299, wherein the glycosidase enzyme molecule is encoded by a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124.

301. 300. The method of any one of claims 254-300, wherein the glycosidase enzyme molecule is encoded by a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence selected from one or more of SEQ ID NOs: 12, 18, 31, 38, 39, 48, 56, 57, 64, 68, 72, 83, 84, 92, 93, 99, 104, 110, and 117.

302. 302. The method of any one of claims 254 to 301, wherein the glycosidase enzyme and / or the glycosidase enzyme molecule is derived from a human intestinal bacterium other than Bifidobacterium.

303. 303. The method of any one of claims 254 to 302, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is derived from a human intestinal bacterium other than Lactobacillus.

304. 304. The method of any one of claims 254 to 303, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is other than alpha-galactosidase.

305. 305. The method of any one of claims 254 to 304, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is other than beta-galactosidase.

306. 306. The method of any one of claims 254-305, wherein the glycosidase enzyme and / or glycosidase enzyme molecule is other than i) alpha-galactosidase; ii) beta-galactosidase, iii) alpha-glucosidase iv) beta-glucosidase, v) alpha-xylosidase, vi) beta-xylosidase, vii) alpha-mannosidase, viii) beta-mannosidase, ix) alpha-fructofuranosidase, and / or x) beta-fructofuranosidase, or other than any combination (e.g., any two, three, four, five, six, seven, or eight) of i), ii), iii), iv), v), vi), vii), viii), viii), ix), and x).

307. 307. The method of any one of claims 254 to 306, wherein the glycan subunit is a sugar monomer selected from glucose, galactose, mannose, fructose, fucose, rhamnose, xylose, and arabinose.

308. 308. The method of any one of claims 254 to 307, wherein the glycan unit is a sugar dimer selected from sucrose, maltose, gentiobiose, lactulose, lactose, raffinose, melibiose, xylobiose, arabinobiose, fructobiose, turanose, cellobiose, mannobiose, galactobiose, sophorose, laminaribiose, and chitobiose.

309. 309. The method of any one of claims 254-308, wherein the glycan unit is a sugar dimer selected from sucrose, isomaltose, maltose, melezitose, gentiobiose, cellobiose, melibiose, raffinose, lactose, lactulose, and palatinose (e.g., those listed in Tables 23, Column E and 24, Column E).

310. The method of any one of claims 254 to 309, wherein the glycan unit is a sugar dimer other than lactose.

311. The method of any one of claims 254 to 310, wherein the glycan unit is a sugar dimer other than lactulose.

312. The method of any one of claims 254 to 311, wherein the conditions that result in the incorporation of a glycan subunit into a glycan polymer are suitable for a condensation reaction to incorporate a monomer into the glycan polymer.

313. The method of any one of claims 254 to 312, wherein the conditions resulting in the incorporation of a glycan subunit into a glycan polymer are suitable for a transglycosylation reaction (e.g., transgalactosylation, transglucosylation, transfructosylation) involving the incorporation of a monomer into the glycan polymer from a dimeric starting material.

314. The method of any one of claims 254 to 313, wherein the conditions that result in the incorporation of glycan subunits into a glycan polymer are suitable for a hydrolysis reaction.

315. The method of any one of claims 254 to 314, wherein the average degree of polymerization (DP) of the glycan preparation is at least about DP2, at least about DP3, at least about DP4, or at least DP5.

316. The method of any one of claims 254 to 315, wherein the average degree of polymerization (DP) of the glycan preparation is about DP2 to DP4, DP2 to DP5, DP2 to DP6, DP3 to DP5, or DP3 to DP6.

317. The method of any one of claims 254 to 316, wherein the average degree of polymerization (DP) of the glycan preparation is about DP2 to DP8, about DP2 to DP10, about DP3 to DP8, or about DP3 to DP10.

318. The method of any one of claims 254 to 317, wherein at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of the glycan polymers of the preparation have a DP of 2 or greater.

319. Any of claims 254 to 318, wherein at least 50%, 60%, 70%, 80%, 90% or at least 95% of the glycan polymers of the preparation have a DP of 3 or greater.

1. The method according to claim 1.

320. 320. The method of any one of claims 254-319, wherein at least 50%, 60%, 70%, 80%, 90% or at least 95% of the glycan polymers of the preparation have a DP of about 2-4, 2-5, 2-6, 2-8, 2-10, 3-5, 3-6, 3-8, or about 3-10.

321. The method of any one of claims 254 to 320, wherein the glycan polymers of the preparation have a degree of branching (DB) of 0.

322. The method of any one of claims 254 to 321, wherein at least 50%, 60%, 70%, 80%, 90% or at least 95% of the glycan polymers in the preparation are branched.

323. The method of any one of claims 254 to 322, wherein no more than 1%, 5%, 10%, 20%, 30%, 40%, or 50% of the glycan polymers of the preparation are branched.

324. The method of claim 322 or 323, wherein the branched glycan polymers of the preparation comprise one or more (e.g., 1, 2, 3, 4, or 5) branch points.

325. The method of any one of claims 254 to 324, wherein the glycan polymers of the preparation comprise alpha-glycosidic bonds, for example, at least about 90%, 95%, 98%, 99%, or 100% of the glycosidic bonds of the glycan polymers of the preparation are alpha-glycosidic bonds.

326. The method of any one of claims 254 to 325, wherein the glycan polymers of the preparation comprise beta-glycosidic bonds, for example, at least about 90%, 95%, 98%, 99%, or 100% of the glycosidic bonds of the glycan polymers of the preparation are beta-glycosidic bonds.

327. 327. The method of any one of claims 254 to 326, wherein the glycan polymers of the preparation comprise alpha- and beta-glycosidic bonds.

328. 328. The method of claim 327, wherein the alpha- to beta-glycosidic bond ratio is 1:1, 1:2, 1:3, 1:4 or 1:

5.

329. 328. The method of claim 327, wherein the beta- to alpha-glycosidic bond ratio is 1:1, 1:2, 1:3, 1:4 or 1:

5.

330. 328. The method of claim 327, wherein the beta- to alpha-glycosidic bond ratio is 1:

4.

331. The method of any one of claims 254 to 330, wherein the alpha- to beta-glycosidic bond ratio of the glycan polymers of the preparation is 0 or about 0.1:1 to 1:5, 1:1 to 1:5, or 1:1 to 1:

4.

332. The method of any one of claims 254 to 331, wherein the glycan polymers of the preparation have a beta- to alpha-glycosidic bond ratio of 0 or about 0.1:1 to 1:5, 1:1 to 1:5, or 1:1 to 1:

4.

333. 333. The method of any one of claims 254 to 332, wherein the glycan polymer comprises one or more glycan units of glucose, galactose, mannose, fructose, fucose, rhamnose, xylose, and / or arabinose.

334. The method of any one of claims 254 to 333, wherein the glycan polymer comprises one or more glycosidic bonds selected from 1,2 glycosidic bonds, 1,3 glycosidic bonds, 1,4 glycosidic bonds, 1,5 glycosidic bonds, or 1,6 glycosidic bonds.

335. The method of claim 334, wherein the glycan polymer preparation comprises at least 20%, 30%, 40%, 50% or at least 60% (mol%) of 1,4 glycosidic bonds.

336. The method of claim 334, wherein the glycan polymer preparation comprises at least 80%, 90%, at least 95%, or 100% (mol%) 1,4 glycosidic bonds.

337. The method of claim 334, wherein the glycan polymer preparation comprises at least 20%, 30%, 40%, 50% or at least 60% (mol%) of 1,6 glycosidic bonds.

338. The method of claim 334, wherein the glycan polymer preparation comprises at least 80%, 90%, at least 95%, or 100% (mol%) 1,6 glycosidic bonds.

339. The method of claim 334, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,2 glycosidic bonds.

340. The method of claim 334, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,3 glycosidic bonds.

341. The method of claim 334, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,4 glycosidic bonds.

342. The method of claim 334, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,6 glycosidic bonds.

343. The method of any one of claims 254 to 342, wherein the glycan polymer is other than galactooligosaccharides (GOS).

344. 334. The method of claim 333, wherein the glycan polymer is other than a galactose homopolymer.

345. The method of claim 333, wherein the glycan polymer preparation is less than 99%, 95%, 90%, 80%, 70%, 60%, or 50% galactose homopolymer.

346. 334. The method of claim 333, wherein the first and second most abundant glycan polymers in the preparation are other than i) galactose homopolymers and / or ii) galactose polymers having a terminal glucose.

347. 347. The method of any one of claims 254-346, wherein the glycan polymer is other than i) fructooligosaccharides (FOS), ii) galactooligosaccharides (GOS), iii) xylooligosaccharides (XOS), iv) isomaltooligosaccharides (IMOS), and v) glucooligosaccharides (GLOS), or any combination (one, two, three, four, or all) of i), ii), iii), iv) and v).

348. 348. The method of any one of claims 254-347, wherein the glycan polymer is other than i) lactosucrose, ii) lactulosucrose, iii) 2-alpha-glucosyllactose, iv) gentiooligosaccharides, v) pectin oligosaccharides, and vi) maltosylfructoside, or any combination (one, two, three, four, five, or all) of i), ii), iii), iv), v) and vi).

349. The method of any one of claims 254 to 348, wherein the plurality of glycan subunits comprises a first and a second glycan subunit, and the first and second glycan subunits have different structures.

350. 350. The method of any one of claims 254 to 349, wherein the plurality of glycan subunits comprises a first and a second glycan subunit, and the first and second glycan subunits have the same structure.

351. 351. The method of any one of claims 254-350, wherein the glycan polymer comprises glucose, mannose, or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond.

352. 352. The method of any one of claims 254-351, wherein the glycan polymer comprises glucose, mannose, or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond.

353. 353. The method of any one of claims 254-352, wherein the glycan polymer comprises xylose, arabinose, fucose, or rhamnose subunits, or a combination thereof, and at least one alpha-glycosidic bond.

354. 354. The method of any one of claims 254-353, wherein the glycan polymer comprises xylose, arabinose, fucose, or rhamnose subunits, or a combination thereof, and at least one beta-glycosidic bond.

355. 355. The method of any one of claims 254 to 354, wherein the glycan polymer comprises glucose or galactose subunits, or a combination thereof, and at least one alpha-glycosidic bond.

356. The method of any one of claims 254 to 355, wherein the glycan polymer comprises glucose or galactose subunits, or a combination thereof, and at least one beta-glycosidic bond.

357. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond, optionally the beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a glu-gal preparation); iv. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a glu-man preparation); and v. the glycan polymer preparation further comprises glycan polymers comprising galactose and mannose (e.g., a glu-gal-man preparation).

358. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a glu-gal preparation); iv. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a glu-man preparation); and v. the glycan polymer preparation further comprises glycan polymers comprising galactose and mannose (e.g., a glu-gal-man preparation).

359. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation comprises at least one beta-glycosidic bond. The method of any one of claims 254 to 306, further comprising one, two, three, or more, e.g., all, of: (i) the glycan polymer preparation further comprises a glycan polymer comprising a glycan polymer comprising a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, or a combination thereof; (ii) the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a gal-glu preparation); (iv) the glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a gal-man preparation); and (v) the glycan polymer preparation further comprises a glycan polymer comprising glucose and mannose (e.g., a gal-man-glu preparation).

360. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one beta-glycosidic linkage, optionally the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a gal-glu preparation); iv. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises glycan polymers comprising mannose (e.g., a gal-man preparation); and v. the glycan polymer preparation further comprises glycan polymers comprising glucose and mannose (e.g., a gal-glu-man preparation).

361. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage, optionally the beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); iv. The glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a man-glu preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising galactose and glucose (e.g., a man-gal-glu preparation).

307. A method according to any one of claims 254 to 306, comprising one, two, three or more, such as all, of:

362. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); iv. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a man-glu preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising galactose and glucose (e.g., a man-gal-glu preparation).

363. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage, optionally wherein the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising fucose (e.g., a gal-fuc preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a gal-man preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising fucose and mannose (e.g., a gal-fuc-man preparation).

364. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one beta-glycosidic bond, optionally the beta-1,3 glycosidic bond, the beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. the glycan polymer preparation further comprises a glycan polymer comprising fucose (e.g., a gal-fuc preparation); v. the glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a gal-man preparation); and vi. the glycan polymer preparation further comprises a glycan polymer comprising fucose and mannose (e.g., a gal-fuc-man preparation).

365. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises fucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage, optionally wherein the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a fuc-gal preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a fuc-man preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising galactose and mannose (e.g., a fuc-gal-man preparation).

366. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises fucose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-1; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a fuc-gal preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising mannose (e.g., a fuc-man preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising galactose and mannose (e.g., a fuc-gal-man preparation).

367. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation has at least one beta- The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises one, two, three, or more, e.g., all, of: iv. the glycan polymer preparation further comprises a glycan polymer comprising a glycosidic linkage, optionally wherein the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. the glycan polymer preparation further comprises a glycan polymer comprising fucose (e.g., a man-fuc preparation); v. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); and vi. the glycan polymer preparation further comprises a glycan polymer comprising galactose and fucose (e.g., a man-gal-fuc preparation).

368. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises mannose and at least one beta-glycosidic bond, optionally the beta-glycosidic bond is a beta-1,3 glycosidic bond, a beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic linkage, optionally an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. The glycan polymer preparation further comprises a glycan polymer comprising fucose (e.g., a man-fuc preparation); v. The glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a man-gal preparation); and vi. The glycan polymer preparation further comprises a glycan polymer comprising galactose and fucose (e.g., a man-gal-fuc preparation).

369. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises one, two, or three of glucose, xylose, and arabinose, and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, an alpha-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; The method of any one of claims 254-306, wherein the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage, optionally wherein the beta-glycosidic linkage is a beta-1,3 glycosidic linkage, a beta-1,4 glycosidic linkage, a beta-1,6 glycosidic linkage, or a combination thereof; iv. the glycan polymer preparation comprises glycan polymers comprising glucose; v. the glycan polymer preparation comprises glycan polymers comprising xylose; and vi. the glycan polymer preparation comprises glycan polymers comprising arabinose.

370. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises one, two, or three of glucose, xylose, and arabinose, and at least one beta-glycosidic bond, optionally the beta-1,3 glycosidic bond, beta-1,4 glycosidic bond, or a combination thereof, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising a beta-1,6 glycosidic bond; iii. The method of any one of claims 254-306, wherein the glycan polymer preparation further comprises glycan polymers comprising at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,2 glycosidic linkage, an alpha-1,3 glycosidic linkage, an alpha-1,4 glycosidic linkage, an alpha-1,6 glycosidic linkage, or a combination thereof; iv. the glycan polymer preparation comprises glycan polymers comprising glucose; v. the glycan polymer preparation comprises glycan polymers comprising xylose; and vi. the glycan polymer preparation comprises glycan polymers comprising arabinose.

371. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising an alpha-1,2 glycosidic bond, an alpha-1,4 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a glu-gal preparation); v. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a glu-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a glu-xyl preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising two or three of galactose, arabinose, and xylose.

372. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic linkages, alpha-1,4 glycosidic linkages, alpha-1,6 glycosidic linkages, or combinations thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage; iv. the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., a gal-glu preparation); v. The method of any one of claims 254-306, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a gal-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a gal-xyl preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising two or three of glucose, arabinose, or xylose.

373. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises one or two of xylose and arabinose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising an alpha-1,2 glycosidic bond, an alpha-1,4 glycosidic bond, an alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation comprises glycan polymers comprising xylose; and v. The method of any one of claims 254 to 306, wherein the glycan polymer preparation comprises one, two, three, or more, for example all, of the following: glycan polymers comprising arabinose.

374. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises arabinose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., an ara-gal preparation); iv. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., an ara-xyl preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., an ara-gal preparation). The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising galactose and xylose (e.g., an ara-gal-xyl preparation);

375. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises galactose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage; iii. the glycan polymer preparation further comprises a glycan polymer comprising arabinose (e.g., a gal-ara preparation); iv. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a gal-xyl preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a gal-xyl preparation). The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising arabinose and xylose (e.g., a gal-ara-xyl preparation);

376. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage; ii. the glycan polymer preparation further comprises a glycan polymer comprising xylose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; iii. the glycan polymer preparation further comprises a glycan polymer comprising galactose (e.g., a xyl-gal preparation); iv. the glycan polymer preparation further comprises a glycan polymer comprising arabinose (e.g., a xyl-ara preparation); and v. the glycan polymer preparation further comprises a glycan polymer comprising arabinose (e.g., a xyl-ara preparation). The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises a glycan polymer comprising galactose and arabinose (e.g., a xyl-ara-gal preparation); The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises one, two, three, or more, for example, all, of the following:

377. The method of any one of claims 254-306, wherein the glycan polymer and / or glycan polymer preparation comprises one, two, or more, e.g., all, of the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally, the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage; and iii. the glycan polymer preparation further comprises a glycan polymer comprising one, two, or three of arabinose, galactose, or xylose.

378. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, and optionally, the average degree of polymerization (DP) of the preparation is DP2-4, DP2-6, DP3-10, or DP3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic linkages, alpha-1,3 glycosidic linkages, alpha-1,4 glycosidic linkages, alpha-1,6 glycosidic linkages, or combinations thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage; and iv. the glycan polymer preparation further comprises glycan polymers comprising one, two, three, or four of galactose, mannose, arabinose, or sialic acid.

307. A method according to any one of claims 254 to 306, comprising one, two, three or more, such as all, of:

379. The method of any one of claims 254-306, wherein the glycan polymer and / or glycan polymer preparation comprises one, two, three, or more, e.g., all, of the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally, the average degree of polymerization (DP) of the preparation is DP2-4, DP2-6, DP3-10, or DP3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage; iii. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a glu-xyl preparation); and iv. the glycan polymer preparation further comprises a glycan polymer comprising one, two, or three of mannose, arabinose, or galactose.

380. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one beta-glycosidic bond, and optionally, the average degree of polymerization (DP) of the preparation is DP2-4, DP2-6, DP3-10, or DP3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, and optionally, the alpha-glycosidic bond is an alpha-1,3 glycosidic bond; iii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises a glycan polymer comprising xylose (e.g., a glu-xyl preparation); and v. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises one, two, three, or more, for example all, of glycan polymers comprising one, two, or three of mannose, arabinose, or galactose.

381. The method of any one of claims 254-306, wherein the glycan polymer and / or glycan polymer preparation comprises one, two, three, or more, e.g., all, of the following characteristics: i. the glycan polymer comprises xylose and at least one alpha-glycosidic linkage, optionally the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and further optionally, the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic linkage; iii. the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a xyl-glu preparation); and iv. the glycan polymer preparation further comprises a glycan polymer comprising one, two, or three of mannose, arabinose, or galactose.

382. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises xylose and at least one beta-glycosidic bond, and optionally, the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises a glycan polymer comprising at least one alpha-glycosidic bond, and optionally, the alpha-glycosidic bond is an alpha-1,3 glycosidic bond; iii. the glycan polymer preparation further comprises a glycan polymer comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises a glycan polymer comprising glucose (e.g., a xyl-glu preparation); and v. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises one, two, three, or more, for example all, of glycan polymers comprising one, two, or three of mannose, arabinose, or galactose.

383. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises glucose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic bond, alpha-1,4 glycosidic bond, alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a glu-xyl preparation); v. The method of any one of claims 254-306, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a glu-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., a glu-gal preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising one, two, or three of xylose, arabinose, or galactose, one, two, three, or more, e.g., all.

384. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises xylose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic bond, alpha-1,4 glycosidic bond, alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., a xyl-glu preparation); v. The method of any one of claims 254 to 306, wherein the glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a xyl-ara preparation); vi. The glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., a xyl-gal preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising one, two, or three of glucose, arabinose, or galactose.

385. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer comprises arabinose and at least one alpha-glycosidic bond, optionally the alpha-glycosidic bond is an alpha-1,3 glycosidic bond, and further optionally the average degree of polymerization (DP) of the preparation is DP2-4, DP2-6, DP3-10, or DP3-15; ii. the glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic bond, alpha-1,4 glycosidic bond, alpha-1,6 glycosidic bond, or a combination thereof; iii. the glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic bond; iv. the glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., an ara-xyl preparation); v. The method of any one of claims 254-306, wherein the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., an ara-glu preparation); vi. the glycan polymer preparation further comprises glycan polymers comprising galactose (e.g., an ara-gal preparation); and vii. the glycan polymer preparation further comprises glycan polymers comprising one, two, or three of xylose, glucose, or galactose.

386. The glycan polymer and / or glycan polymer preparation has the following characteristics: i. the glycan polymer is galactosyl and at least one alpha-glycosidic linkage, optionally wherein the alpha-glycosidic linkage is an alpha-1,3 glycosidic linkage, and optionally wherein the average degree of polymerization (DP) of the preparation is DP 2-4, DP 2-6, DP 3-10, or DP 3-15; ii. The glycan polymer preparation further comprises glycan polymers comprising alpha-1,2 glycosidic linkages, alpha-1,4 glycosidic linkages, alpha-1,6 glycosidic linkages, or combinations thereof; iii. The glycan polymer preparation further comprises glycan polymers comprising at least one beta-glycosidic linkage; iv. The glycan polymer preparation further comprises glycan polymers comprising xylose (e.g., a gal-xyl preparation); v. The glycan polymer preparation further comprises glycan polymers comprising arabinose (e.g., a gal-ara preparation); vi. The method of any one of claims 254-306, wherein the glycan polymer preparation further comprises glycan polymers comprising glucose (e.g., a gal-glu preparation); and vii. The glycan polymer preparation further comprises glycan polymers comprising one, two, or three of xylose, arabinose, or glucose.

387. 363. The method of any of claims 351, 352, or 357-362, wherein the glycan polymer is a substrate for one or more human gut microbial glycosidase enzymes selected from GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, GH13 subfamily 36, GH113, or GH112 CAZy family, e.g., two, three, four, or more.

388. 363. The method of any of claims 351, 352, or 357-362, wherein the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13, GH13 subfamily 9, GH13 subfamily 31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77, or GH94 CAZy family.

389. 370. The method of any one of claims 353, 354, or 363-370, wherein the glycan polymer is a substrate for human gut microbial glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of the following: GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, or GH13 subfamily 14 CAZy family.

390. 370. The method of any one of claims 353, 354, or 363-370, wherein the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, or GH77 CAZy family.

391. The method of any of claims 355, 356, or 371-373, wherein the glycan polymer is a substrate for human gut microbial glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, or GH13 CAZy family.

392. The glycan polymer is GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77, GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, Gh28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, or GH24. The method of any of claims 355, 356, or 371-373, wherein the glycosidase is a substrate for one or more human gut microbial glycosidase enzymes selected from the CAZy family, e.g., two, three, four, or more.

393. The method of any of claims 349, 350, or 374-377, wherein the glycan polymer is a substrate for human gut microbial glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family.

394. 378. The method of any one of claims 349, 350, or 374-377, wherein the glycan polymer is a substrate for glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of the GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92 CAZy family.

395. The method of any one of claims 349, 350, or 378, wherein the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from one or more, for example, two, three, four, or more, of the GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family.

396. The method of any one of claims 349, 350, or 378, wherein the glycan polymer is a substrate for a human gut microbial glycosidase enzyme selected from one or more, e.g., two, three, four, or more, of the GH23, GH24, or GH33 CAZy family.

397. The method of any of claims 349, 350, or 379-382, wherein the glycan polymer is a substrate for human gut microbial glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family.

398. The method of any of claims 349, 350, or 379-382, wherein the glycan polymer is a substrate for human gut microbial glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of the GH2, GH31, GH23, GH13, or GH24 CAZy family.

399. The method of any of claims 349, 350, or 383-386, wherein the glycan polymer is a substrate for human gut microbial glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of the GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family.

400. The method of claims 349, 350, or 383-386, wherein the glycan polymer is a substrate for human gut microbial glycosidase enzymes selected from one or more, e.g., two, three, four, or more, of the GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106 CAZy family.

401. A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, mannose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, GH13 subfamily 36, GH113, or GH112 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from the group consisting of GT5, GH94, GH13 subfamily 9, GH13 subfamily 39, GH13 subfamily 36, GH113, or GH112 CAZy family.

402. The glycan polymers of the preparation are GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13.0, GH13.9, GH13.31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77, GH94 A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, mannose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, the glycan subunits being a substrate for human intestinal microorganisms containing a CAZy family glycosidase enzyme; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from the group consisting of GT2, GT4, GT5, GT35, GT51, GH1, GH2, GH3, GH4, GH13.0, GH13.9, GH13.31, GH18, GH23, GH25, GH28, GH31, GH32, GH36, GH51, GH73, GH77, or GH94 CAZy family.

403. A method for producing a glycan polymer preparation, comprising: providing a plurality of xylose, arabinose, galactose, and / or glucose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are a substrate for human gut microorganisms comprising a glycosidase enzyme of the GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 3, GH13 subfamily 30, GH30 subfamily 2, GH30 subfamily 5, GH43 subfamily 22, GH43 subfamily 8, or GH84 CAZy family.

404. A method for producing a glycan polymer preparation, comprising: providing a plurality of glycan subunits (e.g., monomers or dimers) containing xylose, arabinose, galactose, and / or glucose under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92 CAZy family; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from the GH3, GH106, GH105, GH2, GH20, GH28, GH76, GH97, or GH92 CAZy family.

405. The glycan polymer of the preparation is selected from the GH13 subfamily A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose- and / or sialic acid-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, the glycan subunits being a substrate for human intestinal microorganisms containing a glycosidase enzyme of the GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 19, GH13 subfamily 21, GH23, GH33, GH37, or GH104 CAZy family.

406. A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose- and / or sialic acid-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GH23, GH24, or GH33 CAZy family; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from the GH23, GH24, or GH33 CAZy family.

407. A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, xylose-, mannose-, arabinose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are a substrate for human gut microorganisms comprising a glycosidase enzyme of the GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 20, GH13 subfamily 31, GH13 subfamily 39, GH39, GH43 subfamily 11, GH5 subfamily 44, or GH94 CAZy family.

408. A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose, xylose, mannose, arabinose, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions resulting in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GH2, GH31, GH23, GH13, or GH24 CAZy family; Contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH2, GH31, GH23, GH13, or GH24 CAZy family.

409. A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, xylose-, arabinose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the GH13 subfamily 3, GH13 subfamily 30, GH121, GH15, GH43 subfamily 27, GH43 subfamily 34, or GH43 subfamily 8 CAZy family.

410. A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose-, xylose-, arabinose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from the GH92, GH97, GH76, GH28, GH20, GH105, GH2, GH50, GH3, or GH106 CAZy family.

411. The glycan polymers of the preparation are substrates for human gut microorganisms containing GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, GH13 subfamily 14 CAZy family glycosidase enzymes.

412. The glycan polymers of the preparation are GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77 providing a plurality of glucose-, mannose-, and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation that is a substrate for human gut microorganisms containing CAZy family glycosidase enzymes; A method for producing a glycan polymer preparation, comprising contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the CAZy family.

413. A method for producing a glycan polymer preparation, comprising: providing a plurality of glycan subunits (e.g., monomers or dimers) containing xylose, arabinose, fucose, and / or rhamnose under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, GH13 subfamily 14 CAZy family; contacting the plurality of glycan subunits with a glycosidase enzyme selected from the group consisting of GT11, GT10, GH92, GH51, GH35, GH29, GH28, GH20, GH130, GH13 subfamily 8, and GH13 subfamily 14 CAZy family.

414. A method for producing a glycan polymer preparation, comprising: providing a plurality of glycan subunits, e.g., monomers or dimers, of a substrate in column E of Table 23 under conditions that result in the production of a glycan polymer preparation, e.g., conditions in columns F, G, H, I, J, K, and / or L in the same row as the substrate and glycosidase enzyme; Contacting the plurality of glycan subunits of the substrate with a glycosidase enzyme in column A in the same row as the substrate.

415. The method of claim 414, wherein the glycan polymer preparation has an average DP of about 2 to 4 or about 2 to 5.

416. The method of any one of claims 414 or 415, wherein the glycan polymer preparation comprises at least 20%, 30%, 40%, 50% or at least 60% (mol%) of 1,4 glycosidic bonds.

417. The method of any one of claims 414 or 415, wherein the glycan polymer preparation comprises at least 80%, 90%, at least 95%, or 100% (mol%) 1,4 glycosidic bonds.

418. The method of any one of claims 414 or 415, wherein the glycan polymer preparation comprises at least 20%, 30%, 40%, 50% or at least 60% (mol%) of 1,6 glycosidic bonds.

419. The method of any one of claims 414 or 415, wherein the glycan polymer preparation comprises at least 80%, 90%, at least 95%, or 100% (mol%) 1,6 glycosidic bonds.

420. The method of any one of claims 414 or 415, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,2 glycosidic bonds.

421. The method of any one of claims 414 or 415, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,3 glycosidic bonds.

422. The method of any one of claims 414 or 415, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,4 glycosidic bonds.

423. The method of any one of claims 414 or 415, wherein the glycan polymer preparation contains less than 10%, 5%, less than 1%, or 0% 1,6 glycosidic bonds.

424. The glycosidic bond distribution (mol %) is: a) less than 10% alpha-1,2, less than 10% alpha-1,3, at least 30% alpha-1,4, at least 30% alpha-1,6, less than 5% beta-1,2, less than 5% beta-1,3, and less than 5% beta-1,4 / 1,6; b) less than 5% alpha-1,2, less than 5% alpha-1,3, at least 5% alpha-1,4, less than 5% alpha-1,6, and at least 5% beta-1,2; beta 1,3 at least 1%, beta 1,4 / 1,6 at least 85%, c) alpha-1,2 less than 5%, alpha 1,3 less than 5%, alpha 1,4 less than 5%, alpha 1,6 at least 85%, beta 1,2 less than 5%, beta 1,3 less than 5%, beta 1,4 / 1,6 less than 5%, d) alpha-1,2 less than 10%, alpha 1,3 less than 5%, alpha 1,4 at least 15%, alpha 1,6 at least at least 50%, beta 1,2 less than 5%, beta 1,3 less than 5%, beta 1,4 / 1,6 less than 5%, e) alpha-1,2 less than 5%, alpha 1,3 less than 5%, alpha 1,4 less than 15%, alpha 1,6 at least 85%, beta 1,2 less than 5%, beta 1,3 less than 5%, beta 1,4 / 1,6 less than 5%, f) alpha-1,2 less than 5%, alpha 1,3 less than 5%, alpha 1,4 less than 5%, alpha The method of any of claims 414 or 415, wherein the alpha 1,6 is less than 5%, beta 1,2 is less than 5%, beta 1,3 is less than 5%, and beta 1,4 / 1,6 is at least 85%; g) alpha-1,2 is less than 5%, alpha 1,3 is less than 5%, alpha 1,4 is at least 50%, alpha 1,6 is at least 5%, beta 1,2 is less than 10%, beta 1,3 is less than 5%, and beta 1,4 / 1,6 is at least 10%.

425. The glycan polymers of the preparation are GT2, GT4, GH2, GH23, GH3, GT8, GT51 , GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH 13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77 providing a plurality of xylose-, arabinose-, fucose-, and / or rhamnose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation that is a substrate for human gut microorganisms containing CAZy family glycosidase enzymes; A method for producing a glycan polymer preparation, comprising contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the CAZy family.

426. A method for producing a glycan polymer preparation, comprising: providing a plurality of glucose- and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation, wherein the glycan polymers of the preparation are substrates for human gut microorganisms containing glycosidase enzymes of the GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, and GH13 CAZy family; and contacting the plurality of glycan subunits with a glycosidase enzyme selected from one of the following: GT3, GH97, GH43 subfamily 24, GH27, GH133, GH13 subfamily 8, and GH13 CAZy family.

427. The glycan polymers of the preparation are selected from the group consisting of GT2, GT4, GH2, GH23, GH3, GT8, GT51, GT9, GH1, GH92, GH73, GH31, GH20, GH28, GT25, GT28, GT35, GH18, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT 10, GH77, GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, GT9, GH73, GH31, GH20, Gh28, GT35, GT2 8, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, GH24 providing a plurality of glucose- and / or galactose-containing glycan subunits (e.g., monomers or dimers) under conditions that result in the production of a glycan polymer preparation that is a substrate for human gut microorganisms containing CAZy family glycosidase enzymes; 8, GT0, GH13, GH36, GH97, GH105, GH25, GH4, GH32, GH78, GH29, GH0, GH51, GT10, GH77, GT2, GT4, GH2, GH23, GH3, GT51, GH1, GT8, GH92, G T9, GH73, GH31, GH20, Gh28, GT35, GT28, GH18, GH13, GH97, GH25, GH36, GH4, GH105, GH32, GH78, GH29, GH0, GT25, GH51, GH77, GH88, GH24 A method for producing a glycan polymer preparation, comprising contacting a glycosidase enzyme selected from one of the CAZy family.

428. 428. The method of any one of claims 401-413 or 425-427, wherein the glycosidase enzyme or glycosidase enzyme molecule is other than one or more of GH1, GH2, GH3, GH35, GH42, and GH50.

429. 428. The method of any one of claims 401-413 or 425-427, wherein the glycosidase enzyme or glycosidase enzyme molecule is other than one or more of GH32, GH68, GH100.

430. 428. The method of any one of claims 401-413 or 425-427, wherein the glycosidase enzyme or glycosidase enzyme molecule is other than one or more of GH1, GH2, GH3, GH4, GH5, GH8, GH9, GH10, GH11, GH12, GH13, GH14, GH16, GH26, GH28, GH30, GH31, GH32, GH35, GH42, GH43, GH44, GH50, GH51, GH57, GH62, GH63, GH68, GH70, GH97, GH100, GH116, GH119, GH122.

431. A glycan polymer preparation made, producible, or producible by a method disclosed herein, for example, by a method according to any of claims 254-430.

432. A glycan polymer preparation selected or selectable by a method disclosed herein, for example, by a method according to any of claims 254 to 430.

433. The glycan polymer preparation of claim 431, formulated as a pharmaceutical composition, medical food, dietary supplement, food ingredient, or therapeutic nutritional product.

434. 432. The glycan polymer preparation of claim 431, further comprising an excipient or carrier.

435. A unit dosage form comprising the glycan preparation of any one of claims 431 to 434.

436. 436. The unit dosage form of claim 435, formulated for enteral administration, oral, oral or rectal administration, or tube feeding.

437. 437. The unit dosage form of either claim 435 or 436, formulated as a powder or syrup.

438. 438. The unit dosage form of any one of claims 435 to 437, formulated for timed and / or targeted release in the colon or large intestine.

439. A pharmaceutical composition comprising the glycan polymer preparation of any one of claims 431 to 434.

440. A medical food comprising the glycan polymer preparation of any one of claims 431 to 434.

441. 435. A dietary supplement comprising the glycan polymer preparation of any one of claims 431 to 434.

442. A food ingredient comprising the glycan polymer preparation of any one of claims 431 to 434.

443. A therapeutic nutritional product comprising the glycan polymer preparation of any one of claims 431-434.

444. A reaction mixture produced by the method described herein, for example, any one of claims 254 to 430, in an amount suitable to produce a glycan polymer preparation comprising at least 0.25, 0.5, 1, 5, 10, 20, 50, 100, 200, 300, 400, or 500 kilograms of glycan polymers, and / or at least about 15%, 30%, 45%, 60%, or about 75% (determined on a weight / weight basis as a % of the input glycan subunits). and a glycosidase enzyme molecule (e.g., Table 4 (column 2), Table 23 (column A), Table 24 (column A), or Table 22 (column 1)); or one or more glycosidase enzymes associated with glycotaxa class 1, class 2, class 3, class 4, class 5, class 6, or class 7) suitable for producing the glycan polymer under conditions suitable to obtain a yield of at least one of the following:

445. 445. A reaction mixture according to claim 444 suitable for carrying out a method as described herein, such as any of the methods of claims 254 to 430.

446. 432. A method of making a pharmaceutical composition, medical food, dietary supplement, food ingredient, or therapeutic nutritional product comprising formulating the preparation of claim 431 into the pharmaceutical composition, medical food, dietary supplement, food ingredient, or therapeutic nutritional product.

447. 447. The method of claim 446, comprising dividing the preparation into a plurality of portions, e.g., unit doses or formulations, e.g., at least 10, 100, or at least 1,000 portions.

448. 447. The method of claim 446, comprising combining the preparation with an excipient.

449. 432. A glycan polymer preparation according to claim 431, or a portion thereof.

450. A fraction, e.g., a molecular weight fraction, of a glycan polymer preparation described in claim 431.

451. The molecular weight fraction of claim 450, wherein the fraction comprises an average DP different from that of the glycan preparation, for example, an average DP of about 3, 4, or 5.

452. A method for producing, evaluating, selecting, classifying, or providing a preparation of glycan polymers produced or preparable by the method of any of claims 254 to 430, comprising the steps of: obtaining a candidate preparation; obtaining a value for a parameter of the preparation, for example, a physical parameter, for example, a molecular weight, for example, an average molecular weight or molecular weight distribution, a glycan subunit composition or purity, or a parameter related to a biological property, for example, the ability to regulate the growth of the human gut microorganism, the ability to regulate a microbial metabolite produced by the microorganism, for example, in an ex vivo assay, or the ability to regulate a biomarker, for example, an inflammatory or immune biomarker, a toxic or waste compound, a bacterial compound, for example, in a human subject; and comparing the value with a reference value, thereby producing, evaluating, selecting, classifying, or providing a preparation of glycan polymers.

453. The method of claim 452, comprising the step of performing an assay to obtain said value.

454. 453. The method of claim 452, comprising obtaining the value from another party.

455. The value is compared to a reference value to evaluate the candidate for suitability for use, for example, as a preparation of a glycan polymer, or for formulation into a product or dosage form, for example, a product or dosage form described herein. The method of any of claims 452 to 454.

456. A method for producing a pharmaceutical composition for regulating a target human gut microorganism, comprising the steps of: providing a plurality of glycan subunits; contacting the plurality of glycan subunits with a glycosidase enzyme composition having glycosidase activity present in the target gut microorganism under conditions that result in the incorporation of the glycan subunits into a glycan polymer; optionally purifying the glycan polymer; and formulating the glycan polymer as a pharmaceutical composition for administration to the intestine and regulating the gut microorganism, thereby producing a pharmaceutical composition for regulating the target human gut microorganism.

457. A purified preparation of glycosidase enzyme molecules comprising a glycosidase enzyme encoded by a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124, wherein the glycosidase enzyme is present in a human intestinal microorganism.

458. A vector comprising a nucleic acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124, wherein the nucleic acid encodes a glycosidase enzyme present in a human intestinal microorganism, and the vector can be used to express the glycosidase enzyme.

459. Encoded by a nucleic acid sequence selected from one or more of SEQ ID NOs: 1-124 A reaction mixture comprising a glycosidase enzyme to be synthesized and a substrate for the glycosidase enzyme, e.g., a glycan subunit, e.g., a monomer or dimer, wherein the substrate is present in an amount sufficient to form a glycan polymer, e.g., by condensation.

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