Oligosaccharide compositions and methods of use thereof

Abstract

Aspects of the present disclosure relate to oligosaccharide compositions and methods for preparing the same. Methods of using the oligosaccharide compositions as microbiome metabolic therapies for reducing the levels and / or concentrations of glucose and / or lipids, such as cholesterol, low-density lipoprotein cholesterol, non-esterified free fatty acids and triglycerides, and for treating related diseases are also provided.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 053,528, filed on July 17, 2020, and entitled “Oligosaccharide Compositions and Methods of Use Thereof,” the contents of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present disclosure relates to oligosaccharide compositions and uses thereof. Background Art

[0004] Maintaining or restoring human health presents numerous challenges, many of which are due to the lack of effective treatment options. There is a continuous need for new therapies and treatment options. Summary of the invention

[0005] According to some aspects, provided herein are microbiome metabolic therapies utilizing oligosaccharide compositions that can be used to drive the functional output of intestinal microbiome organs to, for example, treat disease. Some aspects of the disclosure relate to the recognition that oligosaccharide compositions can be used to reduce lipid and / or glucose (e.g., blood) levels in a subject, both of which functional outcomes can be used to treat many diseases and conditions.

[0006] Some aspects of the present disclosure provide an oligosaccharide composition comprising a plurality of oligosaccharides, the composition being characterized by a multiply edited gradient enhanced 1H-13C heteronuclear single quantum coherence (HSQC) NMR spectrum comprising one or more of signals 1, 2, 5, or 6 of the following table, wherein the spectrum is generated using an oligosaccharide composition sample having less than 2% monomers:

[0007]

[0008]

[0009] In some embodiments, the oligosaccharide composition comprises each of HSQC signals 1, 2, 5, and 6. In some embodiments, the oligosaccharide composition further comprises one or more of HSQC signals 3, 4, or 7-12. In some embodiments, the oligosaccharide composition comprises each of HSQC signals 1-12.

[0010] In some embodiments, at least one of the HSQC signals 1-12 of the oligosaccharide composition sample is defined as follows:

[0011]

[0012]

[0013] In some embodiments, the HSQC signals 1-12 of the oligosaccharide composition sample are each further characterized by 1 H integration area and 13 The C integration region is defined as follows:

[0014]

[0015] In some embodiments, the NMR spectrum is enhanced by performing multiple edited gradients on the composition sample. 1 H- 13 C heteronuclear single quantum coherence (HSQC) experiments using an echo-anti-echo scheme with the following pulse sequence diagram, acquisition parameters and processing parameters for coherence selection:

[0016] Pulse sequence diagram

[0017]

[0018] Acquisition parameters

[0019] 1 H carrier frequency = 4ppm

[0020] 13 C carrier frequency = 65ppm

[0021] Number of points in the collection dimension = 596

[0022] Spectral range in acquisition dimension = 6.23ppm to 1.83ppm

[0023] Number of points in indirect dimension = 300 complex points

[0024] Spectral range in indirect dimension = 120ppm to 10ppm

[0025] Loop delay = 1 second

[0026] single bond 1 H- 13 C coupling constant = J CH =146Hz

[0027] Scan times = 8

[0028] Temperature = 298-299K

[0029] Solvent = D2O

[0030] Processing parameters

[0031] Direct dimension window function = Gaussian broadening, 7.66Hz

[0032] Indirect dimension window function = Gaussian widening 26.48Hz

[0033] Processing = 512 complex points in direct dimension, 1024 complex points in indirect dimension

[0034] In some embodiments, the NMR spectrum is obtained by performing HSQC NMR on a sample of the oligosaccharide composition, wherein the sample is dissolved in D2O. In some embodiments, the oligosaccharide composition has been subjected to a demonomerization procedure. In some embodiments, the oligosaccharide composition comprises less than 10%, less than 5%, or less than 2% monomers.

[0035] Some aspects of the present disclosure provide an oligosaccharide composition comprising a plurality of oligosaccharides, each oligosaccharide comprising a plurality of monomeric groups; the plurality of oligosaccharides comprising one or more of the following monomeric groups:

[0036] (2) t-mannopyranose monogroups, which represent 11.47-18.18 mol % of the monomeric groups in the various oligosaccharides;

[0037] (4) t-mannofuranose monogroups, which represent 0.17-0.46 mol % of the monomeric groups in the various oligosaccharides;

[0038] (8) 3-mannofuranose monogroup, which represents 0.07-0.24 mol % of the monomeric groups in the various oligosaccharides;

[0039] (10) 2-mannopyranose and / or 3-mannopyranose monogroups, which represent 3.91-5.93 mol % of the monomeric groups in the various oligosaccharides;

[0040] (15) 6-pyranose monogroup, which represents 8.30-10.84 mol% of the monomer groups in the various oligosaccharides;

[0041] (27) 4,6-mannopyranose and / or 5,6-mannofuranose diyl groups, which represent 1.18-1.82 mol % of the monomer groups in the various oligosaccharides;

[0042] (30) 3,6-pyranose diyl, which represents 1.76-2.40 mol % of the monomeric group in the various oligosaccharides;

[0043] (31) 2,6-pyranose diyl, which represents 1.42-1.84 mol % of the monomeric groups in the various oligosaccharides; and

[0044] (32) 3,6-galactofuranosyl, which represents 0.95–1.41 mol % of the monomeric group in the various oligosaccharides.

[0045] In some embodiments, the plurality of oligosaccharides comprises at least 2, 3, 4, 5, 6, 7, 8 or 9 of the monomeric groups selected from groups (2), (4), (8), (10), (15), (27) and (30)-(32).

[0046] In some embodiments, the plurality of oligosaccharides further comprises one or more of the following monomer groups:

[0047] (5) t-galactofuranose monogroups, which represent 2.96-6.24 mol % of the monomeric groups in the various oligosaccharides;

[0048] (6) t-galactopyranose monogroups, which represent 12.90-17.63 mol % of the monomeric groups in the various oligosaccharides;

[0049] (12) 3-galactopyranose monogroup, which represents 3.52-4.87 mol % of the monomeric groups in the various oligosaccharides;

[0050] (18) 4-galactopyranose and / or 5-galactofuranose monogroups, which represent 2.86-3.57 mol % of the monomeric groups in the various oligosaccharides;

[0051] (22) 2,3-galactofuranosyl, which represents 0.10-0.59 mol % of the monomeric group in the various oligosaccharides;

[0052] (23) 2,3-pyranose diyl, which represents 0.24-0.51 mol % of the monomeric group in the various oligosaccharides;

[0053] (24) 6-galactopyranose monogroup, which represents 9.78-13.08 mol % of the monomeric groups in the various oligosaccharides;

[0054] (25) 3,4-galactopyranose and / or 3,5-galactofuranose and / or 2,3-galactopyranose diyl groups, which represent 0.83-1.32 mol % of the monomer groups in the plurality of oligosaccharides;

[0055] (26) 2,4-mannopyranose and / or 2,5-mannofuranose diyl groups, which represent 0.09-1.15 mol % of the monomer groups in the various oligosaccharides;

[0056] (28) 3,6-mannofuranose diyl, which represents 0.07-0.18 mol % of the monomeric group in the various oligosaccharides;

[0057] (33) 4,6-galactopyranose and / or 5,6-galactofuranosyl groups, which represent 2.38-3.46 mol % of the monomeric groups in the various oligosaccharides;

[0058] (34) 2,6-galactopyranosyldiyl, which represents 1.23–1.84 mol % of the monomeric groups in the various oligosaccharides;

[0059] (35) 3,4,6-mannopyranose and / or 3,5,6-mannofuranose and / or 2,3,6-mannofuranose triyl groups, which represent 0.19-0.48 mol % of the monomer groups in the various oligosaccharides;

[0060] (36) 3,4,6-galactopyranose and / or 3,5,6-galactofuranose and / or 2,3,6-galactofuranose triyl groups, which represent 0.53-1.03 mol % of the monomer groups in the various oligosaccharides;

[0061] (37) 2,3,6-mannopyranose and / or 2,4,6-mannopyranose and / or 2,5,6-mannofuranose triyl groups, which represent 0.43-0.68 mol % of the monomer groups in the plurality of oligosaccharides; and

[0062] (38) 2,3,6-galactopyranose and / or 2,4,6-galactopyranose and / or 2,5,6-galactofuranosyl triyl groups, which represent 0.59-0.93 mol % of the monomer groups in the various oligosaccharides.

[0063] In some embodiments, the plurality of oligosaccharides comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the monomer groups selected from the groups (2), (4)-(6), (8), (10), (12), (15), (18), (22)-(28) and (30)-(38).

[0064] In some embodiments, the plurality of oligosaccharides comprises each of a monomeric group selected from the group consisting of (2), (4)-(6), (8), (10), (12), (15), (18), (22)-(28), and (30)-(38).

[0065] In some embodiments, the plurality of oligosaccharides comprises one or more of the following monomer groups:

[0066] (9) 2-Mannofuranose monogroup, which represents 0.01-1.07 mol % of the monomer group in the various oligosaccharides;

[0067] (11) 2-galactofuranose monogroup, which represents 0.01-4.34 mol% of the monomeric groups in the various oligosaccharides;

[0068] (13) 4-mannopyranose and / or 5-mannofuranose monogroups, which represent 0.01-7.91 mol % of the monomer groups in the plurality of oligosaccharides;

[0069] (14) 3-galactofuranose monogroup, which represents 0.01-4.44 mol% of the monomeric groups in the various oligosaccharides;

[0070] (16) 2-galactopyranose monogroup, which represents 0.01-8.70 mol % of the monomeric groups in the various oligosaccharides;

[0071] (19) 2,3-Mannofuranose diyl, which represents 0.01-0.26 mol % of the monomeric group in the various oligosaccharides;

[0072] (21) 6-Mannofuranose monogroup, which represents 0.01-1.72 mol % of the monomeric group in the various oligosaccharides;

[0073] (39) 2,3,4,6-mannopyranose and / or 2,3,5,6-mannofuranose tetrayl groups, which represent 0.01-0.68 mol % of the monomer groups in the plurality of oligosaccharides; and

[0074] (40) 2,3,4,6-galactopyranose and / or 2,3,5,6-galactofuranosyl, which represent 0.01-0.31 mol % of the monomeric groups in the various oligosaccharides.

[0075] In some embodiments, the plurality of oligosaccharides further comprises one or more of the following monomer groups:

[0076] (9) 2-mannofuranose monogroup, which represents about 0.11 mol % of the monomeric groups in the various oligosaccharides;

[0077] (11) 2-galactofuranose monogroup, which represents about 0.43 mol % of the monomer groups in the various oligosaccharides;

[0078] (13) 4-mannopyranose and / or 5-mannofuranose monogroups, which represent about 1.10 mol % of the monomer groups in the various oligosaccharides;

[0079] (14) 3-galactofuranose monogroup, which represents about 0.35 mol % of the monomeric groups in the various oligosaccharides;

[0080] (16) 2-galactopyranose monogroup, which represents 0.06-6.54 mol % of the monomeric groups in the various oligosaccharides;

[0081] (19) 2,3-Mannofuranose diyl, which represents 0.06-0.21 mol % of the monomeric group in the various oligosaccharides;

[0082] (21) 6-Mannofuranose monogroup, which represents about 0.36 mol % of the monomer groups in the various oligosaccharides;

[0083] (39) 2,3,4,6-mannopyranose and / or 2,3,5,6-mannofuranose tetrayl groups, which represent about 0.12 mol % of the monomer groups in the various oligosaccharides; and

[0084] (40) 2,3,4,6-galactopyranose and / or 2,3,5,6-galactofuranosyl, which represent about 0.04 mol % of the monomeric groups in the various oligosaccharides.

[0085] In some embodiments, the plurality of oligosaccharides comprises each of a monomeric group selected from the group consisting of (2), (4)-(6), (8)-(16), (18), (19), (21)-(28), and (30)-(39).

[0086] In some embodiments, the mole percentage of monomeric groups is determined using a permethylation assay, wherein the permethylation assay comprises gas chromatography-mass spectrometry (GC-MS) analysis.

[0087] In some embodiments, the oligosaccharide composition comprises a plurality of oligosaccharides consisting essentially of formula (I) and formula (II):

[0088]

[0089] Wherein R in formula (I) and formula (II) is independently selected from hydrogen, and formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc), (IId):

[0090]

[0091] wherein R in Formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc) and (IId) is independently as defined in Formula (I) and Formula (II).

[0092] In some embodiments, the average degree of polymerization (DP) of the oligosaccharide composition is from about DP6 to about DP 19. In some embodiments, the average degree of polymerization (DP) of the oligosaccharide composition is from about DP10 to about DP 16. In some embodiments, the average degree of polymerization (DP) of the oligosaccharide composition is from about DP10 to about DP14.

[0093] In some embodiments, the oligosaccharide composition comprises 55% to 95% total dietary fiber (dry basis). Some aspects of the present disclosure provide an oligosaccharide composition comprising a plurality of oligosaccharides, the oligosaccharides comprising

[0094] Formula (I) and formula (II):

[0095]

[0096] Wherein R in formula (I) and formula (II) is independently selected from hydrogen, and formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc), (IId):

[0097]

[0098] wherein R in Formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc) and (IId) are independently as defined in Formula (I) and Formula (II);

[0099] Wherein the oligosaccharide composition is produced by a method comprising:

[0100] (a) forming a reaction mixture comprising galactose monomers and mannose monomers with an acid catalyst comprising positively charged hydrogen ions, wherein the molar ratio of galactose to mannose is about 3:2; and

[0101] (b) promoting the formation of acid-catalyzed oligosaccharides in the reaction mixture by transferring sufficient heat to the reaction mixture to maintain the reaction mixture at its boiling point.

[0102] In some embodiments, step (b) comprises loading the reaction mixture with an acid catalyst comprising positively charged hydrogen ions in an amount such that the molar ratio of positively charged hydrogen ions to the total galactose monomer and mannose monomer content is within a suitable range. In some embodiments, steps (a) and (b) occur simultaneously.

[0103] In some embodiments, step (a) comprises heating the reaction mixture to a temperature in the range of 100°C to 160°C under stirring conditions. In some embodiments, step (a) comprises heating the reaction mixture to a temperature in the range of 130°C to 140°C under stirring conditions. In some embodiments, step (a) comprises heating the reaction mixture at a temperature in the range of 100°C to 160°C under stirring conditions. In some embodiments, step (a) comprises heating the reaction mixture at a temperature in the range of 130°C to 140°C under stirring conditions. In some embodiments, step (a) comprises gradually increasing the temperature (e.g., from room temperature) to about 135°C under suitable conditions to achieve homogeneity and uniform heat transfer. In some embodiments, step (b) comprises maintaining the reaction mixture at atmospheric pressure or vacuum, at a temperature in the range of 130°C to 140°C, under conditions that promote the formation of an acid-catalyzed oligosaccharide composition, until the weight percentage of galactose monomers and mannose monomers in the oligosaccharide composition is in the range of 4-14. In some embodiments, step (b) comprises gradually increasing the temperature (e.g., from room temperature) to about 135°C under suitable conditions to achieve homogeneity and uniform heat transfer.

[0104] In some embodiments, the acid catalyst is a strong acid cation exchange resin having one or more physical and chemical properties according to Table 1 and / or wherein the catalyst comprises >3.0 mmol / g sulfonic acid moieties and <1.0 mmol / g cationic moieties. In some embodiments, the catalyst has a nominal moisture content of 45-50 weight percent.

[0105] In some embodiments, the acid catalyst is a soluble catalyst. In some embodiments, the soluble catalyst is an organic acid. In some embodiments, the soluble catalyst is a weak organic acid. In some embodiments, the soluble catalyst is citric acid.

[0106] In some embodiments, the method further comprises: (c) quenching the reaction mixture, for example with water, while allowing the temperature of the reaction mixture to reach a temperature in the range of 55°C to 95°C (eg, 85°C, 90°C).

[0107] In some embodiments, the method further comprises: (d) separating the oligosaccharide composition from the acid catalyst. In some embodiments, in (d), the separation comprises removing the catalyst by filtration. In some embodiments, (d) comprises cooling the reaction mixture to less than about 85° C. before filtering.

[0108] In some embodiments, the method further comprises: (e) diluting the oligosaccharide composition of (d) with water to a concentration of about 45-55 weight percent; (f) passing the diluted composition through a cation exchange resin; (g) passing the diluted composition through a decolorizing polymer resin; and / or (h) passing the diluted composition through an anion exchange resin; wherein each of (f), (g) and (h) can be performed one or more times in any order.

[0109] In some embodiments, the average degree of polymerization (DP) of the oligosaccharide composition is from about DP6 to about DP19. In some embodiments, the average degree of polymerization (DP) of the oligosaccharide composition is from about DP10 to about DP14. In some embodiments, the composition comprises 55% to 95% total dietary fiber (dry basis). In some embodiments, the oligosaccharide composition comprises less than 15% monomer (DP1).

[0110] Some aspects of the present disclosure provide methods of treating a subject having or suspected of having nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH).

[0111] In some embodiments, a method of treating a subject having or suspected of having non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) comprises administering to the gastrointestinal tract of the subject an effective amount of an oligosaccharide composition, wherein the oligosaccharide composition has an average degree of polymerization of 5-20 and comprises a plurality of oligosaccharides selected from formula (I) and formula (II):

[0112]

[0113] Wherein R in formula (I) and formula (II) is independently selected from hydrogen, and formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc), (IId):

[0114]

[0115]

[0116] wherein R in Formulas (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc) and (IId) is independently as defined in Formula (I) and Formula (II); thereby treating the subject.

[0117] In some embodiments, a method of treating a subject having or suspected of having nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH) comprises administering to the gastrointestinal tract of the subject an effective amount of an oligosaccharide composition described herein.

[0118] Some aspects of the present disclosure provide methods for increasing the relative or absolute concentration of propionic acid in a human subject. In some embodiments, the method for increasing the relative or absolute concentration of propionic acid comprises administering an effective amount of an oligosaccharide composition as described herein to the gastrointestinal tract of the subject. In some embodiments, the method increases the relative or absolute concentration of propionic acid in the gastrointestinal tract of the subject.

[0119] In some embodiments, the relative or absolute concentration of propionic acid is increased by at least 5%, 10%, 20%, or 30% compared to a baseline measurement (eg, where the baseline measurement is determined prior to treatment).

[0120] Some aspects of the present disclosure provide methods of reducing the relative or absolute concentration of lipids in a human subject.In some embodiments, the method of reducing the relative or absolute concentration of lipids in a human subject comprises administering to the gastrointestinal tract of the subject an effective amount of an oligosaccharide composition described herein.

[0121] In some embodiments, the relative or absolute concentration of a lipid is reduced by at least 5%, 10%, 20%, or 30% compared to a baseline measurement (eg, where the baseline measurement is determined prior to treatment).

[0122] In some embodiments, lipid is cholesterol, very low density lipoprotein (VLDL), low density lipoprotein (LDL), non-esterified free fatty acids and / or triglycerides. In some embodiments, cholesterol is liver cholesterol and / or blood cholesterol. In some embodiments, triglycerides are liver triglycerides and / or blood triglycerides.

[0123] Some aspects of the present disclosure provide methods for treating a subject suffering from or suspected of having a disease, disorder or condition associated with elevated lipid and / or glucose levels. In some embodiments, the method for treating a subject suffering from or suspected of having a disease, disorder or condition associated with elevated lipid and / or glucose levels comprises administering an effective amount of an oligosaccharide composition as described herein to the gastrointestinal tract of the subject, thereby treating the subject.

[0124] Some aspects of the present disclosure provide methods for treating a subject suffering from or suspected of having a disease or disorder associated with elevated lipid and / or glucose levels. In some embodiments, the method for treating a subject suffering from or suspected of having a disease or disorder associated with elevated lipid and / or glucose levels comprises administering an effective amount of an oligosaccharide composition to the gastrointestinal tract of the subject, wherein the oligosaccharide composition has an average degree of polymerization of 5-20 and comprises a plurality of oligosaccharides selected from formula (I) and formula (II):

[0125]

[0126] Wherein R in formula (I) and formula (II) is independently selected from hydrogen, and formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc), (IId):

[0127]

[0128]

[0129] wherein R in Formulas (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc) and (IId) is independently as defined in Formula (I) and Formula (II); thereby treating the subject.

[0130] In some embodiments, the disease, disorder, or condition is dyslipidemia, hyperlipidemia (eg, primary or familial hyperlipidemia), or a cardiometabolic disease.

[0131] In some embodiments, the subject suffers from or is suspected of suffering from metabolic syndrome.In some embodiments, the subject suffers from or is at risk of suffering from a disease associated with abnormal lipid and / or glucose levels, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease (CVD), type 2 diabetes (T2D) and chronic kidney disease (CKC).

[0132] In some embodiments, the hyperlipidemia is heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), familial chylomicronemia syndrome, familial combined hyperlipidemia, or familial dysbetalipoproteinemia.

[0133] In some embodiments, the subject is resistant to standard of care medications, e.g., treatment of abnormal levels of lipids, e.g., statins (e.g., statin-refractory subjects or statin non-responders). In some embodiments, the subject is intolerant to standard of care medications, e.g., treatment of abnormal levels of lipids, e.g., statins, e.g., subjects exhibiting statin-associated muscle symptoms (SAMS).

[0134] In some embodiments, the subject is a human subject. In some embodiments, the subject is a neonate (preterm neonate, full-term neonate), an infant under one year old, a toddler (e.g., 1 to 12 years old), a teenager (e.g., 13-19 years old), an adult (e.g., 20-64 years old) or an elderly person (e.g., 65 years old and above).

[0135] In some embodiments, the method includes administering the oligosaccharide composition to the intestine (e.g., large intestine). In some embodiments, the oligosaccharide composition is self-administered to the subject. In some embodiments, the oligosaccharide composition is formulated as a pharmaceutical composition for oral delivery. In some embodiments, the oligosaccharide composition is orally administered to the subject. In some embodiments, the oligosaccharide composition is formulated as a pharmaceutical composition for delivery by a feeding tube. In some embodiments, the oligosaccharide composition is administered to the subject by a feeding tube.

[0136] In some embodiments, the oligosaccharide composition is administered to the subject once a day or twice a day.

[0137] In some embodiments, the oligosaccharide composition is co-administered to the subject with a standard of care therapy. In some embodiments, the standard of care therapy is the administration of a statin.

[0138] In some embodiments, the method reduces the concentration of lipid in the subject. In some embodiments, lipid concentration is reduced by at least 5%, 10%, 20% or 30% relative to baseline measurement (for example, wherein the baseline measurement is determined before treatment). In some embodiments, lipid is cholesterol, very low density lipoprotein (VLDL), low density lipoprotein (LDL), non-esterified free fatty acids and / or triglycerides. In some embodiments, cholesterol is liver cholesterol and / or blood cholesterol. In some embodiments, triglycerides are liver triglycerides and / or blood triglycerides.

[0139] In some embodiments, the methods reduce or alleviate hepatic steatosis and / or hepatic inflammation. In some embodiments, the methods reduce atherosclerotic plaque formation. In some embodiments, the methods support the growth of bacterial genera within the subject's gastrointestinal microbiota that do not promote dysbiosis.

[0140] In some embodiments, method supports the growth of propionic acid producing bacteria group.In some embodiments, method supports Allistipes, Blautia, Parabacteroides, Akkermansia, Ruthenibacterium, rare micrococcus (Subdoligranulum), Bacteroidetes, Rickenellaceae, Tannerellaceae, another branch Bacteroidetes, Parabacteroidetes and / or the growth of Ruminococcaceae.In some embodiments, the method increases the relative abundance of one or more bacterial taxa selected from the group consisting of Allistipes, Blautia, Parabacteroidetes, Akkermansia, Ruthenibacterium, rare micrococcus, Bacteroidetes, Rickenellaceae, Tannerellaceae, another branch Bacteroidetes, Parabacteroidetes and / or Ruminococcaceae. BRIEF DESCRIPTION OF THE DRAWINGS

[0141] Figure 1 A-1C provides data showing the effects of selected oligosaccharide compositions on body weight ( Figure 1 A) Relative weight ( Figure 1 B) and food intake ( Figure 1 C) Graph with minimal impact.

[0142] Figure 2 A-2B provides data showing the effects of selected oligosaccharide compositions ("Selected OS") on fasting blood glucose levels ( Figure 2 A) and insulin ( Figure 2 B) The effect of Figure 2 In A, data sets that do not share the same letter designation (a, b, or c) are statistically different from each other.

[0143] Figure 3 A-3E provides data showing that selected oligosaccharide compositions have an effect on fasting serum total cholesterol ( Figure 3 A) Blood triglycerides ( Figure 3 B), serum non-esterified fatty acids (NEFA) ( Figure 3 C) Liver total cholesterol ( Figure 3 D) and liver triglycerides ( Figure 3 E) A plot of the impact of the . Data sets that do not share the same letter designation (a, b, or c) are statistically different from each other.

[0144] Figure 4 A-4E provides data showing the effects of selected oligosaccharide compositions on liver weight ( Figure 4 A) Liver weight relative to body weight ( Figure 4 B) Hepatic fatty degeneration ( Figure 4 C) Inflammation Figure 4 D) and NAS( Figure 4 E) Plot of the effect of histological score. Data sets that do not share the same letter designation (a, b, or c) are statistically different from each other.

[0145] Figure 5 Multidimensional scaling (MDS) ordination plots of fecal microbiota from high-fat diet (HFD)-induced obese mice treated with selected oligosaccharides (circles), glucose / galactose-based oligosaccharides (crosses), or liraglutide (triangles) 1 week before the start of treatment (light shading) or after 4 weeks of treatment (dark shading) are provided.

[0146] Figure 6 Graphs showing differences in the microbiota of high fat diet (HFD)-induced obese mice treated with selected oligosaccharides, glucose / galactose-based oligosaccharides, or liraglutide relative to control HFD-induced mice (untreated) are provided.

[0147] Figure 7 Provided are representative images of liver histology of high fat diet (HDF)-induced obese mice treated with selected oligosaccharides, glucose / galactose-based oligosaccharides, or liraglutide after 5 weeks of treatment.

[0148] Figure 8 A-8C provides a method showing that the selected oligosaccharide compositions have an effect on the low-density lipoprotein receptor knockout (LDLR) induced obesity in a Western diet (WD) - / - ) Body weight in mouse models ( Figure 8 A) Relative weight ( Figure 8 B) and food intake ( Figure 8 C) Graph of the impact of.

[0149] Fig. 9 A-9C provides a method showing that the selected oligosaccharide composition has an effect on the low density lipoprotein receptor knockout (LDLR) in Western diet (WD) induced obesity. - / - ) mouse model after 15-16 weeks of treatment ( Fig. 9 A) Fasting serum insulin concentration ( Fig. 9 B) and HOMA-IR score ( Fig. 9 C) A plot of the impact of the datasets. Data sets that do not share the same letter designation (a, b, or c) are statistically different from each other.

[0150] Fig.10 A-10H provides a method showing that the selected oligosaccharide composition has an effect on the low density lipoprotein receptor knockout (LDL-Knockout) in Western diet (WD) induced obesity. - / -- / - ) in the mouse model after 15-16 weeks of treatment ( Fig.10 A), total serum cholesterol ( Fig.10 B) Very low density lipoprotein (VLDL) cholesterol ( Fig.10 C) low-density lipoprotein (LDL) cholesterol ( Fig.10 D) High-density lipoprotein (HDL) cholesterol ( Fig.10 E), serum non-esterified fatty acids (NEFA) ( Fig.10 F), liver triglycerides ( Fig.10 G) and liver total cholesterol ( Fig.10 H) Plot of the impact of the datasets. Data sets that do not share the same letter designation (a, b, or c) are statistically different from each other.

[0151] Fig.11 A-11H provides a method showing that the selected oligosaccharide composition has an effect on the low density lipoprotein receptor knockout (LDL-Knockout) in Western diet (WD) induced obesity. - / - ) Liver weight after 15-16 weeks of treatment in the mouse model ( Fig.11 A) Liver weight relative to body weight ( Fig.11 B) Hepatic fatty degeneration ( Fig.11 C) Inflammation Fig.11 D), NAFLD Activity Score ("NAS") ( Fig.11 E) Severity score of aortic sinus plaque ( Fig.11F ), plaque area in the aortic sinus quantified by morphometric analysis of elastin trichome-stained sections ( Fig.11G ) and plaque area in the aortic arch quantified by Oil Red O staining “frontal” analysis ( Fig.11H ) affects the graph. Fig.11 Data sets in A-11E that do not share the same letter designation (a, b, or c) are statistically distinct from each other.

[0152] Fig.12 WDldlr from mice treated with selected oligosaccharides (circles), glucose / galactose-based oligosaccharides (crosses), or fenofibrate (triangles), or untreated (diamonds) one week before treatment (light shading) or after 15 weeks of treatment (dark shading) are provided. - / - Multidimensional scaling (MDS) ordination plot of the fecal microbiome of mice.

[0153] Fig.13 Provided are methods for treating Western diet (WD)-induced obese LDLRs with selected oligosaccharides, glucose / galactose-based oligosaccharides, or liraglutide - / - Representative images of liver histology in mice after 16 weeks of treatment.

[0154] Fig.14 A-14B provides LDLR for Western diet (WD)-induced obesity after 16 weeks of treatment - / - Histological staining of atherosclerotic plaque formation in the aortic arch of mice ( Fig.14 A) and quantization ( Fig.14 B) Image.

[0155] Fig.15 Provided are methods showing that Western diet (WD)-induced obesity LDLR treated with selected oligosaccharides, glucose / galactose-based oligosaccharides or liraglutide - / - Plot of differences in the microbiota of HFD-induced mice relative to control (untreated) mice.

[0156] Figures 16A-16B Provided are graphs showing the production of short chain fatty acids (SCFA) (propionate, acetate and butyrate) in an ex vivo assay in which stool samples from 10 healthy subjects were incubated with selected oligosaccharide compositions ("Selected OS") or a negative control (water). Fig.16A The total amount of SCFA produced is provided (in mM). Fig. 16B The percentage of each unique SCFA (propionate, acetate and butyrate) relative to the total SCFA amount is provided.

[0157] Figures 17A-17B Provided are graphs showing the production of short chain fatty acids (SCFA) (propionate, acetate and butyrate) in an ex vivo assay in which stool samples from 31 overweight subjects with type 2 diabetes (T2D) were incubated with selected oligosaccharide compositions ("Selected OS") or a negative control (water). Fig.17A The total amount of SCFA produced is provided (in mM). Fig. 17B The percentage of each unique SCFA (propionate, acetate and butyrate) relative to the total SCFA amount is provided.

[0158] Figures 18A-18B Provided are methods showing that selected oligosaccharide compositions have an effect on the susceptibility of low-density lipoprotein receptor knockout (LDLR) mice to Western diet (WD)-induced obesity. - / - ) on the production of short-chain fatty acids (SCFA) (propionate, acetate, and butyrate) and succinate in a mouse model. Fig.18A The total amount of SCFA produced is provided (in μM). Fig.18B The percentage of each unique SCFA (propionate, acetate, butyrate) and succinate relative to the total SCFA and succinate amounts is provided.

[0159] Fig.19 A graph showing that selected oligosaccharide compositions promote propionic acid production in bacterial taxa (eg, Bacteroidetes) is provided.

[0160] Figures 20A-20B Representative HSQC NMR spectra of selected oligosaccharide compositions are provided. Fig. 20A The anomeric and nonanomeric regions of the HSQC NMR spectrum are shown. Fig. 20B Representative HSQC NMR spectra of selected oligosaccharide compositions (anomeric region only) with indicated linkages are shown. DETAILED DESCRIPTION OF THE INVENTION

[0162] Compositions and methods described herein are based on the following discovery: oligosaccharide compositions can be used to reduce lipid and / or glucose (e.g., blood) levels in subjects. In some embodiments, oligosaccharide compositions described herein can be used to reduce lipid levels, such as cholesterol, low-density lipoprotein (LDL) non-esterified free fatty acids and triglycerides (e.g., in blood or liver). In some embodiments, oligosaccharide compositions described herein can be used to reduce glucose levels, such as blood sugar. In addition, in some embodiments, compositions and methods described herein can be used to treat non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Some aspects of the present disclosure relate to recognizing that oligosaccharide compositions can be used to increase propionic acid (propionate) (also referred to as propionic acid (propionic acid)) levels in subjects.

[0163] Some aspects of the present disclosure are based on the results of extensive screening work that was performed to identify oligosaccharide compositions that can modulate (e.g., reduce) different types of lipids, lipoproteins, and lipid esters (e.g., cholesterol, LDL, non-esterified free fatty acids, and triglycerides) levels in a subject, as well as sugars such as glucose levels. Hundreds of unique oligosaccharide compositions were determined for their effects on the human gastrointestinal microbiome in an in vitro environment. The oligosaccharide compositions examined in the screening were generated using different sugar monomers (e.g., dextrose monomers, xylose monomers, etc.) and under different reaction temperatures, for different time periods, and / or in the presence of different catalysts.

[0164] From this screening work, the selected oligosaccharide composition is identified as a highly efficient regulator of glucose and / or lipid levels, such as cholesterol, low-density lipoprotein (LDL), non-esterified free fatty acids and triglyceride levels. Therefore, in some embodiments, this oligosaccharide composition is particularly suitable for treating subjects suffering from diseases or disorders associated with elevated lipid and / or glucose levels. In some embodiments, the selected oligosaccharide composition can be used to reduce the lipid (e.g., triglyceride, cholesterol and / or adipose phospholipid) level of subjects in need thereof. In some embodiments, the selected oligosaccharide composition can be used to treat lipid and lipoprotein abnormalities. In some embodiments, the selected oligosaccharide composition can be used to treat dyslipidemia. In some embodiments, the selected oligosaccharide composition can be used to prevent or delay the onset of dyslipidemia. In some embodiments, the selected oligosaccharide composition can be used to treat hyperlipidemia. In some embodiments, the selected oligosaccharide composition can be used to prevent or delay the onset of hyperlipidemia. In some embodiments, hyperlipidemia is primary (or familial), for example, due to genetic reasons. In some embodiments, hyperlipidemia is secondary (or acquired), for example, due to another disease or condition (e.g., caused by lifestyle, diet, age, etc.). In some embodiments, the selected oligosaccharide composition can be used to prevent or delay the onset of diseases associated with abnormal lipid and / or glucose levels, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease (CVD), type 2 diabetes (T2D), and chronic kidney disease (CKC). In some embodiments, the selected oligosaccharide composition can be used to reduce the risk of subjects suffering from diseases associated with abnormal lipid and / or glucose levels, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease (CVD), type 2 diabetes (T2D), and chronic kidney disease (CKC). Further aspects of the present disclosure are provided below, including a description of defined terms.

[0165] definition

[0166] Agitation conditions: As used herein, the term "agitation conditions" refers to conditions that promote or maintain a mixture (e.g., a reaction mixture comprising galactose monomers and mannose monomers) with respect to a substantially uniform or homogeneous state of solid (e.g., solid catalyst) dispersion, heat transfer uniformity, or other similar parameters. Agitation conditions typically include stirring, shaking, and / or mixing the reaction mixture. In some embodiments, agitation conditions may include adding a gas or other liquid to the solution. In some embodiments, agitation conditions are used to maintain a substantially uniform or homogeneous distribution of a catalyst, such as an acid catalyst. In some embodiments, a monosaccharide preparation is heated in the presence of an acid catalyst and under suitable conditions to achieve homogeneity and uniform heat transfer, thereby synthesizing an oligosaccharide composition.

[0167] About: As used herein, the term "approximately" or "about", as applied to one or more values ​​of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that differ from a stated reference value by no more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in any direction (greater or less) unless otherwise stated or obvious from the context (unless the number exceeds 100% of the possible values).

[0168] Effective amount: As used herein, the term "effective amount" refers to the amount or concentration of an oligosaccharide composition that is necessary and sufficient to cause a biological response in a subject or patient, for example. In some embodiments, an effective amount of an oligosaccharide composition is capable of regulating (e.g., increasing or decreasing) the activity or level of an enzyme in a subject. In some embodiments, an effective amount of an oligosaccharide composition is capable of regulating (e.g., increasing or decreasing) the processing of metabolites. In some embodiments, an effective amount of an oligosaccharide composition is capable of regulating (e.g., increasing or decreasing) the concentration or number of at least one microorganism. In some embodiments, an effective amount of an oligosaccharide composition is capable of regulating (e.g., decreasing) the symptoms (e.g., severity or number of symptoms) of a disease associated with elevated pathogen colonization in a subject. In some embodiments, an effective amount of an oligosaccharide composition is capable of reducing the acquisition, colonization and / or reduction of a reservoir of a pathogen (e.g., a drug or antibiotic resistant pathogen, or an MDR pathogen) in a subject. In some embodiments, an effective amount of an oligosaccharide composition is capable of treating a subject with intestinal colonization of a pathogen (e.g., CRE or VRE).

[0169] Galactose monomer: As used herein, the term "galactose monomer" generally refers to the D-isomer of galactose monomer, referred to as D -Galactose.

[0170] Mannose monomer: As used herein, the term "mannose monomer" generally refers to the D-isomer of the mannose monomer, referred to as D -Mannose.

[0171] Monosaccharide preparation: As used herein, the term "monosaccharide preparation" refers to a preparation comprising two or more monosaccharides (eg, galactose monomers and mannose monomers). In some embodiments, the monosaccharide preparation comprises galactose monomers and mannose monomers.

[0172] Oligosaccharide: As used herein, the term "oligosaccharide" (which may be used interchangeably with the term "oligosaccharide" in some cases) refers to a sugar molecule comprising at least two monosaccharides (e.g., galactose monomers, mannose monomers) linked together via glycosidic bonds (degree of polymerization (DP) of at least 2 (e.g., DP2+)). In some embodiments, the oligosaccharide comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten monosaccharide subunits linked by glycosidic bonds. In some embodiments, oligosaccharides in the range of 3-20, 4-16, 5-15, 8-12, 5-25, 10-25, 20-50, 40-80, or 75-100 monosaccharides are linked by glycosidic bonds. In some embodiments, the oligosaccharide comprises at least one 1,2; 1,3; 1,4; and / or 1,6 glycosidic bond. The oligosaccharide can be linear or branched. Oligosaccharides may have one or more glycosidic bonds in the α-configuration and / or one or more glycosidic bonds in the β-configuration.

[0173] Pharmaceutical composition: As used herein, "pharmaceutical composition" refers to a composition having pharmacological activity or other direct effect in alleviating, treating or preventing a disease, and / or its final dosage form or preparation, and is used for humans. Pharmaceutical compositions or pharmaceutical preparations are usually produced under good manufacturing practice (GMP) conditions. Pharmaceutical compositions or preparations can be sterile or non-sterile. If non-sterile, such pharmaceutical compositions or preparations generally meet the microbiological indicators and standards for non-sterile drug products described in the United States Pharmacopoeia (USP) or the European Pharmacopoeia (EP). Any oligosaccharide composition described herein can be formulated into a pharmaceutical composition.

[0174] Subject: As used herein, the term "subject" refers to a human subject or patient. Subjects may include neonates (preterm neonates, full-term neonates), infants under one year old, toddlers (e.g., 1 to 12 years old), adolescents (e.g., 13-19 years old), adults (e.g., 20-64 years old), and elderly people (65 years old and above). In some embodiments, the subject is a child population or a subpopulation thereof, including neonates (birth to 1 month), infants (1 month to 2 years old), developing children (2 to 12 years old), and adolescents (12 to 16 years old). In some embodiments, the subject is a healthy subject. In some embodiments, the subject is a patient with elevated levels of lipids, e.g., cholesterol, LDL, non-esterified free fatty acids, and triglycerides relative to healthy subjects. In some embodiments, the subject has lipid and lipoprotein abnormalities. In some embodiments, the subject has one or more genetic defects associated with abnormal (e.g., elevated) lipid levels, such as familial hypercholesterolemia, familial hyperlipoproteinemia, familial hyperchylomicronemia). In some embodiments, the subject has obtained a disease associated with abnormal (e.g., elevated) lipid levels, such as by lifestyle, diet, or age. In some embodiments, the subject suffers from or is suspected of having dyslipidemia. In some embodiments, the subject is at risk of developing into dyslipidemia. In some embodiments, the subject suffers from or is suspected of having hyperlipidemia. In some embodiments, the subject is at risk of developing into hyperlipidemia. In some embodiments, the subject suffers from primary (or familial) hyperlipidemia. In some embodiments, the subject suffers from secondary (or acquired) hyperlipidemia. In some embodiments, the subject suffers from or is suspected of having or is at risk of suffering from a disease or disease associated with abnormal lipids and / or glucose levels, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease (CVD), type 2 diabetes (T2D), and chronic kidney disease (CKC). In some embodiments, the subject suffers from or is suspected of having or is at risk of suffering from metabolic syndrome, or shows one, two, three, or more symptoms associated with metabolic syndrome. In some embodiments, the subject suffers from or is suspected of having cardiometabolic disorders or liver disease. In some embodiments, the subject has elevated glucose levels (e.g., hyperglycemia). In some embodiments, the subject has elevated insulin levels (e.g., hyperinsulinemia). In some embodiments, the subject has elevated levels of any other type 2 diabetes markers (e.g., hemoglobin A1c (HbA1c), fructosamine, C-reactive protein (CRP)). In some embodiments, the subject exhibits resistance to standard of care medications, such as treatment of abnormal levels of lipids, such as statins (e.g., statin-refractory subjects or statin non-responders).In some embodiments, the subject exhibits intolerance to a standard of care medication, eg, a medication that treats abnormal levels of lipids, such as a statin, such as a subject exhibiting statin associated muscle symptoms (SAMS).

[0175] Treatment methods and treatment: As used herein, the terms "treatment" and "treatment methods" refer to the administration of a 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 symptoms and / or their underlying causes, and / or promote improvement or remediation of damage, and / or prevent the adverse condition, disorder, or disease in an asymptomatic subject who is susceptible to a specific adverse condition, disorder, or disease, or is suspected of suffering from the condition, disorder, or disease, or is at risk of developing such a condition, disorder, or disease. In some embodiments, treating a subject with an oligosaccharide composition reduces the relative or absolute level of lipids, such as cholesterol, LDL, non-esterified free fatty acids, and triglycerides in the subject. In some embodiments, treating a subject with an oligosaccharide composition reduces the relative or absolute level of lipids, such as cholesterol, LDL, non-esterified free fatty acids, and triglycerides in the subject's blood or liver. In some embodiments, treating a subject with an oligosaccharide composition reduces the severity of a disease associated with elevated lipid and / or glucose levels. In some embodiments, treating a subject with an oligosaccharide composition can extend the lifespan of a person suffering from or suspected of having a disease associated with elevated lipid and / or glucose levels. In some embodiments, treatment of a population of subjects with an oligosaccharide composition increases the average lifespan of subjects suffering from or suspected of suffering from a disease associated with elevated lipid and / or glucose levels. In some embodiments, treatment of a subject with an oligosaccharide composition prevents exacerbation, progression or onset of a disease or disorder associated with elevated lipid and / or glucose levels.

[0176] II. Oligosaccharide Composition

[0177] Provided herein are oligosaccharide compositions and methods of using the same for modulating lipid levels in human subjects.

[0178] In one aspect, the oligosaccharide composition provided herein comprises a plurality of oligosaccharides selected from formula (I) and formula (II):

[0179]

[0180] Wherein R in formula (I) and formula (II) is independently selected from hydrogen, and formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc), (IId):

[0181]

[0182]

[0183] wherein R in Formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc) and (IId) is independently as defined in Formula (I) and Formula (II).

[0184] In some embodiments, the oligosaccharide composition is initially heated to a temperature in the range of 100°C to 160°C, 100°C to 120°C, 110°C to 110°C to 130°C, 120°C to 140°C, 130°C to 150°C, or about 135°C. The ratio of galactose monomer to mannose monomer may be 3:2. The heating may be carried out under stirring conditions. The heating may include gradually increasing the temperature (e.g., from room temperature) to about 130°C, about 135°C, about 140°C, about 145°C, or about 150°C under suitable conditions to achieve homogeneity and uniform heat transfer.

[0185] An acid catalyst comprising a positively charged hydrogen ion is added to the preparation (e.g., before heating). In some embodiments, the acid catalyst is a solid catalyst. In some embodiments, the catalyst is a strong acid cation exchange resin with one or more physical and chemical properties according to Table 1. In some embodiments, the catalyst comprises a sulfonic acid moiety>3.0mmol / g and a cationic moiety<1.0mmol / g. In certain embodiments, the catalyst has a nominal moisture content of 45-50 weight percent. In some embodiments, the catalyst is a soluble catalyst, such as an organic acid catalyst. In some embodiments, the catalyst is citric acid, acetic acid, butyric acid or propionic acid. In certain embodiments, the catalyst is added simultaneously with a galactose monomer and a mannose monomer.

[0186] Table 1: Non-limiting examples of strong acid cation exchange resin properties

[0187]

[0188] In some embodiments, after loading the preparation with the catalyst, the resulting reaction mixture is maintained at atmospheric pressure and a temperature in the range of 100°C to 160°C, 100°C to 120°C, 110°C to 130°C, 120°C to 140°C, 130°C to 150°C, or about 135°C under conditions that promote the formation of acid-catalyzed oligosaccharides. In some embodiments, once the weight percentage of the total monomer content in the oligosaccharide composition (the total monomer content includes the amount of galactose monomers and / or mannose monomers) is within the range of 2-14% (optionally 2-5%, 4-8%, 7-10%, 9-14%, or 8-12%), the reaction mixture is quenched. Quenching generally involves diluting the reaction mixture with water (e.g., deionized water) and gradually reducing the temperature of the reaction mixture to 55°C to 95°C. In some embodiments, the water used for quenching is about 95°C. Water can be added to the reaction mixture under conditions sufficient to avoid solidifying the mixture. In certain embodiments, water can be removed from the reaction mixture by evaporation. In some embodiments, the reaction mixture may contain 50-55 weight percent dissolved solids. Finally, in order to obtain a purified oligosaccharide composition, the composition is generally diluted with water to a concentration of about 35-50 weight percent and a temperature of less than about 85°C by the quenched reaction mixture, and then the mixture is passed through a filter or a series of chromatographic resins. In some embodiments, the composition is separated from the acid catalyst. In certain embodiments, the filter used is a 0.45 μm filter. Alternatively, a series of chromatographic resins can be used and generally include cation exchange resins, anion exchange resins and / or decolorizing polymer resins. In some embodiments, any or all types of resins can be used once or multiple times in any order. In some embodiments, the oligosaccharide composition includes water below the level necessary for microbial growth when stored at room temperature.

[0189] In certain embodiments, the average degree of polymerization of all oligosaccharides is in the range of 6-19, optionally 11-16. In some embodiments, the oligosaccharide composition comprises water in the range of 45-55 weight percent. In some embodiments, the oligosaccharide composition comprises oligosaccharides with MWw (weight average molecular weight) (g / mol) in the range of 1807-2985. In some embodiments, the oligosaccharide composition comprises oligosaccharides with MWn (number average molecular weight) (g / mol) in the range of 1322-2073. In some embodiments, the pH of the solution comprising the oligosaccharide composition is in the range of 2.50-3.50. In some embodiments, the oligosaccharide composition comprises oligomers with two or more repeating units (DP2+) in the range of 89-93 weight percent.

[0190] In addition, in some embodiments, the oligosaccharide composition can be de-monomerized. In some embodiments, de-monomerization includes removing residual sugar monomers. In some embodiments, de-monomerization is performed using a chromatographic resin. Therefore, in some embodiments, different compositions can be prepared according to the percentage of monomers present. In some embodiments, the oligosaccharide composition is de-monomerized to a monomer content of about 1%, about 3%, about 5%, about 10%, or about 15%. In some embodiments, the oligosaccharide composition is de-monomerized to a monomer content of about 1-3%, about 3-6%, about 5-8%, about 7-10%, or about 10-15%. In one embodiment, the oligosaccharide composition is de-monomerized to a monomer content of less than 1%. In one embodiment, the oligosaccharide composition is de-monomerized to a monomer content of between about 7% and 10%. In one embodiment, the oligosaccharide composition is de-monomerized to a monomer content of between about 1% and 3%. In one embodiment, de-monomerization is achieved by osmotic separation. In a second embodiment, de-monomerization is achieved by tangential flow filtration (TFF). In a third embodiment, de-monomerization is achieved by ethanol precipitation.

[0191] In some embodiments, the oligosaccharide compositions with different monomer contents may also have different measurements of total dietary fiber, moisture, total dietary fiber (on a dry basis), or dextrose equivalent percentage (DE). In some embodiments, total dietary fiber is measured according to the method of AOAC 2011.25. In some embodiments, moisture is measured at 60°C by using a vacuum oven. In some embodiments, total dietary fiber (on a dry basis) is calculated. In some embodiments, DE percentage is measured according to the Food Chemicals Codex (FCC).

[0192] In some embodiments, the total dietary fiber content of the oligosaccharide composition is 58-65% (by dry weight). In some embodiments, the oligosaccharide composition has a total dietary fiber content of 50-80, 55-80, 60-80, 50-70, 55-70, 60-70, 50-65, 55-65 or 60-65% (by dry basis). In some embodiments, the oligosaccharide composition has a total dietary fiber content of about 50%, about 55%, about 58%, about 60%, about 62% or about 65% (by dry basis). In some embodiments, the oligosaccharide composition has a total dietary fiber content of 50-95% (by dry basis).

[0193] In some embodiments, the oligosaccharide composition has a total reducing sugar content (dextrose equivalent (DE) (dry solids)) of 5-40%.

[0194] In some embodiments, the generation of the oligosaccharide composition according to the method provided herein can be carried out in a batch process or a continuous process. For example, in one embodiment, the oligosaccharide composition is generated in a batch process, wherein the contents of the reactor are placed under stirring conditions (e.g., continuous mixing or blending), and all or a large amount of the reaction products are removed (e.g., separated and / or recovered).

[0195] In certain embodiments, the method using the catalyst is carried out in an aqueous environment. A suitable aqueous solvent is water, which can be obtained from various sources. Generally, in some embodiments, a water source with a lower concentration of ionic species (e.g., sodium, phosphorus, ammonium or magnesium salts) can be used because such ionic species may reduce the effectiveness of the catalyst. In some embodiments where the aqueous solvent is water, the water has less than 10% ionic species (e.g., sodium, phosphorus, ammonium, magnesium salts). In some embodiments where the aqueous solvent is water, the resistivity of the water is at least 0.1 megohm-cm, at least 1 megohm-cm, at least 2 megohm-cm, at least 5 megohm-cm or at least 10 megohm-cm.

[0196] In some embodiments, as the reaction of the method provided herein proceeds, the respective glycosidic coupling of one or more sugar monomers produces water (e.g., released water). In certain embodiments, the method described herein may also include 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. Therefore, in some embodiments, the water content in the reaction mixture can be changed during the reaction, such as removing the released water produced. Suitable methods can be used to remove water (e.g., released water) in the reaction mixture, including, for example, by evaporation, such as by distillation. In some embodiments, the method includes including water in the reaction mixture. In certain embodiments, the method includes removing water from the reaction mixture by evaporation.

[0197] In some embodiments, the ratio of galactose monomers to mannose monomers is about 1:4, 1:3, 1:2, 2:3, 1:1, 3:2, 2:1, 3:1, or 4: 1. In some embodiments, the ratio of galactose monomers to mannose monomers is about 3:2.

[0198] In some embodiments, the monosaccharide preparation comprises about 40-80% galactose monomers and 20-60% mannose monomers. In some embodiments, the monosaccharide preparation comprises about 50-70% galactose monomers and about 30-50% mannose monomers. In some embodiments, the monosaccharide preparation comprises about 60% galactose monomers and about 40% mannose monomers.

[0199] In certain embodiments, the formulation is loaded with an acid catalyst comprising positively charged hydrogen ions. In some embodiments, the acid catalyst is a solid catalyst (e.g., Dowex Marathon C). In some embodiments, the acid catalyst is a soluble catalyst (e.g., citric acid).

[0200] In some embodiments, the molar ratio of positively charged hydrogen ions to the total galactose monomer and mannose monomer content is in a suitable range. In some embodiments, the molar ratio of positively charged hydrogen ions to the total galactose monomer and mannose monomer content is in the range of 0.01 to 0.1, 0.02 to 0.08, 0.03 to 0.06, or 0.05 to 0.06. In some embodiments, the molar ratio of positively charged hydrogen ions to the content of galactose monomers and mannose monomers is in the range of 0.003 to 0.01, 0.005 to 0.02, 0.01 to 0.02, 0.01 to 0.03, 0.02 to 0.03, 0.02 to 0.04, 0.03 to 0.05, 0.03 to 0.08, 0.04 to 0.07, 0.05 to 0.1, 0.05 to 0.2, 0.1 to 0.2, 0.1 to 0.3, or 0.2 to 0.3. In some embodiments, the molar ratio of positively charged hydrogen ions to the total content of galactose monomers and mannose monomers is in the range of 0.050 to 0.052. In some embodiments, the molar ratio of positively charged hydrogen ions to the total content of galactose monomers and mannose monomers is in the range of 0.020 to 0.035.

[0201] In some embodiments, the molar ratio of the soluble acid catalyst (e.g., citric acid catalyst) to the total galactose monomer and mannose monomer content is in a suitable range. In some embodiments, the molar ratio of the soluble acid catalyst (e.g., citric acid catalyst) to the total galactose monomer and mannose monomer content is in the range of 0.01 to 0.1, 0.02 to 0.08, 0.03 to 0.06, or 0.05 to 0.06. In some embodiments, the molar ratio of the soluble acid catalyst (e.g., citric acid catalyst) to the total galactose monomer and mannose monomer content is in the range of 0.003 to 0.01, 0.005 to 0.02, 0.01 to 0.02, 0.01 to 0.03, 0.02 to 0.03, 0.02 to 0.04, 0.03 to 0.05, 0.03 to 0.08, 0.04 to 0.07, 0.05 to 0.1, 0.05 to 0.2, 0.1 to 0.2, 0.1 to 0.3, or 0.2 to 0.3. In some embodiments, the molar ratio of the soluble acid catalyst (e.g., citric acid catalyst) to the total galactose monomer and mannose monomer content is in the range of 0.050 to 0.052. In some embodiments, the molar ratio of soluble acid catalyst (eg, citric acid catalyst) to total galactose monomer and mannose monomer content is in the range of 0.020 to 0.035.

[0202] In some embodiments, water is added to the reaction mixture to quench the reaction by bringing the temperature of the reaction mixture to 100°C or less. In some embodiments, the water used for quenching is deionized water. In some embodiments, the water used for quenching is USP water. In some embodiments, the water has a temperature of about 60°C to about 100°C. In certain embodiments, the water used for quenching is about 95°C. In some embodiments, water is added to the reaction mixture under conditions sufficient to avoid solidifying the mixture.

[0203] The viscosity of the reaction mixture can be measured and / or changed during the reaction. Generally, viscosity refers to the measurement of the internal flow resistance (e.g., "consistency") of a fluid, expressed in centipoise (cP) or Pascal-seconds. In some embodiments, the viscosity of the reaction mixture is between about 100 cP and about 95,000 cP, about 5,000 cP and about 75,000 cP, about 5,000 and about 50,000 cP, or about 10,000 and about 50,000 cP. In certain embodiments, the viscosity of the reaction mixture is between about 50 cP and about 200 cP.

[0204] In some embodiments, the oligosaccharide compositions provided herein can be subjected to one or more additional processing steps. Additional processing steps may include, for example, purification steps. Purification steps may include, for example, separation, denomerization, dilution, concentration, filtration, desalination or ion exchange, chromatographic separation or decolorization or any combination thereof.

[0205] In certain embodiments, the methods described herein further comprise a dilution step. In some embodiments, deionized water is used for dilution. In certain embodiments, USP water is used for dilution. In certain embodiments, after dilution, the oligosaccharide composition comprises water in the range of about 5-75, 25-65, 35-65, 45-55 or 47-53 weight percent. In certain embodiments, after dilution, the oligosaccharide composition comprises water in the range of about 45-55 weight percent.

[0206] In some embodiments, the methods described herein further include a decolorization step. The one or more oligosaccharide compositions produced can be subjected to a decolorization step using an appropriate method, including, for example, treatment with an adsorbent, activated carbon, chromatography (e.g., using an ion exchange resin) and / or filtration (e.g., microfiltration).

[0207] In some embodiments, the one or more oligosaccharide compositions produced are contacted with a material to remove salts, minerals and / or other ionic species. For example, in certain embodiments, the one or more oligosaccharide compositions produced are flowed through an anion exchange column. In other embodiments, the oligosaccharide compositions produced are flowed through an anion / cation exchange column pair.

[0208] In some embodiments, the methods described herein may further include a concentration step. For example, in some embodiments, the oligosaccharide composition may be evaporated (e.g., vacuum evaporation) to produce a concentrated oligosaccharide composition. In other embodiments, the oligosaccharide composition may be subjected to a spray drying step to produce an oligosaccharide powder. In certain embodiments, the oligosaccharide composition may be subjected to an evaporation step and a spray drying step simultaneously. In some embodiments, the oligosaccharide composition may be subjected to a freeze drying (e.g., freeze drying) step to remove water and produce a powdered product.

[0209] In some embodiments, the method described herein also includes a classification step. The oligosaccharide composition prepared and purified can be subsequently 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). In certain embodiments, the oligosaccharide composition is divided into a collection of species representing 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or more than 98% of short (about DP1-2), medium (about DP3-10), long (about DP11-18) or very long (about DP>18).

[0210] In certain embodiments, the prepared oligosaccharide composition is fractionated by adsorption on a carbonaceous material, followed by desorption of the fraction by washing the material with a mixture of an organic solvent at a concentration of 1%, 5%, 10%, 20%, 50% or 100% in water. In one embodiment, the adsorbent material is activated carbon. In another embodiment, the adsorbent material is a mixture of activated carbon and a filler such as diatomaceous earth or Celite 545 (at a ratio of 5%, 10%, 20%, 30%, 40% or 50% by volume or weight).

[0211] In further embodiments, the prepared oligosaccharide composition is separated by a high performance liquid chromatography system. In certain variations, the prepared oligosaccharide composition is separated by ion affinity chromatography, hydrophilic interaction chromatography, or size exclusion chromatography including gel permeation and gel filtration.

[0212] In some embodiments, the catalyst is removed by filtration. In certain embodiments, a 0.45 μm filter is used to remove the catalyst during the filtration process. In other embodiments, low molecular weight materials are removed by filtration. In certain variants, low molecular weight materials can be removed by dialysis, ultrafiltration, diafiltration or tangential flow filtration. In certain embodiments, the filtration is carried out in a static dialysis tubing device. In other embodiments, the filtration is carried out in a dynamic flow filtration system. In other embodiments, the filtration is carried out in a centrifugal force driven filter cartridge. In certain embodiments, the reaction mixture is cooled to less than about 85° C. and then filtered.

[0213] In certain embodiments, the average degree of polymerization of all oligosaccharides is in the range of 6-19. In certain embodiments, the average degree of polymerization of all oligosaccharides is in the range of 11-16. In certain embodiments, the average degree of polymerization of all oligosaccharides is in the range of 9-17. In some embodiments, the average degree of polymerization of all oligosaccharides is in the range of 5-20, 6-19, 11-16, 12-18, 10-17, 7-15, 7-12, 7-10, 7-8, 9-10, 10-11, 11-12, 11-15, 12-13, 12-14, 13-14, 14-15, 15-16, 17-18, 15-20, 3-8, 4-7, or 5-6. In some embodiments, the average degree of polymerization (DP) of all oligosaccharides is about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, or about 19.

[0214] In certain embodiments, the weight percentage of galactose monomers and mannose monomers in the oligosaccharide composition is in the range of 7-12. In certain embodiments, the weight percentage of galactose monomers and mannose monomers in the oligosaccharide composition is in the range of 10-12. In certain embodiments, the weight percentage of galactose monomers and mannose monomers in the oligosaccharide composition is in the range of 12-16. In certain embodiments, the weight percentage of galactose monomers and mannose monomers in the oligosaccharide composition is in the range of 13-15.

[0215] In some embodiments, the oligosaccharide composition is a mixture of polymers of galactose and mannose in a ratio of about 60% and 40% by weight, respectively. In some embodiments, the molecular formula is H-[C6H 9-11 O5] n -OH, wherein the total number of monomer units in a single polymer of the mixture ranges from 2 to about 60 (n=2-60), with an average of about 14.3 monomer units for the mixture. Each monomer unit may be unsubstituted, mono-, di- or tri-substituted with another galactose or mannose unit in any glycosidic isomer.

[0216] In some embodiments, the oligosaccharide composition comprises water in the range of 5-75 weight percent. In some embodiments, the oligosaccharide composition comprises water in the range of 25-65 weight percent. In some embodiments, the oligosaccharide composition comprises water in the range of 35-65 weight percent. In some embodiments, the oligosaccharide composition comprises water in the range of 45-55 weight percent.

[0217] In some embodiments, the oligosaccharide composition comprises oligosaccharides with a MWw (g / mol) in the range of 1807-2985. In some embodiments, the oligosaccharide composition comprises oligosaccharides with a MWw (g / mol) in the range of 1510-3124. In some embodiments, the oligosaccharide composition comprises oligosaccharides with a MWw (g / mol) in the range of 1500-2000, 1700-2200, 1900-2400, 2100-2600, 2300-2800, 2500-3000, or 2700-3200.

[0218] In some embodiments, the oligosaccharide composition comprises oligosaccharides with a MWn (g / mol) in the range of 1322-2073. In some embodiments, the oligosaccharide composition comprises oligosaccharides with a MWn (g / mol) in the range of 1238-2295. In some embodiments, the oligosaccharide composition comprises oligosaccharides with a MWn (g / mol) in the range of 1200-1500, 1300-1600, 1400-1700, 1500-1800, 1600-1900, 1700-2000, 1800-2100, 1900-2200, 2000-2300, or 2100-2500.

[0219] In some embodiments, the pH of the solution comprising the oligosaccharide composition is in the range of 1.50-6.00. In some embodiments, the pH of the solution comprising the oligosaccharide composition is in the range of 1.50-5.00. In some embodiments, the pH of the solution comprising the oligosaccharide composition is in the range of 2.00-4.00. In some embodiments, the pH of the solution comprising the oligosaccharide composition is in the range of 2.50-3.50.

[0220] In some embodiments, the oligosaccharide composition comprises oligosaccharides having a degree of branching in the range of about 10% to about 35%. In some embodiments, the oligosaccharide composition comprises oligosaccharides having a degree of branching in the range of about 15% to about 30%. In some embodiments, the oligosaccharide composition comprises oligosaccharides having a degree of branching in the range of 5-50%, 5-40%, 5-30%, 5-20%, 5-15%, 10-50%, 10-40%, 10-30%, 10-25%, 15-30% or 15-20%.

[0221] In some embodiments, the oligosaccharide composition comprises an oligomer with two or more repeating units (DP2+) within the range of 80-100 weight percent. In some embodiments, the oligosaccharide composition comprises an oligomer with two or more repeating units (DP2+) within the range of 89-93 weight percent. In some embodiments, the oligosaccharide composition comprises an oligomer with two or more repeating units (DP2+) within the range of 85-95 weight percent. In some embodiments, the oligosaccharide composition comprises an oligomer with two or more repeating units (DP2+) within the range of 80-85, 85-87, 86-88, 87-90, 88-91, 89-92, 90-93, 91-94, 92-95, 93-96 or 95-98 weight percent.

[0222] In some embodiments, the oligosaccharide composition has a polydispersity index (PDI) of 1.8-2.4. In some embodiments, the oligosaccharide composition has a polydispersity index (PDI) of 2.0-2.3. In some embodiments, the oligosaccharide composition has a PDI of 1.0-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.7-1.8, 1.8-2.0, 2.0-2.2, 2.2-2.4- or 2.4-2.6. In some embodiments, the oligosaccharide composition has a PDI of about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.1, about 2.2, about 2.3, or about 2.4.

[0223] In some embodiments, the MWw, MWn, PDI, monomer content (DP1) and / or DP2+ values ​​of the oligosaccharides in the oligosaccharide composition are determined using size exclusion chromatography as described in Example 5.

[0224] In some embodiments, the oligosaccharide composition comprises an oligomer having at least three linked monomeric units (DP3+) in the range of 80-95 weight percent. In some embodiments, the oligosaccharide composition comprises an oligomer having at least three linked monomeric units (DP3+) in the range of 80-85, 85-87, 86-88, 87-90, 88-91, 89-92, 90-93, 91-94, or 92-95 weight percent.

[0225] In some embodiments, the oligosaccharide composition comprises 11.1% to 16.0% monomers (DP1). In some embodiments, the oligosaccharide composition comprises 5% to 10%, 7% to 12%, 11% to 18%, 10% to 15% or 12% to 17% monomers (DP1). In some embodiments, the oligosaccharide composition comprises 84% ​​to 89% of oligomers with at least two connected monomer units (DP2+). In some embodiments, the oligosaccharide composition comprises 80% to 81%, 81% to 82%, 82% to 83%, 84% to 85%, 85% to 86%, 86% to 87%, 87% to 88%, 88% to 90%, or 89% to 95% of oligomers with at least two connected monomer units (DP2+). In some embodiments, the oligosaccharide composition comprises 84% ​​to 85%, 85% to 86%, 86% to 87%, 87% to 88%, or 88% to 90% of oligomers having at least three linked monomeric units (DP3+).

[0226] In some embodiments, the oligosaccharide composition comprises less than 0.10% total impurities (excluding monomers). In some embodiments, the oligosaccharide composition comprises less than 0.05% total impurities (excluding monomers). In some embodiments, the oligosaccharide composition comprises less than 0.20%, 0.15%, 0.10% or 0.05% total impurities (excluding monomers). In some embodiments, the oligosaccharide composition comprises less than 0.10% w / w levoglucosan, less than 0.10% w / w glucuronic acid, less than 0.10% w / w lactic acid, less than 0.10% w / w formic acid, less than 0.10% w / w acetic acid, and less than 0.10% w / w hydroxymethylfurfural (HMF). In some embodiments, the oligosaccharide composition comprises undetectable levels of lactic acid, formic acid, levulinic acid and HMF. In some embodiments, the oligosaccharide composition comprises 2.52% w / w citric acid. In some embodiments, the oligosaccharide composition comprises 2.18-2.72% w / w, 2.00-3.00%, 2.00-2.50%, or 2.50-3.00% citric acid.

[0227] In some embodiments, the oligosaccharide composition comprises a MWw of 1807-2985, a MWn of 1322-2073, and / or a PDI of 2.0-2.3.

[0228] In certain embodiments, the oligosaccharide composition analyzed by NMR contains monosaccharide monomers (DP1), i.e., the DP1 component is not removed from the composition prior to NMR analysis. For example, in some embodiments, the oligosaccharide composition analyzed by NMR contains 10%-25% DP1 monomer. In certain embodiments, the composition analyzed by NMR is de-monomerized, i.e., some or all of the DP1 component of the composition is removed prior to NMR analysis, e.g., by the method described in Example 4. For example, in some embodiments, the oligosaccharide composition analyzed by NMR contains between 0.05% and 8% DP1 monomer.

[0229] The oligosaccharide compositions described herein, as well as the oligosaccharide compositions prepared according to the methods described herein, can be characterized and distinguished from the compositions of the prior art using full methylation analysis. See, for example, Zhao, Y., et al. 'Rapid, sensitive structure analysis of oligosaccharides,' PNAS March 4, 1997 94 (5) 1629-1633; Kailemia, MJ, et al. 'Oligosaccharide analysis by mass spectrometry: A review of recent developments,' Anal Chem. 2014 Jan 7; 86 (1): 196–212. Therefore, on the other hand, an oligosaccharide composition is provided herein, which comprises a plurality of oligosaccharides that are minimally digestible by humans, wherein the plurality of oligosaccharides comprises monomeric groups. The molar percentages of different types of monomeric groups in the plurality of oligosaccharides can be quantified using the full methylation assay as described in Example 2. The de-monomerized sample of the composition was subjected to a full methylation assay.

[0230] In some embodiments, the plurality of oligosaccharides comprises two or more monomeric groups selected from groups (1)-(40):

[0231] (1) t-arapyranose monogroup, which represents 0.00-0.12 mol% of the monomer group in the various oligosaccharides;

[0232] (2) t-mannopyranose monogroups, which represent 11.47-18.18 mol % of the monomeric groups in the various oligosaccharides;

[0233] (3) t-glucopyranose monogroups, which represent 0.06-0.18 mol % of the monomer groups in the various oligosaccharides;

[0234] (4) t-mannofuranose monogroups, which represent 0.17-0.46 mol % of the monomeric groups in the various oligosaccharides;

[0235] (5) t-galactofuranose monogroups, which represent 2.96-6.24 mol % of the monomeric groups in the various oligosaccharides;

[0236] (6) t-galactopyranose monogroups, which represent 12.90-17.63 mol % of the monomeric groups in the various oligosaccharides;

[0237] (7) 2-arabinopyranose monogroups, which represent 0.00-0.19 mol % of the monomer groups in the various oligosaccharides;

[0238] (8) 3-mannofuranose monogroup, which represents 0.07-0.24 mol % of the monomeric groups in the various oligosaccharides;

[0239] (9) 2-mannofuranose monogroup, which represents 0.00-1.07 mol % of the monomer group in the various oligosaccharides;

[0240] (10) 2-mannopyranose and / or 3-mannopyranose monogroups, which represent 3.91-5.93 mol % of the monomeric groups in the various oligosaccharides;

[0241] (11) 2-galactofuranose and / or 2-glucopyranose monogroups, which represent 0.00-4.34 mol % of the monomer groups in the plurality of oligosaccharides;

[0242] (12) 3-galactopyranose monogroup, which represents 3.52-4.87 mol % of the monomeric groups in the various oligosaccharides;

[0243] (13) 4-mannopyranose and / or 5-mannofuranose monogroups, which represent 0.00-7.91 mol % of the monomer groups in the plurality of oligosaccharides;

[0244] (14) 3-galactofuranose monogroup, which represents 0.00-4.44 mol% of the monomer group in the various oligosaccharides;

[0245] (15) 6-pyranose monogroup, which represents 8.30-10.84 mol% of the monomer groups in the various oligosaccharides;

[0246] (16) 2-galactopyranose monogroup, which represents 0.00-8.70 mol % of the monomer group in the plurality of oligosaccharides;

[0247] (17) 6-pyranose monogroup, which represents 0.00-0.32 mol % of the monomer group in the various oligosaccharides;

[0248] (18) 4-galactopyranose and / or 5-galactofuranose monogroups, which represent 2.86-3.57 mol % of the monomeric groups in the various oligosaccharides;

[0249] (19) 2,3-Mannofuranose diyl, which represents 0.00-0.26 mol % of the monomeric group in the various oligosaccharides;

[0250] (20) 4-pyranose and / or 5-furanose mono groups, which represent 0.00-0.37 mol % of the monomer groups in the plurality of oligosaccharides;

[0251] (21) 6-Mannofuranose monogroup, which represents 0.00-1.72 mol% of the monomer group in the various oligosaccharides;

[0252] (22) 2,3-galactofuranosyl, which represents 0.10-0.59 mol % of the monomeric group in the various oligosaccharides;

[0253] (23) 2,3-pyranose diyl, which represents 0.24-0.51 mol % of the monomeric group in the various oligosaccharides;

[0254] (24) 6-galactopyranose monogroup, which represents 9.78-13.08 mol % of the monomeric groups in the various oligosaccharides;

[0255] (25) 3,4-galactopyranose and / or 3,5-galactofuranose and / or 2,3-galactopyranose diyl groups, which represent 0.83-1.32 mol % of the monomer groups in the plurality of oligosaccharides;

[0256] (26) 2,4-mannopyranose and / or 2,5-mannofuranose diyl groups, which represent 0.09-1.15 mol % of the monomer groups in the various oligosaccharides;

[0257] (27) 4,6-mannopyranose and / or 5,6-mannofuranose diyl groups, which represent 1.18-1.82 mol % of the monomer groups in the various oligosaccharides;

[0258] (28) 3,6-mannofuranose diyl, which represents 0.07-0.18 mol % of the monomeric group in the various oligosaccharides;

[0259] (29) 2,3,4-pyranose and / or 2,3,5-furanose triyl groups, which represent 0.07-0.14 mol % of the monomer groups in the various oligosaccharides;

[0260] (30) 3,6-pyranose diyl, which represents 1.76-2.40 mol % of the monomeric group in the various oligosaccharides;

[0261] (31) 2,6-pyranose diyl, which represents 1.42-1.84 mol % of the monomeric group in the various oligosaccharides;

[0262] (32) 3,6-galactofuranosyl, which represents 0.95–1.41 mol % of the monomeric group in the various oligosaccharides;

[0263] (33) 4,6-galactopyranose and / or 5,6-galactofuranosyl groups, which represent 2.38-3.46 mol % of the monomeric groups in the various oligosaccharides;

[0264] (34) 2,6-galactopyranosyldiyl, which represents 1.23–1.84 mol % of the monomeric groups in the various oligosaccharides;

[0265] (35) 3,4,6-mannopyranose and / or 3,5,6-mannofuranose and / or 2,3,6-mannofuranose triyl groups, which represent 0.19-0.48 mol % of the monomer groups in the various oligosaccharides;

[0266] (36) 3,4,6-galactopyranose and / or 3,5,6-galactofuranose and / or 2,3,6-galactofuranose triyl groups, which represent 0.53-1.03 mol % of the monomer groups in the various oligosaccharides;

[0267] (37) 2,3,6-mannopyranose and / or 2,4,6-mannopyranose and / or 2,5,6-mannofuranose triyl groups, which represent 0.43-0.68 mol % of the monomer groups in the various oligosaccharides;

[0268] (38) 2,3,6-galactopyranose and / or 2,4,6-galactopyranose and / or 2,5,6-galactofuranosyl triyl groups, which represent 0.59-0.93 mol % of the monomer groups in the various oligosaccharides;

[0269] (39) 2,3,4,6-mannopyranose and / or 2,3,5,6-mannofuranose tetrayl groups, which represent 0.00-0.68 mol % of the monomer groups in the plurality of oligosaccharides; and

[0270] (40) 2,3,4,6-galactopyranose and / or 2,3,5,6-galactofuranosyl, which represent 0.00-0.31 mol % of the monomeric groups in the various oligosaccharides.

[0271] The presence of t-arabinopyranose, t-glucopyranose, 2-arabinopyranose, 2-furanose, 6-glucopyranose, 4-glucopyranose, 5-furanose, 2,3,4-glucopyranose and / or 2,3,5-furanose monomer groups may be due to epimerization and / or trace contamination.

[0272] In some embodiments, about 20-45% of the total glycosidic bonds in the oligosaccharide composition are 1,2 glycosidic bonds. In some embodiments, about 28% of the total glycosidic bonds in the oligosaccharide composition are 1,2 glycosidic bonds. In some embodiments, 15-50%, 15-40%, 15-30%, 15-20%, 20-40%, 20-30%, 25-50%, 25-30%, or 30-45% of the total glycosidic bonds in the oligosaccharide composition are 1,2 glycosidic bonds.

[0273] In some embodiments, about 15-42% of the total glycosidic bonds in the oligosaccharide composition are 1,3 glycosidic bonds. In some embodiments, about 22% of the total glycosidic bonds in the oligosaccharide composition are 1,3 glycosidic bonds. In some embodiments, 10-50%, 15-40%, 15-30%, 15-25%, 10-40%, 10-30%, 10-25%, 15-30%, or 15-20% of the total glycosidic bonds in the oligosaccharide composition are 1,3 glycosidic bonds.

[0274] In some embodiments, about 10-27% of the total glycosidic bonds in the oligosaccharide composition are 1,4 glycosidic bonds. In some embodiments, about 15% of the total glycosidic bonds in the oligosaccharide composition are 1,4 glycosidic bonds. In some embodiments, 5-35%, 10-30%, 10-25%, 10-20%, 5-20%, 5-15%, or 20-30% of the total glycosidic bonds in the oligosaccharide composition are 1,4 glycosidic bonds.

[0275] In some embodiments, about 31-60% of the total glycosidic bonds in the oligosaccharide composition are 1,6 glycosidic bonds. In some embodiments, about 41% of the total glycosidic bonds in the oligosaccharide composition are 1,6 glycosidic bonds. In some embodiments, 25-60%, 25-50%, 25-40%, 30-60%, 30-50%, 30-40%, or 35-45% of the total glycosidic bonds in the oligosaccharide composition are 1,6 glycosidic bonds.

[0276] In some embodiments, the oligosaccharide composition comprises 16-64% total furanose. In some embodiments, the oligosaccharide composition comprises 30% total furanose. In some embodiments, the oligosaccharide composition comprises 15-65%, 15-50%, 15-40%, 20-40%, 25-35% or 25-40% total furanose.

[0277] In some embodiments, the oligosaccharide composition comprises at least one mannofuranose or mannopyranose group and at least one galactofuranose or galactopyranose group.

[0278] In some embodiments, an oligosaccharide composition is provided that includes a plurality of oligosaccharides comprising the monomer groups (1)-(40) in the mole percentages shown in Table 2.

[0279] Table 2. Full methylation data

[0280]

[0281]

[0282]

[0283] *The presence of t-arabinopyranose, t-glucopyranose, 2-arabinopyranose, 2-furanose, 6-glucopyranose, 4-glucopyranose, 5-furanose, 2,3,4-glucopyranose and / or 2,3,5-furanose monomer groups may be due to epimerization and / or trace contamination.

[0284] In certain embodiments, the oligosaccharide composition does not contain monomers. In other embodiments, the oligosaccharide composition comprises monomers.

[0285] In some embodiments, an oligosaccharide composition is provided, comprising a plurality of oligosaccharides, the oligosaccharides comprising monomeric groups (1)-(40) or consisting essentially of monomeric groups (1)-(40), as described herein. In some embodiments, an oligosaccharide composition is provided, comprising a plurality of oligosaccharides, the oligosaccharides comprising at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) monomeric groups selected from groups (1)-(40) and at least one (e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) corresponding molar percentages as shown in Table 2. In some embodiments, an oligosaccharide composition is provided, comprising a plurality of oligosaccharides, the oligosaccharides comprising monomeric groups (1)-(40) or consisting essentially of monomeric groups (1)-(40), the molar percentages of which are as shown in Table 2.

[0286] The oligosaccharide compositions described herein and prepared according to the methods described herein can be characterized using two-dimensional heteronuclear NMR and distinguished from prior art compositions. Thus, in another aspect, an oligosaccharide composition comprising a plurality of oligosaccharides that are minimally digestible in humans is provided, the composition being characterized by multiple edited gradient enhanced 1 H- 13 The C heteronuclear single quantum coherence (HSQC) NMR spectrum comprises one or more of signals 1-12 of the following table, wherein the spectrum was generated using a sample of an oligosaccharide composition having less than 2% monomer:

[0287]

[0288]

[0289] In some embodiments, multiple editing gradients are used to enhance 1 H- 13 The composition characterized by a C heteronuclear single quantum coherence (HSQC) NMR spectrum comprising one or more of signals 1-12 has 1 H integration area and 13 The C integration area is defined as follows:

[0290]

[0291]

[0292] As used herein, "heteronuclear single quantum coherence (HSQC)" is used interchangeably with "heteronuclear single quantum correlation (HSQC)".

[0293] In some embodiments, the NMR spectrum is enhanced by performing multiple edited gradients on the oligosaccharide composition sample. 1 H- 13 C heteronuclear single quantum coherence (HSQC) experiments using an echo-anti-echo scheme with the following pulse sequence diagram, acquisition parameters and processing parameters for coherence selection:

[0294] Pulse sequence diagram

[0295]

[0296] Acquisition parameters

[0297] 1 H carrier frequency = 4ppm

[0298] 13 C carrier frequency = 65ppm

[0299] Number of points in the collection dimension = 596

[0300] Spectral range in acquisition dimension = 6.23ppm to 1.83ppm

[0301] Number of points in indirect dimension = 300 complex points

[0302] Spectral range in indirect dimension = 120ppm to 10ppm

[0303] Loop delay = 1 second

[0304] single bond 1 H- 13 C coupling constant = J CH =146Hz

[0305] Scan times = 8

[0306] Temperature = 298-299K

[0307] Solvent = D2O

[0308] Processing parameters

[0309] Direct dimension window function = Gaussian broadening, 7.66Hz

[0310] Indirect dimension window function = Gaussian widening 26.48Hz

[0311] Processing = 512 complex points in direct dimension, 1024 complex points in indirect dimension

[0312] In certain embodiments, the NMR spectra are obtained by subjecting a sample of the composition to HSQC NMR, wherein the sample is a solution in a deuterated solvent. Suitable deuterated solvents include deuterated acetonitrile, deuterated acetone, deuterated methanol, D2O, and mixtures thereof. In particular embodiments, the deuterated solvent is D2O. In addition, in some embodiments, the oligosaccharide composition characterized by HSQC NMR has been subjected to a de-monomerization procedure such that the oligosaccharide composition comprises less than 10% monomers (e.g., less than 8%, 6%, 5%, 4%, 2%, or 1% monomers).

[0313] In certain embodiments, the NMR spectra are obtained using the conditions described in Example 8.

[0314] Exemplary oligosaccharide compositions can be prepared according to the procedures described herein.

[0315] III. How to use

[0316] As described herein, oligosaccharide compositions can be used to regulate lipid levels, such as cholesterol, LDL, non-esterified free fatty acids and triglyceride levels. In some embodiments, oligosaccharide compositions as described herein can be used to reduce lipid levels, such as cholesterol, LDL, non-esterified free fatty acids and triglyceride levels. In some embodiments, oligosaccharide compositions as described herein can be used to reduce glucose levels and / or insulin levels.

[0317] In some embodiments, the oligosaccharide compositions described herein can be used to reduce the lipid, glucose and / or insulin levels of a subject suffering from or suspected of having a disease, disorder or condition associated with elevated lipid and / or glucose levels. In some embodiments, the selected oligosaccharide composition is administered to the subject in an amount that effectively reduces the lipid (e.g., triglyceride, cholesterol and / or adipose phospholipid) (e.g., in the blood or liver) level of a subject in need. In some embodiments, the selected oligosaccharide composition is administered to the subject in an amount that effectively reduces the glucose or insulin level (e.g., in the blood) of a subject in need. In some embodiments, the oligosaccharide composition is administered to the subject in an amount that effectively reduces the glucose or insulin level (e.g., in the blood) of a subject in need. In some embodiments, the oligosaccharide composition is administered to a subject suffering from or suspected of having a disease, disorder or condition associated with elevated lipid and / or glucose levels, and the disease, disorder or condition can be effectively treated.

[0318] On the one hand, the method of administering the oligosaccharide composition described herein can effectively treat hepatic steatosis (e.g., in subjects showing fatty liver disease (FLD, such as NAFLD)). Hepatic steatosis is considered a reversible condition in which large vacuoles of triglyceride fat accumulate in hepatocytes through a steatosis process (e.g., abnormal retention of intracellular lipids). Although there are many causes, fatty liver can be considered a single disease. Fatty liver occurs worldwide, for example, in subjects with excessive alcohol intake and obesity (with or without effects on insulin resistance). This condition is also associated with other diseases that affect fat metabolism. When the fat metabolism process is disrupted, fat accumulates excessively in the liver, resulting in fatty liver. In some embodiments, the accumulation of fat is accompanied by progressive liver inflammation (hepatitis) called steatohepatitis. This more serious condition may be referred to as alcoholic steatohepatitis or non-alcoholic steatohepatitis (NASH).

[0319] In some aspects, the method of administering the oligosaccharide composition described herein can effectively treat non-alcoholic fatty liver disease (NAFLD). NAFLD is a disease in which too much fat is retained in the liver (and the excessive fat is not the result of alcohol abuse). In some embodiments, the method of administering the oligosaccharide composition described herein can effectively treat non-alcoholic steatohepatitis (NASH), a fatty liver disease. In addition to the excessive fat retained in the liver, patients with NASH also have liver inflammation and liver damage. In some embodiments, patients with NASH also have liver fibrosis or scarring. In some embodiments, patients with NASH also suffer from cirrhosis (e.g., permanent liver damage). In some embodiments, patients with NASH are at risk of developing liver cancer. In some embodiments, patients with NAFLD or NASH suffer from one or more coexisting diseases, such as obesity, extreme obesity, or type 2 diabetes.

[0320] The data described herein demonstrate that the selected oligosaccharide composition can be used to treat non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). For example, the data in Example 11 show that the selected oligosaccharide composition causes liver steatosis, inflammation, liver triglyceride concentration, liver weight and inflammation, fibrosis, oxidative stress and apoptosis marker genes to be reduced in the liver; each is an indicator that the selected oligosaccharide composition can be used to treat NAFLD / NASH. In addition, it is believed that insulin resistance plays an important role in the onset of NAFLD and NASH. The selected oligosaccharide composition causes fasting blood insulin to decrease (see Example 11), indicating that the selected oligosaccharide can treat NAFLD / NASH. Finally, the selected oligosaccharide has been shown to regulate markers associated with NAFLD and NASH, including lowering blood and liver cholesterol (especially LDL-C), reducing atherosclerosis formation and reducing weight gain (see Example 11).

[0321] In some embodiments, administering an oligosaccharide composition to a subject can effectively treat lipid and lipoprotein abnormalities. In some embodiments, administering an oligosaccharide composition to a subject can effectively treat dyslipidemia. In some embodiments, administering an oligosaccharide composition to a subject can effectively treat hyperlipidemia. In some embodiments, administering an oligosaccharide composition to a subject can effectively treat hyperlipidemia, such as primary (or familial) hyperlipidemia or secondary (or acquired) hyperlipidemia. In some embodiments, administering an oligosaccharide composition to a subject can effectively prevent or delay the onset of diseases associated with abnormal lipid and / or glucose levels, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease (CVD), type 2 diabetes (T2D) and chronic kidney disease (CKC). In some embodiments, administering an oligosaccharide composition to a subject can effectively reduce the risk of the subject suffering from diseases associated with abnormal lipid and / or glucose levels, such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cardiovascular disease (CVD), type 2 diabetes (T2D) and chronic kidney disease (CKC). In some embodiments, administering an oligosaccharide composition to a subject can effectively treat metabolic syndrome. In some embodiments, administering an oligosaccharide composition to a subject can effectively treat a subject who suffers from or is suspected of having metabolic syndrome or is at risk of developing metabolic syndrome, or a subject who exhibits one, two, three or more symptoms associated with metabolic syndrome. In some embodiments, administering an oligosaccharide composition to a subject can effectively treat a subject who exhibits one or more genetic defects associated with abnormal (e.g., elevated) lipid levels, such as primary (or familial) hyperlipidemia, familial hypercholesterolemia, familial hyperlipoproteinemia, familial hyperchylomicronemia. In some embodiments, administering an oligosaccharide composition to a subject can effectively treat a subject who exhibits secondary (or acquired) hyperlipidemia. In some embodiments, administering an oligosaccharide composition to a subject is effective in treating a subject with a cardiometabolic disorder or liver disease. In some embodiments, administering an oligosaccharide composition to a subject is effective in treating a subject who exhibits resistance to standard of care medications, such as abnormal levels of lipids, such as statins (e.g., statin-refractory subjects or statin non-responders). In some embodiments, administering an oligosaccharide composition to a subject is effective in treating a subject who exhibits intolerance to standard of care medications, such as abnormal levels of lipids, such as statins, e.g., subjects exhibiting statin-associated muscle symptoms (SAMS).

[0322] In some embodiments, there is provided a method for regulating microbial community composition and / or microbial community metabolic output in a subject, e.g., regulating the environment, e.g., regulating (e.g., reducing) lipid levels or glucose or insulin levels. In some embodiments, the oligosaccharide composition is administered in an effective amount to regulate the microbial community and change the gastrointestinal environment (e.g., change pH, change lactic acid, change microbial density, etc.).

[0323] In some embodiments, administration of the oligosaccharide composition to a subject results in a decrease in lipid levels in the subject (e.g., in the subject's blood, plasma, or liver) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of the oligosaccharide composition to a subject results in a decrease in lipid levels in the subject (e.g., in the subject's blood, plasma, or liver) by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0324] In some embodiments, administration of an oligosaccharide composition to a subject results in a reduction in cholesterol (e.g., low-density lipoprotein (LDL)) levels in the subject (e.g., in the subject's blood, plasma, or liver) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of an oligosaccharide composition to a subject results in a reduction in cholesterol levels in the subject (e.g., in the subject's blood, plasma, or liver) by about 25% relative to a reference measurement. In some embodiments, administration of the oligosaccharide composition to a subject results in a decrease in cholesterol levels in the subject (e.g., in the subject's blood, plasma, or liver) by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0325] In some embodiments, administering an oligosaccharide composition to a subject results in a reduction in the ratio of low-density lipoprotein (LDL) relative to high-density lipoprotein (HDL). In some embodiments, administering an oligosaccharide composition to a subject results in a reduction in the ratio of LDL to HDL by 2, 3, 4, 5, 6, 7, 8, 9, 10 or more factors. In some embodiments, administering an oligosaccharide composition to a subject results in a reduction in the ratio of LDL to HDL by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175% or 150-200%.

[0326] In some embodiments, administration of an oligosaccharide composition to a subject results in a decrease in phospholipid (e.g., lysophosphatidic acid (LPA)) levels in the subject (e.g., in the subject's blood, plasma, or liver) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of an oligosaccharide composition to a subject results in a decrease in phospholipid levels in the subject (e.g., in the subject's blood, plasma, or liver) by about 25% relative to a reference measurement. In some embodiments, administration of the oligosaccharide composition to a subject results in a decrease in phospholipid levels in the subject (e.g., in the subject's blood, plasma, or liver) by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0327] In some embodiments, administration of the oligosaccharide composition to a subject results in a decrease in the level of non-esterified free fatty acids in the subject (e.g., in the subject's blood, plasma, or liver) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of the oligosaccharide composition to a subject results in a decrease in the level of non-esterified free fatty acids in the subject (e.g., in the subject's blood, plasma, or liver) by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0328] In some embodiments, administration of the oligosaccharide composition to a subject results in a reduction in triglyceride levels in the subject (e.g., in the liver or blood of the subject) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of the oligosaccharide composition to a subject results in a reduction in triglyceride levels in the subject (e.g., in the liver or blood of the subject) by about 15% relative to a reference measurement. In some embodiments, administration of the oligosaccharide composition to a subject results in a decrease in triglyceride levels in the subject (e.g., in the liver or blood of the subject) by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0329] In some embodiments, administration of an oligosaccharide composition to a subject results in a reduction in glucose levels in the subject (e.g., in the blood or plasma of the subject) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of an oligosaccharide composition to a subject results in a reduction in glucose levels in the subject (e.g., in the blood or plasma of the subject) by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0330] In some embodiments, administration of an oligosaccharide composition to a subject results in a reduction in insulin levels in the subject (e.g., in the blood or plasma of the subject) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of an oligosaccharide composition to a subject results in a reduction in insulin levels in the subject (e.g., in the blood or plasma of the subject) by at least 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0331] In some embodiments, administering an oligosaccharide composition to a subject results in an increase in short-chain fatty acids (e.g., propionate, acetate, butyrate). In some embodiments, administering an oligosaccharide composition to a subject results in an increase in propionic acid. In some embodiments, administering an oligosaccharide composition to a subject results in an increase in succinic acid. In some embodiments, administering an oligosaccharide composition to a subject results in an increase in the level or concentration of propionic acid in the subject (e.g., in the subject's blood, plasma, or liver) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200%, relative to a reference measurement (e.g., a measurement obtained before administering the composition). In some embodiments, administration of the oligosaccharide composition to a subject results in an increase in the level or concentration of propionic acid in the subject (e.g., in the subject's blood, plasma, or liver) by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0332] In some embodiments, the concentration of short chain fatty acids is performed using gas chromatography with flame ionization detection (GC-FID).

[0333] In some embodiments, administration of the oligosaccharide composition to a subject enriches the subject's gastrointestinal microbiome for species belonging to the genera Allistipes, Blautia, Parabacteroides, Akkermansia, Ruthenibacterium, Micrococcus uncommonus, Bacteroides, and / or Butyrivibrio. In some embodiments, administration of the oligosaccharide composition to a subject depletes the subject's gastrointestinal microbiome for species belonging to the genera Anaerotruncus, Dorea, Staphylococcus, Streptococcus, and / or Enterococcus.

[0334] In some embodiments, the oligosaccharide compositions described herein are effective for treating metabolic syndrome. As described herein, metabolic syndrome (MetS) describes a group of metabolic abnormalities associated with obesity or severe overweight status (e.g., obesity) in a subject. Metabolic abnormalities associated with MetS include insulin resistance, hypertension, dyslipidemia (e.g., low high-density lipoprotein cholesterol, hypertriglyceridemia), and central obesity (abdominal fat accumulation). Subjects with metabolic syndrome have an increased risk of diabetes (e.g., type 2 diabetes), cardiovascular disease (CVD), hyperlipidemia, and liver disease (e.g., cirrhosis, fatty liver disease (e.g., NAFLD or NASH)). Subjects with metabolic syndrome or related diseases typically have elevated levels of inflammation. In some embodiments, the oligosaccharide composition can reduce the severity of at least one metabolic abnormality associated with MetS or eliminate at least one metabolic abnormality associated with MetS.

[0335] In some embodiments, a subject with metabolic syndrome has three or more of the following characteristics: (1) a large waist (35 inches (89 cm) or more for women and 40 inches (102 cm) or more for men); (2) high triglyceride levels (150 milligrams per deciliter (mg / dL), or 1.7 millimoles per liter (mmol / L), or more of blood triglycerides); (3) reduced high-density lipoprotein (HDL) cholesterol levels (less than 40 mg / dL (1.04 mmol / L) for men or less than 50 mg / dL (1.3 mmol / L) for women); (4) elevated blood pressure (130 / 85 millimeters of mercury (mm Hg) or more; and / or (5) elevated fasting blood glucose (100 mg / dL (5.6 mmol / L) or more). Waist circumference, blood cholesterol levels, blood pressure, and blood glucose levels can be measured using any method known in the art.

[0336] In some embodiments, a subject suitable for treatment with an oligosaccharide composition described herein is diagnosed as having metabolic syndrome when three or more of the following are observed in the subject (e.g., by a health or medical provider): (1) if the subject is a female, the subject has a waist circumference of 35 inches or greater (e.g., 35 inches, 38 inches, 40 inches, 42 inches, 45 inches, 48 ​​inches, 50 inches, 52 inches, 58 inches, or 60 inches or greater), or if the subject is a male, a waist circumference of 40 inches or greater (e.g., 40 inches, 42 inches, 45 inches, 48 ​​inches, 50 inches, 52 inches, 58 inches, or 60 inches or greater). inch, 65 inches, or 70 inches or more); (2) the subject has a blood triglyceride level of 150 mg / dL or more (e.g., 150 mg / dL, 155 mg / dL, 160 mg / dL, 165 mg / dL, 170 mg / dL, 175 mg / dL, 180 mg / dL, 185 mg / dL, 190 mg / dL, 200 mg / dL, 225 mg / dL, 250 mg / dL, 275 mg / dL, 300 mg / dL, 350 mg / dL, 400 mg / dL, 450 mg / dL, or 500 mg / dL or more) or 1.7 mmol / dL. L, 2.0mmol / L, 2.5mmol / L, 3.0mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L or 13mmol / L or more); (3) if the subject is a female, the subject's blood HDL cholesterol level is 50mg / dL or less (e.g., 50mg / dL, 45mg / dL, 40mg / dL, 35mg / dL, 30mg / dL, 25mg / dL, 20mg / dL, 15mg / dL dL, 10 mg / dL, or 5 mg / dL or less) or 1.3 mmol / L or less (e.g., 1.3 mmol / L, 1.16 mmol / L, 1.04 mmol / L, 0.9 mmol / L, 0.8 mmol / L, 0.65 mmol / L, 0.5 mmol / L, 0.4 mmol / L, 0.25 mmol / L, or 0.15 mmol / L or less), or if the subject is male, the blood HDL cholesterol level is 40 mg / dL or less (e.g., 40 mg / dL, 35 mg / dL, 30 mg / dL, 25 mg / dL, 20 mg / dL).(4) the subject's blood pressure is 130 / 85 mm Hg or higher (e.g., systolic blood pressure is 130 mm Hg, 135 mm Hg, 140 mm Hg, 145 mm Hg, 150 mm Hg, 160 mm Hg, 170 mm Hg, 180 mm Hg, 190 mm Hg, or 200 mm Hg or higher; diastolic blood pressure is 85 mm Hg, 90 mm Hg, 95 mm Hg, 100 mm Hg, 110 mm Hg, 120 mm Hg, or 130 mm Hg). Hg or higher); and / or (5) the subject's fasting blood glucose is 100 mg / dL or higher (e.g., 100 mg / dL, 110 mg / dL, 120 mg / dL, 126 mg / dL, 130 mg / dL, 140 mg / dL, 150 mg / dL, 160 mg / dL, 165 mg / dL, 170 mg / dL, 180 mg / dL, or 190 mg / dL or higher) or 5.6 mmol / L or higher (e.g., 5.6 mmol / L, 6.1 mmol / L, 6.7 mmol / L, 7.2 mmol / L, 7.8 mmol / L, 8.3 mmol / L, 8.9 mmol / L, 9.2 mmol / L, 9.4 mmol / L, 10 mmol / L, or 10.6 mmol / L or higher).

[0337] In some embodiments, the oligosaccharide composition as described herein can be administered to treat lipid or lipoprotein abnormalities in a subject, or to alleviate symptoms associated with lipid or lipoprotein abnormalities in a subject. In one embodiment, a method for treating lipid or lipoprotein abnormalities in a subject in need thereof is provided, comprising administering to the subject an oligosaccharide composition as described herein in an amount effective to treat lipid or lipoprotein abnormalities.

[0338] In some embodiments, the oligosaccharide composition as described herein can be administered to treat dyslipidemia in a subject, or to alleviate symptoms associated with dyslipidemia in a subject. In one embodiment, a method of treating dyslipidemia in a subject in need thereof is provided, comprising administering to the subject an oligosaccharide composition as described herein in an amount effective to treat dyslipidemia.

[0339] In some embodiments, the oligosaccharide composition as described herein can be administered to treat hyperlipidemia in a subject, or to alleviate symptoms associated with hyperlipidemia in a subject. In one embodiment, a method of treating hyperlipidemia in a subject in need thereof is provided, comprising administering to the subject an oligosaccharide composition as described herein in an amount effective to treat hyperlipidemia.

[0340] In some embodiments, oligosaccharide compositions as described herein can be used to treat the metabolic syndrome of a subject, or to alleviate the symptoms associated with metabolic syndrome of a subject. In one embodiment, there is provided a method for treating metabolic syndrome in a subject in need, including applying oligosaccharide compositions as described herein to the subject in an amount that effectively treats metabolic syndrome. In some embodiments, the method further includes co-administering standard of care treatment (e.g., delivering statins and / or changing lifestyles). In some embodiments, the method includes administering oligosaccharide compositions while carrying out standard of care treatment (e.g., delivering statins and / or changing lifestyles).

[0341] In some embodiments, an oligosaccharide composition as described herein can be administered to treat a subject who exhibits statin intolerance or is refractory to statins. In one embodiment, a method of treating a subject who exhibits statin intolerance or is refractory to statins is provided, comprising administering to the subject an oligosaccharide composition as described herein in an amount effective to treat the subject who exhibits statin intolerance or is refractory to statins.

[0342] In some embodiments, relative to a reference measurement (e.g., a measurement obtained before applying the composition), administering an oligosaccharide composition to a subject (e.g., a subject with metabolic syndrome) causes the subject's waist circumference to be reduced by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150% or 200%. In some embodiments, relative to a reference measurement, administering an oligosaccharide composition to a subject (e.g., a subject with metabolic syndrome) causes the subject's waist circumference to be reduced by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175% or 150-200%.

[0343] In some embodiments, administration of the oligosaccharide composition to a subject (e.g., a subject with metabolic syndrome) results in a decrease in the subject's blood and / or liver triglyceride levels by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of an oligosaccharide composition to a subject (e.g., a subject with metabolic syndrome) results in a decrease in the subject's blood and / or liver triglyceride levels by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0344] In some embodiments, administration of an oligosaccharide composition to a subject (e.g., a subject with metabolic syndrome) results in an increase in the subject's blood and / or liver high-density lipoprotein (HDL) levels by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, or 200% relative to a reference measurement (e.g., a measurement obtained prior to administration of the composition). In some embodiments, administration of an oligosaccharide composition to a subject (e.g., a subject with metabolic syndrome) results in an increase in the subject's blood and / or liver HDL levels by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% relative to a reference measurement.

[0345] In some embodiments, the compositions and methods described herein can be used to treat metabolic syndrome symptoms in subjects. In some embodiments, the compositions and methods described herein can treat at least one feature (e.g., one, two, three, four, or five or more features) of a subject with metabolic syndrome associated with metabolic syndrome (e.g., large waist, high triglyceride levels, reduced HDL cholesterol levels, elevated blood pressure and / or elevated fasting blood glucose levels). In some embodiments, if the symptom is adjusted so that it is more similar to the measured value or level of the symptom in a healthy subject without metabolic syndrome, the symptom is treated or alleviated.

[0346] For example, in some embodiments, the compositions and methods described herein can be used to treat at least one (e.g., one, two, three or four or more) symptom of metabolic abnormality associated with metabolic syndrome, for example, obesity, cardiovascular disease (CVD), diabetes (e.g., type 2 diabetes), hyperlipidemia and / or fatty liver disease (e.g., non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH)). In some embodiments, the compositions and methods can regulate (e.g., reduce) obesity or obesity symptoms of a subject. In some embodiments, the compositions and methods can regulate (e.g., reduce) cardiovascular disease or symptoms of cardiovascular disease of a subject. In some embodiments, the compositions and methods can regulate (e.g., reduce) diabetes (e.g., type 2 diabetes) or symptoms of diabetes of a subject. In some embodiments, the compositions and methods can regulate (e.g., reduce) fatty liver disease (e.g., NAFLD or NASH) or symptoms of fatty liver disease of a subject.

[0347] In one aspect, the methods of administering the oligosaccharide compositions described herein are effective for treating cardiovascular disease. Cardiovascular disease (CVD) is a class of diseases involving the heart or blood vessels, and can include coronary artery disease (CAD), such as angina and myocardial infarction (e.g., heart attack), stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmias, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolic disease, venous thrombosis.

[0348] In some embodiments, subjects suitable for treatment with the oligosaccharide compositions described herein have or are suspected of having cardiovascular disease, which may be manifested as increased blood pressure, increased lipid levels, increased cholesterol, chest pain, arrhythmias, and heart failure. Conditions associated with cardiovascular disease include chronic kidney disease; hypercholesterolemia; hypertension (e.g., increased blood pressure); diabetes (e.g., increased blood sugar); hyperlipidemia (e.g., increased blood cholesterol); (undiagnosed) celiac disease; atherosclerosis; coronary heart disease; and obesity. Coronary artery disease, stroke, and peripheral arterial disease involve atherosclerosis, which may be caused by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive drinking. In some embodiments, administration of the oligosaccharide compositions described herein can effectively treat the risk of at least one condition associated with cardiovascular disease (e.g., hypercholesterolemia, atherosclerosis, familial hypercholesterolemia, and coronary heart disease).

[0349] In some embodiments, oligosaccharide compositions can be applied to improve cardiac function. In one embodiment, a method for treating cardiovascular disease in a subject in need is provided, including applying oligosaccharide compositions as described herein to a subject in an amount that effectively improves cardiac function. In one embodiment, a method for treating cardiovascular disease in a subject in need is provided, including applying oligosaccharide compositions as described herein to a subject in an amount that effectively reduces or stabilizes plaque formation. In some embodiments, treatment includes combined administration of oligosaccharide compositions and standard care treatment (e.g., delivery of statins, lipid-lowering drugs, and / or lifestyle changes). In some embodiments, the method includes administering oligosaccharide compositions while standard care treatment (e.g., delivery of statins, lipid-lowering drugs, and / or lifestyle changes). In some embodiments, the statins co-administered with the oligosaccharide compositions are atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, or simvastatin. In some embodiments, lipid-lowering drugs are fibrates, PCSK9 inhibitors, or ω-3 fatty acids. In some embodiments, the lifestyle modification is a change in overall health level, nutritional diet, alcohol use, or tobacco use.

[0350] In some embodiments, administration of the oligosaccharide composition results in a reduction in the prevalence, risk and / or incidence of abnormal heart problems (e.g., myocardial infarction, hypertensive heart disease, arrhythmias), e.g., 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200%, relative to a reference subject (e.g., a subject not receiving the oligosaccharide composition). For example, in some embodiments, a subject administered an oligosaccharide composition is 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175%, or 150-200% less likely to experience a myocardial infarction following administration of the composition relative to a reference subject.

[0351] In one aspect, the methods described herein can be used to treat obesity. Obesity is a medical condition in which excess body fat accumulates to a degree that may negatively affect health. In some embodiments, obesity may be caused by or associated with genetic disorders, endocrine disorders, medications, or psychiatric disorders. On average, obesity shortens life expectancy by six to seven years: a body mass index (BMI) of 30-35 kg / m 2It may shorten life expectancy by two to four years, and severe obesity (BMI>40kg / m 2 ) may shorten life expectancy by ten years. In some embodiments, subjects suitable for treatment with the oligosaccharide compositions described herein are obese and have a body mass index of more than 30 kg / m 2 body mass index (BMI).

[0352] In some embodiments, the administration of the oligosaccharide composition treats obesity. In some embodiments, relative to a reference subject (e.g., a subject who has not received the oligosaccharide composition), the administration of the oligosaccharide composition causes the subject's BMI to decrease by 1-10%, 5-20%, 10-25%, 20-40%, 30-50%, 40-60%, 50-70%, 60-80%, 70-90%, 80-100%, 90-110%, 100-125%, 110-150%, 125-175% or 150-200%.

[0353] In some embodiments, treatment includes administering an oligosaccharide composition in combination with a standard of care treatment (e.g., delivering a statin and / or changing a lifestyle). In some embodiments, the method includes administering an oligosaccharide composition simultaneously with a standard of care treatment (e.g., delivering a statin and / or changing a lifestyle).

[0354] In some embodiments, a subject suitable for treatment with an oligosaccharide composition described herein has fatty liver disease and has a baseline level of serum alanine aminotransferase (AST) levels that are higher than aspartate aminotransferase (ALT).

[0355] In some embodiments, the oligosaccharides of the present invention can be administered to improve liver function and / or reduce hepatic steatosis. In one embodiment, a method for treating hepatic steatosis (e.g., fatty liver disease) in a subject in need thereof is provided, comprising administering a composition to the subject in an amount effective to reverse the accumulation of triglyceride vacuoles in the liver. In some embodiments, treating liver disease comprises administering an oligosaccharide composition in combination with standard of care treatment.

[0356] On the one hand, the compositions and methods described herein can be used to treat insulin resistance and diabetes, such as type 2 diabetes. Diabetes is a metabolic disease associated with abnormally elevated blood sugar levels caused by reduced or failed insulin production and / or reduced insulin sensitivity and function. Pancreatic β cells produce insulin hormone, which helps cells absorb glucose to provide energy. When less or no insulin is produced, or when cells become less sensitive to insulin, diabetes occurs. Type 1 diabetes is associated with failure to produce insulin when β cells are destroyed by T cell autoimmunity. Individuals with type 1 diabetes are usually genetically susceptible to the disease, although dietary and lifestyle factors also contribute to the occurrence and severity of the disease (e.g., unhealthy diets rich in carbohydrates and sugars, lack of exercise, sedentary lifestyles, etc.). Type 2 diabetes is caused by insulin resistance and reduced insulin production. Obesity caused by a poor diet and a sedentary lifestyle significantly increases an individual's chance of developing type 2 diabetes. Inflammation that causes β cell destruction is often associated with type 1 and type 2 diabetes. If left untreated, diabetes can lead to disorders such as diabetic nephropathy, retinopathy, neuropathy, cardiovascular disease, and ulcers.

[0357] In some embodiments, the subject suitable for treatment with the oligosaccharide composition described herein has type 2 diabetes and a blood glucose level of 126 mg / dl (7 mmol / 1) or higher. In some embodiments, the subject suitable for treatment with the oligosaccharide composition described herein has type 2 diabetes, diabetic symptoms and two random blood glucose tests exceeding 200 mg / dl (11.1 mmol / 1); or hemoglobin A1c test exceeds 6.5% on two different days.

[0358] In some embodiments, the oligosaccharides of the present invention can be administered to reduce or alleviate insulin resistance and / or treat or prevent diabetes (e.g., type 1 or type 2 diabetes). In one embodiment, a method for treating diabetes (e.g., type 1 or type 2 diabetes) in a subject in need is provided, including administering a composition to a subject with an amount that effectively reduces the subject's blood sugar level, reduces glucose absorption, increases the amount of insulin (e.g., by increasing the insulin produced by the pancreas), increases the subject's sensitivity to insulin, and / or reduces resistance to insulin. In some embodiments, treating insulin resistance and / or diabetes includes administering an oligosaccharide composition in combination with standard care treatment (e.g., delivering insulin and / or changing lifestyle).

[0359] In some embodiments, the subject in need of the oligosaccharide composition as described herein is a statin-refractory patient (also referred to as a statin-resistant patient). In some embodiments, a statin-refractory patient is a patient who takes a statin but fails to reach a target LDL-C level. Statin-refractory patients may have an increased risk of cardiovascular disease. In some embodiments, statin-refractory patients are receiving or have received a higher dose of statins than control patients, thereby increasing the risk of side effects.

[0360] In some embodiments, the subject of the oligosaccharide composition as described herein is a patient who is intolerant to statins. Patients who are intolerant to statins may not be able to tolerate the sufficiently high concentration of statins required to reach the target LDL-C level. In some embodiments, patients who are intolerant to statins have one or more muscle symptoms associated with statins, including myopathy (myalgia, myositis, rhabdomyolysis), elevated liver function enzymes, or new-onset diabetes. In some embodiments, patients who are intolerant to statins have low body mass index, women, advanced age, Asians, comorbidities (e.g., hypothyroidism, liver and kidney disease, and rheumatism), vitamin D deficiency, alcoholism, consumption of grapefruit juice (1qt / day or 0.95L / day), major surgery or perioperative period, excessive physical activity, receiving another lipid-lowering treatment with a history of myopathy, a history of CK elevation, a family history of myopathy, and a family history of myopathy when receiving another lipid-lowering drug. In some embodiments, the statin-intolerant patient is taking concomitant medications such as fibrates, cyclosporine, antifungals, macrolide antibiotics, amiodarone, verapamil, and anti-HIV drugs - protease inhibitors.

[0361] In one aspect, the compositions and methods described herein can be used to treat familial hypercholesterolemia, hyperlipidemia, heterozygous familial hypercholesterolemia (HeFH), and homozygous familial hypercholesterolemia (HoFH). In some embodiments, the oligosaccharides of the present invention can be administered to reduce or alleviate the symptoms of familial hypercholesterolemia, hyperlipidemia, heterozygous familial hypercholesterolemia (HeFH), and / or homozygous familial hypercholesterolemia (HoFH) in a subject.

[0362] Familial hypercholesterolemia is a hereditary disorder characterized by very high cholesterol levels in the blood. Patients with familial hypercholesterolemia are at high risk of coronary artery disease at a young age. In some embodiments, familial hypercholesterolemia can also cause health and aesthetic problems related to the accumulation of excess cholesterol in tissues other than the heart and blood vessels, such as tendon xanthomas, xanthelasmata, and arcus cornealis. In most countries, it is estimated that one in 200 to one in 250 people suffer from familial hypercholesterolemia, and it is considered the most common hereditary disorder affecting the heart and blood vessels. Mutations in the APOB, LDLR, LDLRAP1, or PCSK9 genes can cause familial hypercholesterolemia. Changes in the LDLR gene (encoding low-density lipoprotein receptors) are the most common cause of this disorder. Low-density lipoprotein receptors bind to low-density lipoproteins (LDL) and act as the main carrier of cholesterol in the blood. These receptors play a key role in regulating cholesterol levels by removing LDL from the bloodstream. Some LDLR gene mutations reduce the number of low-density lipoprotein receptors produced in cells. Other mutations destroy the ability of the receptor to remove low-density lipoprotein from the bloodstream. Therefore, people with LDLR gene mutations have very high blood cholesterol levels. In some embodiments, familial hypercholesterolemia is caused by mutations in APOB, LDLRAP1 or PCSK9 genes. Therefore, in some embodiments, the oligosaccharide compositions described herein can be used to treat patients with mutations in LDLR, APOB, LDLRAP1 and / or PCSK9 genes. However, in some embodiments, patients with familial hypercholesterolemia do not have mutations in one of these genes.

[0363] On the one hand, compositions and methods described herein can be used for preventing or treating hyperlipoproteinemia. In some embodiments, hyperlipoproteinemia is classified phenotypically according to Fredrickson classification (Fredrickson classification). In some embodiments, hyperlipoproteinemia is classified as type 1, type 2, type 3, type 4 or type 5 hyperlipoproteinemia. In some embodiments, hyperlipoproteinemia is classified by origin basis (e.g., primary / genetic basis; or secondary / acquired). In some embodiments, type I hyperlipoproteinemia is lipoprotein lipase deficiency (type Ia), due to lipoprotein lipase (LPL) lacking or changing apolipoprotein C2. In some embodiments, type I hyperlipoproteinemia is familial apolipoprotein CII deficiency (type Ib), a disease caused by lacking lipoprotein lipase activators. In some embodiments, type I hyperlipoproteinemia is chylomicronemia caused by circulating lipoprotein lipase inhibitors (type Ic). In some embodiments, patients with type I hyperlipoproteinemia have complications including retinal vein occlusion, acute pancreatitis, fatty degeneration and organomegaly, and retinal lipidemia. In some embodiments, type II hyperlipoproteinemia is type IIa and may be sporadic (due to dietary factors), polygenic, or familial (due to mutations in the LDL receptor gene (0.2% of the population) or the ApoB gene (0.2%) on chromosome 19). The familial form is characterized by tendon xanthomas, xanthelasmas, and premature cardiovascular disease. Type II hyperlipoproteinemia patients may have elevated LDL cholesterol levels in the blood (e.g., due to lack of uptake of LDL particles). Individuals may present with a unique set of physical features, such as xanthelasmas (yellow fat deposits under the skin, usually appearing in the nasal part of the eye), tendon and tubercular xanthelasmas, arcus juvenilis (graying of the eyes is usually characteristic of the elderly), arterial murmurs, claudication, and atherosclerosis. In some embodiments, the total serum cholesterol level of patients with type II hyperlipoproteinemia is two to three times the normal level, as well as increased LDL cholesterol. Patients with type IIb hyperlipoproteinemia (e.g., including familial combined hyperlipoproteinemia (FCH), lysosomal acid lipase deficiency (cholesteryl ester storage disease) or secondary combined hyperlipoproteinemia) may have high VLDL levels, for example, due to substrates, including overproduction of triglycerides, acetyl-CoA, and increased synthesis of B-100. Type III hyperlipoproteinemia is caused by high chylomicrons and IDL (intermediate density lipoprotein), and patients with type III may have elevated cholesterol-rich VLDL (β-VLDL), hypercholesterolemia (usually 8-12mmol / L) and / or hypertriglyceridemia (usually 5-20mmol / L).Type IV hyperlipoproteinemia (or familial hypertriglyceridemia) is an autosomal dominant condition. People with type IV may have high triglyceride levels. Type V hyperlipoproteinemia, also called mixed familial hyperlipoproteinemia or mixed hyperlipidemia, is similar to type I but is associated with high VLDL in addition to chylomicrons and may also be associated with glucose intolerance and hyperuricemia.

[0364] Some patients with hyperlipoproteinemia also have hyperlipidemia, a common form of hypercholesterolemia (elevated cholesterol levels) characterized by elevated LDL and triglyceride concentrations, often with decreased HDL. It is the most common inherited lipid disorder, occurring in approximately 1 in 200 people. Almost one in five people who develop coronary heart disease before the age of 60 have this disorder. Elevated triglyceride levels (>5mmol / l) are usually due to an increase in very low-density lipoproteins (VLDL), a class of lipoproteins that are prone to atherosclerosis. Familial combined hyperlipidemia (FCH) is a familial occurrence of this disorder, probably caused by a reduction in LDL receptors and an increase in ApoB. FCH is extremely common in people with other conditions such as metabolic syndrome ("Syndrome X", which includes type II diabetes, hypertension, central obesity, and CH). Excessive production of free fatty acids by various tissues leads to increased synthesis of VLDL in the liver. Initially, most of the VLDL is converted to LDL until this mechanism is saturated, after which VLDL levels rise.

[0365] In one aspect, the compositions and methods described herein can be used to prevent or treat familial chylomicronemia syndrome, a disease characterized by elevated triglyceride levels. In some embodiments, the oligosaccharides of the present invention can be administered to reduce triglyceride levels in subjects with familial chylomicronemia syndrome. Familial chylomicronemia syndrome, also known as familial lipoprotein lipase deficiency, is a genetic disorder that disrupts the normal breakdown of body fat. Subjects with familial lipoprotein lipase deficiency usually develop signs and symptoms before the age of 10, with 25% of subjects developing symptoms by the age of 1 year. The first symptom of this condition is usually abdominal pain, which can range from mild to severe. Abdominal pain is usually caused by inflammation of the pancreas (pancreatitis), which can become chronic and even life-threatening. Patients may also have an enlarged liver and spleen. About half of individuals with familial lipoprotein lipase deficiency develop small yellow fat deposits under the skin, called eruptive xanthomas. Mutations in the LPL gene (encoding lipoprotein lipase) cause familial lipoprotein lipase deficiency. The mutations that cause familial lipoprotein lipase deficiency result in reduced or eliminated lipoprotein lipase activity, which prevents the enzyme from effectively breaking down triglycerides. As a result, triglycerides attached to lipoproteins accumulate in the blood and tissues, causing the signs and symptoms of familial lipoprotein lipase deficiency. Treatment is required to maintain plasma triglyceride concentrations below 1000 mg / dL. Maintaining triglyceride levels below 2000 mg / dL prevents recurrences of abdominal pain.

[0366] The compounds and compositions provided herein can be used to regulate the method of bacterial groups (e.g., 1, 2, 3, 4, 5 or more groups) present in the subject's microbiome. In some embodiments, the regulation includes changes in the structure of the microbiome, such as changes in the relative composition of the taxonomic group or changes in the relative abundance of the taxonomic group, for example, relative to another taxonomic group or relative to what will be observed without regulation. In other embodiments, the regulation includes changes in the function of the microbiome, such as changes in gene expression, changes in gene copy number, the overall abundance of DNA, the level of gene products (e.g., RNA or protein), or the metabolic output of the microbiome, or changes in host functional pathways (e.g., changes in gene expression, gene product levels, or metabolic output of host cells or host processes). The method for regulating the microbial taxa disclosed in WO2016 / 122889 and WO 2016 / 172657 is applicable to the methods described herein, which are incorporated herein by reference.

[0367] The methods described herein include administering to a subject a composition described herein, for example, including an oligosaccharide composition described herein, in an amount effective to regulate a taxonomic group. In some embodiments, when the composition is administered, the abundance of the bacterial taxonomic group may increase relative to other taxonomic groups (or relative to from one time point to another time point), and the increase may be at least 5%, 10%, 25%, 50%, 75%, 100%, 250%, 500%, 750% increase or at least 1000% increase. When the composition is administered, the abundance of the bacterial taxonomic group may also decrease relative to other taxonomic groups (or relative to from one time point to another time point), and the reduction may be at least 5%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99% reduction, or at least 99.9% reduction. The administration of the composition can regulate the abundance of desired and / or undesired bacterial taxonomic groups in the subject's gastrointestinal microbiome.

[0368] In some embodiments, a composition described herein (e.g., including an oligosaccharide composition described herein) modulates (e.g., significantly increases or significantly decreases) the growth (and total number) (or significantly increases or significantly decreases the relative representation / abundance) of the total (gastrointestinal) community of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) bacterial taxa.

[0369] In some embodiments, a composition described herein (e.g., including an oligosaccharide composition described herein) significantly increases the growth, e.g., the total number or relative representation / abundance of the total (gastrointestinal) population of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) commensal bacterial taxa.

[0370] In some embodiments, a composition described herein (e.g., comprising an oligosaccharide composition described herein) significantly reduces the growth, e.g., the total number or relative representation / abundance of a total (gastrointestinal) population of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) bacterial taxa.

[0371] In some embodiments, the oligosaccharide compositions described herein modulate the growth, e.g., the total number or relative representation / abundance of a total (gastrointestinal) community of cholesterol-consuming bacterial taxa and species, e.g., at a higher ratio than a reference bacterial taxa or species.

[0372] In some embodiments, the oligosaccharide composition is formulated into a powder, such as for reconstitution (e.g., in water) for oral administration. In some embodiments, the oligosaccharide composition is formulated into a solid form (e.g., chewable tablets or soft candies) for oral administration. In some embodiments, the oligosaccharide composition is formulated into a pharmaceutical composition for delivery by a feeding tube. In some embodiments, the oligosaccharide composition is formulated into a pharmaceutical composition for delivery by total parenteral nutrition (TPN).

[0373] Oligosaccharide compositions can be administered to subjects every day, every week, every two weeks or every month. In some embodiments, the compositions are administered to subjects more than once a day (e.g., 2, 3 or 4 times a day). In some embodiments, the compositions are administered to subjects more than once a week (e.g., 2, 3 or 4 times a week). In some embodiments, the compositions are administered to subjects once or twice a day for one, two, three or four consecutive weeks. In some embodiments, the compositions are administered to subjects for a long time. In some embodiments, the compositions are administered to subjects for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months in a row.

[0374] In some embodiments, the composition is administered to the subject according to the following schedule: 10-40 grams total on any or all days of days 1-14 of the treatment regimen; 20-100 grams total on any or all days of days 15-30 of the treatment regimen.

[0375] In some embodiments, the effective amount of oligosaccharide is a total of 5-200 grams, 5-150 grams, 5-100 grams, 5-75 grams, 5-50 grams, 5-25 grams, 10-50 grams, 25-50 grams, 30-60 grams, 50-75 grams, 50-100 grams, 18-72 grams, or 36-72 grams administered per day.

[0376] The oligosaccharide compositions of the present disclosure are well tolerated by the subject (e.g., the oligosaccharide compositions do not cause or cause minimal discomfort in the subject, such as the production of gas or gastrointestinal discomfort). In some embodiments, a total daily dose of 5-200 grams, 5-150 grams, 5-100 grams, 5-75 grams, 5-50 grams, 5-25 grams, 10-50 grams, 25-50 grams, 30-60 grams, 50-75 grams, 50-100 grams, 18-72 grams, or 36-72 grams is well tolerated by the subject. Any dose of the oligosaccharide composition as described herein administered to the subject once or in a single dose can be well tolerated by the subject.

[0377] In some embodiments, the amount of the oligosaccharide composition administered to a subject in a single or single dose is better tolerated by the subject than a similar amount of commercial low digestibility sugars such as oligofructose (FOS). It is known in the art that commercially available low digestibility sugars are poorly tolerated in subjects (see, e.g., Grabitske, HA, Critical Reviews in Food Science and Nutrition, 49: 327-360 (2009)), for example, at high doses. For example, a tolerability study of FOS showed that 20 grams of FOS per day can cause mild gastrointestinal symptoms, and 30 grams of FOS per day can cause severe discomfort and gastrointestinal symptoms.

[0378] In some embodiments, the oligosaccharide compositions described herein are co-administered with symbiotic or probiotic bacterial groups and generally recognized as safe bacteria (GRAS) or known symbiotic or probiotic microorganisms. In some embodiments, the symbiotic or probiotic bacterial groups (or their preparations) can be administered to the subject before or after the oligosaccharide composition is administered to the subject. In some embodiments, the probiotic or symbiotic bacterial taxa (or their preparations) can be administered to the subject simultaneously with the administration of the oligosaccharide composition to the subject.

[0379] In some embodiments, the oligosaccharide compositions described herein are administered with a fecal microbiota transplant (FMT).

[0380] Symbiotic or beneficial bacteria are also referred to as probiotics. Probiotics can include metabolites produced by probiotic bacteria during fermentation. These metabolites can be released into a fermentation medium, such as into a host organism (e.g., a subject), or they can be stored in bacteria. "Probiotic bacteria" include bacteria, bacterial homogenates, bacterial proteins, bacterial extracts, bacterial fermentation supernatants, and combinations thereof, which, for example, when administered in therapeutic doses, play a beneficial role in a host animal.

[0381] Useful probiotics include at least one bacteria that produces lactic acid and / or acetic acid and / or propionic acid, for example, by decomposing carbohydrates such as glucose and lactose to produce lactic acid and / or acetic acid and / or propionic acid microorganisms. Preferably, probiotic bacteria are lactic acid bacteria. In embodiments, lactic acid bacteria include Lactobacillus, Leuconostoc, Pediococcus, Streptococcus and Bifidobacterium. Suitable probiotic bacteria may also include other bacteria that beneficially affect the host by improving the balance of host intestinal bacteria, such as but not limited to yeasts such as Saccharomyces, Debaromyces, Candida, Pichia and Torulopsis, molds such as Aspergillus, Rhizopus, Mucor and Penicillium. ium, and Torulopsis, and other bacteria such as, but not limited to, Bacteroides, Clostridium, Fusobacterium, Melissococcus, Propionibacterium, Enterococcus, Lactococcus, Staphylococcus, Peptostreptococcus, Bacillus, Pediococcus, Micrococcus, Leuconostoc, Weissella, Aerococcus, and Oenococcus, and combinations thereof.

[0382] Non-limiting examples of lactic acid bacteria that can be used in the present disclosure include Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetyllacti, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bifidum, Lactobacillus casei, Lactobacillus, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus thermophilus, Lactobacillus fermentii, Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus breve, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium lactis, Bifidobacterium breve, Bifidobacterium adolescentis and Pediococcus cerevisiae and combinations thereof, in particular Lactobacillus, Bifidobacterium and combinations thereof.

[0383] Commensal and probiotic bacteria that are particularly useful in the present disclosure include those that are derived from humans (or from the mammal to which the probiotic bacteria are being administered) (for human administration), are non-pathogenic to the host, are resistant to technological processes (i.e., can remain viable and active during processing and in the delivery vehicle), are resistant to gastric acid and bile toxicity, adhere to intestinal epithelial tissue, have the ability to colonize the gastrointestinal tract, produce antimicrobial substances, modulate the host's immune response, and affect metabolic activities (e.g., cholesterol assimilation, lactase activity, vitamin production).

[0384] The commensal and probiotic bacteria can be used as a single strain or a combination of strains, where the total number of bacteria in a dose of probiotic bacteria ranges from about 1 x 10 3 About 1x 10 14 , or from about 1 x 10 to about 1 x 10 12 , or from about 1x10 7 About 1x 10 11 CFU.

[0385] Symbiotic or probiotic bacteria may be formulated with the oligosaccharide composition, while the probiotic bacteria are alive but in a "dormant" or lethargic state. Once freeze-dried, the live culture or cultures of the probiotic bacteria are treated to minimize exposure to moisture (which will rejuvenate the culture), since once rejuvenated, the culture may experience high morbidity unless quickly cultured in a high moisture environment or medium. Additionally, the culture is treated to reduce potential exposure to high temperatures (especially in the presence of moisture) to reduce morbidity.

[0386] The probiotic bacteria may be used in a powdered dry form. The probiotic bacteria may also be administered in the oligosaccharide composition or in a separate oligosaccharide composition, simultaneously with the oligosaccharide composition or at a different time.

[0387] Other suitable probiotics include Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium infantis.

[0388] In embodiments, commensal bacterial taxa that can be used and / or combined with the oligosaccharide compositions described herein include Akkermansia, Anaerococcus, Bacteroides, Bifidobacterium (including Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium breve, and Bifidobacterium infantis), Blautia, Clostridium, Corynebacterium, Dialister, Eubacterium, Faecalibacterium, Finegoldi a), Fusobacterium, Lactobacillus (including Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus bifidum, Lactobacillus lactis, Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus thermophilus, Lactobacillus crispatus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus sporogenes and Lactobacillus bulgaricus), Peptococcus, Peptostreptococcus, Peptoniphilus, Prevotella, Roseburia, Ruminococcus, Staphylococcus, and / or Streptococcus (including Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetyllactosus, Streptococcus thermophilus).

[0389] In embodiments, commensal bacterial taxa, such as GRAS strains, that can be used in and / or in combination with the oligosaccharide compositions described herein include Bacillus coagulans GBI-30, 6086; Bifidobacterium animalis lactic acid subsp. BB-12; Bifidobacterium breve Yakult; Bifidobacterium infantis 35624; Bifidobacterium animalis lactic acid subsp. UNO 19 (DR10); Bifidobacterium longum BB536; Escherichia coli M-17; Escherichia coli Nissle 1917; Lactobacillus acidophilus DDS-1; Lactobacillus acidophilus LA-5; Lactobacillus acidophilus NCFM; Lactobacillus casei DN114-001 (immune / defense Lactobacillus casei); Lactobacillus casei CRL431; Lactobacillus casei F19; Lactobacillus paracasei Stl l (or NCC2461); Lactobacillus johnsonii (Lactobacillus johnsonii johnsonii)Lai (Lactobacillus LCI, Lactobacillus johnsonii NCC533); Lactococcus lactis L1A; Lactobacillus plantarum 299V; Lactobacillus reuteri ATTC 55730 (Lactobacillus reuteri SD2112); Lactobacillus rhamnosus ATCC 53013; Lactobacillus rhamnosus LB21; Saccharomyces cerevisiae (boulardii) lyo; a mixture of Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14; a mixture of Lactobacillus acidophilus NCFM and Bifidobacterium lactis BB-12 or BL-04; a mixture of Lactobacillus acidophilus CL1285 and Lactobacillus casei; and a mixture of Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011 and / or Lactobacillus rhamnosus GG (LGG).

[0390] IV. Kit

[0391] Kits are also contemplated. For example, a kit may include a unit dosage form of an oligosaccharide composition, and a package insert containing instructions for using the composition to treat, for example, a disease associated with hyperammonemia, for example, a urea cycle disorder or hepatic encephalopathy. In some embodiments, the composition is provided in the form of a dry powder. In some embodiments, the composition is provided in the form of a solution, powder, or tablet. The kit includes an oligosaccharide composition in a suitable package for use by a subject in need. Any composition described herein may be packaged in the form of a kit. The kit may include an oligosaccharide composition in an amount sufficient for the entire treatment process or a portion of the treatment process. The dose of the oligosaccharide composition may be packaged separately, or the oligosaccharide composition may be provided in bulk, or a combination thereof. Therefore, in one embodiment, the kit provides a single dose of an oligosaccharide composition corresponding to a dosing point in a treatment regimen in a suitable package, wherein the dose is packaged in one or more packets.

[0392] The kit may also include written materials, such as instructions, expected results, testimonials, explanations, warnings, clinical data, information for use by health care professionals, etc. In one embodiment, the kit contains a label or other information indicating that the kit is to be used only under the guidance of a health care professional. The container may also include a spoon, syringe, bottle, cup, applicator or other measuring or serving device. Example

[0393] Example 1. Production of short-chain fatty acids (SCFA) in healthy and patient stool samples in the presence of oligosaccharide compositions

[0394] Hundreds of different oligosaccharide compositions were tested for their ability to modulate (e.g., reduce) metabolite levels in healthy and patient stool samples. A library of synthetic oligosaccharide compositions was screened in an ex vivo assay to test their effects on metabolite production by gut microbiota from (1) healthy subjects, (2) overweight subjects, (3) obese subjects, and (4) subjects with type 2 diabetes. Stool samples were collected from donors, frozen, and stored at -80°C until use. The frozen stool samples were transferred to an anaerobic chamber, allowed to thaw, and homogenized in phosphate-buffered saline containing 15% glycerol to a final concentration of 20% solids. The 20% stool slurry was then filtered to remove large debris, aliquoted, removed from the anaerobic chamber and immediately frozen on dry ice and then stored at -80°C. On the day of the experiment, an aliquot of each 20% stool slurry was thawed and diluted into bacterial growth medium in an anaerobic chamber to a final 1%. The 1% slurry solution was then dispensed into the wells of a 96-deep well plate containing sterile water (negative control) or oligosaccharide preparations (final concentration 0.5%). The plates were incubated anaerobically at 37°C for 45 hours. After the 45-hour incubation period, the culture supernatant was collected and SCFAs were quantified by gas chromatography with a flame ionization detector (GC-FID).

[0395] One of hundreds of oligosaccharide compositions tested, such as the selected oligosaccharide composition described throughout this document (ie, produced by the methods in Examples 2-4), was determined to significantly reduce lipid levels in obese, overweight, and type 2 diabetic subjects.

[0396] Example 2. Production of selected oligosaccharide compositions from galactose and mannose using soluble acid catalysts at 10 kg scale

[0397] A procedure for synthesizing the selected oligosaccharide composition as described in Example 1 was developed on a 10 kg scale. 5.46 kg of anhydrous galactose, 3.64 kg of anhydrous mannose, 0.50 kg of citric acid monohydrate acid catalyst and 0.71 kg of water were added to a reaction vessel (22L Littleford-Day horizontal plow mixer). A distillation condenser unit was connected to the reactor. The contents were stirred at about 30 RPM, and the vessel temperature was gradually raised to about 135°C at atmospheric pressure over 2.5-3 hours. The mixture was kept at this temperature for 80-90 minutes, after which heating was stopped and preheated water was gradually added to the reaction mixture at a rate of 60 mL / min until the temperature of the reactor contents dropped to 120°C, then at 150 mL / min until the temperature of the reactor contents dropped to 110°C, then at 480 mL / min until a total of 7.8-8.0 kg of water was added and the temperature of the reactor contents dropped to below 100°C. An additional 0.4-0.58 kg of water was added to the reactor for further dilution. The reaction mixture was discharged from the vessel to obtain 16 kg of crude oligosaccharides (about 52-53 wt%) as an aqueous solution.

[0398] The oligosaccharide composition was prepared by passing it through a cation exchange resin ( Monosphere 88H) column, two decolorizing polymer resins ( OptiPore SD-2) columns and anion exchange resins ( The purified material having a concentration of about 40 wt % was then concentrated by vacuum rotary evaporation to a final concentration of about 75 wt % solids to obtain a purified oligosaccharide composition.

[0399] Example 3. Production of 100 g scale oligosaccharide composition from galactose and mannose using soluble acid catalyst

[0400] A procedure for synthesizing the selected oligosaccharide composition as described in Example 1 was developed on a 100 gram scale. 60 g of galactose, 40 g of mannose and an amount of water sufficient to achieve a starting concentration of 91% dissolved solids were added to a reaction vessel (1 L three-necked round bottom flask). The reaction vessel was equipped with a heating jacket equipped with an overhead stirrer. The probe thermocouple was arranged in the vessel through a diaphragm so that the probe tip was located above the stirring blade and did not contact the reaction vessel wall. Prior to the addition of the catalyst, the reaction vessel was equipped with a condenser in the reflux position.

[0401] The procedure used citric acid (1.5-3% w / w) as a catalyst and deionized water for quenching. After the catalyst was added, the reaction vessel was equipped with a distillation station to remove excess water throughout the course of the reaction.

[0402] The temperature controller was set to the target temperature (130 to 140°C) and as the syrup temperature reached the target temperature at ambient (atmospheric) pressure, stirring of the vessel contents began to promote uniform heat transfer and melting of the sugar solids.

[0403] After the catalyst was added, the reaction was held at the target temperature with continuous mixing for approximately 4 hours, as determined by following the reaction by HPLC. Next, the heat was turned off while maintaining constant stirring.

[0404] The reaction was then quenched by slowly adding about 60 mL of deionized (DI) water (room temperature) to dilute and cool the product mixture to achieve a target final concentration of 60-70 wt% dissolved solids. Typically, as the oligosaccharide composition is cooled and diluted, the rate of water addition is implemented to control the mixture viscosity.

[0405] Example 4. Production of 100 g scale oligosaccharide composition from galactose and mannose using a solid polymer catalyst

[0406] A procedure for synthesizing the selected oligosaccharide composition as described in Example 1 was developed on a 100 gram scale. 60 g of galactose, 40 g of mannose and water in an amount sufficient to reach a starting concentration of 91% dissolved solids were added to a reaction vessel (1 L three-necked round bottom flask). The reaction vessel was equipped with a heating jacket equipped with an overhead stirrer. The probe thermocouple was set in the vessel by a diaphragm so that the probe tip was located above the stirring blade and did not contact the wall of the reaction vessel. Prior to the addition of the catalyst, the reaction vessel was equipped with a condenser in the reflux position.

[0407] The procedure also uses an acidic oligomerization catalyst (Dowex Marathon C) (3-5% w / w) and deionized water for quenching. In some cases, the catalyst is processed in wet form, for example, at a nominal moisture content of 45–50 wt% H2O. The exact catalyst moisture content is typically determined on a per-experimental basis, for example, using a moisture analysis balance (e.g., Mettler-Toledo MJ-33). After adding the catalyst, the reaction vessel is set in a distillation position to remove excess water throughout the reaction.

[0408] The temperature controller was set to the target temperature (130 to 140°C) and stirring of the contents of the vessel was begun to promote uniform heat transfer and melting of the sugar solids while bringing the temperature of the syrup to the target temperature at ambient (atmospheric) pressure.

[0409] After the catalyst was added, the reaction was maintained at the target temperature with continuous mixing for about 4 hours, as determined by following the reaction by HPLC. Next, the heat was turned off while maintaining constant stirring.

[0410] The reaction was then quenched by slowly adding about 60 mL of deionized (DI) water (room temperature) to dilute and cool the product mixture to a target final concentration of 60-70 wt% dissolved solids. Typically, the rate of water addition is controlled to control the mixture viscosity as the oligosaccharide composition cools and dilutes.

[0411] After dilution, the oligosaccharide composition is cooled to about 60° C. The catalyst is then removed by vacuum filtration through a 100 micron mesh or sintered glass filter to obtain a final oligosaccharide composition of about 40° Bx.

[0412] Example 5. De-monomerization procedure

[0413] After treatment with an ion exchange resin (e.g., as described herein), each batch of oligosaccharide composition produced in Examples 2-4 was concentrated on a rotary evaporator to about 50 Brix (measured by a Brix refractometer). The resulting syrup (200 mg) was loaded onto a Teledyne ISCO RediSep Rf Gold Amine column (11 grams of stationary phase) using a thick syringe. Other similar chromatographic columns, such as Biotage SNAP KP-NH Cartridges, can also be used. The sample was purified on a Biotage Isolera equipped with an ELSD detector (using a 20 / 80 to 50 / 50 (v / v) deionized water / ACN mobile phase gradient elution over 55 column volumes). Other flash chromatography systems, such as Teledyne ISCO Rf, can also be used. The flow rate is set according to the manufacturer's specifications for the chromatographic column and system. After the ~20 column volumes of the monomer fraction are completely eluted, the mobile phase is set to 100% water until the rest of the oligosaccharide composition is eluted and collected. The monomer-free fractions were concentrated by rotary evaporation to give the demonomerized product.

[0414] Example 6. Size Exclusion Chromatography

[0415] The weight average molecular weight (MWw), number average molecular weight (MWn) and polydispersity index (PDI) of batches of selected oligosaccharide compositions as described in Example 1 and produced according to the methods described in Examples 2-4 were determined by SEC HPLC.

[0416] method

[0417] These methods involve the use of an Agilent 1100 with a refractive index (RI) detector equipped with the following two columns in series: Shodex OHpak SB-802HQ, 8.0 x 300 mm, 8 μm, P / N F6429100 and Shodex OHpakSB-803HQ, 8.0 x 300 mm, 6 μm, P / N F6429102. Equivalent columns known in the art may also be used.

[0418] The mobile phase (0.1 M NaNO3) was prepared by weighing 34 g of NaNO3 (ACS grade reagent) and dissolving in 2000 mL of deionized (DI) water (from MiliQ water filter). The solution was filtered through a 0.2 μm filter.

[0419] Polymer standard solutions (10.0 mg / mL) were prepared by weighing 20 mg of standard into individual 20 mL scintillation vials and then adding 2.0 mL of DI water to each vial.

[0420] Sample A was prepared in duplicate. About 300 mg of the oligosaccharide composition sample was weighed into a 20 mL scintillation vial and 10 mL of DI water was added. The solutions were mixed and filtered through an Acrodisc 25 mm syringe filter with a 0.2 μm polyethersulfone membrane. Sample B was prepared in duplicate. About 210 mg of the oligosaccharide sample was weighed into a 20 mL scintillation vial and 10 mL of DI water was added. The solutions were mixed and filtered through an Acrodisc 25 mm syringe filter with a 0.2 μm polyethersulfone membrane.

[0421] At least 2 hours before running the samples, the flow rate was set to 0.9 mL / min, the column temperature and RI detector were both set to 40 °C, and the RI detector purge was turned on.

[0422] Prior to running the samples where the injection volume for all samples was 10 μL and the run time was 28 minutes, the detector purge was turned off and the pump was run at 0.9 mL / min until an acceptable baseline was obtained.

[0423] Run a blank sample consisting of DI water. Run a sample of each standard. Run Sample A. Run Sample B.

[0424] The peak between 15 and 22 minutes was integrated. The integration of the monomer and broad peak (product) is shown in the sample chromatogram. The calibration curve fit type in Empower 3 software was set to 3rd order. The molecular weight distribution and polydispersity of the broad peak were calculated using Empower 3 software. The Mw, Mn, and polydispersity of the product peak (DP2+) are reported.

[0425] result

[0426] Thirty-five batches of the selected oligosaccharide composition produced at a 10 kg scale using the method described in Example 2 were analyzed using the above SEC method. Small batches of the selected oligosaccharide composition produced by the method described in Examples 3 and 4 were demonomerized.

[0427] The assayed batch of the oligosaccharide composition produced according to Example 2 contained oligosaccharides having an average MWw of 2329 g / mol (range 1807-2985 g / mol), an average MWn of 1767 g / mol (range 1322-2073 g / mol), and an average PDI of 2.1 (range 2.0-2.3). The assayed batch also contained an average DP2+ of 90.1% (range 89.2-92.7) and an average degree of polymerization (DP) of 14.3 (range 11.1-18.4).

[0428] Example 7. SEC HPLC method for determination of impurities

[0429] Batches and samples of selected oligosaccharide compositions as produced by the method in Example 2 were determined by SEC HPLC for the presence of residual organic acid impurities and related substances.

[0430] method

[0431] These methods involve the use of an Agilent 1100 with a refractive index (RI) detector equipped with a guard column (Bio-Rad MicroGuard Cation H+ Cartridge, PIN 125-0129 or equivalent) and a Bio-Rad Aminex HPX-87H, 300 x 7.8 mm, 9 μm, PIN 125-0140 column, or equivalent.

[0432] The mobile phase (25 mM H2SO4 in water) was prepared by filling a bottle with 2000 mL of deionized water and slowly adding 2.7 mL of H2SO4. The solution was filtered through a 0.2 μm filter.

[0433] Prepare the standard solution by measuring 50 ± 2 mg of the reference standard in a 100-mL volumetric flask, adding the mobile phase to the 100-mL mark and mixing thoroughly.

[0434] Samples of selected oligosaccharide compositions (Sample A) were prepared in duplicate. Approximately 1000 mg of oligosaccharide sample was weighed into a 10 mL volumetric flask and the mobile phase was added to the mark. The solution was mixed and filtered through a PES syringe filter with a 0.2 μm polyethersulfone membrane.

[0435] Samples of selected oligosaccharide compositions (Sample B) were prepared in duplicate. Approximately 700 mg of oligosaccharide sample was weighed into a 10 mL volumetric flask and the mobile phase was added to the mark. The solution was mixed and filtered through a PES syringe filter with a 0.2 μm polyethersulfone membrane.

[0436] The flow rate was set to 0.65 mL / min at least 2 hours before running samples (column temperature set to 50 °C and RI detector temperature set to 50 °C, with RI detector purge turned on).

[0437] Prior to running samples, where all samples had an injection volume of 50 μL and a run time of 40 minutes, the detector purge was turned off and the pump was run at 0.65 mL / min until an acceptable baseline was obtained.

[0438] Run a blank consisting of deionized water. Run the standards, sample A, and sample B independently.

[0439] Peaks at 7.5 min (glucuronic acid), 9.4 min (maleic acid), 11.3 min (levoglucosan), 11.9 min (lactic acid), 13.1 min (formic acid), 14.2 min (acetic acid), 15.5 min (levulinic acid), 31.8 min (hydroxymethylfurfural, HMF), and 8.3 min (glucose) were integrated. The calibration curve fit type in Empower 3 software was set to third order.

[0440] result

[0441] Eighteen batches of selected oligosaccharides produced by the method of Example 2 were tested using the method described above. A sample of the selected oligosaccharide composition contained 2.52% w / w citric acid (range 2.18-2.72% w / w) and undetectable levels of lactic acid, formic acid, levulinic acid and HMF.

[0442] Example 8. HSQC NMR analysis procedure using a Bruker NMR machine

[0443] HSQC NMR spectra of selected samples of the oligosaccharide compositions described in Example 1 and produced as described in Example 2 were measured using a Bruker NMR machine according to the following protocol.

[0444] method

[0445] Sample preparation:

[0446] 25 mg of previously lyophilized solid sample of oligosaccharide composition was dissolved in 300 μL D2O (containing 0.1% acetone as internal standard). The solution was then placed in a 3 mm NMR tube.

[0447] NMR experiments:

[0448] Each sample was analyzed at 499.83MHz (125.69MHz 13C), equipped with an XDB broadband probe, a Z-axis gradient, tuned to 13C, and run at 25°C in a Bruker NMR. Using an echo-reverse echo scheme for coherent selection, multiple edited gradient-enhanced 1H-13C heteronuclear single quantum coherence (HSQC) experiments were performed on each sample. The following pulse sequence diagrams and acquisition and processing parameters were used to obtain the NMR spectrum of each sample:

[0449] Pulse sequence diagram

[0450]

[0451] Acquisition parameters

[0452] 1H carrier frequency = 4ppm

[0453] 13C carrier frequency = 65ppm

[0454] Number of points in the collection dimension = 596

[0455] Spectral range in acquisition dimension = 6.23ppm to 1.83ppm

[0456] Number of points in indirect dimension = 300 complex points

[0457] Spectral range in indirect dimension = 120ppm to 10ppm

[0458] Loop delay = 1 second

[0459] single bond 1 H- 13 C coupling constant = JCH = 146 Hz

[0460] Scan times = 8

[0461] Temperature = 298-299K

[0462] Solvent = D2O

[0463] Processing parameters

[0464] Direct dimension window function = Gaussian broadening, 7.66Hz

[0465] Indirect dimension window function = Gaussian widening 26.48Hz

[0466] Processing = 512 complex points in direct dimension, 1024 complex points in indirect dimension

[0467] Spectral analysis

[0468] The resulting spectra were analyzed using the MNova software package from Mestrelab Research (Santiago de Compostela, Spain). The spectra were referenced to the internal acetone signal (1H–2.22 ppm; 13C–30.8 ppm) and phased using the Regions2D method in the F2 and F1 dimensions. Apodization using 90-degree offset sinusoids was applied in both the F2 and F1 dimensions. For each spectrum, individual signals (CH correlations) were quantified by integrating the respective peaks using a “predefined integration region” with an elliptical integration shape. The resulting table of integration regions and values ​​from the spectra was normalized to a sum of 100 so that the value represents a percentage of the total. The peak integration regions were chosen to avoid monomer-related peaks.

[0469] result

[0470] Six batches of selected oligosaccharide compositions (having an average DP of 14.0 (± 1.7)) produced according to the method described in Example 2 were analyzed using the above-described NMR method. In general, these batches contained the following NMR peak signals (Table 3).

[0471] Table 3. HSQC NMR peaks of selected oligosaccharide compositions

[0472]

[0473] The relative size (AUC) of each peak collected from the NMR spectrum of the selected oligosaccharide composition produced according to the method described in Example 2 was further determined as follows:

[0474]

[0475]

[0476] Representative HSQC NMR spectra of selected oligosaccharide compositions are provided in Fig. 20A In the figure, the anomeric and nonanomeric regions are indicated.

[0477] Dimers from commercial sources of Carbosynth Inc were used as reference materials to further characterize and assign selected HSQC NMR peaks in the anomeric region belonging to the selected oligosaccharide composition. The assignment was based on the strategy described in "1H NMR structural-reporter-group concept" (Leeuwen et al., Carbohydrate Research 343 (2008), 1114-1119). The dimers used in the analysis included: Galp-b (1-6) -Galp, Galp-a (1-6) -Galp, Galp-b (1-4) -Galp, Galp-a (1-4) -Galp, Galp-b (1-3) -Galp, Galp-a (1-3) -Galp, Galp-b (1-2) -Galp and Galp-a (1-2) -Galp. The junction abundance was compared with the full methylation data and cross-referenced with literature values ​​and internal control oligosaccharide compositions. Fig. 20B Representative HSQC NMR spectra of selected oligosaccharide compositions (anomeric region only) with indicated linkages are provided.

[0478] Example 9. Determination of glycosidic bond distribution using permethylation analysis

[0479] Determination of the glycosidic bond distribution of selected oligosaccharide composition samples as produced by the method in Example 2 was performed using permethylation analysis according to the protocol described below. The samples were demonomerized prior to permethylation analysis.

[0480] The reagents used included methanol, acetic acid, sodium borodeuteride, sodium carbonate, dichloromethane, isopropanol, trifluoroacetic acid (TFA), and acetic anhydride. The apparatus included a heating module, a drying device, a gas chromatograph equipped with a capillary column and equipped with a RID / MSD detector and a 30-meter (RESTEK). All derivations were performed in a fume hood.

[0481] Production of alditol acetate

[0482] A. Standard Preparation

[0483] Prepare 1 mg / mL solutions of the following standard analytes: arabinose, rhamnose, fucose, xylose, mannose, galactose, glucose, and inositol. Prepare the standards by mixing 50 μL of each of arabinose, xylose, fucose, glucose, mannose, and galactose with 20 μL of inositol in a vial. The standards are then lyophilized.

[0484] B. Sample Preparation

[0485] Each sample was prepared by mixing 100-500 μg of the selected oligosaccharide composition (weighed on an analytical balance) with 20 μg (20 μL) of inositol in a vial.

[0486] C. Hydrolysis

[0487] 200 μL of 2M trifluoroacetic acid (TFA) was added to the sample. The vial containing the sample was capped and incubated on a heating block at 121°C for 2 hours. After 2 hours, the sample was removed from the heating block and cooled to room temperature. The sample was then dried with N2 / air. 200 μL of IPA (isopropanol) was added and dried again with N2 / air. This hydrolysis step (adding TFA at 121°C for 2 hours; washing with isopropanol) was repeated twice.

[0488] Standards were hydrolyzed using TFA similarly as described for the samples.

[0489] D. Reduction and acetylation

[0490] Prepare a 10 mg / mL sodium borodeuteride solution in 1M ammonium hydroxide. Add 200 μL of this solution to the sample. Then incubate the sample at room temperature for at least one hour or overnight. After incubation with the sodium borodeuteride solution, add 5 drops of glacial acetic acid to the sample, followed by 5 drops of methanol. The sample is then dried. Add 500 μL of 9:1 MeOH:HOAc to the sample, then dry (repeat twice). Then add 500 μL of MeOH to the sample, then dry (repeat once). This creates a crusty white residue on the side of the sample vial.

[0491] Then add 250 μL of acetic anhydride to the sample vial and vortex the sample until dissolved. Add 230 μL of concentrated TFA to the sample and incubate the sample at 50°C for 20 minutes. Remove the sample from the heat source and cool to room temperature. Add about 1 mL of isopropanol and dry the sample. Then, add about 200 μL of isopropanol and dry the sample again. Then add about 1 mL of 0.2 M sodium carbonate to the sample and mix gently. Finally, add about 2 mL of dichloromethane to the sample, which is then vortexed and briefly centrifuged. Discard the aqueous top layer. Add 1 mL of water and vortex the sample and briefly centrifuge. Repeat this step, then remove the organic layer (bottom) and transfer to another vial. Use N2 / air to concentrate the sample to a final volume of about 100 μL. Then inject 1 μL of the final sample into the GC-MS.

[0492] GC temperature program SP2330 was used for GC-MS analysis. The initial temperature was 80°C and the initial time was 2.0 minutes. The first heating rate was 30°C / min, the final temperature was 170°C and the final time was 0.0 minutes. The second heating rate was 4°C / min, the final temperature was 240°C and the final time was 20.0 minutes.

[0493] Hakomori Methylation Analysis of Glycosyl Linkages of Polysaccharides and Oligosaccharides

[0494] A. Preparation of NaOH Base

[0495] In a glass screw top tube, combine 100 μL of the 50 / 50 NaOH solution and 200 μL of anhydrous MeOH. Use a plastic pipette for the NaOH and a glass pipette for the MeOH. Vortex the solution briefly, add about 4 mL of anhydrous DMSO, and then vortex the solution again. Centrifuge the tube to concentrate the solution, then aspirate the DMSO and salts from the precipitate. Repeat the first two steps about four times to remove all water from the precipitate. Remove any white residue from the sides of the tube. Once all residue has been removed and the precipitate is clear, add about 1 mL of anhydrous DMSO and vortex the solution. The base is then ready to use. Prepare the base fresh each time it is needed.

[0496] B. Full methylation

[0497] Each sample was prepared by mixing 600-1000 μg of the selected oligosaccharide composition (weighed on an analytical balance) with 200 μL DMSO. The samples were stirred overnight until the oligosaccharide composition dissolved.

[0498] An equal amount of NaOH base (400 μL) was added to the sample, and the sample was then returned to the blender and mixed thoroughly for 10 minutes. 100 μL of iodomethane (CH3I) was added to the sample. The sample was mixed on the blender for 20 minutes, and the previous steps (addition of NaOH base and iodomethane) were then repeated.

[0499] About 2mL ultrapure water is added to the sample and the sample is fully mixed to make it turbid. The tip of the pipette is placed in the sample solution at the bottom of the test tube and bubble CH3I with a very small amount of air. As the CH3I bubbled, the sample became clear. The pipette was moved around the solution to ensure that all CH3I had disappeared. Then about 2mL of dichloromethane was added, and the solution was fully mixed by vortexing for 30 seconds. The sample was then centrifuged and the water layer on the top was removed. About 2mL of water was added and the sample was mixed, then centrifuged briefly, and then the water layer on the top was removed. Repeated addition of dichloromethane and water. Remove the organic bottom layer and transfer to another tube and dry with N2. Use Alditol Acetate to continue analysis.

[0500] C. Hydrolysis

[0501] 200 μL of 2M trifluoroacetic acid (TFA) was added to the sample. The vial containing the sample was capped and incubated on a heating block at 121°C for 2 hours. After 2 hours, the sample was removed from the heating block and cooled to room temperature. The sample was then dried with N2 / air. 200 μL of IPA (isopropanol) was added and dried again with N2 / air. This hydrolysis step (adding TFA at 121°C for 2 hours; washing with isopropanol) was repeated twice.

[0502] D. Reduction and acetylation

[0503] Prepare a 10 mg / mL sodium borodeuteride solution in 1M ammonium hydroxide. Add 200 μL of this solution to the sample. Then incubate the sample at room temperature for at least one hour or overnight. After incubation with the sodium borodeuteride solution, add 5 drops of glacial acetic acid to the sample, followed by 5 drops of methanol. The sample is then dried. Add 500 μL of 9:1 MeOH:HOAc to the sample, then dry (repeat twice). Then add 500 μL of MeOH to the sample, then dry (repeat once). This creates a crusty white residue on the side of the sample vial.

[0504] Then 250 μL of acetic anhydride is added to the sample vial, and the sample is vortexed until dissolved. 230 μL of concentrated TFA is added to the sample, and the sample is incubated at 50°C for 20 minutes. The sample is removed from the heat source and cooled to room temperature. About 1 mL of isopropanol is added, and the sample is dried. Then, about 200 μL of isopropanol is added, and the sample is dried again. Then about 1 mL of 0.2 M sodium carbonate is added to the sample and mixed gently. Finally, about 2 mL of dichloromethane is added to the sample, which is then vortexed and briefly centrifuged. The aqueous top layer is discarded. 1 mL of water is added and the sample is vortexed and briefly centrifuged. Repeat this step, then remove the organic layer (bottom) and transfer to another vial. The sample is concentrated to a final volume of about 100 μL using N2 / air. Then 1 μL of the final sample is injected into the GC-MS.

[0505] GC temperature program SP2330 was used for GC-MS analysis. The initial temperature was 80°C and the initial time was 2.0 minutes. The first heating rate was 30°C / min, the final temperature was 170°C and the final time was 0.0 minutes. The second heating rate was 4°C / min, the final temperature was 240°C and the final time was 20.0 minutes.

[0506] result

[0507] The above method was used to collect permethylation data for six batches of de-monomerized oligosaccharide compositions produced by the method described in Example 2. Each batch was analyzed in duplicate. The data related to the groups present in these six batches of de-monomerized oligosaccharide compositions are provided below:

[0508]

[0509]

[0510]

[0511] Example 10. Therapeutic and microbiome effects of selected oligosaccharide compositions in a high fat diet (HFD)-induced obese mouse model

[0512] The therapeutic and microbiome effects of the selected oligosaccharide compositions were tested in a validated high-fat diet (HFD)-induced obese mouse model (Winzell MS and Ahrén B, Diabetes, 2004), which is thought to recapitulate many of the metabolic pathologies found in humans, including obesity, hyperglycemia, glucose intolerance, hyperinsulinemia, insulin resistance, hypercholesterolemia, hepatic steatosis, and mild hepatic inflammation. Liraglutide, a GLP1 analog used in patients with type 2 diabetes to improve glycemic control and reduce the risk of major adverse cardiovascular events, was used as a positive control.

[0513] Six-week-old male C57Bl / 6N mice were fed a high-fat diet (HFD) (60 kcal% fat, ResearchDiets D12492) for 8 weeks. After 8 weeks, the mice were assigned to one of four treatment groups (12 animals per group, 4 animals per cage)—(1) no treatment (NT), (2) selected oligosaccharides (generated as described in Example 2), (3) glucose / galactose-based oligosaccharide composition, and (4) liraglutide—for the next 5 weeks. Mice in the NT group were fed the same HFD throughout the 5 weeks. Mice in the oligosaccharide groups (#2 and #3) were fed a HFD supplemented with 7.5% w / w oligosaccharide composition for 5 weeks. The oligosaccharide composition treatment diet was modified to provide the mice with the same caloric value as the HFD mice. Mice in the liraglutide group were fed a HFD and administered liraglutide (0.2 mg / kg SC, QD) throughout the 5 weeks. The body weight of each animal was measured weekly before and throughout the treatment period. The food intake of each cage was measured for 4 days per week during the treatment period and averaged as the food intake per mouse per day. Fresh fecal samples were collected within one week before the start of treatment and at week 5 after the start of treatment. For each fecal collection period, a fresh fecal pellet was collected for each animal on three different days. Fecal samples were sent for shallow shotgun sequencing to characterize the changes in the intestinal microbiota caused by the selected oligosaccharides, glucose / galactose-based oligosaccharides or liraglutide. On the day of the end of treatment, after 5 weeks of treatment and overnight fasting, retro-orbital blood was collected under anesthesia for quantitative analysis of serum total cholesterol (TC), serum non-esterified fatty acids (NEFA), serum insulin and whole blood triglycerides (TG) to characterize the effects of the treatment groups on hyperlipidemia and hyperinsulinemia. The animals were then euthanized and their whole livers were dissected and weighed. For each animal, separate liver sections (~100 mg each) were collected and frozen for quantification of liver TG, liver TC and RNAseq transcriptomic analysis. One liver lobe was also preserved in 10% neutral buffered formalin (NBF) for embedding, sectioning and H&E staining, and then scored for the severity of steatosis (0 to 3), lobular inflammation (0 to 3) and hepatocyte ballooning (0 to 2) using a validated non-alcoholic fatty liver disease histological scoring system (Kleiner et al., Hepatology, 2005).

[0514] like Figure 1 As shown in A, 1B and 1C, the two oligosaccharide compositions in this experiment did not seem to have a significant effect on body weight and food intake. Liraglutide reduced body weight by 15%. These data indicate that the two oligosaccharide compositions were well tolerated by HFD-induced obese mice. Compared with no treatment (NT), the selected oligosaccharide compositions and liraglutide reduced fasting blood glucose ( Figure 2A). Compared with NT, the selected oligosaccharides and the glucose / galactose based oligosaccharide combination reduced serum total cholesterol (TC) by 21% and 29%, and blood triglycerides (TG) by 16% and 12%, respectively ( Figure 3 A, 3B). Liraglutide also reduced serum total cholesterol (TC) (-41%) ( Figure 3 A), but does not affect blood triglycerides (TG) ( Figure 3 B). The selected oligosaccharide combination was the only treatment that showed a decrease in serum non-esterified fatty acids (NEFA) (-28%) compared to NT ( Figure 3 C). In summary, the lipid data showed that the selected oligosaccharide composition reduced all three major lipids (TC, TG and NEFA) compared to NT in HFD-induced obese mice.

[0515] In addition to blood lipids, the selected oligosaccharides also reduced total liver cholesterol (-27%) ( Figure 3 D) and liver triglycerides (-51%) ( Figure 3 E). These effects were not observed for glucose / galactose-based oligosaccharides. Liraglutide lowered hepatic triglycerides but not total cholesterol ( Figure 3 D, 3E). The effects of the selected oligosaccharide composition and liraglutide on liver fat were confirmed by their reduction in liver weight (-31% and -55% absolute liver weight, -27% and -24% relative liver weight, respectively, compared to NT) ( Figure 4 A, 4B) and a decrease in the pathological score of hepatic steatosis compared with NT (-1 and -3 points, respectively) ( Figure 4 C). In addition, the selected oligosaccharide combination and liraglutide significantly reduced liver inflammation pathology scores compared to NT (-1 point for both). No hepatocellular ballooning was observed in this model, with scores of zero for all treatments. Finally, the selected oligosaccharide combination significantly reduced the overall non-alcoholic fatty liver activity score (NAS), calculated as the sum of hepatic steatosis, lobular inflammation, and hepatocellular ballooning (-1.5 points) ( Figure 4 E) Representative images of liver histology in different treatment groups. Figure 7 As shown, a reduction in NAFLD pathology was demonstrated in animals treated with the selected oligosaccharide compositions.

[0516] Consistent with its effect on hepatic fat accumulation, the selected oligosaccharides downregulated the hepatic expression of genes involved in lipogenesis and lipid storage compared to NT (Table 4). For example, the selected oligosaccharides reduced the expression of Scdl, a gene encoding the stearoyl-CoA desaturase-1 protein, a key enzyme in fatty acid metabolism and a limiting step in the formation of monounsaturated fatty acids. The selected oligosaccharides also reduced the hepatic expression of marker genes for inflammation, liver injury and fibrosis (Table 4), such as genes encoding serum amyloid A1 and 2 (SAA1, SAA2), two liver-derived proteins that are elevated in chronic inflammation or liver injury. The expression of Cxcl10, a proinflammatory cytokine associated with the severity of lobular inflammation, which is an independent risk factor for NASH patients and an effective biomarker for the development of heart failure and left ventricular dysfunction, was also reduced by the selected oligosaccharides. In addition, the profibrotic marker Col1a1, encoding the major component of type I collagen, was also downregulated by the selected oligosaccharides.

[0517] Consistent with their hepatic cholesterol-lowering effects, the selected oligosaccharides upregulated genes controlled by sterol regulatory element binding protein 2 (SREBP-2), a transcription factor activated by low levels of cholesterol in the liver, such as cholesterol biosynthesis genes controlled by SREBP-2 (Table 4). The lack of upregulation of genes related to bile acid synthesis, hepatic cholesterol efflux, or bile acid efflux, accompanied by a decrease in hepatic and serum cholesterol, suggests that the selected oligosaccharides may act by limiting cholesterol absorption by the intestinal epithelium. This reduction in cholesterol absorption by the selected oligosaccharides may be mediated by microbiome factors that downregulate the expression, stability, or activity of the cholesterol transporter Npc1l1 in intestinal epithelial cells. The reduction in cholesterol intestinal absorption by the selected oligosaccharides may contribute to the observed decrease in hepatic cholesterol and deplete hepatic oxysterols, the endogenous agonists of the liver X receptor (LXR). LXR is a transcription factor that controls the expression of lipogenic genes, including genes that are downregulated by the selected oligosaccharides and are involved in fatty acid synthesis (e.g., Scd1 and Elovl6). The reduction in fatty acid synthesis mediated by reduced LXR activity could explain the reduction in hepatic triglycerides observed with the selected oligosaccharides. In addition, the reduction in hepatic fatty acids would reduce the number of reactive oxygen species (ROS), a well-known proinflammatory factor and inducer of proinflammatory cytokines (e.g., TNFa), generated by fatty acid β-oxidation and associated oxidative stress. The reduction in fatty acid β-oxidation was confirmed by the downregulation of Cpt1a, Acox1, and Acox3 (Table 4) by the selected oligosaccharides, three genes encoding enzymes involved in fatty acid β-oxidation. The reduction in ROS generated by fatty acid β-oxidation may be the mechanism by which the selected oligosaccharides inhibit inflammation and reduce markers of liver inflammation and fibrosis.

[0518] Table 4. Effects of selected oligosaccharide compositions on gene expression

[0519]

[0520]

[0521] Shallow gun sequencing of stool samples collected before and at the end of the treatment period showed that the selected oligosaccharides had a measurable effect on the gut microbiome. Figure 5 The multidimensional scaling (MDS) ordination plot of the microbiome sequencing data shown shows the correlation between the selected oligosaccharides ( Figure 5 The gut microbiota composition of animals treated with NT (circles in the figure) was different from their baseline state and from the gut microbiota and glucose / galactose-based oligosaccharides ( Figure 5 ) or liraglutide ( Figure 5 triangle in the figure).

[0522] Furthermore, it was shown that the selected oligosaccharides had a greater impact on the composition of the intestinal microbiota than glucose / galactose-based oligosaccharides and liraglutide. Figure 6 The Bray-Curtis dissimilarity analysis presented in was further verified. Figure 6 It was shown that animals treated with the selected oligosaccharides had more different intestinal microbial communities at the end of the treatment period compared to the other treatment groups. The intestinal microbial communities of animals treated with the selected oligosaccharides were also more different from their original state (before treatment) compared to the other treatment groups. The changes in the composition of the intestinal microbiome of mice triggered by the selected oligosaccharides included enrichment of Bacteroides, Blautia, Parabacteroides, Akkermansia, Butyrivibrio, Ruthenibacterium, and rare micrococci, as well as depletion of Anaerotruncus, Enterococcus, Dorea, and Streptococcus (Table 5). Importantly, Bacteroides and Parabacteroides were depleted in patients with atherosclerosis, while Dorea, Streptococcus, and Enterococcus were enriched in these patients. Therefore, the selected oligosaccharides have the potential to keep the intestinal microbiome away from dysbiosis and restore the intestinal microbiome of patients with cardiovascular disease. Furthermore, Akkermansia is a genus associated with reduced obesity, diabetes, and inflammation, supporting the potential use of selected oligosaccharides to treat these conditions.

[0523] Table 5. Changes in the number of bacterial genera in the fecal microbiota of HFD-induced obese mice treated with selected oligosaccharides after 4 weeks of treatment

[0524]

[0525]

[0526]

[0527] The selected oligosaccharide composition showed a significant effect in reducing all major blood lipids in the HFD-induced obese mouse model (TC, TG, NEFA), which was associated with the improvement of liver pathology associated with non-alcoholic fatty liver disease (liver steatosis and inflammation). This was confirmed by the reduction in liver expression of lipogenic genes, inflammatory markers, and fibrogenic genes caused by the selected oligosaccharide composition. These data suggest that the selected oligosaccharide composition may be useful for the treatment of acquired or inherited hyperlipidemia and may help prevent related diseases such as NAFLD, NASH, and cardiovascular disease.

[0528] Example 11. Therapeutic and microbiome effects of selected oligosaccharide compositions in a genetic model of Western diet (WD)-induced obesity in mice

[0529] In the validated Western diet (WD)-induced obese low-density lipoprotein receptor knockout (LDLR - / - The therapeutic and microbiome effects of the selected oligosaccharide compositions were tested in a 1:1 mouse model, which is thought to recapitulate many of the metabolic pathologies found in humans, including obesity, hyperglycemia, glucose intolerance, hyperinsulinemia, insulin resistance, severe hypercholesterolemia, hypertriglyceridemia, atherosclerosis, hepatic steatosis, and hepatic inflammation. Fenofibrate, a peroxisome proliferator receptor α (PPAR) activator used in patients with hypercholesterolemia, hypertriglyceridemia, or mixed dyslipidemia to reduce elevated low-density lipoprotein (LDL), total cholesterol (TC), or triglycerides (TG), was used as a positive control.

[0530] 9-11 weeks old male C57BL / 6J ldlr - / -Mice were assigned to one of four treatment groups (12 animals per group, 4 animals per cage) - (1) no treatment (NT), (2) selected oligosaccharides (generated as described in Example 2), (3) glucose / galactose-based oligosaccharide composition and (4) fenofibrate - for 16 weeks. Mice in the NT group were fed a Western diet (WD) (40 kcal% fat, 1.5% cholesterol, Research Diets D12079B). Mice in the oligosaccharide composition groups (#2 and #3) were fed a WD supplemented with 7.5% w / w of the respective composition. The oligosaccharide composition treatment diets were modified to provide the mice with the same caloric value as those mice on WD (no oligosaccharide composition). Mice in the fenofibrate group were fed a WD supplemented with fenofibrate (100 mg / kg / day). The body weight of each animal was measured weekly before and throughout the treatment period. The food intake of each cage was measured for 4 days per week during the treatment period and averaged as the food intake per mouse per day. Fresh fecal samples were collected within one week before the start of treatment and at week 15 after the start of treatment. For each fecal collection period, one fresh fecal pellet was collected for each animal on three different days. Fecal samples were sent for shallow gun sequencing to characterize the changes in the intestinal microbiota caused by the two oligosaccharide compositions or fenofibrate. On the day of the end of treatment, after 16 weeks of treatment and overnight fasting, retro-orbital blood was collected under anesthesia for quantification of serum very low-density lipoprotein (VLDL), serum low-density lipoprotein (LDL), serum high-density lipoprotein (HDL), serum total cholesterol (TC), serum non-esterified fatty acids (NEFA), serum insulin and whole blood triglycerides (TG) to characterize the effects of the selected oligosaccharide compositions, glucose / galactose-based oligosaccharide compositions and fenofibrate on hyperlipidemia and hyperinsulinemia. The animals were then euthanized and their whole livers were dissected and weighed. For each animal, individual liver sections (~100 mg each) were collected and frozen for quantification of liver TG and liver TC. One liver lobe was also preserved in 10% neutral buffered formalin (NBF) for embedding, sectioning, and H&E staining, and then the severity of steatosis, lobular inflammation, and hepatocellular ballooning was scored using a validated nonalcoholic fatty liver histological scoring system (Kleiner et al., Hepatology, 2005). The aortic arch was also dissected and preserved in 10% NBF after perfusion with phosphate buffered saline (PBS). The surface of the aortic arch was stained with Oil Red O to quantify the surface area of ​​atherosclerotic plaques.

[0531] like Figure 8 As shown in A and 8B, the selected oligosaccharide composition had a small effect on weight gain. At the same time, fenofibrate reduced weight gain by 44% compared to NT. The oligosaccharide composition and fenofibrate had no effect on food intake ( Figure 8C) These data suggest that WD-induced obesity - / - The two oligosaccharide compositions were well tolerated by mice. The selected oligosaccharide compositions did not reduce fasting blood glucose ( Fig. 9 A). Similar to fenofibrate, the selected oligosaccharide composition reduced fasting insulin (-80%) and homeostasis model assessment of insulin resistance (HOMA-IR) (-78%) compared to NT ( Fig. 9 B-9C), indicating that the selected oligosaccharide composition reduces WD-induced obesity ldlr - / - Insulin resistance in mice. The selected oligosaccharide composition was the only treatment group that significantly reduced serum TC (-43%) compared with NT ( Fig.10 B). The selected oligosaccharide composition also provided the greatest effect on lowering serum VLDL (-58%; significant by t-test) and LDL (-39%) compared to NT ( Fig.10 C-10D); and significantly reduced serum HDL (-47%) compared with NT ( Fig.10 E). In addition, the selected oligosaccharides decreased serum NEFA (-16%; significant by t-test) ( Fig.10 F). In addition to its dramatic effects on serum TC and LDL, the selected oligosaccharide composition reduced liver TC (-39% compared to NT) ( Fig.10 H) and significantly reduced liver TG (-37%) compared with NT ( Fig.10 G). The effect of the selected oligosaccharide composition on liver fat was demonstrated by its effect on reducing liver weight ( Fig.11 A, 11B) and a decreased pathological score of hepatic steatosis (-1 point) compared with NT ( Fig.11 In addition, the selected oligosaccharide composition significantly reduced the liver inflammation pathology score (-1 point) compared with NT ( Fig.11 D). No hepatocellular ballooning was observed in this model, with a score of zero for all treatments. The selected oligosaccharide composition also significantly reduced the overall nonalcoholic fatty liver activity score (NAS), calculated as the sum of hepatic steatosis, lobular inflammation, and hepatocellular ballooning (-2.5 points) ( Fig.11 E). Finally, the selected oligosaccharide composition also resulted in a significant reduction in the overall plaque severity score of the aortic sinus (-2 points) ( Fig.11F ), while in the aortic sinus (reduced by 38% relative to untreated) ( Fig.11G ) and aortic arch (38% reduction relative to untreated) ( Fig.11H ) showed a significant reduction in atherosclerotic plaque formation. Representative images of liver histology in different groups are shown in Fig.13 shown. Fig.13The reduction of NAFLD pathology in animals treated with the selected oligosaccharides was demonstrated. In addition to its beneficial effects on the liver, the selected oligosaccharides also reduced the formation of atherosclerotic plaques in the aortic arch (-64%) ( Fig.14 A-14B), consistent with its effect of lowering serum total cholesterol and LDL.

[0532] Shallow gun sequencing of stool samples collected before, during and at the end of treatment showed that the selected oligosaccharides had a strong impact on the gut microbial community. Fig.12 The multidimensional scaling (MDS) ordination plot of the microbiome sequencing data shown shows the correlation between the selected oligosaccharides ( Fig.12 The composition of the gut microbiota of animals treated with NT ( Fig.12 Diamonds in the middle), glucose / galactose-based oligosaccharides ( Fig.12 in the cross) or fenofibrate ( Fig.12 The gut microbiome of an animal (the triangle in the middle). This is Fig.15 The Bray-Curtis dissimilarity analysis presented in confirms that Fig.15 It was shown that at the end of the treatment period, the intestinal microbial communities of animals treated with the selected oligosaccharides were less similar than those of the other treatment groups compared to the NT group. The changes in the composition of the intestinal microbiota triggered by the selected oligosaccharides included enrichment of Allistipes, Blautia, Parabacteroides, Akkermansia, Ruthenibacterium, and Micrococcus uncommonus and depletion of Anaerotruncus, Dorsa, Staphylococcus, Streptococcus, and Enterococcus (Table 6). Importantly, Parabacteroides was depleted in patients with atherosclerosis, while Dorsa, Streptococcus, and Enterococcus were enriched in these patients. Therefore, the selected oligosaccharides have the potential to move the intestinal microbiota away from dysbiosis and restore the intestinal microbiota in patients with cardiovascular disease. In addition, Akkermansia is a genus associated with reducing obesity, diabetes, and inflammation, which supports the use of the selected oligosaccharides to treat these conditions.

[0533] Fig.19The taxa that were significantly enriched at the phylum, family, genus, and species level in mice treated with the selected oligosaccharide compositions compared to the untreated (NT) group at the end of the study (adjusted p-value < 0.05, Mann-Whitney U test with FDR correction). Many of these enriched bacterial taxa are considered to be propionic acid-producing bacterial taxa (e.g., Bacteroides, Muscicapaceae, Tannerellaceae, Alternaria, Parabacteroides, and to a lesser extent Ruminococcaceae) or acetic acid-producing taxa (e.g., Blautia producta). The selected oligosaccharide compositions also enriched Akkermansia muciniphila and Parasartella (belonging to the Proteobacteria phylum, recently shown to be associated with LDL-C reduction in humans).

[0534] Table 6. Treatment of WD-induced obese LDLR with selected oligosaccharides after 15 weeks of treatment - / - Changes in the number of bacterial genera in the fecal microbiome of mice

[0535]

[0536] LDLR fed in WD - / - Short-chain fatty acids (SCFA) were quantified in the cecal contents of mice. - / - Treatment of mice with the selected oligosaccharide combination increased total acetate and propionate levels by 126% and 342% ( Fig.18A A small increase in butyrate was also observed, although not significant. In addition, the selected oligosaccharide composition reduced the fractional proportions of acetate and butyrate by 42% and 12%, respectively, and increased the fractional proportion of propionate by 84% compared to NT ( Fig.18B ). This suggests that the selected oligosaccharide composition induced the enrichment of propionic acid-producing bacteria in vivo. The selected oligosaccharide composition also significantly increased the total amount and proportion of succinate, by 547% and 169%, respectively. Succinate is a key intermediate in the "succinate pathway" of propionic acid formation, which is the main propionic acid biosynthetic pathway in the large intestine, for example, for taxa belonging to the phylum Bacteroidetes. As expected, fenofibrate did not change the total amount or proportion of SCFA or succinate.

[0537] Effect of selected oligosaccharide compositions on WD-induced obese LDLR - / -The mouse model shows the effect of reducing blood TC, VLDL and LDL, which is related to improving the liver pathology related to non-alcoholic fatty liver disease (liver steatosis and inflammation) and reducing atherosclerotic plaque formation. In addition, the selected oligosaccharide composition also reduces insulin and HOMA-IR in the animal model. In addition, these data show that the selected oligosaccharide composition increases the amount of propionic acid (and / or propionic acid-producing bacteria) in the body. These data show that the selected oligosaccharide composition can be used to treat acquired or hereditary hyperlipidemia and prevent related diseases, such as atherosclerosis, coronary heart disease, stable and unstable angina, hypertension and related comorbidities, such as metabolic syndrome, hyperinsulinemia, insulin resistance and type 2 diabetes. These data further show that the selected oligosaccharide composition can be used to treat non-alcoholic fatty liver disease (NAFLD) and non-alcoholic fatty hepatitis (NASH). Reductions in hepatic steatosis, inflammation, hepatic triglyceride concentrations, liver weight, and hepatic expression of marker genes for inflammation, fibrosis, oxidative stress, and apoptosis are indicators that the selected oligosaccharide composition may be useful in treating NAFLD or NASH.

[0538] Example 12: Evaluation of selected oligosaccharide compositions in fecal slurries

[0539] The ability of the selected oligosaccharide compositions produced by the methods described in Examples 2-4 to increase the production of SCFAs (propionate / propionic acid, acetate / acetic acid and butyrate / butyric acid) was evaluated by fecal slurries of ten healthy patients and thirty-four overweight patients diagnosed with type 2 diabetes (T2D).

[0540] Fecal samples from healthy patients and overweight T2D patients were collected and stored at -80°C. To prepare fecal material for ex vivo assays, aliquots of a 20% w / v slurry in phosphate buffered saline (PBS) and glycerol were thawed in a COY anaerobic chamber. The slurry was then further diluted into a 1% solution of Mega Media (MM). The medium was filter sterilized using a 0.2 μm filter and stored in an anaerobic chamber before use to allow any dissolved oxygen to dissipate.

[0541] In healthy patients, the median propionic acid concentration in fecal samples was 13.1 mM higher in samples incubated with the selected oligosaccharide composition compared to negative controls ( Fig.16A ). Fermentation of the selected oligosaccharide composition with these fecal samples also resulted in a median increase of 12.0 mM acetate, but it did not increase butyrate concentration in a statistically significant manner. The selected oligosaccharide composition selectively increased the production of propionate as a fraction of total SCFA. Propionate accounted for 22.8% of the total SCFA produced in the negative control samples, while it increased to 40.4% of SCFA in the samples incubated with the selected oligosaccharides ( Fig. 16B). In addition, although the selected oligosaccharide composition also increased total acetic acid production, it did not increase the acetic acid concentration proportionally. This fermentation product selectivity suggests that the selected oligosaccharide composition may be mainly metabolized by propionic acid-producing bacteria in healthy patients.

[0542] Thirty-four overweight subjects diagnosed with T2D were men and women aged 18 to 70 years with a BMI between 25 and 45 kg / m 2 Similar to what was observed for samples from healthy subjects, the selected oligosaccharide composition resulted in a median increase in propionic acid concentration of 11.6 mM in fecal samples from these patients compared to negative controls ( Fig.17A ). It also increased the acetate concentration by 12.5 mM and slightly increased the butyrate concentration. In addition, the selected oligosaccharide composition increased the fractional propionate concentration to 30.0% of the total SCFA compared to 16.3% in the negative control ( Fig. 17B ). There was no increase in the fractional concentrations of acetate or butyrate. These results suggest that in overweight subjects with T2D, a population reported to have a disrupted gut microbiome, the selected oligosaccharide composition may be metabolized primarily by propionic acid-producing bacteria.

[0543] Equivalent forms and terms

[0544] The disclosure illustratively described herein may be appropriately practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Thus, for example, in each case herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with any of the other two terms. The terms and expressions that have been adopted are used as descriptive terms, rather than restrictive, and it is not intended to exclude any equivalents of the features shown and described or portions thereof when such terms and expressions are used, but it should be recognized that various modifications may be made within the scope of the present disclosure. Therefore, it should be understood that although the present disclosure has been specifically disclosed through preferred embodiments, optional features, those skilled in the art may make modifications and variations with the aid of the concepts belonging to the present disclosure, and such modifications and variations may be considered to be within the scope of the present disclosure.

[0545] In addition, where features or aspects of the disclosure are described in terms of Markush groups or other groupings of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other grouping.

[0546] Unless otherwise noted or clearly contradictory to the context, in the context of describing the present invention (especially in the context of the following claims), the use of the terms "one", "a kind of" and "the / said" and similar references should be interpreted as covering the singular and plural. Unless otherwise noted, the terms "comprise", "have", "include" and "contain" should be interpreted as open terms (i.e., meaning "including but not limited to"). Unless otherwise noted herein, the description of the numerical range herein is only intended to be used as a shorthand method for referring to each individual value falling within the range, respectively, and each individual value is incorporated into the specification as if it is separately described herein. Unless otherwise noted herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary languages ​​(e.g., "such as") provided herein is only intended to better illustrate the present invention, and is not intended to limit the scope of the present invention. Any language in the specification should not be interpreted as indicating any unclaimed elements that are essential for implementing the present invention.

[0547] Embodiments of the invention are described herein. Variations of those embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description.

[0548] The inventors expect that those skilled in the art will appropriately adopt such variations, and the inventors wish to practice the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalent forms of the subject matter recorded in the appended claims as permitted by applicable law. Moreover, unless otherwise indicated herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof. Using only routine experiments, those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be covered by the appended claims.

Claims

1. An oligosaccharide composition comprising a plurality of oligosaccharides, the composition being characterized by a multiply edited gradient enhanced 1H-13C heteronuclear single quantum coherence (HSQC) NMR spectrum comprising each of signals 1-12 of the following table, wherein the spectrum is generated using an oligosaccharide composition sample having less than 2% monomers: wherein signals 1-12 are each further characterized by 1 H integration area and 13 The C integration region is defined as follows: The oligosaccharide composition comprises a plurality of oligosaccharides consisting essentially of formula (I) and formula (II): Wherein R in formula (I) and formula (II) is independently selected from hydrogen, and formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc), (IId): wherein R in formula (Ia), (Ib), (Ic), (Id), (IIa), (IIb), (IIc) and (IId) is independently as defined in formula (I) and formula (II), Wherein the oligosaccharide composition is produced by a method comprising: (a) forming a reaction mixture comprising galactose monomers and mannose monomers with an acid catalyst comprising positively charged hydrogen ions, wherein the molar ratio of galactose to mannose is about 3:2; and (b) promoting the formation of acid-catalyzed oligosaccharides in the reaction mixture by transferring sufficient heat to the reaction mixture to maintain the reaction mixture at its boiling point.

2. The oligosaccharide composition of claim 1, wherein at least one of signals 1-12 is defined as follows:

3. The oligosaccharide composition of claim 1, wherein the NMR spectrum is enhanced by multiple editing gradients of the composition sample. 1 H- 13 C heteronuclear single quantum coherence (HSQC) experiments using an echo-anti-echo scheme with the following pulse sequence diagram, acquisition parameters and processing parameters for coherence selection: Pulse sequence diagram Acquisition parameters 1 H carrier frequency = 4ppm 13 C carrier frequency = 65ppm Number of points in the collection dimension = 596 Spectral range in acquisition dimension = 6.23ppm to 1.83ppm Number of points in indirect dimension = 300 complex points Spectral range in indirect dimension = 120ppm to 10ppm Loop delay = 1 second single bond 1 H- 13 C coupling constant = J CH =146Hz Scan times = 8 Temperature = 298-299K Solvent = D2O Processing parameters Direct dimension window function = Gaussian broadening, 7.66Hz Indirect dimension window function = Gaussian widening 26.48Hz Processing = 512 complex points in the direct dimension, 1024 complex points in the indirect dimension.

4. The oligosaccharide composition of claim 1, wherein the NMR spectrum is obtained by performing HSQC NMR on a sample of the oligosaccharide composition, wherein the sample is dissolved in D2O.

5. The oligosaccharide composition of claim 1, wherein the oligosaccharide composition has been subjected to a demonomerization procedure.

6. The oligosaccharide composition of claim 1, wherein the oligosaccharide composition comprises less than 10% monomers.

7. The oligosaccharide composition of claim 1, wherein the oligosaccharide composition comprises less than 5% monomers.

8. The oligosaccharide composition of claim 1, wherein the oligosaccharide composition comprises less than 2% monomers.

9. The oligosaccharide composition of claim 1, wherein the average degree of polymerization (DP) of the oligosaccharide composition is from about DP6 to about DP19.

10. The oligosaccharide composition of claim 1, wherein the average degree of polymerization (DP) of the oligosaccharide composition is from about DP10 to about DP16.

11. The oligosaccharide composition of claim 1, wherein the average degree of polymerization (DP) of the oligosaccharide composition is from about DP10 to about DP14.

12. The oligosaccharide composition of claim 1, wherein the composition comprises 55% to 95% total dietary fiber (dry basis).

13. The oligosaccharide composition of claim 1, wherein each oligosaccharide comprises a plurality of monomer groups; The plurality of oligosaccharides comprises one or more of the following monomer groups: (2) t-mannopyranose monogroups, which represent 11.47-18.18 mol % of the monomeric groups in the various oligosaccharides; (4) t-mannofuranose monogroups, which represent 0.17-0.46 mol % of the monomeric groups in the various oligosaccharides; (8) 3-mannofuranose monogroup, which represents 0.07-0.24 mol % of the monomeric groups in the various oligosaccharides; (10) 2-mannopyranose and / or 3-mannopyranose monogroups, which represent 3.91-5.93 mol % of the monomeric groups in the various oligosaccharides; (15) 6-pyranose monogroup, which represents 8.30-10.84 mol% of the monomer groups in the various oligosaccharides; (27) 4,6-mannopyranose and / or 5,6-mannofuranose diyl groups, which represent 1.18-1.82 mol % of the monomer groups in the various oligosaccharides; (30) 3,6-pyranose diyl, which represents 1.76-2.40 mol % of the monomeric group in the various oligosaccharides; (31) 2,6-pyranose diyl, which represents 1.42-1.84 mol % of the monomeric groups in the various oligosaccharides; and (32) 3,6-galactofuranosyl, which represents 0.95–1.41 mol % of the monomeric group in the various oligosaccharides.

14. The oligosaccharide composition of claim 13, wherein the plurality of oligosaccharides comprises at least 2, 3, 4, 5, 6, 7, 8 or 9 of the monomeric groups selected from groups (2), (4), (8), (10), (15), (27) and (30)-(32).

15. The oligosaccharide composition of claim 13, further comprising one or more of the following monomer groups: (5) t-galactofuranose monogroups, which represent 2.96-6.24 mol % of the monomeric groups in the various oligosaccharides; (6) t-galactopyranose monogroups, which represent 12.90-17.63 mol % of the monomeric groups in the various oligosaccharides; (12) 3-galactopyranose monogroup, which represents 3.52-4.87 mol % of the monomeric groups in the various oligosaccharides; (18) 4-galactopyranose and / or 5-galactofuranose monogroups, which represent 2.86-3.57 mol % of the monomeric groups in the various oligosaccharides; (22) 2,3-galactofuranosyl, which represents 0.10-0.59 mol % of the monomeric group in the various oligosaccharides; (23) 2,3-pyranose diyl, which represents 0.24-0.51 mol % of the monomeric group in the various oligosaccharides; (24) 6-galactopyranose monogroup, which represents 9.78-13.08 mol % of the monomeric groups in the various oligosaccharides; (25) 3,4-galactopyranose and / or 3,5-galactofuranose and / or 2,3-galactopyranose diyl groups, which represent 0.83-1.32 mol % of the monomer groups in the plurality of oligosaccharides; (26) 2,4-mannopyranose and / or 2,5-mannofuranose diyl groups, which represent 0.09-1.15 mol % of the monomer groups in the various oligosaccharides; (28) 3,6-mannofuranose diyl, which represents 0.07-0.18 mol % of the monomeric group in the various oligosaccharides; (33) 4,6-galactopyranose and / or 5,6-galactofuranosyl groups, which represent 2.38-3.46 mol % of the monomeric groups in the various oligosaccharides; (34) 2,6-galactopyranosyldiyl, which represents 1.23–1.84 mol % of the monomeric groups in the various oligosaccharides; (35) 3,4,6-mannopyranose and / or 3,5,6-mannofuranose and / or 2,3,6-mannofuranose triyl groups, which represent 0.19-0.48 mol % of the monomer groups in the various oligosaccharides; (36) 3,4,6-galactopyranose and / or 3,5,6-galactofuranose and / or 2,3,6-galactofuranose triyl groups, which represent 0.53-1.03 mol % of the monomer groups in the various oligosaccharides; (37) 2,3,6-mannopyranose and / or 2,4,6-mannopyranose and / or 2,5,6-mannofuranose triyl groups, which represent 0.43-0.68 mol % of the monomer groups in the plurality of oligosaccharides; and (38) 2,3,6-galactopyranose and / or 2,4,6-galactopyranose and / or 2,5,6-galactofuranosyl triyl groups, which represent 0.59-0.93 mol % of the monomer groups in the various oligosaccharides.

16. The oligosaccharide composition of claim 15, wherein the plurality of oligosaccharides comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of the monomer groups selected from the group consisting of (2), (4)-(6), (8), (10), (12), (15), (18), (22)-(28) and (30)-(38).

17. The oligosaccharide composition of claim 15, wherein the plurality of oligosaccharides comprises each of a monomeric group selected from the group consisting of (2), (4)-(6), (8), (10), (12), (15), (18), (22)-(28), and (30)-(38).

18. The oligosaccharide composition of claim 13, further comprising one or more of the following monomer groups: (9) 2-Mannofuranose monogroup, which represents 0.01-1.07 mol % of the monomer group in the various oligosaccharides; (11) 2-galactofuranose monogroup, which represents 0.01-4.34 mol% of the monomeric groups in the various oligosaccharides; (13) 4-mannopyranose and / or 5-mannofuranose monogroups, which represent 0.01-7.91 mol % of the monomer groups in the plurality of oligosaccharides; (14) 3-galactofuranose monogroup, which represents 0.01-4.44 mol% of the monomeric groups in the various oligosaccharides; (16) 2-galactopyranose monogroup, which represents 0.01-8.70 mol % of the monomeric groups in the various oligosaccharides; (19) 2,3-Mannofuranose diyl, which represents 0.01-0.26 mol % of the monomeric group in the various oligosaccharides; (21) 6-Mannofuranose monogroup, which represents 0.01-1.72 mol % of the monomeric group in the various oligosaccharides; (39) 2,3,4,6-mannopyranose and / or 2,3,5,6-mannofuranose tetrayl groups, which represent 0.01-0.68 mol % of the monomer groups in the plurality of oligosaccharides; and (40) 2,3,4,6-galactopyranose and / or 2,3,5,6-galactofuranosyl, which represent 0.01-0.31 mol % of the monomeric groups in the various oligosaccharides.

19. The oligosaccharide composition of claim 13, further comprising one or more of the following monomer groups: (9) 2-mannofuranose monogroup, which represents about 0.11 mol % of the monomeric groups in the various oligosaccharides; (11) 2-galactofuranose monogroup, which represents about 0.43 mol % of the monomer groups in the various oligosaccharides; (13) 4-mannopyranose and / or 5-mannofuranose monogroups, which represent about 1.10 mol % of the monomer groups in the various oligosaccharides; (14) 3-galactofuranose monogroup, which represents about 0.35 mol % of the monomeric groups in the various oligosaccharides; (16) 2-galactopyranose monogroup, which represents 0.06-6.54 mol % of the monomeric groups in the various oligosaccharides; (19) 2,3-Mannofuranose diyl, which represents 0.06-0.21 mol % of the monomeric group in the various oligosaccharides; (21) 6-Mannofuranose monogroup, which represents about 0.36 mol % of the monomer groups in the various oligosaccharides; (39) 2,3,4,6-mannopyranose and / or 2,3,5,6-mannofuranose tetrayl groups, which represent about 0.12 mol % of the monomer groups in the various oligosaccharides; and (40) 2,3,4,6-galactopyranose and / or 2,3,5,6-galactofuranosyl, which represent about 0.04 mol % of the monomeric groups in the various oligosaccharides.

20. The oligosaccharide composition of claim 18, wherein the plurality of oligosaccharides comprises each of a monomeric group selected from the group consisting of (2), (4)-(6), (8)-(16), (18), (19), (21)-(28) and (30)-(39).

21. The oligosaccharide composition of claim 13, wherein the mole percentage of monomeric groups is determined using a permethylation assay, wherein the permethylation assay comprises gas chromatography-mass spectrometry (GC-MS) analysis.

22. The oligosaccharide composition of claim 1, wherein step (b) comprises loading the reaction mixture with an acid catalyst comprising positively charged hydrogen ions in an amount such that the molar ratio of the positively charged hydrogen ions to the total galactose monomer and mannose monomer content is within a suitable range.

23. The oligosaccharide composition of claim 1, wherein steps (a) and (b) occur simultaneously.

24. The oligosaccharide composition of claim 1, wherein step (a) comprises heating the reaction mixture to a temperature in the range of 100°C to 160°C under stirring conditions.

25. The oligosaccharide composition of claim 24, wherein step (a) comprises heating the reaction mixture to a temperature in the range of 130°C to 140°C under stirring conditions.

26. The oligosaccharide composition of claim 1, wherein step (a) comprises heating the reaction mixture at a temperature in the range of 100°C to 160°C under stirring conditions.

27. The oligosaccharide composition of claim 26, wherein step (a) comprises heating the reaction mixture at a temperature in the range of 130°C to 140°C under stirring conditions.

28. The oligosaccharide composition of claim 1, wherein step (a) comprises gradually increasing the temperature to about 135°C under suitable conditions to achieve homogeneity and uniform heat transfer.

29. The oligosaccharide composition of claim 1, wherein step (b) comprises maintaining the reaction mixture at atmospheric pressure or vacuum at a temperature in the range of 130°C to 140°C under conditions that promote acid-catalyzed formation of the oligosaccharide composition until the weight percent of galactose monomers and mannose monomers in the oligosaccharide composition is in the range of 4-14.

30. The oligosaccharide composition of claim 1, wherein step (b) comprises gradually increasing the temperature to about 135°C under suitable conditions to achieve homogeneity and uniform heat transfer.

31. The oligosaccharide composition of claim 1, wherein the acid catalyst is a strong acid cation exchange resin having one or more physical and chemical properties according to Table 1 and / or wherein the catalyst comprises >3.0 mmol / g sulfonic acid moieties and <1.0 mmol / g cationic moieties.

32. The oligosaccharide composition of claim 31, wherein the catalyst has a nominal moisture content of 45-50 weight percent.

33. The oligosaccharide composition of claim 1, wherein the acid catalyst is a soluble catalyst.

34. The oligosaccharide composition of claim 33, wherein the soluble catalyst is an organic acid.

35. The oligosaccharide composition of claim 33, wherein the soluble catalyst is a weak organic acid.

36. The oligosaccharide composition of claim 33, wherein the soluble catalyst is citric acid.

37. The oligosaccharide composition of claim 1, wherein the method further comprises: (c) quenching the reaction mixture while allowing the temperature of the reaction mixture to reach a temperature in the range of 55°C to 95°C.

38. The oligosaccharide composition of claim 37, wherein the method further comprises: (d) separating the oligosaccharide composition from the acid catalyst.

39. The oligosaccharide composition of claim 38, wherein in (d), the separating comprises removing the catalyst by filtration.

40. The oligosaccharide composition of claim 38, wherein (d) comprises cooling the reaction mixture to less than about 85°C prior to filtering.

41. The oligosaccharide composition of claim 38, wherein the method further comprises: (e) diluting the oligosaccharide composition of (d) with water to a concentration of about 45-55 weight percent; (f) passing the diluted composition through a cation exchange resin; (g) passing the diluted composition through a decolorizing polymer resin; and / or (h) passing the diluted composition through an anion exchange resin; Each of (f), (g) and (h) may be performed once or multiple times in any order.

42. The oligosaccharide composition of claim 1, wherein the average degree of polymerization (DP) of the oligosaccharide composition is from about DP6 to about DP19.

43. The oligosaccharide composition of claim 1, wherein the average degree of polymerization (DP) of the oligosaccharide composition is from about DP10 to about DP14.

44. The oligosaccharide composition of claim 1, wherein the oligosaccharide composition comprises 55% to 95% total dietary fiber (dry basis).

45. The oligosaccharide composition of claim 1, wherein the oligosaccharide composition comprises less than 15% monomer (DP1).

46. ​​Use of an oligosaccharide composition according to any one of claims 1 to 45 in the preparation of a medicament for treating a subject suffering from or suspected of suffering from non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

47. The use of claim 46, wherein the subject has or is suspected of having NAFLD.

48. The use of claim 46, wherein the subject has or is suspected of having NASH.

49. Use of an oligosaccharide composition according to any one of claims 1 to 45 in the preparation of a medicament for reducing the relative or absolute concentration of lipids in a human subject.

50. The use of claim 49, wherein the medicament reduces the relative or absolute concentration of lipids in the gastrointestinal tract of a subject.

51. The use of claim 49, wherein the relative or absolute concentration of lipid is reduced by at least 5%, 10%, 20% or 30% compared to a baseline measurement, wherein the baseline measurement is determined prior to treatment.

52. The use of claim 49, wherein the lipid is cholesterol, very low density lipoprotein (VLDL), low density lipoprotein (LDL), non-esterified free fatty acids and / or triglycerides.

53. The use of claim 52, wherein the cholesterol is liver cholesterol and / or blood cholesterol.

54. The use of claim 52, wherein the triglycerides are liver triglycerides and / or blood triglycerides.

55. Use of an oligosaccharide composition according to any one of claims 1 to 45 in the preparation of a medicament for treating a subject suffering from or suspected of suffering from dyslipidemia or hyperlipidemia.

56. The use of claim 55, wherein the hyperlipidemia is primary or familial hyperlipidemia.

57. The use of any one of claims 46-56, wherein the subject has or is at risk of developing non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or type 2 diabetes (T2D).

58. The use of claim 55, wherein the hyperlipidemia is heterozygous familial hypercholesterolemia (HeFH), homozygous familial hypercholesterolemia (HoFH), familial chylomicronemia syndrome, familial combined hyperlipidemia, or familial dysbetalipoproteinemia.

59. The use of any one of claims 46-56, wherein the subject is resistant to statins.

60. The use of any one of claims 46-56, wherein the subject is intolerant to statins.

61. The use of any one of claims 46-56, wherein the subject is a human subject.

62. The use of claim 61, wherein the subject is a newborn, an infant under one year old, a toddler, a teenager, an adult, or an elderly person.

63. The use of any one of claims 46-56, wherein the oligosaccharide composition is administered to the intestine.

64. The use of any one of claims 46-56, wherein the oligosaccharide composition is self-administered to a subject.

65. The use of any one of claims 46-56, wherein the oligosaccharide composition is formulated as a pharmaceutical composition for oral delivery.

66. The use of any one of claims 46-56, wherein the oligosaccharide composition is orally administered to a subject.

67. The use of any one of claims 46-56, wherein the oligosaccharide composition is formulated as a pharmaceutical composition for delivery via a feeding tube.

68. The use of any one of claims 46-56, wherein the oligosaccharide composition is administered to the subject via a feeding tube.

69. The use of any one of claims 46-56, wherein the oligosaccharide composition is administered to the subject once a day or twice a day.

70. The use of any one of claims 46-56, wherein the oligosaccharide composition is co-administered to the subject with standard of care treatment.

71. The use of claim 70, wherein the standard of care treatment is administration of a statin.

72. The use of any one of claims 46-56, wherein the medicament reduces the concentration of lipids in a subject.

73. The use of claim 72, wherein lipid concentration is reduced by at least 5%, 10%, 20% or 30% relative to a baseline measurement, wherein the baseline measurement is determined prior to treatment.

74. The use of claim 73, wherein the lipid is cholesterol, very low density lipoprotein (VLDL), low density lipoprotein (LDL), non-esterified free fatty acids and / or triglycerides.

75. The use of claim 74, wherein the cholesterol is liver cholesterol and / or blood cholesterol.

76. The use of claim 74, wherein the triglycerides are liver triglycerides and / or blood triglycerides.

77. The use of any one of claims 46-56, wherein the medicament reduces or alleviates liver steatosis and / or liver inflammation.

78. The use of any one of claims 46-56, wherein the medicament reduces atherosclerotic plaque formation.

79. The use of any one of claims 46-56, wherein the medicament supports the growth of bacterial genera that do not promote dysbiosis within the subject's gastrointestinal microbiota.

80. The use of any one of claims 46-56, wherein the medicament supports the growth of propionic acid producing bacterial taxa.

81. The use of any one of claims 46-56, wherein the medicament supports the growth of Blautia, Akkermansia, Ruthenibacterium, Micrococcus, Bacteroidetes, Muscicapaceae, Tannerellaceae, Alternaria, and / or Ruminococcaceae.

82. The use of any one of claims 46-56, wherein the medicament increases the relative abundance of one or more bacterial taxa selected from the group consisting of Blautia, Akkermansia, Ruthenibacterium, Micrococcus, Bacteroidetes, Muscicapaceae, Tannerellaceae, Alternaria, and / or Ruminococcaceae.

83. The use of any one of claims 46-56, wherein the medicament supports the growth of Parabacteroides or increases the relative abundance of Parabacteroides.

84. The use of any one of claims 46-56, wherein the medicament reduces or shrinks liver fibrosis.

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