Method for supporting gastrointestinal homeostasis

Abstract

To provide nutritional compositions containing animal feed for preventing and / or treating gastrointestinal barrier dysfunction.SOLUTION: Provided is a synthetic oligosaccharide preparation for use in treatment, amelioration and / or prevention of at least one symptom caused by coccidiosis vaccination in coccidiosis vaccinated birds. The synthetic oligosaccharide preparation comprises at least n fractions (DP1 to DPn fractions) of oligosaccharides, each having a distinct degree of polymerization selected from 1 to n, n being an integer greater than or equal to 2, each of the DP1 and DP2 fractions containing 0.5% to 90% anhydro-subunit-containing oligosaccharides in relative abundance as determined by mass spectrometry, the at least one symptom being selected from decreased ileal nutrient absorption, tissue damage, increased acute phase liver proteins, decreased lymphocytes, and increased inflammation.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Related Applications]

[0001] 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 entire disclosure of each of which is incorporated herein by reference.

[0002] [Background]

[0002] The gastrointestinal system of an animal is the largest surface area interface between the internal anatomical structure of the animal and the external environment. It is involved in the digestion and absorption of nutrients and the provision of an effective barrier against environmental pathogens. The gastrointestinal tract (GIT) strongly cooperates with the immune system of the host animal and there is an intestinal microbiota of the animal that includes various microorganisms (bacteria, fungi, molds, viruses, etc.) that inhabit the digestive tract.

[0003]

[0003] In healthy animals, the gastrointestinal system maintains homeostasis through complex interactions of the immune function of the host animal, the physiological functions of the intestinal mucosal layer and endothelium, and the biochemistry of the intestinal microbiota. Mucin and mucus production by intestinal goblet cells, non-specific immune system activity, chemokine and cytokine mediation and inflammatory regulation, and tight junctions between cells of the intestinal inner layer promote healthy absorption of nutrients and defense against pathogens and toxins.

[0004]

[0004] Disruption of gastrointestinal homeostasis results in negative health and nutritional consequences for the animal. For example, disruption of a healthy barrier function allows movement of intestinal contents into the host circulation, enabling, for example, pathogens and toxins to penetrate the mucosal and endothelial layers and negatively affect the host animal. Inflammation and / or damage of epithelial intestinal cells reduces the animal's ability to absorb nutrients. For production animals, maintaining a healthy gastrointestinal system of gastrointestinal homeostasis indicates inadequate nutrition and health outcomes, resulting in reduced weight gain, reduced feed efficiency, reduced meat yield, reduced meat quality, and increased mortality. For companion animals, disruption of gastrointestinal homeostasis can reduce quality of life and overall health. Accordingly, there is a significant need to provide nutritional compositions, including animal feeds, that prevent and / or treat gastrointestinal barrier dysfunction.

[0005] [Summary]

[0005] In one aspect, a method of treating and preventing gastrointestinal barrier dysfunction in an animal, comprising administering to the animal a nutritional composition comprising a basal nutritional composition and a synthetic oligosaccharide preparation, wherein at least n fractions of the oligosaccharides each have a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), n is an integer greater than 3, and each of the DP1 and DP2 fractions independently comprises an oligosaccharide containing anhydro subunits in a relative abundance of about 0.5% to about 15% as determined by mass spectrometry, thereby treating and preventing the gastrointestinal barrier dysfunction. A method is provided herein.

[0006]

[0006] In some embodiments, the permeability of the animal's gastrointestinal barrier is reduced compared to the permeability of the animal's gastrointestinal barrier prior to administering the synthetic oligosaccharide preparation.

[0007]

[0007] In some embodiments, the decrease is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% decrease compared to the permeability of the gastrointestinal barrier of the animal before administering the synthetic oligosaccharide preparation. In some embodiments, the decrease 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% decrease compared to the permeability of the gastrointestinal barrier of the animal before administering the synthetic oligosaccharide preparation.

[0008]

[0008] In some embodiments, the decrease is a greater decrease compared to the decrease in the permeability of the gastrointestinal barrier of an equivalent control animal administered a nutritional composition without the synthetic oligosaccharide preparation. In some embodiments, the decrease is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% greater compared to the decrease in the permeability of the gastrointestinal barrier of an equivalent control animal administered a nutritional composition without the synthetic oligosaccharide preparation.

[0009]

[0009] In some embodiments, the permeability of the gastrointestinal barrier of the animal decreases compared to the permeability of the gastrointestinal barrier of the animal before administering the synthetic oligosaccharide preparation.

[0010]

[0010] In some embodiments, the decrease is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% decrease compared to the permeability of the gastrointestinal barrier of the animal before administering the synthetic oligosaccharide preparation. In some embodiments, the decrease 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% decrease compared to the permeability of the gastrointestinal barrier of the animal before administering the synthetic oligosaccharide preparation.

[0011]

[0011] 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.

[0012]

[0012] In some embodiments, the method further comprises evaluating the mucosal morphology of the gastrointestinal barrier. In some embodiments, the evaluation comprises determining the length of the villi, the length of the crypts, the level of inflammatory cell infiltration, or any combination thereof.

[0013]

[0013] In some embodiments, the permeability of the gastrointestinal barrier is determined from a blood, fecal, or urine sample from the animal. In some embodiments, the permeability is measured by determining the level of a tracer orally administered to the animal in the sample. In some embodiments, the tracer is a non-digestible sugar, polyethylene glycol (PEG), fluorescently labeled dextran, or a radioisotope. In some embodiments, the permeability is measured by determining the level of at least one microbial species in the blood, fecal, or urine sample.

[0014]

[0014] In some embodiments, the permeability is measured by determining the level of an antibody that binds to at least one microbial species in the sample. In some embodiments, the at least one microbial species is present in the gastrointestinal tract of the animal.

[0015]

[0015] In some embodiments, the decrease in the permeability of the gastrointestinal barrier is mediated directly by the synthetic oligosaccharide preparation. In some embodiments, the decrease in the permeability of the gastrointestinal barrier is mediated indirectly by the synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation is processed in vivo into at least one secondary species. In some embodiments, the synthetic oligosaccharide preparation is processed in vivo by components of the gastrointestinal microbiota 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 decrease in gastrointestinal permeability is mediated directly by the at least one secondary species.

[0016]

[0016] In some embodiments, the gastrointestinal barrier dysfunction is a hyperpermeable gastrointestinal barrier. In some embodiments, the hyperpermeable gastrointestinal barrier allows the movement of intestinal contents from the luminal space into the circulation of the animal. In some embodiments, the intestinal contents include food particles, microorganisms, toxins, or any combination thereof.

[0017]

[0017] 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 a decrease in mucus synthesis, a decrease in mucin synthesis, a decrease in mucus secretion, a decrease in mucin secretion, a decrease in nutrient absorption, an increase in inflammation, a decrease in resistance to infection, a decrease in the proliferation of intestinal epithelial cells, a decrease in the maturation of intestinal epithelial cells, an increase in immune cells in the gastrointestinal tract, an increase in the levels of pro-inflammatory cytokines or chemokines in the blood or gastrointestinal tract, irritable bowel syndrome, inflammatory bowel syndrome, rectal inflammation, systemic infection, systemic inflammation, malnutrition, a decrease in weight gain, weight loss, an increase in feed requirement, a decrease in feed efficiency, hair loss, fecal incontinence, or diarrhea, compared to an equivalent control animal lacking the gastrointestinal barrier dysfunction.

[0018]

[0018] In some embodiments, the animal has an infection. In some embodiments, the gastrointestinal barrier dysfunction is related to 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.

[0019]

[0019] 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 Hammondia infection. In some embodiments, the coccidiosis infection is an Eimeria infection. In some embodiments, the Eimeria infection is an E. mivati, E. tenella, E. acervulina, or E. maxima infection. In some embodiments, the coccidiosis infection is a Toxoplasma infection. In some embodiments, the Toxoplasma infection is a Toxoplasma gondii infection. In some embodiments, the coccidiosis infection is a 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.

[0020]

[0020] 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 a Hammondia infection. In some embodiments, the Hammondia infection is a Hammondia spp infection. In some embodiments, the coccidiosis infection is an Eimeria acervuline, Eimeria maxima, Eimeria mitis, Eimeria tenella, Toxoplasma gondii, Hammondia spp., Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Isospora canis, Isospora ohioensis, Isospora burrosi, or Isospora felis infection.

[0021]

[0021] In some embodiments, the infection is a bacterial infection. In some embodiments, the bacterial infection is a Staphylococcal infection, a Shigella infection, a Campylobacter infection, a Salmonella infection, an Escherichia infection, or a Yersinia infection.

[0022]

[0022] 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 liver protein APG (α-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, compared to an equivalent animal without the infection.

[0023]

[0023] In some embodiments, the animal has a decreased gastrointestinal barrier inflammation compared to the gastrointestinal barrier inflammation before administration of the synthetic oligosaccharide preparation. In some embodiments, the animal has a decreased gastrointestinal barrier inflammation compared to the gastrointestinal barrier inflammation of an equivalent control animal administered a nutritional composition without the synthetic oligosaccharide preparation. 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.

[0024]

[0024] In some embodiments, the animal shows a decrease in CD8+ T cells, an increase in CD4+ T cells, an increase in the level of circulating diamine oxidase, or a decrease in the level of circulating antibodies (e.g., IgA), compared to the animal before administration of the synthetic oligosaccharide preparation. In some embodiments, the animal shows a decrease in CD8+ T cells, an increase in CD4+ T cells, an increase in the level of circulating diamine oxidase, or a decrease in the level of circulating antibodies (e.g., IgA), compared to an equivalent control animal administered a nutritional composition without the synthetic oligosaccharide preparation.

[0025]

[0025] In some embodiments, the animal exhibits an increase in nutrient absorption through the gastrointestinal barrier as compared to the gastrointestinal barrier before administering the synthetic oligosaccharide preparation. In some embodiments, the animal exhibits an increase in nutrient absorption through the gastrointestinal barrier as compared to the gastrointestinal barrier of an equivalent control animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation.

[0026]

[0026] 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, NPC1L1, 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.

[0027]

[0027] In some embodiments, the animal has an increased body weight as compared to the body weight of the animal before administration of the synthetic oligosaccharide preparation. In some embodiments, the body weight of the animal increases by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% as compared to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. 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% as compared to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the increase in body weight is a greater increase as compared to the increase in body weight of an equivalent control animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation. In some embodiments, the increase in body weight is an increase that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% greater than the increase in body weight of an equivalent control animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation. In some embodiments, the increase in body weight is an increase that 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 of an equivalent control animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation.

[0028] In some embodiments, the animal has an increased feed efficiency compared to the feed efficiency of the animal before administration of the synthetic oligosaccharide preparation. In some embodiments, the feed efficiency of the animal is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% increased compared to the feed efficiency of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. 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% compared to the body weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the increase in feed efficiency is a greater increase compared to an equivalent subject animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation. 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 of an equivalent control animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation. 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 of the equivalent control animal administered the equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation.

[0029]

[0029] In some embodiments, the animal has a reduced feed conversion ratio (FCR) compared to the animal's FCR before administration of the synthetic oligosaccharide preparation. In some embodiments, the animal's feed conversion ratio is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% reduced compared to the animal's feed conversion ratio before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the animal's feed conversion ratio (FCR) 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% compared to the animal's body weight before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the animal has the reduction in feed conversion ratio that is a greater reduction compared to an equivalent subject animal administered an equivalent nutritional composition without the synthetic oligosaccharide preparation. In some embodiments, the reduction in feed conversion ratio is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% greater than the reduction in the feed conversion ratio of the equivalent control animal administered an equivalent nutritional composition without the synthetic oligosaccharide preparation. In some embodiments, the reduction in feed conversion ratio is about 1 - 40%, 1 - 30%, 1 - 20%, 1 - 10%, 1 - 5%, 1 - 4%, 1 - 3% or 1 - 2% greater than the reduction in the feed conversion ratio of the equivalent control animal administered the equivalent nutritional composition without the synthetic oligosaccharide preparation.

[0030]

[0030] In some embodiments, the average lifespan or survival rate of the animal is increased compared to an equivalent subject animal administered an equivalent nutritional composition without the synthetic oligosaccharide preparation.

[0031]

[0031] 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.

[0032]

[0032] 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 per day. In some embodiments, the administration comprises providing the animal with the nutritional composition comprising the synthetic oligosaccharide preparation for free intake. In some embodiments, the animal ingests at least a portion of the nutritional composition comprising the synthetic oligosaccharide preparation over at least one, two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, forty, fifty, sixty, ninety or one hundred and twenty 24-hour periods.

[0033]

[0033] In some embodiments, the nutritional composition comprises at least 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. In some embodiments, 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. In some embodiments, the nutritional composition comprises about 500 ppm 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.

[0034]

[0034] In some embodiments, the animal is poultry, fish, sheep, cattle, calves, buffalo, bison, pigs, cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigs, ferrets, chinchillas, hamsters, mice, rats, fish, shrimp or birds.

[0035]

[0035] 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 chicken, a layer chicken or a breeder chicken.

[0036]

[0036] In some embodiments, the animal is a pig. In some embodiments, the pig is a young pig, a growing pig or a finishing pig.

[0037]

[0037] In some embodiments, the animal is a fish. In some embodiments, the fish is a salmon, a tilapia or a tropical fish.

[0038]

[0038] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basic nutritional composition is a basic animal feed.

[0039]

[0039] In some embodiments, the relative abundance is determined by LC-MS / MS.

[0040]

[0040] In some embodiments, the relative abundance of the oligosaccharide in at least 5, 10, 20 or 30 DP fractions decreases monotonically with its degree of polymerization. In some embodiments, the relative abundance of the oligosaccharide in each of the n fractions decreases monotonically with its degree of polymerization.

[0041]

[0041] 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, 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.

[0042]

[0042] In some embodiments, the DP2 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of 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%.

[0043]

[0043] In some embodiments, the DP2 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 5% to about 10%. In some embodiments, the DP2 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 1% to about 10%. In some embodiments, the DP2 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.5% to about 10%. In some embodiments, the DP2 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 2% to about 12%.

[0044]

[0044] In some embodiments, the DP1 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of 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%. In some embodiments, the DP1 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 2% to about 12%. In some embodiments, the DP1 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 1% to about 10%. In some embodiments, the DP1 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.5% to about 10%. In some embodiments, the DP1 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 5% to about 10%.

[0045]

[0045] In some embodiments, the DP3 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of 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%. In some embodiments, the DP3 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 2% to about 12%. In some embodiments, the DP3 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 1% to about 10%. In some embodiments, the DP3 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.5% to about 10%. In some embodiments, the DP3 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 5% to about 10%.

[0046]

[0046] In some embodiments, the oligosaccharide preparation contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 2% to about 12%. In some embodiments, the oligosaccharide preparation contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.5% to about 10%. In some embodiments, the oligosaccharide preparation contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 1% to about 10%. In some embodiments, the oligosaccharide preparation contains anhydro-subunit-containing oligosaccharides in a relative abundance of about 5% to about 10%.

[0047]

[0047] In some embodiments, the DP2 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.6%, more than 0.8%, more than 1.0%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11% or more than 12%.

[0048]

[0048] In some embodiments, the DP1 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.6%, more than 0.8%, more than 1.0%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11% or more than 12%.

[0049]

[0049] In some embodiments, the DP3 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.6%, more than 0.8%, more than 1.0%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11% or more than 12%.

[0050]

[0050] In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.5%, 0.6%, more than 0.8%, more than 1.0%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11% or more than 12%.

[0051]

[0051] In some embodiments, the oligosaccharide preparation has a DP1 fraction content of about 1 wt% to about 40 wt%, as determined by liquid chromatography.

[0052]

[0052] In some embodiments, the oligosaccharide preparation has a DP2 fraction content of about 1 wt% to about 35 wt%, as determined by liquid chromatography.

[0053]

[0053] In some embodiments, the oligosaccharide preparation has a DP3 fraction content of about 1 wt% to about 30 wt%, as determined by liquid chromatography.

[0054]

[0054] In some embodiments, the oligosaccharide preparation has a DP4 fraction content of about 0.1 wt% to about 20 wt%, as determined by liquid chromatography.

[0055]

[0055] In some embodiments, the oligosaccharide preparation has a DP5 fraction content of about 0.1 wt% to about 15 wt%, as determined by liquid chromatography.

[0056]

[0056] In some embodiments, the ratio of the DP2 fraction to the DP1 fraction is from about 0.02 to about 0.40 as determined by liquid chromatography.

[0057]

[0057] In some embodiments, the ratio of the DP3 fraction to the DP2 fraction is from about 0.01 to about 0.30 as determined by liquid chromatography.

[0058]

[0058] In some embodiments, the aggregate content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, less than 40% or less than 30% as determined by liquid chromatography.

[0059]

[0059] In some embodiments, the oligosaccharide preparation comprises at least 103, at least 104, at least 105, at least 106 or at least 109 different oligosaccharide species.

[0060]

[0060] In some embodiments, two or more independent oligosaccharides comprise different anhydro subunits.

[0061]

[0061] In some embodiments, each of the anhydro-subunit-containing oligosaccharides comprises one or more anhydro subunits that are thermally dehydrated products of monosaccharides.

[0062]

[0062] In some embodiments, the oligosaccharide preparation comprises one or more anhydro subunits selected from anhydro-glucose, anhydro-galactose, anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-glucose, anhydro-idose, anhydro-talose, anhydro-fructose, anhydro-ribose, anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose and anhydro-xylose.

[0063]

[0063] In some embodiments, the oligosaccharide preparation comprises one or more anhydro-glucose, anhydro-galactose, anhydro-mannose or anhydro-fructose subunits.

[0064]

[0064] In some embodiments, the DP1 fraction comprises 1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranose anhydro subunits. In some embodiments, the DP1 fraction comprises both 1,6-anhydro-β-D-glucofuranose anhydro subunits and 1,6-anhydro-β-D-glucopyranose anhydro subunits.

[0065]

[0065] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is from 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 the oligosaccharide preparation. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 the oligosaccharide preparation. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in the oligosaccharide preparation.

[0066]

[0066] In some embodiments, the DP2 fraction comprises at least 5 anhydro-subunit-containing oligosaccharides. In some embodiments, the DP2 fraction comprises about 5 to 10 anhydro-subunit-containing oligosaccharides.

[0067]

[0067] 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-hydroxycyclopenta-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosenone; cyclic hydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxymethylfurfural (5-hmf).

[0068]

[0068] In some embodiments, more than 50%, 60%, 70%, 80%, 90%, 95% or 99% of the anhydro-subunit-containing oligosaccharides contain a chain-terminal anhydro-subunit.

[0069]

[0069] In some embodiments, the oligosaccharide preparation has a weight-average molecular weight of about 300 to about 5000 g / mol, as determined by high performance liquid chromatography (HPLC). In some embodiments, the oligosaccharide preparation has a weight-average molecular weight of about 300 to about 2500 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a weight-average molecular weight of about 500 to about 2000 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a weight-average molecular weight of about 500 to about 1500 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a number-average molecular weight of about 300 to about 5000 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a number-average molecular weight of about 300 to about 2500 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a number-average molecular weight of about 500 to about 2000 g / mol, as determined 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.

[0070]

[0070] 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.

[0071]

[0071] In one aspect, a method of treating and preventing infection in an animal, comprising administering to the animal a nutritional composition comprising a basal nutritional composition and a synthetic oligosaccharide preparation, wherein at least n fractions of the oligosaccharide each have a distinct degree of polymerization selected from 1 to n (DP1 to DPn fractions), n is an integer greater than 3, and each of the DP1 and DP2 fractions independently comprises an oligosaccharide containing anhydro subunits in a relative abundance of about 0.5% to about 15%, as determined by mass spectrometry, thereby treating and preventing the infection. A method is provided herein.

[0072]

[0072] In some embodiments, the level of at least one immune cell in a sample from the animal is increased as compared to the level of the at least one immune cell in a sample from the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the increase is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% increase as compared to the level of the at least one immune cell prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the increase is about 1-40%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3% or 1-2% increase as compared to the level of the at least one immune cell prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0073]

[0073] In some embodiments, the level of at least one immune cell in a sample from the animal is increased as compared to the level of the at least one immune cell in a sample from an equivalent control animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation. In some embodiments, the increase is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% increase as compared to the level of the at least one immune cell in the sample from the equivalent control animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation. In some embodiments, the increase is about 1-40%, 1-35%, 1-30%, 1-25%, 1-20%, 1-15%, 1-10%, 1-5%, 1-4%, 1-3% or 1-2% as compared to the level of the at least one immune cell in the sample from the equivalent control animal administered an equivalent nutritional composition that does not comprise the synthetic oligosaccharide preparation.

[0074]

[0074] In some embodiments, the at least one immune cell is a neutrophil, eosinophil, basophil, mast cell, T cell, B cell, macrophage, monocyte, granulocyte or dendritic cell. In some embodiments, the immune cell is a phagocyte. In some embodiments, the phagocyte is a neutrophil, eosinophil, basophil, mast cell, macrophage, monocyte or dendritic cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a granulocyte.

[0075]

[0075] In some embodiments, the level of at least one pro-inflammatory cytokine in the sample from the animal increases as compared to the level of the at least one pro-inflammatory cytokine in the sample from the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the level of at least one pro-inflammatory cytokine in the sample from the animal increases as compared to the level of the at least one pro-inflammatory cytokine in the sample from an equivalent control animal administered an equivalent nutritional composition without the synthetic oligosaccharide preparation. In some embodiments, the at least one pro-inflammatory cytokine is IL1, IL-12, IL18, TNFA, IFNG, GM-CSF, IL-1β, IL6, RANTES, MCP1, IL8, MIP-1α, MIP-1β, lymphotactin, fractalkine or GRO / KC.

[0076]

[0076] In some embodiments, the permeability of the gastrointestinal barrier of the animal is decreased as compared to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation. In some embodiments, the decrease is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% decrease as compared to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation, or the decrease 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% decrease as compared to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation. In some embodiments, the decrease is a greater decrease as compared to the decrease in the permeability of the gastrointestinal barrier of an equivalent control animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, the decrease is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% greater than the decrease in the permeability of the gastrointestinal barrier of an equivalent control animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation.

[0077]

[0077] In some embodiments, the permeability of the gastrointestinal barrier of the animal is decreased as compared to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation. In some embodiments, the decrease is at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% decrease as compared to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation, or the decrease 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% decrease as compared to the permeability of the gastrointestinal barrier of the animal before administration of the synthetic oligosaccharide preparation.

[0078]

[0078] In some embodiments, the gastrointestinal barrier dysfunction is related to or caused by an infection. In some embodiments, the infection is a parasitic, bacterial, fungal, or viral infection.

[0079]

[0079] 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 Hammondia infection. In some embodiments, the coccidiosis infection is an Eimeria infection. In some embodiments, the Eimeria infection is an E. mivati, E. tenella, E. acervulina, or E. maxima infection. In some embodiments, the coccidiosis infection is a Toxoplasma infection. In some embodiments, the Toxoplasma infection is a Toxoplasma gondii infection. In some embodiments, the coccidiosis infection is a 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. 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 a Hammondia infection.In some embodiments, the Hammondia infection is an infection with Hammondia spp. In some embodiments, the coccidiosis infection is an infection with Eimeria acervuline, Eimeria maxima, Eimeria mitis, Eimeria tenella, Toxoplasma gondii, Hammondia spp., Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Isospora canis, Isospora ohioensis, Isospora burrosi or Isospora felis.

[0080]

[0080] In some embodiments, the infection is a bacterial infection. In some embodiments, the bacterial infection is a Staphylococcal infection, a Shigella infection, a Campylobacter infection, a Salmonella infection, an Escherichia infection or a Yersinia infection.

[0081]

[0081] 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 1, 2, 3, 4 or 5 times a day. In some embodiments, the administration comprises providing the animal with the nutritional composition comprising the synthetic oligosaccharide preparation for free 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 24-hour periods.

[0082]

[0082] In some embodiments, the nutritional composition comprises at least 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. In some embodiments, 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. In some embodiments, the nutritional composition comprises about 500 ppm 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.

[0083]

[0083] In some embodiments, the animal is poultry, fish, sheep, cattle, beef cattle, buffalo, bison, pigs, cats, dogs, rabbits, goats, guinea pigs, donkeys, camels, horses, pigs, ferrets, gerbils, hamsters, mice, rats, fish, shrimp or birds.

[0084]

[0084] In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken, a turkey, a duck or a goose. In some embodiments, the chicken is a broiler chicken, a layer chicken or a breeder chicken.

[0085]

[0085] In some embodiments, the animal is a pig. In some embodiments, the pig is a young pig, a growing pig or a finishing pig.

[0086]

[0086] In some embodiments, the animal is a fish. In some embodiments, the fish is a salmon, a tilapia or a tropical fish.

[0087]

[0087] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basic nutritional composition is a basic animal feed.

[0088]

[0088] In some embodiments, the relative abundance is determined by LC-MS / MS.

[0089]

[0089] In some embodiments, the relative abundance of the oligosaccharide in at least 5, 10, 20 or 30 DP fractions decreases monotonically with its degree of polymerization. In some embodiments, the relative abundance of the oligosaccharide in each of the n fractions decreases monotonically with its degree of polymerization.

[0090]

[0090] 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, 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.

[0091]

[0091] In some embodiments, the DP2 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of 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%.

[0092]

[0092] In some embodiments, the DP2 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 5% to about 10%. In some embodiments, the DP2 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 1% to about 10%. In some embodiments, the DP2 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 0.5% to about 10%. In some embodiments, the DP2 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 2% to about 12%.

[0093]

[0093] In some embodiments, the DP1 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of 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%. In some embodiments, the DP1 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 2% to about 12%. In some embodiments, the DP1 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 1% to about 10%. In some embodiments, the DP1 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 0.5% to about 10%. In some embodiments, the DP1 fraction contains oligosaccharides containing anhydro subunits in a relative abundance of about 5% to about 10%.

[0094]

[0094] In some embodiments, the DP3 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of 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%. In some embodiments, the DP3 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 2% to about 12%. In some embodiments, the DP3 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 1% to about 10%. In some embodiments, the DP3 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 0.5% to about 10%. In some embodiments, the DP3 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 5% to about 10%.

[0095]

[0095] In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 2% to about 12%. In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 0.5% to about 10%. In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 1% to about 10%. In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of from about 5% to about 10%.

[0096]

[0096] In some embodiments, the DP2 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.6%, more than 0.8%, more than 1.0%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, or more than 12%.

[0097]

[0097] In some embodiments, the DP1 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.6%, more than 0.8%, more than 1.0%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, or more than 12%.

[0098]

[0098] In some embodiments, the DP3 fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.6%, more than 0.8%, more than 1.0%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, or more than 12%.

[0099]

[0099] In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 0.5%, 0.6%, 0.8%, 1.0%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12%.

[0100]

[0100] In some embodiments, the oligosaccharide preparation has a DP1 fraction content of about 1 wt% to about 40 wt%, as determined by liquid chromatography.

[0101]

[0101] In some embodiments, the oligosaccharide preparation has a DP2 fraction content of about 1 wt% to about 35 wt%, as determined by liquid chromatography.

[0102]

[0102] In some embodiments, the oligosaccharide preparation has a DP3 fraction content of about 1 wt% to about 30 wt%, as determined by liquid chromatography.

[0103]

[0103] In some embodiments, the oligosaccharide preparation has a DP4 fraction content of about 0.1 wt% to about 20 wt%, as determined by liquid chromatography.

[0104]

[0104] In some embodiments, the oligosaccharide preparation has a DP5 fraction content of about 0.1 wt% to about 15 wt%, as determined by liquid chromatography.

[0105]

[0105] In some embodiments, the ratio of the DP2 fraction to the DP1 fraction is determined by liquid chromatography and is from about 0.02 to about 0.40.

[0106]

[0106] In some embodiments, the ratio of the DP3 fraction to the DP2 fraction is determined by liquid chromatography and is from about 0.01 to about 0.30.

[0107]

[0107] In some embodiments, the aggregate content of the DP1 and DP2 fractions in the oligosaccharide preparation is determined by liquid chromatography and is less than 50%, less than 40% or less than 30%.

[0108]

[0108] In some embodiments, the oligosaccharide preparation comprises at least 103, at least 104, at least 105, at least 106 or at least 109 different oligosaccharide species.

[0109]

[0109] In some embodiments, two or more independent oligosaccharides comprise different anhydro subunits.

[0110]

[0110] In some embodiments, each of the anhydro-subunit-containing oligosaccharides comprises one or more anhydro subunits that are thermally dehydrated products of monosaccharides.

[0111]

[0111] In some embodiments, the oligosaccharide preparation comprises one or more anhydro subunits selected from anhydro-glucose, anhydro-galactose, anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-glucose, anhydro-idose, anhydro-talose, anhydro-fructose, anhydro-ribose, anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose and anhydro-xylose.

[0112]

[0112] In some embodiments, the oligosaccharide preparation comprises one or more anhydro-glucose, anhydro-galactose, anhydro-mannose, or anhydro-fructose subunits.

[0113]

[0113] In some embodiments, the DP1 fraction comprises 1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranose anhydro subunits. In some embodiments, the DP1 fraction comprises both 1,6-anhydro-β-D-glucofuranose anhydro subunits and 1,6-anhydro-β-D-glucopyranose anhydro subunits.

[0114]

[0114] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is from 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 the oligosaccharide preparation. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 the oligosaccharide preparation. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in the oligosaccharide preparation.

[0115]

[0115] In some embodiments, the DP2 fraction comprises at least 5 anhydro-subunit-containing oligosaccharides. In some embodiments, the DP2 fraction comprises about 5 to 10 anhydro-subunit-containing oligosaccharides.

[0116]

[0116] 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-hydroxycyclopenta-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosenone; cyclic hydroxylated lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxymethylfurfural (5-hmf).

[0117]

[0117] In some embodiments, more than 50%, 60%, 70%, 80%, 90%, 95% or 99% of the anhydro-subunit-containing oligosaccharides contain a chain-terminal anhydro-subunit.

[0118]

[0118] In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 300 to about 5000 g / mol, as determined by high performance liquid chromatography (HPLC). In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 300 to about 2500 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 500 to about 2000 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a weight average molecular weight of about 500 to about 1500 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 300 to about 5000 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 300 to about 2500 g / mol, as determined by HPLC. In some embodiments, the oligosaccharide preparation has a number average molecular weight of about 500 to about 2000 g / mol, as determined 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.

[0119]

[0119] 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.

[0120]

[0120] The present disclosure is based, at least in part, on the discovery that oligosaccharides containing one or more anhydro subunits reduce the permeability of the gastrointestinal barrier. Accordingly, the present disclosure features, inter alia, a method for preventing gastrointestinal dysfunction in an animal, comprising administering an oligosaccharide preparation described herein.

[0121] In some embodiments, the relative abundance of the oligosaccharides in at least the 5, 10, 20 or 30 DP fractions of the oligosaccharide preparation decreases monotonically with its degree of polymerization. In some embodiments, the relative abundance of the oligosaccharides in each of the n fractions of the oligosaccharide preparation decreases monotonically with its 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, 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.

[0122] In some embodiments, at least one fraction of the oligosaccharide preparation comprises oligosaccharides containing anhydro subunits 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 oligosaccharides containing anhydro subunits 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 oligosaccharides containing anhydro subunits 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, at least one fraction of the oligosaccharide preparation comprises oligosaccharides containing anhydro subunits in a relative abundance of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2%. In some embodiments, the oligosaccharide preparation comprises oligosaccharides containing anhydro subunits in a relative abundance of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2%. In some embodiments, each fraction of the oligosaccharide preparation comprises oligosaccharides containing anhydro subunits in a relative abundance of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2%. In some embodiments, at least one fraction of the oligosaccharide comprises oligosaccharides containing anhydro subunits in a relative abundance of more 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%.In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more 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%. In some embodiments, each fraction of the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more 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%. In some embodiments, at least one fraction of the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 20%, 21%, 22%, 23%, 24% or 25%. In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 20%, 21%, 22%, 23%, 24% or 25%. In some embodiments, each fraction of the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of more than 20%, 21%, 22%, 23%, 24% or 25%. 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-containing oligosaccharides have only one anhydro-subunit.

[0123] In some embodiments, the oligosaccharide preparation has a DP1 fraction content of 1 to 40% in relative abundance. In some embodiments, the oligosaccharide preparation has a DP2 fraction content of 1 to 35% in relative abundance. In some embodiments, the oligosaccharide preparation has a DP3 fraction content of 1 to 30% in relative abundance. In some embodiments, the oligosaccharide preparation has a DP4 fraction content of 0.1 to 20% in relative abundance. In some embodiments, the oligosaccharide preparation includes a DP5 fraction content of 0.1 to 15% in relative abundance. In some embodiments, the oligosaccharide preparation includes a DP2 fraction and a DP1 fraction, wherein the ratio of the DP2 fraction to the DP1 fraction is 0.02 to 0.40 in relative abundance. In some embodiments, the oligosaccharide preparation includes 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 to 0.30 in relative abundance. In some embodiments, the oligosaccharide preparation includes a DP1 fraction and a DP2 fraction, wherein the aggregate content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 50, 30 or 10% in relative abundance. In some embodiments, the oligosaccharide preparation includes at least 25, 50, 75, 100, 103, 104, 105, 106, 109, 110, 120, 150 or 200 different oligosaccharide species.

[0124] In some embodiments, at least two independent oligosaccharides of the oligosaccharide preparation comprise different anhydro subunits. In some embodiments, the oligosaccharide preparation comprises at least one anhydro subunit that is a reversible thermal dehydration product of a monosaccharide. In some embodiments, the oligosaccharide preparation comprises at least one anhydro-glucose, anhydro-galactose, anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-glucose, anhydro-idose, anhydro-talose, anhydro-fructose, anhydro-ribose, anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose or anhydro-xylose subunit. In some embodiments, the oligosaccharide preparation comprises at least one anhydro-glucose, anhydro-galactose, anhydro-mannose or anhydro-fructose subunit.

[0125] In some embodiments, the oligosaccharide preparation comprises at least one 1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranose subunit. In some embodiments, the oligosaccharide preparation comprises at least one 1,6-anhydro-β-D-glucofuranose subunit and at least one 1,6-anhydro-β-D-glucopyranose anhydro subunit. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose in the oligosaccharide preparation is about 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-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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-glucofuranose 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-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 each fraction.In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 each fraction of the oligosaccharide preparation. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in each fraction of the oligosaccharide preparation. 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 the group consisting of 1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranose.

[0126]

[0126] In some embodiments, the oligosaccharide preparation comprises at least one anhydro subunit that is 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-hydroxycyclopenta-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosenone; cyclic hydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxymethylfurfural (5-hmf). In some embodiments, about 0.1% to 5%, 0.1% to 2% or 0.1% to 1% of the anhydro subunits in the oligosaccharide preparation are caramelization products.

[0127]

[0127] In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the anhydro-subunit-containing oligosaccharides in the oligosaccharide preparation contain a chain-terminal anhydro-subunit.

[0128]

[0128] In some embodiments, the weight-average molecular weight of the oligosaccharide preparation is about 300 - 5000 g / mol, 500 - 5000 g / mol, 700 - 5000 g / mol, 500 - 2000 g / mol, 700 - 2000 g / mol, 700 - 1500 g / mol, 300 - 1500 g / mol, 300 - 2000 g / mol, 400 - 1300 g / mol, 400 - 1200 g / mol, 400 - 1100 g / mol, 500 - 1300 g / mol, 500 - 1200 g / mol, 500 - 1100 g / mol, 600 - 1300 g / mol, 600 - 1200 g / mol or 600 - 1100 g / mol. In some embodiments, the number-average molecular weight of the oligosaccharide preparation is about 300 - 5000 g / mol, 500 - 5000 g / mol, 700 - 5000 g / mol, 500 - 2000 g / mol, 700 - 2000 g / mol, 700 - 1500 g / mol, 300 - 1500 g / mol, 300 - 2000 g / mol, 400 - 1000 g / mol, 400 - 900 g / mol, 400 - 800 g / mol, 500 - 900 g / mol or 500 - 800 g / mol. In some embodiments, the weight-average molecular weight of the oligosaccharide preparation is about 2000 - 2800 g / mol, 2100 - 2700 g / mol, 2200 - 2600 g / mol, 2300 - 2500 g / mol or 2320 - 2420 g / mol. In some embodiments, the number-average molecular weight of the oligosaccharide preparation is about 1000 - 2000 g / mol, 1100 - 1900 g / mol, 1200 - 1800 g / mol, 1300 - 1700 g / mol, 1400 - 1600 g / mol or 1450 - 1550 g / mol.

[0129]

[0129] In one aspect, a method for treating and preventing gastrointestinal barrier dysfunction in an animal, comprising administering to the animal a nutritional composition comprising a basal nutritional composition and a synthetic oligosaccharide preparation, the synthetic oligosaccharide preparation comprising at least n fractions of oligosaccharides (DP1 - DPn fractions) each having a distinct degree of polymerization selected from 1 to n, where n is an integer greater than 2; and each fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of 1% to 90% as measured by mass spectrometry. A method is provided herein.

[0130]

[0130] In some embodiments, the permeability of the gastrointestinal barrier is lower than that of an animal administered a nutritional composition without the synthetic oligosaccharide preparation. In some embodiments, the gastrointestinal barrier dysfunction is hyperpermeability (leaky gut).

[0131]

[0131] 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% lower than that of an animal administered a nutritional composition without 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% lower than that of an animal administered a nutritional composition without the synthetic oligosaccharide preparation.

[0132]

[0132] 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 species. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the animal's gastrointestinal microbiota. In some embodiments, the component is a gastrointestinal bacterium. In some embodiments, the reduction in gastrointestinal permeability is directly mediated by one or more of the secondary species.

[0133]

[0133] In some embodiments, an animal is less likely to develop gastrointestinal barrier dysfunction compared to an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation.

[0134]

[0134] In some embodiments, an animal has a higher body weight than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, an animal has a higher feed efficiency than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, an animal has a lower feed requirement rate than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation.

[0135]

[0135] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cattle, beef cattle, buffalo, bison, pig (e.g., young pig, growing / finishing pig), cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird or human. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler, laying or breeding), duck, goose or turkey. In some embodiments, the animal is a pig (e.g., young pig, growing / finishing pig).

[0136]

[0136] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basic animal feed.

[0137]

[0137] A method for promoting goblet cell proliferation in an animal, comprising administering to the animal a nutritional composition comprising a basal nutritional composition and an oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1 - DPn fractions) each having a distinct degree of polymerization selected from 1 to n, where n is an integer greater than 2; and each fraction comprises 1% - 90% anhydro subunit-containing oligosaccharides by relative abundance measured by mass spectrometry. A method is provided herein.

[0138]

[0138] In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is higher compared to the level of goblet cells in the gastrointestinal tract of an animal administered a nutritional composition that does not contain the synthetic oligosaccharide preparation. 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 administered a nutritional composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, the level of goblet cells in the gastrointestinal tract of the animal is 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% greater than the level of goblet cells in a nutritional composition that does not contain the synthetic oligosaccharide preparation.

[0139]

[0139] In some embodiments, the increase in goblet cell levels is mediated directly by the synthetic oligosaccharide preparation. In some embodiments, the increase in goblet cell levels is mediated indirectly by the synthetic oligosaccharide preparation. In some embodiments, the oligosaccharide preparation is processed in vivo into one or more secondary species. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the animal's gastrointestinal microbiota. In some embodiments, the component is a gastrointestinal bacterium. In some embodiments, the increase in the number of goblet cells is mediated directly by one or more of the secondary species.

[0140]

[0140] In some embodiments, the animal has a higher body weight than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, the animal has a higher feed efficiency than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, the animal has a lower feed requirement than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation.

[0141]

[0141] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cattle, beef cattle, buffalo, bison, pig (e.g., piglet, growing / finishing pig), cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird or human. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler, laying or breeding), duck, goose or turkey. In some embodiments, the animal is a pig (e.g., piglet, growing / finishing pig). In some embodiments, the animal is livestock. In some embodiments, the animal is a companion animal.

[0142]

[0142] In some embodiments, the nutrient composition is an animal feed composition. In some embodiments, the basal nutrient composition is a basic animal feed.

[0143]

[0143] A method for promoting mucus production in the gastrointestinal tract of an animal, comprising administering to the animal a nutritional composition comprising a basal nutritional composition and an oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1 - DPn fractions) each having a distinct degree of polymerization selected from 1 to n, where n is an integer greater than 2; and each fraction comprises anhydro subunit-containing oligosaccharides in a relative abundance of 1% to 90% as measured by mass spectrometry. A method is provided herein.

[0144]

[0144] In some embodiments, the level of mucus in the gastrointestinal tract of the animal is higher compared to that in an animal administered a nutritional composition that does not contain the synthetic oligosaccharide preparation. 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% higher compared to the level of mucus in the gastrointestinal tract of an animal administered a nutritional composition that does not contain the synthetic oligosaccharide preparation. 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% higher compared to the level of mucus in the gastrointestinal tract of an animal administered a nutritional composition that does not contain the synthetic oligosaccharide preparation.

[0145]

[0145] In some embodiments, the method comprises obtaining a gastrointestinal sample from the animal. In some embodiments, the sample is a gastrointestinal tissue (e.g., 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 the protein component of the mucus. In some embodiments, the protein is mucin.

[0146]

[0146] In some embodiments, the increase in mucus level is directly mediated by the mucin oligosaccharide preparation. In some embodiments, the increase in mucus level is indirectly mediated by the synthetic oligosaccharide preparation. In some embodiments, the oligosaccharide preparation is processed into one or more secondary species in vivo. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the animal's gastrointestinal microbiota. In some embodiments, the component is a gastrointestinal bacterium. In some embodiments, the increase in mucus level is directly mediated by one or more of the secondary species.

[0147]

[0147] In some embodiments, the animal has a higher body weight than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, the animal has a higher feed efficiency than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, the animal has a lower feed requirement than an animal administered a nutrient composition that does not contain the synthetic oligosaccharide preparation.

[0148]

[0148] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cattle, beef cattle, buffalo, bison, pig (e.g., piglet, growing / finishing pig), cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird or human. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler, laying or breeding), duck, goose or turkey. In some embodiments, the animal is a pig (e.g., piglet, growing / finishing pig). In some embodiments, the animal is a livestock animal. In some embodiments, the animal is a companion animal.

[0149]

[0149] In some embodiments, the nutrient composition is an animal feed composition. In some embodiments, the basal nutrient composition is a basic animal feed.

[0150] A method of treating and preventing animal infections, comprising administering to an animal a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1-DPn fractions) each having a distinct degree of polymerization selected from 1 to n, n is an integer greater than 2, and each fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of about 1% to about 90% as measured by mass spectrometry. A method is provided herein.

[0151] In some embodiments, the level of one or more types of immune cells in the gastrointestinal tract of an animal is higher compared to the level in the gastrointestinal tract of an animal administered a nutritional composition that does not contain the synthetic oligosaccharide preparation.

[0152] In some embodiments, the immune cells are phagocytic. In some embodiments, the immune cells are granulocytes. In some embodiments, the level of granulocyte phagocytosis in the gastrointestinal tract of an animal is higher compared to the level in the gastrointestinal tract of an animal administered a nutritional composition that does not contain the synthetic oligosaccharide preparation. In some embodiments, the level of immune cells or immune cell activity is directly mediated by the synthetic oligosaccharide preparation.

[0153] 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 in vivo into one or more secondary species. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the animal's gastrointestinal microbiota. In some embodiments, the component is a gastrointestinal bacterium. In some embodiments, the level of immune cells or immune cell activity is directly mediated by one or more of the secondary species.

[0154]

[0154] In some embodiments, the level of one or more pro-inflammatory cytokines or chemokines in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation. In some embodiments, the level of one or more anti-inflammatory cytokines or chemokines in the gastrointestinal tract of an animal is low compared to the level in the gastrointestinal tract of an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation.

[0155]

[0155] In some embodiments, the permeability of the gastrointestinal barrier of an animal is low compared to the permeability of the gastrointestinal barrier of an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation.

[0156]

[0156] In some embodiments, the level of mucus in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation.

[0157]

[0157] In some embodiments, the level of goblet cells in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation.

[0158]

[0158] In some embodiments, the animal has a higher body weight than an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation. In some embodiments, the animal has a higher feed efficiency than an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation.

[0159]

[0159] In some embodiments, the animal has a lower feed requirement rate than an animal administered a nutritional composition that does not contain a synthetic oligosaccharide preparation.

[0160]

[0160] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cattle, beef cattle, buffalo, bison, pig (e.g., piglet, growing / finishing pig), cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, chinchilla, hamster, mouse, rat, bird or human. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler, laying or breeding), duck, goose or turkey. In some embodiments, the animal is a pig (e.g., piglet, growing / finishing pig). In some embodiments, the animal is livestock. In some embodiments, the animal is a companion animal.

[0161]

[0161] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basic animal feed.

[0162]

[0162] In one aspect, the present disclosure is based, at least in part, on the discovery that oligosaccharides containing one or more anhydro subunits reduce the permeability of the gastrointestinal barrier. Accordingly, in one aspect, the present disclosure features, inter alia, a method for treating gastrointestinal dysfunction in an animal, comprising administering an oligosaccharide preparation disclosed herein.

[0163]

[0163] In one aspect, provided herein is a method for treating gastrointestinal barrier dysfunction in an animal, comprising administering to an animal having gastrointestinal barrier dysfunction a nutritional composition comprising a basal nutritional composition and a synthetic oligosaccharide preparation, wherein the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1 - DPn fractions), each having a distinct degree of polymerization selected from 1 to n, where n is an integer greater than 2; and each fraction comprises 1% - 90% anhydro subunit-containing oligosaccharides by relative abundance measured by mass spectrometry.

[0164]

[0164] In some embodiments, the permeability of the animal's gastrointestinal barrier is lower compared to the permeability of the animal's gastrointestinal barrier prior to administration of the nutritional preparation comprising the synthetic oligosaccharide preparation.

[0165]

[0165] In some embodiments, the method includes that gastrointestinal barrier dysfunction includes hyperpermeability (leaky gut). In some embodiments, the permeability of the gastrointestinal barrier enables the movement of intestinal contents (e.g., food particles, microorganisms, toxins) from the luminal space into the animal's circulation. 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% lower than the permeability of the animal's gastrointestinal barrier prior to 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% lower than the permeability of the animal's gastrointestinal barrier prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0166]

[0166] In some embodiments, the decrease in gastrointestinal permeability is directly mediated by the synthetic oligosaccharide preparation. In some embodiments, the decrease 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 species. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the animal's gastrointestinal microbiota. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the decrease in gastrointestinal permeability is directly mediated by one or more of the secondary species.

[0167]

[0167] In some embodiments, one or more symptoms of gastrointestinal barrier dysfunction are improved. In some embodiments, the one or more symptoms are a decrease in mucus synthesis, a decrease in mucin synthesis, a decrease in mucus secretion, a decrease in mucin secretion, a decrease in nutrient absorption, an increase in inflammation, a decrease in resistance to infection, a decrease in the proliferation of intestinal epithelial cells, a decrease in the maturation of intestinal epithelial cells, malnutrition, a decrease in weight gain, an increase in feed requirement rate, a decrease in feed efficiency, and an increase in mortality.

[0168]

[0168] In some embodiments, the animal has a weight higher than the weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the animal has a feed efficiency higher than the feed efficiency of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the animal has a feed requirement rate lower than the feed requirement rate of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0169]

[0169] In some embodiments, the animal is a livestock animal. In some embodiments, the animal is a companion animal. In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), a poultry (e.g., chicken, turkey), an aquatic product (e.g., shrimp), a sheep, a cow, a beef cattle, a buffalo, a bison, a pig (e.g., a young pig, a growing / finishing pig), a cat, a dog, a rabbit, a goat, a guinea pig, a donkey, a camel, a horse, a pigeon, a ferret, a gerbil, a hamster, a mouse, a rat, a fish, a bird or a human. In some embodiments, the animal is a poultry. In some embodiments, the animal is a chicken (e.g., a broiler, a layer, a breeder), a turkey, a duck or a goose. In some embodiments, the animal is a pig (e.g., a young pig, a growing / finishing pig).

[0170]

[0170] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basal nutritional composition is a basal animal feed.

[0171]

[0171] A method of treating an infection, comprising administering to an animal a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation, the synthetic oligosaccharide preparation comprising at least n fractions of oligosaccharides (DP1 - DPn fractions) each having a different degree of polymerization selected from 1 to n, where n is an integer greater than 2; and each fraction comprises anhydro-subunit-containing oligosaccharides in a relative abundance of 1% - 90% as measured by mass spectrometry, is provided.

[0172]

[0172] In some embodiments, the level of one or more types of immune cells in the gastrointestinal tract of the animal is higher compared to the level in the gastrointestinal tract of the animal prior to administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0173]

[0173] In some embodiments, the immune cells are phagocytic. 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 compared 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 immune cells or immune cell activity is directly mediated by the synthetic oligosaccharide preparation.

[0174]

[0174] 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 in vivo into one or more secondary species. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the animal's gastrointestinal microbiota. In some embodiments, the components are gastrointestinal bacteria. In some embodiments, the increase in the level of mucus is directly mediated by one or more of the secondary species.

[0175]

[0175] In some embodiments, the level of one or more pro-inflammatory cytokines or chemokines in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the level of one or more anti-inflammatory cytokines or chemokines in the gastrointestinal tract of an animal is low compared to the level in the gastrointestinal tract of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation.

[0176]

[0176] In some embodiments, the permeability of the gastrointestinal barrier of an animal is low compared to the permeability of the gastrointestinal barrier of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the level of mucus in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the level of goblet cells in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation.

[0177]

[0177] In some embodiments, the animal has a body weight higher than the body weight of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the animal has a feed efficiency higher than the feed efficiency of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the animal has a feed requirement rate lower than the feed requirement rate of the animal prior to administration of a nutritional composition comprising a synthetic oligosaccharide preparation.

[0178]

[0178] In some embodiments, the animal is a livestock animal. In some embodiments, the animal is a companion animal. In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), a poultry (e.g., chicken, turkey), an aquatic product (e.g., shrimp), a sheep, a cow, a beef cattle, a buffalo, a bison, a pig (e.g., a young pig, a growing / finishing pig), a cat, a dog, a rabbit, a goat, a guinea pig, a donkey, a camel, a horse, a pigeon, a ferret, a gerbil, a hamster, a mouse, a rat, a fish, a bird or a human. In some embodiments, the animal is a poultry. In some embodiments, the animal is a chicken (e.g., a broiler, a layer, a breeder), a turkey, a duck or a goose. In some embodiments, the animal is a pig (e.g., a young pig, a growing / finishing pig).

[0179]

[0179] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the basic nutritional composition is a basic animal feed.

[0180]

[0180] A method of enhancing or inducing an immune response, comprising administering to an animal a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation, the synthetic oligosaccharide preparation comprising at least n fractions of oligosaccharides (DP1 - DPn fractions) each having a distinct degree of polymerization selected from 1 to n, where n is an integer greater than 2; and each fraction comprises 1% - 90% anhydro-subunit-containing oligosaccharides by relative abundance measured by mass spectrometry, is provided herein.

[0181]

[0181] In some embodiments, the level of one or more types of immune cells in the gastrointestinal tract of the animal is higher compared to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0182]

[0182] In some embodiments, the level of one or more types of immune cells in the gastrointestinal tract of the animal is higher compared to the level in the gastrointestinal tract of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0183]

[0183] In some embodiments, the immune cells are phagocytic. In some embodiments, the immune cells are granulocytes. In some embodiments, the level of granulocyte phagocytosis in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of the animal before administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the level of immune cells or immune cell activity is directly mediated by the synthetic oligosaccharide preparation.

[0184]

[0184] 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 in vivo into one or more secondary species. In some embodiments, the synthetic oligosaccharide preparation is processed by components of the animal's gastrointestinal microbiota. In some embodiments, the component is a gastrointestinal bacterium. In some embodiments, the increase in the level of mucus is directly mediated by one or more of the secondary species.

[0185]

[0185] In some embodiments, the level of one or more pro-inflammatory cytokines or chemokines in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of the animal before administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the level of one or more anti-inflammatory cytokines or chemokines in the gastrointestinal tract of an animal is low compared to the level in the gastrointestinal tract of the animal before administration of a nutritional composition comprising a synthetic oligosaccharide preparation.

[0186]

[0186] In some embodiments, the permeability of the animal's gastrointestinal barrier is low compared to the permeability of the animal's gastrointestinal barrier before administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the level of mucus in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of the animal before administration of a nutritional composition comprising a synthetic oligosaccharide preparation. In some embodiments, the level of goblet cells in the gastrointestinal tract of an animal is high compared to the level in the gastrointestinal tract of the animal before administration of a nutritional composition comprising a synthetic oligosaccharide preparation.

[0187]

[0187] In some embodiments, the animal has a weight that is higher than the weight of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the animal has a feed efficiency that is higher than the feed efficiency of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation. In some embodiments, the animal has a feed requirement that is lower than the feed requirement of the animal before administration of the nutritional composition comprising the synthetic oligosaccharide preparation.

[0188]

[0188] In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), seafood (e.g., shrimp), sheep, cattle, beef cattle, buffalo, bison, pig (e.g., piglet, growing / finishing pig), cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, bird or human. In some embodiments, the animal is poultry. In some embodiments, the animal is a chicken (e.g., broiler, layer, breeder), turkey, duck or goose. In some embodiments, the animal is a pig (e.g., piglet, growing / finishing pig).

[0189]

[0189] Additional aspects and advantages of the present disclosure will be readily apparent to those of ordinary skill in the art from the following detailed description, where only exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0190] [Incorporation by reference]

[0190] All publications, patents, and patent applications cited in this specification are incorporated 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 the incorporated publications and patents or patent applications conflict with the disclosure contained herein, the specification is intended to supersede and / or take precedence over such conflicting material.

[0191]

[0191] 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 from the following detailed description which illustrates exemplary embodiments in which the principles of the invention are utilized, and from the appended drawings (also referred to herein as "figures" and "FIGs").

Brief Description of the Drawings

[0192]

[0192]

Figure 1

[0193]

Figure 2

[0194]

Figure 3

[0195]

Figure 4

[0196]

Figure 5

[0197]

Figure 6

[0198]

Figure 7

[0199]

Figure 8A

[0199]

Figure 8B

[0200]

Figure 9A

Figure 9B

[0201]

Figure 10

[0202]

Figure 11

[0203]

Figure 12

[0204]

Figure 13

[0205]

Figure 14

[0206]

Figure 15

[0207]

Figure 16

[0208]

Figure 17

[0209]

Figure 18

[0210]

Figure 19

[0211]

Figure 20

[0212]

Figure 21

[0213]

Figure 22

[0214]

Figure 23

[0215]

Figure 24

[0216]

Figure 25

[0217]

Figure 26

[0218]

Figure 27A

[0218]

Figure 27B

[0218]

Figure 27C

[0219]

Figure 28A

[0219]

Figure 28B

[0220]

Figure 29A

[0220]

Figure 29B

[0221]

Figure 30

[0222]

Figure 31

[0223]

Figure 32A

[0223]

Figure 32B

[0224]

Figure 33A

[0224]

Figure 33B

[0224]

Figure 33C

[0225]

Figure 34A

[0225]

Figure 34B

[0225]

Figure 34C

[0226]

Figure 35A

[0226]

Figure 35B

[0226]

Figure 35C

[0227]

Figure 36A

[0227]

Figure 36B

[0227]

Figure 36C

[0228]

Figure 37A

[0228]

Figure 37B

[0229]

Figure 38A

[0229]

Figure 38B

[0230]

Figure 39A

[0230]

Figure 39B

[0231]

Figure 40A

[0231]

Figure 40B

[0232]

Figure 41

[0233]

Figure 42

[0234]

Figure 43

[0193] [Detailed Description]

[0235] The following description and examples explain the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein and can itself change. Those skilled in the art will understand that there are many embodiments and modifications of the present disclosure and that they are included within its scope.

[0194]

[0236] All terms are intended to be understood as understood by those skilled in the art. Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present disclosure pertains.

[0195]

[0237] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0196]

[0238] The various features of the present disclosure may be described in relation to a single embodiment, but the features may be provided separately or in any suitable combination. Conversely, the present disclosure may be described herein in relation to separate embodiments for clarity, but the present disclosure may also be implemented in a single embodiment.

[0197]

[0239] The following definitions supplement the definitions in the art and are directed to this application and do not pertain, for example, to patents or applications of the same owner, whether related or unrelated. Any methods and materials similar or equivalent to those described herein can be used to carry out the tests of the present disclosure, but the preferred materials and methods are described herein. Accordingly, the terms used herein are for the purpose of merely describing a particular embodiment and are not intended to be limiting.

[0198] [I. Definitions]

[0240] The terms used in this specification are for the purpose of describing particular cases only and are not intended to be limiting. As used herein, the singular forms "a", "an", and "the" also include the plural forms unless the context clearly dictates otherwise. Further, the terms "comprising", "comprises", "having", "has", "including", or any variation thereof are used in either the detailed description and / or the claims, and such terms are intended to be inclusive in the same manner as the term "including".

[0199]

[0241] The terms "include", "included", "including", etc. are understood to have the meaning ascribed to them in the United States Patent Law; that is, they mean "comprise", "comprised", "comprising", etc., and are intended to be inclusive or open-ended and do not exclude additional, unrecited elements or method steps; the terms "consisting essentially of" and "consisting essentially of" have the meaning ascribed to them in the United States Patent Law; that is, they allow for elements not expressly recited, but exclude elements found in the prior art or elements that affect the basic or novel characteristics of the invention.

[0200]

[0242] The term "and / or" used in phrases such as "A and / or B" in this specification is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" used in phrases such as "A, B and / or C" is intended to include 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.

[0201]

[0243] When ranges are used herein with respect to physical properties such as molecular weight or chemical properties such as chemical formula, all combinations and subcombinations of the ranges and specific embodiments therein are intended to be included. The term "about" when referring to a numerical value or numerical range means that the referenced numerical value or numerical range is an approximation within experimental variability (or within statistical experimental error), and thus, in some cases, the numerical value or numerical range may vary by 1% to 15% of the recited numerical value or numerical range. In some embodiments, the term "about" means within 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05% of a given value or range.

[0202]

[0244] As used herein, "administering" includes providing to an animal a synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition described herein such that the animal can freely ingest the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition. In such embodiments, the animal ingests a portion of the synthetic oligosaccharide preparation, nutritional composition, or animal feed composition. In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is provided to the animal such that the animal can freely ingest the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition. In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is administered to the animal as a prescribed diet. In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is administered to the animal via manual feeding, such as oral syringe feeding, tube feeding, etc. In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is administered orally to the animal, e.g., freely or manually. In some embodiments, the animal ingests a portion of the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition at least once every 24 hours or every other day for 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 another liquid, and the animal ingests a portion of the oligosaccharide preparation by drinking the liquid. In some embodiments, the oligosaccharide is provided to the animal via drinking water. In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is freely ingested.

[0203]

[0245] As used herein, the feed conversion ratio (FCR) refers to the ratio of the feed input (e.g., that ingested by the animal) to the livestock production, where the livestock product is the desired livestock product. For example, the livestock product for dairy animals is milk, while the livestock product for animals raised for meat is body mass.

[0204]

[0246] As used herein, "feed efficiency" refers to the ratio of livestock production to the amount of feed input (e.g., that ingested by an animal), where the livestock product is the desired livestock product.

[0205]

[0247] As used herein, the term "anhydro subunit" refers to the thermal dehydration product of a monosaccharide (or monosaccharide subunit) or a caramelized sugar product. For example, an "anhydro subunit" can be an anhydro-monosaccharide such as anhydro-glucose. As another example, an "anhydro subunit" can be capable of binding to one or more normal or anhydro-monosaccharide subunits via a glycosidic bond.

[0206]

[0248] The term "oligosaccharide" refers to a compound containing a monosaccharide or two or more monosaccharide subunits linked by a glycosidic bond. As such, oligosaccharides include normal monosaccharides; anhydro-monosaccharides; or compounds containing two or more monosaccharide subunits, where one or more of the monosaccharide subunits are optionally and independently replaced by one or more anhydro subunits. Oligosaccharides can be functionalized. As used herein, the term "oligosaccharide" encompasses all species of oligosaccharides, where each of the monosaccharide subunits in the oligosaccharide is independently and optionally functionalized and / or replaced by its corresponding anhydro-monosaccharide subunit.

[0207]

[0249] As used herein, the term "oligosaccharide preparation" refers to a preparation containing at least one oligosaccharide.

[0208]

[0250] As used herein, the term "gluco-oligosaccharide" refers to a compound containing glucose or two or more glucose monosaccharide subunits linked by a glycosidic bond. As such, gluco-oligosaccharides include glucose; anhydro-glucose; or a compound containing two or more glucose monosaccharide subunits linked by a glycosidic bond, wherein one or more of said glucose monosaccharide subunits are each optionally and independently replaced by anhydro-glucose subunits.

[0209]

[0251] As used herein, the term "galacto-oligosaccharide" refers to a compound containing galactose or two or more galactose monosaccharide subunits linked by a glycosidic bond. As such, galacto-oligosaccharides include galactose; anhydro-galactose; or a compound containing two or more galactose monosaccharide subunits linked by a glycosidic bond, wherein at least one monosaccharide subunit is optionally replaced by an anhydro-galactose subunit.

[0210]

[0252] As used herein, the term "gluco-galacto-oligosaccharide preparation" refers to a composition produced from a complete or incomplete sugar condensation reaction of glucose and galactose. Thus, in some embodiments, a gluco-galacto-oligosaccharide preparation includes a gluco-oligosaccharide, a galacto-oligosaccharide, a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by a glycosidic bond, or a combination thereof. In some embodiments, a gluco-galacto-oligosaccharide preparation includes a gluco-oligosaccharide and a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by a glycosidic bond. In some embodiments, a gluco-galacto-oligosaccharide preparation includes a galacto-oligosaccharide and a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by a glycosidic bond. In some embodiments, a gluco-galacto-oligosaccharide preparation includes a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by a glycosidic bond.

[0211]

[0253] As used herein, the terms "monosaccharide unit" and "monosaccharide subunit" are used interchangeably. A "monosaccharide subunit" refers to a monosaccharide monomer of an oligosaccharide. For an oligosaccharide having a degree of polymerization of 1, the oligosaccharide may be referred to as a monosaccharide subunit or a monosaccharide. For an oligosaccharide having a degree of polymerization of 2 or more, its monosaccharide subunits are linked via glycosidic bonds.

[0212]

[0254] As used herein, the term "normal monosaccharide" refers to a monosaccharide that does not contain an anhydro subunit. The term "normal disaccharide" refers to a disaccharide that does not contain an anhydro subunit. Thus, the term "normal subunit" refers to a subunit that is not an anhydro subunit.

[0213]

[0255] As used herein, the term "relative abundance" or "abundance" refers to the abundance of a species, based on how common or rare the species is. For example, a DP1 fraction containing anhydro-subunit-containing oligosaccharides with a relative abundance of 10% refers to a plurality of DP1 oligosaccharides where 10% of the DP1 oligosaccharides are anhydro-monosaccharides. For example, with respect to a particular DP fraction of oligosaccharides, the relative abundance can be determined by suitable analytical instruments, such as mass spectrometry and liquid chromatography, e.g., LC-MS / MS, GC-MS, HPLC-MS, and MALDI-MS. In some embodiments, the relative abundance is determined by integrating the area under the peak of a chromatograph (e.g., LC-MS / MS, GC-MS, and HPLC-MS) corresponding to the fraction of interest. In some embodiments, the relative abundance is determined by peak intensity (e.g., MALDI-MS). In some embodiments, the relative abundance is determined by a combination of analytical methods such as gravimetric measurement after separation by liquid chromatography.

[0214]

[0256] As used herein, the terms "anhydro DPn oligosaccharide", "anhydro DPn species", or "oligosaccharide having DPn anhydro-subunits" refer to oligosaccharides having a degree of polymerization of n and containing one or more anhydro-subunits. Thus, anhydroglucose is a DP1 anhydro-subunit containing an oligosaccharide, and cellotriosan is a DP3 anhydro-subunit containing an oligosaccharide.

[0215]

[0257] As used herein, the singular forms "a", "and", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "an oligosaccharide" includes one or more oligosaccharides (or plural oligosaccharides) and equivalents thereof known to those skilled in the art.

[0216] [II. Oligosaccharide Preparation]

[0258] This specification provides an oligosaccharide preparation suitable for use in a nutritional composition. In some embodiments, the oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, where n is an integer greater than 2. In some embodiments, n is an integer of 3 or more. In some embodiments, each of the 1 to n fractions in the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides in a relative abundance of 1% to 90% as measured by mass spectrometry. In some embodiments, the relative abundance of the oligosaccharides in each fraction decreases monotonically with its degree of polymerization.

[0217]

[0259] In some embodiments, n is an integer of 3 or more. In some embodiments, n is an integer within 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 contains anhydro-subunit-containing oligosaccharides in a relative abundance of 0.1% to 90% as measured by mass spectrometry, LC-MS / MS or GC-MS. In some embodiments, each of the 1 to n fractions in the oligosaccharide preparation independently contains anhydro-subunit-containing oligosaccharides in an amount of about 0.1% to about 15%. In some embodiments, each of the 1 to n fractions in the oligosaccharide preparation independently contains anhydro-subunit-containing oligosaccharides in an amount of about 0.5% to about 15%. In some embodiments, the DP1 and DP2 fractions each independently contain anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.1% to about 15% as measured by mass spectrometry such as MALDI-MS, LC-MS / MS or GC-MS. In some embodiments, the DP1 and DP2 fractions each independently contain anhydro-subunit-containing oligosaccharides in an amount of about 0.5% to about 15%. In some embodiments, the DP1 and DP2 fractions each independently contain anhydro-subunit-containing oligosaccharides in a relative abundance 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 the oligosaccharides in each fraction decreases monotonically with its degree of polymerization.

[0218]

[0260] In some embodiments, the oligosaccharide preparation is a synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a process that does not require a living organism. In some embodiments, the synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a process that does not require an enzyme. In some embodiments, the synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a chemical process. In certain embodiments, the synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by the condensation of sugars.

[0219] [A. Usefulness of Oligosaccharides as Prebiotics]

[0261] Disclosed herein is an oligosaccharide preparation comprising anhydro-sugar components and / or sugar dehydration product components that exhibit complex functional regulation of microbial communities such as the animal gut microbiota. The oligosaccharide preparation regulates the utilization of fermentable carbon by the microflora and provides the usefulness of directing the metabolic flux towards beneficial species, thus providing health or nutritional benefits mediated by the microbiota.

[0220]

[0262] Indigestible carbohydrates can function as prebiotics by providing a fermentable carbon source for the microbial community. For example, a diet rich in soluble plant fiber has been confirmed to have the ability to nourish the gut microflora. Furthermore, bifidobacterium prebiotics support the growth of bifidobacteria (e.g., members of the genus Bifidobacterium), and lactic acid bacterium prebiotics support the growth of Lactobacillus species.

[0221]

[0263] Prebiotic fibers can be fermented into beneficial chemical species such as short-chain fatty acids (SCFAs). Examples of prebiotic fibers include resistant starch, cellulose, pectins such as ramnogalactan, arabinogalactan, arabinan, hemicelluloses such as arabinoxylan, xyloglucan, glucomannan, galactomannan, xylans such as corn cob oligosaccharides, β-glucans such as cereal β-glucan, yeast β-glucan, bacterial β-glucan, polyfructans such as inulin and levan, and gums such as alginate. Inulin is a common bifidogenic prebiotic fiber.

[0222]

[0264] In other cases, prebiotics act by preventing the ability of pathogenic bacteria to colonize and thus infect the host organism through anti-adhesion mechanisms such as competitive binding at cell surface receptor sites. Certain galacto-oligosaccharides provide effective prevention of adhesion of various enteropathogenic organisms such as Escherichia species.

[0223]

[0265] Prebiotics are typically provided to host animals by incorporation into the diet, and on that they show a dose-dependent response (up to at least the saturation threshold). For example, providing a high dose of a bifidobacterium prebiotic such as inulin tends to significantly increase the population of Bifidobacterium species. A high dose of inulin corresponds to a higher production of SCFAs by fermentation. This is because prebiotics provide a metabolic carbon source and more carbon is converted into more fermentation products. Similarly, providing a higher dose of anti-adhesion prebiotics brings the possibility of competitively binding to surface receptor sites.

[0224]

[0266] Certain carbohydrate species containing modified monomer subunits can potentially affect how the microbial system utilizes other carbohydrates that are otherwise available as prebiotic sources. For example, such carbohydrate species can be modified carbohydrate species that regulate the bacterial starch utilization system (SUS), i.e., the proteins involved in cell surface recognition, glycoside cleavage, and import of starch metabolites.

[0225]

[0267] Carbohydrate compositions that can complexly regulate the animal microbiota have utility as feed additives for improving animal health and nutrition through their effects on the animal microbiome. For example, regulation of butyrate production by the gut microbiota promotes a healthy gut mucosa, barrier function, and confers health benefits to animals via an anti-inflammatory effect. Regulation of propionate production affects the metabolic energy extracted from the animal diet via increased gluconeogenesis. The associated microbiota includes, for example, the microbiota of the ileum, jejunum, and cecum and / or feces of poultry, pigs, dogs, cats, horses, or the microbiota of ruminants such as cattle, cows, sheep. Other microbiota includes skin flora, nasal microbiota.

[0226]

[0268] Furthermore, the oligosaccharide preparations disclosed herein are advantageous in that, due to the presence of anhydro-subunits, they can be selectively analyzed and quantified in complex nutritional compositions such as complete animal feeds. It is commercially useful to evaluate the presence and / or concentration of feed additives such as oligosaccharide preparations. Such evaluation is carried out for quality control purposes and can determine whether the additive is blended uniformly with the basal nutritional composition to provide a final nutritional composition containing the additive at the intended dosage or inclusion level.

[0227]

[0269] However, the nutritional composition itself contains a large and diverse number of carbohydrate structures (e.g., starch, plant fiber, and pectin). Therefore, it is particularly difficult to distinguish a small amount of oligosaccharide-based feed additive from the vast ocean of other carbohydrates present as the base of the nutritional composition. Because of this, the oligosaccharide preparation disclosed herein provides a means to distinguish itself from other carbohydrate sources in the nutritional composition via anhydro-subunits.

[0228] [B. Degree of Polymerization (DP) Distribution]

[0270] In some embodiments, the oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1 - DPn fractions), each having a different degree of polymerization selected from 1 to n. In some embodiments, the oligosaccharide preparation comprises n fractions of oligosaccharides (DP1 - 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 anhydro-monosaccharides. As another example, in some embodiments, the DP1 fraction comprises glucose, galactose, fructose, 1,6-anhydro-β-D-glucofuranose, 1,6-anhydro-β-D-glucopyranose, or any combination thereof. As yet another example, in some embodiments, the DP2 fraction comprises one or more normal disaccharides and one or more anhydro-subunit-containing disaccharides. In some embodiments, the DP2 fraction comprises lactose.

[0229]

[0271] 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, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 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 about 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, 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, 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.

[0230]

[0272] The degree of polymerization distribution of the oligosaccharide preparation can be determined by any suitable analytical method and instrument including, but not limited to, end-group method, osmotic pressure (osmotic pressure measurement), ultracentrifugation, viscosity measurement, light scattering method, 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 degree of polymerization distribution can be determined and / or detected by mass spectrometry such as matrix-assisted laser desorption ionization (MALDI)-MS, liquid chromatography (LC)-MS, or gas chromatography (GC)-MS. As another example, the degree of polymerization distribution can be determined and / or detected by SEC such as gel permeation chromatography (GPC). As yet another example, the degree of polymerization distribution can be determined and / or detected by HPLC, FFF, or A4F. In some embodiments, the degree of polymerization distribution is determined and / or detected by MALDI-MS. In some embodiments, the degree of polymerization distribution is determined and / or detected by GC-MS or LC-MS. In some embodiments, the degree of polymerization distribution is determined and / or detected by SEC. In some embodiments, the degree of polymerization distribution is determined and / or detected by HPLC. In some embodiments, the degree of polymerization distribution is determined and / or detected by a combination of analytical instruments such as MALDI-MS and SEC. In some embodiments, the degree of polymerization of the oligosaccharide preparation can be determined based on its molecular weight and molecular weight distribution. For example, FIG. 2 shows a MALDI-MS spectrum showing the degree of polymerization of various fractions and the presence of anhydro-subunit-containing oligosaccharides (-18 g / mol MW offset peak) in all observed fractions.

[0231]

[0273] In some embodiments, the relative abundance of oligosaccharides in the majority of the fractions decreases monotonically with its degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in fractions less than 6, less than 5, less than 4, or less than 2 of the oligosaccharide preparation does not decrease monotonically with its degree of polymerization.

[0232]

[0274] In some embodiments, the relative abundance of the oligosaccharide 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 its degree of polymerization. In some embodiments, the relative abundance of the oligosaccharide 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 its degree of polymerization. In some embodiments, the relative abundance of the oligosaccharide in at least 5, at least 10, at least 20 or at least 30 DP fractions decreases monotonically with its degree of polymerization. In some embodiments, the relative abundance of the oligosaccharide in at least 5, at least 10, at least 20 or at least 30 consecutive DP fractions decreases monotonically with its degree of polymerization.

[0233]

[0275] In some embodiments, the relative abundance of the oligosaccharide in each of the n fractions decreases monotonically with its degree of polymerization. For example, FIG. 15 provides an example of a DP distribution in which the relative abundance of the oligosaccharide in each of the n fractions decreases monotonically with its DP. For example, in some embodiments, only the relative abundance of the oligosaccharide in the DP3 fraction does not decrease monotonically with its degree of polymerization, i.e., the relative abundance of the oligosaccharide in the DP3 fraction is lower than the relative abundance of the oligosaccharide in the DP4 fraction. In some embodiments, the relative abundance of the oligosaccharide in the DP2 fraction is lower than the relative abundance of the oligosaccharide in the DP3 fraction. For example, FIG. 16 shows a degree of polymerization distribution in which the relative abundance of the oligosaccharide in the DP2 fraction does not decrease monotonically with its degree of polymerization.

[0234]

[0276] In some embodiments, the oligosaccharide preparation described herein has 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 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 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.

[0235]

[0277] In some embodiments, the oligosaccharide preparation described herein has 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 relative abundance. In some embodiments, the oligosaccharide preparation has 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 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.

[0236]

[0278] In some embodiments, the oligosaccharide preparation described herein has 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 relative abundance. In some embodiments, the oligosaccharide preparation has 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 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.

[0237]

[0279] In some embodiments, the oligosaccharide preparation described herein has 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 relative abundance. In some embodiments, the oligosaccharide preparation has a DP4 fraction content of about 1% to about 15%, about 1% to about 10% or about 1% to about 5% by weight or relative abundance. In some embodiments, the oligosaccharide preparation described herein has 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 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 relative abundance. In some embodiments, the content of the DP4 fraction and / or the DP5 fraction is determined by MALDI-MS. In some embodiments, the content of the DP4 fraction and / or the DP5 fraction is determined by HPLC. In some embodiments, the content of the DP4 fraction and / or the DP5 fraction is determined by LC-MS / MS or GC-MS.

[0238]

[0280] In some embodiments, the ratio of the DP2 fraction to the DP1 fraction in the oligosaccharide preparation is from about 0.01 to about 0.8, from about 0.02 to about 0.7, from about 0.02 to about 0.6, from about 0.02 to about 0.5, from about 0.02 to about 0.4, from about 0.02 to about 0.3, from about 0.02 to about 0.2, from about 0.1 to about 0.6, from about 0.1 to about 0.5, from about 0.1 to about 0.4, or from about 0.1 to about 0.3, in terms of their weight or relative abundance. In some embodiments, the ratio of the DP2 fraction to the DP1 fraction in the oligosaccharide preparation is from about 0.02 to about 0.4, in terms of their weight or relative abundance.

[0239]

[0281] In some embodiments, the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation is from about 0.01 to about 0.7, from about 0.01 to about 0.6, from about 0.01 to about 0.5, from about 0.01 to about 0.4, from about 0.01 to about 0.3, or from about 0.01 to about 0.2, in terms of their weight or relative abundance. In some embodiments, the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation is from about 0.01 to about 0.3, in terms of their weight or relative abundance.

[0240]

[0282] In some embodiments, the aggregate content of the 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%, in terms of weight or relative abundance. In some embodiments, the aggregate content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, less than 30%, or less than 10%, in terms of weight or relative abundance.

[0241]

[0283] In some embodiments, the oligosaccharide preparation described herein has an average DP value in the range of 2 to 10. In some embodiments, the oligosaccharide preparation has an average DP value of from about 2 to about 8, from about 2 to about 5, or from about 2 to about 4. In some embodiments, the oligosaccharide preparation has an average DP value of about 3.5. The average DP value can be determined by SEC or elemental analysis.

[0242] [C. anhydro subunit level]

[0284] In some embodiments, each of the n fractions of the oligosaccharide independently comprises an anhydro subunit level. For example, in some embodiments, the DP1 fraction comprises anhydro subunit-containing oligosaccharides at a relative abundance of 10%, and the DP2 fraction comprises anhydro subunit-containing oligosaccharides at a relative abundance of 15%. For another example, in some embodiments, the DP1, DP2, and DP3 fractions each comprise anhydro subunit-containing oligosaccharides at relative abundances of 5%, 10%, and 2%, respectively. In other embodiments, two or more fractions of the oligosaccharide may comprise the same level of anhydro subunit-containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fractions each comprise anhydro subunit-containing oligosaccharides at a relative abundance of about 5%.

[0243]

[0285] In some embodiments, each of the 1 to n fractions in the oligosaccharide preparation described herein independently comprises anhydro subunit-containing oligosaccharides at a relative abundance 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 preparation independently comprises anhydro subunit-containing oligosaccharides at a relative abundance 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 the DP1, DP2, and / or DP3 fractions. In some embodiments, GC-MS is used to determine the relative abundance of oligosaccharides in the DP1, DP2, and / or DP3 fractions. In some embodiments, MALDI-MS is used to determine the relative abundance of oligosaccharides in DP fractions of DP4 and above. In some embodiments, the relative abundance of a particular fraction is determined by integrating the area under the peak of the LC-MS / MS chromatogram designated as corresponding to that fraction. In some embodiments, the relative abundance of a particular fraction is determined by integrating the area under the peak of the GC-MS chromatogram designated as corresponding to that fraction.

[0244]

[0286] The level of the anhydro subunit can be determined 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 the anhydro subunit is determined, at least in part, by a mass spectrometry method such as MALDI-MS. In some embodiments, the level of the anhydro subunit is determined, at least in part, by NMR. In some embodiments, the level of the anhydro-subunit-containing oligosaccharide is determined, at least in part, by HPLC. In some embodiments, the level of the anhydro-subunit-containing oligosaccharide is determined by MALDI-MS as indicated by the -18 g / mol MW offset peak in FIG. 2. In some embodiments, the presence and type of the anhydro subunit species can be determined and / or detected by NMR as shown in Example 11, FIGS. 3 and 4. In some embodiments, the relative abundance of the anhydro-subunit-containing oligosaccharide is determined by MALDI-MS. In some embodiments, the relative abundance of the anhydro-subunit-containing oligosaccharide is determined by LC-MS / MS as shown in FIGS. 33A-33C, 34A-34C, 35A-35C, and 36A-36C. In some embodiments, the relative abundance of the anhydro-subunit-containing oligosaccharide is determined by GC-MS as shown in FIGS. 37A-37B, 38A-38B, 39A-39B, and 40A-40B.

[0245]

[0287] In some embodiments, at least one fraction of the oligosaccharide preparation described herein comprises anhydro subunit-containing oligosaccharides in a relative abundance of 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%. In some embodiments, at least one fraction of the oligosaccharide preparation described herein comprises anhydro subunit-containing oligosaccharides in a relative abundance of 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%. In other embodiments, at least one fraction of the oligosaccharide preparation described herein comprises anhydro subunit-containing oligosaccharides in a relative abundance of more than 0.5%, more than 0.8%, more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80%. In other embodiments, at least one fraction of the oligosaccharide preparation described herein comprises anhydro subunit-containing oligosaccharides in a relative abundance of more than 20%, more than 21%, more than 22%, more than 23%, more than 24%, more than 25%, more than 26%, more than 27%, more than 28%, more than 29%, or more than 30%. In some embodiments, at least one fraction (such as DP1, DP2, and / or DP3) of the oligosaccharide preparation comprises anhydro subunit-containing oligosaccharides in a relative abundance of 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%.In some embodiments, at least one fraction of the oligosaccharide preparation (such as DP1, DP2, and / or DP3) comprises anhydro subunit-containing oligosaccharides in a relative abundance of 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%. In some embodiments, at least one fraction of the oligosaccharide preparation (such as DP1, DP2, and / or DP3) comprises anhydro subunit-containing oligosaccharides in a relative abundance of 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 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 1% to about 10%, 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%. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently comprise anhydro subunit-containing oligosaccharides in a relative abundance ranging from 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% as measured by mass spectrometry, LC-MS / MS, or GC-MS. In some embodiments, the DP1 and DP2 fractions each independently comprise anhydro subunit-containing oligosaccharides in a relative abundance of from about 0.5% to about 15% as measured by mass spectrometry, LC-MS / MS, or GC-MS.

[0246]

[0288] In some embodiments, each fraction of the oligosaccharide preparation described herein contains anhydro-subunit-containing oligosaccharides in a relative abundance of 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%. In some embodiments, each fraction of the oligosaccharide preparation described herein contains anhydro-subunit-containing oligosaccharides in a relative abundance of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2%. In other embodiments, each fraction of the oligosaccharide preparation described herein contains anhydro-subunit-containing oligosaccharides in a relative abundance of more 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%. In other embodiments, each fraction of the oligosaccharide preparation described herein contains anhydro-subunit-containing oligosaccharides in a relative abundance of more than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30%. In some embodiments, each fraction of the oligosaccharide preparation described herein contains anhydro-subunit-containing oligosaccharides in a relative abundance of 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%. In some embodiments, each fraction of the oligosaccharide preparation described herein contains anhydro-subunit-containing oligosaccharides in a relative abundance of 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%.In some embodiments, each fraction of the oligosaccharide preparation described herein comprises anhydro subunit-containing oligosaccharides in a relative abundance of about 0.1% to about 90%, about 0.1% to about 15%, about 0.5% to about 90%, about 0.5% to about 80%, about 0.5% to about 70%, about 0.5% to about 60%, about 0.5% to about 50%, about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 20%, about 0.5% to about 10%, about 0.5% 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 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%.

[0247]

[0289] In some embodiments, the oligosaccharide preparation 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% anhydro-subunit-containing oligosaccharide in relative abundance. In some embodiments, the oligosaccharide preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit-containing oligosaccharide in relative abundance. In other embodiments, the oligosaccharide preparation comprises more than 0.5%, more than 0.8%, more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, more than 15%, more than 16%, more than 17%, more than 18%, more than 19%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% anhydro-subunit-containing oligosaccharide in relative abundance. In other embodiments, the oligosaccharide preparation comprises more than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% anhydro-subunit-containing oligosaccharide in 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%, 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% anhydro-subunit-containing oligosaccharide in 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% anhydro-subunit-containing oligosaccharide in relative abundance.In some embodiments, the oligosaccharide preparation comprises anhydro subunit-containing oligosaccharides in a relative abundance of about 0.1% to about 90%, about 0.1% to about 15%, about 0.5% to about 90%, about 0.5% to about 80%, about 0.5% to about 70%, about 0.5% to about 60%, about 0.5% to about 50%, about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 20%, about 0.5% to about 10%, about 0.5% 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 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%.

[0248]

[0290] In some embodiments, the DP1 fraction of the oligosaccharide preparation described herein comprises oligosaccharides containing anhydro subunits in a relative abundance of 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%. In some embodiments, the DP1 fraction of the oligosaccharide preparation described herein comprises oligosaccharides containing anhydro subunits in a relative abundance of 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%. In some embodiments, the DP1 fraction of the oligosaccharide preparation described herein comprises oligosaccharides containing anhydro subunits in a relative abundance of 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%. In some embodiments, the DP1 fraction of the oligosaccharide preparation described herein comprises oligosaccharides containing anhydro subunits in a relative abundance 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 preparation described herein comprises oligosaccharides containing anhydro subunits in a relative abundance of about 0.5% to about 10%. In some embodiments, the relative abundance of the oligosaccharides containing anhydro subunits is determined by a mass spectrometry method such as MALDI-MS. In some embodiments, the relative abundance of the oligosaccharides containing anhydro subunits is determined by LC-MS / MS. In some embodiments, the relative abundance of the oligosaccharides containing anhydro subunits is determined by GC-MS.

[0249]

[0291] In some embodiments, the DP2 fraction of the oligosaccharide preparation 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% anhydro-subunit-containing oligosaccharide in relative abundance. In some embodiments, the DP2 fraction of the oligosaccharide preparation described herein comprises more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, or more than 15% anhydro-subunit-containing oligosaccharide in relative abundance. In some embodiments, the DP2 fraction of the oligosaccharide preparation 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% anhydro-subunit-containing oligosaccharide in relative abundance. In some embodiments, the DP2 fraction of the oligosaccharide preparation described herein comprises 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 of anhydro-subunit-containing oligosaccharide in relative abundance. In some embodiments, the DP2 fraction of the oligosaccharide preparation described herein comprises about 5% to about 10% anhydro-subunit-containing oligosaccharide in relative abundance. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharide is determined by a mass spectrometry method such as MALDI-MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharide is determined by LC-MS / MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharide is determined by GC-MS.

[0250]

[0292] In some embodiments, the DP3 fraction of the oligosaccharide preparation described herein contains oligosaccharides containing anhydro subunits in a relative abundance of 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%. In some embodiments, the DP3 fraction of the oligosaccharide preparation described herein contains oligosaccharides containing anhydro subunits in a relative abundance of more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, or more than 15%. In some embodiments, the DP3 fraction of the oligosaccharide preparation described herein contains oligosaccharides containing anhydro subunits in a relative abundance of 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%. In some embodiments, the DP3 fraction of the oligosaccharide preparation described herein contains oligosaccharides containing anhydro subunits in a relative abundance 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 preparation described herein contains oligosaccharides containing anhydro subunits in a relative abundance of about 0.5% to about 10%. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined by a mass spectrometry method such as MALDI-MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined by LC-MS / MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined by GC-MS.

[0251]

[0293] In some embodiments, the anhydro-subunit-containing oligosaccharide contains one or more anhydro-subunits. For example, the DP1 anhydro-subunit-containing oligosaccharide contains one anhydro-subunit. In some embodiments, the DPn anhydro-subunit-containing oligosaccharide may contain from 1 to n anhydro-subunits. For example, in some embodiments, the DP2 anhydro-subunit-containing oligosaccharide contains one or two anhydro-subunits. In some embodiments, each oligosaccharide in the oligosaccharide preparation independently contains 0, 1, or 2 anhydro-subunits. 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-containing oligosaccharides have only one anhydro-subunit. In some embodiments, more than 99%, 95%, 90%, 85%, or 80% of the anhydro-subunit-containing oligosaccharides have only one anhydro-subunit.

[0252]

[0294] In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or each fraction of the oligosaccharide preparation each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anhydro-subunits linked via glycosidic bonds, where the glycosidic bonds linking each anhydro-subunit are independently selected. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or each fraction of the oligosaccharide preparation each contain 1, 2, or 3 anhydro-subunits linked via glycosidic bonds, where the glycosidic bonds linking each anhydro-subunit are independently selected. In some embodiments, more than 50%, 60%, 70%, 80%, 90%, or 99% of the oligosaccharides in the oligosaccharide preparation or each fraction contain 1, 2, or 3 anhydro-subunits linked via glycosidic bonds, where the glycosidic bonds linking each anhydro-subunit are independently selected. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or each fraction contain 1 anhydro-subunit linked via a glycosidic bond. In some embodiments, more than 50%, more than 60%, more than 70%, more than 80%, 90%, or more than 99% of the oligosaccharides in the oligosaccharide preparation or each fraction contain 1 anhydro-subunit linked via a glycosidic bond.

[0253] [D. Anhydro-Subunit Species]

[0295] In some embodiments, the oligosaccharide preparation contains different species of anhydro-subunits. In some embodiments, exemplary anhydro-subunit-containing oligosaccharides are shown in FIGS. 42, 30, and 31. In some embodiments, the oligosaccharide preparation contains one or more anhydro-subunits that are thermally dehydrated products of monosaccharides, i.e., anhydro-monosaccharide subunits. In some embodiments, the oligosaccharide preparation contains one or more anhydro-subunits that are reversible thermally dehydrated products of monosaccharides.

[0254]

[0296] An anhydro-monosaccharide (or anhydro-monosaccharide subunit) should be understood to refer to the thermal dehydration product of one or more species of monosaccharides. For example, in some embodiments, anhydro-glucose refers to 1,6-anhydro-β-D-glucopyranose (levoglucosan) or 1,6-anhydro-β-D-glucofuranose. In some embodiments, a plurality of anhydro-glucoses refers to a plurality of 1,6-anhydro-β-D-glucopyranoses (levoglucosan), a plurality of 1,6-anhydro-β-D-glucofuranoses, a plurality of other thermal dehydration products of glucose, or any combination thereof. Similarly, in some embodiments, a plurality of anhydro-galactoses refers to a plurality of any thermal dehydration products of galactose or any combination thereof.

[0255]

[0297] In some embodiments, the oligosaccharide preparations described herein include one or more anhydro-glucose, anhydro-galactose, anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-glulose, anhydro-idose, anhydro-talose, anhydro-fructose, anhydro-ribose, anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, anhydro-xylose, or any combination of these subunits. In some embodiments, the oligosaccharide preparation includes one or more anhydro-glucose, anhydro-galactose, anhydro-mannose, or anhydro-fructose subunits. In some embodiments, the oligosaccharide preparations described herein include 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 (cellobiosan), 1,6-anhydro-β-D-cellotriose (cellotriosan), 1,6-anhydro-β-D-cellotetraose (cellotetraosan), 1,6-anhydro-β-D-cellopentaose (cellopentaosan), and 1,6-anhydro-β-D-maltose (maltosan).

[0256]

[0298] In some embodiments, the oligosaccharide preparation includes one or more 1,6-anhydro-β-D-glucofuranose subunits. In some embodiments, the oligosaccharide preparation includes one or more 1,6-anhydro-β-D-glucopyranose (levoglucosan) subunits. For example, FIG. 42 shows two DP1 anhydro-subunit-containing oligosaccharides (levoglucosan and 1,6-anhydro-β-D-glucofuranose) and one DP2 anhydro-subunit-containing oligosaccharide (anhydro-cellobiose).

[0257]

[0299] The presence and level of anhydro subunit species can vary based on the feed sugars used to produce the oligosaccharide. For example, in some embodiments, gluco-oligosaccharides contain anhydro-glucose subunits, galacto-oligosaccharides contain anhydro-galactose subunits, and gluco-galacto-oligosaccharides contain anhydro-glucose and anhydro-galactose subunits.

[0258]

[0300] In some embodiments, the oligosaccharide preparation contains both 1,6-anhydro-β-D-glucofuranose anhydro subunits and 1,6-anhydro-β-D-glucopyranose anhydro subunits. 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 the group consisting of 1,6-anhydro-β-D-glucofuranose 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-glucofuranose. 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.

[0259]

[0301] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 the preparation. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 the preparation. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in the preparation.

[0260]

[0302] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is 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 each fraction. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 each fraction. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in each fraction.

[0261]

[0303] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 at least one fraction. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose 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 at least one fraction. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in at least one fraction.

[0262]

[0304] In some embodiments, the oligosaccharide preparation described herein comprises anhydro subunit-containing DP2 oligosaccharides. In some embodiments, the oligosaccharide preparation comprises anhydro-lactose, anhydro-sucrose, anhydro-cellobiose, or combinations thereof. In some embodiments, the oligosaccharide preparation comprises about 2 to 20, 2 to 15, 5 to 20, 5 to 15, or 5 to 10 species of DP2 anhydro subunit-containing oligosaccharides. In some embodiments, the oligosaccharide preparation described herein does not contain cellobiose or contains cellobiose at undetectable levels.

[0263]

[0305] In some embodiments, the oligosaccharide preparation described herein comprises one or more anhydro subunits that are caramelization products. In some embodiments, an oligosaccharide preparation comprising one or more anhydro subunits 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-hydroxycyclopenta-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosenone; cyclic hydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxymethylfurfural (5-hmf). In some embodiments, the oligosaccharide preparation comprises a 5-hmf anhydro subunit.

[0264]

[0306] In some embodiments, in at least one of the oligosaccharide preparation or DP fraction, the anhydro subunit that is a caramelization product is not more abundant than the anhydro subunit that is a thermal dehydration product of a monosaccharide. In some embodiments, in at least one of the oligosaccharide preparation or fraction, the anhydro subunit that is a caramelization product is more abundant than the anhydro subunit that is a thermal dehydration product of a monosaccharide. In some embodiments, in at least one of the oligosaccharide preparation or fraction, the anhydro subunit that is a caramelization product and the anhydro subunit that is a thermal dehydration product of a monosaccharide have similar abundance ratios.

[0265]

[0307] In some embodiments, from about 0.01% to about 50%, from about 0.01% to about 40%, from about 0.01% to about 30%, from about 0.01% to about 20%, from about 0.01% to about 10%, from about 0.01% to about 5%, from about 0.01% to about 4%, from about 0.01% to about 3%, from about 0.01% to about 2%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, from about 0.1% to about 50%, from about 0.1% to about 40%, from about 0.1% to about 30%, from about 0.1% to about 20%, from about 0.1% to about 10%, from about 0.1% to about 5%, from about 0.1% to about 4%, from about 0.1% to about 3%, from about 0.1% to about 2%, from about 0.1% to about 1% or from about 0.1% to about 0.5% of the anhydro subunits in the oligosaccharide preparations described herein are caramelization products. In some embodiments, from about 0.1% to about 5%, from about 0.1% to about 2% or from about 0.1% to about 1% of the anhydro subunits in the oligosaccharide preparations are caramelization 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 anhydro subunits in the oligosaccharide preparations are caramelization products.

[0266]

[0308] 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 at least one fraction (e.g., DP1, DP2 and / or DP3 fraction) of the 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 anhydro subunits in at least one fraction (e.g., DP1, DP2 and / or DP3) of the preparation are caramelization 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 anhydro subunits in at least one fraction of the preparation are caramelization products. In some embodiments, less than 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the anhydro subunits in the DP1, DP2 and / or DP3 fractions of the oligosaccharide preparations described herein are caramelization products.

[0267]

[0309] In some embodiments, from about 0.01% to about 50%, from about 0.01% to about 40%, from about 0.01% to about 30%, from about 0.01% to about 20%, from about 0.01% to about 10%, from about 0.01% to about 5%, from about 0.01% to about 4%, from about 0.01% to about 3%, from about 0.01% to about 2%, from about 0.01% to about 1%, from about 0.01% to about 0.5%, from about 0.1% to about 50%, from about 0.1% to about 40%, from about 0.1% to about 30%, from about 0.1% to about 20%, from about 0.1% to about 10%, from about 0.1% to about 5%, from about 0.1% to about 4%, from about 0.1% to about 3%, from about 0.1% to about 2%, from about 0.1% to about 1% or from about 0.1% to about 0.5% of the anhydro subunits in each fraction of the oligosaccharide preparation described herein is a caramelized product. In some embodiments, from about 0.1% to about 5%, from about 0.1% to about 2% or from about 0.1% to about 1% of the anhydro subunits in each fraction of the preparation is a caramelized product. In some embodiments less than, 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 anhydro subunits in each fraction of the preparation is a caramelized product.

[0268]

[0310] In some embodiments, each of the oligosaccharides in the oligosaccharide preparation described herein independently and optionally contains an anhydro subunit. In some embodiments, two or more independent oligosaccharides contain the same or different anhydro subunits. In some embodiments, two or more independent oligosaccharides contain different anhydro subunits. For example, in some embodiments, the oligosaccharide preparation contains a DP1 anhydro subunit-containing oligosaccharide containing 1,6-anhydro-β-D-glucopyranose and a DP2 anhydro subunit-containing oligosaccharide containing a 1,6-anhydro-β-D-glucofuranose subunit. In some embodiments, one or more of the oligosaccharides in the oligosaccharide preparation contains two or more of the same or different anhydro subunits.

[0269]

[0311] In some embodiments, in any fraction of the oligosaccharide preparation having a degree of polymerization of 2 or more (i.e., the DP2 - DPn fraction), anhydro subunits can be bound to one or more normal or anhydro subunits. In some embodiments, in the DP2 - DPn fraction, at least one anhydro subunit is bound to one, two, or three other normal or anhydro subunits. In some embodiments, in the DP2 - DPn fraction, at least one anhydro subunit is bound to one or two normal subunits. In some embodiments, in the DP2 - DPn fraction, at least one anhydro subunit is bound to one normal subunit. In some embodiments, in any of the DP2 - DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the anhydro subunits are bound to one normal subunit. In some embodiments, in each of the DP2 - DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the anhydro subunits are bound to one normal subunit.

[0270]

[0312] In some embodiments, in any fraction of the oligosaccharide preparation having a degree of polymerization of 2 or more (i.e., the DP2-DPn fraction), the anhydro subunit may be located at the chain end of the oligosaccharide. In some embodiments, in any fraction of the oligosaccharide preparation having a degree of polymerization of 3 or more (i.e., the DP3-DPn fraction), the anhydro subunit may be located at a position other than the chain end of the oligosaccharide. In some embodiments, in the DP2-DPn fraction, at least one of the anhydro subunits is located at the chain end of the oligosaccharide. In some embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% of the anhydro subunits in the DP2-DPn fraction are located at the chain end of the oligosaccharide. In some embodiments, more than 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the anhydro subunits in the oligosaccharide preparation are located at the chain end of the oligosaccharide. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the anhydro subunit-containing oligosaccharides contain a chain-end anhydro subunit. In some embodiments, more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the anhydro subunit-containing oligosaccharides contain a chain-end anhydro subunit.

[0271] [E. Glycosidic bond]

[0313] In some embodiments, the oligosaccharide preparations described herein contain various glycosidic bonds. The type and distribution of the glycosidic bonds can depend on the source and manufacturing method of the oligosaccharide preparation. In some embodiments, the type and distribution of the various glycosidic bonds can be determined and / or detected by any suitable method known in the art, such as NMR. For example, in some embodiments, the glycosidic bond is 1 H NMR, 13Determined and / or detected by 13C 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 bond is at least partially 1 Determined and / or detected by 1H NMR. In some embodiments, the glycosidic bond is at least partially 13 Determined and / or detected by 13C NMR. In some embodiments, the glycosidic bond is at least partially 2D 1 1H, 13 1H-13C-HSQC NMR.

[0272]

[0314] In some embodiments, the oligosaccharide preparations described herein include one or more α-(1,2) glycosidic bonds, α-(1,3) glycosidic bonds, α-(1,4) glycosidic bonds, α-(1,6) glycosidic bonds, β-(1,2) glycosidic bonds, β-(1,3) glycosidic bonds, β-(1,4) glycosidic bonds, β-(1,6) glycosidic bonds, α-(1,1)-α glycosidic bonds, α-(1,1)-β glycosidic bonds, β-(1,1)-β glycosidic bonds, or combinations thereof.

[0273]

[0315] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution in which the α-(1,6) glycosidic bond is 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 50 mol%, about 15% to about 45 mol%, about 15% 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%.

[0274]

[0316] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution in which the α-(1,3) glycosidic bond is 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%.

[0275]

[0317] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution with an α-(1,2) glycosidic bond 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%.

[0276]

[0318] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution with an α-(1,4) glycosidic bond 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%. In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution with an α-(1,4) glycosidic bond 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%.

[0277]

[0319] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution with a β-(1,6) glycosidic bond 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%.

[0278]

[0320] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution in which the β-(1,4) glycosidic bond is 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%.

[0279]

[0321] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution in which the β-(1,2) glycosidic bond is 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%. In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution in which the β-(1,2) glycosidic bond is 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%.

[0280]

[0322] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution in which the β-(1,3) glycosidic bond is 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%. In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution in which the β-(1,3) glycosidic bond is 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%.

[0281]

[0323] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution different from that of the non-synthetic oligosaccharide preparation. For example, in some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution different from that of the basal nutritional composition. In some embodiments, the basal nutritional composition includes natural carbohydrate sources such as starch and plant fiber. Some of the natural carbohydrate sources have a high proportion of α-(1,4), α-(1,6) and / or β-(1,6) glycosidic bonds. Thus, in some embodiments, the oligosaccharide preparation has a lower proportion of α-(1,4) glycosidic bonds than the basal nutritional composition. In some embodiments, the oligosaccharide preparation has a lower proportion of α-(1,6) glycosidic bonds than the basal nutritional composition. In other embodiments, the oligosaccharide preparation has a higher proportion of α-(1,6) glycosidic bonds than the basal nutritional composition. In some embodiments, the oligosaccharide preparation has a lower proportion of β-(1,6) glycosidic bonds than the basal nutritional composition. In some embodiments, the oligosaccharide preparation contains glycosidic bonds that are not easily digested or hydrolyzed by enzymes.

[0282]

[0324] Specifically, in some embodiments, the 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 linkage type distribution of the oligosaccharide preparation described herein are 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 than those of the basal nutritional composition. In some embodiments, the 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 linkage type distribution of the oligosaccharide preparation are 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% higher than those of the basal nutritional composition.

[0283]

[0325] It should be understood by those skilled in the art that certain types of glycosidic linkages may not be applicable to oligosaccharides containing certain types of monosaccharides. For example, in some embodiments, the oligosaccharide preparation contains α-(1,2) glycosidic linkages and α-(1,6) glycosidic linkages. In other embodiments, the oligosaccharide preparation contains α-(1,2) glycosidic linkages and β-(1,3) glycosidic linkages. In some embodiments, the oligosaccharide preparation contains α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages and β-(1,6) glycosidic linkages. In some embodiments, the oligosaccharide preparation contains 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 and β-(1,6) glycosidic linkages.

[0284] [F. Molecular weight]

[0326] The molecular weight and molecular weight distribution of the oligosaccharide preparation can be determined by any suitable analytical means and equipment, such as end group method, osmotic pressure (osmometry), ultracentrifugation, viscosity measurement, light scattering method, 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.

[0285]

[0327] In some embodiments, the oligosaccharide preparations described herein have a weight average molecular weight of about 100 to about 10000 g / mol, about 200 to about 8000 g / mol, about 300 to about 5000 g / mol, about 500 to about 5000 g / mol, about 700 to about 5000 g / mol, about 900 to about 5000 g / mol, about 1100 to about 5000 g / mol, about 1300 to about 5000 g / mol, about 1500 to about 5000 g / mol, about 1700 to about 5000 g / mol, about 300 to about 4500 g / mol, about 500 to about 4500 g / mol, about 700 to about 4500 g / mol, about 900 to about 4500 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 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 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, about 2300 to about 2500 g / mol, or about 2320 to about 2420 g / mol.In some embodiments, the weight average molecular weight of the oligosaccharide preparation is from about 2000 to about 2800 g / mol, from about 2100 to about 2700 g / mol, from about 2200 to about 2600 g / mol, from about 2300 to about 2500 g / mol or from about 2320 to about 2420 g / mol. In some embodiments, the oligosaccharide preparation has a weight average molecular weight in the range from at least 500 g / mol, 750 g / mol, 1000 g / mol or 1500 g / mol to a maximum of 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 preparation described herein is determined by HPLC according to Example 9.

[0286]

[0328] In some embodiments, the oligosaccharide preparations described herein have a molecular weight 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 1,100 to about 4,500 g / mol, about 1,300 to about 4,500 g / mol, about 1,500 to about 4,500 g / mol, about 1,700 to about 4,500 g / mol, about 1,900 to about 4,500 g / mol, about 300 to about 4,000 g / mol, about 500 to about 4,000 g / mol, about 700 to about 4,000 g / mol, about 900 to about 4,000 g / mol, about 1,100 to about 4,000 g / mol, about 1,300 to about 4,000 g / mol, about 1,500 to about 4,000 g / mol, about 1,700 to about 4,000 g / mol, about 1,900 to about 4,000 g / mol, about 300 to about 3,000 g / mol, about 500 to about 3,000 g / mol, about 700 to about 3,000 g / mol, about 900 to about 3,000 g / mol, about 1,100 to about 3,000 g / mol, about 1,300 to about 3,000 g / mol, about 1,500 to about 3,000 g / mol, about 1,700 to about 3,000 g / mol, about 1,900 to about 3,000 g / mol, about 2,100 to about 3,000 g / mol, about 300 to about 2,500 g / mol, about 500 to about 2,500 g / mol, about 700 to about 2,500 g / mol, about 900 to about 2,500 g / mol, about 1,100 to about 2,500 g / mol, about 1,300 to about 2,500 g / mol, about 1,500 to about 2,500 g / mol, about 1,700 to about 2,500 g / mol, about 1,900 to about 2,500 g / mol, about 2,100 to about 2,500 g / mol, about 300 to about 2,000 g / mol, about 500 to about 300 - 2,000 g / mol, about 700 to about 2,000 g / mol, about 900 to about 2,000 g / mol, about 1,100 to about 2,000 g / mol, about 300 to about 1,500 g / mol, about 500 to about 1,500 g / mol, about 700 to about 1,500 g / mol, about 900 to about 1,500 g / mol, about 1,100 to about 1,500 g / mol, about 1,300 to about 1,500 g / mol, about 1,000 to about 2,000 g / mol, about 1,100 to about 1,900 g / mol, about 1,200 to about 1,800 g / mol,It has a number average molecular weight of 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 to 1550 g / mol. In some embodiments, the oligosaccharide preparation has a number average molecular weight in the range of at least 500 g / mol, 750 g / mol, 1000 g / mol, or 1500 g / mol to a maximum of 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 preparation described herein is determined by HPLC according to Example 9.,

[0287] [G. Types of Oligosaccharides]

[0329] The types of oligosaccharides present in the oligosaccharide preparation may depend on the types of one or more feed sugars. For example, in some embodiments, when the feed sugar contains glucose, the oligosaccharide preparation contains gluco-oligosaccharides. For example, in some embodiments, when the feed sugar contains galactose, the oligosaccharide preparation contains galacto-oligosaccharides. For another example, in some embodiments, when the feed sugar contains galactose and glucose, the oligosaccharide preparation contains gluco-galacto-oligosaccharides.

[0288]

[0330] In some embodiments, the oligosaccharide preparation described herein contains one or more species of monosaccharide subunits. In some embodiments, the oligosaccharide preparation contains oligosaccharides having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different species of monosaccharide subunits.

[0289]

[0331] In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 1, 2, 3, or 4 different species of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 1, 2, or 3 different species of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 3 different species of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 2 different species of monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises oligosaccharides having 1 species of monosaccharide subunit.

[0290]

[0332] In some embodiments, the oligosaccharide preparation comprises different oligosaccharide species in which each oligosaccharide molecule independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different species of monosaccharide subunits. In some embodiments, the oligosaccharide preparations described herein are 10 2 、10 3 、10 4 、10 5 or more different oligosaccharide species. In some embodiments, some of the oligosaccharides in the preparation comprise 1 species of monosaccharide subunit and some of the other oligosaccharides in the same preparation comprise 2 or more species of monosaccharide subunits. For example, in some embodiments, when the feed sugar is glucose galactose, the oligosaccharide preparation can include oligosaccharides that contain only glucose subunits, oligosaccharides that contain only galactose subunits, oligosaccharides that contain both glucose and galactose subunits in various ratios, or any combination thereof.

[0291]

[0333] In some embodiments, any or all of the n fractions of the oligosaccharide preparation comprise different oligosaccharide species subunits in which each oligosaccharide independently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different species of monosaccharide subunits. In some embodiments, some of the oligosaccharides in a fraction of the preparation comprise 1 species of monosaccharide subunit and some of the other oligosaccharides in the same fraction of the preparation comprise 2 or more species of monosaccharide subunits.

[0292]

[0334] In some embodiments, the oligosaccharide preparations described herein include one or more monosaccharide subunits selected from the group consisting of trioses, tetroses, pentoses, hexoses, heptoses, and any combination thereof, wherein each of said triose, tetrose, pentose, hexose or heptose subunit is independently and optionally functionalized and / or substituted with one of its corresponding anhydro subunits. In some embodiments, the corresponding anhydro subunit is the thermal dehydration product of the monosaccharide subunit. In some embodiments, the corresponding anhydro subunit is the caramelization product of the monosaccharide subunit.

[0293]

[0335] In some embodiments, the oligosaccharide preparations described herein include pentose subunits, hexose subunits or any combination thereof, wherein each of said pentose or hexose subunit is independently and optionally functionalized and / or substituted with one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparation includes hexose subunits, wherein each of said hexose subunits is independently and optionally functionalized and / or substituted with one of its corresponding anhydro subunits.

[0294]

[0336] As used herein, tetrose refers to a monosaccharide having 4 carbon atoms such as erythrose, threose, and erythrulose. As used herein, pentose refers to a monosaccharide having 5 carbon atoms such as arabinose, lyxose, ribose, and xylose. As used herein, hexose refers to a monosaccharide having 6 carbon atoms such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose. As used herein, heptose refers to a monosaccharide having 7 carbon atoms such as sedoheptulose and mannoheptulose.

[0295]

[0337] In some embodiments, the oligosaccharide preparations described herein include glucose subunits, where at least one glucose subunit is optionally replaced with an anhydro-glucose subunit. In some embodiments, the oligosaccharide preparations described herein include galactose subunits, where at least one galactose subunit is optionally replaced with an anhydro-galactose subunit. In some embodiments, the oligosaccharide preparations described herein include galactose and glucose subunits, where at least one galactose subunit or at least one glucose subunit is optionally replaced with one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparations described herein include fructose and glucose subunits, where at least one fructose subunit or at least one glucose subunit is optionally replaced with one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparations described herein include mannose and glucose subunits, where at least one mannose subunit or at least one glucose subunit is optionally replaced with one of its corresponding anhydro subunits.

[0296]

[0338] In some embodiments, the oligosaccharide preparations described herein include glucogalacto-oligosaccharide preparations, gluco-oligosaccharide preparations, galacto-oligosaccharide preparations, fructo-oligosaccharide preparations, manno-oligosaccharide preparations, arabino-oligosaccharide preparations, xylo-oligosaccharide preparations, glucofructo-oligosaccharide preparations, glucomanno-oligosaccharide preparations, glucarabino-oligosaccharide preparations, glucoxylo-oligosaccharide preparations, galactofructo-oligosaccharide preparations, galactomanno-oligosaccharide preparations, galactoarabino-oligosaccharide preparations, galactoxylo-oligosaccharide preparations, fructomanno-oligosaccharide preparations, fructoarabino-oligosaccharide preparations, fructoxylo-oligosaccharide preparations, mannoarabino-oligosaccharide preparations, mannooxylo-oligosaccharide preparations, arabinoxylosaccharide preparations, galactoarabino-xylo-oligosaccharide preparations, fructogalacto-xylo-oligosaccharide preparations, arabinofructomanno-xylo-oligosaccharide preparations, glucofructogalactoarabino-oligosaccharide preparations, fructoglucoarabinomanno-xylo-oligosaccharide preparations, glucogalactofructomanno-arabino-xylo-oligosaccharide preparations, or any combination thereof; wherein each of the monosaccharide subunits within the preparation is independently and optionally functionalized and / or substituted with one of its corresponding anhydro subunits.

[0297]

[0339] In certain embodiments, the oligosaccharide preparations described herein include greater than 99% by weight glucose subunits. In some embodiments, the oligosaccharide preparation includes only glucose subunits.

[0298]

[0340] In some embodiments, the oligosaccharide preparations described herein include from about 45% to 55% by weight glucose subunits and from about 55% to 45% by weight galactose subunits. In some particular embodiments, the oligosaccharide preparation includes about 50% by weight glucose and 50% by weight galactose subunits.

[0299]

[0341] In some embodiments, the oligosaccharide preparation described herein comprises from about 80% to about 95% glucose subunits and from about 20% to about 5% mannose subunits by weight. In some embodiments, the oligosaccharide preparation comprises from about 85% to about 90% glucose subunits and from about 15% to about 10% mannose subunits by weight.

[0300]

[0342] In some embodiments, the oligosaccharide preparation described herein comprises from about 80% to about 95% glucose subunits and from about 20% to about 5% galactose subunits by weight. In some embodiments, the oligosaccharide preparation comprises from about 85% to about 90% glucose subunits and from about 15% to about 10% galactose subunits by weight.

[0301]

[0343] In some embodiments, the oligosaccharide preparation described herein comprises from about 80% to about 95% glucose subunits, from 0% to about 8% galactose subunits and from about 5% to about 20% mannose subunits by weight. In some embodiments, the oligosaccharide preparation comprises from about 80% to about 90% glucose subunits, from about 1% to about 5% galactose subunits and from about 10% to about 15% mannose subunits by weight.

[0302]

[0344] In some embodiments, the oligosaccharide preparation described herein comprises from about 1 wt% to about 100 wt%, from about 50 wt% to about 100 wt%, from about 80 wt% to about 98 wt% or from about 85 wt% to about 95 wt% or any range therebetween of glucose subunits. In some embodiments, galactose subunits are present in the oligosaccharide preparation described herein in an amount from about 0 wt% to about 90 wt%, from about 1 wt% to about 50 wt%, from about 2 wt% to about 20 wt% or from about 5 wt% to about 15 wt% or any range therebetween. In some embodiments, mannose subunits are present in the oligosaccharide preparation described herein in an amount from about 0 wt% to about 90 wt%, from about 1 wt% to about 50 wt%, from about 2 wt% to about 20 wt% or from about 5 wt% to about 15 wt% or any range therebetween.

[0303]

[0345] In some embodiments, the oligosaccharide preparation described herein has a composition of monosaccharide subunits shown in Table 29.

[0304]

Table 1

[0305] [H.D type vs. L type]

[0346] In some embodiments, at least one monosaccharide subunit in the oligosaccharide is of the L-type. In some embodiments, at least one monosaccharide subunit in the oligosaccharide is of the D-type. In some embodiments, the monosaccharide subunits in the oligosaccharide preparation described herein are in a naturally abundant form, such as D-glucose, D-xylose, and L-arabinose.

[0306]

[0347] In some embodiments, the oligosaccharide preparation described herein contains a mixture of L-type and D-type monosaccharide subunits. In some embodiments, the ratio of L-type to D-type or D-type to L-type monosaccharide subunits 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.

[0307] [I. Functionalized oligosaccharides]

[0348] In some embodiments, one or more oligosaccharides in the preparation are independently functionalized. The functionalized oligosaccharide can be produced by combining 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 for the purposes of their disclosures).

[0308]

[0349] In some embodiments, the functionalized compound comprises one or more acidic groups (e.g., -COOH), hydroxyl groups, N-containing groups (e.g., -CN, -NO2, and -N(R a )2, where Ra is a hydrogen group, alkyl group, alkenyl group, alkynyl group, haloalkyl group, heteroalkyl group, cycloalkyl group, aryl group, heterocycloalkyl group, or heteroaryl group), S-containing groups (e.g., thiol and sulfate), halides (e.g., -Cl), P-containing groups (e.g., phosphate), or any combination thereof. In some embodiments, the functionalized compound is attached to at least one monosaccharide subunit by an ether bond, ester bond, oxygen-sulfur bond, amine bond, or oxygen-phosphorus bond. In some embodiments, one or more functionalized compounds are attached to the monosaccharide subunit via a single bond. In some embodiments, at least one functionalized compound is attached to one or two oligosaccharides via two or more bonds.

[0309]

[0350] For each oligosaccharide in the oligosaccharide preparation, each of the described embodiments is independent and can be combined as if every combination were separately described; thus, it should be understood that any combination of embodiments is included in the present disclosure. For example, the various embodiments can be grouped 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 subunit species; (iv) the position of the anhydro subunit; (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 bond; and (x) L-type versus D-type. Accordingly, the described oligosaccharide preparations contain multiple oligosaccharides of different species. In some embodiments, the oligosaccharide preparations described herein have at least 10, 10 2 、10 3 、10 4 、10 5 、10 6, 10 7 , 10 8 , 10 9 or 10 10 and contains different oligosaccharide species. In some embodiments, the preparation contains at least 10 3 , 10 4 , 10 5 , 10 6 or 10 9 and contains different oligosaccharide species. In some embodiments, the preparation contains at least 10 3 different oligosaccharide species.

[0310] [III. Method for Producing Oligosaccharide Preparation]

[0351] In one aspect, provided herein is a method for producing an oligosaccharide preparation. In some embodiments, provided herein is a method for producing an oligosaccharide preparation suitable for use in a nutritional composition such as an animal feed composition or for direct feeding to an animal. In one aspect, provided herein is a method for producing an oligosaccharide preparation comprising heating an aqueous composition comprising one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerization, wherein the catalyst is (+)-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, 3-(1-pyridinio)-1-propanesulfonate; 2-(2-pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl-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(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; methanediazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; trifluoromethanesulfonic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline;Selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan, the oligosaccharide preparation contains at least n fractions of oligosaccharides each having a distinct degree of polymerization selected from 1 (DP1 fraction) to n (DPn fraction), where n is an integer greater than 2. A method is provided.;

[0311]

[0352] 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, the polymerization of the feed sugar is achieved by step-growth polymerization. In some embodiments, the polymerization of the feed sugar is achieved by polycondensation.;

[0312] [A. Feed Sugar]

[0353] In some embodiments, the method for producing the oligosaccharide preparation described herein includes heating one or more types of feed sugars. In some embodiments, the one or more types of feed sugars include monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or any mixture thereof.;

[0313]

[0354] 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.;

[0314]

[0355] In some embodiments, one or more feed sugars include disaccharides such as lactose, sucrose, and cellobiose. In some embodiments, one or more feed sugars include trisaccharides such as maltotriose or raffinose. In certain embodiments, one or more feed sugars include glucose, mannose, galactose, xylose, maltodextrin, arabinose, or galactose, or any combination thereof. In certain embodiments, one or more feed sugars include sugar syrups such as corn syrup. In some embodiments, one or more feed sugars include glucose and lactose. In some embodiments, one or more feed sugars include glucose and sucrose.

[0315]

[0356] In some embodiments, the type of feed sugar can affect the resulting manufactured oligosaccharide preparation. For example, in some variations where all of the one or more feed sugars are glucose, the resulting oligosaccharide preparation includes a gluco-oligosaccharide preparation. In other embodiments, when all of the one or more feed sugars are mannose, the resulting oligosaccharide preparation includes a manno-oligosaccharide preparation. In some embodiments, when one or more feed sugars include glucose and galactose, the resulting oligosaccharide preparation includes a gluco-galacto-oligosaccharide preparation. In yet other embodiments, when one or more feed sugars include xylose, glucose, and galactose, the resulting oligosaccharide preparation includes a gluco-galacto-xyl-oligosaccharide preparation.

[0316]

[0357] In some embodiments, each of the one or more feed sugars can independently be in its anhydrous or hydrated form. In some embodiments, the one or more feed sugars include glucose, galactose, fructose, mannose, or any combination thereof, and each of glucose, galactose, fructose, or mannose can independently be in its monohydrate or anhydrous form. 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 its anhydrous form and at least one sugar in its hydrated form.

[0317]

[0358] In some embodiments, the one or more feed sugars can be provided as a sugar solution in which the sugar is combined with water and fed to the reactor. In some embodiments, the sugar is fed to the reactor in solid form and can be combined with water within the reactor. In some embodiments, the one or more feed sugars are combined and mixed together before adding water. In other embodiments, the one or more feed sugars are combined with water and then mixed.

[0318]

[0359] In some embodiments, the method includes combining 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 combining a mixture of sugars (e.g., monosaccharides, disaccharides, and / or trisaccharides) with a catalyst to produce an oligosaccharide preparation. In other embodiments, the method includes combining a mixture of a sugar and a sugar alcohol with a catalyst to produce an oligosaccharide preparation.

[0319]

[0360] In some embodiments, 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, an amino sugar refers to a sugar molecule in which a hydroxyl group is replaced by an amine group. Exemplary amino sugars include, but are not limited to, N-acetyl-d-glucosamine, mannosamine, neuraminic acid, muramic acid, N-acetyl-neuramin, N-acetyl-muram, N-acetyl-galactosamine, N-acetyl-mannosamine, N-glycolylneuramin, acarbiosin, D-glucosamine, and D-galactosamine.

[0320]

[0361] In an embodiment, a 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), ursonic acids (such as neuraminic acid and ketodeoxyoctulosonic acid), uronic acids (such as glucuronic acid, galacturonic acid, and iduronic acid), and aldaric acids (such as tartaric acid, mucic acid, and saccharic acid).

[0321]

[0362] In some embodiments, a sugar alcohol refers to a polyol derived from a sugar. Exemplary sugar alcohols include, but are not limited to, ethylene glycol, arabinitol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, and volemitol.

[0322]

[0363] In some embodiments, a sugar amide refers to a sugar molecule containing a -C(=O)-N- group. In an embodiment, a sugar ether refers to a sugar molecule containing an ether bond such as a glucoside.

[0323]

[0364] In some embodiments, the functionalized or modified sugars include glucosamine, N-acetylglucosamine, glucuronic acid, galacturonic acid, glucitol, xylitol, mannitol, sorbitol. In some embodiments, one or more feed sugars include deoxysugars such as fucose, rhamnose, deoxyribose or fuculose.

[0324]

[0365] In some embodiments, the method for producing the oligosaccharide preparation described herein is carried out on a gram scale. In some embodiments, the method for producing the oligosaccharide preparation described herein is carried out on a scale of more than one kilogram. Thus, in some embodiments, the method includes heating an aqueous composition comprising one or more feed sugars in an amount of more than 0.5 kg, more than 1 kg, more than 2 kg, more than 3 kg, more than 4 kg, more than 5 kg, more than 6 kg, more than 7 kg, more than 9 kg, more than 10 kg, more than 100 kg or more than 1000 kg. In some embodiments, it includes heating an aqueous composition comprising one or more feed sugars in an amount of 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10, 100, 1000 or 1500 kg or less. In some embodiments, it includes heating an aqueous composition comprising one or more feed sugars in an amount of 1 kg or less.

[0325] [B. Catalyst]

[0366] In some embodiments, the catalyst provided herein comprises one or more acids. In some embodiments, the catalyst provided herein comprises a mineral acid, a carboxylic acid, an amino acid, a sulfonic acid, a boronic acid, a phosphonic acid, a phosphinic acid, sulfuric acid, phosphoric acid, poly(styrene sulfonic acid-co-vinylbenzyl-imidazolium sulfate-co-divinylbenzene), poly(styrene sulfonic 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, diphenylphosphinic acid, hexylphosphonic acid, methylphosphonic acid, phenylphosphinic acid, phenylphosphonic acid, tert-butylphosphonic acid, (SS)-VAPOL hydrogen phosphate, 6-quinolinesulfonic acid, 3-(1-pyridinio)-1-propanesulfonate, 2-(2-pyridinyl)ethanesulfonic acid, 3-(2-pyridyl)-5,6-diphenyl-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, butanoic acid, glutamic acid, 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, methanediazide, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, perfluorobutanesulfonic acid, 6-sulfoquinovose, trifluoromethanesulfonic acid, 2-aminoethanesulfonic acid, benzoic acid, chloroacetic acid, trifluoroacetic acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, lauric acid, palmitic acid, stearic acid, arachidic acid, aspartic acid, glutamic acid, serine, threonine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine,It contains methionine; phenylalanine; tyrosine; tryptophan; polymeric acid; carbon-supported acid; or any combination thereof.

[0326]

[0367] In some embodiments, the catalyst provided herein is (+)-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, 3-(1-pyridinio)-1-propanesulfonate; 2-(2-pyridyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl-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(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; methaniazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; trifluoromethanesulfonic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic 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.

[0327]

[0368] 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 (SS)-VAPOL hydrogen phosphate. In some embodiments, the catalyst provided herein is 6-quinolinesulfonic acid. In some embodiments, the catalyst provided herein is 3-(1-pyridinio)-1-propanesulfonate. In some embodiments, the catalyst provided herein is 2-(2-pyridinyl)ethanesulfonic acid. In some embodiments, the catalyst provided herein is 3-(2-pyridyl)-5,6-diphenyl-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-cysteic acid monohydrate. In some embodiments, the catalyst provided herein is poly(styrene sulfonic acid-co-divinylbenzene). In some embodiments, the catalyst provided herein is lysine.

[0328]

[0369] 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 an embodiment, the catalyst is naphthalene-1-sulfonic acid. In some embodiments, the catalyst is some embodiments, and the catalyst is methaniazide. In some, it is 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 caproic acid. In some embodiments, the catalyst is enanthic acid. In some embodiments, the catalyst is caprylic acid. In some embodiments, the catalyst is pelargonic 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.

[0329]

[0370] In some embodiments, the catalyst provided herein is a polymer catalyst or a carbon-supported catalyst disclosed in International Publication No. WO 2016 / 122887 (which is incorporated herein by reference in its entirety for all purposes).

[0330]

[0371] 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% of one or more feed sugars by dry weight. In some embodiments, the catalyst provided herein is present in an amount of about 1% to 2% of one or more feed sugars by dry weight. 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% of one or more feed sugars by dry weight.

[0331]

[0372] 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% of the aqueous composition by dry weight. In some embodiments, the catalyst provided herein is present in an amount of about 1% to 2% of the aqueous composition by dry weight. In some embodiments, the catalyst provided herein 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 the aqueous composition by dry weight.

[0332]

[0373] In some embodiments, the catalyst provided herein is a combination of two or more different catalysts. In some embodiments, the catalyst includes recyclable catalysts such as resins and polymer catalysts and non-recyclable catalysts. In some embodiments, when the catalyst includes at least two different catalysts, each of the catalysts is present in the amounts provided herein. In other embodiments, when the catalyst includes at least two different catalysts, the at least two different catalysts are present as aggregates in the amounts provided herein.

[0333]

[0374] 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 a wet form such as an aqueous solution. In some embodiments, the catalyst is combined with one or more feed sugars before adding water. In other embodiments, the catalyst is dissolved in water before combining with the one or more feed sugars. In some embodiments, the methods provided herein include producing an aqueous composition by combining one or more feed sugars in dehydrated form and a catalyst in wet form (e.g., an aqueous solution).

[0334] [C. Addition of Water]

[0375] In some embodiments, a method of manufacturing an oligosaccharide preparation comprises 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 bound water, for example, in a sugar monohydrate or dihydrate. In some embodiments, all of the water in the aqueous composition is added as bound water in a monosaccharide monohydrate such as glucose monohydrate. In certain embodiments, all or part of the water in the aqueous composition is added together with a catalyst, i.e., via a catalyst solution.

[0335] [D. Water content]

[0376] As the method of manufacturing the oligosaccharide preparation proceeds, water can be generated by the reaction. For example, in some embodiments, water is generated (i) together with the formation of a glycosidic bond, (ii) together with the formation of an anhydro subunit, or (iii) by other mechanisms or sources. Since both sugar condensation and dehydration reactions involve water, in some embodiments, the water content affects the composition of the oligosaccharide preparation.

[0336]

[0377] Furthermore, in some embodiments, the water content affects the viscosity of the aqueous composition, which in turn can affect the effectiveness of mixing of the aqueous composition. For example, in some embodiments, an overly viscous aqueous composition can result in an undesirable non-uniform catalyst distribution in the aqueous composition. Moreover, in some embodiments, if the water content is very low, the aqueous composition may solidify and effective mixing may be hindered. On the other hand, in other embodiments, if the water content is extremely high, the sugar condensation reaction may be hindered and the level of anhydro subunits may decrease. Accordingly, the present disclosure describes a water content suitable for the manufacture of an oligosaccharide preparation.

[0337]

[0378] In some embodiments, the method for producing the oligosaccharide preparation described herein includes 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, the 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 total weight. In some embodiments, the aqueous composition comprises about 9% water by total weight. However, it should be understood that the amount of water in the aqueous composition can be adjusted based on the reaction conditions and the particular catalyst used. In some embodiments, the water content in the aqueous composition as disclosed above is measured at the start of the reaction, for example, before heating the feed sugar. In some embodiments, the water content in the aqueous composition as disclosed above is measured at the end of the polymerization or condensation reaction. In some embodiments, the water content in the aqueous composition as disclosed above is measured as the average water content at the start and at the end of the reaction.

[0338]

[0379] In certain embodiments, the methods described herein may further include monitoring, over a period of time, the water content present in the aqueous composition and / or the ratio of water to sugar or catalyst. In some embodiments, the method may further include removing at least a portion of the water in the aqueous composition, for example, by distillation. Water can 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.

[0339]

[0380] In some embodiments, the oligosaccharide preparations described herein are hygroscopic. Thus, in some embodiments, the hygroscopicity of the feed sugars and oligosaccharides formed by polymerization can affect the rate at which water can be removed from the aqueous composition.

[0340]

[0381] In some embodiments, the method described herein includes removing at least a portion of the water in the aqueous composition such that the water content in 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% by total weight. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition such 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 such 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 such 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 such that the water content in the aqueous composition at the end of the polymerization and / or condensation reaction is the water content disclosed above. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition such that the water content in the aqueous composition at the start of the polymerization and / or condensation reaction is the water content disclosed above. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition such that the average water content in the aqueous composition at the start and end of the polymerization and / or condensation reaction is within the range disclosed above. In some embodiments, the method includes removing at least a portion of the water in the aqueous composition such that the water content in the aqueous composition is maintained within the range disclosed above over the course of the polymerization and / or condensation reaction.

[0341]

[0382] In some embodiments, the method described herein includes adding at least a portion of water to an aqueous composition such that the water content in 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% by total weight. In some embodiments, the method includes adding at least a portion of water to an aqueous composition such 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 adding at least a portion of water to an aqueous composition such 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 adding at least a portion of water to an aqueous composition such that the water content in 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 an aqueous composition such that the water content in the aqueous composition at the end of the polymerization and / or condensation reaction is the water content disclosed above. In some embodiments, the method includes adding at least a portion of water to an aqueous composition such that the water content in the aqueous composition at the start of the polymerization and / or condensation reaction is the water content disclosed above. In some embodiments, the method includes adding at least a portion of water to an aqueous composition such that the average water content in the aqueous composition at the start and end of the polymerization and / or condensation reaction is within the range disclosed above. In some embodiments, the method includes adding at least a portion of water to an aqueous composition such that the water content in the aqueous composition is maintained within the range disclosed above over the course of the polymerization and / or condensation reaction.

[0342]

[0383] In some embodiments, the degree of polymerization of the oligosaccharides and / or the amount and type of anhydro subunits within the oligosaccharide preparation can be adjusted by regulating or controlling the water content present in the aqueous composition over the course of the manufacturing process. For example, in some embodiments, the degree of polymerization of the oligosaccharides and / or the amount and type of anhydro subunits increases by reducing the water content.

[0343]

[0384] Accordingly, in some embodiments, the methods described herein can include in-process control (IPC) of the water content, which can include monitoring the water content, maintaining the water content, increasing the water content, decreasing the water content, or any combination thereof. In some embodiments, the IPC process includes maintaining the water content while the aqueous composition is heated to the temperatures 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 ranges by adding water to or removing water from the aqueous composition, or both. In some embodiments, the method includes maintaining the water content within the disclosed ranges by distillation. In some embodiments, the method includes maintaining the water content within the disclosed ranges by vacuum distillation. In some embodiments, the method includes maintaining the water content within the disclosed ranges by distillation at atmospheric pressure.

[0344]

[0385] In some embodiments, the water content of the aqueous composition is maintained in the 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 the 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.

[0345]

[0386] The water content of the aqueous composition can be determined by various analytical methods and instruments. In some embodiments, the water content is determined by an evaporation method (e.g., loss on drying), a distillation method, or a chemical reaction method (e.g., Karl Fischer titration). In some embodiments, the water content is determined by an analytical instrument such as a moisture meter. In some embodiments, the water content is determined by Karl Fischer titration.

[0346]

[0387] 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, infrared spectroscopy, near-infrared spectroscopy, conductivity, viscosity, density, torque mixing, or energy mixing. 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 a flow valve.

[0347]

[0388] Although not bound by theory, the water content during the sugar polymerization and / or condensation reaction is thought to be able to affect the level of anhydro subunits in the oligosaccharide preparations described herein. For example, as shown in Figure 30, in some embodiments, a higher water content correlates with a lower level of anhydro subunits. In some embodiments, a lower reaction temperature may correlate with a lower level of anhydro subunit content.

[0348] [E. Temperature]

[0389] In some embodiments, the degree of polymerization of the oligosaccharides and / or the amount and type of anhydro subunits within the oligosaccharide preparation can be adjusted by regulating the temperature to which the aqueous composition is heated. In some embodiments, the method of manufacturing the oligosaccharide preparations described herein includes 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 method of manufacturing the oligosaccharide preparation includes 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 manufacturing the oligosaccharide preparation includes heating the aqueous composition to a temperature of about 135 °C to about 145 °C. In other embodiments, the method of manufacturing the oligosaccharide preparation includes heating the aqueous composition to a temperature of about 125 °C to about 135 °C.

[0349] [F. Reaction Time]

[0390] In some embodiments, the method of manufacturing the oligosaccharide preparations described herein includes heating the aqueous composition for a sufficient time. In some embodiments, the degree of polymerization of the oligosaccharides produced according to the methods described herein can be adjusted by the reaction time.

[0350]

[0391] In some embodiments, sufficient time is defined as a significant amount of time. For example, in some embodiments, sufficient 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, sufficient time is from about 1 to about 24 hours, from about 1 to about 16 hours, from about 1 to about 8 hours, from about 1 to about 4 hours, from about 1 to about 3 hours, from about 1 to about 2 hours, from about 2 to about 12 hours, from about 2 to about 10 hours, from about 2 to about 8 hours, from about 2 to about 6 hours, from about 2 to about 4 hours, from about 3 to about 8 hours, from about 3 to about 6 hours, from about 3 to about 5 hours, or from about 3 to about 4 hours.

[0351]

[0392] In some embodiments, sufficient time is determined by measuring one or more chemical or physical properties of the oligosaccharide preparation, such as water content, viscosity, molecular weight, anhydro subunit content, and / or degree of polymerization distribution.

[0352]

[0393] In some embodiments, the molecular weight of the oligosaccharide preparation is monitored during polymerization. In some embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to achieve the number average molecular weight or weight average molecular weight described herein. In certain embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to achieve a number average molecular weight in the range of 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, about 300 to about 2000 g / mol, about 400 to about 1000 g / mol, about 400 to about 900 g / mol, about 400 to about 800 g / mol, about 500 to about 900 g / mol, or about 500 to about 800 g / mol. In certain embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to achieve a number average molecular weight of about 500 to about 2000 g / mol. In certain embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to achieve a weight average molecular weight in the range of 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, about 300 to about 2000 g / mol, about 400 to about 1300 g / mol, about 400 to about 1200 g / mol, about 400 to about 1100 g / mol, about 500 to about 1300 g / mol, about 500 to about 1200 g / mol, about 500 to about 1100 g / mol, about 600 to about 1300 g / mol, about 600 to about 1200 g / mol, or about 600 to about 1100 g / mol. In certain embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to achieve a weight average molecular weight of about 700 to about 3000 g / mol.

[0353]

[0394] In some embodiments, the sufficient time is the time required for the aqueous composition to reach reaction equilibrium at each reaction temperature. Thus, in some embodiments, the method includes heating the aqueous composition for a time sufficient for 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.

[0354]

[0395] In certain embodiments, equilibrium is determined by measuring the number average or weight average molecular weight of the aqueous composition. In some embodiments, equilibrium is determined by the number or weight average molecular weight of the aqueous composition that does not essentially change over time. In some embodiments, equilibrium is determined by the change in the number or weight average molecular weight of the aqueous composition that is less than a specific percentage over a certain period. In some embodiments, the molecular weight of the aqueous composition is measured by HPLC or SEC.

[0355]

[0396] In some embodiments, equilibrium is determined by the change in the number or weight average molecular weight of the aqueous composition that is less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a certain period. In some embodiments, equilibrium is determined by the change in the number or weight average molecular weight of the aqueous composition over 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, equilibrium is determined by the change in the weight average molecular weight of the aqueous composition that is less than 15% over 1 hour.

[0356]

[0397] 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 that does not essentially change over time. In some embodiments, equilibrium is determined by the change in the viscosity of the aqueous composition that is less than a specific percentage over a certain period. In some embodiments, the viscosity of the aqueous composition is measured by a viscometer or rheometer.

[0357]

[0398] In some embodiments, the equilibrium is determined by a change in the viscosity of the aqueous composition that is less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a certain period of time. In some embodiments, the equilibrium is determined by a change in the viscosity of the aqueous composition over 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, the equilibrium is determined by a change in the viscosity of the aqueous composition that is less than 15% over 1 hour.

[0358]

[0399] In certain embodiments, the equilibrium is determined by measuring the DP distribution of the aqueous composition. In some embodiments, the equilibrium is determined by the DP distribution of the aqueous composition that does not substantially change over time. In some embodiments, the change in the DP distribution of the aqueous composition is determined by calculating a series of Kms, where

Number

[0359]

[0400] In some embodiments, the concentration of the oligosaccharide in the DP1, DPm-1, and DPm fractions is determined by SEC, HPLC, FFF, A4F, mass spectrometry, or any other suitable method. In some embodiments, the concentration of the oligosaccharide in the DP1, DPm-1, and DPm fractions is determined by SEC, such as GPC. In some embodiments, the concentration of the oligosaccharide in the DP1, DPm-1, and DPm fractions is determined by mass spectrometry, such as GC-MS, LC-MS / MS, and MALDI-MS. In some embodiments, the concentration of the oligosaccharide in the DP1, DPm-1, and DPm fractions is determined by HPLC. In some embodiments, the concentration of water is determined by an evaporation method (e.g., loss on drying), a distillation method, or a chemical reaction method (e.g., Karl Fischer titration). In some embodiments, the concentration of water is determined by any suitable analytical instrument, such as a moisture meter.

[0360]

[0401] In some embodiments, the method includes 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 values. In some embodiments, the method includes 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 values. In some embodiments, the method includes calculating about 3, 4, 5, 6, 7, 8, 9, 10, or 15 Km values. In some embodiments, the method includes calculating K2-K4, K2-K5, K2-K6, K2-K7, K2-K8, K2-K9, K2-K10, K2-K11, K2-K12, K2-K13, K2-K14, K2-K15, K3-K5, K3-K6, K3-K7, K3-K8, K3-K9, K3-K10, K3-K11, K3-K12, K3-K13, K3-K14, or K3-K15. In certain embodiments, the method includes calculating K2-K4 or K3-K5.

[0361]

[0402] In some embodiments, the value of Km depends on temperature, moisture concentration, and / or the amount and type of sugar supplied. In some embodiments, Km is from about 0.1 to about 100, from about 0.1 to about 90, from about 0.1 to about 80, from about 0.1 to about 70, from about 0.1 to about 60, from about 0.1 to about 50, from about 0.1 to about 40, from about 0.1 to about 30, from about 0.1 to about 25, from about 0.1 to about 20, or from about 0.1 to about 15. In some embodiments, Km is from about 1 to about 100, from about 1 to about 90, from about 1 to about 80, from about 1 to about 70, from about 1 to about 60, from about 1 to about 50, from about 1 to about 40, from about 1 to about 30, from about 1 to about 25, from about 1 to about 20, from about 1 to about 15, from about 1 to about 10, from about 5 to about 50, from about 5 to about 40, from about 5 to about 30, from about 5 to about 20, from about 5 to about 15, or from about 5 to about 10. In some particular embodiments, Km is from about 1 to about 15 or from about 5 to about 15.

[0362]

[0403] In some embodiments, for a calculated series of Kms, the mean, standard deviation, and / or relative standard deviation are determined. As used herein, relative standard deviation is expressed as a percentage and is obtained by multiplying the standard deviation by 100 and dividing this product by the mean.

[0363]

[0404] In some embodiments, equilibrium is determined by the relative standard deviation of a series of Kms that is 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%. In some embodiments, equilibrium is determined by the relative standard deviation of a series of Kms that is less than 15%, less than 10%, or less than 5%.

[0364] [G. Post - reaction process]

[0405] In some embodiments, the method for producing the oligosaccharide preparation described herein further comprises one or more additional processing steps after heating the aqueous composition at a certain temperature for a sufficient time. In some embodiments, the additional processing steps include, for example, separation (such as chromatographic separation), dilution, concentration, drying, filtration, desalting, extraction, decolorization, or any combination thereof. For example, in some embodiments, the method includes a dilution step and a decolorization step. In some embodiments, the method includes a filtration step and a drying step.

[0365]

[0406] In some embodiments, the method includes a dilution step of adding water to the oligosaccharide preparation to make a syrup of the oligosaccharide preparation. In some embodiments, the concentration of the oligosaccharide preparation in the syrup 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 rather solidifies the oligosaccharide preparation. In some embodiments, the method includes a filtration step. In some embodiments, the method includes recycling the catalyst by filtration.

[0366]

[0407] In some embodiments, the method described herein further comprises a decolorization step. In some embodiments, for example, any method known in the art such as treatment by an absorbent, activated carbon, chromatography (e.g., using an ion exchange resin), hydrogenation, and / or filtration (e.g., microfiltration) can be used to carry out the decolorization step.

[0367]

[0408] In some embodiments, the oligosaccharide preparation is contacted with a material for removing salts, minerals, and / or other ionic species. In certain embodiments, the oligosaccharide preparation passes through an anion / cation exchange column pair. In one embodiment, the anion exchange column contains a weakly basic exchange resin in the form of hydroxide, and the cation exchange column contains a strongly acidic exchange resin in the protonated form.

[0368]

[0409] In some embodiments, the method includes a concentration step. In some embodiments, the concentration step produces an oligosaccharide preparation with 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.

[0369]

[0410] In some embodiments, the method includes an isolation step in which at least a portion of the oligosaccharide preparation is separated. In some embodiments, the isolation step includes crystallization, precipitation, filtration (e.g., vacuum filtration), and centrifugation, or any combination thereof.

[0370]

[0411] In some embodiments, the method includes a separation step. In some embodiments, the separation step includes 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 sugar, or from both. In some embodiments, the separation step includes filtration, chromatography, solubility differences, precipitation, extraction, or centrifugation.

[0371] [H. Reactor]

[0412] The methods described herein may include the use of one or more reactors suitable for sugar condensation, taking into account reaction temperature, pH, pressure, and other factors. In some embodiments, one or more suitable reactors include fed-batch stirred reactors, batch stirred reactors, continuous flow stirred reactors, continuous plug flow column reactors, attrition reactors, or reactors using stirring induced by an electromagnetic field. In some embodiments, one or more suitable reactors include reactors described in Ryu, S.K., and Lee, J.M., Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25:53-65 (1983); Gusakov, A.V., and Sinitsyn, A.P., Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol., 7:346-352 (1985); Gusakov, A.V., Sinitsyn, A.P., Davydkin, I.Y., Davydkin, V.Y., Protas, O.V., 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, Acta Scientiarum. Technology, 25:33-38 (2003).

[0372]

[0413] In some embodiments, one or more suitable reactors include fluidized bed reactors, upflow blanket reactors, fixed bed reactors, or extruder reactors for hydrolysis and / or fermentation. In some embodiments, one or more suitable reactors include open reactors, closed reactors, or both. In some embodiments, if the method includes a continuous process, one or more suitable reactors may include continuous mixers such as screw mixers.

[0373] [I. Process]

[0414] In some embodiments, the method for producing the oligosaccharide preparation described herein includes a batch process, a continuous process, or both. In some embodiments, the method for producing the oligosaccharide preparation includes a batch process. For example, in some embodiments of the batch process, the production of subsequent batches of the oligosaccharide preparation is not started until the current batch is completed. In some embodiments, during the batch process, all or a substantial amount of the oligosaccharide preparation is removed from the reactor. In some embodiments, during the batch process, all of the feed sugars and catalyst are combined in the reactor before the aqueous composition is heated to the described temperature or before polymerization is induced. In some embodiments, during the batch process, the feed sugars are added before, after, or simultaneously with the addition of the catalyst.

[0374]

[0415] In some embodiments, the batch process is a fed-batch process, where not all of the feed sugars are added to the reactor at the same time. In some embodiments of the fed-batch process, at least a portion of the feed sugars are added to the reactor during polymerization or after the aqueous composition has been heated to the described temperature. In some embodiments of the fed-batch process, at least 10 wt%, 20 wt%, 30 wt%, 40 wt%, 50 wt%, or 60 wt% of the feed sugars are added to the reactor during polymerization or after the aqueous composition has been heated to the described temperature.

[0375]

[0416] In some embodiments, a method for producing an oligosaccharide preparation includes a continuous process. For example, in some embodiments of a continuous process, the contents of the reactor flow continuously throughout the reactor. In some embodiments, combining the feed sugar with the catalyst and removing at least a portion of the oligosaccharide preparation are carried out simultaneously.

[0376]

[0417] In some embodiments, a method for producing an oligosaccharide preparation includes a single-vessel or multi-vessel process. For example, in some embodiments of a single-vessel process, the polymerization is carried out in a single reaction vessel. For another example, in some embodiments of a multi-vessel process, the polymerization is carried out in two or more reaction vessels. In some embodiments of a multi-vessel process, the method includes two, three, or more than three reaction vessels. In some embodiments of a multi-vessel process, the method includes a combining step of combining the polymerization products from two or more reactors.

[0377] [IV. Nutritional Composition Containing Oligosaccharide Preparation]

[0418] Provided herein is a nutritional composition comprising an oligosaccharide preparation. In certain embodiments, provided herein is a nutritional composition comprising the described oligosaccharide preparation, wherein the presence and / or concentration of the oligosaccharide preparation in the nutritional composition can be selectively determined and / or detected. An oligosaccharide preparation that exhibits complex functional regulation of the microbiota can be an important component of a nutritional composition. Thus, the presence and / or concentration of the oligosaccharide preparation 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. Thus, the provided nutritional composition is advantageous from the perspective of quality control and manufacturing purposes because the presence and / or concentration of the oligosaccharide preparation can be selectively determined and / or detected. For example, in some embodiments, the presence and concentration of the oligosaccharide preparation can be determined and / or detected by measuring a signal related to anhydro-subunit-containing oligosaccharides.

[0378]

[0419] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the nutritional composition comprises a basal nutritional composition.

[0379] [A. Basal nutritional composition]

[0420] In some embodiments, the basal nutritional composition comprises a carbohydrate source different from the oligosaccharide preparation. For example, in some embodiments, the basal nutritional composition comprises a naturally occurring carbohydrate source such as starch and plant fiber. In some embodiments, the basal nutritional composition comprises starch. In some embodiments, the basal nutritional composition comprises plant fiber.

[0380]

[0421] In some embodiments, the basal nutritional composition comprises one or more carbohydrate sources derived from seeds, roots, tubers, corn, tapioca, arrowroot, wheat, rice, potatoes, sweet potatoes, sago, beans (e.g., broad beans, lentils, mung beans, peas and chickpeas), corn, cassava or other starchy foods (e.g., acorns, arrowroot, arracacha, bananas, barley, breadnut, buckwheat, canna, colocasia, katakuri, kudzu, malanga, millet, oats, oca, potato, sorghum, rye, taro, chestnuts, water chestnuts and yams).

[0381]

[0422] In some embodiments, the basal nutritional composition comprises one or more carbohydrate sources derived from leguminous plants (e.g., peas, soybeans, mung beans, green beans, and other beans), oats, rye, chia, barley, fruits (e.g., figs, avocados, plums, prunes, berries, bananas, apple peels, quince, and pears), vegetables (e.g., broccoli, carrots, cauliflower, zucchini, celery, prickly pear cactus, and Jerusalem artichokes), root tubers, root vegetables (e.g., sweet potatoes and onions), the husk of oat seeds, seeds (e.g., flaxseeds), nuts (e.g., almonds), whole grain foods, wheat, corn bran, lignans, or any combination thereof. In some embodiments, the basal nutritional composition comprises one or more plant fibers derived from wheat bran, beet pulp, fuzzy cottonseed, soybean hulls, or any combination thereof.

[0382]

[0423] In some embodiments, the basal 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, less than 1 ppm of anhydro-subunits or anhydro-subunit-containing oligosaccharides. In some embodiments, the basal nutritional composition comprises less than 50 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm of anhydro-subunits or anhydro-subunit-containing oligosaccharides. In some embodiments, the basal nutritional composition is essentially free of anhydro-subunits.

[0383]

[0424] In some embodiments, the basal nutritional composition does not contain detectable levels of anhydro subunits. Depending on the detection or determination method, anhydro subunit levels below a particular threshold may not be detectable. For example, in some embodiments, detectable levels of anhydro subunits refer to anhydro subunits or anhydro subunit-containing oligosaccharides of 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 in the basal nutritional composition.

[0384]

[0425] In some embodiments, the basal nutritional composition contains a plurality of oligosaccharides. In some embodiments, the basal nutritional composition contains a glycosidic bond distribution different from that of the oligosaccharide preparation. For example, in some embodiments, the basal nutritional composition contains a higher proportion of α-(1,4) glycosidic bonds than the oligosaccharide preparation. In some embodiments, glycosidic bonds such as α-(1,4) glycosidic bonds in the basal nutritional composition are digested by one or more enzymes. In some embodiments, the glycosidic bonds in the basal nutritional composition are more readily digestible and / or hydrolyzable than the glycosidic bonds in the oligosaccharide preparation.

[0385]

[0426] In some embodiments, the level of α-(1,2) glycosidic bond, α-(1,3) glycosidic bond, α-(1,6) glycosidic bond, β-(1,2) glycosidic bond, β-(1,3) glycosidic bond, β-(1,4) glycosidic bond or β-(1,6) glycosidic bond in the basal 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 each glycosidic bond in the oligosaccharide preparation. In some embodiments, the level of α-(1,2) glycosidic bond, α-(1,3) glycosidic bond, α-(1,6) glycosidic bond, β-(1,2) glycosidic bond, β-(1,3) glycosidic bond, β-(1,4) glycosidic bond or β-(1,6) glycosidic bond in the basal nutritional composition is at least 10% lower than the level of each glycosidic bond in the oligosaccharide preparation.

[0386]

[0427] In some embodiments, the level of α-(1,4) glycosidic bond in the basal 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 bond in the oligosaccharide preparation. In some embodiments, the level of α-(1,4) glycosidic bond in the basal nutritional composition is at least 10% higher than the level of α-(1,4) glycosidic bond in the oligosaccharide preparation.

[0387] [B. Animal Feed Composition]

[0428] Depending on the animal species and age, the nutritional composition may contain the oligosaccharide preparation and the basic nutritional composition in different ratios. For example, the oligosaccharide preparation can be combined with the basic nutritional composition in various ratios suitable for the animal species and age. 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, 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 3000 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.

[0388]

[0429] 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.

[0389]

[0430] In some embodiments, depending on the animal species and age, the nutritional composition may further contain protein, minerals (such as copper, calcium, and zinc), salts, essential amino acids, vitamins, and / or antibiotics.

[0390]

[0431] Also provided herein is a method of administering to an animal a nutritional composition comprising a basal nutritional composition and the oligosaccharide preparation of the present disclosure. In some embodiments, the animal is selected from beef cattle (e.g., beef and dairy cattle), pigs, aquatic animals, and poultry. In some embodiments, the animal is a pig such as a sow, piglet, and castrated pig. In other embodiments, the animal is a poultry such as chicken, duck, turkey, goose, quail, and hen. In an embodiment, the poultry is a broiler, breeder, or layer. In some embodiments, the animal is an aquatic animal such as salmon, catfish, bass, eel, tilapia, flounder, shrimp, and crab. In some embodiments, the nutritional composition is administered to the animal in a dry form, liquid form, paste, or a combination thereof. In some embodiments, the form of administration, feeding rate, and feeding schedule can vary depending on the animal species and age.

[0391] [C. Method for Producing a Nutritional Composition]

[0432] The present specification provides a method for producing a nutritional composition, including combining an oligosaccharide preparation with a basal nutritional composition. In some embodiments, the oligosaccharide preparation includes anhydro-subunit-containing oligosaccharides. In some embodiments, the oligosaccharide preparation includes a glycosidic linkage type distribution different from that of the basal nutritional composition.

[0392]

[0433] In some embodiments, the oligosaccharide preparation is a synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation includes at least n fractions of oligosaccharides (DP1 - DPn fractions), each having a distinct degree of polymerization selected from 1 to n. In some embodiments, n is an integer of 2 or more. In some embodiments, n is an integer of 3 or more. In some embodiments, n is an integer of 3 or more. 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 - DPn fractions includes anhydro-subunit-containing oligosaccharides in a relative abundance of 0.1% - 90% as measured by mass spectrometry. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently include anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.1% - about 15% or about 0.5% - about 10% as measured by mass spectrometry. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently include anhydro-subunit-containing oligosaccharides in a relative abundance in the range from 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% as measured by mass spectrometry. In some embodiments, the relative abundance of the oligosaccharides in each of the n fractions decreases monotonically with its degree of polymerization. In some embodiments, the relative abundance of the oligosaccharides in at least 5, 10, 20 or 30 DP fractions decreases monotonically with its degree of polymerization.

[0393]

[0434] In some embodiments, a method of manufacturing a nutritional composition includes mixing an oligosaccharide preparation with a basal nutritional composition. For example, in some embodiments, the mixing can be performed by an industrial blender and / or mixer such as a drum blender, a double cone blender, a ribbon blender, a V blender, a shear mixer, and a paddle mixer.

[0394]

[0435] In some embodiments, a method of manufacturing a nutritional composition further includes the quality control process described herein. In some embodiments, the quality control process described herein includes determining a signal level in a sample of the nutritional composition and calculating the concentration of the oligosaccharide preparation in the nutritional composition based on the signal level. In some embodiments, the quality control process described herein includes 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 process described herein includes 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 and 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 related to the degree of polymerization (DP) distribution of the oligosaccharide, or (iii) is related to an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond of the oligosaccharide.

[0395]

[0436] Furthermore, in some embodiments, the method of manufacturing the nutritional composition further comprises mixing the oligosaccharide preparation with the basal 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 includes adding 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 includes adding additional basal nutritional composition to the nutritional composition or removing a portion of the basal nutritional composition from the nutritional composition. In some specific embodiments, adjusting the level of the oligosaccharide preparation includes adding additional oligosaccharide preparation to the nutritional composition.

[0396] [D. Animal feed premix]

[0437] In some embodiments, the nutritional composition includes an animal feed premix comprising the described oligosaccharide preparation.

[0397]

[0438] In some embodiments, the animal feed premix includes a carrier material that can be combined with the oligosaccharide preparation to produce the animal feed premix. In some embodiments, the carrier material can be any material in a dry or liquid form suitable for combination with the oligosaccharide preparation in the nutritional composition. In some embodiments, the carrier material includes hulls such as dried distillers grains, clay, vermiculite, diatomaceous earth, ground rice hulls and ground oat hulls, silica such as feed grade silica gel and feed grade fumed silica, 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 rice hulls or ground oat hulls.

[0398]

[0439] In some embodiments, the animal feed premix is produced by combining a carrier material with an oligosaccharide preparation, both in dry form. In some embodiments, the animal feed premix is produced by combining a carrier material with an oligosaccharide preparation (one of two is in dry form). In some embodiments, the animal feed premix is produced by combining a carrier material with an oligosaccharide preparation, both in liquid form. For example, in some embodiments, the oligosaccharide preparation in liquid form refers to an oligosaccharide in solution, such as an aqueous solution of oligosaccharide such as syrup.

[0399]

[0440] In some embodiments, the animal feed premix is produced by combining a carrier material with a syrup containing an oligosaccharide preparation. In some embodiments, the concentration of the oligosaccharide preparation in the syrup is at least 40 wt%, at least 45 wt%, at least 50 wt%, at least 55 wt%, at least 60 wt%, at least 65 wt%, at least 70 wt%, at least 75 wt% or at least 80 wt%. In some embodiments, the concentration of the oligosaccharide preparation in the syrup is about 40 wt% - 80 wt%, 50 wt% - 75 wt% or 60 wt% - 70 wt%.

[0400]

[0441] In some embodiments, the animal feed premix is in powder (e.g., free-flowing powder), slash, slurry, pellet form or liquid form. In some embodiments, the animal feed premix has a moisture content of less than 40 wt%, 30 wt%, 20 wt%, 15 wt%, 10 wt% or 5 wt%. In some embodiments, the animal feed premix has a moisture content of less than 10 wt% or 5 wt%. In some embodiments, the animal feed premix has a moisture content of more than 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt% or 30 wt%. In further 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 the moisture content to the described range.

[0401]

[0442] In some embodiments, depending on the particular application, the animal feed premix includes various levels of the oligosaccharide preparation. In some embodiments, the animal feed premix includes 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 includes up to 50%, 60%, 70%, 80%, 90%, 95% or 99% of the oligosaccharide preparation by dry weight.

[0402]

[0443] In some embodiments, the animal feed premix or carrier material further includes other animal nutrients such as minerals, fats and proteins. In some embodiments, the carrier material or animal feed premix includes copper, zinc or both. In some embodiments, the carrier material or animal feed premix includes an ionophore or a coccidiostat. In some embodiments, the carrier material or animal feed premix includes an antibiotic. In some embodiments, the carrier material includes a carbohydrate source. In some embodiments, the carbohydrate source in the carrier material does not include anhydro subunits. In some embodiments, the carbohydrate source in the carrier material includes a glycosidic bond type distribution different from that of the oligosaccharide preparation.

[0403]

[0444] Thus, in some embodiments, a method of manufacturing a nutritional composition includes combining an animal feed premix with a basal nutritional composition.

[0404] [V. Method of Pro...

Claims

1. A synthetic oligosaccharide preparation for use in the treatment, improvement and / or prevention of at least one symptom caused by coccidiosis vaccination in birds vaccinated with a coccidiosis vaccine, wherein the synthetic oligosaccharide preparation comprises oligosaccharides of at least n fractions (DP1 to DPn fractions) each having a distinct degree of polymerization selected from 1 to n, where n is an integer of 3 or more, each of the DP1 fraction and the DP2 fraction contains anhydro-subunit-containing oligosaccharides in a relative abundance of 0.5% to 15% as determined by mass spectrometry, the at least one symptom is selected from a decrease in ileal nutrient absorption, tissue damage, an increase in acute-phase liver proteins, a decrease in T helper cells, and an increase in inflammation, a synthetic oligosaccharide preparation.

2. The synthetic oligosaccharide preparation according to claim 1, wherein the tissue damage is selected from liver tissue damage and / or ileal tissue damage.

3. The synthetic oligosaccharide preparation according to claim 1, wherein the acute-phase liver protein is acute-phase liver α-glycoprotein.

4. The synthetic oligosaccharide preparation according to any one of claims 1 to 3, wherein the synthetic oligosaccharide preparation is contained in a nutritional composition administered to the bird.

5. The synthetic oligosaccharide preparation according to claim 4, wherein the synthetic oligosaccharide preparation is contained in the nutritional composition at a content rate of at least 50 ppm.

6. The synthetic oligosaccharide preparation according to any one of claims 1 to 5, wherein the bird is a poultry.

7. The synthetic oligosaccharide preparation according to claim 6, wherein the poultry is selected from chicken, duck, goose, turkey, pigeon or quail.

8. The synthetic oligosaccharide preparation according to claim 6, wherein the poultry is a chicken.

9. The synthetic oligosaccharide preparation according to any one of claims 1 to 8, wherein 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, 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.

10. At least one fraction of the synthetic oligosaccharide preparation contains an anhydro-subunit-containing oligosaccharide 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%, and / or Each fraction of the synthetic oligosaccharide preparation contains an anhydro-subunit-containing oligosaccharide in a relative abundance of more than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, or 14%. The synthetic oligosaccharide preparation according to any one of claims 1 to 9.

11. The synthetic oligosaccharide preparation has a weight average molecular weight of 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, and / or The synthetic oligosaccharide preparation according to any one of claims 1 to 10, having a number average molecular weight of 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.

12. The synthetic oligosaccharide preparation according to any one of claims 1 to 11, wherein the synthetic oligosaccharide preparation is fed to the bird simultaneously with the diet of the bird inoculated with the coccidiosis vaccine.

13. The synthetic oligosaccharide preparation according to any one of claims 1 to 12, wherein the relative abundance of the oligosaccharide in each of the n fractions of the synthetic oligosaccharide preparation decreases monotonically with its degree of polymerization.

Images