Ways to support gastrointestinal homeostasis

Abstract

The present disclosure relates to methods for preventing or treating gastrointestinal barrier dysfunction by feeding animals with feed additives that modulate the intestinal microbiome. The present disclosure also relates to methods for preventing or treating infection by feeding animals with feed additives that modulate the intestinal microbiome.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 757,500, filed on November 8, 2018, and U.S. Provisional Patent Application No. 62 / 757,465, filed on November 8, 2018, the disclosures of each of which are hereby incorporated by reference in their entireties. Background Art

[0003] An animal's gastrointestinal system is the largest surface area interface between its internal anatomy and the outside world. It is responsible for digestion and absorption of nutrients and provides an effective barrier against environmental pathogens. The gastrointestinal tract (GIT) strongly interacts with the host animal's immune system and is home to the animal's intestinal microbiome, which includes a variety of microorganisms (bacteria, fungi, molds, viruses, etc.) that reside in the digestive tract.

[0004] In healthy animals, the gastrointestinal system maintains homeostasis through a complex interplay between the host animal's immune function, the physiology of the intestinal mucosal layer and endothelium, and the biochemistry of the gut microbiome. Mucin and mucus production by intestinal goblet cells, nonspecific immune system activity, chemokine- and cytokine-mediated regulation of inflammation, and tight junctions between intestinal lining cells promote healthy nutrient absorption and protection against pathogens and toxins.

[0005] Disruption of gastrointestinal homeostasis leads to negative health and nutritional consequences for animals. For example, disruption of healthy barrier function enables intestinal contents to translocate into the host circulation, for example allowing pathogens and toxins to penetrate the mucosa and endothelium and negatively impact the host animal. Inflammation and / or damage to intestinal epithelial cells reduces the ability of the animal to absorb nutrients. For production animals, maintaining healthy gastrointestinal homeostasis results in poor nutritional and health outcomes for the gastrointestinal system, leading to reduced weight gain, reduced feed efficiency, reduced meat production, lower meat quality, and higher mortality. For companion animals, disruption of gastrointestinal homeostasis may reduce quality of life and overall health. Therefore, there is a great need to provide nutritional compositions, including animal feeds, for preventing and / or treating gastrointestinal barrier dysfunction. Summary of the Invention

[0006] In one aspect, provided herein is a method for treating or preventing gastrointestinal barrier dysfunction in an animal in need thereof, the method comprising: administering to the animal a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation, each having at least n oligosaccharide fractions (DP1 to DPn fractions) of different degrees of polymerization selected from 1 to n, wherein n is an integer greater than 3; and wherein each of the DP1 and DP2 fractions independently comprises from about 0.5% to about 15% anhydrosubunit-containing oligosaccharides in relative abundance as determined by mass spectrometry, thereby treating or preventing the gastrointestinal barrier dysfunction.

[0007] In some embodiments, the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal prior to said administering of the synthetic oligosaccharide preparation.

[0008] In some embodiments, the reduction is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% relative to the permeability of the gastrointestinal barrier of the animal prior to the administration of the synthetic oligosaccharide formulation. In some embodiments, the reduction is about 0.5%-30%, 0.5%-20%, 0.5%-10%, 0.5%-5%, 0.5%-4%, 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 10%-4%, 1%-3%, or 1%-2% relative to the permeability of the gastrointestinal barrier of the animal prior to the administration of the synthetic oligosaccharide formulation.

[0009] In some embodiments, the reduction is a greater reduction than the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the reduction is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% greater than the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0010] In some embodiments, the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal prior to said administering of the synthetic oligosaccharide preparation.

[0011] In some embodiments, the reduction is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% relative to the permeability of the gastrointestinal barrier of the animal prior to the administration of the synthetic oligosaccharide formulation. In some embodiments, the reduction is about 0.5%-30%, 0.5%-20%, 0.5%-10%, 0.5%-5%, 0.5%-4%, 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 10%-4%, 1%-3%, or 1%-2% relative to the permeability of the gastrointestinal barrier of the animal prior to the administration of the synthetic oligosaccharide formulation.

[0012] In some embodiments, the permeability of the gastrointestinal barrier is determined from a sample of the gastrointestinal barrier from the animal. In some embodiments, the permeability is measured by histological analysis, staining, or any combination thereof.

[0013] In some embodiments, the method further comprises evaluating the mucosal morphology of the gastrointestinal barrier. In some embodiments, the evaluation comprises measuring villus length, crypt length, inflammatory cell infiltration level, or any combination thereof.

[0014] In some embodiments, the permeability of the gastrointestinal barrier is measured from a blood, feces, or urine sample from the animal. In some embodiments, the permeability is measured by measuring the level of a tracer administered orally to the animal in the sample. In some embodiments, the tracer is an indigestible sugar, polyethylene glycol (PEG), fluorescently labeled dextran, or a radioactive isotope. In some embodiments, the permeability is measured by measuring the level of at least one microbial species in the blood, feces, or urine sample.

[0015] In some embodiments, the permeability is measured by determining the level of antibodies in the sample that bind to at least one microbial species. In some embodiments, the at least one microbial species is present in the gastrointestinal tract of the animal.

[0016] In some embodiments, the reduction in permeability of the gastrointestinal barrier is directly mediated by the synthetic oligosaccharide preparation. In some embodiments, the reduction in permeability of the gastrointestinal barrier is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation is processed in vivo into at least one secondary substance. In some embodiments, the synthetic oligosaccharide preparation is processed in vivo by components of the gastrointestinal microbiome of the animal. In some embodiments, the synthetic oligosaccharide preparation is processed in vivo by bacteria present in the gastrointestinal tract of the animal. In some embodiments, the reduction in gastrointestinal permeability is directly mediated by the at least one secondary substance.

[0017] In some embodiments, the gastrointestinal barrier dysfunction is a hyperpermeable gastrointestinal barrier. In some embodiments, the hyperpermeable gastrointestinal barrier allows intestinal contents to translocate from the intraluminal space into the animal's circulation. In some embodiments, the intestinal contents include food particles, microorganisms, toxins, or any combination thereof.

[0018] In some embodiments, at least one symptom associated with the gastrointestinal barrier dysfunction is improved or prevented. In some embodiments, the at least one symptom is reduced mucus synthesis, reduced mucin synthesis, reduced mucus secretion, reduced mucin secretion, reduced nutrient absorption, increased inflammation, reduced resistance to infection, reduced intestinal epithelial cell proliferation, reduced intestinal epithelial cell maturation, increased immune cells in the gastrointestinal tract, increased levels of proinflammatory cytokines or chemokines in the blood or gastrointestinal tract, irritable bowel syndrome, inflammatory bowel syndrome, rectal inflammation, systemic infection, systemic inflammation, malnutrition, reduced weight gain, weight loss, increased feed conversion rate, reduced feed efficiency, hair loss, fecal inconsistency, or diarrhea relative to a comparable control animal lacking the gastrointestinal barrier dysfunction.

[0019] In some embodiments, the animal has an infection. In some embodiments, the gastrointestinal barrier dysfunction is associated with or caused by an infection. In some embodiments, the infection is a parasitic, bacterial, fungal, or viral infection. In some embodiments, the infection is a parasitic infection.

[0020] In some embodiments, the parasitic infection is coccidiosis infection. In some embodiments, the coccidiosis infection is Eimeria infection, Toxoplasma infection, Cryptosporidium infection, Isospora infection or Hammondia infection. In some embodiments, the coccidiosis infection is Eimeria infection. In some embodiments, the Eimeria infection is E.mivati ​​(E.mivati), E.tenella (E.tenella), E.acervulina (E.acervulina) or E.maxima (E.maxima) infection. In some embodiments, the coccidiosis infection is Toxoplasma infection. In some embodiments, the Toxoplasma infection is Toxoplasma gondii (Toxoplasmagondii). In some embodiments, the coccidiosis infection is Cryptosporidium infection. In some embodiments, the Cryptosporidium infection is a Cryptosporidium parvum, Cryptosporidium muris, or Cryptosporidium hominis infection. In some embodiments, the coccidiosis infection is an Isospora infection.

[0021] In some embodiments, the Isospora infection is an Isospora canis, Isospora ohioensis, Isospora burrosi, or Isospora felis infection. In some embodiments, the coccidiosis infection is an infection with the genus Hammondia. In some embodiments, the infection with the genus Hammondia is an infection with a species of Hammondia. In some embodiments, the coccidiosis infection is an infection with Eimeria acervulina, Eimeria maxima, Eimeria mitis, Eimeria tenella, Toxoplasma gondii, Hammondia species, Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Isospora canis, Isospora ohioensis, Isospora burrosi, or Isospora felis.

[0022] In some embodiments, the infection is a bacterial infection. In some embodiments, the bacterial infection is a Staphylococcus infection, a Shigella infection, a Campylobacter infection, a Salmonella infection, an Escherichia infection, or a Yersinia infection.

[0023] In some embodiments, the infection causes an increase in gastrointestinal inflammation, a decrease in the number of goblet cells in the gastrointestinal tract of the animal, a decrease in mucus secretion in the gastrointestinal tract of the animal, a decrease in the length of the villi of the gastrointestinal barrier of the animal, damage to the villi of the gastrointestinal barrier of the animal, an increase in the level of immune cells in the gastrointestinal tract of the animal, an increase in the level of CD8+ T cells in the gastrointestinal tract of the animal, an increase in the level of the liver protein APG (alpha glycoprotein), an increase in the level of circulating antibodies, an increase in the level of circulating IgA antibodies, or a decrease in the level of circulating diamine oxidase, or any combination thereof, relative to a comparable animal that does not have the infection.

[0024] In some embodiments, the animal has reduced inflammation of the gastrointestinal barrier relative to inflammation of the gastrointestinal barrier prior to the administration of the synthetic oligosaccharide formulation. In some embodiments, the animal has reduced inflammation of the gastrointestinal barrier relative to inflammation of the gastrointestinal barrier of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the inflammation is measured by an increase in the level of at least one anti-inflammatory cytokine. In some embodiments, the anti-inflammatory cytokine is IL1B, IL4, IL10, IL-6, IL-11, IL-13, IL1RA, or TGF-β. In some embodiments, the anti-inflammatory cytokine is IL1B, IL4, or IL10.

[0025] In some embodiments, the animal exhibits a decrease in CD8+ T cells, an increase in CD4+ T cells, an increase in circulating diamine oxidase levels, or a decrease in circulating antibody levels (e.g., IgA) relative to the animal prior to administering the synthetic oligosaccharide formulation. In some embodiments, the animal exhibits a decrease in CD8+ T cells, an increase in CD4+ T cells, an increase in circulating diamine oxidase levels, or a decrease in circulating antibody levels (e.g., IgA) relative to a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0026] In some embodiments, the animal exhibits increased nutrient absorption across the gastrointestinal barrier relative to the gastrointestinal barrier prior to administering the synthetic oligosaccharide formulation. In some embodiments, the animal exhibits increased nutrient absorption across the gastrointestinal barrier relative to the gastrointestinal barrier of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0027] In some embodiments, the nutrient absorption is measured by an increase in the level of at least one gastrointestinal protein associated with nutrient absorption. In some embodiments, the protein is SI (sucrase-isomaltase), SLC5A10, SLC34A1, SLC2A2, SLC34A2, SLC23A1, SLC23A2, SLC5A8, SLC16A3, SLC4, SLC4A9, SLC4A2, SLC4A3, NPC1L1, C6orf58, DDC, MCT1, MCT4, NaS1, DTDST, PAT1, DRA, CLD, SAT1, SUT2, SGLT1, GLUT2, B 0 AT1, ATB0+, SIT1, TAUT, EAAC1, ASCT2, SN1, SN2, PEPT1, SNAT2, GLYT1, y+LAT1, y+LAT2, CD36, LFABP, NP C1L1, ABCG5, ABCG8, SVCT1, SVCT2, SMVT, GIF, AMN, CUBL, MRP1, FOLT, PCFT, FOLR1, OAT10, RFVT1, RFVT2, THTR1, THTR2, VDR, DCYTB, DMT1, HCP1, FPN1, HEPH, HAMP, ZIP4, ZIP11, ZIP8, ZIP14A, ZIP14B, ZnT1, ZnT2, CTR1, SLC3A1, SLC1A4, ALPI, C17orf78, MUC17, DEFA5, RBP2, DEFA6, MLN, MEP1B, LCT, TM4SF20 or FABP6. In some embodiments, the protein is SI, SLC5A10 or SLC34A1.

[0028] In some embodiments, the animal has an increased body weight relative to the body weight of the animal before administration of the synthetic oligosaccharide formulation. In some embodiments, the body weight of the animal increases by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the body weight of the animal increases by about 1%-40%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 1%-4%, 1%-3% or 1%-2% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the increase in body weight is a greater increase relative to the increase in body weight in a comparable control animal that is administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the increase in body weight is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% greater than the increase in body weight in a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the increase in body weight is about 1%-40%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 1%-4%, 1%-3% or 1%-2% greater than the increase in body weight in a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation.

[0029] In some embodiments, the animal has increased feed efficiency relative to the feed efficiency of the animal before administration of the synthetic oligosaccharide formulation. In some embodiments, the feed efficiency of the animal is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% relative to the feed efficiency of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the feed efficiency of the animal is increased by about 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-4%, 1%-3% or 1%-2% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the increase in feed efficiency is a greater increase relative to a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the increase in feed efficiency is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% greater than the increase in feed efficiency in the comparable control animals administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the increase in feed efficiency is about 1%-40%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% greater than the increase in feed efficiency in the comparable control animals administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation.

[0030] In some embodiments, the animal has a reduced feed conversion ratio (FCR) relative to the animal's feed conversion ratio (FCR) before administration of the synthetic oligosaccharide formulation. In some embodiments, the feed conversion ratio of the animal is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% relative to the animal's feed conversion ratio before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the feed conversion ratio (FCR) of the animal is reduced by about 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-4%, 1%-3% or 1%-2% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the reduction in feed conversion ratio of the animal is a greater reduction relative to a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the reduction in feed conversion ratio is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% greater relative to the reduction in feed conversion ratio in the comparable control animals administered the comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the reduction in feed conversion ratio is greater than about 1%-40%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% relative to the reduction in feed conversion ratio in the comparable control animals administered the comparable nutritional composition lacking the synthetic oligosaccharide formulation.

[0031] In some embodiments, the life expectancy or survival rate of the animal is increased relative to a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

[0032] In some embodiments, the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal in an amount sufficient to treat or prevent the gastrointestinal barrier dysfunction.

[0033] In some embodiments, the nutritional composition comprising the synthetic oligosaccharide formulation is administered to the animal for at least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days. In some embodiments, the nutritional composition comprising the synthetic oligosaccharide formulation is administered to the animal at least once, twice, three times, four times, or five times per day. In some embodiments, the administering comprises providing the nutritional composition comprising the synthetic oligosaccharide formulation to the animal for ad libitum ingestion. In some embodiments, the animal ingests at least a portion of the nutritional composition comprising the synthetic oligosaccharide formulation over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 90, or 120 twenty-four hour periods.

[0034] In some embodiments, the nutritional composition comprises at least 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm, 700ppm, 800ppm, 900ppm, 1000ppm, 1500ppm or 2000ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm, 700ppm, 800ppm, 900ppm, 1000ppm, 1500ppm or 2000ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 500ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 100 ppm-2000 ppm, 100 ppm-1500 ppm, 100 ppm-1000 ppm, 100 ppm-900 ppm, 100 ppm-800 ppm, 100 ppm-700 ppm, 100 ppm-600 ppm, 100 ppm-500 ppm, 100 ppm-400 ppm, 100 ppm-300 ppm, 100 ppm-200 ppm, 200 ppm-1000 ppm, 200 ppm-800 ppm, 200 ppm-700 ppm, 200 ppm-600 ppm, 200 ppm-500 ppm, 300 ppm-1000 ppm, 300 ppm-700 ppm, 300 ppm-600 ppm, or 300 ppm-500 ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 300 ppm-600 ppm of the synthetic oligosaccharide preparation.

[0035] In some embodiments, the animal is poultry, seafood, sheep, cow, cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, shrimp, or bird.

[0036] In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken, turkey, duck, or goose. In some embodiments, the chicken is a broiler, laying hen, or breeder.

[0037] In some embodiments, the animal is a pig. In some embodiments, the pig is a nursery pig, a growing pig, or a finishing pig.

[0038] In some embodiments, the animal is a fish. In some embodiments, the fish is salmon, tilapia, or tropical fish.

[0039] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0040] In some embodiments, the relative abundance is determined by LC-MS / MS.

[0041] In some embodiments, the relative abundance of oligosaccharides in at least 5, 10, 20 or 30 DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with its degree of polymerization.

[0042] In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0043] In some embodiments, the DP2 fraction comprises less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharides by relative abundance.

[0044] In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 5% to about 10%. In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 1% to about 10%. In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 0.5% to about 10%. In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 2% to about 12%.

[0045] In some embodiments, the DP1 fraction comprises less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 2% to about 12% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 1% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 0.5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance.

[0046] In some embodiments, the DP3 fraction comprises less than 15%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 2% to about 12% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 1% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 0.5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance.

[0047] In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 2% to about 12% by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 0.5% to about 10% by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 1% to about 10% by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 5% to about 10% by relative abundance.

[0048] In some embodiments, the DP2 fraction comprises greater than 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11% or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0049] In some embodiments, the DP1 fraction comprises greater than 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0050] In some embodiments, the DP3 fraction comprises greater than 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0051] In some embodiments, the oligosaccharide preparation comprises greater than 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11% or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0052] In some embodiments, the oligosaccharide preparation has a DP1 fraction content of about 1% to about 40% by weight as determined by liquid chromatography.

[0053] In some embodiments, the oligosaccharide preparation has a DP2 fraction content of about 1% to about 35% by weight as determined by liquid chromatography.

[0054] In some embodiments, the oligosaccharide preparation has a DP3 fraction content of about 1% to about 30% by weight as determined by liquid chromatography.

[0055] In some embodiments, the oligosaccharide preparation has a DP4 fraction content of about 0.1% to about 20% by weight as determined by liquid chromatography.

[0056] In some embodiments, the oligosaccharide preparation has a DP5 fraction content of about 0.1% to about 15% by weight as determined by liquid chromatography.

[0057] In some embodiments, the ratio of the DP2 fraction to the DP1 fraction is about 0.02 to about 0.40 as determined by liquid chromatography.

[0058] In some embodiments, the ratio of the DP3 fraction to the DP2 fraction is about 0.01 to about 0.30 as determined by liquid chromatography.

[0059] In some embodiments, the combined content of DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, less than 40%, or less than 30%, as determined by liquid chromatography.

[0060] In some embodiments, the oligosaccharide preparation comprises at least 10<3>, at least 10<4>, at least 10<5>, at least 10<6>, or at least 10<9> different oligosaccharide species.

[0061] In some embodiments, two or more separate oligosaccharides comprise different anhydro subunits.

[0062] In some embodiments, each of the anhydrosubunit-containing oligosaccharides comprises one or more anhydrosubunits that are thermal dehydration products of monosaccharides.

[0063] In some embodiments, the oligosaccharide preparation comprises one or more anhydrosubunits selected from the group consisting of anhydroglucose, anhydrogalactose, anhydromannose, anhydroallose, anhydroaltrose, anhydrogulose, anhydroidose, anhydrotalose, anhydrofructose, anhydroribose, anhydroarabinose, anhydrorhamnose, anhydrolyxose, and anhydroxylose.

[0064] In some embodiments, the oligosaccharide preparation comprises one or more anhydroglucose, anhydrogalactose, anhydromannose, or anhydrofructose subunits.

[0065] In some embodiments, the DP1 fraction comprises 1,6-anhydro-β-D-glucopyranose or 1,6-anhydro-β-D-glucopyranose anhydrosubunits. In some embodiments, the DP1 fraction comprises both 1,6-anhydro-β-D-glucopyranose and 1,6-anhydro-β-D-glucopyranose anhydrosubunits.

[0066] In some embodiments, in the oligosaccharide preparation, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose is about 10:1 to 1:10, about 9:1 to about 1:10, about 8:1 to about 1:10, about 7:1 to about 1:10, about 6:1 to about 1:10, about 5:1 to about 1:10, about 4:1 to about 1:10, about 3:1 to about 1:10, about 2:1 to about 1:10, about 10:1 to about 1:9, about 10:1 to about 1:8, about 10:1 to about 1:7, about 10:1 to about 1:6, about 10:1 to about 1:5, about 10:1 to about 1:4, about 10:1 to about 1:3, about 10:1 to about 1:2, or about 1:1 to about 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in the oligosaccharide preparation is about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1: 10. In some embodiments, the ratio of 1,6-anhydro-β-D-furanose to 1,6-anhydro-β-D-glucopyranose in the oligosaccharide preparation is about 2: 1.

[0067] In some embodiments, the DP2 fraction comprises at least 5 species of anhydrosubunit-containing oligosaccharides. In some embodiments, the DP2 fraction comprises about 5 to 10 species of anhydrosubunit-containing oligosaccharides.

[0068] In some embodiments, the oligosaccharide preparation comprises one or more sugar caramelization products. In some embodiments, the sugar caramelization products are selected from the group consisting of: methanol; ethanol; furan; methylglyoxal; 2-methylfuran; vinyl acetate; glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol; 3-furanmethanol; 2-hydroxycyclopent-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosone; cyclic hydroxylactone; 1,4,3,6-dianhydro-α-D-pyranose; dianhydropyranose; and 5-hydroxymethylfurfural (5-hmf).

[0069] In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the anhydrosubunit-containing oligosaccharides comprise chain-end anhydrosubunits.

[0070] In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 300 to about 5000g / mol as measured by high performance liquid chromatography (HPLC). In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 300 to about 2500g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 500 to about 2000g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 500 to about 1500g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 300 to about 5000g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 300 to about 2500g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 500 to about 2000g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 500 to about 1500 g / mol as determined by HPLC.

[0071] In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 2000 to about 2800 g / mol. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 1000 to about 2000 g / mol.

[0072] In one aspect, provided herein is a method for treating or preventing an infection in an animal in need thereof, the method comprising: administering to the animal a nutritional composition comprising a base nutritional composition and a synthetic oligosaccharide preparation, each having at least n oligosaccharide fractions (DP1 to DPn fractions) of different degrees of polymerization selected from 1 to n, wherein n is an integer greater than 3; and wherein each of the DP1 and DP2 fractions independently comprises from about 0.5% to about 15% anhydrosubunit-containing oligosaccharides by relative abundance as determined by mass spectrometry, thereby treating or preventing the infection.

[0073] In some embodiments, the level of at least one immune cell in a sample from the animal is increased relative to the level of at least one immune cell in a sample from the animal before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the increase is an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% relative to the level of at least one immune cell before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the increase is an increase of about 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% relative to the level of at least one immune cell before administration of the nutritional composition comprising the synthetic oligosaccharide formulation.

[0074] In some embodiments, the level of at least one immune cell in a sample from the animal is increased relative to the level of at least one immune cell in a sample from a comparable control animal that has been administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the increase is an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% relative to the level of at least one immune cell in a sample from the comparable control animal that has been administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the increase is an increase of about 1%-40%, 1%-35%, 1%-30%, 1%-25%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-4%, 1%-3%, or 1%-2% relative to the level of at least one immune cell in a sample from the comparable control animal that has been administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation.

[0075] In some embodiments, the at least one immune cell is a neutrophil, an eosinophil, a basophil, a mast cell, a T cell, a B cell, a macrophage, a monocyte, a granulocyte, or a dendritic cell. In some embodiments, the immune cell is a phagocyte. In some embodiments, the phagocyte is a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, a monocyte, or a dendritic cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a granulocyte.

[0076] In some embodiments, the level of at least one proinflammatory cytokine in a sample from the animal is increased relative to the level of at least one proinflammatory cytokine in a sample from the animal before administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the level of at least one proinflammatory cytokine in a sample from the animal is increased relative to the level of at least one proinflammatory cytokine in a sample from a comparable control animal that has been administered a comparable nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the at least one proinflammatory cytokine is IL1, IL-12, IL18, TNFA, IFNG, GM-CSF, IL-1β, IL6, RANTES, MCP1, IL8, MIP-1α, MIP-1β, lymphotactin, fractal chemokine, or GRO / KC.

[0077] In some embodiments, the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal prior to said administering of the synthetic oligosaccharide preparation. In some embodiments, the reduction is at least about a 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% reduction relative to the permeability of the gastrointestinal barrier of the animal prior to said administration of the synthetic oligosaccharide formulation; or the reduction is about a 0.5%-30%, 0.5%-20%, 0.5%-10%, 0.5%-5%, 0.5%-4%, 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 10%-4%, 1%-3%, or 1%-2% reduction relative to the permeability of the gastrointestinal barrier of the animal prior to said administration of the synthetic oligosaccharide formulation. In some embodiments, the reduction is a greater reduction than the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the reduction is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or 60% greater than the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0078] In some embodiments, the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal before the administration of the synthetic oligosaccharide formulation. In some embodiments, the reduction is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% relative to the permeability of the gastrointestinal barrier of the animal before the administration of the synthetic oligosaccharide formulation. In some embodiments, the reduction is a reduction of about 0.5%-30%, 0.5%-20%, 0.5%-10%, 0.5%-5%, 0.5%-4%, 0.5%-3%, 0.5%-2%, 0.5%-1%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 10%-4%, 1%-3%, or 1%-2% relative to the permeability of the gastrointestinal barrier of the animal prior to the administration of the synthetic oligosaccharide formulation.

[0079] In some embodiments, the gastrointestinal barrier dysfunction is associated with or caused by an infection. In some embodiments, the infection is a parasitic, bacterial, fungal or viral infection.

[0080] In some embodiments, the infection is a parasitic infection. In some embodiments, the parasitic infection is a coccidiosis infection. In some embodiments, the coccidiosis infection is an Eimeria infection, a Toxoplasma infection, a Cryptosporidium infection, an Isospora infection, or a Hammondella infection. In some embodiments, the coccidiosis infection is an Eimeria infection. In some embodiments, the Eimeria infection is an Eimeria desmodus, an Eimeria tenella, an Eimeria acervulina, or an Eimeria maxima infection. In some embodiments, the coccidiosis infection is a Toxoplasma infection. In some embodiments, the Toxoplasma infection is Toxoplasma gondii. In some embodiments, the coccidiosis infection is a Cryptosporidium infection. In some embodiments, the Cryptosporidium infection is an Cryptosporidium parvum, a Cryptosporidium muris, or a Cryptosporidium hominis infection. In some embodiments, the coccidiosis infection is an Isospora infection. In some embodiments, the Isospora infection is an Isospora canis, Isospora ohioensis, Isospora brorosi, or Isospora felis infection. In some embodiments, the coccidiosis infection is an infection with the genus Hammondella. In some embodiments, the infection with the genus Hammondella is an infection with a species of Hammondella. In some embodiments, the coccidiosis infection is an infection with Eimeria acervulina, Eimeria maxima, Eimeria multocida, Eimeria tenella, Toxoplasma gondii, a species of Hammondella, Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Isospora canis, Isospora ohioensis, Isospora brorosi, or Isospora felis.

[0081] In some embodiments, the infection is a bacterial infection. In some embodiments, the bacterial infection is a Staphylococcus infection, a Shigella infection, a Campylobacter infection, a Salmonella infection, an Escherichia infection, or a Yersinia infection.

[0082] In some embodiments, the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal in an amount sufficient to treat or prevent the infection. In some embodiments, the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal for at least 1, 7, 10, 14, 30, 45, 60, 90 or 120 days. In some embodiments, the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal at least once, twice, three times, four times or five times a day. In some embodiments, the administration comprises providing the nutritional composition comprising the synthetic oligosaccharide preparation to the animal for ad libitum intake. In some embodiments, the animal ingests at least a portion of the nutritional composition comprising the synthetic oligosaccharide preparation over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 90 or 120 twenty-four hour periods.

[0083] In some embodiments, the nutritional composition comprises at least 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm, 700ppm, 800ppm, 900ppm, 1000ppm, 1500ppm or 2000ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 100ppm, 200ppm, 300ppm, 400ppm, 500ppm, 600ppm, 700ppm, 800ppm, 900ppm, 1000ppm, 1500ppm or 2000ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 500ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 100 ppm-2000 ppm, 100 ppm-1500 ppm, 100 ppm-1000 ppm, 100 ppm-900 ppm, 100 ppm-800 ppm, 100 ppm-700 ppm, 100 ppm-600 ppm, 100 ppm-500 ppm, 100 ppm-400 ppm, 100 ppm-300 ppm, 100 ppm-200 ppm, 200 ppm-1000 ppm, 200 ppm-800 ppm, 200 ppm-700 ppm, 200 ppm-600 ppm, 200 ppm-500 ppm, 300 ppm-1000 ppm, 300 ppm-700 ppm, 300 ppm-600 ppm, or 300 ppm-500 ppm of the synthetic oligosaccharide preparation. In some embodiments, the nutritional composition comprises about 300 ppm-600 ppm of the synthetic oligosaccharide preparation.

[0084] In some embodiments, the animal is poultry, seafood, sheep, cow, cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, shrimp, or bird.

[0085] In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken, turkey, duck, or goose. In some embodiments, the chicken is a broiler, laying hen, or breeder.

[0086] In some embodiments, the animal is a pig. In some embodiments, the pig is a nursery pig, a growing pig, or a finishing pig.

[0087] In some embodiments, the animal is a fish. In some embodiments, the fish is salmon, tilapia, or tropical fish.

[0088] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0089] In some embodiments, the relative abundance is determined by LC-MS / MS.

[0090] In some embodiments, the relative abundance of oligosaccharides in at least 5, 10, 20 or 30 DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with its degree of polymerization.

[0091] In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0092] In some embodiments, the DP2 fraction comprises less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharides by relative abundance.

[0093] In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 5% to about 10%. In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 1% to about 10%. In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 0.5% to about 10%. In some embodiments, the DP2 fraction comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 2% to about 12%.

[0094] In some embodiments, the DP1 fraction comprises less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 2% to about 12% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 1% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 0.5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction comprises about 5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance.

[0095] In some embodiments, the DP3 fraction comprises less than 15%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 2% to about 12% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 1% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 0.5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction comprises about 5% to about 10% anhydrosubunit-containing oligosaccharides by relative abundance.

[0096] In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 2% to about 12% by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 0.5% to about 10% by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 1% to about 10% by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharides in an amount of about 5% to about 10% by relative abundance.

[0097] In some embodiments, the DP2 fraction comprises greater than 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11% or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0098] In some embodiments, the DP1 fraction comprises greater than 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0099] In some embodiments, the DP3 fraction comprises greater than 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0100] In some embodiments, the oligosaccharide preparation comprises greater than 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11% or greater than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

[0101] In some embodiments, the oligosaccharide preparation has a DP1 fraction content of about 1% to about 40% by weight as determined by liquid chromatography.

[0102] In some embodiments, the oligosaccharide preparation has a DP2 fraction content of about 1% to about 35% by weight as determined by liquid chromatography.

[0103] In some embodiments, the oligosaccharide preparation has a DP3 fraction content of about 1% to about 30% by weight as determined by liquid chromatography.

[0104] In some embodiments, the oligosaccharide preparation has a DP4 fraction content of about 0.1% to about 20% by weight as determined by liquid chromatography.

[0105] In some embodiments, the oligosaccharide preparation has a DP5 fraction content of about 0.1% to about 15% by weight as determined by liquid chromatography.

[0106] In some embodiments, the ratio of the DP2 fraction to the DP1 fraction is about 0.02 to about 0.40 as determined by liquid chromatography.

[0107] In some embodiments, the ratio of the DP3 fraction to the DP2 fraction is about 0.01 to about 0.30 as determined by liquid chromatography.

[0108] In some embodiments, the combined content of DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, less than 40%, or less than 30%, as determined by liquid chromatography.

[0109] In some embodiments, the oligosaccharide preparation comprises at least 10<3>, at least 10<4>, at least 10<5>, at least 10<6>, or at least 10<9> different oligosaccharide species.

[0110] In some embodiments, two or more separate oligosaccharides comprise different anhydro subunits.

[0111] In some embodiments, each of the anhydrosubunit-containing oligosaccharides comprises one or more anhydrosubunits that are thermal dehydration products of monosaccharides.

[0112] In some embodiments, the oligosaccharide preparation comprises one or more anhydrosubunits selected from the group consisting of anhydroglucose, anhydrogalactose, anhydromannose, anhydroallose, anhydroaltrose, anhydrogulose, anhydroidose, anhydrotalose, anhydrofructose, anhydroribose, anhydroarabinose, anhydrorhamnose, anhydrolyxose, and anhydroxylose.

[0113] In some embodiments, the oligosaccharide preparation comprises one or more anhydroglucose, anhydrogalactose, anhydromannose, or anhydrofructose subunits.

[0114] In some embodiments, the DP1 fraction comprises 1,6-anhydro-β-D-glucopyranose or 1,6-anhydro-β-D-glucopyranose anhydrosubunits. In some embodiments, the DP1 fraction comprises both 1,6-anhydro-β-D-glucopyranose and 1,6-anhydro-β-D-glucopyranose anhydrosubunits.

[0115] In some embodiments, in the oligosaccharide preparation, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose is about 10:1 to 1:10, about 9:1 to about 1:10, about 8:1 to about 1:10, about 7:1 to about 1:10, about 6:1 to about 1:10, about 5:1 to about 1:10, about 4:1 to about 1:10, about 3:1 to about 1:10, about 2:1 to about 1:10, about 10:1 to about 1:9, about 10:1 to about 1:8, about 10:1 to about 1:7, about 10:1 to about 1:6, about 10:1 to about 1:5, about 10:1 to about 1:4, about 10:1 to about 1:3, about 10:1 to about 1:2, or about 1:1 to about 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in the oligosaccharide preparation is about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1: 10. In some embodiments, the ratio of 1,6-anhydro-β-D-furanose to 1,6-anhydro-β-D-glucopyranose in the oligosaccharide preparation is about 2: 1.

[0116] In some embodiments, the DP2 fraction comprises at least 5 species of anhydrosubunit-containing oligosaccharides. In some embodiments, the DP2 fraction comprises about 5 to 10 species of anhydrosubunit-containing oligosaccharides.

[0117] In some embodiments, the oligosaccharide preparation comprises one or more sugar caramelization products. In some embodiments, the sugar caramelization products are selected from the group consisting of: methanol; ethanol; furan; methylglyoxal; 2-methylfuran; vinyl acetate; glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol; 3-furanmethanol; 2-hydroxycyclopent-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosone; cyclic hydroxylactone; 1,4,3,6-dianhydro-α-D-pyranose; dianhydropyranose; and 5-hydroxymethylfurfural (5-hmf).

[0118] In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the anhydrosubunit-containing oligosaccharides comprise chain-end anhydrosubunits.

[0119] In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 300 to about 5000g / mol as measured by high performance liquid chromatography (HPLC). In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 300 to about 2500g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 500 to about 2000g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 500 to about 1500g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 300 to about 5000g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 300 to about 2500g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 500 to about 2000g / mol as measured by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 500 to about 1500 g / mol as determined by HPLC.

[0120] In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 2000 to about 2800 g / mol. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 1000 to about 2000 g / mol.

[0121] The present disclosure is based, at least in part, on the discovery that oligosaccharides comprising one or more anhydro subunits reduce the permeability of the gastrointestinal barrier. Thus, the present disclosure relates, inter alia, to methods of preventing gastrointestinal dysfunction in animals comprising administering the oligosaccharide formulations described herein.

[0122] In some embodiments, the relative abundance of oligosaccharides in at least 5, 10, 20, or 30 DP fractions of the oligosaccharide preparation decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions of the oligosaccharide preparation decreases monotonically with their degree of polymerization. In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0123] In some embodiments, at least one fraction of the oligosaccharide preparation comprises anhydro subunit-containing oligosaccharides in a relative abundance of less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2%. In some embodiments, the oligosaccharide preparation comprises anhydro subunit-containing oligosaccharides in a relative abundance of less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2%. In some embodiments, each fraction of the oligosaccharide preparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydro subunit oligosaccharide. In some embodiments, at least one fraction of the oligosaccharide preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydro subunit oligosaccharide. In some embodiments, the oligosaccharide preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydro subunit oligosaccharide. In some embodiments, each fraction of the oligosaccharide preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydro subunit-containing oligosaccharides by relative abundance. In some embodiments, at least one fraction of the oligosaccharides comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70% or 80% anhydro subunit-containing oligosaccharides by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydro subunit oligosaccharides in an amount greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the oligosaccharides. In some embodiments, each fraction of the oligosaccharide preparation comprises anhydro subunit oligosaccharides in an amount greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the oligosaccharides.In some embodiments, at least one fraction of the oligosaccharide preparation comprises anhydro subunit oligosaccharides greater than 20%, 21%, 22%, 23%, 24% or 25% by relative abundance. In some embodiments, the oligosaccharide preparation comprises anhydro subunit oligosaccharides greater than 20%, 21%, 22%, 23%, 24% or 25% by relative abundance. In some embodiments, each fraction of the oligosaccharide preparation comprises anhydro subunit oligosaccharides greater than 20%, 21%, 22%, 23%, 24% or 25% by relative abundance. In some embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the anhydro subunit oligosaccharides have only one anhydro subunit.

[0124] In some embodiments, the oligosaccharide preparation has a DP1 fraction content of 1% to 40% by relative abundance. In some embodiments, the oligosaccharide preparation has a DP2 fraction content of 1% to 35% by relative abundance. In some embodiments, the oligosaccharide preparation has a DP3 fraction content of 1% to 30% by relative abundance. In some embodiments, the oligosaccharide preparation has a DP4 fraction content of 0.1% to 20% by relative abundance. In some embodiments, the oligosaccharide preparation comprises a DP5 fraction content of 0.1% to 15% by relative abundance. In some embodiments, the oligosaccharide preparation comprises a DP2 fraction and a DP1 fraction, wherein the ratio of the DP2 fraction to the DP1 fraction is 0.02-0.40 by relative abundance. In some embodiments, the oligosaccharide preparation comprises a DP3 fraction and a DP2 fraction, wherein the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation is 0.01-0.30 by relative abundance. In some embodiments, the oligosaccharide preparation comprises a DP1 fraction and a DP2 fraction, wherein the combined content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, 30%, or 10% by relative abundance. In some embodiments, the oligosaccharide preparation comprises at least 25, 50, 75, 100, 103, 104, 105, 106, 109, 110, 120, 150, or 200 different oligosaccharide species.

[0125] In some embodiments, at least two independent oligosaccharides of the oligosaccharide preparation comprise different dehydrated subunits. In some embodiments, the oligosaccharide preparation comprises at least one dehydrated subunit that is a reversible thermal dehydration product of a monosaccharide. In some embodiments, the oligosaccharide preparation comprises at least one dehydrated glucose, dehydrated galactose, dehydrated mannose, dehydrated allose, dehydrated altrose, dehydrated gulose, dehydrated idose, dehydrated talose, dehydrated fructose, dehydrated ribose, dehydrated arabinose, dehydrated rhamnose, dehydrated lyxose or dehydrated xylose subunit. In some embodiments, the oligosaccharide preparation comprises at least one dehydrated glucose, dehydrated galactose, dehydrated mannose or dehydrated fructose subunit.

[0126] In some embodiments, the oligosaccharide preparation comprises at least one 1,6-anhydro-β-D-glucopyranose or 1,6-anhydro-β-D-glucopyranose subunit. In some embodiments, the oligosaccharide preparation comprises at least one 1,6-anhydro-β-D-glucopyranose subunit and at least one 1,6-anhydro-β-D-glucopyranose anhydrosubunit. In some embodiments, in the oligosaccharide preparation, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in the oligosaccharide preparation is about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 8, 1: 9, or 1: 10. In some embodiments, the ratio of 1,6-anhydro-β-D-furanose to 1,6-anhydro-β-D-glucopyranose in the oligosaccharide preparation is about 2: 1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in each fraction is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in each fraction of the oligosaccharide preparation is about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 8, 1: 9, or 1: 10. In some embodiments, the ratio of 1,6-anhydro-β-D-furanose to 1,6-anhydro-β-D-glucopyranose in each fraction of the oligosaccharide preparation is about 2: 1.In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the anhydro subunits in the oligosaccharide preparation are selected from 1,6-anhydro-β-D-glucopyranose and 1,6-anhydro-β-D-glucopyranose.

[0127] In some embodiments, the oligosaccharide preparation comprises at least one anhydrous subunit as a sugar caramelization product. In some embodiments, the sugar caramelization product is selected from the group consisting of: methanol; ethanol; furan; methylglyoxal; 2-methylfuran; vinyl acetate; glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol; 3-furanmethanol; 2-hydroxycyclopent-2-ene-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levuloglucosone; cyclic hydroxylactone; 1,4,3,6-dianhydro-α-D-pyranose; dianhydropyranose; and 5-hydroxymethylfurfural (5-hmf). In some embodiments, about 0.1% to 5%, 0.1% to 2%, or 0.1% to 1% of the anhydrous subunits in the oligosaccharide preparation are caramelization products.

[0128] In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the anhydrosubunit-containing oligosaccharides in the oligosaccharide preparation comprise chain-end anhydrosubunits.

[0129] In some embodiments, the weight average molecular weight of the oligosaccharide preparation is about 300 to 5000 g / mol, 500 to 5000 g / mol, 700 to 5000 g / mol, 500 to 2000 g / mol, 700 to 2000 g / mol, 700 to 1500 g / mol, 300 to 1500 g / mol, 300 to 2000 g / mol, 400 to 1300 g / mol, 400 to 1200 g / mol, 400 to 1100 g / mol, 500 to 1300 g / mol, 500 to 1200 g / mol, 500 to 1100 g / mol, 600 to 1300 g / mol, 600 to 1200 g / mol, or 600 to 1100 g / mol. In some embodiments, the number average molecular weight of the oligosaccharide preparation is about 300 to 5000 g / mol, 500 to 5000 g / mol, 700 to 5000 g / mol, 500 to 2000 g / mol, 700 to 2000 g / mol, 700 to 1500 g / mol, 300 to 1500 g / mol, 300 to 2000 g / mol, 400 to 1000 g / mol, 400 to 900 g / mol, 400 to 800 g / mol, 500 to 900 g / mol, or 500 to 800 g / mol. In some embodiments, the weight average molecular weight of the oligosaccharide preparation is about 2000 to 2800 g / mol, 2100 to 2700 g / mol, 2200 to 2600 g / mol, 2300 to 2500 g / mol, or 2320 to 2420 g / mol. In some embodiments, the number average molecular weight of the oligosaccharide preparation is about 1000 to 2000 g / mol, 1100 to 1900 g / mol, 1200 to 1800 g / mol, 1300 to 1700 g / mol, 1400 to 1600 g / mol, or 1450 to 1550 g / mol.

[0130] In one aspect, provided herein is a method for preventing gastrointestinal barrier dysfunction in an animal, the method comprising: administering to the animal a nutritional composition comprising a basal nutritional composition and a synthetic oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 2; wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides in relative abundance as measured by mass spectrometry.

[0131] In some embodiments, the gastrointestinal barrier is less permeable than the gastrointestinal barrier of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation. In some embodiments, the gastrointestinal barrier dysfunction is hyperpermeability (leaky gut).

[0132] In some embodiments, the permeability of the gastrointestinal barrier is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% less than the permeability of the gastrointestinal barrier of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation. In some embodiments, the permeability of the gastrointestinal barrier is about 0.5%-40%, 0.5%-30%, 0.5%-20%, 0.5%-10%, 0.5%-5%, 0.5%-1%, 1%-40%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 1%-2%, 5%-40%, 5%-30%, 5%-20%, 5%-10%, 10%-40%, 10%-30%, or 10%-20% less than the permeability of the gastrointestinal barrier of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

[0133] In some embodiments, the reduction in gastrointestinal permeability is directly mediated by the synthetic oligosaccharide preparation. In some embodiments, the reduction in gastrointestinal permeability is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the oligosaccharide preparation is processed in vivo into one or more secondary substances. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the gastrointestinal microbiota of the animal. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the reduction in gastrointestinal permeability is directly mediated by one or more of the secondary substances.

[0134] In some embodiments, the animal has a reduced likelihood of developing gastrointestinal barrier dysfunction relative to an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

[0135] In some embodiments, the animal has a higher body weight relative to the body weight of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the animal has a higher feed efficiency relative to the feed efficiency of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the animal has a lower feed conversion ratio relative to the feed conversion ratio of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0136] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cows, cattle, buffalo, bison, pigs (e.g., nursery pigs, growing / finishing pigs), cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigeons, ferrets, gerbils, hamsters, mice, rats, birds, or humans. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler chickens, laying hens, breeders), turkeys, ducks, or geese. In some embodiments, the animal is a pig (e.g., nursery pigs, growing / finishing pigs).

[0137] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0138] Provided herein are methods of promoting goblet cell proliferation in an animal, the method comprising: administering to the animal a nutritional composition comprising a base nutritional composition and an oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions) each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 2; wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides by relative abundance as measured by mass spectrometry.

[0139] In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is greater relative to the level of goblet cells in the gastrointestinal tract of an animal that was administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% greater than the level of goblet cells in the gastrointestinal tract of an animal that was administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is greater by about 0.5%-20%, 0.5%-15%, 0.5%-10%, 0.5%-5%, 0.5%-1%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-2%, 5%-20%, 5%-15%, or 5%-10% relative to the level of goblet cells in a nutritional composition lacking the synthetic oligosaccharide preparation.

[0140] In some embodiments, the increase in goblet cell levels is directly mediated by the synthetic oligosaccharide preparation. In some embodiments, the increase in goblet cell levels is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the oligosaccharide preparation is processed in vivo into one or more secondary substances. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the gastrointestinal microbiota of the animal. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the increase in goblet cell number is directly mediated by one or more of the secondary substances.

[0141] In some embodiments, the animal has a higher body weight relative to the body weight of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the animal has a higher feed efficiency relative to the feed efficiency of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the animal has a lower feed conversion ratio relative to the feed conversion ratio of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0142] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cows, cattle, buffalo, bison, pigs (e.g., nursery pigs, growing / finishing pigs), cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigeons, ferrets, gerbils, hamsters, mice, rats, birds, or humans. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler chickens, laying hens, breeders), turkeys, ducks, or geese. In some embodiments, the animal is a pig (e.g., nursery pigs, growing / finishing pigs). In some embodiments, the animal is livestock. In some embodiments, the animal is a companion animal.

[0143] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0144] Provided herein are methods of promoting mucus production in the gastrointestinal tract of an animal, the method comprising: administering to the animal a nutritional composition comprising a base nutritional composition and an oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 2; wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides by relative abundance as measured by mass spectrometry.

[0145] In some embodiments, the level of mucus in the gastrointestinal tract of the animal is greater relative to an animal that was administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the level of mucus is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% greater relative to the level of mucus in the gastrointestinal tract of an animal that was administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the level of mucus is at least about 0.5%-20%, 0.5%-15%, 0.5%-10%, 0.5%-5%, 0.5%-2%, 0.5%-1%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-2%, 5%-20%, 5%-15%, or 5%-10% greater relative to the level of mucus in the gastrointestinal tract of an animal that was administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0146] In some embodiments, the method comprises obtaining a gastrointestinal sample from the animal. In some embodiments, the sample is a gastrointestinal tissue (e.g., a cecal biopsy) or a fecal sample. In some embodiments, the method comprises detecting the level of mucus in the sample. In some embodiments, the level of mucus is detected by detecting a protein component of the mucus. In some embodiments, the protein is mucin.

[0147] In some embodiments, the increase in mucus levels is directly mediated by the synthetic oligosaccharide preparation. In some embodiments, the increase in mucus levels is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the oligosaccharide preparation is processed in vivo into one or more secondary substances. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the gastrointestinal microbiota of the animal. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the increase in mucus levels is directly mediated by one or more of the secondary substances.

[0148] In some embodiments, the animal has a higher body weight relative to the body weight of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the animal has a higher feed efficiency relative to the feed efficiency of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the animal has a lower feed conversion ratio relative to the feed conversion ratio of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0149] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cows, cattle, buffalo, bison, pigs (e.g., nursery pigs, growing / finishing pigs), cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigeons, ferrets, gerbils, hamsters, mice, rats, birds, or humans. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler chickens, laying hens, breeders), turkeys, ducks, or geese. In some embodiments, the animal is a pig (e.g., nursery pigs, growing / finishing pigs). In some embodiments, the animal is livestock. In some embodiments, the animal is a companion animal.

[0150] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0151] Provided herein are methods for preventing infection in an animal, the method comprising: administering to the animal a nutritional composition comprising a base nutritional composition and a synthetic oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 2; wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides by relative abundance as measured by mass spectrometry.

[0152] In some embodiments, the levels of one or more types of immune cells are greater in the gastrointestinal tract of the animal relative to the levels in the gastrointestinal tract of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

[0153] In some embodiments, the immune cells are phagocytes. In some embodiments, the immune cells are granulocytes. In some embodiments, the level of granulocyte phagocytosis in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the level of immune cells or immune cell activity is directly mediated by the synthetic oligosaccharide formulation.

[0154] In some embodiments, the level of immune cells or immune cell activity is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation is processed into one or more secondary substances in vivo. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the gastrointestinal microbiome of the animal. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the level of immune cells or immune cell activity is directly mediated by one or more of the secondary substances.

[0155] In some embodiments, the level of one or more pro-inflammatory cytokines or chemokines in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of an animal that was administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the level of one or more anti-inflammatory cytokines or chemokines in the gastrointestinal tract of the animal is lower relative to the level in the gastrointestinal tract of an animal that was administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0156] In some embodiments, the gastrointestinal barrier of the animal is less permeable relative to the permeability of the gastrointestinal barrier of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

[0157] In some embodiments, the level of mucus in the gastrointestinal tract of the animal is greater relative to the level in the gastrointestinal tract of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

[0158] In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is greater relative to the level in the gastrointestinal tract of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

[0159] In some embodiments, the animal has a higher body weight relative to the body weight of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation. In some embodiments, the animal has a higher feed efficiency relative to the feed efficiency of an animal administered a nutritional composition lacking the synthetic oligosaccharide formulation.

[0160] In some embodiments, the animal has a lower feed conversion ratio relative to the feed conversion ratio of an animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

[0161] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cows, cattle, buffalo, bison, pigs (e.g., nursery pigs, growing / finishing pigs), cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigeons, ferrets, gerbils, hamsters, mice, rats, birds, or humans. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler chickens, laying hens, breeders), turkeys, ducks, or geese. In some embodiments, the animal is a pig (e.g., nursery pigs, growing / finishing pigs). In some embodiments, the animal is livestock. In some embodiments, the animal is a companion animal.

[0162] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0163] In one aspect, the present disclosure is based, at least in part, on the discovery that oligosaccharides comprising one or more anhydro subunits reduce the permeability of the gastrointestinal barrier. Thus, in one aspect, the present disclosure relates, inter alia, to a method for treating gastrointestinal dysfunction in an animal comprising administering an oligosaccharide formulation as described herein.

[0164] Provided herein are methods for treating gastrointestinal barrier dysfunction in animals, the methods comprising: administering to an animal suffering from gastrointestinal barrier dysfunction a nutritional composition comprising a base nutritional composition and a synthetic oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 2; wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides by relative abundance as measured by mass spectrometry.

[0165] In some embodiments, the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal prior to administration of the nutritional formulation comprising the synthetic oligosaccharide formulation.

[0166] In some embodiments, the method includes that the gastrointestinal barrier dysfunction is too permeable (leaky gut). In some embodiments, the permeability of the gastrointestinal barrier allows intestinal contents (e.g., food particles, microorganisms, toxins) to translocate from the intraluminal space into the circulation of the animal. In some embodiments, the permeability of the gastrointestinal barrier is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% less than the permeability of the gastrointestinal barrier of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the permeability of the gastrointestinal barrier is about 0.5%-40%, 0.5%-30%, 0.5%-20%, 0.5%-10%, 0.5%-5%, 0.5%-1%, 1%-40%, 1%-30%, 1%-20%, 1%-10%, 1%-5%, 1%-2%, 5%-40%, 5%-30%, 5%-20%, 5%-10%, 10%-40%, 10%-30%, or 10%-20% less than the permeability of the gastrointestinal barrier of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation.

[0167] In some embodiments, the reduction in gastrointestinal permeability is directly mediated by the synthetic oligosaccharide preparation. In some embodiments, the reduction in gastrointestinal permeability is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation is processed in vivo into one or more secondary substances. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the gastrointestinal microbiota of the animal. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the reduction in gastrointestinal permeability is directly mediated by one or more of the secondary substances.

[0168] In some embodiments, one or more symptoms of gastrointestinal barrier dysfunction are improved. In some embodiments, the one or more symptoms are reduced mucus synthesis, reduced mucin synthesis, reduced mucus secretion, reduced mucin secretion, reduced nutrient absorption, increased inflammation, reduced resistance to infection, reduced intestinal epithelial cell proliferation, reduced intestinal epithelial cell maturation, malnutrition, reduced weight gain, increased feed conversion rate, reduced feed efficiency, and increased mortality.

[0169] In some embodiments, the animal has a higher body weight relative to the animal's body weight prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the animal has a higher feed efficiency relative to the animal's feed efficiency prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the animal has a lower feed conversion ratio relative to the animal's feed conversion ratio prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation.

[0170] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cows, cattle, buffalo, bison, pigs (e.g., nursery pigs, growing / finishing pigs), cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigeons, ferrets, gerbils, hamsters, mice, rats, birds, or humans. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler chickens, laying hens, breeders), turkeys, ducks, or geese. In some embodiments, the animal is a pig (e.g., nursery pigs, growing / finishing pigs).

[0171] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0172] Provided herein are methods of treating an infection, the methods comprising administering to the animal a nutritional composition comprising a base nutritional composition and a synthetic oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions) each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 2; wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides by relative abundance as measured by mass spectrometry.

[0173] In some embodiments, the levels of one or more types of immune cells are greater in the gastrointestinal tract of the animal relative to the levels in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0174] In some embodiments, the immune cells are phagocytes. In some embodiments, the immune cells are granulocytes. In some embodiments, the level of granulocyte phagocytosis in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of immune cells or immune cell activity is directly mediated by the synthetic oligosaccharide preparation.

[0175] In some embodiments, the level of immune cells or immune cell activity is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation is processed into one or more secondary substances in vivo. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the gastrointestinal microbiota of the animal. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the increase in mucus levels is directly mediated by one or more of the secondary substances.

[0176] In some embodiments, the level of one or more pro-inflammatory cytokines or chemokines in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of one or more anti-inflammatory cytokines or chemokines in the gastrointestinal tract of the animal is lower relative to the level in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0177] In some embodiments, the permeability of the gastrointestinal barrier of the animal is lower relative to the permeability of the gastrointestinal barrier of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of mucus in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0178] In some embodiments, the animal has a higher body weight relative to the animal's body weight prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the animal has a higher feed efficiency relative to the animal's feed efficiency prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the animal has a lower feed conversion ratio relative to the animal's feed conversion ratio prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation.

[0179] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cows, cattle, buffalo, bison, pigs (e.g., nursery pigs, growing / finishing pigs), cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigeons, ferrets, gerbils, hamsters, mice, rats, birds, or humans. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler chickens, laying hens, breeders), turkeys, ducks, or geese. In some embodiments, the animal is a pig (e.g., nursery pigs, growing / finishing pigs).

[0180] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0181] Provided herein are methods of enhancing or inducing an immune response, the methods comprising: administering to the animal a nutritional composition comprising a base nutritional composition and a synthetic oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 2; wherein each fraction comprises 1% to 90% anhydrosubunit-containing oligosaccharides by relative abundance as measured by mass spectrometry.

[0182] In some embodiments, the levels of one or more types of immune cells are greater in the gastrointestinal tract of the animal relative to the levels in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0183] In some embodiments, the levels of one or more types of immune cells are greater in the gastrointestinal tract of the animal relative to the levels in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0184] In some embodiments, the immune cells are phagocytes. In some embodiments, the immune cells are granulocytes. In some embodiments, the level of granulocyte phagocytosis in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of immune cells or immune cell activity is directly mediated by the synthetic oligosaccharide preparation.

[0185] In some embodiments, the level of immune cells or immune cell activity is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation is processed into one or more secondary substances in vivo. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the gastrointestinal microbiota of the animal. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the increase in mucus levels is directly mediated by one or more of the secondary substances.

[0186] In some embodiments, the level of one or more pro-inflammatory cytokines or chemokines in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of one or more anti-inflammatory cytokines or chemokines in the gastrointestinal tract of the animal is lower relative to the level in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0187] In some embodiments, the permeability of the gastrointestinal barrier of the animal is lower relative to the permeability of the gastrointestinal barrier of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of mucus in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is higher relative to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0188] In some embodiments, the animal has a higher body weight relative to the animal's body weight prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the animal has a higher feed efficiency relative to the animal's feed efficiency prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation. In some embodiments, the animal has a lower feed conversion ratio relative to the animal's feed conversion ratio prior to administration of the nutritional composition comprising the synthetic oligosaccharide formulation.

[0189] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cows, cattle, buffalo, bison, pigs (e.g., nursery pigs, growing / finishing pigs), cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigeons, ferrets, gerbils, hamsters, mice, rats, birds, or humans. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler chickens, laying hens, breeders), turkeys, ducks, or geese. In some embodiments, the animal is a pig (e.g., nursery pigs, growing / finishing pigs).

[0190] Further aspects and advantages of the present disclosure will readily become apparent to those skilled in the art from the following detailed description, in which only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative rather than restrictive.

[0191] Incorporated by Reference

[0192] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0193] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description and accompanying drawings (also referred to herein as "figures" and "FIG.") which set forth illustrative embodiments utilizing the principles of the invention, wherein:

[0194] Figure 1 shows the oligosaccharide preparation of Example 9.7 1 H.13 Part of the C-HSQC NMR spectrum.

[0195] Figure 2 shows the MALDI-MS spectrum of the oligosaccharide preparation from Example 9.7, demonstrating the presence of the anhydro subunit.

[0196] FIG3 shows the ID of the dehydrated DP1 fraction isolated from the oligosaccharides of Example 9. 1 H-proton NMR spectrum.

[0197] FIG4 shows the 1D APT of the dehydrated DP1 fraction isolated from the oligosaccharide of Example 9 13 C-NMR spectrum.

[0198] Figure 5 presents GC-MS chromatograms (TIC and XIC (m / z 229) plots) showing the DP2 and dehydrated DP2 components of oligosaccharide preparation 2.9 after derivatization.

[0199] Figure 6 shows the oligosaccharide preparation with caramelized anhydro subunits 1 H. 13 C-HSQC spectrum.

[0200] Figure 7 shows a portion of a MALDI-MS spectrum comparing oligosaccharide preparations from Example 9 at different laser energies.

[0201] Figure 8A shows LC-MS / MS detection of anhydro DP2 species (at concentrations ranging from 1 to 80 μg / mL) of oligosaccharide preparations in water.Figure 8B shows a linear calibration curve generated by the LC-MS / MS detection of Figure 8A.

[0202] FIG9 shows the quantification of the dehydrated DP2 content of various control and treated dietary compositions.

[0203] FIG10 shows the 2D-1H JRES NMR spectrum of the glucose oligosaccharide sample containing anhydrosubunits.

[0204] Figure 11 is a representative sample of glucose oligosaccharide containing anhydrosubunits 1 H. 13 C-HSQC NMR spectrum with relevant resonances and assignments for bonding distribution.

[0205] Figure 12 shows three oligosaccharides containing anhydro subunits 1 Superposition of H DOSY spectra.

[0206] FIG13 shows a comparison of 1,6-anhydro-β-D-glucose (DP1-18), 1,6-anhydro-β-D-cellobiose (DP2-18), and anhydrosubunit-containing oligosaccharide samples.

[0207] FIG14 shows mass chromatograms of anhydrosubunit-containing oligosaccharides (top) and digested anhydrosubunit-containing oligosaccharides (bottom) under selected multiple reaction monitoring (MRM).

[0208] Figure 15 shows a plot of relative abundance versus degree of polymerization (DP) for the oligosaccharides of Example 9. The plot shows that the oligosaccharide preparation has a monotonically decreasing DP distribution.

[0209] Figure 16 shows a plot of relative abundance versus degree of polymerization for the oligosaccharides of Example 9. The plot shows that the oligosaccharide preparation has a non-monotonic decreasing DP distribution.

[0210] FIG17 shows a graphical depiction of a healthy gastrointestinal barrier and a leaky gastrointestinal barrier.

[0211] FIG18 shows a graphical depiction of a device for assessing healthy intestinal barrier integrity.

[0212] FIG. 19 is a flow cytometric graph showing granulocytes with and without ingested pathogens.

[0213] Figure 20 is a graph showing the fold change in expression of immune and inflammatory cytokines and chemokines in Peyer's Patch tissue for selected genes that had at least a 1.5-fold increase in expression.

[0214] Figure 21 shows the chamber used for in vitro evaluation of direct and indirect effects of oligosaccharide preparations on intestinal barrier function disrupted by rhamnolipid apical stressor.

[0215] Figure 22 is a bar graph showing the fold change in expression of IL1B, IL4, IL10, and SLC5A10 in birds fed a diet containing 500 ppm of the oligosaccharides of Example 9.3 (filled bars) compared to a control diet (open bars). The data show increased expression of genes associated with immunity, anti-inflammatory responses, and nutrient absorption in birds fed the oligosaccharides of Example 9.3.

[0216] Figure 23 is a bar graph showing relative gene expression of SLC34A2 in treatment groups AB as detailed in Table 26. The data demonstrate that the oligosaccharides from Examples 9.2 and 9.3 result in improved ileal absorption of phosphorus as indicated by analysis of increased SLC34A2 gene expression.

[0217] Figure 24 is a microscopic image of ileum tissue from birds in Treatment Group B and Treatment Group D (groups as defined in Table 26). Ileum histology revealed significantly fewer inflammatory lesions and improved villus length in broiler chickens fed the diet containing the oligosaccharides of Example 9.2.

[0218] Figure 25 is a bar graph showing the percentage of goblet cells in treatment groups A, B and D (as defined in Table 26). The data show that oligosaccharides increased goblet cell counts in broiler chickens challenged with inflammation (Group B).

[0219] Figure 26 is a bar graph showing the percentage of lymphocytes from birds in treatment groups A, B, C and D (as defined in Table 26). The data show that broiler chickens treated with selected oligosaccharides of Example 9 promoted an increase in T helper cells (measured as a percentage of total lymphocytes) when subjected to an acute inflammatory challenge.

[0220] Figure 27A is a microscopic image of liver tissue from broilers fed a diet containing the oligosaccharides of Example 9.3, with healthy liver tissue (left) and tissue exhibiting inflammatory damage (right). Figure 27B shows a bar graph of histological injury scores for liver samples from broilers fed in treatment groups A, B or C (as defined in Table 26). The data show that treatment group C (feed containing the oligosaccharides of Example 9.3) provides reduced liver damage. Figure 27C shows the APG levels in broilers in treatment groups A, B and C (as defined in Table 26). The data show that acute phase liver protein AGP decreases when subjected to inflammatory attack.

[0221] Figure 28A is a bar graph showing IgA levels in blood samples from broiler chickens in treatment groups A, B, and D on day 21. The data show that broiler chickens in treatment group D exhibited an accelerated immune response on day 21. Figure 28B is a bar graph showing IgA levels in blood samples from broiler chickens in treatment groups A, B, and D on day 28. The data show that when subjected to an acute inflammatory challenge on day 14, broiler chickens exhibited a faster recovery toward the baseline of control group A (day 28).

[0222] Figure 29A is a bar graph showing diamine oxidase levels in blood samples from broiler chickens in treatment groups A, B, and D on day 21. The data show that when subjected to an acute inflammatory challenge on day 14, the broiler chickens exhibited a faster recovery toward the control group A baseline compared to birds not fed oligosaccharides. Figure 29B is a bar graph showing levels in blood samples from broiler chickens in treatment groups A, B, and D on day 28. The data show that when subjected to an acute inflammatory challenge on day 14, the broiler chickens exhibited a faster recovery toward the control group A baseline compared to birds not fed oligosaccharides.

[0223] FIG30 shows two DP1 anhydrosubunit-containing oligosaccharides and one DP2 anhydrosubunit-containing oligosaccharide.

[0224] FIG31 shows anhydrosubunit-containing oligosaccharide (cellotriosan).

[0225] Figure 32A shows a MALDI-MS spectrum of an oligosaccharide preparation from Example 2, demonstrating the presence of anhydro subunits. Figure 32B shows a MALDI-MS spectrum of an oligosaccharide preparation from Example 2, demonstrating the presence of anhydro subunits.

[0226] Figure 33A shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 1. Figure 33B shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 1. Figure 33C shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 1.

[0227] Figure 34A shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 3. Figure 34B shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 3. Figure 34C shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 3.

[0228] Figure 35A shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 4. Figure 35B shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 4. Figure 35C shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 4.

[0229] Figure 36A shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 7. Figure 36B shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 7. Figure 36C shows LC-MS / MS detection of dehydrated DP2, dehydrated DP1, and DP2 species of the oligosaccharide preparation of Example 7.

[0230] Figure 37A shows GC-MS spectra of DP1, dehydrated DP1, DP2 and dehydrated DP2 fractions of the oligosaccharide preparation of Example 1. Figure 37B shows an enlarged view of the DP2 and dehydrated DP2 fractions shown in Figure 37A.

[0231] Figure 38A shows GC-MS spectra of DP1, dehydrated DP1, DP2 and dehydrated DP2 fractions of the oligosaccharide preparation of Example 3. Figure 38B shows an enlarged view of the DP2 and dehydrated DP2 fractions shown in Figure 38A.

[0232] Figure 39A shows GC-MS spectra of DP1, dehydrated DP1, DP2 and dehydrated DP2 fractions of the oligosaccharide preparation of Example 4. Figure 39B shows an enlarged view of DP2 and dehydrated DP2 fractions shown in Figure 39A.

[0233] Figure 40A shows GC-MS spectra detection of DP1, dehydrated DP1, DP2 and dehydrated DP2 fractions of the oligosaccharide preparation of Example 7. Figure 40B shows an enlarged view of DP2 and dehydrated DP2 fractions shown in Figure 40A.

[0234] FIG41 shows the effects of reaction temperature, water content and reaction time on the content of DP2 anhydrosubunit-containing oligosaccharides in the oligosaccharide preparation compared to the oligosaccharide preparation according to Example 2.

[0235] FIG42 shows the NMR assignments of 1,6-anhydro-β-D-glucopyranose and 1,6-anhydro-β-D-glucopyranose.

[0236] Figure 43 shows a comparison of MALDI-MS spectra of oligosaccharide preparations from Example 9 at different laser energies. DETAILED DESCRIPTION

[0237] The following description and examples illustrate embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein, as these can vary. Those skilled in the art will recognize that there are many embodiments and modifications of the present disclosure that are encompassed within the scope of the present disclosure.

[0238] All terms are intended to be understood as they would be understood by one skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates.

[0239] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0240] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

[0241] The following definitions supplement the definitions in the art and are for the present application and should not be attributed to any relevant or irrelevant situation, such as any jointly owned patent or application. Although any method and material similar or equivalent to those methods and materials described herein can be used in the practice for testing this disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only used to describe the purpose of specific embodiments and are not intended to be restrictive.

[0242] I. Definition

[0243] The terminology used herein is for descriptive purposes only and is not intended to be limiting. Unless the context clearly indicates otherwise, as used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0244] It should be understood that terms such as “comprises,” “comprised,” “comprising,” and the like have the meaning ascribed to them in U.S. patent law; that is, they mean “includes,” “included,” “including,” and the like, and are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps; and that terms such as “consisting essentially of” and “consists essentially of,” and the like have the meaning ascribed to them in U.S. patent law; that is, they allow for elements not expressly recited, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention.

[0245] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0246] When range is used herein for physical property (such as molecular weight) or chemical property (such as chemical formula), it is intended to include all combinations and subcombinations of scope and specific embodiments thereof.When referring to a numeral or numerical range, the term "about" means that the numeral or numerical range mentioned is an approximate value within experimental variation (or within statistical experimental error), and therefore, the numeral or numerical range will change between 1% and 15% of the stated numeral or numerical range in some cases. In some embodiments, the term "about" means in 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of a given value or scope.

[0247] As used herein, the term "administering" includes providing an animal with a synthetic oligosaccharide preparation, a nutrient composition, a liquid or an animal feed composition as described herein so that the animal can ingest the synthetic oligosaccharide preparation, the nutrient composition, the liquid or the animal feed composition. In such embodiments, the animal ingests a portion of the synthetic oligosaccharide preparation, the nutrient composition or the animal feed composition. In some embodiments, a synthetic oligosaccharide preparation, a nutrient composition, a liquid or an animal feed composition is provided to the animal so that the animal can ingest the synthetic oligosaccharide preparation, the nutrient composition, the liquid or the animal feed composition at will. In some embodiments, a synthetic oligosaccharide preparation, a nutrient composition, a liquid or an animal feed composition is administered to the animal as a prescribed diet. In some embodiments, a synthetic oligosaccharide preparation, a nutrient composition, a liquid or an animal feed composition is administered to the animal via artificial feeding (e.g., oral syringe feeding, tube feeding, etc.). In some embodiments, a synthetic oligosaccharide preparation, a nutrient composition, a liquid or an animal feed composition is, for example, administered orally to the animal at will or manually. In some embodiments, the animal ingests a portion of the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition every 24 hours or every 24 hours for at least 7 days, 14 days, 21 days, 30 days, 45 days, 60 days, 75 days, 90 days, or 120 days. In some embodiments, the oligosaccharide preparation can be dissolved in water or other liquids, and the animal ingests a portion of the oligosaccharide preparation by drinking the liquid. In some embodiments, the oligosaccharides are provided to the animal via its drinking water. In some embodiments, the oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is consumed at will.

[0248] As used herein, the term "feed conversion ratio (FCR)" refers to the ratio of feed mass input (e.g., consumed by an animal) to animal output, where the animal output is the desired animal product. For example, the animal output of dairy animals is milk, while the animal output of animals raised for meat is body weight.

[0249] As used herein, "feed efficiency" refers to the ratio of animal output to feed mass input (eg, consumed by the animal), where the animal output is the desired animal product.

[0250] As used herein, the term "anhydro subunit" refers to a thermal dehydration product of a monosaccharide (or monosaccharide subunit) or a sugar caramelization product. For example, the "anhydro subunit" can be an anhydromonosaccharide, such as anhydroglucose. As another example, the "anhydro subunit" can be linked to one or more conventional or anhydromonosaccharide subunits via a glycosidic linkage.

[0251] The term "oligosaccharide" refers to a monosaccharide or a compound containing two or more monosaccharide subunits linked by glycosidic bonds. Thus, oligosaccharides include conventional monosaccharides; anhydromonosaccharides; or compounds containing two or more monosaccharide subunits, wherein one or more monosaccharide subunits are optionally independently replaced by one or more anhydrosubunits. Oligosaccharides can be functionalized. As used herein, the term oligosaccharide encompasses all types of oligosaccharides, wherein each of the monosaccharide subunits in the oligosaccharide is independently and optionally functionalized and / or replaced by its corresponding anhydromonosaccharide subunit.

[0252] As used herein, the term "oligosaccharide preparation" refers to a preparation comprising at least one oligosaccharide.

[0253] As used herein, the term "glucose oligosaccharide" refers to glucose or a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds. Thus, glucose oligosaccharides include glucose; anhydroglucose; or a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds, wherein one or more of the glucose monosaccharide subunits are each optionally and independently replaced by anhydroglucose subunits.

[0254] As used herein, the term "galacto-oligosaccharide" refers to galactose or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds. Thus, galacto-oligosaccharides include galactose; anhydrogalactose; or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds, wherein at least one monosaccharide subunit is optionally replaced by an anhydrogalactose subunit.

[0255] As used herein, the term "glucose-galactose oligosaccharide preparation" refers to a composition produced by the complete or incomplete sugar condensation reaction of glucose and galactose. Therefore, in some embodiments, the glucose-galactose oligosaccharide preparation comprises glucose oligosaccharides, galactose oligosaccharides, a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits connected by glycosidic bonds, or a combination thereof. In some embodiments, the glucose-galactose oligosaccharide preparation comprises glucose oligosaccharides and a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits connected by glycosidic bonds. In some embodiments, the glucose-galactose oligosaccharide preparation comprises galactose oligosaccharides and a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits connected by glycosidic bonds. In some embodiments, the glucose-galactose oligosaccharide preparation comprises a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits connected by glycosidic bonds.

[0256] As used herein, the terms "monosaccharide unit" and "monosaccharide subunit" are used interchangeably. "Monosaccharide subunit" refers to a monosaccharide monomer in an oligosaccharide. For oligosaccharides with a degree of polymerization of 1, the oligosaccharide can be referred to as a monosaccharide subunit or a monosaccharide. For oligosaccharides with a degree of polymerization of 2 or higher, the monosaccharide subunits are linked via glycosidic bonds.

[0257] As used herein, the term "conventional monosaccharide" refers to a monosaccharide that does not contain an anhydro subunit. The term "conventional disaccharide" refers to a disaccharide that does not contain an anhydro subunit. Therefore, the term "conventional subunit" refers to a subunit that is not an anhydro subunit.

[0258] As used herein, the term "relative abundance" or "abundance" refers to the abundance of a species in terms of how common or rare the species is. For example, a DP1 fraction containing 10% anhydrosubunit oligosaccharides in relative abundance may refer to a plurality of DP1 oligosaccharides, wherein 10% of the DP1 oligosaccharides are anhydromonosaccharides. The relative abundance of a certain DP fraction, for example, of oligosaccharides, can be determined by suitable analytical instruments (e.g., mass spectrometry and liquid chromatography, such as LC-MS / MS, GC-MS, HPLC-MS, and MALDI-MS). In some embodiments, relative abundance is determined by integrating the area under the peak of a chromatogram (e.g., LC-MS / MS, GC-MS, and HPLC-MS) corresponding to the fraction of interest. In some embodiments, relative abundance is determined by peak intensity (e.g., MALDI-MS). In some embodiments, relative abundance is determined by a combination of analytical methods (e.g., gravimetric determination after separation by liquid chromatography).

[0259] As used herein, the term "anhydro DPn oligosaccharide," "anhydro DPn species," or "DPn anhydrosubunit-containing oligosaccharide" refers to an oligosaccharide having a degree of polymerization of n and comprising one or more anhydrosubunits. Thus, anhydroglucose is a DP1 anhydrosubunit-containing oligosaccharide, and cellotriose is a DP3 anhydrosubunit-containing oligosaccharide.

[0260] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents and reference to "the oligosaccharide" includes reference to one or more oligosaccharides (or oligosaccharides) and equivalents thereof known to those skilled in the art, and so forth.

[0261] II. Oligosaccharide Preparations

[0262] Disclosed herein are oligosaccharide preparations suitable for use in nutritional compositions. In some embodiments, the oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than or equal to 2. In some embodiments, n is an integer greater than 2. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation comprises 1% to 90% anhydrosubunit-containing oligosaccharides in relative abundance as measured by mass spectrometry. In some embodiments, the relative abundance of oligosaccharides in each fraction decreases monotonically with its degree of polymerization.

[0263] In some embodiments, n is an integer greater than or equal to 3. In some embodiments, n is an integer in the range of 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50. In some embodiments, each of the 1 to n fractions in the oligosaccharide preparation independently comprises 0.1% to 90% of anhydro subunit-containing oligosaccharides as measured by mass spectrometry or by LC-MS / MS or GC-MS in relative abundance. In some embodiments, each of the 1 to n fractions in the oligosaccharide preparation independently comprises about 0.1% to about 15% anhydro subunit-containing oligosaccharides. In some embodiments, each of the 1 to n fractions in the oligosaccharide preparation independently comprises about 0.5% to about 15% anhydro subunit-containing oligosaccharides. In some embodiments, DP1 and DP2 fractions each independently comprise anhydro subunit oligosaccharides in an amount of about 0.1% to about 15% as measured by mass spectrometry (such as MALDI-MS) or by LC-MS / MS or GC-MS. In some embodiments, DP1 and DP2 fractions each independently comprise anhydro subunit oligosaccharides in an amount of about 0.5% to about 15% as measured by mass spectrometry LC-MS / MS or GC-MS. In some embodiments, DP1 and DP2 fractions each independently comprise anhydro subunit oligosaccharides in an amount of about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.8%, 1%, 2% or 3% to about 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% as measured by mass spectrometry LC-MS / MS or GC-MS. In some embodiments, the relative abundance of oligosaccharides in each fraction decreases monotonically with its degree of polymerization.

[0264] In some embodiments, the oligosaccharide preparation is a synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation refers to a variety of oligosaccharides produced by a method that does not require a living organism. In some embodiments, the synthetic oligosaccharide preparation refers to a variety of oligosaccharides produced by a method that does not require an enzyme. In some embodiments, the synthetic oligosaccharide preparation refers to a variety of oligosaccharides produced by a chemical method. In certain embodiments, the synthetic oligosaccharide preparation refers to a variety of oligosaccharides produced by the condensation of sugars.

[0265] A. Prebiotic Effects of Oligosaccharides

[0266] Disclosed herein are oligosaccharide preparations comprising dehydrated sugar components and / or sugar dehydration product components that exhibit complex functional modulation of microbial communities, such as animal intestinal microbiomes. The oligosaccharide preparations provide the utility of modulating the utilization of fermentable carbon by microflora and directing metabolic flux to beneficial substances, thereby providing microbiome-mediated health or nutritional benefits.

[0267] Indigestible carbohydrates can act as prebiotics by providing a fermentable carbon source to the microbial community. For example, the ability of a diet rich in soluble plant fiber to nourish the intestinal microflora has been identified. In addition, bifidogenic prebiotics support the growth of bifidobacteria (e.g., members of the genus Bifidobacterium), while lactogenic prebiotics support the growth of Lactobacillus species.

[0268] Prebiotic fibers can be fermented into beneficial chemicals, such as short-chain fatty acids (SCFAs). Prebiotic fibers include: resistant starch; cellulose; pectins, such as rhamnogalactan, arabinogalactan, and arabinan; hemicelluloses, such as arabinoxylan, xyloglucan, glucomannan, and galactomannan; xylans, such as corncob oligosaccharides; b-glucans, such as cereal b-glucans, yeast b-glucans, and bacterial b-glucans; polyfructose, such as inulin and fructans; and gums, such as alginates. Inulin is a common bifidogenic prebiotic fiber.

[0269] In other cases, prebiotics act by hindering the ability of pathogenic bacteria to migrate and thereby infect the host organism via anti-adhesion mechanisms such as competitive binding to cell surface receptor sites. Certain galacto-oligosaccharides provide effective anti-adhesion to various enteropathogenic organisms such as Escherichia species.

[0270] Prebiotics are typically provided to host animals by incorporation into the diet, where they exhibit a dose-dependent response (at least to a saturation threshold). For example, providing higher doses of bifidogenic prebiotics (such as inulin) tends to provide greater increases in Bifidobacterium species populations. Higher doses of inulin correspond to higher production of SCFAs through fermentation. This is because the prebiotic provides a metabolic carbon source, and more carbon is converted to more fermentation products. Similarly, providing higher doses of anti-adhesive prebiotics offers the potential for competitive binding to surface receptor sites.

[0271] Certain carbohydrate species comprising modified monomer subunits may affect how a microbial system utilizes other carbohydrates that could otherwise be used by the microbial system as a prebiotic source. For example, such carbohydrate species may be modified carbohydrate species that modulate the bacterial starch utilization system (SUS), a protein responsible for cell surface recognition, glycoside cleavage, and import of starch metabolites.

[0272] Carbohydrate compositions capable of complex modulation of animal microbiota have utility as feed additives that improve animal health and nutrition via their effects on the animal microbiome. For example, modulation of butyrate production by the intestinal microflora confers animal health benefits by promoting a healthy intestinal mucosa, barrier function, and via anti-inflammatory effects. Modulation of propionic acid production affects the metabolic energy extracted from the animal diet via increased gluconeogenesis. Relevant microbial communities include, for example, the ileum, jejunum, and cecum and / or fecal microbiota in poultry, pigs, dogs, cats, horses, or the ruminant microbiota of cattle, cows, sheep, and the like. Other microbial communities include skin microbiota, nasal microbiota, and the like.

[0273] In addition, the oligosaccharide preparations disclosed herein are advantageous because, due to the presence of the dehydrated subunits, they can be selectively analyzed and quantified in complex nutritional compositions (such as complete animal feeds). It has commercial utility for determining the presence and / or concentration of feed additives (such as oligosaccharide preparations). Such determinations can be performed for quality control purposes to determine whether the additive is consistently blended with the base nutritional composition to provide a final nutritional composition containing the desired dosage or inclusion level of the additive.

[0274] However, nutritional compositions themselves contain a large and diverse number of carbohydrate structures (e.g., starch, plant fibers, and pectin). Therefore, it is particularly challenging to distinguish a small amount of oligosaccharide-based feed additives from the vast array of other carbohydrates that form the basis of nutritional compositions. Therefore, the oligosaccharide preparations disclosed herein provide a means of distinguishing themselves from other carbohydrate sources in nutritional compositions through the dehydrated subunits.

[0275] B. Degree of Polymerization (DP) Distribution

[0276] In some embodiments, the oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n. In some embodiments, the oligosaccharide preparation comprises n oligosaccharide fractions (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n. For example, in some embodiments, the DP1 fraction comprises one or more monosaccharides and / or one or more anhydromonosaccharides. As another example, in some embodiments, the DP1 fraction comprises glucose, galactose, fructose, 1,6-anhydro-β-D-furanose, 1,6-anhydro-β-D-pyranose, or any combination thereof. As yet another example, in some embodiments, the DP2 fraction comprises one or more conventional disaccharides and one or more anhydrosubunit-containing disaccharides. In some embodiments, the DP2 fraction comprises lactose.

[0277] In some embodiments, n is at least 2, at least 3, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, or at least 100. In some embodiments, n is 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.In some embodiments, n is less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, less than 26, less than 27, less than 28, less than 29, less than 30, less than 31, less than 32, less than 33, less than 34, less than 35, less than 36, less than 37, less than 38, less than 39, less than 40, less than 41, less than 42, less than 43, less than 44, less than 45, less than 46, less than 47, less than 48, less than 49, less than 50, less than 51, less than 52, less than 53, less than 54, less than 55, less than 56, less than 57, less than 58, less than 59, less than 60, less than 61, less than 62, less than 63, less than 64, less than 65, less than 66, less than 67, less than 68, less than 69, less than 70, less than 71, less than 72, less than 73, less than 74, less than 75 4. Less than 55, less than 56, less than 57, less than 58, less than 59, less than 60, less than 61, less than 62, less than 63, less than 64, less than 65, less than 66, less than 67, less than 68, less than 69, less than 70, less than 71, less than 72, less than 73, less than 74, less than 75, less than 76, less than 77, less than 78, less than 79, less than 80, less than 81, less than 82, less than 83, less than 84, less than 85, less than 86, less than 87, less than 88, less than 89, less than 90, less than 91, less than 92, less than 93, less than 94, less than 95, less than 96, less than 97, less than 98, less than 99 or less than 100. In some embodiments, n is 2 to 100, 5 to 90, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 15 to 60, 15 to 50, 15 to 45, 15 to 40, 15 to 35, or 15 to 30.

[0278] The distribution of the degree of polymerization of oligosaccharide preparations can be measured by any suitable analytical method and instrument, including but not limited to end group method, osmotic pressure (osmotic pressure determination method), ultracentrifugation, viscosity measurement, light scattering, size exclusion chromatography (SEC), SEC-MALLS, field flow fractionation (FFF), asymmetric flow field flow fractionation (A4F), high performance liquid chromatography (HPLC) and mass spectrometry (MS). For example, the distribution of degree of polymerization can be measured and / or detected by mass spectrometry (such as matrix assisted laser desorption / ionization (MALDI)-MS, liquid chromatography (LC)-MS or gas chromatography (GC)-MS). For another example, the distribution of degree of polymerization can be measured and / or detected by SEC (such as gel permeation chromatography (GPC)). As another example again, the distribution of degree of polymerization can be measured and / or detected by HPLC, FFF or A4F. In some embodiments, the distribution of degree of polymerization is measured and / or detected by MALDI-MS. In some embodiments, the distribution of degree of polymerization is measured and / or detected by GC-MS or LC-MS. In some embodiments, the distribution of degree of polymerization is measured and / or detected by SEC. In some embodiments, the distribution of degree of polymerization is measured and / or detected by HPLC. In some embodiments, the distribution of degree of polymerization is measured and / or detected by the combination of analytical instruments (such as MALDI-MS and SEC). In some embodiments, the degree of polymerization of oligosaccharide preparations can be measured based on its molecular weight and molecular weight distribution. For example, Fig. 2 shows a MALDI-MS spectrum, which shows the degree of polymerization of various fractions and the presence of anhydrous subunit oligosaccharides (-18g / mol MW offset peak) in all observed fractions.

[0279] In some embodiments, the relative abundance of oligosaccharides in most fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in less than 6, less than 5, less than 4, less than 3 or less than 2 fractions of an oligosaccharide preparation does not decrease monotonically with its degree of polymerization.

[0280] In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 consecutive DP fractions decreases monotonically with their degree of polymerization.

[0281] In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with its degree of polymerization. For example, Figure 15 provides an example of a DP distribution, wherein the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with its DP. For example, in some embodiments, only the relative abundance of oligosaccharides in the DP3 fraction does not decrease monotonically with its degree of polymerization, i.e., the relative abundance of oligosaccharides in the DP3 fraction is lower than the relative abundance of oligosaccharides in the DP4 fraction. In some embodiments, the relative abundance of oligosaccharides in the DP2 fraction is lower than the relative abundance of oligosaccharides in the DP3 fraction. For example, Figure 16 shows a degree of polymerization distribution, wherein the relative abundance of oligosaccharides in the DP2 fraction does not decrease monotonically with its degree of polymerization.

[0282] In some embodiments, the oligosaccharide preparations described herein have a DP1 fraction content of about 1% to about 50%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 5% to about 50%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 50%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, or about 10% to about 15%, by weight or by relative abundance. In some embodiments, the oligosaccharide preparation has a DP1 fraction content of about 10% to about 35%, about 10% to about 20%, or about 10% to about 15% by weight or by relative abundance. In some embodiments, the content of the DP1 fraction is determined by MALDI-MS. In some embodiments, the content of the DP1 fraction is determined by HPLC. In some embodiments, the content of the DP1 fraction is determined by LC-MS / MS or GC-MS.

[0283] In some embodiments, the oligosaccharide preparations described herein have a DP2 fraction content of about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% by weight or by relative abundance. In some embodiments, the oligosaccharide preparations have a DP2 fraction content of about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% by weight or by relative abundance. In some embodiments, the content of the DP2 fraction is determined by MALDI-MS. In some embodiments, the content of the DP2 fraction is determined by HPLC. In some embodiments, the content of the DP2 fraction is determined by LC-MS / MS or GC-MS.

[0284] In some embodiments, the oligosaccharide preparations described herein have a DP3 fraction content of about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% by weight or by relative abundance. In some embodiments, the oligosaccharide preparations have a DP3 fraction content of about 1% to about 15%, about 1% to about 10%, about 5% to about 15%, or about 5% to about 10% by weight or by relative abundance. In some embodiments, the content of the DP3 fraction is determined by MALDI-MS. In some embodiments, the content of the DP3 fraction is determined by HPLC. In some embodiments, the content of the DP3 fraction is determined by LC-MS / MS or GC-MS.

[0285] In some embodiments, the oligosaccharide preparations described herein have a DP4 fraction content of about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% by weight or by relative abundance. In some embodiments, the oligosaccharide preparations described herein have a DP4 fraction content of about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% by weight or by relative abundance. In some embodiments, the oligosaccharide preparations described herein have a DP5 fraction content of about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% by weight or by relative abundance. In some embodiments, the oligosaccharide preparation has a DP5 fraction content of about 1% to about 10% or about 1% to about 5% by weight or by relative abundance. In some embodiments, the content of the DP4 and / or DP5 fraction is determined by MALDI-MS. In some embodiments, the content of the DP4 and / or DP5 fraction is determined by HPLC. In some embodiments, the content of the DP4 and / or DP5 fraction is determined by LC-MS / MS or GC-MS.

[0286] In some embodiments, the ratio of the DP2 fraction to the DP1 fraction in the oligosaccharide preparation is about 0.01 to about 0.8, about 0.02 to about 0.7, about 0.02 to about 0.6, about 0.02 to about 0.5, about 0.02 to about 0.4, about 0.02 to about 0.3, about 0.02 to about 0.2, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, or about 0.1 to about 0.3. In some embodiments, the ratio of the DP2 fraction to the DP1 fraction in the oligosaccharide preparation is about 0.02 to about 0.4, by weight or relative abundance.

[0287] In some embodiments, the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation is about 0.01 to about 0.7, about 0.01 to about 0.6, about 0.01 to about 0.5, about 0.01 to about 0.4, about 0.01 to about 0.3, or about 0.01 to about 0.2, in some embodiments, the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation is about 0.01 to about 0.3, in terms of their weight or relative abundance.

[0288] In some embodiments, the combined content of DP1 and DP2 fractions in the oligosaccharide preparation is less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% by weight or by relative abundance. In some embodiments, the combined content of DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, less than 30%, or less than 10% by weight or by relative abundance.

[0289] In some embodiments, the oligosaccharide preparations described herein have an average DP value in the range of 2 to 10. In some embodiments, the oligosaccharide preparations have an average DP value of about 2 to about 8, about 2 to about 5, or about 2 to about 4. In some embodiments, the oligosaccharide preparations have an average DP value of about 3.5. The average DP value can be determined by SEC or by elemental analysis.

[0290] C. Dehydrated subunit levels

[0291] In some embodiments, each of the n oligosaccharide fractions independently comprises anhydrosubunit levels. For example, in some embodiments, the DP1 fraction comprises 10% anhydrosubunit-containing oligosaccharides by relative abundance, and the DP2 fraction comprises 15% anhydrosubunit-containing oligosaccharides by relative abundance. For another example, in some embodiments, the DP1, DP2, and DP3 fractions each comprise 5%, 10%, and 2% anhydrosubunit-containing oligosaccharides by relative abundance, respectively. In other embodiments, two or more oligosaccharide fractions may comprise similar levels of anhydrosubunit-containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fractions each comprise approximately 5% anhydrosubunit-containing oligosaccharides by relative abundance.

[0292] In some embodiments, each of the 1 to n fractions in the oligosaccharide preparations described herein independently comprises anhydro subunit oligosaccharides in an amount of about 0.1% to 15% as measured by mass spectrometry LC-MS / MS or GC-MS. In some embodiments, each of the 1 to n fractions in the oligosaccharide preparations independently comprises anhydro subunit oligosaccharides in an amount of about 0.5% to 15% as measured by mass spectrometry LC-MS / MS or GC-MS. In some embodiments, LC-MS / MS is used to determine the relative abundance of oligosaccharides in DP1, DP2, and / or DP3 fractions. In some embodiments, GC-MS is used to determine the relative abundance of oligosaccharides in DP1, DP2, and / or DP3 fractions. In some embodiments, MALDI-MS is used to determine the relative abundance of oligosaccharides in DP4 fractions or higher DP fractions. In some embodiments, the relative abundance of a fraction is determined by integrating the area under the peak of the LC-MS / MS chromatogram designated as corresponding to the fraction. In some embodiments, the relative abundance of a fraction is determined by integrating the area under the peak of the GC-MS chromatogram designated as corresponding to the fraction.

[0293] The level of anhydro subunits can be measured by any suitable analytical method (such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, HPLC, FFF, A4F or any combination thereof). In some embodiments, the level of anhydro subunits is measured at least in part by mass spectrometry (such as MALDI-MS). In some embodiments, the level of anhydro subunits is measured at least in part by NMR. In some embodiments, the level of anhydro subunit-containing oligosaccharides is measured at least in part by HPLC. In some embodiments, the level of anhydro subunit-containing oligosaccharides is measured by MALDI-MS, as shown by the -18 g / mol MW shift peak in Figure 2. In some embodiments, the presence and type of anhydro subunits can be measured and / or detected by NMR, as shown in Example 11, Figure 3 and Figure 4. In some embodiments, the relative abundance of anhydro subunit-containing oligosaccharides is measured by MALDI-MS. In some embodiments, the relative abundance of anhydro subunit-containing oligosaccharides is determined by LC-MS / MS, as shown in Figures 33A to 33C, Figures 34A to 34C, Figures 35A to 35C, and Figures 36A to 36C. In some embodiments, the relative abundance of anhydro subunit-containing oligosaccharides is determined by GC-MS, as shown in Figures 37A to 37B, Figures 38A to 38B, Figures 39A to 39B, and Figures 40A to 40B.

[0294] In some embodiments, at least one fraction of the oligosaccharide preparations described herein comprises less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharide. In some embodiments, at least one fraction of the oligosaccharide preparations described herein comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, or less than 2% anhydrosubunit-containing oligosaccharide. In other embodiments, at least one fraction of the oligosaccharide preparations described herein comprises greater than 0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 16%, greater than 17%, greater than 18%, greater than 19%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80% anhydrosubunit-containing oligosaccharides by relative abundance. In other embodiments, at least one fraction of the oligosaccharide preparations described herein comprises greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, or greater than 30% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, at least one fraction of the oligosaccharide preparation (such as DP1, DP2 and / or DP3) comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, 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%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25% or about 30% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, at least one fraction of the oligosaccharide preparation (such as DP1, DP2 and / or DP3) comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% anhydrosubunit-containing oligosaccharides by relative abundance.In some embodiments, at least one fraction of the oligosaccharide preparation (e.g., DP1, DP2, and / or DP3) comprises, by relative abundance, from about 0.1% to about 90%, from about 0.5% to about 90%, from about 0.5% to about 80%, from about 0.5% to about 70%, from about 0.5% to about 60%, from about 0.5% to about 50%, from about 0.5% to about 40%, from about 0.5% to about 30%, from about 0.5% to about 20%, from about 0.5% to about 10%, from about 0.5% to about 15%, from about 0.5% to about 2 ...15%, from about 0.5% to about 15%, from about 0.5% to about 15%, from about 0.5% to about 15%, from about 0.5% to about 15%, from about 0.5% to about 15%, from about From about 1% to about 9%, about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 1% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, or about 5% to about 10% anhydrosubunit-containing oligosaccharides. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently comprise anhydrosubunit-containing oligosaccharides in a range of about 0.1%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5% to about 8%, 9%, 10%, 11%, 12% or 15% relative abundance as measured by mass spectrometry LC-MS / MS or GC-MS. In some embodiments, the DP1 and DP2 fractions each independently comprise anhydrosubunit-containing oligosaccharides in a range of about 0.5% to about 15% relative abundance as measured by mass spectrometry or by LC-MS / MS or GC-MS.

[0295] In some embodiments, each fraction of the oligosaccharide preparations described herein comprises less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3% or less than 2% anhydrosubunit-containing oligosaccharide. In some embodiments, each fraction of the oligosaccharide preparations described herein comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2% anhydrosubunit-containing oligosaccharide by relative abundance. In other embodiments, each fraction of the oligosaccharide preparations described herein comprises an anhydro subunit oligosaccharide greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70% or 80% by relative abundance. In other embodiments, each fraction of the oligosaccharide preparations described herein comprises an anhydro subunit oligosaccharide greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% by relative abundance. In some embodiments, each fraction of the oligosaccharide preparations described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, 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%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, each fraction of the oligosaccharide preparations described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% anhydrosubunit-containing oligosaccharides by relative abundance.In some embodiments, each fraction of the oligosaccharide preparations described herein comprises, by relative abundance, from about 0.1% to about 90%, from about 0.1% to about 15%, from about 0.5% to about 90%, from about 0.5% to about 80%, from about 0.5% to about 70%, from about 0.5% to about 60%, from about 0.5% to about 50%, from about 0.5% to about 40%, from about 0.5% to about 30%, from about 0.5% to about 20%, from about 0.5% to about 10%, from about 0.5% to about 25%, from about 0.5% to about 30%, from about 0.5% to about 40%, from about 0.5% to about 50%, from about 0.5% to about 60%, from about 0.5% to about 70%, from about 0.5% to about 80%, from about 0.5% to about 90%, from about 0.5% to about 10%, from about 0.5% to about 15 ... From about 0.5% to about 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%, from about 0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about 2% to about 9%, from about 2% to about 8%, from about 2% to about 7%, from about 2% to about 6%, from about 2% to about 5%, from about 2% to about 4%, from about 2% to about 3%, or from about 5% to about 10% anhydrosubunit-containing oligosaccharides.

[0296] In some embodiments, the oligosaccharide preparations described herein comprise less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharide. In some embodiments, the oligosaccharide preparations comprise less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydrosubunit-containing oligosaccharide by relative abundance. In other embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharide at a relative abundance of greater than 0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 16%, greater than 17%, greater than 18%, greater than 19%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70% or greater than 80%. In other embodiments, the oligosaccharide preparation comprises anhydrosubunit-containing oligosaccharide at a relative abundance of greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%. In some embodiments, the oligosaccharide preparation comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, 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%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the oligosaccharide preparation comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% anhydrosubunit-containing oligosaccharides by relative abundance.In some embodiments, the oligosaccharide preparation comprises, by relative abundance, from about 0.1% to about 90%, from about 0.1% to about 15%, from about 0.5% to about 90%, from about 0.5% to about 80%, from about 0.5% to about 70%, from about 0.5% to about 60%, from about 0.5% to about 50%, from about 0.5% to about 40%, from about 0.5% to about 30%, from about 0.5% to about 20%, from about 0.5% to about 10%, from about 0.5% to about 15%. or about 5% to about 10% anhydrosubunit-containing oligosaccharides.

[0297] In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharide. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15% anhydrosubunit-containing oligosaccharide. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, 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%, about 19%, or about 20% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides in an amount of about 0.1% to about 15%, about 0.1% to about 20%, about 0.5% to about 20%, 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides in an amount of about 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the relative abundance of anhydrosubunit-containing oligosaccharides is determined by LC-MS / MS. In some embodiments, the relative abundance of anhydrosubunit-containing oligosaccharides is determined by GC-MS.

[0298] In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharide. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15% anhydrosubunit-containing oligosaccharide. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, 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%, about 19% or about 20% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides in an amount of about 0.1% to about 15%, about 0.1% to about 20%, about 0.5% to about 20%, 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 0.5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides in an amount of about 5% to about 10%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 0.5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the relative abundance of anhydrosubunit-containing oligosaccharides is determined by LC-MS / MS. In some embodiments, the relative abundance of anhydrosubunit-containing oligosaccharides is determined by GC-MS.

[0299] In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% anhydrosubunit-containing oligosaccharide. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15% anhydrosubunit-containing oligosaccharide. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, 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%, about 19% or about 20% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides in an amount of about 0.1% to about 15%, about 0.1% to about 20%, about 0.5% to about 20%, 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides in an amount of about 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the relative abundance of anhydrosubunit-containing oligosaccharides is determined by LC-MS / MS. In some embodiments, the relative abundance of anhydrosubunit-containing oligosaccharides is determined by GC-MS.

[0300] In some embodiments, the anhydrosubunit-containing oligosaccharide comprises one or more anhydrosubunits. For example, the DP1 anhydrosubunit-containing oligosaccharide comprises one anhydrosubunit. In some embodiments, the DPn anhydrosubunit-containing oligosaccharide may comprise 1 to n anhydrosubunits. For example, in some embodiments, the DP2 anhydrosubunit-containing oligosaccharide comprises one or two anhydrosubunits. In some embodiments, each oligosaccharide in the oligosaccharide preparation independently comprises zero, one, or two anhydrosubunits. In some embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the anhydrosubunit-containing oligosaccharides have only one anhydrosubunit. In some embodiments, more than 99%, 95%, 90%, 85%, or 80% of the anhydrosubunit-containing oligosaccharides have only one anhydrosubunit.

[0301] In some embodiments, one or more oligosaccharides in an oligosaccharide preparation or in each fraction of an oligosaccharide preparation include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 dehydrated subunits connected via glycosidic bonds, wherein the glycosidic bonds connecting each dehydrated subunit are independently selected. In some embodiments, one or more oligosaccharides in an oligosaccharide preparation or in each fraction of an oligosaccharide preparation include 1, 2 or 3 dehydrated subunits connected via glycosidic bonds, wherein the glycosidic bonds connecting each dehydrated subunit are independently selected. In some embodiments, more than 50%, 60%, 70%, 80%, 90% or 99% of oligosaccharides in an oligosaccharide preparation or in each fraction include 1, 2 or 3 dehydrated subunits connected via glycosidic bonds, wherein the glycosidic bonds connecting each dehydrated subunit are independently selected. In some embodiments, one or more oligosaccharides in an oligosaccharide preparation or in each fraction include 1 dehydrated subunit connected via glycosidic bonds. In some embodiments, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 99% of the oligosaccharides in the oligosaccharide preparation or in each fraction comprise one anhydro subunit linked via a glycosidic bond.

[0302] D. Anhydro subunit types

[0303] In some embodiments, the oligosaccharide preparation comprises different types of anhydro subunits. In some embodiments, exemplary anhydro subunit-containing oligosaccharides are shown in Figures 42, 30, and 31. In some embodiments, the oligosaccharide preparation comprises one or more anhydro subunits that are thermal dehydration products of monosaccharides, i.e., anhydromonosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises one or more anhydro subunits that are reversible thermal dehydration products of monosaccharides.

[0304] It should be understood that anhydromonosaccharides (or anhydromonosaccharide subunits) refer to thermal dehydration products of one or more types of monosaccharides. For example, in some embodiments, anhydroglucose refers to 1,6-anhydro-β-D-pyranose glucose (levoglucosan) or 1,6-anhydro-β-D-furanose glucose. In some embodiments, a plurality of anhydroglucose refers to a plurality of 1,6-anhydro-β-D-pyranose glucose (levoglucosan), a plurality of 1,6-anhydro-β-D-furanose glucose, a plurality of other thermal dehydration products of glucose, or any combination thereof. Similarly, in some embodiments, a plurality of anhydrogalactose refers to a plurality of any thermal dehydration products of galactose, or any combination thereof.

[0305] In some embodiments, the oligosaccharide preparations as described herein comprise one or more dehydrated glucose, dehydrated galactose, dehydrated mannose, dehydrated allose, dehydrated altrose, dehydrated gulose, dehydrated idose, dehydrated talose, dehydrated fructose, dehydrated ribose, dehydrated arabinose, dehydrated rhamnose, dehydrated lyxose, dehydrated xylose, or any combination of these subunits. In some embodiments, the oligosaccharide preparations comprise one or more dehydrated glucose, dehydrated galactose, dehydrated mannose, or dehydrated fructose subunits. In some embodiments, the oligosaccharide preparations described herein comprise one or more of: 1,6-anhydro-3-O-β-D-glucopyranosyl-β-D-glucopyranose, 1,6-anhydro-3-O-α-D-glucopyranosyl-β-D-glucopyranose, 1,6-anhydro-2-O-β-D-glucopyranosyl-β-D-glucopyranose, 1,6-anhydro-2-O-α-D-glucopyranosyl-β-D-glucopyranose , 1,6-anhydro-β-D-cellobiose (cellobiose), 1,6-anhydro-β-D-cellotriose (cellotriose), 1,6-anhydro-β-D-cellotetraose (cellotetraosan), 1,6-anhydro-β-D-cellopentaose (ellopentaosan), and 1,6-anhydro-β-D-maltose (maltosan).

[0306] In some embodiments, the oligosaccharide preparation comprises one or more 1,6-anhydro-β-D-glucopyranose subunits. In some embodiments, the oligosaccharide preparation comprises one or more 1,6-anhydro-β-D-glucopyranose (levoglucosan) subunits. For example, Figure 42 shows two DP1 anhydrosubunit-containing oligosaccharides (levoglucosan and 1,6-anhydro-β-D-glucopyranose) and one DP2 anhydrosubunit-containing oligosaccharide (anhydrocellobiose).

[0307] The presence and level of anhydro subunit species can vary according to the feed sugar used to make oligosaccharides. For example, in some embodiments, glucose oligosaccharides comprise anhydroglucose subunits, galactose oligosaccharides comprise anhydrogalactose subunits, and glucose-galactose oligosaccharides comprise anhydroglucose and anhydrogalactose subunits.

[0308] In some embodiments, the oligosaccharide preparation comprises two anhydro subunits, 1,6-anhydro-β-D-glucopyranose and 1,6-anhydro-β-D-glucopyranose. In some embodiments, at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the anhydro subunits are selected from 1,6-anhydro-β-D-glucopyranose and 1,6-anhydro-β-D-glucopyranose. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the anhydro subunits are 1,6-anhydro-β-D-glucopyranose. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the anhydro subunits are 1,6-anhydro-β-D-glucopyranose.

[0309] In some embodiments, in the formulation, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in the formulation is about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 8, 1: 9, or 1: 10. In some embodiments, the ratio of 1,6-anhydro-β-D-furanose to 1,6-anhydro-β-D-glucopyranose in the formulation is about 2: 1.

[0310] In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in each fraction is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose in each fraction is about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 8, 1: 9, or 1: 10. In some embodiments, the ratio of 1,6-anhydro-β-D-furanose to 1,6-anhydro-β-D-glucopyranose in each fraction is about 2: 1.

[0311] In some embodiments, in at least one fraction, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, in at least one fraction, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-furanose is about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 8, 1: 9, or 1: 10. In some embodiments, in at least one fraction, the ratio of 1,6-anhydro-β-D-furanose to 1,6-anhydro-β-D-glucopyranose is about 2: 1.

[0312] In some embodiments, oligosaccharide preparations as herein described comprise DP2 containing anhydro subunit oligosaccharides. In some embodiments, oligosaccharide preparations comprise anhydrolactose, anhydrosucrose, anhydrocellobiose or a combination thereof. In some embodiments, oligosaccharide preparations comprise about 2 to 20, 2 to 15, 5 to 20, 5 to 15 or 5 to 10 kinds of DP2 containing anhydro subunit oligosaccharides. In some embodiments, oligosaccharide preparations as herein described do not comprise polycellobiose or do not comprise detectable levels of polycellobiose.

[0313] In some embodiments, the oligosaccharide preparations described herein include one or more dehydrated subunits as sugar caramelization products. In some embodiments, the oligosaccharide preparations include one or more dehydrated subunits as sugar caramelization products selected from the group consisting of methanol, ethanol, furan, methylglyoxal, 2-methylfuran, vinyl acetate, glycolaldehyde, acetic acid, acetol, furfural, 2-furanmethanol, 3-furanmethanol, 2-hydroxycyclopent-2-ene-1-one, 5-methylfurfural, 2 (5H) -furanone, 2-methylcyclopentenolone, levulinone, cyclic hydroxylactone, 1,4,3,6-dianhydro-α-D-pyranose glucose, dianhydropyranose glucose, and 5-hydroxymethylfurfural (5-hmf). In some embodiments, the oligosaccharide preparations include 5-hmf dehydrated subunits.

[0314] In some embodiments, in an oligosaccharide preparation or in at least one DP fraction, anhydro subunits that are caramelization products are less abundant than anhydro subunits that are thermal dehydration products of monosaccharides. In some embodiments, in an oligosaccharide preparation or in at least one fraction, anhydro subunits that are caramelization products are more abundant than anhydro subunits that are thermal dehydration products of monosaccharides. In some embodiments, in an oligosaccharide preparation or in at least one fraction, anhydro subunits that are caramelization products and anhydro subunits that are thermal dehydration products of monosaccharides have similar abundance.

[0315] In some embodiments, about 0.01% to about 50%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3%, about 0.01% to about 2%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, or about 0.1% to about 0.5% of the anhydro subunits in the oligosaccharide preparations described herein are caramelization products. In some embodiments, about 0.1% to about 5%, about 0.1% to about 2%, or about 0.1% to about 1% of the anhydrous subunits in the oligosaccharide preparation are caramelized products. In some embodiments, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the anhydrous subunits in the oligosaccharide preparation are caramelized products.

[0316] In some embodiments, about 0.01% to about 50%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3%, about 0.01% to about 2%, about 0.01% to about 3 ... In some embodiments, about 0.1% to about 1%, about 0.01% to about 0.5%, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, or about 0.1% to about 0.5% of the anhydrous subunits are caramelized products. In some embodiments, about 0.1% to about 5%, about 0.1% to about 2%, or about 0.1% to about 1% of the anhydrous subunits in at least one fraction of the preparation (e.g., DP1, DP2, and / or DP3) are caramelized products. In some embodiments, less than 50%, 40%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the anhydrous subunits in at least one fraction of the preparation are charred products. In some embodiments, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the anhydrous subunits in the DP1, DP2 and / or DP3 fractions of the oligosaccharide preparations described herein are caramelized products.

[0317] In some embodiments, each fraction of the oligosaccharide preparations described herein comprises about 0.01% to about 50%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3%, about 0.01% to about 2%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, or about 0.1% to about 0.5% of the anhydrous subunits are caramelized products. In some embodiments, about 0.1% to about 5%, about 0.1% to about 2%, or about 0.1% to about 1% of the anhydrous subunits in each fraction of the preparation are caramelized products. In some embodiments, less than 50%, less than 40%, less than 30%, less than 20%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the anhydrosubunits in each fraction of the preparation are caramelization products.

[0318] In some embodiments, each oligosaccharide in the oligosaccharide preparations described herein independently and optionally comprises an anhydro subunit. In some embodiments, two or more independent oligosaccharides comprise the same or different anhydro subunits. In some embodiments, two or more independent oligosaccharides comprise different anhydro subunits. For example, in some embodiments, the oligosaccharide preparation comprises DP1 anhydro subunit-containing oligosaccharide, which comprises 1,6-anhydro-β-D-pyranose glucose subunits; and DP2 anhydro subunit-containing oligosaccharide, which comprises 1,6-anhydro-β-D-furanose glucose subunits. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation comprise two or more identical or different anhydro subunits.

[0319] In some embodiments, in any fraction (i.e., DP2 to DPn fractions) where the degree of polymerization of the oligosaccharide preparation is equal to or greater than 2, the dehydrated subunit may be connected to one or more conventional or dehydrated subunits. In some embodiments, in the DP2 to DPn fractions, at least one dehydrated subunit is connected to one, two, or three other conventional or dehydrated subunits. In some embodiments, in the DP2 to DPn fractions, at least one dehydrated subunit is connected to one or two conventional subunits. In some embodiments, in the DP2 to DPn fractions, at least one dehydrated subunit is connected to a conventional subunit. In some embodiments, in any one of the DP2 to DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the dehydrated subunits are connected to a conventional subunit. In some embodiments, in each of the DP2 to DPn fractions, greater than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the anhydro subunits are linked to one conventional subunit.

[0320] In some embodiments, in any fraction (i.e., DP2 to DPn fractions) of the oligosaccharide preparation having a degree of polymerization equal to or greater than 2, the anhydro subunit may be located at the end of the oligosaccharide chain. In some embodiments, in any fraction (i.e., DP3 to DPn fractions) of the oligosaccharide preparation having a degree of polymerization equal to or greater than 3, the anhydro subunit may be located at a position that is not the end of the oligosaccharide chain. In some embodiments, in the DP2 to DPn fractions, at least one anhydro subunit is located at the end of the oligosaccharide chain. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the anhydro subunits in the DP2 to DPn fractions are located at the end of the oligosaccharide chain. In some embodiments, greater than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the anhydro subunits in the oligosaccharide preparation are located at the end of the oligosaccharide chain. In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the anhydro subunit-containing oligosaccharides contain chain-terminal anhydro subunits. In some embodiments, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the anhydro subunit-containing oligosaccharides contain chain-terminal anhydro subunits.

[0321] E. Glycosidic linkage

[0322] In some embodiments, the oligosaccharide preparations described herein contain a variety of glycosidic linkages. The type and distribution of glycosidic linkages may depend on the source and manufacturing method of the oligosaccharide preparation. In some embodiments, the type and distribution of various glycosidic linkages may be determined and / or detected by any suitable method known in the art (e.g., NMR). For example, in some embodiments, the glycosidic linkages are determined by 1 HNMR, 13 C NMR, 2D NMR (such as 2D JRES, HSQC, HMBC, DOSY, COSY, ECOSY, TOCSY, NOESY or ROESY) or any combination thereof. In some embodiments, the glycosidic linkage is at least partially determined by 1 H NMR is used to determine and / or detect. In some embodiments, the glycosidic linkages are at least partially determined by 13 C NMR is used to determine and / or detect. In some embodiments, the glycosidic linkages are at least partially determined by 2D 1 H. 13 C-HSQC NMR for determination and / or detection.

[0323] In some embodiments, the oligosaccharide preparations described herein comprise one or more α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,4) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, β-(1,6) glycosidic linkages, α-(1,1)-α glycosidic linkages, α-(1,1)-β glycosidic linkages, β-(1,1)-β glycosidic linkages, or any combination thereof.

[0324] In some embodiments, the oligosaccharide preparation has about 0 to about 60 mol%, about 5% to about 55 mol%, about 5% to about 50 mol%, about 5% to about 45 mol%, about 5% to about 40 mol%, about 5% to about 35 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 10% to about 60 mol%, about 10% to about 55 mol%, about 10% to about 50 mol%, about 10% to about 45 mol%, about 10% to about 40 mol%, about 10% to about 35 mol%, about 15% to about 60 mol%, about 15% to about 55 mol%, about 15% to about 5 % to about 40 mol%, about 15% to about 35 mol%, about 20% to about 60 mol%, about 20% to about 55 mol%, about 20% to about 50 mol%, about 20% to about 45 mol%, about 20% to about 40 mol%, about 20% to about 35 mol%, about 25% to about 60 mol%, about 25% to about 55 mol%, about 25% to about 50 mol%, about 25% to about 45 mol%, about 25% to about 40 mol%, or about 25% to about 35 mol% of α-(1,6) glycosidic linkages.

[0325] In some embodiments, the oligosaccharide preparation has a glycosidic linkage type distribution of about 0 to about 50 mol%, about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 5% to about 40 mol%, about 5% to about 35 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 10% to about 40 mol%, about 10% to about 35 mol%, about 10% to about 20 mol%, about 15% to about 40 mol%, about 15% to about 35 mol%, about 15% to about 30 mol%, about 15% to about 25 mol%, or about 15% to about 20 mol% of α-(1,3) glycosidic linkages.

[0326] In some embodiments, the oligosaccharide preparation has a glycosidic linkage type distribution of about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 2% to about 30 mol%, about 2% to about 25 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, about 3% to about 30 mol%, about 3% to about 25 mol%, about 3% to about 20 mol%, about 3% to about 15 mol%, about 3% to about 10 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 5% to about 15 mol%, or about 5% to about 10 mol% of α-(1,2) glycosidic linkages.

[0327] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution of about 0 to about 40 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, or about 0 to about 5 mol% of α-(1,4) glycosidic linkages. In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution of less than 40 mol%, less than 30 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, less than 9 mol%, less than 8 mol%, less than 7 mol%, less than 6 mol%, less than 5 mol%, less than 4 mol%, less than 3 mol%, or less than 2 mol% of α-(1,4) glycosidic linkages.

[0328] In some embodiments, the oligosaccharide preparation has a glycosidic linkage type distribution of about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 2% to about 30 mol%, about 2% to about 25 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 5% to about 15 mol%, about 5% to about 10 mol%, about 8% to about 30 mol%, about 8% to about 25 mol%, about 8% to about 20 mol%, about 8% to about 15 mol%, or about 10% to about 15 mol% of β-(1,6) glycosidic linkages.

[0329] In some embodiments, the oligosaccharide preparation has a glycosidic linkage type distribution of about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 2% to about 30 mol%, about 2% to about 25 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, about 3% to about 30 mol%, about 3% to about 25 mol%, about 3% to about 20 mol%, about 3% to about 15 mol%, about 3% to about 10 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 5% to about 15 mol%, or about 5% to about 10 mol% of β-(1,4) glycosidic linkages.

[0330] In some embodiments, the oligosaccharide preparation has a glycosidic linkage type distribution of about 0 to about 40 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 0 to about 5 mol%, about 1% to about 20 mol%, about 1% to about 15 mol%, about 1% to about 10 mol%, about 1% to about 5 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, or about 2% to about 5 mol% of β-(1,2) glycosidic linkages. In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution of less than 40 mol%, less than 30 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, less than 9 mol%, less than 8 mol%, less than 7 mol%, less than 6 mol%, less than 5 mol%, less than 4 mol%, less than 3 mol% or less than 2 mol% β-(1,2) glycosidic bonds.

[0331] In some embodiments, the oligosaccharide preparation has a glycosidic linkage type distribution of about 0 to about 40 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 0 to about 5 mol%, about 1% to about 20 mol%, about 1% to about 15 mol%, about 1% to about 10 mol%, about 1% to about 5 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, or about 2% to about 5 mol% of β-(1,3) glycosidic linkages. In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution of less than 40 mol%, less than 30 mol%, less than 20 mol%, less than 15 mol%, less than 10 mol%, less than 9 mol%, less than 8 mol%, less than 7 mol%, less than 6 mol%, less than 5 mol%, less than 4 mol%, less than 3 mol% or less than 2 mol% β-(1,3) glycosidic bonds.

[0332] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution that is different from the glycosidic bond type distribution of the non-synthetic oligosaccharide preparation. For example, in some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution that is different from the glycosidic bond type distribution of the basic nutritional composition. In some embodiments, the basic nutritional composition comprises a natural carbohydrate source, such as starch and plant fiber. Some of the natural carbohydrate sources have a high percentage of α-(1,4), α-(1,6) and / or β-(1,6) glycosidic linkages. Therefore, in some embodiments, the oligosaccharide preparation has a lower percentage of α-(1,4) glycosidic linkages than the basic nutritional composition. In some embodiments, the oligosaccharide preparation has a lower percentage of α-(1,6) glycosidic linkages than the basic nutritional composition. In other embodiments, the oligosaccharide preparation has a higher percentage of α-(1,6) glycosidic linkages than the basic nutritional composition. In some embodiments, the oligosaccharide preparation has a lower percentage of β-(1,6) glycosidic linkages than the basic nutritional composition. In some embodiments, the oligosaccharide preparation comprises glycosidic linkages that are not easily digested or hydrolyzed by enzymes.

[0333] Specifically, in some embodiments, the oligosaccharide preparations described herein have at least 50 mol%, at least 40 mol%, at least 30 mol%, at least 20 mol%, at least 15 mol%, at least 10 mol%, at least 5 mol%, at least 2 mol% or at least 1 mol% lower α-(1,2), α-(1,3), α-(1,4), α-(1,6), β-(1,2), β-(1,3), β-(1,4) and / or β-(1,6) glycosidic linkages in the glycosidic bond type distribution than the basic nutritional composition. In some embodiments, the oligosaccharide preparation has at least 50 mol%, at least 40 mol%, at least 30 mol%, at least 20 mol%, at least 15 mol%, at least 10 mol%, at least 5 mol%, at least 2 mol%, or at least 1 mol% more α-(1,2), α-(1,3), α-(1,4), α-(1,6), β-(1,2), β-(1,3), β-(1,4), and / or β-(1,6) glycosidic linkages in the glycosidic bond type distribution than the base nutritional composition.

[0334] It will be understood by those skilled in the art that certain types of glycosidic linkages may not be suitable for oligosaccharides containing certain types of monosaccharides. For example, in some embodiments, the oligosaccharide preparation comprises α-(1,2) glycosidic linkages and α-(1,6) glycosidic linkages. In other embodiments, the oligosaccharide preparation comprises α-(1,2) glycosidic linkages and β-(1,3) glycosidic linkages. In some embodiments, the oligosaccharide preparation comprises α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, and β-(1,6) glycosidic linkages. In some embodiments, the oligosaccharide preparation comprises α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, and β-(1,6) glycosidic linkages.

[0335] F. Molecular weight

[0336] The molecular weight and molecular weight distribution of the oligosaccharide preparation can be determined by any suitable analytical means and instrument (such as end group method, osmotic pressure (osmotic pressure determination method), ultracentrifugation, viscosity measurement, light scattering, SEC, SEC-MALLS, FFF, A4F, HPLC and mass spectrometry). In some embodiments, the molecular weight and molecular weight distribution are determined by mass spectrometry (such as MALDI-MS, LC-MS or GC-MS). In some embodiments, the molecular weight and molecular weight distribution are determined by size exclusion chromatography (SEC) (such as gel permeation chromatography (GPC)). In other embodiments, the molecular weight and molecular weight distribution are determined by HPLC. In some embodiments, the molecular weight and molecular weight distribution are determined by MALDI-MS.

[0337] In some embodiments, the oligosaccharide preparations described herein have a g / mol of about 100 to about 10,000 g / mol, about 200 to about 8,000 g / mol, about 300 to about 5,000 g / mol, about 500 to about 5,000 g / mol, about 700 to about 5,000 g / mol, about 900 to about 5,000 g / mol, about 1,100 to about 5,000 g / mol, about 1,300 to about 5,000 g / mol, about 1,500 to about 5,000 g / mol, about 1,700 to about 5,000 g / mol, about 300 to about 4,500 g / mol, about 500 to about 4,500 g / mol, about 700 to about 4,500 g / mol, about 900 to about 4,500 g / mol, g / mol, about 100 to about 4500 g / mol, about 1300 to about 4500 g / mol, about 1500 to about 4500 g / mol, about 1700 to about 4500 g / mol, about 1900 to about 4500 g / mol, about 300 to about 4000 g / mol, about 500 to about 4000 g / mol, about 700 to about 4000 g / mol, about 900 to about 4000 g / mol, about 1100 to about 4000 g / mol, about 1300 to about 4000 g / mol, about 1500 to about 4000 g / mol, about 1700 to about 4000 g / mol, about 1900 to about 4000 g / mol , about 300 to about 3000 g / mol, about 500 to about 3000 g / mol, about 700 to about 3000 g / mol, about 900 to about 3000 g / mol, about 1100 to about 3000 g / mol, about 1300 to about 3000 g / mol, about 1500 to about 3000 g / mol, about 1700 to about 3000 g / mol, about 1900 to about 3000 g / mol, about 2100 to about 3000 g / mol, about 300 to about 2500 g / mol, about 500 to about 2500 g / mol, about 700 to about 2500 g / mol, about 900 to about 2500 g / mol, about 1100 to about 250 g / mol, about 1500 to about 1500 g / mol, about 1700 to about 2500 g / mol, about 1900 to about 2500 g / mol, about 2100 to about 2500 g / mol, about 300 to about 1500 g / mol, about 500 to about 1500 g / mol, about 700 to about 1500 g / mol, about 900 to about 1500 g / mol, about 1100 to about 1500 g / mol, about 1300 to about 1500 g / mol, about 2000 to about 2800 g / mol, about 2100 to about 2700 g / mol, about 2200 to about 2600 g / mol,In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 2000 to about 2800 g / mol, about 2100 to about 2700 g / mol, about 2200 to about 2600 g / mol, about 2300 to about 2500 g / mol, or about 2320 to about 2420 g / mol. In some embodiments, the oligosaccharide preparation has a weight average molecular weight ranging from at least 500 g / mol, 750 g / mol, 1000 g / mol, or 1500 g / mol to at most 1750 g / mol, 2000 g / mol, 2250 g / mol, 2500 g / mol, or 3000 g / mol. In some embodiments, the weight average molecular weight of the oligosaccharide preparations described herein is determined by HPLC according to Example 9.

[0338] In some embodiments, the oligosaccharide preparations described herein have a saturated fat content of about 100 to about 10,000 g / mol, about 200 to about 8,000 g / mol, about 300 to about 5,000 g / mol, about 500 to about 5,000 g / mol, about 700 to about 5,000 g / mol, about 900 to about 5,000 g / mol, about 1,100 to about 5,000 g / mol, about 1,300 to about 5,000 g / mol, about 1,500 to about 5,000 g / mol, about 1,700 to about 5,000 g / mol, about 300 to about 4,500 g / mol, about 500 to about 4,500 g / mol, about 700 to about 4,500 g / mol, about 900 to about 4,500 g / mol, about 1100 to about 4500 g / mol, about 1300 to about 4500 g / mol, about 1500 to about 4500 g / mol, about 1700 to about 4500 g / mol, about 1900 to about 4500 g / mol, about 300 to about 4000 g / mol, about 500 to about 4000 g / mol, about 700 to about 4000 g / mol, about 900 to about 4000 g / mol, about 1100 to about 4000 g / mol, about 1300 to about 4000 g / mol, about 1500 to about 4000 g / mol, about 1700 to about 4000 g / mol, about 1900 to about 4000 g / mol, about 300 to about 3000 g / mol, about 500 to about 3000 g / mol, about 700 to about 3000 g / mol, about 900 to about 3000 g / mol, about 1100 to about 3000 g / mol, about 1300 to about 3000 g / mol, about 1500 to about 3000 g / mol, about 1700 to about 3000 g / mol, about 1900 to about 3000 g / mol, about 2100 to about 3000 g / mol, about 300 to about 2500 g / mol, about 500 to about 2500 g / mol, about 700 to about 2500 g / mol, about 900 to about 2500 g / mol, about 1100 to about 2500 g / mol, about 1300 to about 2500 g / mol, about g / mol, about 1500 to about 2500 g / mol, about 1700 to about 2500 g / mol, about 1900 to about 2500 g / mol, about 2100 to about 2500 g / mol, about 300 to about 2000 g / mol, about 500 to about 300 to 2000 g / mol, about 700 to about 2000 g / mol, about 900 to about 2000 g / mol, about 1100 to about 2000 g / mol, about 300 to about 1500 g / mol, about 500 to about 1500 g / mol, about 700 to about 1500 g / mol, about 900 to about 1500 g / mol, about 1100 to about 1500 g / mol, about 1300 to about 1500 g / mol,In some embodiments, the number average molecular weight of the oligosaccharide preparation is about 1000 to about 2000 g / mol, about 1100 to about 1900 g / mol, about 1200 to about 1800 g / mol, about 1300 to about 1700 g / mol, about 1400 to about 1600 g / mol, or about 1450 to about 1550 g / mol. In some embodiments, the number average molecular weight of the oligosaccharide preparation is about 1000 to about 2000 g / mol, about 1100 to about 1900 g / mol, about 1200 to about 1800 g / mol, about 1300 to about 1700 g / mol, 1400 to 1600 g / mol, or 1450-1550 g / mol. In some embodiments, the oligosaccharide preparations have a number average molecular weight ranging from at least 500 g / mol, 750 g / mol, 1000 g / mol, or 1500 g / mol to at most 1750 g / mol, 2000 g / mol, 2250 g / mol, 2500 g / mol, or 3000 g / mol. In some embodiments, the number average molecular weight of the oligosaccharide preparations described herein is determined by HPLC according to Example 9.

[0339] G. Types of Oligosaccharides

[0340] The kind of oligosaccharide present in the oligosaccharide preparation can depend on the type of described one or more feed sugars.For example, in some embodiments, when feed sugar includes glucose, oligosaccharide preparation comprises glucose oligosaccharide.For example, in some embodiments, when feed sugar includes galactose, oligosaccharide preparation comprises galactose oligosaccharide.For another example, in some embodiments, when feed sugar includes galactose and glucose, oligosaccharide preparation comprises glucose-galactose oligosaccharide.

[0341] In some embodiments, oligosaccharide preparations as described herein comprise one or more types of monosaccharide subunits. In some embodiments, oligosaccharide preparations comprise oligosaccharides with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different types of monosaccharide subunits.

[0342] In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 1, 2, 3 or 4 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 1, 2 or 3 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 3 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 2 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises one type of monosaccharide subunit.

[0343] In some embodiments, the oligosaccharide preparation comprises different types of oligosaccharides, wherein each oligosaccharide molecule independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation described herein comprises 10 2 , 10 3 , 10 4 , 10 5 In some embodiments, the oligosaccharide preparation comprises one or more different types of oligosaccharides. In some embodiments, some oligosaccharides in the preparation comprise one type of monosaccharide subunit, and some other oligosaccharides in the same preparation comprise two or more types of monosaccharide subunits. For example, in some embodiments, when the feed sugar is glucose and galactose, the oligosaccharide preparation can comprise oligosaccharides that only comprise glucose subunits, oligosaccharides that only comprise galactose subunits, oligosaccharides that comprise different ratios of glucose and galactose subunits, or any combination thereof.

[0344] In some embodiments, any or all of the n fractions of an oligosaccharide preparation comprise different species of oligosaccharide subunits, wherein each oligosaccharide independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different species of monosaccharide subunits. In some embodiments, some oligosaccharides in the fractions of the preparation comprise one species of monosaccharide subunit, and some other oligosaccharides in the same fraction of the preparation comprise two or more species of monosaccharide subunits.

[0345] In some embodiments, the oligosaccharide preparations described herein comprise one or more monosaccharide subunits selected from the group consisting of trisaccharides, tetrasaccharides, pentoses, hexoses, heptoses, and any combination thereof, wherein each of the trisaccharide, tetrasaccharide, pentose, hexose, or heptose subunits is independently and optionally functionalized and / or replaced by one of its corresponding anhydro subunits. In some embodiments, the corresponding anhydro subunit is a thermal dehydration product of a monosaccharide subunit. In some embodiments, the corresponding anhydro subunit is a caramelization product of a monosaccharide subunit.

[0346] In some embodiments, the oligosaccharide preparations described herein comprise pentose subunits, hexose subunits, or any combination thereof, wherein each of the pentose or hexose subunits is independently and optionally functionalized and / or replaced by one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparations comprise hexose subunits, wherein each of the hexose subunits is independently and optionally replaced by one of its corresponding anhydro subunits.

[0347] As used herein, tetrasaccharides refer to monosaccharides with four carbon atoms, such as erythrose, threose, and erythrulose. As used herein, pentoses refer to monosaccharides with five carbon atoms, such as arabinose, lyxose, ribose, and xylose. As used herein, hexoses refer to monosaccharides with six carbon atoms, such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose. As used herein, heptoses refer to monosaccharides with seven carbon atoms, such as sedoheptulose and mannoheptulose.

[0348] In some embodiments, oligosaccharide preparations as described herein include glucose subunits, wherein at least one glucose subunit is optionally replaced by anhydroglucose subunits. In some embodiments, oligosaccharide preparations as described herein include galactose subunits, wherein at least one galactose subunit is optionally replaced by anhydrogalactose subunits. In some embodiments, oligosaccharide preparations as described herein include galactose and glucose subunits, wherein at least one galactose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydro subunits. In some embodiments, oligosaccharide preparations as described herein include fructose and glucose subunits, wherein at least one fructose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydro subunits. In some embodiments, oligosaccharide preparations as described herein include mannose and glucose subunits, wherein at least one mannose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydro subunits.

[0349] In some embodiments, the oligosaccharide preparations described herein include glucose-galactose oligosaccharide preparations, glucose oligosaccharide preparations, galactose oligosaccharide preparations, fructose oligosaccharide preparations, mannose oligosaccharide preparations, arabinose oligosaccharide preparations, xylose oligosaccharide preparations, glucose-fructose oligosaccharide preparations, glucose-mannose oligosaccharide preparations, glucose-arabinose oligosaccharide preparations, glucose-xylose oligosaccharide preparations, galactose-fructose oligosaccharide preparations, galactose-mannose oligosaccharide preparations, galactose-arabinose oligosaccharide preparations, galactose-xylose oligosaccharide preparations, fructose-mannose oligosaccharide preparations, fructose-arabinose oligosaccharide preparations, fructose-xylose oligosaccharide preparations, A mannose-arabinose oligosaccharide preparation, a mannose-xylose oligosaccharide preparation, an arabinose-xylose oligosaccharide preparation, a galactose-arabinose-xylose oligosaccharide preparation, a fructose-galactose-xylose oligosaccharide preparation, an arabinose-fructose-mannose-xylose oligosaccharide preparation, a glucose-fructose-galactose-arabinose oligosaccharide preparation, a fructose-glucose-arabinose-mannose-xylose oligosaccharide preparation, a glucose-galactose-fructose-mannose-arabinose-xylose oligosaccharide preparation, or any combination thereof; wherein each of the monosaccharide subunits within the preparation is independently and optionally functionalized and / or replaced with one of its corresponding anhydro subunits.

[0350] In certain embodiments, the oligosaccharide preparations described herein comprise greater than 99% glucose subunits by weight. In some embodiments, the oligosaccharide preparations comprise only glucose subunits.

[0351] In some embodiments, the oligosaccharide preparations described herein comprise about 45% to 55% glucose subunits and about 55% to 45% galactose subunits by weight. In some specific embodiments, the oligosaccharide preparations comprise about 50% glucose subunits and 50% galactose subunits by weight.

[0352] In some embodiments, the oligosaccharide preparations described herein comprise about 80% to 95% glucose subunits and about 20% to 5% mannose subunits by weight. In some embodiments, the oligosaccharide preparations comprise about 85% to 90% glucose subunits and about 15% to 10% mannose subunits by weight.

[0353] In some embodiments, the oligosaccharide preparations described herein comprise about 80% to 95% glucose subunits and about 20% to 5% galactose subunits by weight. In some embodiments, the oligosaccharide preparations comprise about 85% to 90% glucose subunits and about 15% to 10% galactose subunits by weight.

[0354] In some embodiments, the oligosaccharide preparations described herein comprise about 80% to 95% glucose subunits, 0% to 8% galactose subunits, and 5% to 20% mannose subunits by weight. In some embodiments, the oligosaccharide preparations comprise about 80% to 90% glucose subunits, 1% to 5% galactose subunits, and 10% to 15% mannose subunits by weight.

[0355] In some embodiments, the oligosaccharide preparations described herein comprise glucose subunits in the range of about 1 wt% to about 100 wt%, about 50 wt% to about 100 wt%, about 80 wt% to about 98 wt% or about 85 wt% to about 95 wt% or any range therebetween. In some embodiments, the galactose subunit is present in the oligosaccharide preparations described herein in the range of about 0 wt% to about 90 wt%, about 1 wt% to about 50 wt%, about 2 wt% to about 20 wt% or about 5 wt% to about 15 wt% or any range therebetween. In some embodiments, the mannose subunit is present in the oligosaccharide preparations described herein in the range of about 0 wt% to about 90 wt%, about 1 wt% to about 50 wt%, about 2 wt% to about 20 wt% or about 5 wt% to about 15 wt% or any range therebetween.

[0356] In some embodiments, the oligosaccharide preparations described herein have a composition of monosaccharide subunits as shown in Table 29.

[0357] Table 29. Exemplary composition of oligosaccharide formulations

[0358]

[0359]

[0360] HD mode and L mode

[0361] In some embodiments, at least one monosaccharide subunit in the oligosaccharide is in the L form. In some embodiments, at least one monosaccharide subunit in the oligosaccharide is in the D form. In some embodiments, the monosaccharide subunits in the oligosaccharide preparations described herein are in their naturally abundant forms, such as D-glucose, D-xylose, and L-arabinose.

[0362] In some embodiments, the oligosaccharide preparations described herein comprise a mixture of monosaccharide subunits in the L form and the D form. In some embodiments, the ratio of monosaccharide subunits in the L form to the D form or the D form to the L form is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:14, about 1:16, about 1:18, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, about 1:100, or about 1:150.

[0363] I. Functionalized Oligosaccharides

[0364] In some embodiments, one or more oligosaccharides in the formulation are independently functionalized. Functionalized oligosaccharides can be produced by, for example, merging one or more sugars with one or more functionalized compounds in the presence of a catalyst. Methods for producing functionalized oligosaccharides are described in WO 2012 / 118767, WO 2014 / 031956, and WO / 2016 / 122887, which are hereby incorporated by reference in their entirety and with respect to their disclosures.

[0365] In some embodiments, the functionalized compound comprises one or more acid groups (e.g., -COOH), hydroxyl groups, or N-containing groups (e.g., -CN, -NO2, and -N(R a )2, where R aIn some embodiments, the functionalized compound is linked to at least one monosaccharide subunit via an ether, ester, oxygen-sulfur, amine, or oxygen-phosphorus bond. In some embodiments, one or more functionalized compounds are linked to a monosaccharide subunit via a single bond. In some embodiments, at least one functionalized compound is linked to one or two oligosaccharides via two or more bonds.

[0366] It should be understood that for each oligosaccharide in the oligosaccharide preparation, each of the embodiments described is independent and combinable, just as if each combination were listed separately; therefore, the present disclosure covers any combination of embodiments. For example, the various embodiments can be divided into several categories, including but not limited to (i) the presence or absence of anhydro subunits; (ii) the number and level of anhydro subunits; (iii) the type of anhydro subunits; (iv) the position of the anhydro subunits; (v) the degree of polymerization; (vi) the molecular weight; (vii) the presence or absence of any functional groups; (viii) the type of oligosaccharide; (ix) the type of glycosidic linkage; and (x) L form versus D form. Thus, the oligosaccharide preparations described contain a plurality of oligosaccharides of different types. In some embodiments, the oligosaccharide preparations described herein contain at least 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 In some embodiments, the formulation comprises at least 10 3 , 10 4 , 10 5 , 10 6 or 10 9 In some embodiments, the formulation comprises at least 10 3 Different types of oligosaccharides.

[0367] III. Methods for producing oligosaccharide preparations

[0368] In one aspect, provided herein are methods for making oligosaccharide preparations. In some embodiments, provided herein are methods for making oligosaccharide preparations suitable for use in nutritional compositions (such as animal feed compositions) or for feeding directly to animals. In one aspect, provided herein are methods for making oligosaccharide preparations, the methods comprising heating an aqueous composition comprising one or more feed sugars and a catalyst to a temperature sufficient to induce polymerization and for a time sufficient to induce polymerization, wherein the catalyst is selected from: (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate; α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid; butylphosphonic acid; biphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; Phosphonic acid; phenylphosphonic acid; tert-butylphosphonic acid; SS)-VAPOL hydrogen phosphate; 6-quinolinesulfonic acid; 3-(1-pyrido)-1-propanesulfonate; 2-(2-pyridyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-biphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-cysteic acid monohydrate; poly(phenylene glycol) olefinsulfonic acid-co-divinylbenzene); lysine; ethanedisulfonic acid; ethanesulfonic acid; isethionic acid; homocysteic acid; HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid; methanesulfonic acid; methylsulfonic acid hydrazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; trifluoromethanesulfonic acid; 2-amino ethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; hexanoic acid; heptanoic acid; octanoic acid; nonanoic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan, and wherein the oligosaccharide preparation comprises at least n oligosaccharide fractions each having a different degree of polymerization selected from 1 (DP1 fraction) to n (DPn fraction), wherein n is an integer greater than 2.

[0369] In some embodiments, n is an integer greater than or equal to 3. In some embodiments, n is an integer in the range of 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, polymerization of the feed sugar is achieved by step-growth polymerization. In some embodiments, polymerization of the feed sugar is achieved by condensation polymerization.

[0370] A. Feed sugar

[0371] In some embodiments, the methods of making oligosaccharide preparations described herein comprise heating one or more types of feed sugars. In some embodiments, the one or more types of feed sugars comprise monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or any mixture thereof.

[0372] In some embodiments, the one or more feed sugars include glucose. In some embodiments, the one or more feed sugars include glucose and galactose. In some embodiments, the one or more feed sugars include glucose, xylose and galactose. In some embodiments, the one or more feed sugars include glucose and mannose. In some embodiments, the one or more feed sugars include glucose and fructose. In some embodiments, the one or more feed sugars include glucose, fructose and galactose. In some embodiments, the one or more feed sugars include glucose, galactose and mannose.

[0373] In some embodiments, the one or more feed sugars include disaccharides such as lactose, sucrose and cellobiose. In some embodiments, the one or more feed sugars include trisaccharides such as maltotriose or raffinose. In certain embodiments, the one or more feed sugars include glucose, mannose, galactose, xylose, maltodextrin, arabinose or galactose or any combination thereof. In certain embodiments, the one or more feed sugars include syrup such as corn syrup. In some embodiments, the one or more feed sugars include glucose and lactose. In some embodiments, the one or more feed sugars include glucose and sucrose.

[0374] In some embodiments, the type of feed sugar can affect the oligosaccharide preparation of the obtained manufacture.For example, in some modifications in which the one or more feed sugars are all glucose, the oligosaccharide preparation obtained includes glucose oligosaccharide preparation.In other embodiments, when the one or more feed sugars are all mannose, the oligosaccharide preparation obtained includes mannose oligosaccharide preparation.In some embodiments, when the one or more feed sugars include glucose and galactose, the oligosaccharide preparation obtained includes glucose-galactose oligosaccharide preparation.In other embodiments, when the one or more feed sugars include xylose, glucose and galactose, the oligosaccharide preparation obtained includes glucose-galactose-xylose oligosaccharide preparation.

[0375] In some embodiments, each of the one or more feed sugars can be independently in the form of its dehydrate or hydrate. In some embodiments, the one or more feed sugars include glucose, galactose, fructose, mannose or any combination thereof, and wherein each of glucose, galactose, fructose or mannose is independently in the form of its monohydrate or dehydrate. In some embodiments, the one or more feed sugars include monosaccharide monohydrates, such as glucose monohydrate. In some embodiments, the one or more feed sugars include sugar dihydrates, such as trehalose dihydrate. In some embodiments, the one or more feed sugars include at least one sugar in the form of its dehydrate and at least one sugar in the form of its hydrate.

[0376] In some embodiments, the one or more feed sugars can be provided as a sugar solution, wherein the sugar is combined with water and fed into the reactor. In some embodiments, the sugar can be fed into the reactor in solid form and combined with water in the reactor. In some embodiments, the one or more feed sugars are combined and mixed before adding water. In other embodiments, the one or more feed sugars are combined into water and mixed thereafter.

[0377] In some embodiments, the method includes merging two or more feed sugars with a catalyst to produce an oligosaccharide preparation. In some embodiments, the two or more feed sugars include glucose, galactose, fructose, mannose, lactose or any combination thereof. In some embodiments, the method includes merging a mixture of sugar (e.g., monosaccharide, disaccharide and / or trisaccharide) with a catalyst to produce an oligosaccharide preparation. In other embodiments, the method includes merging a mixture of sugar and sugar alcohol with a catalyst to produce an oligosaccharide preparation.

[0378] In some embodiments, the one or more feed sugars include functionalized or modified sugars. Functionalized or modified sugars can include amino sugars, sugar acids, sugar alcohols, sugar amides, sugar ethers or any combination thereof. In some embodiments, amino sugars refer to sugar molecules in which hydroxyl groups are replaced by amino groups. Exemplary amino sugars include but are not limited to N-acetyl-d-glucosamine, mannosamine, neuraminic acid, muramic acid, N-acetyl-neuramine (neuramin), N-acetyl-muramic acid (muramic), N-acetyl-galactosamine, N-acetyl-mannosamine (mannosa), N-glycolylneuraminic acid (N-glycolylneuram), acarviosin (acarviosin), D-glucosamine and D-galactosamine.

[0379] In embodiments, sugar acid refers to a sugar having a carboxyl group. Exemplary sugar acids include, but are not limited to, aldonic acids (such as glyceric acid, xylonic acid, gluconic acid, and ascorbic acid), ketonic acids (such as neuraminic acid and ketodeoxyoctulonic acid), uronic acids (such as glucuronic acid, galacturonic acid, and iduronic acid), and saccharic acids (such as tartaric acid, mucic acid, and saccharic acid).

[0380] In some embodiments, sugar alcohol refers to a polyol derived from a sugar. Exemplary sugar alcohols include, but are not limited to, ethylene glycol, arabitol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, and volemitol.

[0381] In some embodiments, sugar amide refers to a sugar molecule containing a -C(=O)-N- group. In some embodiments, sugar ether refers to a sugar molecule containing an ether linkage, such as a glucoside.

[0382] In some embodiments, the functionalized or modified sugars include glucosamine, N-acetylglucosamine, glucuronic acid, galacturonic acid, glucitol, xylitol, mannitol, sorbitol. In some embodiments, the one or more feed sugars include deoxy sugars such as fucose, rhamnose, deoxyribose or fuculose.

[0383] In some embodiments, the method for manufacturing oligosaccharide preparations as herein described is carried out on a gram scale. In some embodiments, the method for manufacturing oligosaccharide preparations as herein described is carried out on a kilogram or higher scale. Therefore, in some embodiments, the method comprises heating an aqueous composition, and the aqueous composition comprises one or more feed sugars of an amount exceeding 0.5, exceeding 1, exceeding 2, exceeding 3, exceeding 4, exceeding 5, exceeding 6, exceeding 7, exceeding 9, exceeding 10, exceeding 100 or exceeding 1000kg. In some embodiments, the method comprises heating an aqueous composition, and the aqueous composition comprises one or more feed sugars of an amount exceeding 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10, 100, 1000 or 1500kg. In some embodiments, the method comprises heating an aqueous composition, and the aqueous composition comprises one or more feed sugars of an amount exceeding 1kg.

[0384] B. Catalyst

[0385] In some embodiments, the catalysts provided herein include one or more acids. In some embodiments, the catalysts provided herein include mineral acids; carboxylic acids; amino acids; sulfonic acids; boric acids; phosphonic acids; phosphinic acids; sulfuric acid; phosphoric acid; poly(styrenesulfonic acid-co-vinylbenzyl-imidazolium sulfate-co-divinylbenzene); poly(styrenesulfonic acid-co-divinylbenzene); (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate; α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid; Butylphosphonic acid; biphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; SS)-VAPOL hydrogen phosphate; 6-quinolinesulfonic acid; 3-(1-pyrido)-1-propanesulfonate; 2-(2-pyridyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-biphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate; bis(4-methoxyphenyl)phosphinic acid ; Phenyl(3,5-xylyl)phosphinic acid; L-cysteic acid monohydrate; Acetic acid; Propionic acid; Butyric acid; Glutamic acid; Lysine; Ethanedisulfonic acid; Ethansulfonic acid; Isethionic acid; Homocysteic acid; HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 2-Hydroxy-3-morpholinopropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid; Methanesulfonic acid; Methylsulfonic acid hydrazide; Naphthalene- 1-Aminoethanesulfonic acid; 1-aminoethanesulfonic acid; 1-aminobutane ...

[0386] In some embodiments, the catalysts provided herein include: (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate; α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; SS)-VAPOL hydrogen phosphate; 6-quinolinesulfonic acid acid; 3-(1-pyrido)-1-propanesulfonate; 2-(2-pyridyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-biphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diyl-hydrogen phosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-sulfoalanine monohydrate; poly(styrenesulfonic acid- co-divinylbenzene); lysine; ethanedisulfonic acid; ethanesulfonic acid; isethionic acid; homocysteic acid; HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid; methanesulfonic acid; methylsulfonic acid hydrazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; Perfluorobutanesulfonic acid; 6-sulfoquinovose; trifluoromethanesulfonic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; hexanoic acid; heptanoic acid; octanoic acid; nonanoic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan; or any combination thereof.

[0387] In some embodiments, the catalyst provided herein is (+)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is 2-pyridinesulfonic acid. In some embodiments, the catalyst provided herein is 3-pyridinesulfonic acid. In some embodiments, the catalyst provided herein is 8-hydroxy-5-quinolinesulfonic acid hydrate. In some embodiments, the catalyst provided herein is α-hydroxy-2-pyridinemethanesulfonic acid. In some embodiments, the catalyst provided herein is (β)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is butylphosphonic acid. In some embodiments, the catalyst provided herein is diphenylphosphinic acid. In some embodiments, the catalyst provided herein is hexylphosphonic acid. In some embodiments, the catalyst provided herein is methylphosphonic acid. In some embodiments, the catalyst provided herein is phenylphosphinic acid. In some embodiments, the catalyst provided herein is phenylphosphonic acid. In some embodiments, the catalyst provided herein is tert-butylphosphonic acid. In some embodiments, the catalyst provided herein is S(-VAPOL) hydrogen phosphate. In some embodiments, the catalyst provided herein is 6-quinolinesulfonic acid. In some embodiments, the catalyst provided herein is 3-(1-pyrido)-1-propane sulfonate. In some embodiments, the catalyst provided herein is 2-(2-pyridyl)ethane sulfonic acid. In some embodiments, the catalyst provided herein is 3-(2-pyridyl)-5,6-biphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate. In some embodiments, the catalyst provided herein is 1,1'-binaphthyl-2,2'-diyl-hydrogen phosphate. In some embodiments, the catalyst provided herein is bis(4-methoxyphenyl)phosphinic acid. In some embodiments, the catalyst provided herein is phenyl(3,5-xylyl)phosphinic acid. In some embodiments, the catalyst provided herein is L-sulfoalanine monohydrate. In some embodiments, the catalyst provided herein is poly(styrenesulfonic acid-to-divinylbenzene). In some embodiments, the catalyst provided herein is lysine.

[0388] In some embodiments, the catalyst is ethanedisulfonic acid. In some embodiments, the catalyst is ethanesulfonic acid. In some embodiments, the catalyst is isethionic acid. In some embodiments, the catalyst is homocysteic acid. In some embodiments, the catalyst is HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)). In some embodiments, the catalyst is HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the catalyst is 2-hydroxy-3-morpholinopropanesulfonic acid. In some embodiments, the catalyst is 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the catalyst is methanesulfonic acid. In some embodiments, the catalyst is naphthalene-1-sulfonic acid. In some embodiments, the catalyst is methylsulfonic acid hydrazide. In some naphthalene-2-sulfonic acid. In some embodiments, the catalyst is perfluorobutanesulfonic acid. In some embodiments, the catalyst is 6-sulfoquinovose. In some embodiments, the catalyst is trifluoromethanesulfonic acid. In some embodiments, the catalyst is 2-aminoethanesulfonic acid. In some embodiments, the catalyst is benzoic acid. In some embodiments, the catalyst is chloroacetic acid. In some embodiments, the catalyst is trifluoroacetic acid. In some embodiments, the catalyst is hexanoic acid. In some embodiments, the catalyst is heptanoic acid. In some embodiments, the catalyst is octanoic acid. In some embodiments, the catalyst is nonanoic acid. In some embodiments, the catalyst is lauric acid. In some embodiments, the catalyst is palmitic acid. In some embodiments, the catalyst is stearic acid. In some embodiments, the catalyst is arachidic acid. In some embodiments, the catalyst is aspartic acid. In some embodiments, the catalyst is glutamic acid. In some embodiments, the catalyst is serine. In some embodiments, the catalyst is threonine. In some embodiments, the catalyst is glutamine. In some embodiments, the catalyst is cysteine. In some embodiments, the catalyst is glycine. In some embodiments, the catalyst is proline. In some embodiments, the catalyst is alanine. In some embodiments, the catalyst is valine. In some embodiments, the catalyst is isoleucine. In some embodiments, the catalyst is leucine. In some embodiments, the catalyst is methionine. In some embodiments, the catalyst is phenylalanine. In some embodiments, the catalyst is tyrosine. In some embodiments, the catalyst is tryptophan.

[0389] In some embodiments, the catalyst provided herein is a polymerization catalyst or a carbon-supported catalyst disclosed in WO 2016122887, which is hereby incorporated by reference in its entirety and with respect to its disclosure.

[0390] In some embodiments, the catalyst provided herein is present in an amount of about 0.01% to about 5%, about 0.02% to about 4%, about 0.03% to about 3%, or about 0.05% to about 2% by dry weight of the one or more feed sugars. In some embodiments, the catalyst provided herein is present in an amount of about 1% to 2% by dry weight of the one or more feed sugars. In some embodiments, the catalyst provided herein is present in an amount of about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0% by dry weight of the one or more feed sugars.

[0391] In some embodiments, provided herein is a catalyst that is present in an amount of about 0.01% to about 5%, about 0.02% to about 4%, about 0.03% to about 3%, or about 0.05% to about 2% of an aqueous composition by dry weight. In some embodiments, provided herein is a catalyst that is present in an amount of about 1% to 2% of an aqueous composition by dry weight. In some embodiments, provided herein is a catalyst that is present in an amount of about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0% of an aqueous composition by dry weight.

[0392] In some embodiments, provided herein is a combination of two or more different catalysts. In some embodiments, catalyst includes recyclable catalysts (such as resins and polymerization catalysts) and non-recyclable catalysts. In some embodiments, when catalyst includes at least two different catalysts, every kind of catalyst exists with the amount provided herein. In other embodiments, when catalyst includes at least two different catalysts, the at least two different catalysts add up to exist with the amount provided herein.

[0393] In some embodiments, the catalyst is added to the aqueous composition in dry form. In other embodiments, the catalyst is added to the aqueous composition in wet form (e.g., in an aqueous solution). In some embodiments, the catalyst is merged with the one or more feed sugars before adding water. In other embodiments, the catalyst is dissolved in water before it is merged with the one or more feed sugars. In some embodiments, the method provided herein includes producing an aqueous composition by merging the one or more feed sugars in the form of a dehydrate and the catalyst in wet form (e.g., as an aqueous solution).

[0394] C. Adding water

[0395] In some embodiments, the method for manufacturing oligosaccharide preparation includes adding water to form an aqueous composition. In some embodiments, all or part of the water in the aqueous composition is added as free water. In other embodiments, all of the water in the aqueous composition is added as bonded water (for example, in sugar monohydrate or dihydrate). In some embodiments, all of the water in the aqueous composition is added as bonded water in a monosaccharide monohydrate (such as glucose monohydrate). In certain embodiments, all or part of the water in the aqueous composition is added along with a catalyst (that is, via a catalyst solution).

[0396] D. Water content

[0397] As the method for manufacturing the oligosaccharide preparation proceeds, water may be produced by reaction. For example, in some embodiments, water is produced (i) as a result of the formation of glycosidic bonds, (ii) as a result of the formation of dehydrating subunits, or (iii) by other mechanisms or sources. Since both sugar condensation reactions and dehydration reactions involve water, in some embodiments, water content affects the composition of the oligosaccharide preparation.

[0398] In addition, in some embodiments, water content affects the viscosity of the aqueous composition, which in turn may affect the mixing effectiveness of the aqueous composition. For example, in some embodiments, an overly viscous aqueous composition may result in an undesirable uneven catalyst distribution in the aqueous composition. In addition, in some embodiments, very low water content may result in the solidification of the aqueous composition, which prevents effective mixing. On the other hand, in some other embodiments, extremely high water content may hinder sugar condensation reactions and reduce the level of dehydrated subunits. Therefore, the present disclosure describes suitable water contents for the manufacture of oligosaccharide preparations.

[0399] In some embodiments, the methods of making oligosaccharide preparations described herein comprise forming and / or heating an aqueous composition. In some embodiments, the aqueous composition comprises from about 0% to about 80%, from about 0% to about 70%, from about 0% to about 60%, from about 0% to about 50%, from about 0% to about 40%, from about 0% to about 35%, from about 0% to about 30%, from about 0% to about 25%, from about 0% to about 20%, from about 0% to about 19%, from about 0% to about 18%, from about 0% to about 17%, from about 0% to about 16%, from about 0% to about 15%, from about 0% to about 14%, from about 0% to about 13%, from about 0% to about 12%, from about 0% to about 11%, from about 0% to about 10%, from about 0% to about 9%, from about 0% to about 8%, from about 0% to about 7%, from about 0% to about 6%, from about 0% to about 5%, from about 0% to about 4%, from about 0% to about 3%, from about 0% to about 2%, or from about 0% to about 1% water by total weight. In some embodiments, the aqueous composition comprises from about 1% to about 20%, from about 1% to about 18%, from about 1% to about 16%, from about 1% to about 14%, from about 1% to about 12%, from about 1% to about 10%, from about 1% to about 8%, from about 1% to about 6%, or from about 1% to about 4% water by total weight. In some embodiments, the aqueous composition comprises from about 3% to about 16%, from about 3% to about 14%, from about 3% to about 12%, from about 3% to about 10%, from about 3% to about 8%, from about 3% to about 6%, from about 5% to about 16%, from about 5% to about 14%, from about 5% to about 12%, from about 5% to about 10%, from about 7% to about 16%, from about 7% to about 14%, from about 7% to about 12%, from about 7% to about 10%, or from about 8% to about 10% water by total weight. In some embodiments, aqueous composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15% water by gross weight. In some embodiments, aqueous composition comprises about 9% water by gross weight. However, it should be understood that the amount of water in the aqueous composition can be adjusted based on reaction conditions and the specific catalyst used. In some embodiments, the water content in the aqueous composition as disclosed above is measured when the reaction starts (for example, before heating the feed sugar). In some embodiments, the water content in the aqueous composition as disclosed above is measured when polymerization or condensation reaction finishes. In some embodiments, the water content in the aqueous composition as disclosed above is measured as the average water content when the reaction starts and the reaction finishes.

[0400] In certain embodiments, the methods described herein may further comprise monitoring the amount of water present in the aqueous composition and / or the ratio of water to sugar or catalyst over a period of time. In some embodiments, the methods further comprise removing at least a portion of the water from the aqueous composition, for example by distillation. Water may be removed from the aqueous composition using any method known in the art, including, for example, vacuum filtration, vacuum distillation, heating, steam, hot air, and / or evaporation.

[0401] In some embodiments, the oligosaccharide preparations described herein are hygroscopic.Thus, in some embodiments, the hygroscopicity of the feed sugars and the oligosaccharides formed in the polymerization may affect the rate at which water is removed from the aqueous composition.

[0402] In some embodiments, the methods described herein comprise removing at least a portion of the water in the aqueous composition such that the water content of the aqueous composition is from about 1% to about 20%, from about 1% to about 18%, from about 1% to about 16%, from about 1% to about 14%, from about 1% to about 12%, from about 1% to about 10%, from about 1% to about 8%, from about 2% to about 16%, from about 2% to about 14%, from about 2% to about 12%, from about 2% to about 10%, from about 2% to about 8%, from about 2% to about 6%, from about 4% to about 16%, from about 4% to about 14%, from about 4% to about 12%, from about 4% to about 10%, from about 4% to about 8%, from about 6% to about 16%, from about 6% to about 12%, from about 6% to about 10%, or from about 6% to about 8% by total weight. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition so that the water content in the aqueous composition is about 2% to about 10%, about 2% to about 8%, or about 4% to about 8% by total weight. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition so that the water content in the aqueous composition is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by total weight. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition so that the water content in the aqueous composition is about 4% to about 8% by total weight. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition so that at the end of the polymerization reaction and / or condensation reaction, the water content in the aqueous composition is the water content as disclosed above. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition so that at the beginning of the polymerization reaction and / or condensation reaction, the water content in the aqueous composition is the water content as disclosed above. In some embodiments, the method comprises removing at least a portion of the water in the aqueous composition such that the average water content in the aqueous composition at the beginning and end of the polymerization reaction and / or condensation reaction is within the range disclosed above. In some embodiments, the method comprises removing at least a portion of the water in the aqueous composition such that the water content in the aqueous composition remains within the range disclosed above throughout the polymerization reaction and / or condensation reaction.

[0403] In some embodiments, the methods described herein comprise adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 6% to about 16%, about 6% to about 12%, about 6% to about 10%, or about 6% to about 8%. In some embodiments, the methods comprise adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 2% to about 10%, about 2% to about 8%, or about 4% to about 8% by total weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition so that the water content of the aqueous composition is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% by total weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition so that the water content of the aqueous composition is about 4% to about 8% by total weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition so that at the end of the polymerization reaction and / or condensation reaction, the water content of the aqueous composition is the water content disclosed above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition so that at the beginning of the polymerization reaction and / or condensation reaction, the water content of the aqueous composition is the water content disclosed above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition so that the average water content of the aqueous composition at the beginning and end of the polymerization reaction and / or condensation reaction is within the range disclosed above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition remains within the range disclosed above throughout the polymerization reaction and / or condensation reaction.

[0404] In some embodiments, the degree of polymerization of the oligosaccharide and / or the amount and type of anhydro subunits within the oligosaccharide preparation can be adjusted by adjusting or controlling the water content present in the aqueous composition throughout the manufacturing process. For example, in some embodiments, the degree of polymerization of the oligosaccharide and the amount of anhydro subunits are increased by reducing the water content.

[0405] Therefore, in some embodiments, the methods described herein include in-process control (IPC) of water content, which may include monitoring water content, maintaining water content, increasing water content, reducing water content, or any combination thereof. In some embodiments, the IPC process includes maintaining water content while heating the aqueous composition to a temperature as described herein. In some embodiments, the method includes maintaining the water content for a time sufficient to induce polymerization. In some embodiments, the method includes maintaining the water content within the disclosed range by adding water or removing water from the aqueous composition, or both. In some embodiments, the method includes maintaining the water content within the disclosed range by distillation. In some embodiments, the method includes maintaining the water content within the disclosed range by vacuum distillation. In some embodiments, the method includes maintaining the water content within the disclosed range by distillation at atmospheric pressure.

[0406] In some embodiments, the water content of the aqueous composition is maintained in a range of about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 6% to about 16%, about 6% to about 12%, about 6% to about 10%, or about 6% to about 8% by total weight. In some embodiments, the water content of the aqueous composition is maintained in a range of about 2% to about 10%, about 2% to about 8%, or about 4% to about 8% by total weight. In some embodiments, the water content of the aqueous composition is maintained in the range of about 2% to about 8% by total weight.

[0407] The water content of the aqueous composition can be measured by a variety of analytical methods and instruments. In some embodiments, the water content is measured by an evaporation method (e.g., loss on drying technique), a distillation method, or a chemical reaction method (e.g., Karl Fischer titration). In some embodiments, the water content is measured by an analytical instrument such as a moisture analyzer. In some embodiments, the water content is measured by Karl Fischer titration.

[0408] In some embodiments, the water content of the aqueous composition is measured during the reaction and used to implement in-process control (IPC) of the water content. In certain embodiments, the water content of the reaction is measured by Karl-Fischer titration, IR spectroscopy, NIR spectroscopy, conductivity, viscosity, density, mixing torque or mixing energy. In some embodiments, the measurement of the water content of the reaction is used to control a device that actively adjusts the water content of the reaction, such as a water addition pump or flow valve.

[0409] Without being bound by theory, it is believed that the water content during the sugar polymerization reaction and / or condensation reaction can affect the level of anhydro subunits in the oligosaccharide preparations described herein. For example, as shown in Figure 30, in some embodiments, higher water content is associated with lower levels of anhydro subunits. In some embodiments, lower reaction temperatures can be associated with lower levels of anhydro subunit content.

[0410] E. Temperature

[0411] In some embodiments, the degree of polymerization of the oligosaccharide and / or the amount and type of anhydro subunits within the oligosaccharide preparation can be adjusted by adjusting the temperature to which the aqueous composition is heated. In some embodiments, the methods of making an oligosaccharide preparation described herein comprise heating the aqueous composition to a temperature of about 80°C to about 250°C, about 90°C to about 200°C, about 100°C to about 200°C, about 100°C to about 180°C, about 110°C to about 170°C, about 120°C to about 160°C, about 130°C to about 150°C, or about 135°C to about 145°C. In some embodiments, the methods of making an oligosaccharide preparation comprise heating the aqueous composition to a temperature of about 100°C to about 200°C, about 100°C to about 180°C, about 110°C to about 170°C, about 120°C to about 160°C, about 130°C to about 150°C, or about 135°C to about 145°C. In some embodiments, the method of making an oligosaccharide formulation comprises heating the aqueous composition to a temperature of about 135° C. to about 145° C. In other embodiments, the method of making an oligosaccharide formulation comprises heating the aqueous composition to a temperature of about 125° C. to about 135° C.

[0412] F. Reaction time

[0413] In some embodiments, the methods of making oligosaccharide preparations described herein comprise heating the aqueous composition for a sufficient time. In some embodiments, the degree of polymerization of the oligosaccharides made according to the methods described herein can be adjusted by reaction time.

[0414] In some embodiments, enough time is specified by hours.For example, in some embodiments, enough time is at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours or at least 10 hours.In some embodiments, enough time is about 1 to about 24 hours, about 1 to about 16 hours, about 1 to about 8 hours, about 1 to about 4 hours, about 1 to about 3 hours, about 1 to about 2 hours, about 2 to about 12 hours, about 2 to about 10 hours, about 2 to about 8 hours, about 2 to about 6 hours, about 2 to about 4 hours, about 3 to about 8 hours, about 3 to about 6 hours, about 3 to about 5 hours or about 3 to about 4 hours.

[0415] In some embodiments, sufficient time is determined by measuring one or more chemical or physical properties of the oligosaccharide preparation (eg, water content, viscosity, molecular weight, anhydro subunit content, and / or distribution of degree of polymerization).

[0416] In some embodiments, the molecular weight of the oligosaccharide preparation is monitored during polymerization. In some embodiments, the method includes heating the aqueous composition to be enough to make the aqueous composition reach the time of number-average molecular weight or weight-average molecular weight as described herein. In certain embodiments, the method includes heating the aqueous composition to be enough to make the aqueous composition reach the time of number-average molecular weight in the following range: about 300 to about 5000g / mol, about 500 to about 5000g / mol, about 700 to about 5000g / mol, about 500 to about 2000g / mol, about 700 to about 2000g / mol, about 700 to about 1500g / mol, about 300 to about 1500g / mol, about 300 to about 2000g / mol, about 400 to about 1000g / mol, about 400 to about 900g / mol, about 400 to about 800g / mol, about 500 to about 900g / mol or about 500 to about 800g / mol. In certain embodiments, the method comprises heating the aqueous composition for a time sufficient to allow the aqueous composition to reach a number average molecular weight of about 500 to about 2000 g / mol. In certain embodiments, the method comprises heating the aqueous composition for a time sufficient to allow the aqueous composition to reach a weight average molecular weight in the following ranges: about 300 to about 5000 g / mol, about 500 to about 5000 g / mol, about 700 to about 5000 g / mol, about 500 to about 2000 g / mol, about 700 to about 2000 g / mol, about 700 to about 1500 g / mol, about 300 to about 1500 g / mol. In some embodiments, the method comprises heating the aqueous composition for a time sufficient to allow the aqueous composition to reach a weight average molecular weight of about 700 to about 3000 g / mol.

[0417] In some embodiments, sufficient time is the time required for the aqueous composition to reach reaction equilibrium at the corresponding reaction temperature. Thus, in some embodiments, the method comprises heating the aqueous composition for a time sufficient to allow the aqueous composition to reach equilibrium. For example, in some embodiments, equilibrium is determined by measuring the molecular weight, viscosity, or DP distribution of the aqueous composition.

[0418] In certain embodiments, equilibrium is determined by measuring the number average molecular weight or weight average molecular weight of the aqueous composition. In some embodiments, equilibrium is determined by the number average molecular weight or weight average molecular weight of the aqueous composition remaining substantially unchanged over time. In some embodiments, equilibrium is determined by a change in the number average molecular weight or weight average molecular weight of the aqueous composition of less than a certain percentage over a period of time. In some embodiments, the molecular weight of the aqueous composition is measured by HPLC or SEC.

[0419] In some embodiments, equilibrium is determined by a change in the number average molecular weight or weight average molecular weight of the aqueous composition of less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a period of time. In some embodiments, equilibrium is determined by a change in the number average molecular weight or weight average molecular weight of the aqueous composition over a period of 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, equilibrium is determined by a change in the weight average molecular weight of the aqueous composition of less than 15% over a period of 1 hour.

[0420] In certain embodiments, equilibrium is determined by measuring the viscosity of the aqueous composition. In some embodiments, equilibrium is determined by the viscosity of the aqueous composition remaining substantially unchanged over time. In some embodiments, equilibrium is determined by a change in the viscosity of the aqueous composition of less than a certain percentage over a period of time. In some embodiments, the viscosity of the aqueous composition is measured using a viscometer or a rheometer.

[0421] In some embodiments, equilibrium is determined by a change in the viscosity of the aqueous composition of less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a period of time. In some embodiments, equilibrium is determined by a change in the viscosity of the aqueous composition of less than 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, equilibrium is determined by a change in the viscosity of the aqueous composition of less than 15% over a period of 1 hour.

[0422] In certain embodiments, equilibrium is determined by measuring the DP distribution of the aqueous composition. In some embodiments, equilibrium is determined by a DP distribution of the aqueous composition that remains substantially unchanged over time. In some embodiments, changes in the DP distribution of the aqueous composition are determined by calculating a series of Km values, wherein Wherein [HO] represents the molar water concentration (mol / L), and [DP1], [DPm-1], and [DPm] represent the molar concentration (mol / L) of oligosaccharides in the DP1, DPm-1, and DPm fractions, respectively. For example, according to the above formula, K2 is equal to [DP2][HO] / [DP1][DP1]. In some embodiments, m is an integer greater than 1 and less than n. In some embodiments, m is an integer greater than 1 and less than or equal to n. In some embodiments, m is equal to n. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0423] In some embodiments, the concentration of oligosaccharides in DP1, DPm-1 and DPm fractions is measured by SEC, HPLC, FFF, A4F, mass spectrometry or any other suitable method. In some embodiments, the concentration of oligosaccharides in DP1, DPm-1 and DPm fractions is measured by SEC (such as GPC). In some embodiments, the concentration of oligosaccharides in DP1, DPm-1 and DPm fractions is measured by mass spectrometry (such as GC-MS, LC-MS / MS and MALDI-MS). In some embodiments, the concentration of oligosaccharides in DP1, DPm-1 and DPm fractions is measured by HPLC. In some embodiments, water concentration is measured by evaporation method (for example, loss on drying technique), distillation method or by chemical reaction method (for example, Karl Fischer titration). In some embodiments, water concentration is measured by any suitable analytical instrument (such as moisture analyzer).

[0424] In some embodiments, the method comprises calculating a series of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 Km numbers. In some embodiments, the method comprises calculating a series of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 15 Km numbers. In some embodiments, the method comprises calculating about 3, 4, 5, 6, 7, 8, 9, 10, or 15 Km numbers. In some embodiments, the method comprises calculating K2 to K4, K2 to K5, K2 to K6, K2 to K7, K2 to K8, K2 to K9, K2 to K10, K2 to K11, K2 to K12, K2 to K13, K2 to K14, K2 to K15, K3 to K5, K3 to K6, K3 to K7, K3 to K8, K3 to K9, K3 to K10, K3 to K11, K3 to K12, K3 to K13, K3 to K14, or K3 to K15. In certain embodiments, the method comprises calculating K2 to K4 or K3 to K5.

[0425] In some embodiments, the value of Km depends on the temperature, water concentration and / or the amount and type of feed sugar. In some embodiments, Km is about 0.1 to about 100, about 0.1 to about 90, about 0.1 to about 80, about 0.1 to about 70, about 0.1 to about 60, about 0.1 to about 50, about 0.1 to about 40, about 0.1 to about 30, about 0.1 to about 25, about 0.1 to about 20 or about 0.1 to about 15. In some embodiments, Km is about 1 to about 100, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, about 5 to about 15 or about 5 to about 10. In some embodiments, Km is from about 1 to about 15 or from about 5 to about 15.

[0426] In some embodiments, the mean, standard deviation and / or relative standard deviation of a series of calculated Km's are determined. As used herein, the relative standard deviation is expressed as a percentage and is obtained by multiplying the standard deviation by 100 and dividing the product by the mean.

[0427] In some embodiments, equilibrium is determined by a relative standard deviation of less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of a series of Km. In some embodiments, equilibrium is determined by a relative standard deviation of less than 15%, less than 10%, or less than 5% of a series of Km.

[0428] G. Post-reaction steps

[0429] In some embodiments, the method for manufacturing the oligosaccharide preparation described herein further comprises one or more additional processing steps after the aqueous composition is heated at a certain temperature and for a sufficient time. In some embodiments, the additional processing steps include, for example, separation (such as chromatographic separation), dilution, concentration, drying, filtration, demineralization, extraction, decolorization, or any combination thereof. For example, in some embodiments, the method comprises a dilution step and a decolorization step. In some embodiments, the method comprises a filtration step and a drying step.

[0430] In some embodiments, the method includes a dilution step in which water is added to the oligosaccharide preparation to prepare a slurry of the oligosaccharide preparation. In some embodiments, the concentration of the oligosaccharide preparation in the slurry is about 5% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, or about 15% to about 25%. In other embodiments, the method does not include a dilution step, but solidifies the oligosaccharide preparation. In some embodiments, the method includes a filtration step. In some embodiments, the method includes recycling the catalyst by filtration.

[0431] In some embodiments, the methods described herein further comprise a decolorization step. In some embodiments, the oligosaccharide preparation can be subjected to a decolorization step using any method known in the art, including, for example, treatment with an absorbent activated carbon, chromatography (e.g., using an ion exchange resin), hydrogenation, and / or filtration (e.g., microfiltration).

[0432] In some embodiments, the oligosaccharide preparation is contacted with a material to remove salts, minerals, and / or other ionic species. In certain embodiments, the oligosaccharide preparation is flowed through an anion / cation exchange column pair. In one embodiment, the anion exchange column contains a weak base exchange resin in the hydroxide form, and the cation exchange column contains a strong acid exchange resin in the protonated form.

[0433] In some embodiments, the method includes a concentration step. In some embodiments, the concentration step produces an oligosaccharide preparation with an increased concentration. For example, in some embodiments, the concentration step includes evaporation (e.g., vacuum evaporation), drying (e.g., freeze drying and spray drying) or any combination thereof.

[0434] In some embodiments, the method comprises an isolating step, wherein at least a portion of the oligosaccharide preparation is isolated. In some embodiments, the isolating step comprises crystallization, precipitation, filtration (e.g., vacuum filtration), and centrifugation, or any combination thereof.

[0435] In some embodiments, the method comprises a separation step. In some embodiments, the separation step comprises separating at least a portion of the oligosaccharide preparation from at least a portion of the catalyst, from at least a portion of the unreacted feed sugars, or from both. In some embodiments, the separation step comprises filtration, chromatography, differential solubility, precipitation, extraction, or centrifugation.

[0436] H. Reactor

[0437] Taking into account reaction temperature, pH, pressure and other factors, the methods described herein may include the use of one or more reactors suitable for sugar condensation. In some embodiments, the one or more suitable reactors include a fed-batch stirred reactor, a stirred batch reactor, a continuous flow stirred reactor, a continuous plug flow column reactor, an attrition reactor, or a reactor with stirring caused by an electromagnetic field. In some embodiments, the one or more suitable reactors include those described in Ryu, SK and Lee, JM, Bioconversion of wastecellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65 (1983); Gusakov, AV and Sinitsyn, AP, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. TechnoL, 7: 346-352 (1985); Gusakov, AV, Sinitsyn, AP, Davydkin, IY, Davydkin, VY, Protas, OV, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol., 56: 141-153 (1996); or Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, Optimal control in fed-batch reactor for the cellobiose hydrolysis, ActaScientiarum. Technology, 25:33-38 (2003).

[0438] In some embodiments, the one or more suitable reactors include fluidized beds, upflow blankets, immobilized or extruder type reactors for hydrolysis and / or fermentation. In some embodiments, the one or more suitable reactors include open reactors, closed reactors, or both. In some embodiments, where the method comprises a continuous process, the one or more suitable reactors may include a continuous mixer, such as a spiral mixer.

[0439] I. Craftsmanship

[0440] In some embodiments, the method for manufacturing oligosaccharide preparation as herein described comprises batch process, continuous process or both.In some embodiments, the method for manufacturing oligosaccharide preparation comprises batch process.For example, in some embodiments of batch process, the manufacture of subsequent batches of oligosaccharide preparation does not begin until the current batch is completed.In some embodiments, during batch process, all or a large amount of oligosaccharide preparations are removed from the reactor.In some embodiments, during batch process, before the aqueous composition is heated to the described temperature or before inducing polymerization, all feed sugars and catalysts are merged in the reactor.In some embodiments, during batch process, feed sugars are added before, after or simultaneously with the addition of catalyst.

[0441] In some embodiments, the batch process is a fed-batch process, wherein not all of the feed sugar is added to the reactor simultaneously. In some embodiments of the fed-batch process, at least a portion of the feed sugar is added to the reactor during polymerization or after the aqueous composition is heated to the described temperature. In some embodiments of the fed-batch process, at least 10%, 20%, 30%, 40%, 50%, or 60% by weight of the feed sugar is added to the reactor during polymerization or after the aqueous composition is heated to the described temperature.

[0442] In some embodiments, the method for manufacturing oligosaccharide preparation comprises continuous process.For example, in some embodiments of continuous process, the content of reactor flows through reactor continuously.In some embodiments, carry out simultaneously the merging of feed sugar and catalyst and the removal of at least a portion of oligosaccharide preparation.

[0443] In some embodiments, the method for making an oligosaccharide preparation comprises a single pot or a multi-pot process. For example, in some embodiments of a single pot process, polymerization is carried out in a single reactor. For another example, in some embodiments of a multi-pot process, polymerization is carried out in more than one reactor. In some embodiments of a multi-pot process, the method comprises 2, 3 or more reactors. In some embodiments of a multi-pot process, the method comprises a merging step in which the polymerization products from two or more reactors are combined.

[0444] IV. Nutritional Compositions Containing Oligosaccharide Preparations

[0445] Provided herein are nutritional compositions comprising oligosaccharide preparations. In certain embodiments, provided herein are nutritional compositions comprising described oligosaccharide preparations, wherein the presence and / or concentration of oligosaccharide preparations in the nutritional composition can be selectively determined and / or detected. Oligosaccharide preparations that exhibit complex functional regulation of microbial communities can be important components of the nutritional composition. Therefore, the presence and / or concentration of oligosaccharide preparations in the nutritional composition can be one of the factors that need to be measured in the quality control and manufacturing process of the nutritional composition. Therefore, the nutritional composition provided is advantageous in terms of quality control and manufacturing purposes because the presence and / or concentration of oligosaccharide preparations can be selectively determined and / or detected. For example, in some embodiments, the presence and concentration of oligosaccharide preparations can be determined and / or detected by measuring the signal associated with dehydrated subunit oligosaccharides.

[0446] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the nutritional composition comprises a basal nutritional composition.

[0447] A. Basic Nutritional Composition

[0448] In some embodiments, the basic nutritional composition comprises a carbohydrate source other than the oligosaccharide preparation. For example, in some embodiments, the basic nutritional composition comprises a naturally occurring carbohydrate source, such as starch and plant fiber. In some embodiments, the basic nutritional composition comprises starch. In some embodiments, the basic nutritional composition comprises plant fiber.

[0449] In some embodiments, the base nutritional composition comprises one or more carbohydrates derived from seeds, roots, tubers, corn, tapioca, arrowroot, wheat, rice, potato, sweet potato, sago, legumes (e.g., beans, lentils, mung beans, peas, and chickpeas), maize, cassava, or other starchy foods (e.g., acorns, arrowroot, Peruvian carrots, bananas, barley, breadfruit, buckwheat, canna, taro, katakuri, kudzu, yellow taro, millet, oats, tuberous sorrel, Polynesian arrowroot, sorghum, rye, taro, chestnuts, water chestnuts, and yams).

[0450] In some embodiments, the basic nutritional composition comprises one or more carbohydrates derived from legumes (e.g., peas, soybeans, lupines, green beans, and other legumes), oats, rye, chia, barley, fruits (e.g., figs, avocados, plums, prunes, berries, bananas, apple peels, quinces, and pears), vegetables (e.g., broccoli, carrots, cauliflower, zucchini, celery, nopal, and Jerusalem artichokes), tubers, root vegetables (e.g., sweet potatoes and onions), psyllium husks, seeds (e.g., flaxseed), nuts (e.g., almonds), whole grains, wheat, corn bran, lignans, or any combination thereof. In some embodiments, the basic nutritional composition comprises one or more plant fibers derived from wheat bran, sugar beet pulp, cottonseed hair, soybean hulls, or any combination thereof.

[0451] In some embodiments, the basic nutritional composition comprises less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm of anhydro subunits or anhydro subunit-containing oligosaccharides. In some embodiments, the basic nutritional composition comprises less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm of anhydro subunits or anhydro subunit-containing oligosaccharides. In some embodiments, the basic nutritional composition is substantially free of anhydro subunits.

[0452] In some embodiments, the base nutritional composition does not have detectable levels of anhydro subunits. Depending on the method of detection or assay, levels of anhydro subunits below a certain threshold may be undetectable. For example, in some embodiments, a detectable level of anhydro subunits may refer to at least 1000 ppm, at least 500 ppm, at least 400 ppm, at least 300 ppm, at least 200 ppm, at least 100 ppm, at least 50 ppm, at least 10 ppm, at least 5 ppm, or at least 1 ppm of anhydro subunits or anhydro subunit-containing oligosaccharides in the base nutritional composition.

[0453] In some embodiments, the basic nutritional composition comprises a plurality of oligosaccharides. In some embodiments, the basic nutritional composition comprises a distribution of glycosidic bond types that is different from that of the oligosaccharide preparation. For example, in some embodiments, the basic nutritional composition comprises a higher percentage of α-(1,4) glycosidic linkages than the oligosaccharide preparation. In some embodiments, the glycosidic linkages (such as α-(1,4) glycosidic linkages) in the basic nutritional composition are digestible by one or more enzymes. In some embodiments, the glycosidic linkages in the basic nutritional composition are more easily digested and / or hydrolyzed than the glycosidic linkages in the oligosaccharide preparation.

[0454] In some embodiments, the level of α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, or β-(1,6) glycosidic linkages in the base nutritional composition is at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% lower than the level of the corresponding glycosidic linkages in the oligosaccharide preparation. In some embodiments, the level of α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, or β-(1,6) glycosidic linkages in the base nutritional composition is at least 10% lower than the level of the corresponding glycosidic linkages in the oligosaccharide preparation.

[0455] In some embodiments, the level of α-(1,4) glycosidic linkages in the base nutritional composition is at least 50%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, or at least 2% higher than the level of α-(1,4) glycosidic linkages in the oligosaccharide preparation. In some embodiments, the level of α-(1,4) glycosidic linkages in the base nutritional composition is at least 10% higher than the level of α-(1,4) glycosidic linkages in the oligosaccharide preparation.

[0456] B. Animal Feed Composition

[0457] Depending on the type and age of the animal, the nutritional composition may comprise the oligosaccharide preparation and the base nutritional composition in different ratios. For example, the oligosaccharide preparation may be combined with the base nutritional composition in various ratios appropriate to the type and age of the animal. In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration of about 1 to about 10000 ppm, about 1 to about 5000 ppm, about 1 to about 3000 ppm, about 1 to about 2000 ppm, about 1 to about 1500 ppm, about 1 to about 1000 ppm, about 1 to about 500 ppm, about 1 to about 250 ppm, about 1 to about 100 ppm, about 10 to about 5000 ppm, about 10 to about 3000 ppm, about 10 to about 2000 ppm, about 10 to about 1500 ppm, about 10 to about 1000 ppm, about 10 to about 500 ppm, about 10 to about 250 ppm, about 10 to about 100 ppm, about 50 to about 5000 ppm, about 50 to about 3000 ppm, about 50 to about 2000 ppm, about 50 to about 1500 ppm, about 50 to about 1000 ppm, about 50 to about 500 ppm, about 50 to about 250 ppm 0 ppm, about 50 to about 100 ppm, about 100 to about 5000 ppm, about 100 to about 3000 ppm, about 100 to about 2000 ppm, about 100 to about 1500 ppm, about 100 to about 1000 ppm, about 100 to about 500 ppm, about 100 to about 400 ppm, about 100 to about 300 ppm, about 100 to about 200 ppm, about 200 to about 5000 ppm, about 200 to about 3 ppm, about 200 to about 2500 ppm, about 200 to about 2000 ppm, about 200 to about 1500 ppm, about 200 to about 1000 ppm, about 200 to about 500 ppm, about 500 to about 5000 ppm, about 500 to about 3000 ppm, about 500 to about 2500 ppm, about 500 to about 2000 ppm, about 500 to about 1500 ppm, or about 500 to about 1000 ppm. In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration of about 1 to about 5000 ppm, about 1 to about 1000 ppm, about 1 to about 500 ppm, about 10 to about 5000 ppm, about 10 to about 2000 ppm, about 10 to about 1000 ppm, about 10 to about 500 ppm, about 10 to about 250 ppm, about 10 to about 100 ppm, about 50 to about 5000 ppm, about 50 to about 2000 ppm, about 50 to about 1000 ppm, about 50 to about 500 ppm, about 50 to about 250 ppm, or about 50 to about 100 ppm.In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration of about 1 to about 5000 ppm, about 10 to about 1000 ppm, about 10 to about 500 ppm, or about 50 to about 500 ppm.

[0458] In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration greater than 10 ppm, greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, greater than 300 ppm, greater than 400 ppm, greater than 500 ppm, greater than 600 ppm, greater than 1000 ppm, or greater than 2000 ppm. In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration greater than 10 ppm, greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, or greater than 500 ppm.

[0459] In some embodiments, depending on the type and age of the animal, the nutritional composition may also include protein, minerals (such as copper, calcium, and zinc), salts, essential amino acids, vitamins, and / or antibiotics.

[0460] Also provided herein is a method for administering a nutritional composition comprising a basic nutritional composition and a disclosed oligosaccharide preparation to an animal. In some embodiments, the animal is selected from cattle (e.g., beef cattle and dairy cows), pigs, aquatic animals, and poultry. In some embodiments, the animal is a pig, such as a sow, a piglet, and a barrow. In other embodiments, the animal is poultry, such as a chicken, a duck, a turkey, a goose, a quail, and a hen. In embodiments, poultry is a broiler, a breeder, or a laying hen. In some embodiments, the animal is an aquatic animal, such as salmon, catfish, sea bass, eel, tilapia, flounder, shrimp, and crab. In some embodiments, the nutritional composition is administered to the animal in a dry form, a liquid form, a paste, or a combination thereof. In some embodiments, the administration form, feeding rate, and feeding schedule can vary according to the type and age of the animal.

[0461] C. Methods of Producing Nutritional Compositions

[0462] The present invention provides a method for making a nutritional composition, comprising: combining an oligosaccharide preparation with a base nutritional composition. In some embodiments, the oligosaccharide preparation comprises anhydro subunit-containing oligosaccharides. In some embodiments, the oligosaccharide preparation comprises a glycosidic bond type distribution that is different from the glycosidic bond type distribution of the base nutritional composition.

[0463] In some embodiments, the oligosaccharide preparation is a synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions) each having a different degree of polymerization selected from 1 to n. In some embodiments, n is an integer greater than or equal to 2. In some embodiments, n is an integer greater than 2. In some embodiments, n is an integer greater than or equal to 3. In some embodiments, n is an integer in the range of 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40 or 50. In some embodiments, each of the DP1 to DPn fractions comprises 0.1% to 90% anhydrosubunit-containing oligosaccharides as measured by mass spectrometry in relative abundance. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently comprise anhydro subunit oligosaccharides as measured by mass spectrometry in an amount of about 0.1% to about 15% or about 0.5% to about 10% relative abundance. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently comprise anhydro subunit oligosaccharides as measured by mass spectrometry in an amount of about 0.1%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5% to about 8%, 9%, 10%, 11%, 12%, 15% or 20% relative abundance. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with its degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, 10, 20 or 30 DP fractions decreases monotonically with its degree of polymerization.

[0464] In some embodiments, the method of making the nutritional composition comprises mixing the oligosaccharide formulation with the base nutritional composition. For example, in some embodiments, the mixing can be performed by industrial blenders and / or mixers such as drum blenders, double cone blenders, ribbon blenders, V-blenders, shear mixers, and paddle mixers.

[0465] In some embodiments, the method of manufacturing the nutritional composition further comprises a quality control step as described herein. In some embodiments, the quality control step as described herein comprises determining the level of a signal in a sample of the nutritional composition, and calculating the concentration of the oligosaccharide preparation in the nutritional composition based on the level of the signal. In some embodiments, the quality control step as described herein comprises detecting a signal in a sample of the nutritional composition by an analytical instrument, and accepting or rejecting a batch of the nutritional composition based on the presence or absence of the signal. In some embodiments, the quality control step as described herein comprises detecting the presence or absence of a first signal in a first sample of the nutritional composition and a second signal in a second sample of the nutritional composition by an analytical instrument, and comparing the first signal to the second signal. In some embodiments, the signal, the first signal and / or the second signal (i) indicates one or more anhydro subunit-containing oligosaccharides, (ii) is associated with the degree of polymerization (DP) distribution of the oligosaccharides, or (iii) is associated with the α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages or β-(1,6) glycosidic linkages of the oligosaccharides.

[0466] In addition, in some embodiments, the method of manufacturing the nutritional composition comprises further mixing the oligosaccharide preparation with the basic nutritional composition, adjusting the level of the oligosaccharide preparation, or a combination thereof after performing the quality control step. In some embodiments, adjusting the level of the oligosaccharide preparation comprises adding an additional oligosaccharide preparation to the nutritional composition or removing a portion of the oligosaccharide preparation from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide preparation comprises adding an additional basic nutritional composition to the nutritional composition or removing a portion of the basic nutritional composition from the nutritional composition. In some specific embodiments, adjusting the level of the oligosaccharide preparation comprises adding an additional oligosaccharide preparation to the nutritional composition.

[0467] D. Animal feed premixes

[0468] In some embodiments, the nutritional composition comprises an animal feed premix containing the described oligosaccharide formulations.

[0469] In some embodiments, the animal feed premix comprises a carrier material that can be combined with the oligosaccharide formulation to produce the animal feed premix. In some embodiments, the carrier material can be any material in dry or liquid form that is suitable for combining with the oligosaccharide formulation in the nutritional composition. In some embodiments, the carrier material includes dried distiller's grains, clay, vermiculite, diatomaceous earth, shells (such as ground rice husks and ground oat husks), silicon dioxide (such as feed grade silica gel and feed grade fumed silicon dioxide), corn (such as corn gluten feed, corn gluten meal and ground corn) or any combination thereof. In some embodiments, the carrier material is ground corn. In other embodiments, the carrier material is ground rice husks or ground oat husks.

[0470] In some embodiments, the animal feed premix is ​​produced by merging a carrier material with an oligosaccharide preparation (both in dry form). In some embodiments, the animal feed premix is ​​produced by merging a carrier material with an oligosaccharide preparation (one of which is in dry form). In some embodiments, the animal feed premix is ​​produced by merging a carrier material with an oligosaccharide preparation (both in liquid form). For example, in some embodiments, an oligosaccharide preparation in liquid form refers to an oligosaccharide in solution, such as an aqueous solution of oligosaccharides, such as a slurry.

[0471] In some embodiments, the animal feed premix is ​​produced by combining a carrier material with a slurry comprising an oligosaccharide formulation. In some embodiments, the concentration of the oligosaccharide formulation in the slurry is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% by weight. In some embodiments, the concentration of the oligosaccharide formulation in the slurry is about 40% to 80%, 50% to 75%, or 60% to 70% by weight.

[0472] In some embodiments, the animal feed premix is ​​in the form of a powder (e.g., a flowable powder), a slurry, a slurry, a pellet, or a liquid. In some embodiments, the animal feed premix has a moisture content of less than 40%, 30%, 20%, 15%, 10%, or 5% by weight. In some embodiments, the animal feed premix has a moisture content of less than 10% or 5% by weight. In some embodiments, the animal feed premix has a moisture content greater than 5%, 10%, 15%, 20%, 25%, or 30% by weight. In other embodiments, the moisture content of the animal feed premix is ​​adjusted to any described range. For example, in some embodiments, the animal feed premix is ​​dried to increase its moisture content to the described range.

[0473] In some embodiments, the animal feed premix comprises different levels of the oligosaccharide preparation, depending on the specific application. In some embodiments, the animal feed premix comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the oligosaccharide preparation by dry weight. In some embodiments, the animal feed premix comprises up to 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the oligosaccharide preparation by dry weight.

[0474] In some embodiments, the animal feed premix or carrier material further comprises other animal nutrients, such as minerals, fats, and proteins. In some embodiments, the carrier material or animal feed premix comprises copper, zinc, or both. In some embodiments, the carrier material or animal feed premix comprises ionophores or other anticoccidial drugs. In some embodiments, the carrier material or animal feed premix comprises antibiotics. In some embodiments, the carrier material comprises a carbohydrate source. In some embodiments, the carbohydrate source in the carrier material does not comprise anhydro subunits. In some embodiments, the carbohydrate source in the carrier material comprises a glycosidic bond type distribution that is different from the glycosidic bond type distribution of the oligosaccharide preparation.

[0475] Thus, in some embodiments, a method of making a nutritional composition comprises combining an animal feed premix with a base nutritional composition.

[0476] V. Methods of providing oligosaccharide preparations to animals

[0477] In some embodiments, the methods described herein include providing an oligosaccharide preparation to an animal. In some variations, the animal is treated by being fed or providing an oligosaccharide preparation. In some embodiments, an oligosaccharide preparation of expected specific dose is provided to the animal. The specific dose can be, for example, as the mass of the oligosaccharide preparation consumed by the animal per unit time (e.g., grams / day), or as the mass of the oligosaccharide preparation consumed by the animal per unit time (e.g., mg oligosaccharide / kg body weight / day) per unit animal body weight. In certain embodiments, the specific dose of the oligosaccharide preparation is 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000 or 10000 mg / kg / day. In some embodiments, the mass of the oligosaccharide preparation is measured as the DP1+ content based on dry solids. In some embodiments, the mass of the oligosaccharide preparation is measured as the DP2+ content based on dry solids.

[0478] In some embodiments, the oligosaccharide preparation is provided to the animal by oral administration via the nutritional composition. In some embodiments, the nutritional composition is formulated as an oligosaccharide preparation containing a fixed inclusion concentration or level. The oligosaccharide inclusion concentration or level in the nutritional composition can be quantified by, for example, the mass fraction of the oligosaccharide preparation based on the total mass of the final feed or nutritional composition. In some embodiments, the inclusion concentration or level is measured as parts per million (ppm) of oligosaccharides based on dry solids based on the final nutritional composition as is. In some embodiments, the concentration of the oligosaccharide preparation is measured as the mass fraction of the DP1+ species based on dry solids. In some embodiments, the concentration of the oligosaccharide preparation is measured as the mass fraction of the DP2+ species based on dry solids.

[0479] Those skilled in the art will be aware of various methods and techniques for determining the concentration of an oligosaccharide formulation in a nutritional composition or final feed to achieve a desired specific dose. For example, average daily feed intakes that vary with age are established for different species of broiler chickens, and these average daily feed intakes can be used by a nutritionist or veterinarian to determine the desired inclusion level in the final feed.

[0480] In some embodiments, the oligosaccharide formulation is provided to the animal by oral administration via consumption of liquids. In some embodiments, the oligosaccharide formulation is provided via drinking water. In some embodiments, the concentration of the oligosaccharide formulation in the drinking water is selected to provide the animal with an expected specific dose of the oligosaccharide formulation.

[0481] VI. Gastrointestinal barrier dysfunction

[0482] In some embodiments, the methods described herein include preventing or treating a dysfunction of the gastrointestinal barrier of an animal. The gastrointestinal barrier of an animal includes a layer of mucus and a layer of epithelial enterocytes (including, for example, goblet cells). The intestinal epitheli...

Claims

1. Use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation in the preparation of a medicament for treating or preventing gastrointestinal barrier dysfunction in an animal in need thereof, The synthetic oligosaccharide preparation comprises: at least n oligosaccharide fractions (DP1 to DPn fractions) each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 3; and wherein each of the DP1 and DP2 fractions independently comprises 0.5% to 15% anhydrosubunit-containing oligosaccharides by relative abundance as determined by mass spectrometry; wherein the synthetic oligosaccharide preparation is obtainable by reacting glucose with a catalyst selected from 2-pyridinesulfonic acid and (+)-camphor-10-sulfonic acid; The gastrointestinal barrier dysfunction is treated or prevented by increasing transendothelial electrical resistance.

2. The use according to claim 1, wherein the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

3. Use according to claim 2, wherein said reduction is a reduction of at least 0.5% relative to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

4. The use according to claim 2, wherein the reduction is a reduction of 0.5% to 30% relative to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

5. Use according to any one of claims 2 to 4, wherein the reduction is a greater reduction compared to the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

6. Use according to claim 5, wherein the reduction is a reduction that is at least 0.5% greater than the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

7. The use according to any one of claims 2 to 4, wherein the permeability of the gastrointestinal barrier is determined from a sample of the gastrointestinal barrier from the animal.

8. The use according to claim 7, wherein the permeability is measured by histological analysis, staining or any combination thereof.

9. The use according to claim 7, wherein the treatment or prevention further comprises evaluating the mucosal morphology of the gastrointestinal barrier.

10. The use according to claim 9, wherein the evaluation comprises measuring villus length, crypt length, inflammatory cell infiltration level, or any combination thereof.

11. The use according to any one of claims 2 to 4, wherein the permeability of the gastrointestinal barrier is determined from a blood, feces or urine sample from the animal.

12. The use according to claim 11, wherein the permeability is measured by determining the level of a tracer orally administered to the animal in the sample.

13. The use according to claim 12, wherein the tracer is a non-digestible sugar, polyethylene glycol (PEG), fluorescently labeled dextran or a radioisotope.

14. The use according to claim 11, wherein the permeability is measured by determining the level of at least one microbial species in the blood, stool or urine sample.

15. The use according to claim 11, wherein the permeability is measured by determining the level of antibodies that bind to at least one microbial species in the sample.

16. Use according to claim 14 or 15, wherein the at least one microbial species is present in the gastrointestinal tract of the animal.

17. Use according to any one of claims 2 to 4, wherein the reduction in the permeability of the gastrointestinal barrier is directly mediated by the synthetic oligosaccharide preparation.

18. Use according to any one of claims 2 to 4, wherein the reduction in the permeability of the gastrointestinal barrier is indirectly mediated by the synthetic oligosaccharide preparation.

19. The use according to claim 18, wherein the synthetic oligosaccharide preparation is processed in vivo into at least one secondary substance.

20. The use according to claim 19, wherein the synthetic oligosaccharide preparation is processed in vivo by components of the gastrointestinal microbiome of the animal.

21. The use according to claim 20, wherein the synthetic oligosaccharide preparation is processed in vivo by bacteria present in the gastrointestinal tract of the animal.

22. Use according to any one of claims 19 to 21, wherein the reduction in gastrointestinal permeability is directly mediated by the at least one secondary substance.

23. The use according to any one of claims 1 to 4, wherein the gastrointestinal barrier dysfunction is a hyperpermeable gastrointestinal barrier.

24. The use according to claim 23, wherein the hyperpermeable gastrointestinal barrier allows translocation of intestinal contents from the intraluminal space into the circulation of the animal.

25. The use according to claim 24, wherein the intestinal contents comprise food particles, microorganisms, toxins or any combination thereof.

26. The use according to any one of claims 1 to 4, wherein at least one symptom associated with the gastrointestinal barrier dysfunction is improved or prevented.

27. The use of claim 26, wherein the at least one symptom is decreased mucus synthesis, decreased mucin synthesis, decreased mucus secretion, decreased mucin secretion, decreased nutrient absorption, increased inflammation, decreased resistance to infection, decreased intestinal epithelial cell proliferation, decreased intestinal epithelial cell maturation, increased immune cells in the gastrointestinal tract, increased levels of proinflammatory cytokines or chemokines in the blood or gastrointestinal tract, irritable bowel syndrome, inflammatory bowel syndrome, rectal inflammation, systemic infection, systemic inflammation, malnutrition, decreased weight gain, weight loss, increased feed conversion rate, decreased feed efficiency, hair loss, stool inconsistency, or diarrhea relative to a comparable control animal lacking the gastrointestinal barrier dysfunction.

28. The use of any one of claims 1-4, wherein the animal has an infection.

29. The use according to any one of claims 1 to 4, wherein the gastrointestinal barrier dysfunction is associated with or caused by an infection.

30. The use according to claim 29, wherein the infection is a parasitic, bacterial, fungal or viral infection.

31. The use according to claim 30, wherein the infection is a parasitic infection.

32. The use according to claim 31 , wherein the parasitic infection is a coccidiosis infection.

33. The use according to claim 32, wherein the coccidiosis infection is Eimeria spp. Eimeria ) infection, Toxoplasma infection, Cryptosporidium infection, Isospora infection, or Hammondia infection.

34. Use according to claim 33, wherein the coccidiosis infection is an Eimeria infection.

35. The use according to claim 34, wherein the Eimeria infection is Eimeria metamorphosis ( E. mivati ), Eimeria tenella ( E. tenella ), Eimeria acervulina ( E. acervulina ) or Eimeria maxima ( E. maxima )Infect.

36. The use according to claim 33, wherein the coccidiosis infection is a Toxoplasma infection.

37. The use according to claim 36, wherein the Toxoplasma infection is Toxoplasma gondii.

38. The use according to claim 33, wherein the coccidiosis infection is a Cryptosporidium infection.

39. The use of claim 38, wherein the Cryptosporidium infection is Cryptosporidium parvum, Cryptosporidium muris, or Cryptosporidium hominis infection.

40. The use according to claim 33, wherein the coccidiosis infection is an Isospora infection.

41. The use of claim 40, wherein the Isospora infection is an Isospora canis, Isospora ohioensis, Isospora burrosi, or Isospora felis infection.

42. The use according to claim 33, wherein the coccidiosis infection is a Hammondella infection.

43. The use according to claim 42, wherein the Hammondella infection is a Hammondella species infection.

44. The use of claim 32, wherein the coccidiosis infection is an infection with Eimeria acervulina, Eimeria maxima, Eimeria mitis, Eimeria tenella, Toxoplasma gondii, Hammondella species, Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Isospora canis, Isospora ohioensis, Isospora borrosii, or Isospora felis.

45. The use of claim 31 , wherein the infection is a bacterial infection.

46. ​​The use according to claim 45, wherein the bacterial infection is a Staphylococcus infection 、 Shigella spp. ( Shigella ) infection, Campylobacter spp. ( Campylobacter ) infection, Salmonella spp. ( Salmonella ) infection, Escherichia ( Escherichia ) infection or Yersinia spp. ( Yersinia )Infect.

47. The use of claim 28, wherein the infection causes an increase in gastrointestinal inflammation, a decrease in the number of goblet cells in the gastrointestinal tract of the animal, a decrease in mucus secretion in the gastrointestinal tract of the animal, a decrease in the length of the villi of the gastrointestinal barrier of the animal, damage to the villi of the gastrointestinal barrier of the animal, an increase in the level of immune cells in the gastrointestinal tract of the animal, an increase in the level of CD8+ T cells in the gastrointestinal tract of the animal, an increase in the level of the liver protein APG, an increase in the level of circulating antibodies, an increase in the level of circulating IgA antibodies, or a decrease in the level of circulating diamine oxidase, or any combination thereof, relative to a comparable animal that does not have the infection.

48. The use of any one of claims 1-4, wherein the animal has reduced inflammation of the gastrointestinal barrier relative to inflammation of the gastrointestinal barrier prior to administration of the synthetic oligosaccharide preparation.

49. The use of any one of claims 1-4, wherein the animal has reduced inflammation of the gastrointestinal barrier relative to inflammation of the gastrointestinal barrier of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

50. The use of claim 48, wherein the inflammation is measured by an increase in the level of at least one anti-inflammatory cytokine.

51. The use according to claim 50, wherein the anti-inflammatory cytokine is IL1B, IL4, IL10, IL-6, IL-11, IL-13, IL1RA or TGF-β.

52. The use according to claim 51, wherein the anti-inflammatory cytokine is IL1B, IL4 or IL10.

53. The use of any one of claims 1-4, wherein the animal exhibits a decrease in CD8+ T cells, an increase in CD4+ T cells, an increase in circulating diamine oxidase levels, or a decrease in circulating antibody levels relative to the animal prior to administration of the synthetic oligosaccharide preparation.

54. The use of any one of claims 1-4, wherein the animal exhibits a decrease in CD8+ T cells, an increase in CD4+ T cells, an increase in circulating diamine oxidase levels, or a decrease in circulating antibody levels relative to a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

55. The use of any one of claims 1-4, wherein the animal exhibits increased nutrient absorption across the gastrointestinal barrier relative to the gastrointestinal barrier prior to administration of the synthetic oligosaccharide formulation.

56. The use of any one of claims 1-4, wherein the animal exhibits increased nutrient absorption across the gastrointestinal barrier relative to the gastrointestinal barrier of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

57. The use of claim 55, wherein the nutrient absorption is measured by an increase in the level of at least one gastrointestinal protein associated with nutrient absorption.

58. The use according to claim 57, wherein the protein is SI (sucrase-isomaltase), SLC5A10, SLC34A1, SLC2A2, SLC34A2, SLC23A1, SLC23A2, SLC5A8, SLC16A3, SLC4, SLC4A9, SLC4A2, SLC4A3, NPC1L1, C6orf58, DDC, MCT1, MCT4, NaS1, DTDST, PAT1, DRA, CLD, SAT1, SUT2, SGLT1, GLUT2, B 0 AT1, ATB0+, SIT1, TAUT, EAAC1, ASCT2, SN1, SN2, PEPT1, SNAT2, GLYT1, y+LAT1, y+LAT2, CD36, LFABP, NP C1L1, ABCG5, ABCG8, SVCT1, SVCT2, SMVT, GIF, AMN, CUBL, MRP1, FOLT, PCFT, FOLR1, OAT10, RFVT1, RFVT2, THTR1, THTR2, VDR, DCYTB, DMT1, HCP1, FPN1, HEPH, HAMP, ZIP4, ZIP11, ZIP8, ZIP14A, ZIP14B, ZnT1, ZnT 2. CTR1, SLC3A1, SLC1A4, ALPI, C17orf78, MUC17, DEFA5, RBP2, DEFA6, MLN, MEP1B, LCT, TM4SF20 or FABP6.

59. The use according to claim 58, wherein the protein is S1, SLC5A10 or SLC34A1.

60. The use of any one of claims 1-4, wherein the animal has increased body weight relative to the animal's body weight before administration of the synthetic oligosaccharide preparation.

61. The use according to claim 60, wherein the body weight of the animal is increased by at least 1% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

62. The use according to claim 60, wherein the body weight of the animal is increased by 1%-40% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

63. The use of claim 60, wherein the increase in body weight is a greater increase relative to the increase in body weight in a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

64. The use of claim 63, wherein the increase in body weight is at least 1% greater than the increase in body weight in a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

65. The use of claim 63, wherein the increase in body weight is a 1%-40% greater increase relative to the increase in body weight in a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

66. The use of any one of claims 1-4, wherein the animal has increased feed efficiency relative to the feed efficiency of the animal before administration of the synthetic oligosaccharide preparation.

67. The use of claim 66, wherein the feed efficiency of the animal is increased by at least 1% relative to the feed efficiency of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

68. The use according to claim 66, wherein the feed efficiency of the animal is increased by 1% to 40% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

69. The use of claim 66, wherein the increase in feed efficiency is a greater increase relative to a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

70. The use of claim 69, wherein the increase in feed efficiency is at least 1% greater than the increase in feed efficiency in the comparable control animal administered the comparable nutritional composition lacking the synthetic oligosaccharide formulation.

71. The use of claim 69, wherein the increase in feed efficiency is a 1%-40% greater increase relative to the increase in feed efficiency in the comparable control animal administered the comparable nutritional composition lacking the synthetic oligosaccharide formulation.

72. The use of any one of claims 1-4, wherein the animal has a reduced feed conversion ratio (FCR) relative to the animal's FCR before administration of the synthetic oligosaccharide formulation.

73. Use according to claim 72, wherein the feed conversion ratio of the animal is reduced by at least 1% relative to the feed conversion ratio of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

74. The use according to claim 72, wherein the feed conversion ratio (FCR) of the animal is reduced by 1%-40% relative to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

75. The use of claim 72, wherein the reduction in feed conversion ratio experienced by the animal is a greater reduction relative to a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

76. The use of claim 75, wherein the reduction in feed conversion ratio is a reduction that is at least 1% greater relative to the reduction in feed conversion ratio in the comparable control animal administered the comparable nutritional composition lacking the synthetic oligosaccharide preparation.

77. The use of claim 75, wherein said reduction in feed conversion ratio is a 1%-40% greater reduction relative to said reduction in feed conversion ratio in said comparable control animal administered said comparable nutritional composition lacking said synthetic oligosaccharide preparation.

78. The use of any one of claims 1-4, wherein the life expectancy or survival rate of the animal is increased relative to a comparable control animal administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

79. The use of any one of claims 1-4, wherein the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal in an amount sufficient to treat or prevent the gastrointestinal barrier dysfunction.

80. Use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation for the preparation of a medicament for treating or preventing an Eimeria infection in an animal in need thereof, wherein the synthetic oligosaccharide preparation comprises at least n oligosaccharide fractions (DP1 to DPn fractions) each having a different degree of polymerization selected from 1 to n, wherein n is an integer greater than 3; and wherein each of the DP1 and DP2 fractions independently comprises 0.5% to 15% anhydrosubunit-containing oligosaccharides by relative abundance as determined by mass spectrometry; and wherein the synthetic oligosaccharide preparation is obtainable by reacting glucose with a catalyst selected from 2-pyridinesulfonic acid and (+)-camphor-10-sulfonic acid; Thereby treating or preventing the Eimeria infection.

81. The use of claim 80, wherein the level of the at least one immune cell in a sample from the animal is increased relative to the level of the at least one immune cell in a sample from the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

82. Use according to claim 81, wherein said increase is an increase of at least 1% relative to said level of said at least one immune cell before administration of said nutritional composition comprising said synthetic oligosaccharide preparation.

83. The use according to claim 81, wherein said increase is an increase of 1%-40% relative to said level of said at least one immune cell before administration of said nutritional composition comprising said synthetic oligosaccharide preparation.

84. The use of any one of claims 80-83, wherein the level of the at least one immune cell in a sample from the animal is increased relative to the level of the at least one immune cell in a sample from a comparable control animal that has been administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

85. The use of claim 84, wherein said increase is an increase of at least 1% relative to said level of said at least one immune cell in said sample from said comparable control animal that has been administered a comparable nutritional composition lacking said synthetic oligosaccharide preparation.

86. The use of claim 84, wherein said increase is an increase of 1%-40% relative to said level of said at least one immune cell in said sample from said comparable control animal that has been administered a comparable nutritional composition lacking said synthetic oligosaccharide preparation.

87. The use of any one of claims 80-83, wherein the at least one immune cell is a neutrophil, an eosinophil, a basophil, a mast cell, a T cell, a B cell, a macrophage, a monocyte, a granulocyte, or a dendritic cell.

88. The use according to any one of claims 80-83, wherein the immune cell is a phagocyte.

89. The use according to claim 88, wherein the phagocyte is a neutrophil, eosinophil, basophil, mast cell, macrophage, monocyte or dendritic cell.

90. The use according to claim 87, wherein the immune cell is a T cell.

91. The use according to claim 87, wherein the immune cell is a granulocyte.

92. The use of any one of claims 80-83, wherein the level of the at least one pro-inflammatory cytokine in a sample from the animal is increased relative to the level of the at least one pro-inflammatory cytokine in a sample from the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

93. The use of any one of claims 80-83, wherein the level of the at least one pro-inflammatory cytokine in a sample from the animal is increased relative to the level of the at least one pro-inflammatory cytokine in a sample from a comparable control animal that has been administered a comparable nutritional composition lacking the synthetic oligosaccharide preparation.

94. The use according to claim 92, wherein the at least one proinflammatory cytokine is IL1, IL-12, IL18, TNFA, IFNG, GM-CSF, IL-1β, IL6, RANTES, MCP1, IL8, MIP-1α, MIP-1β, lymphocyte chemotactic factor, fractal chemokine or GRO / KC.

95. The use of any one of claims 80-83, wherein the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

96. The use of claim 95, wherein said reduction is a reduction of at least 0.5% relative to the permeability of the gastrointestinal barrier of said animal prior to said administration of said synthetic oligosaccharide formulation; or said reduction is a reduction of 0.5%-30% relative to the permeability of the gastrointestinal barrier of said animal prior to said administration of said synthetic oligosaccharide formulation.

97. The use of claim 95, wherein the reduction is a greater reduction compared to the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

98. The use of claim 97, wherein the reduction is a reduction that is at least 0.5% greater than the reduction in gastrointestinal barrier permeability of a comparable control animal administered a nutritional composition lacking the synthetic oligosaccharide preparation.

99. The use of any one of claims 80-83, wherein the permeability of the gastrointestinal barrier of the animal is reduced relative to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

100. The use of claim 99, wherein the reduction is a reduction of at least 0.5% relative to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

101. The use of claim 99, wherein the reduction is a 0.5%-30% reduction relative to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

102. The use of claim 80, wherein the Eimeria infection is Eimeria desmodiosum, Eimeria tenella, Eimeria acervulina, Eimeria maxima infection, or Eimeria mitis.

103. The use of any one of claims 80-83, wherein the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal in an amount sufficient to treat or prevent the Eimeria infection.

104. The use of any one of claims 1-4 and 80-83, wherein the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal for at least 1 day.

105. The use of any one of claims 1-4 and 80-83, wherein the nutritional composition comprising the synthetic oligosaccharide preparation is administered to the animal at least once, twice, three times, four times or five times per day.

106. The use of any one of claims 1-4 and 80-83, wherein the nutritional composition comprising the synthetic oligosaccharide preparation is provided to the animal for ad libitum ingestion.

107. The use of claim 106, wherein the animal ingests at least a portion of the nutritional composition comprising the synthetic oligosaccharide preparation over at least one twenty-four hour period.

108. The use of any one of claims 1-4 and 80-83, wherein the nutritional composition comprises at least 100 ppm of the synthetic oligosaccharide preparation.

109. The use of any one of claims 1-4 and 80-83, wherein the nutritional composition comprises about 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000 ppm, 1500 ppm or 2000 ppm of the synthetic oligosaccharide preparation.

110. The use according to claim 109, wherein the nutritional composition comprises about 500 ppm of the synthetic oligosaccharide preparation.

111. The use according to any one of claims 1-4 and 80-83, wherein the nutritional composition comprises 100 ppm to 2000 ppm of the synthetic oligosaccharide preparation.

112. The use according to any one of claims 1-4 and 80-83, wherein the nutritional composition comprises 300 ppm to 600 ppm of the synthetic oligosaccharide preparation.

113. The use of any one of claims 1-4 and 80-83, wherein the animal is poultry, seafood, sheep, cattle, bison, pig, cat, dog, rabbit, goat, donkey, camel, horse, ferret, mouse, or rat.

114. The use according to any one of claims 1-4 and 80-83, wherein the animal is a buffalo, a pigeon, a guinea pig, a fish, a shrimp or a bird.

115. The use of any one of claims 1-4 and 80-83, wherein the animal is a gerbil or a cow.

116. The use of any one of claims 1-4 and 80-83, wherein the animal is a hamster.

117. The use according to claim 113, wherein the animal is poultry.

118. The use according to claim 117, wherein the animal is a chicken, a turkey, a duck or a goose.

119. The use according to claim 118, wherein the chicken is a broiler chicken, a layer chicken or a breeder chicken.

120. The use according to claim 113, wherein the animal is a pig.

121. The use according to claim 120, wherein the pig is a nursery pig, a growing pig or a finishing pig.

122. The use according to claim 113, wherein the animal is a fish.

123. The use according to claim 122, wherein the fish is salmon, tilapia or tropical fish.

124. The use of any one of claims 1-4 and 80-83, wherein the nutritional composition is an animal feed composition.

125. The use according to any one of claims 1-4 and 80-83, wherein the basal nutritional composition is a basal animal feed.

126. The use of any one of claims 1-4 and 80-83, wherein the relative abundance is determined by LC-MS / MS.

127. The use according to any one of claims 1-4 and 80-83, wherein the relative abundance of oligosaccharides in at least 5, 10, 20 or 30 DP fractions decreases monotonically with their degree of polymerization.

128. The use according to any one of claims 1-4 and 80-83, wherein the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with its degree of polymerization.

129. The use of any one of claims 1-4 and 80-83, wherein n is at least 4.

130. The use of any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises less than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

131. The use according to any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises 5% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

132. The use according to any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises 1% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

133. The use according to any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises 0.5% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

134. The use according to any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises 2% to 12% anhydrosubunit-containing oligosaccharides in relative abundance.

135. The use of any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises less than 12% anhydrosubunit-containing oligosaccharides by relative abundance.

136. The use according to any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises 2% to 12% anhydrosubunit-containing oligosaccharides in relative abundance.

137. The use according to any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises 1% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

138. The use according to any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises 0.5% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

139. The use according to any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises 5% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

140. The use according to any one of claims 1-4 and 80-83, wherein the DP3 fraction comprises less than 15% anhydrosubunit-containing oligosaccharides by relative abundance.

141. The use according to any one of claims 1-4 and 80-83, wherein the DP3 fraction comprises 2% to 12% anhydrosubunit-containing oligosaccharides in relative abundance.

142. The use according to any one of claims 1-4 and 80-83, wherein the DP3 fraction comprises 1% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

143. The use according to any one of claims 1-4 and 80-83, wherein the DP3 fraction comprises 0.5% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

144. The use according to any one of claims 1-4 and 80-83, wherein the DP3 fraction comprises 5% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

145. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises 2% to 12% anhydrosubunit-containing oligosaccharides by relative abundance.

146. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises 0.5% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

147. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises 1% to 10% anhydrosubunit-containing oligosaccharides in relative abundance.

148. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises 5% to 10% anhydrosubunit-containing oligosaccharides by relative abundance.

149. The use of any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises anhydrosubunit-containing oligosaccharides in a relative abundance greater than 0.6%.

150. The use according to any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises anhydrosubunit-containing oligosaccharides in a relative abundance greater than 0.6%.

151. The use of any one of claims 1-4 and 80-83, wherein the DP3 fraction comprises anhydrosubunit-containing oligosaccharides in a relative abundance greater than 0.6%.

152. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises greater than 0.5% anhydrosubunit-containing oligosaccharides by relative abundance.

153. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a DP1 fraction content of 1 to 40% by weight as determined by liquid chromatography.

154. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a DP2 fraction content of 1 to 35% by weight as determined by liquid chromatography.

155. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a DP3 fraction content of 1 to 30% by weight as determined by liquid chromatography.

156. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a DP4 fraction content of 0.1 to 20% by weight as determined by liquid chromatography.

157. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a DP5 fraction content of 0.1 to 15% by weight as determined by liquid chromatography.

158. The use of any one of claims 1-4 and 80-83, wherein the ratio of the DP2 fraction to the DP1 fraction is 0.02 to 0.40 as determined by liquid chromatography.

159. The use of any one of claims 1-4 and 80-83, wherein the ratio of the DP3 fraction to the DP2 fraction is 0.01 to 0.30 as determined by liquid chromatography.

160. The use according to any one of claims 1-4 and 80-83, wherein the combined content of DP1 and DP2 fractions in the oligosaccharide preparation is less than 50% as determined by liquid chromatography.

161. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises at least 103 different oligosaccharide species.

162. The use of any one of claims 1-4 and 80-83, wherein two or more independent oligosaccharides comprise different anhydro subunits.

163. The use of any one of claims 1-4 and 80-83, wherein each of the anhydrosubunit-containing oligosaccharides comprises one or more anhydrosubunits that are thermal dehydration products of monosaccharides.

164. The use according to any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises one or more anhydrosubunits selected from the group consisting of anhydroglucose, anhydrogalactose, anhydromannose, anhydroallose, anhydroaltrose, anhydrogulose, anhydroidose, anhydrotalose, anhydrofructose, anhydroribose, anhydroarabinose, anhydrorhamnose, anhydrolyxose, and anhydroxylose.

165. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises one or more anhydroglucose, anhydrogalactose, anhydromannose or anhydrofructose subunits.

166. The use according to any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises 1,6-anhydro-β-D-glucopyranose or 1,6-anhydro-β-D-glucopyranose anhydrosubunits.

167. The use according to any one of claims 1-4 and 80-83, wherein the DP1 fraction comprises two anhydro subunits, 1,6-anhydro-β-D-glucopyranose and 1,6-anhydro-β-D-glucopyranose.

168. The use according to claim 167, wherein in the oligosaccharide preparation, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-glucopyranose is 10:1 to 1:

10.

169. The method of claim 167, wherein the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-glucopyranose in the oligosaccharide preparation is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:

10.

170. The use according to claim 166, wherein in the oligosaccharide preparation, the ratio of 1,6-anhydro-β-D-glucopyranose to 1,6-anhydro-β-D-glucopyranose is 2:

1.

171. The use of any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises at least 5 species of anhydrosubunit-containing oligosaccharides.

172. The use of any one of claims 1-4 and 80-83, wherein the DP2 fraction comprises 5 to 10 species of anhydrosubunit-containing oligosaccharides.

173. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation comprises one or more sugar caramelization products.

174. The use according to claim 173, wherein the sugar caramelization product is selected from the group consisting of: methanol; ethanol; furan; methylglyoxal; 2-methylfuran; vinyl acetate; glycolaldehyde; acetic acid; acetol; furfural; 2-furylmethanol; 3-furylmethanol; 2-hydroxycyclopent-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levuloglucosone; cyclic hydroxylactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxymethylfurfural (5-hmf).

175. The use of any one of claims 1-4 and 80-83, wherein greater than 50% of the anhydrosubunit-containing oligosaccharides comprise a chain-end anhydrosubunit.

176. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a weight average molecular weight of 300 to 5000 g / mol as determined by high performance liquid chromatography (HPLC).

177. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a weight average molecular weight of 300 to 2500 g / mol as determined by HPLC.

178. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a weight average molecular weight of 500 to 2000 g / mol as determined by HPLC.

179. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a weight average molecular weight of 500 to 1500 g / mol as determined by HPLC.

180. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a number average molecular weight of 300 to 5000 g / mol as determined by HPLC.

181. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a number average molecular weight of 300 to 2500 g / mol as determined by HPLC.

182. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a number average molecular weight of 500 to 2000 g / mol as determined by HPLC.

183. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a number average molecular weight of 500 to 1500 g / mol as determined by HPLC.

184. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a weight average molecular weight of 2000 to 2800 g / mol.

185. The use of any one of claims 1-4 and 80-83, wherein the oligosaccharide preparation has a number average molecular weight of 1000 to 2000 g / mol.

Images