EA044964B1 - METHODS AND APPLICATION OF A NUTRIENT COMPOSITION CONTAINING A SYNTHETIC OLIGOSACCHARIDE PREPARATION - Google Patents

Description

Related applications

This application claims benefit from U.S. Provisional Patent Application No.

62/757,500, filed November 8, 2018, and U.S. Provisional Patent Application No. 62/757,465, filed November 8, 2018, the disclosures of each of which are incorporated herein by reference in their entirety.

Prior Art

The animal's gastrointestinal system is the interface with the largest area between the animal's internal organs and the external environment. It is responsible for the digestion and absorption of nutrients and provides an effective barrier against environmental pathogens. The gastrointestinal (GI) tract interacts closely with the host animal's immune system and is home to the animal's gut microbiome, which consists of various microorganisms (bacteria, fungi, molds, viruses, etc.) that live in the digestive tract.

In healthy animals, the gastrointestinal system maintains homeostasis through a complex interaction between the host animal's immune function, the physiology of the intestinal mucosa and endothelium, and the biochemistry of the gut microbiome. Mucin and mucus production by intestinal goblet cells, nonspecific immune system activity, chemokine- and cytokine-mediated modulation of inflammation, and tight junctions between intestinal mucosal cells contribute to healthy nutrient absorption and protection from pathogens and toxins.

Disruption of the homeostasis of the gastrointestinal tract leads to negative consequences for the health and nutrition of the animal. For example, disruption of healthy barrier function allows intestinal contents to move into the host's bloodstream, for example, allowing pathogens and toxins to penetrate the mucosa and endothelial layer and adversely affect the host animal. Inflammation and/or damage to intestinal epithelial cells reduces the animal's ability to absorb nutrients. In production animals, disruption of healthy gastrointestinal homeostasis by the gastrointestinal system exhibits poor nutritional and health outcomes, resulting in reduced weight gain, decreased nutritional efficiency, decreased meat yield, poorer meat quality, and higher mortality. For pets, disruption of gastrointestinal homeostasis can impair quality of life and overall health. Therefore, there is a significant need for nutritional compositions, including animal feeds, that prevent and/or treat gastrointestinal barrier dysfunction.

Summary of the invention

The present invention is directed to a method of treating or preventing gastrointestinal barrier dysfunction in an animal in need thereof, comprising administering a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation to the animal, wherein the synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has varying degrees of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3, and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydro subunits, according to relative abundance determined by mass spectrometry.

The present invention is also directed to a method of treating or preventing an infection in an animal in need thereof, comprising administering a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation to the animal, wherein the synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3, and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydro subunits, by relative prevalence determined by mass spectrometry.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation in an animal, wherein the synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3, and wherein each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydro subunits, as determined by mass spectrometry, for the treatment or prevention of dysfunction gastrointestinal barrier in an animal in need of it.

According to the invention, the permeability of the animal's gastrointestinal barrier is reduced compared to the permeability of the animal's gastrointestinal barrier before the use of the synthetic oligosaccharide drug.

According to the invention, the reduction is a reduction of at least 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25% or 30% relative to the permeability of the animal's gastrointestinal barrier before using a synthetic oligosaccharide drug.

According to the invention, the permeability of the gastrointestinal barrier is determined using a sample of blood, feces or urine of an animal.

According to the invention, permeability is measured by determining the level of at least one microbial species in a sample of blood, feces or urine.

According to the invention, dysfunction of the gastrointestinal barrier is associated with or caused by infection.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation in an animal, wherein the synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3, and where each of fractions DP1 and DP2 independently comprises from 0.5 to 15% oligosaccharides containing anhydro subunits, as determined by mass spectrometry, for the treatment or prevention of infection in an animal needing it.

According to the invention, the level of at least one immune cell in a sample from an animal is increased relative to the level of at least one immune cell in a sample from an animal before the use of a nutritional composition that contains a synthetic oligosaccharide preparation.

According to the invention, the increase is an increase of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% relative to the level of at least one immune cell before administration of the nutritional composition containing a synthetic oligosaccharide drug.

According to the invention, the level of at least one pro-inflammatory cytokine in a sample from an animal is increased relative to the level of at least one pro-inflammatory cytokine in a sample from an animal before the use of a nutritional composition containing a synthetic oligosaccharide preparation.

According to the invention, dysfunction of the gastrointestinal barrier is associated with or caused by infection.

Incorporation by reference

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application had been specifically and individually designated for inclusion by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, this specification is intended to supersede and/or take precedence over any such conflicting material.

Brief description of the figures

The new features of the invention are set out in detail in the accompanying claims. A better understanding of the features and advantages of the present invention will be obtained with reference to the following detailed description, which sets forth illustrative embodiments employing the principles of the invention and the accompanying drawings (also figures and figs in the present invention), wherein:

fig. 1 illustrates part of the 1H, 13C-HSQC NMR spectrum of the oligosaccharide drug from Example 9.7;

fig. 2 illustrates the MALDI-MS spectrum of the oligosaccharide preparation from Example 9.7, which demonstrates the presence of anhydro subunits;

fig. 3 illustrates the 1D D-proton NMR spectrum of the anhydro-DP1 fraction isolated from the oligosaccharide of Example 9;

fig. 4 illustrates the ID APT 13C-NMR spectrum of the anhydro-DP1 fraction isolated from the oligosaccharide of Example 9;

fig. 5 shows GC-MS chromatograms (TIC and XIC plots (m/z 229)) showing DP2 and anhydro-DP2 components for oligosaccharide drug 2.9 after derivatization;

fig. 6 illustrates the 1H, 13C-HSQC spectrum of an oligosaccharide drug with an anhydro-subunit from caramelization;

fig. 7 illustrates a portion of MALDI-MS spectra comparing the oligosaccharide formulation of Example 9 at various laser energies;

fig. 8A shows LC-MS/MS detection of anhydro-DP2 forms at concentrations of 1-80 μg/ml oligosaccharide drug in water; fig. 8B illustrates the linear calibration curve obtained from the LC-MS/MS detection of FIG. 8A;

fig. 9 shows quantification of anhydro-DP2 content in various control and treated nutritional compositions;

fig. 10 illustrates a 2D-1H JRES NMR spectrum of a sample of gluco-oligosaccharides containing anhydro subunits;

fig. 11 is a typical 1H, 13C-HSQC NMR spectrum of a sample of gluco-oligosaccharides containing anhydro subunits, with the corresponding resonances and assignments used to assign bonds;

fig. 12 illustrates an overlay of 1H DOSY spectra of three oligosaccharides containing anhydrosubunits;

fig. 13 illustrates a comparison of 1,6-anhydro-β-D-glucose (DP1-18), 1,6-anhydro-β-O-coelobiose

- 2 044964 (DP2-18) and a sample of oligosaccharides containing anhydro subunits;

fig. 14 illustrates mass chromatograms of anhydro-subunit-containing oligosaccharides (top) and digested anhydro-subunit-containing oligosaccharides (bottom) under selective multiple reaction monitoring (MRM);

fig. 15 shows a plot of relative content versus degree of polymerization (DP) of the oligosaccharide from Example 9; the graph shows that the oligosaccharide drug has a monotonically decreasing DP distribution;

fig. 16 shows a graph of relative abundance versus degree of polymerization of the oligosaccharide from Example 9; the graph shows that the oligosaccharide drug has a non-monotonically decreasing DP distribution;

fig. 17 is a graphical representation of a healthy gastrointestinal barrier and a leaky gastrointestinal barrier;

fig. 18 is a graphical representation of a device used to assess the integrity of a healthy intestinal barrier;

fig. 19 is a flow cytogram showing granulocytes with and without ingested pathogens;

fig. 20 is a graph showing fold change in expression of immune and inflammatory cytokines and chemokines in Peyer's patch tissue for selected genes with at least a 1.5-fold increase in expression;

fig. 21 shows a chamber for in vitro assessment of the direct and indirect effects of oligosaccharide drugs on intestinal barrier dysfunction caused by a rhamnolipid apical stressor;

fig. 22 is a bar graph showing the fold change in expression of IL1B, IL4, IL10 and SLC5A10 in birds fed the 500 ppm oligosaccharide diets of Example 9.3 (solid bars) compared to the control diet (white bars); data show increased expression of genes associated with immunity, anti-inflammatory response and nutrient absorption in birds fed the oligosaccharide from Example 9.3;

fig. 23 is a histogram showing the relative expression of the SLC34A2 gene in experimental groups A to B, as detailed in Table. 26; data demonstrate that the oligosaccharides of Examples 9.2 and 9.3 lead to improved ileal phosphorus absorption, as demonstrated by increased SLC34A2 gene expression analysis;

fig. 24 is a microscopic image of ileal tissue of birds in experimental groups B and D (groups defined in Table 26); Ileal histology revealed significantly less inflammation and increased villous length in broilers fed the diet containing the oligosaccharides of Example 9.2;

fig. 25 is a histogram showing the percentage of goblet cells in experimental groups A, B and D (as defined in Table 26); data show that oligosaccharides increase the number of goblet cells in broilers exposed to inflammatory infection (group B);

fig. 26 is a bar graph showing the percentage of lymphocytes from birds in experimental groups A, B, C and D (as defined in Table 26); data show that broiler chickens fed selected oligosaccharides from Example 9 were stimulated to increase the number of T helper cells, measured as a percentage of total lymphocytes, when exposed to an acute inflammatory stimulus;

fig. 27A is a microscopic image of liver tissue from broiler chickens fed the diet containing the oligosaccharide of Example 9.3, with healthy liver tissue (left) and tissue showing inflammatory lesions (right); fig. 27B shows a histological score histogram of lesions in liver samples from broilers from experimental groups A, B or C (as defined in Table 26); data show that in experimental group C (with food containing the oligosaccharide from example 9.3) there was a decrease in liver damage; fig. 27C shows the AGP level of broiler chickens in experimental groups A, B and C (as determined in Table 26); data show decreased acute-phase hepatic protein AGP with inflammatory challenge;

fig. 28A is a bar graph showing the level of IgA in blood samples of broiler chickens in experimental groups A, B and D on day 21; data show that broiler chickens in experimental group D exhibited an accelerated immune response at 21 days; fig. 28B is a bar graph showing the level of IgA in blood samples of broiler chickens in experimental groups A, B and D on day 28; data show that broiler chickens showed faster recovery relative to the baseline of control group A (day 28) when exposed to an acute inflammatory stimulus on day 14;

fig. 29A is a bar graph showing the level of diamine oxidase in blood samples of broiler chickens in experimental groups A, B and D on day 21; data show that broiler chickens showed faster recovery relative to baseline

- 3 044964 level of control group A than birds not receiving oligosaccharide when exposed to an acute inflammatory stimulus on day 14; fig. 29B is a bar graph showing the level in blood samples of broiler chickens in experimental groups A, B and D on day 28; data show that broiler chickens showed faster recovery to control group A baseline than non-oligosaccharide-treated birds when exposed to an acute inflammatory stimulus on day 14;

fig. 30 shows two anhydro-subunit-containing oligosaccharides from the DP1 fraction and one from the DP2 fraction;

fig. 31 illustrates an oligosaccharide containing anhydro subunits (cellotriosan);

fig. 32A shows the MALDI-MS spectrum of the oligosaccharide preparation from Example 2, which demonstrates the presence of anhydro subunits; fig. 32B illustrates the MALDI-MS spectrum of the oligosaccharide preparation from Example 2, which demonstrates the presence of anhydro subunits;

fig. 33A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 1; fig. 33B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 1; fig. 33C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 1;

fig. 34A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 3; fig. 34B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 3; fig. 34C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 3.

fig. 35A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 4; fig. 35B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 4; fig. 35C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 4;

fig. 36A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 7; fig. 36B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 7; fig. 36C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 7;

fig. 37A illustrates the GC/MS detection spectrum with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 1; fig. 37B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 37A;

fig. 38A illustrates the GC/MS detection spectrum with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 3; fig. 38B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 38A;

fig. 39A illustrates the GC/MS detection spectrum with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 4; fig. 39B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 39A;

fig. 40A illustrates the GC/MS detection spectrum of the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 7; fig. 40B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 40A;

fig. 41 illustrates the effect of reaction temperature, water content and reaction time on the amount of oligosaccharides containing DP2 anhydro subunits in oligosaccharide preparations compared to the oligosaccharide preparation according to Example 2;

fig. 42 illustrates NMR assignments of 1,6-anhydro-beta-D-glucofuranose and 1,6-anhydro-beta-Dglucopyranose;

fig. 43 illustrates MALDI-MS spectra comparing the oligosaccharide formulation of Example 9 at different laser energies.

Detailed Description of the Invention

The following description and examples illustrate in detail embodiments of the present invention. It should be understood that this present invention is not limited to the specific embodiments described in the present invention, and as such may vary. Those skilled in the art will appreciate that there are numerous variations and modifications of this present invention that fall within its scope.

All terms are intended to be understood as they would be understood by one skilled in the art. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one skilled in the art to which the invention pertains.

The section headings used in the present invention are for organizational purposes only and should not be construed as limiting the subject matter described.

Although various features of the present invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present invention may be described herein in the context of individual embodiments for the sake of clarity, the present invention may also be practiced in a single embodiment.

The following definitions are in addition to those in the art and are specific to the current application and are not intended to apply to any related or unrelated case, such as any general patent or application. Although any methods and materials similar or equivalent to those described herein may be practiced for testing the present invention, preferred materials and methods are described herein. Accordingly, the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting.

I. Definitions.

The terminology used in the present invention is intended to describe specific cases only and is not intended to be limiting. The singular forms used herein are intended to include the plural unless the context clearly indicates otherwise. Moreover, to the extent that the terms including, includes, having, has, with, or variations thereof are used in the detailed description and/or claims, such terms are intended to be included in the same manner as the term containing.

It is clear that terms such as contains, containing, etc. have the meaning ascribed to them under the United States Patent Laws; those. they mean includes, included, including, etc. and are intended to include or extend and do not exclude additional, not listed elements or steps of the method; and that such terms as consisting essentially of and consisting essentially of have the meaning ascribed to them in the United States Patent Law; that is, they admit elements not explicitly listed, but exclude elements that appear in the prior art or that affect the essential or novel characteristics of the invention.

The term and/or, as used herein in a phrase such as A and/or B, is intended to include both A and B; A or B; A (separately); and B (separately). Likewise, the term and/or used in a phrase such as A, B and/or C is intended to cover 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 (separately); B (separately); and C (separately).

When ranges of physical properties, such as molecular weight, or chemical properties, such as chemical formulas, are used in the present invention, all combinations and subcombinations of ranges and specific embodiments are intended to be included therein. The term approximately when referring to a number or numerical range means that the stated number or numerical range is approximate within experimental variability (or within statistical experimental error), and thus the number or numerical range will in some cases vary from 1% to 15% of the specified number or number range. In some embodiments, the term approximately means within 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, or 0.05% of the set value or range.

As used herein, the term use includes providing the synthetic oligosaccharide preparation, nutritional composition, liquid or animal feed composition described herein to an animal such that the animal can consume the synthetic oligosaccharide preparation, nutritional composition, liquid or animal feed composition. In such embodiments, the animal ingests a portion of the synthetic oligosaccharide drug, nutritional composition, or animal feed composition. In some embodiments, the animal is provided with a synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition such that the animal can consume the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition as desired. In some embodiments, the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition is administered to said animal as a prescribed diet. In some embodiments, the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition is administered to said animal through manual feeding, such as oral syringe feeding, tube feeding, etc. In some embodiments, the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition is administered orally to the animal, such as by self-administration or hand feeding. In some embodiments, the animal ingests a portion of the synthetic oligosaccharide drug, nutritional composition, liquid, or animal feed composition every 24 hours or every second day for at least 7 days, 14 days, 21 days, 30 days, 45 days, 60 days , 75 days, 90 days or 120 days. IN

- 5 044964 In some embodiments, the oligosaccharide drug may be dissolved in water or other liquid, and the animal absorbs some of the oligosaccharide drug by drinking the liquid.

In some embodiments, the oligosaccharide is delivered to the animal through drinking water. In some embodiments, the oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is consumed as desired.

As used herein, the term feed conversion ratio (FCR) refers to the ratio of the mass of feed consumed (eg, consumed by an animal) to the animal product, wherein the animal product is the target animal product. For example, the animal product for dairy animals is milk, while the animal product for meat animals is body weight.

As used herein, the term nutritional efficiency refers to the ratio of the animal product to the amount of feed consumed (eg, consumed by the animal), the animal product being the target animal product.

As used herein, the term anhydro-subunit refers to the thermal dehydration product of a monosaccharide (or monosaccharide subunit) or the caramelization product of a sugar. For example, the anhydro subunit may be an anhydro-monosaccharide such as anhydro-glucose. As another example, an anhydro subunit may be linked to one or more regular or anhydro-monosaccharide subunits via a glycosidic bond.

The term oligosaccharide refers to a monosaccharide or compound containing two or more monosaccharide subunits linked by glycosidic bonds. Essentially, an oligosaccharide includes a conventional monosaccharide, an anhydro-monosaccharide, or a compound containing two or more monosaccharide subunits, where one or more monosaccharide subunits are optionally independently replaced by one or more anhydro-subunits. The oligosaccharide may be functionalized. As used herein, the term oligosaccharide covers all types of oligosaccharides wherein each monosaccharide subunit in the oligosaccharide is independently and optionally functionalized and/or replaced by a corresponding anhydromonosaccharide subunit.

As used herein, the term oligosaccharide drug refers to a drug that contains at least one oligosaccharide.

As used herein, the term gluco-oligosaccharide refers to glucose or a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds. Essentially, a gluco-oligosaccharide includes glucose, anhydro-glucose, or a compound containing two or more glucose monosaccharide subunits linked by glycosidic bonds, each of said glucose monosaccharide subunits being optionally and independently replaced by an anhydro-glucose subunit.

As used herein, the term galacto-oligosaccharide refers to galactose or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds. Essentially, a galacto-oligosaccharide includes galactose, anhydro-galactose, or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds, wherein each of said galactose monosaccharide subunits is optionally and independently replaced by an anhydro-galactose subunit.

As used herein, the term gluco-galacto-oligosaccharide preparation refers to a composition that is obtained by the reaction of complete or partial condensation of the sugars glucose and galactose. Accordingly, in some embodiments, the gluco-galactose-oligosaccharide preparation includes gluco-oligosaccharides, galacto-oligosaccharides, compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds, or a combination thereof. In some embodiments, the gluco-galactose-oligosaccharide preparation includes gluco-oligosaccharides and compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds. In some embodiments, the gluco-galactose-oligosaccharide preparation includes galacto-oligosaccharides and compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds. In some embodiments, the glucogalactose-oligosaccharide preparation includes compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds.

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

As used herein, the term regular monosaccharide refers to a monosaccharide that does not contain an anhydro subunit. The term regular disaccharide refers to a disaccharide that does not contain an anhydro subunit. Accordingly, the term regular subunit refers to a subunit that is not an anhydro subunit.

The term relative abundance or prevalence as used herein refers to the prevalence of a species in terms of how common or rare the species is. For example, the DP1 fraction comprising 10% of oligosaccharides containing anhydrosaccharides, by relative abundance, may refer to a plurality of DP1 oligosaccharides, where 10% of DP1 oligosaccharides are anhydromonosaccharides. The relative abundance, for example, for a certain fraction of DP oligosaccharides, can be determined using suitable analytical tools, for example, mass spectrometry and liquid chromatography, such as LC-MS/MS, GC-MS, HPLC-MS and MALDI-MS. In some embodiments, relative abundance is determined by integrating the area under the peaks on chromatographs (eg, LC-MS/MS, GC-MS, and HPLC-MS) that correspond to the fractions of interest. In some embodiments, relative abundance is determined from peak intensities (eg, MALDI-MS). In some embodiments, the relative abundance is determined by a combination of analytical methods, such as determination of mass after separation using liquid chromatography.

As used herein, the term anhydro-DPn oligosaccharide, anhydro-DPn species, or anhydro-DPn subunit-containing oligosaccharide refers to an oligosaccharide that has a degree of polymerization n and includes anhydro subunits. Essentially, anhydroglucose is an oligosaccharide containing the DP1 anhydro subunit, and cellotriosan is an oligosaccharide containing the DP3 anhydro subunit.

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

II. Oligosaccharide drug.

The present invention discloses oligosaccharide preparations suitable for use in nutritional compositions. In some embodiments, said oligosaccharide preparation contains at least n oligosaccharide fractions, each having a different degree of polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer equal to 2 or more. In some embodiments, n is an integer greater than 2. In some embodiments, each of the fractions 1 to n in the oligosaccharide preparation independently comprises from 0.1% to 90% anhydro subunit-containing oligosaccharides, as measured by relative abundance mass spectrometry. In some embodiments, the relative abundance of oligosaccharides in each fraction decreases monotonically with the degree of polymerization.

In some embodiments, n is an integer equal to 3 or more. In some embodiments, n is an integer ranging from 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 fractions 1 to n in the oligosaccharide preparation independently comprises from 0.1% to 90% oligosaccharides containing anhydro subunits by relative abundance measured by mass spectrometry or LC-MS/MS or GC-MS. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.1 to 15% oligosaccharides containing anhydro subunits. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.5 to 15% oligosaccharides containing anhydro subunits. In some embodiments, each fraction from DP1 and DP2 independently comprises from about 0.1 to 15% anhydro subunit-containing oligosaccharides as measured by relative abundance by mass spectrometry, such as MALDI-MS, or LC-MS/MS or GC-MS . In some embodiments, each fraction from DP1 and DP2 independently comprises from about 0.5 to 15% oligosaccharides containing anhydro subunits. In some embodiments, each fraction of DP1 and DP2 independently comprises 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% anhydro subunit-containing oligosaccharides, by relative abundance measured by mass spectrometry, LC-MS/MS, or GC -MS. In some embodiments, the relative abundance of oligosaccharides in each fraction decreases monotonically with the degree of polymerization.

In some embodiments, the oligosaccharide drug is a synthetic oligosaccharide drug. In some embodiments, a synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a process that does not require living organisms. In some embodiments, a synthetic oligosaccharide preparation refers to a variety of oligosaccharides produced through a process that does not require enzymes. In some embodiments, a synthetic oligosaccharide preparation refers to a variety of chemically produced oligosaccharides. In some embodiments, a synthetic oligosaccharide drug refers to a variety of oligosaccharides obtained by condensation of sugars.

A. Prebiotic value of oligosaccharides.

The present invention discloses oligosaccharide preparations containing anhydro-sugar components and/or sugar dehydration product components that exhibit complex functional modulation of a microbial community, such as the animal gut microbiome. Oligosaccharide formulations provide benefits for regulating fermentable carbon utilization by microflora and direct metabolic flux to beneficial species, thereby providing microbiome-mediated improvements in health or nutrition.

Indigestible carbohydrates can act as prebiotics, providing a fermentable carbon source for the microbial community. For example, diets rich in soluble plant fiber have been identified by their ability to nourish gut microflora. In addition, bifidogenic prebiotics support the growth of bifidobacteria (for example, members of the genus Bifidobacterium), and lactogenic prebiotics support the growth of Lactobacillus species.

Prebiotic fibers can be fermented into beneficial chemicals such as short-chain fatty acids (SCFAs). Prebiotic fibers include resistant starches; cellulose; pectins such as rhamnogalactans, arabinogalactans, arabinans; hemicelluloses such as arabinoxylans, xyloglucans, glucomannans, galactomannans; xylans such as corn cob oligosaccharides; b-glucans such as cereal b-glucans, yeast b-glucans, bacterial b-glucans; polyfructans such as inulin and levan; and gums such as alginate. Inulin is a common bifidogenic prebiotic fiber.

In other cases, prebiotics act by interfering with the ability of pathogenic bacteria to establish themselves and thus infect the host, through countermeasures such as competitive binding of cell surface receptors. Some galacto-oligosaccharides effectively inhibit the adhesion of various enteropathogenic organisms, such as Escherichia species.

Prebiotics are typically administered to the animal host by inclusion in the diet, where they exhibit a dose-dependent response (at least up to the saturation threshold). For example, administration of a higher dose of a bifidogenic prebiotic such as inulin tends to increase the population of Bifidobacterium species more. Higher doses of inulin correspond to higher production of SCFAs through fermentation. This is because the prebiotic is a source of metabolic carbon, and more carbon is converted into a more fermentable product. Similarly, the use of a higher dose of anti-adhesion prebiotic provides the potential for competitive binding of surface receptor sites.

Certain types of carbohydrates containing modified monomeric subunits may influence the way microbial systems utilize other carbohydrates otherwise available to them as a source of prebiotics. For example, such types of carbohydrates may be modified types of carbohydrates that modulate the bacterial starch utilization system (SUS), that is, proteins responsible for cell surface recognition, glycosidic degradation and import of starch metabolites.

Carbohydrate compositions capable of complex modulation of animal microbiota can be used as feed additives that improve animal health and nutrition through effects on the animal microbiota. For example, modulation of butyrate production by the gut microbiota provides health benefits to the animal by promoting healthy intestinal mucosa, barrier function, and providing anti-inflammatory effects. Modulation of propionic acid production affects the metabolic energy extracted from the animal's diet by increasing gluconeogenesis. Suitable microbial communities include, for example, the ileal, jejunal, cecal and/or fecal microbiota of poultry, pigs, dogs, cats, horses, or the ruminant microbiota of cattle, cows, sheep, etc. Other microbial communities include skin microflora, nasal microflora, etc.

In addition, the oligosaccharide preparations described in the present invention have the advantage that they can be selectively analyzed and quantified in a complex nutritional composition such as animal feed due to the presence of anhydro subunits. It is commercially useful to analyze the presence and/or concentration of feed additives such as oligosaccharide preparations. Such analysis may be performed for quality control purposes to determine whether the supplement has been properly mixed with the base nutritional composition to produce a final nutritional composition containing the supplement at the intended dose or inclusion level.

However, the nutritional compositions themselves contain a large amount and variety of carbohydrate structures (eg, starch, plant fibers and pectins). It is therefore particularly difficult to distinguish small quantities of oligosaccharide-based feed additives from the vast quantities of other carbohydrates present as the basis of the nutritional composition. Thus, the oligosaccharide preparation described herein provides a means of distinguishing from other carbohydrate sources in a nutritional composition via anhydro subunits.

B. Distribution by degree of polymerization (DP).

In some embodiments, the oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 to n (fractions DP1-DPn). In some embodiments, the oligosaccharide preparation contains n oligosaccharide fractions, where each fraction has a different degree of polymerization selected from 1 to n (fractions DP1-DPn). For example, in some embodiments, the DP1 fraction contains one or more monosaccharides and/or one or more anhydro-monosaccharides. As another example, in some embodiments, the DP1 fraction includes glucose, galactose, fructose, 1,6-anhydro-β-O-glucofuranose, 1,6-anhydro-β-D-glucopyranose, or any combination thereof. As another example, in some embodiments, the DP2 fraction contains one or more regular disaccharides and one or more anhydro subunit-containing disaccharides. In some embodiments, the DP2 fraction contains lactose.

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 implementation n is 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29,

30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 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 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 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 , from 15 to 60, from 15 to 50, from 15 to 45, from 15 to 40, from 15 to 35 or from 15 to 30.

The distribution of the degree of polymerization of an oligosaccharide preparation can be determined by any suitable analytical method and equipment, including the end group method, osmotic pressure (osmometry), ultracentrifugation, viscosity measurement, light scattering method, size exclusion chromatography (SEC), SEC-MALLS, flow-through force field fractionation ( PFP), asymmetric force field flow fractionation (A4F), high performance liquid chromatography (HPLC) and mass spectrometry (MS), but not limited to. For example, the distribution of the degree of polymerization can be determined and/or detected using mass spectrometry, such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI)-MS, liquid chromatography (LC)-MS, or gas chromatography (GC)-MS. In another example, the distribution of the degree of polymerization can be determined and/or detected using SEC, such as gel permeation chromatography (GPC). As another example, the distribution of the degree of polymerization can be determined and/or detected using HPLC, PFP or A4F. In some embodiments, the degree of polymerization distribution is determined

- 9 044964 and/or detected using MALDI-MS. In some embodiments, the degree of polymerization distribution is determined and/or detected using GC-MS or LC-MS. In some embodiments, the degree of polymerization distribution is determined and/or detected using SEC. In some embodiments, the degree of polymerization distribution is determined and/or detected by HPLC. In some embodiments, the polymerization degree distribution is determined and/or detected using a combination of analytical tools such as MALDI-MS and SEC. In some embodiments, the degree of polymerization of an oligosaccharide drug can be determined based on its molecular weight and molecular weight distribution. For example, FIG. 2 shows a MALDI-MS spectrum that illustrates the degrees of polymerization of the various fractions and the presence of oligosaccharides containing anhydrous subunits (peak offset -18 g/mol MW) in all observed fractions.

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

In some embodiments, the relative abundance of oligosaccharides is 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 In at least 45 or at least 50 fractions, DP decreases monotonically with the degree of polymerization. In some embodiments, the relative abundance of oligosaccharides is 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 in at least 45 or at least 50 successive fractions, DP decreases monotonically with the degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 DP fractions decreases monotonically with degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 consecutive DP fractions decreases monotonically with the degree of polymerization.

In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with the degree of polymerization. For example, FIG. 15 is an example of a DP distribution where the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with DP. For example, in some embodiments, only the relative abundance of oligosaccharides in the DP3 fraction does not decrease monotonically with the degree of polymerization, i.e. the relative abundance of oligosaccharides in the DP3 fraction is lower than the relative abundance of oligosaccharides in the DP4 fraction. In some embodiments, the relative abundance of oligosaccharides in the DP2 fraction is lower than the relative abundance of oligosaccharides in the DP3 fraction. For example, FIG. 16 illustrates the distribution according to the degree of polymerization, in which the relative abundance of oligosaccharides in the DP2 fraction does not decrease monotonically with the degree of polymerization.

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

In some embodiments, the oligosaccharide preparation described herein has a DP2 fraction content of about 1% to 35%, about 1% to 30%, about 1% to 25%, about 1% to 20%, about 1% up to 15%, about 1% to 10%, about 5% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, or about 5% to 10% by weight or relative abundance. In some embodiments, the oligosaccharide preparation has a DP2 fraction content of about 5% to 25%, about 5% to 20%, about 5% to 15%, or about 5% to 10% by weight or relative abundance. In some embodiments, the DP2 fraction is determined by MALDIMS. In some embodiments, the DP2 fraction is determined by HPLC. In some embodiments, the DP2 fraction is determined using LC-MS/MS or

- 10 044964

GC-MS.

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

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

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

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

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

In some embodiments, the oligosaccharide drug described in the present invention has an average DP value ranging from 2 to 10. In some embodiments, the oligosaccharide drug has an average DP value from about 2 to 8, about 2 to 5, or about 2 to 4 In some embodiments, the oligosaccharide preparation has an average DP value of about 3.5. The average DP value can be determined using SEC or elemental analysis.

C. Level of anhydro subunits.

In some embodiments, each of the n oligosaccharide fractions in an oligosaccharide preparation described herein independently comprises a level of anhydro subunits. For example, in some embodiments, fraction DP1 comprises about 10% anhydro-subunit-containing oligosaccharides by relative abundance, and fraction DP2 comprises about 15% anhydro-subunit-containing oligosaccharides by relative abundance. In another example, in some embodiments, each fraction of DP1, DP2, and DP3 contains about 5%, about 10%, and about 2% anhydrosubunit-containing oligosaccharides, by relative abundance, respectively. In some embodiments, two or more oligosaccharide fractions contain similar levels of anhydro subunit-containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fractions each contain approximately 5% anhydro subunit-containing oligosaccharides, by relative abundance.

In some embodiments, each of fractions 1 to n as described herein

- 11 044964 bretene oligosaccharide preparation independently includes from about 0.1 to 15% oligosaccharides containing anhydro subunits, by relative abundance measured by mass spectrometry, LC-MS/MS or GC-MS. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.5 to 15% anhydro subunit-containing oligosaccharides, as measured by mass spectrometry, LC-MS/MS, or GC-containing oligosaccharides. 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 or LC-MS/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 the DP4 or higher DP fraction. In some embodiments, the relative abundance of a particular fraction is determined by integrating the area under the LC-MS/MS chromatogram peaks that are designated as corresponding to that fraction. In some embodiments, the relative abundance of a particular fraction is calculated by integrating the area under the GC-MS chromatogram peaks that are designated as corresponding to that fraction.

The level of anhydro subunits can be determined and/or detected by any suitable analytical methods, such as nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, HPLC, PFP, A4F, or any combination thereof. In some embodiments, the level of oligosaccharides containing anhydro subunits is determined, at least in part, by mass spectrometry, such as MALDI-MS. In some embodiments, the level of anhydrosubunits is determined, at least in part, using NMR. In some embodiments, the level of oligosaccharides containing anhydro subunits is determined, at least in part, by HPLC. In some embodiments, the level of oligosaccharides containing anhydro subunits is determined using MALDI-MS, as indicated by the peaks offset at -18 g/mol MW in FIG. 2. In some embodiments, the presence and type of anhydro subunit species is determined and/or detected by NMR, as illustrated in Example 11 of FIG. 3 and fig. 4. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using MALDI-MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using LC-MS/MS, as shown in FIG. 33A-33C, 34A-34C, 35A-35C and 36A-36C. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using GC-MS, as shown in FIG. 37A-37B, 38A-38B, 39A-39B and 40A-40B.

In some embodiments, at least one fraction of an 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 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% of oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, at least one fraction of an oligosaccharide preparation described herein comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, or less than 2 % of oligosaccharides containing anhydro subunits, by relative abundance. In other embodiments, at least one fraction of an oligosaccharide preparation described herein comprises greater than 0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6 %, 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% oligosaccharides containing anhydro subunits, by relative abundance. In other embodiments, at least one fraction of an oligosaccharide preparation described herein comprises greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28 %, more than 29% or more than 30% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, at least one fraction (such as DP1, DP2, and/or DP3) of the oligosaccharide drug contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0. 5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 1.5%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17% , about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30% anhydro subunit-containing oligosaccharides, by relative abundance. In some embodiments, at least one fraction (such as DP1, DP2, and/or DP3) of the oligosaccharide drug contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0. 5%, approximately 0.6%, approximately 0.7%, approximately 0.8%, with

- 12 044964 approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9% or approximately 10% oligosaccharides, containing anhydrosubunits, according to relative abundance. In some embodiments, at least one fraction (such as DP1, DP2, and/or DP3) of the oligosaccharide drug contains from about 0.1% to 90%, about 0.5% to 90%, about 0.5% to 80%, about 0.5% to 70%, about 0.5% to 60%, about 0.5% to 50%, about 0.5% to 40%, about 0.5% to 30%, about 0.5% to 20%, about 0.5% to 10%, about 0.5% to 9%, about 0.5% to 8%, about 0.5% to 7%, from about 0.5% to 6% from about 0.5% to 5%, from about 0.5% to 4%, from about 0.5% to 3%, from about 0.5% to 2 %, about 1% to 10%, about 2% to 9%, about 2% to 8%, about 2% to 7%, about 2% to 6%, about 2% to 5%, about 2% to 4%, about 2% to 3%, or about 5% to 10% of oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, each of the oligosaccharide preparation fractions DP1 and DP2 independently comprises anhydro subunit-containing oligosaccharides 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% by relative abundance measured by mass spectrometry, LC-MS/MS, or GC-MS. In some embodiments, the DP1 and DP2 fractions each independently comprise from about 0.5 to 15% anhydro subunit-containing oligosaccharides as measured by relative abundance by mass spectrometry or LC-MS/MS or GC-MS.

In some embodiments, each fraction of an 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 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% oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, each fraction of an oligosaccharide preparation described herein contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro subunit-containing oligosaccharides, by relative abundance. In other embodiments, each fraction of the oligosaccharide preparation described herein contains greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro subunit-containing oligosaccharides, by relative abundance. In other embodiments, each fraction of the oligosaccharide preparation described herein contains greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% anhydrous oligosaccharides. -subunits, by relative abundance. In some embodiments, each fraction of an oligosaccharide preparation described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 1.5%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7% , approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30% anhydro subunit-containing oligosaccharides, by relative abundance. In some embodiments, each fraction of an oligosaccharide preparation described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, approximately 0.8%, approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9% or approximately 10% of oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, each fraction of an oligosaccharide preparation described herein comprises from about 0.1% to 90%, about 0.1% to 15%, about 0.5% to 90%, about 0.5% to 80 %, from about 0.5% to 70%, from about 0.5% to 60%, from about 0.5% to 50%, from about 0.5% to 40%, from about 0.5% to 30 %, about 0.5% to 20%, about 0.5% to 10%, about 0.5% to 9%, about 0.5% to 8%, about 0.5% to 7 %, about 0.5% to 6%, about 0.5% to 5%, about 0.5% to 4%, about 0.5% to 3%, about 0.5% to 2 %, about 2% to 9%, about 2% to 8%, about 2% to 7%, about 2% to 6%, about 2% to 5%, about 2% to 4%, about 2% to 3% or about 5% to 10% of oligosaccharides containing anhydro subunits, by relative abundance.

In some embodiments, the oligosaccharide preparation described herein contains 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 4%, less than 3%, less than 2%, or less than 1% oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, the oligosaccharide preparation contains less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro subunit-containing oligosaccharides, by relative race

- 13 044964 dimensions. In other embodiments, the oligosaccharide preparation contains greater than 0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8% , 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 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% of oligosaccharides containing anhydro subunits, by relative abundance. In other embodiments, the oligosaccharide preparation contains greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% anhydro subunit-containing oligosaccharides, by relative abundance . In some embodiments, the oligosaccharide preparation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10% , approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, about 24%, about 25%, or about 30% of oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, the oligosaccharide preparation contains about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, approximately 0.9%, approximately 1%, approximately 1.5%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9% or approximately 10% of oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, the oligosaccharide preparation comprises about 0.1% to 90%, about 0.1 to 15%, about 0.5% to 90%, about 0.5% to 80%, about 0. 5% to 70%, about 0.5% to 60%, about 0.5% to 50%, about 0.5% to 40%, about 0.5% to 30%, about 0. 5% to 20%, about 0.5% to 10%, about 0.5% to 9%, about 0.5% to 8%, about 0.5% to 7%, about 0. 5% to 6%, about 0.5% to 5%, about 0.5% to 4%, about 0.5% to 3%, about 0.5% to 2%, about 2% to 9%, about 2% to 8%, about 2% to 7%, about 2% to 6%, about 2% to 5%, about 2% to 4%, about 2% to 3 % or approximately 5% to 10% of oligosaccharides containing anhydro subunits, by relative abundance.

In some embodiments, the DP1 fraction of an oligosaccharide preparation described herein contains 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 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% oligosaccharides containing anhydrous -subunits, by relative abundance. In some embodiments, the DP1 fraction of an oligosaccharide preparation described herein comprises greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, 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% oligosaccharides containing anhydrous -subunits, by relative abundance. In some embodiments, the DP1 fraction of an oligosaccharide drug described herein contains 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% oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, the DP1 fraction of an oligosaccharide preparation described herein contains from about 0.1% to 15%, about 0.1% to 20%, about 0.5% to 20%, about 0.5% to 10 %, about 0.5 to 15%, about 1% to 20%, about 1% to 15%, about 1% to 10%, about 2% to 14%, about 3% to 13% , about 4% to 12%, about 5% to 11%, about 5% to 10%, about 6% to 9%, or about 7% to 8% anhydro subunit-containing oligosaccharides, by relative abundance , or at any intervals between them. In some embodiments, the DP1 fraction of an oligosaccharide preparation described herein comprises about 5% to 10% anhydro subunit-containing oligosaccharides, by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using mass spectrometry, such as MALDI-MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using GC-MS.

In some embodiments, the DP2 fraction of an oligosaccharide preparation described herein contains 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 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% oligosaccharides containing anhydrous -subunits, by relative abundance. In some embodiments, the DP2 fraction of an oligosaccharide preparation described herein includes more than 0.1%, more

- 14 044964

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 9%, more than 10%, more than 11%, more than 12%, more than 13%, more than 14%, or more than 15% oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, the DP2 fraction of an oligosaccharide drug described herein contains 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% oligosaccharides, containing anhydro subunits, according to relative abundance. In some embodiments, the DP2 fraction of an oligosaccharide preparation described herein contains from about 0.1% to 15%, about 0.1% to 20%, about 0.5% to 20%, about 0.5% to 10 %, about 0.5 to 15%, about 1% to 20%, about 1% to 15%, about 1% to 10%, about 2% to 14%, about 3% to 13% , about 4% to 12%, about 5% to 11%, about 5% to 10%, about 6% to 9%, or about 7% to 8% of oligosaccharides containing anhydro subunits, relative prevalence, or any range in between. In some embodiments, the DP2 fraction of an oligosaccharide preparation described herein comprises from about 5% to 10% anhydro subunit-containing oligosaccharides, by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using mass spectrometry, such as MALDI-MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using GC-MS.

In some embodiments, the DP3 fraction of an oligosaccharide preparation described herein contains 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 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% oligosaccharides containing anhydrous -subunits, by relative abundance. In some embodiments, the DP3 fraction of an oligosaccharide drug described herein comprises greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, 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% oligosaccharides containing anhydrous -subunits, by relative abundance. In some embodiments, the DP3 fraction of an oligosaccharide drug described herein contains 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% oligosaccharides containing anhydro subunits, by relative abundance. In some embodiments, the DP3 fraction of an oligosaccharide preparation described herein contains from about 0.1% to 15%, about 0.1% to 20%, about 0.5% to 20%, about 0.5% to 10 %, about 0.5 to 15%, about 1% to 20%, about 1% to 15%, about 1% to 10%, about 2% to 14%, about 3% to 13% , about 4% to 12%, about 5% to 11%, about 5% to 10%, about 6% to 9%, or about 7% to 8% anhydro subunit-containing oligosaccharides, by relative abundance , or any range in between. In some embodiments, the DP3 fraction of an oligosaccharide preparation described herein contains from about 5% to 10% anhydro subunit-containing oligosaccharides, by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using mass spectrometry, such as MALDI-MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydro subunits is determined using GC-MS.

In some embodiments, the anhydro subunit-containing oligosaccharide includes one or more anhydro subunits. For example, the anhydro subunit-containing oligosaccharide DP1 includes 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 includes one or two anhydro subunits. In some embodiments, each oligosaccharide in the oligosaccharide preparation independently contains zero, one, or two anhydro subunits. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% oligosaccharides , containing anhydro subunits, have only one anhydro subunit. In some embodiments, more than 99%, 95%, 90%, 85%, or 80% of the anhydro subunit-containing oligosaccharides contain only one anhydro subunit.

In some embodiments, one or more oligosaccharides in an oligosaccharide

- 15 044964 drug or in each fraction of the oligosaccharide drug contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 anhydro-subunits, each of which is linked through a glycosidic bond, where the glycosidic bond connecting each anhydro- subunit, independently selected. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation in or each fraction of the oligosaccharide preparation contain 1, 2, or 3 anhydro-subunits, each linked by a glycosidic bond, wherein the glycosidic bond linking each anhydro-subunit is independently selected. In some embodiments, more than 50%, 60%, 70%, 80%, 90%, or 99% of the oligosaccharides in the oligosaccharide preparation or in each fraction contain 1, 2, or 3 anhydro subunits, each of which is linked by a glycosidic bond, wherein the glycosidic bond , binding each anhydro subunit was selected independently. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or in each fraction contain 1 anhydro subunit linked by a glycosidic linkage. In some embodiments, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 99% of the oligosaccharides in the oligosaccharide preparation or in each fraction contain 1 anhydro subunit linked through a glycosidic linkage.

D. Types of anhydro subunits.

In some embodiments, the oligosaccharide preparation contains various types of anhydro subunits. In some embodiments, exemplary oligosaccharides containing anhydro subunits are illustrated in FIG. 42, fig. 30 and fig. 31. In some embodiments, the oligosaccharide preparation contains one or more anhydro-subunits, which are products of thermal dehydration of monosaccharides, that is, anhydro-monosaccharide subunits. In some embodiments, the oligosaccharide preparation contains one or more anhydrosubunits that are products of the reversible thermal dehydration of monosaccharides.

It should be understood that anhydro-monosaccharide (or anhydro-monosaccharide subunit) refers to one or more types of thermal dehydration products of a monosaccharide. For example, in some embodiments, anhydroglucose refers to 1,6-anhydro-O-glucopyranose (levoglucosan) or 1,6-anhydro-O-glucofuranose. In some embodiments, the plurality of anhydroglucoses refers to a plurality of 1,6-anhydro-β-O-glucopyranoses (levoglucosans), a plurality of 1,6anhydro-β-O-glucofuranoses, a plurality of other thermal dehydration products of glucose, or any combination thereof. Likewise, in some embodiments, the term anhydrogalactose plurality refers to the plurality of any thermal dehydration products of galactose or any combination thereof.

In some embodiments, an oligosaccharide preparation as described in the present invention contains one or more subunits of anhydro-glucose, anhydro-galactose, anhydromannose, anhydro-allose, anhydro-altrose, anhydro-gulose, anhydro-indose, anhydro-talose, anhydrofructose, anhydro-ribose, anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, anhydro-xylose, or any combination of these subunits. In some embodiments, the oligosaccharide preparation contains one or more anhydro-glucose, anhydro-galactose, anhydromannose, or anhydro-fructose subunits. In some embodiments, the oligosaccharide preparation as described in the present invention contains one or more of 1,6-anhydro-3-O-b-Oglucopyranosyl-β-D-glucopyranose, 1,6-anhydro-3-O-α- D-glucopyranosyl-β-D-glucopyranoses, 1,6-anhydro-2-O-β-D-glucopyranosyl-β-D-glucopyranoses, 1,6-anhydro-2-O-α-D-glucopyranosyl-β-D-glucopyranoses, 1,6-anhydro-O-cellobiose (cellobiosan), 1,6-anhydro-O-cellotriose (cellotriosan), 1,6-anhydro-O-cellotetraose (cellotetraosan), 1,6-anhydro -β-O-cellopentaose (cellopentaosane) and 1,6-anhydro-β-O-maltose (maltosan).

In some embodiments, the oligosaccharide preparation contains one or more 1,6-anhydro-sO-glucofuranose subunits. In some embodiments, the oligosaccharide preparation contains one or more 1,6-anhydro-β-D-glucopyranose (levoglucosan) subunits. For example, FIG. 42 illustrates two DP1 oligosaccharides containing anhydro-subunits (levoglucosan and 1,6-anhydro-β-O-glucofuranose) and a DP2 oligosaccharide containing anhydro-subunits (anhydro-cellobiose).

The presence and level of anhydro subunit species may vary depending 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.

In some embodiments, the oligosaccharide preparation contains both 1,6-anhydro-β-Oglucofuranose 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% anhydro subunits selected from the group consisting of 1,6-anhydro-β-O-glucofuranose and 1,6-anhydro-β-O-glucopyranose. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the anhydro subunits are anhydro subunits 1 ,6-anhydro-O-glucofuranoses. In some embodiments, at least

1%, 5%, 10%, 20%, 30%, 40%, 50% or 60% of the anhydro subunits are 1,6-anhydro-glucopyranose.

In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, from 7:1 to 1:10, from 6:1 to 1:10, from 5:1 to 1:10, from 4:1 to 1:10, from 3:1 to 1:10, from 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-O-glucofuranose to 1,6-anhydro-O-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-O-glucopyranose in the formulation is approximately 2:1.

In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, from 7:1 to 1:10, from:1 to 1:10, from 5:1 to 1:10, from 4:1 to 1:10, from 3:1 to 1:10, from 2:1 to 1:10, from 10:1 to 1:9, from 10:1 to 1:8, from 10:1 to 1:7, from 10:1 to 1:6, from 10:1 to 1:5, from 10:1 to 1:4, from 10:1 to 1:3, from 10:1 to 1:2 or from 1:1 to 3:1 in each fraction. In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-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: 9 or 1:10 in each fraction. In some embodiments, the ratio of 1,6-anhydro-β-O-glucofuranose to 1,6-anhydro-β-O-glucopyranose is approximately 2:1 in each fraction.

In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, from 7:1 to 1:10, from:1 to 1:10, from 5:1 to 1:10, from 4:1 to 1:10, from 3:1 to 1:10, from 2:1 to 1:10, from 10:1 to 1:9, from 10:1 to 1:8, from 10:1 to 1:7, from 10:1 to 1:6, from 10:1 to 1:5, from 10:1 to 1:4, from 10:1 to 1:3, from 10:1 to 1:2 or from 1:1 to 3:1 in at least one fraction. In some embodiments, the ratio of 1,6-anhydro-O-glucofuranose to 1,6-anhydro-O-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, 1:9 or 1:10 in at least one faction. In some embodiments, the ratio of 1,6-anhydro-β-O-glucofuranose to 1,6-anhydro-β-O-glucopyranose is about 2:1 in at least one fraction.

In some embodiments, the oligosaccharide preparation described herein includes OP2 oligosaccharides containing anhydro subunits. In some embodiments, the oligosaccharide preparation contains anhydro-lactose, anhydro-sucrose, anhydrocellobiose, or a combination thereof. In some embodiments, the oligosaccharide preparation includes about 2 to 20, 2 to 15, 5 to 20, 5 to 15, or 5 to 10 OP2 species of oligosaccharides containing anhydro subunits. In some embodiments, the oligosaccharide preparation described in the present invention does not contain cellobiosan or does not contain a detectable level of cellobiosan.

In some embodiments, the oligosaccharide preparation described herein contains one or more anhydro subunits, which are the caramelization products of sugar. In some embodiments, the oligosaccharide preparation contains one or more anhydro subunits, which are products of caramelization of sugar, selected from the group consisting of methanol; ethanol; furan; methylglyoxal; 2-methylfuran; vinyl acetate; glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol; 3-furanmethanol; 2hydroxycyclopent-2-en-1-one; 5-methylfurfural; 2(5H)-furanone; 2-methylcyclopentenolone; levoglucosenone; cyclic hydroxyl lactone; 1,4,3,6-dianhydro-a-O-glucopyranoses; dianhydroglucopyranoses; and 5-hydroxymethylfurfural (5-hmf). In some embodiments, the oligosaccharide preparation contains 5-hmf anhydro subunits.

In some embodiments, in the oligosaccharide preparation or at least one of the OP fractions, anhydro subunits that are products of caramelization are less abundant than anhydro subunits that are products of reversible thermal dehydration of the monosaccharide. In some embodiments, in the oligosaccharide preparation or at least one of the fractions, anhydro subunits that are products of caramelization are more abundant than anhydro subunits that are products of reversible thermal dehydration of the monosaccharide. In some embodiments, in the oligosaccharide preparation or at least one of the fractions, the anhydro subunits that are products of caramelization and the anhydro subunits that are products of reversible thermal dehydration of the monosaccharide are of similar abundance.

In some embodiments, about 0.01% to 50%, about 0.01% to 40%, about 0.01% to 30%, about 0.01% to 20%, about 0.01% to 10%, from about 0.01% to 5%, from about 0.01% to 4%, from about 0.01% to 3%, from about 0.01% to 2%, from about

- 17 044964

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

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

In some embodiments, about 0.01% to 50%, about 0.01% to 40%, about 0.01% to 30%, about 0.01% to 20%, about 0.01% to 10%, from about 0.01% to 5%, from about 0.01% to 4%, from about 0.01% to 3%, from about 0.01% to 2%, from about 0.01% up to 1%, from about 0.01 from about 0.1% to 0.5%, from about 0.1% to 50%, from about 0.1% to 40%, from about 0.1% to 30% , from about 0.1% to 20%, from about 0.1% to 10%, from about 0.1% to 5%, from about 0.1% to 4%, from about 0.1% to 3%, about 0.1% to 2%, about 0.1% to 1%, or about 0.1% to 0.5% of the anhydro subunits in each fraction of the oligosaccharide preparation described herein are caramelization products. In some embodiments, about 0.1% to 5%, about 0.1% to 2%, or about 0.1% to 1% of the anhydro subunits in each formulation fraction are caramelization products. In some embodiments, less than 50%, less than 40%, less than 30%, less than 20%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than more than 12%, less than 11%, less 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than more than 2% or less than 1% of anhydro-subunits in each fraction of the drug are caramelization products.

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 includes a DP1 anhydro-subunit-containing oligosaccharide that includes 1,6-anhydro-β-O-glucopyranose, and a DP2 anhydro-subunit-containing oligosaccharide that includes 1,6-anhydro-β- D-glucofuranose subunit. In some embodiments, one or more oligosaccharides in an oligosaccharide preparation contain two or more of the same or different anhydro subunits.

In some embodiments, in any fraction of the oligosaccharide preparation that has a degree of polymerization of 2 or more (ie, the DP2-DPn fraction), the anhydro subunit may be associated with one or more regular or anhydro subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydro subunit is associated with one, two, or three other regular or anhydro subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydro subunit is associated with one or two regular subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydro subunit is associated with one regular subunit. In some embodiments, in any of the DP2-DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the anhydrosubunits are associated with a single regular subunit. In some embodiments, each

- 18 044964 from the DP2-DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40% or 30% of the anhydro subunits are associated with one regular subunit.

In some embodiments, in any fraction of the oligosaccharide preparation that has a degree of polymerization of 2 or more (ie, the DP2-DPn fraction), the anhydro subunit may be located at the end of the oligosaccharide chain. In some embodiments, in any fraction of the oligosaccharide preparation that has a degree of polymerization of 3 or more (ie, the DP3-DPn fraction), the anhydro subunit may be located at a position that is not the end of the oligosaccharide chain. In some embodiments, in the DP2-DPn fractions, at least one subunit is located at the end of the oligosaccharide chain. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% anhydrous -subunits in the DP2-DPn fractions are located at the end of the oligosaccharide chain. 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 end of the oligosaccharide chain. In some embodiments, more than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the anhydro subunit-containing oligosaccharides include an anhydro subunit at the end of the chain. In some embodiments, more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anhydro subunit-containing oligosaccharides include anhydro- subunit at the end of the chain.

E. Glycosidic bonds.

In some embodiments, the oligosaccharide preparation described herein contains multiple glycosidic linkages. The type and distribution of glycosidic bonds may depend on the source and method of production of the oligosaccharide drug. In some embodiments, the type and distribution of various glycosidic linkages can be determined and/or detected by any suitable methods known in the art, such as NMR. For example, in some embodiments, glycosidic linkages are determined and/or detected using 1H-NMR, 13C-NMR, 2D NMR such as 2D JRES, HSQC, HMBC, DOSY, COZY, ECOSY, TOCSY, NOESY or ROESY, or any of these combinations. In some embodiments, glycosidic linkages are determined and/or detected, at least in part, by ¹ H-NMR. In some embodiments, glycosidic linkages are determined and/or detected, at least in part, by 13C-NMR. In some embodiments, glycosidic linkages are determined and/or detected, at least in part, by 2D 1H, 13C-HSQC NMR.

In some embodiments, an oligosaccharide preparation described herein contains one or more α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,4) glycosidic linkages, 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, a-(1,1 )-α-glycosidic bonds, α-(1,1)-β-glycosidic bonds, β-(1,1)-β-glycosidic bonds, or any combination thereof.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 60 mole%, about 5% to 55 mole%, about 5% to 50 mole%, about 5% to 45 mole%, from about 5% to 40 mol.%, from about 5% to 35 mol.%, from about 5% to 30 mol.%, from about 5% to 25 mol.%, from about 10% to 60 mol.%, from about 10% to 55 mol.%, from about 10% to 50 mol.%, from about 10% to 45 mol.%, from about 10% to 40 mol.%, from about 10% to 35 mol.%, from about 15% to 60 mol.%, from about 15% to 55 mol.%, from about 15% to 50 mol.%, from about 15% to 45 mol.%, from about 15% to 40 mol.%, from about 15% to 35 mol.%, from about 20% to 60 mol.%, from about 20% to 55 mol.%, from about 20% to 50 mol.%, from about 20% to 45 mol.%, from about 20% to 40 mol.%, from about 20% to 35 mol.%, from about 25% to 60 mol.%, from about 25% to 55 mol.%, from about 25% to 50 mol.%, about 25% to 45 mol%, about 25% to 40 mol%, or about 25% to 35 mol% α-(1,6) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 50 mole%, about 0 to 40 mole%, about 0 to 35 mole%, about 0 to 30 mole%, about 0 to 25 mol.%, from about 0 to 20 mol.%, from about 5% to 40 mol.%, from about 5% to 35 mol.%, from about 5% to 30 mol.% from about 5% to 25 mol.%, about 5% to 20 mol.%, about 10% to 40 mol.%, about 10% to 35 mol.%, about 10% to 20 mol.% about 15% to 40 mol.% .%, about 15% to 35 mol.%, about 15% to 30 mol.%, about 15% to 25 mol.%, or about 15% to 20 mol.% α-(1,3) glycosidic connections.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 35 mole%, about 0 to 30 mole%, about 0 to 25 mole%. mol.%, about 0 to 20 mol.%, about 0 to 15 mol.%, about 0 to 10 mol.%, about 2% to 30 mol.%, about 2% to 25 mol.% , about 2% to 20 mol.%, about 2% to 15 mol.%, about 2% to 10 mol.%, about 3% to 30 mol.%, about 3% to 25 mol.% , about 3% to 20 mol.%, about 3% to 15 mol.%, about 3% to 10 mol.%, about 5% to 30 mol.%, about 5% to 25 mol.% , from about 5% to 20 mol.%, about 19 044964 from about 5% to 15 mol.%, or from about 5% to 10 mol.% α-(1,2) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of about 0 to 40 mole%, about 0 to 30 mole%, about 0 to 25 mole%, about 0 to 20 mole%, about 0 up to 15 mol.%, about 0 to 10 mol.%, or about 0 to 5 mol.% α-(1,4) glycosidic linkages. In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution of less than 40 mol.%, less than 30 mol.%, less than 20 mol.%, less than 15 mol.%, less than 10 mol.%, less than 9 mol.%, less than 8 mol. .%, less than 7 mol.%, less than 6 mol.%, less than 5 mol.%, less than 4 mol.%, less than 3 mol.% or less than 2 mol.% α-(1,4) glycosidic bonds.

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

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

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

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

In some embodiments, the oligosaccharide preparations have a glycosidic linkage type distribution that is different from the glycosidic linkage type distribution in non-synthetic oligosaccharide preparations. For example, in some embodiments, the oligosaccharide preparations have a distribution of glycosidic linkage types that differs from that of the base nutritional compositions. In some embodiments, the basic nutritional compositions contain a natural source of carbohydrates, such as starch and plant fiber. Some of the natural carbohydrate sources have a high percentage of α-(1,4), α-(1,6) and/or β-(1,6) glycosidic linkages. Accordingly, in some embodiments, the oligosaccharide preparations have a lower percentage of α-(1,4) glycosidic linkages than the base nutritional composition. In some embodiments, the oligosaccharide preparations have a lower percentage of α-(1,6) glycosidic linkages than the base nutritional composition. In other embodiments, the oligosaccharide preparations have a higher percentage of α-(1,6) glycosidic linkages than the base nutritional composition. In some embodiments, the oligosaccharide preparations have a lower percentage of β-(1,6) glycosidic linkages than the base nutritional composition. In some embodiments, the oligosaccharide preparation contains glycosidic linkages that are difficult to cleave or hydrolyze by enzymes.

Specifically, in some embodiments, α-(1,2), α-(1,3), α-(1,4), α-(1,6), β-(1,2), β-( 1.3),

- 20 044964 β-(1,4) and/or β-(1,6) glycosidic bonds in the glycosidic bond type distribution of the oligosaccharide preparations described in the present invention by 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 in the main nutritional composition. In some embodiments, 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 bonds in the distribution of glycosidic bond type of oligosaccharide preparations by at least 50 mol.%, at least 40 mol.%, at least by 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 in the main nutritional composition.

One skilled in the art will appreciate that certain types of glycosidic linkages may not be applicable to oligosaccharides containing a certain type of monosaccharide. 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 bonds, β-(1,3) glycosidic bonds, β-(1,4) glycosidic bonds, and β-(1,6) glycosidic bonds.

F. Molecular weight.

The molecular weight and molecular weight distribution of the oligosaccharide preparations described in the present invention can be determined by any suitable analytical means and instruments, such as the end group method, osmotic pressure (osmometry), ultracentrifugation, viscosity measurement, light scattering method, SEC, SEC-MALLS, PFP , A4F, HPLC and mass spectrometry. In some embodiments, molecular weight and molecular weight distribution are determined by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. In some embodiments, molecular weight and molecular weight distribution are determined using size exclusion chromatography (SEC), such as gel permeation chromatography (GPC). In other embodiments, the molecular weight and molecular weight distribution are determined using HPLC. In some embodiments, molecular weight and molecular weight distribution are determined using MALDI-MS.

In some embodiments, the oligosaccharide drug described herein has a weight average molecular weight of about 100 to 10,000 g/mol, about 200 to 8,000 g/mol, about 300 to 5,000 g/mol, about 500 to 5,000 g/mol, about 700 to 5000 g/mol, about 900 to 5000 g/mol, about 1100 to 5000 g/mol, about 1300 to 5000 g/mol, about 1500 to 5000 g/mol, about 1700 to 5000 g/mol, about 300 to 4500 g/mol, about 500 to 4500 g/mol, about 700 to 4500 g/mol, about 900 to 4500 g/mol, about 1100 to 4500 g/mol, about 1300 to 4500 g/mol, about 1500 to 4500 g/mol, about 1700 to 4500 g/mol, about 1900 to 4500 g/mol, about 300 to 4000 g/mol, about 500 to 4000 g /mol, from about 700 to 4000 g/mol, from about 900 to 4000 g/mol, from about 1100 to 4000 g/mol, from about 1300 to 4000 g/mol, from about 1500 to 4000 g/mol, from about 1700 to 4000 g/mol, about 1900 to 4000 g/mol, about 300 to 3000 g/mol, about 500 to 3000 g/mol, about 700 to 3000 g/mol, about 900 to 3000 g/ mole, from about 1100 to 3000 g/mol, from about 1300 to 3000 g/mol, from about 1500 to 3000 g/mol, from about 1700 to 3000 g/mol, from about 1900 to 3000 g/mol, from about 2100 up to 3000 g/mol, about 300 to 2500 g/mol, about 500 to 2500 g/mol, about 700 to 2500 g/mol, about 900 to 2500 g/mol, about 1100 to 2500 g/mol , approximately from 1300 to

2500 g/mol, about 1500 to 2500 g/mol, about 1700 to 2500 g/mol, about 1900 to

2500 g/mol, about 2100 to 2500 g/mol, about 300 to 1500 g/mol, about 500 to 1500 g/mol, about 700 to 1500 g/mol, about 900 to 1500 g/mol, from approximately 1100 to

1500 g/mol, about 1300 to 1500 g/mol, about 2000 to 2800 g/mol, about 2100 to

2700 g/mol, about 2200 to 2600 g/mol, about 2300 to 2500 g/mol, or about 2320 to 2420 g/mol. In some embodiments, the weight average molecular weight of the oligosaccharide drug is about 2000 to 2800 g/mol, about 2100 to 2700 g/mol, about 2200 to 2600 g/mol, about 2300 to 2500 g/mol, or about 2320 up to 2420 g/mol. In some embodiments, the oligosaccharide drug has a weight average molecular weight ranging from at least 500 g/mol, 750 g/mol, 1000 g/mol, or 1500 g/mol to no more than 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 drug described herein is determined by HPLC according to Example 9.

- 21 044964

In some embodiments, the oligosaccharide drug described herein has a number average molecular weight of about 100 to 10,000 g/mol, about 200 to 8,000 g/mol, about 300 to 5,000 g/mol, about 500 to 5,000 g/mol, about 700 to 5000 g/mol, about 900 to 5000 g/mol, about 1100 to 5000 g/mol, about 1300 to 5000 g/mol, about 1500 to 5000 g/mol, about 1700 to 5000 g/mol, about 300 to 4500 g/mol, about 500 to 4500 g/mol, about 700 to 4500 g/mol, about 900 to 4500 g/mol, about 1100 to 4500 g/mol, about 1300 to 4500 g/mol, about 1500 to 4500 g/mol, about 1700 to 4500 g/mol, about 1900 to 4500 g/mol, about 300 to 4000 g/mol, about 500 to 4000 g /mol, from about 700 to 4000 g/mol, from about 900 to 4000 g/mol, from about 1100 to 4000 g/mol, from about 1300 to 4000 g/mol, from about 1500 to 4000 g/mol, from about 1700 to 4000 g/mol, about 1900 to 4000 g/mol, about 300 to 3000 g/mol, about 500 to 3000 g/mol, about 700 to 3000 g/mol, about 900 to 3000 g/ mole, from about 1100 to 3000 g/mol, from about 1300 to 3000 g/mol, from about 1500 to 3000 g/mol, from about 1700 to 3000 g/mol, from about 1900 to 3000 g/mol, from about 2100 up to 3000 g/mol, about 300 to 2500 g/mol, about 500 to 2500 g/mol, about 700 to 2500 g/mol, about 900 to 2500 g/mol, about 1100 to 2500 g/mol , about 1300 to 2500 g/mol, about 1500 to 2500 g/mol, about 1700 to 2500 g/mol, about 1900 to 2500 g/mol, about 2100 to 2500 g/mol, about 300 to 2000 g/mol, about 500 to 2000 g/mol, about 700 to 2000 g/mol, about 900 to 2000 g/mol, about 1100 to 2000 g/mol, about 300 to 1500 g/mol, about 500 to 1500 g/mol, about 700 to 1500 g/mol, about 900 to 1500 g/mol, about 1100 to 1500 g/mol, about 1300 to 1500 g/mol, about 1000 to 2000 g/mol, about 1100 to 1900 g/mol, about 1200 to 1800 g/mol, about 1300 to 1700 g/mol, about 1400 to 1600 g/mol, or about 1450 to 1550 g/mol. In some embodiments, the number average molecular weight of the oligosaccharide drug is about 1000 to 2000 g/mol, about 1100 to 1900 g/mol, about 1200 to 1800 g/mol, about 1300 to 1700 g/mol, about 1400 to 1600 g/mol or about 1450 to 1550 g/mol. In some embodiments, the oligosaccharide drug has a number average molecular weight ranging from at least 500 g/mol, 750 g/mol, 1000 g/mol, or 1500 g/mol to no more than 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 an oligosaccharide drug described herein is determined by HPLC according to Example 9.

G. Types of oligosaccharides.

The types of oligosaccharides present in the oligosaccharide preparation may depend on the type of one or more feed sugars. For example, in some embodiments, the oligosaccharide preparations contain a gluco-oligosaccharide when the feed sugars contain glucose. For example, in some embodiments, the oligosaccharide preparations contain a galacto-oligosaccharide when the feed sugars contain galactose. In another example, in some embodiments, the oligosaccharide preparations contain gluco-galacto-oligosaccharides, where feed sugars include galactose and glucose.

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

In some embodiments, the oligosaccharide preparation contains oligosaccharides with 1, 2, 3, or 4 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation includes oligosaccharides with 1, 2, or 3 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation contains oligosaccharides with 3 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation includes oligosaccharides with 2 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation includes one type of monosaccharide subunit.

In some embodiments, the oligosaccharide preparation includes various types of oligosaccharides, with each oligosaccharide molecule independently comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of monosaccharide subunits. In some embodiments, the oligosaccharide preparation described herein contains 10 ² , 10 ³ , 10 ⁴ , 105 or more different types of oligosaccharides. In some embodiments, some of the oligosaccharides in the preparation contain one type of monosaccharide subunit, and some of the other oligosaccharides in the same preparation contain two or more types of monosaccharide subunits. For example, in some embodiments, when the feed sugars are glucose and galactose, the oligosaccharide preparation may include oligosaccharides that contain only glucose subunits, oligosaccharides that contain only galactose subunits, oligosaccharides that contain glucose and galactose subunits in varying ratios, or any a combination of them.

In some embodiments, any or all n fractions of the oligosaccharide preparation contain different types of oligosaccharide subunits, wherein each oligosaccharide independently includes 1, 2,

3, 4, 5, 6, 7, 8, 9 or 10 different types of monosaccharide subunits. In some embodiments, some of the oligosaccharides in the drug fraction contain one type of monosaccharide subunit, and some of the other oligosaccharides in the same drug fraction contain two or more types of monosaccharide subunits.

In some embodiments, the oligosaccharide preparation described herein contains one or more monosaccharide subunits selected from the group consisting of triose, tetrose, pentose, hexose, heptose, and any combination thereof, wherein each is triose, tetrose, pentose, hexose, or heptose subunits independently and, if necessary, functionalized and/or replaced by one of its corresponding anhydro subunits. In some embodiments, the corresponding anhydro subunit is the product of reversible thermal dehydration of a monosaccharide subunit. In some embodiments, the corresponding anhydro subunit is a caramelization product of a monosaccharide subunit.

In some embodiments, the oligosaccharide preparation described herein comprises pentose subunits, hexose subunits, or any combination thereof, wherein each of the pentose or hexose subunits is independently and optionally functionalized and/or replaced by one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparation contains hexose subunits, wherein each of said hexose subunits is independently and optionally replaced by one of its corresponding anhydro subunits.

In the context of the present invention, tetrose refers to a monosaccharide with four carbon atoms, such as erythrose, threose and erythrulose. In the context of the present invention, pentose refers to a monosaccharide with five carbon atoms, such as arabinose, lyxose, ribose and xylose. In the context of the present invention, hexose refers to a monosaccharide with six carbon atoms, such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose and tagatose. In the context of the present invention, heptose refers to a monosaccharide with seven carbon atoms, such as sedoheptulose and mannoheptulose.

In some embodiments, the oligosaccharide preparation described herein comprises a glucose subunit, wherein at least one glucose subunit is optionally replaced by an anhydroglucose subunit. In some embodiments, the oligosaccharide preparation described herein comprises a galactose subunit, wherein at least one galactose subunit is optionally replaced by an anhydrogalactose subunit. In some embodiments, the oligosaccharide preparation described herein contains galactose and glucose subunits, wherein at least one galactose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparation described herein contains fructose and glucose subunits, wherein at least one fructose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparation described herein comprises mannose and glucose subunits, wherein at least one mannose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydro subunits.

In some embodiments, the oligosaccharide drug described herein includes a gluco-galactose-oligosaccharide drug, a gluco-oligosaccharide drug, a galacto-oligosaccharide drug, a fructo-oligosaccharide drug, a manno-oligosaccharide drug, an arabino-oligosaccharide drug, a xylo-oligosaccharide drug, a gluco-oligosaccharide drug. fructo-oligosaccharide drug, gluco-manno-oligosaccharide drug, gluco-arabino-oligosaccharide drug, glucoxylo-oligosaccharide drug, galacto-fructo-oligosaccharide drug, manno-oligosaccharide drug, galacto-arabino-oligosaccharide drug, galacto-xylo-oligosaccharide drug, fructo-manno-oligosaccharide drug, fructo-arabino-oligosaccharide drug, fructo-xyloo-oligosaccharide drug, manno-arabino-oligosaccharide drug, manno-xylo-oligosaccharide drug, arabino-xylo-oligosaccharide drug, galacto-arabino-xylo-oligosaccharide drug, fructo -galacto-xylo-oligosaccharide drug, arabino-fructo-manno-xylo-oligosaccharide drug, gluco-fructo-galacto-arabino-oligosaccharide drug, fructo-gluco-arabino-manno-xyloo-oligosaccharide drug, gluco-galacto-fructo-manno-arabino -xylo-oligosaccharide drug, or any combination thereof; where each of the monosaccharide subunits in the composition of the drug is independently and, if necessary, functionalized and/or replaced by one of its corresponding anhydrous subunits.

In some embodiments, the oligosaccharide preparation described herein contains more than 99% glucose subunits by weight. In some embodiments, the oligosaccharide preparation contains only glucose subunits.

In some embodiments, the oligosaccharide described in the present invention

- 23 044964 the preparation contains from about 45% to 55% glucose subunits and from about 55% to 45% galactose subunits by weight. In some embodiments, the oligosaccharide preparation contains about 50% glucose subunits and 50% galactose subunits by weight.

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

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

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

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

In some embodiments, the oligosaccharide preparation described herein has a monosaccharide subunit composition as shown in Table. 29.

Table 29

Approximate composition of oligosaccharide preparations

No. of oligosaccharide composition Glucose and anhydroglucose subunits (wt.%) Galactose and anhydrogalactose subunits (wt.%) Mannose and anhydromannose subunits (wt.%) Fructose and anhydrofructose subunits (wt.%) 1 87.5 12.5 0 0 2 100 0 0 0 3 85 2.5 12.5 0 4 87.5 0 12.5 0 5 50 50 0 0 6 75 0 25 0 7 9 6 0 0 8 90 0 10 0 9 95 5 0 0 10 97.5 2.5 0 0 eleven 85 5 10 0 12 85 1.5 13.5 0 13 80 10 10 0 14 85 0 15 0 15 85 15 0 0 16 87.5 0 0 12.5

H. D-shape and L-shape.

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

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

I. Functionalized oligosaccharides.

In some embodiments, one or more oligosaccharides in an oligosaccharide preparation described herein are independently functionalized. Functionalized oligosaccharides can be prepared, for example, by combining one or more sugars with one or more functionalizing compounds in the presence of a catalyst. Methods for preparing functionalized oligosaccharides are described in WO 2012/118767, WO 2014/031956 and WO/2016/122887, which are incorporated herein by reference in their entirety and for their disclosure.

In some embodiments, the functionalizing compound contains one or more acidic groups (e.g., -COOH), hydroxyl groups, or N-containing groups (e.g., -CN, -NO2 _, and -N(Ra) ₂ , where Ra represents hydrogen, alkyl , alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, aryl, heterocycloalkyl or heteroaryl group), S-containing groups (for example, thiol and sulfate), halides (for example, -Cl), P-containing groups (for example, phosphate) or any their combination. In some embodiments, the functionalizing compound is linked to at least one monosaccharide subunit via an ether, ester, oxygen-sulfur, amine, or oxygen-phosphorus bond. In some embodiments, one or more functionalizing compounds are linked to the monosaccharide subunit through a single bond. In some embodiments, the at least one functionalizing compound is linked to one or two oligosaccharides by two or more linkages.

It should be understood that for each oligosaccharide in the oligosaccharide preparation, each of the described embodiments is independent and can be combined as if each combination were listed separately; thus, any combination of embodiments is covered by the present disclosure. For example, various embodiments may be grouped into several categories, which include (i) the presence or absence of an anhydro subunit; (ii) the amount and level of the anhydro subunit; (iii) type of anhydro subunit species; (iv) location of the anhydro subunit; (v) degree of polymerization; (vi) molecular weight; (vii) the presence or absence of any functional groups; (viii) type of oligosaccharide; (ix) type of glycosidic bond and (x) L- or D-form; but are not limited to them. Accordingly, the described oligosaccharide preparation includes a variety of oligosaccharides of different types. In some embodiments, the oligosaccharide preparation described herein contains at least 10, 102, 103, ¹⁰⁴ , 105, 106, ¹⁰⁷ , 108, 109, or ¹⁰¹⁰ different types of oligosaccharides. In some embodiments, the formulation contains at least 103, ¹⁰⁴ , 105, 106, or 109 different types of oligosaccharides. In some embodiments, the formulation contains at least 103 different types of oligosaccharides.

II I. Methods for the production of oligosaccharide preparations.

In one aspect, the present invention provides methods for the production of oligosaccharide preparations. In some embodiments, the present invention provides methods for producing oligosaccharide preparations suitable for use in a nutritional composition, such as an animal feed composition, or for feeding directly to an animal. In one aspect, the present invention provides methods for producing an oligosaccharide preparation comprising heating an aqueous composition containing one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerization, wherein the catalyst is selected from the group consisting of: (+) -camphor-10-sulfonic acid; 2-pyridine sulfonic acid; 3-pyridine sulfonic acid; 8-hydroxy-5-quinoline sulfonic acid hydrate; αhydroxy-2-pyridinemethanesulfonic acid; (c)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; (S)-VAPOL hydrogen phosphate; 6-quinoline sulfonic acid, 3-(1-pyridino)-1-propanesulfonate; 2-(2pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diylhydrogenphosphate; bis(4methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-cysteine acid monohydrate; poly(styrenesulfonic acid-co-divinylbenzene); lysine; ethanesulfonic acid; ethanesulfonic acid; isethionic acid; homocysteine acid; HEPBS (N-(2hydroxyethyl)piperazine-M'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(N

- 25 044964 morpholino)ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6sulfoquinovoses; triflic 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; valina; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan, and wherein the oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has a different degree of polymerization selected from 1 (fraction DP1) to n (fraction DPn), where n is an integer equal to 2 or more.

In some embodiments, n is an integer equal to 3 or more. In some embodiments, n is an integer ranging from 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, polymerization of feed sugars is achieved by step polymerization. In some embodiments, polymerization of feed sugars is achieved by polycondensation.

A. Feed sugar.

In some embodiments, the method for producing oligosaccharide preparations described in the present invention includes heating one or more types of feed sugars. In some embodiments, the one or more feed sugars comprise monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or any mixtures thereof.

In some embodiments, the one or more feed sugars comprise glucose. In some embodiments, the one or more feed sugars comprise glucose and galactose. In some embodiments, the one or more feed sugars comprise glucose, xylose, and galactose. In some embodiments, the one or more feed sugars comprise glucose and mannose. In some embodiments, the one or more feed sugars comprise glucose and fructose. In some embodiments, the one or more feed sugars comprise glucose, fructose, and galactose. In some embodiments, the one or more feed sugars comprise glucose, galactose, and mannose.

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

In some embodiments, the type of feed sugars may influence the resulting oligosaccharide preparations. For example, in some embodiments, when one or more feed sugars are glucose, the resulting oligosaccharide preparations include glucooligosaccharide preparations. In other embodiments, when one or more feed sugars are mannose, the resulting oligosaccharide preparations include manno-oligosaccharide preparations. In some embodiments, where one or more feed sugars comprise glucose and galactose, the resulting oligosaccharide preparations include glucogalacto-oligosaccharide preparations. In still other embodiments, where one or more feed sugars include xylose, glucose and galactose, the resulting oligosaccharide preparations include gluco-galacto-xylo-oligosaccharide preparations.

In some embodiments, each of the one or more feed sugars may independently be in its dehydrated or hydrated form. In some embodiments, the one or more feed sugars comprise glucose, galactose, fructose, mannose, or any combination thereof, and wherein each of glucose, galactose, fructose, or mannose is independently in its monohydrate or dehydrated form. In some embodiments, the one or more feed sugars comprise a monosaccharide monohydrate, such as glucose monohydrate. In some embodiments, the one or more feed sugars comprise a saccharide dihydrate, such as trehalose dihydrate. In some embodiments, the one or more feed sugars comprise at least one sugar in its dehydrated form and at least one sugar in its hydrated form.

In some embodiments, one or more feed sugars may be provided as a sugar solution where the sugars are combined with water and loaded into a reactor. In some embodiments, the sugars may be supplied to the reactor in solid form and combined with water in the reactor. In some embodiments, one or more feed sugars are combined and mixed before water is added. In some embodiments, one or more feed sugars are combined in water and then mixed.

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 (eg, monosaccharides, disaccharides, and/or trisaccharides) with a catalyst to produce an oligosaccharide preparation. In other embodiments, the method includes combining a mixture of sugars and sugar alcohols with a catalyst to produce an oligosaccharide preparation.

In some embodiments, the one or more feed sugars comprise functionalized or modified sugars.

Functionalized or modified sugars may include amino sugars, sugar acids, sugar alcohols, sugar amides, sugar ethers, or any combination thereof. In some embodiments, amino sugars refer to sugar molecules in which the hydroxyl group is replaced by an amino group. Examples of amino sugars include N-acetyl-D-glucosamine, mannosamine, neuramic acid, muramic acid, N-acetylneuraminic acid, N-acetylmuramic acid, N-acetyl-galactosamine, N-acetyl-mannose, N-glycolylneuraminic acid, acarviosine, D-glucosamine and D-galactosamine, but not limited to.

In some embodiments, sugar acids refer to sugars with a carboxyl group. Examples of sugar acids include aldonic acids (such as glyceric acid, xylonic acid, gluconic acid and ascorbic acid), ulosonic acids (such as neuraminic acid and ketodeoxyoctulosonic acid), uronic acids (such as glucuronic acid, galacturonic acid and iduronic acid) and aldaric acids. acids (such as, but not limited to, tartaric acid, mucinic acid and sugar acid).

In some embodiments, sugar alcohols refer to sugar-derived polyols. Examples of sugar alcohols include, but are not limited to, ethylene glycol, arabitol, glycerin, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, and volemitol.

In some embodiments, sugar amides refer to sugar molecules that contain a -C(=O)-N- group. In some embodiments, sugar ethers refer to sugar molecules that contain an ester bond, such as glucosides.

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

In some embodiments, the method of producing an oligosaccharide preparation described in the present invention is carried out on a gram scale. In some embodiments, the method of producing an oligosaccharide preparation described in the present invention is carried out on a kilogram or larger scale. Accordingly, in some embodiments, the method includes heating an aqueous composition containing one or more feed sugars in an amount greater than 0.5, greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 9, more 10, more than 100 or more than 1000 kg. In some embodiments, the method includes heating an aqueous composition containing one or more feed sugars in an amount of not more than 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10, 100, 1000 or 1500 kg. In some embodiments, the method includes heating an aqueous composition containing one or more feed sugars in an amount greater than 1 kg.

B. Catalysts.

In some embodiments, the catalyst provided herein includes one or more acids. In some embodiments, the catalyst provided herein includes a mineral acid, a carboxylic acid; amino acid; sulfonic acid; boronic acid; phosphonic acid; phosphinic acid; sulfuric acid; phosphoric acid; poly(styrenesulfonic acid-co-vinylbenzyl-imidazolium sulfate-co-divinylbenzene); poly(styrenesulfonic acid-co-divinylbenzene); (+)-camphor-10-sulfonic acid; 2pyridine sulfonic acid; 3-pyridine sulfonic acid; 8-hydroxy-5-quinoline sulfonic acid hydrate; a-hydroxy-2-pyridinemethanesulfonic acid; (c)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; (S)-VAPOL-hydrogen phosphate; 6-quinoline sulfonic acid; 3-(1-pyridino)-1-propanesulfonate; 2-(2pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diylhydrogen phosphate; bis(4methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-cysteine acid monohydrate; acetic acid; propionic acid; butanoic acid; glutamic acid; lysine, ethanedisulfonic acid; ethanesulfonic acid; isethionic acid; homocysteine acid; HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2

- 27 044964 hydroxyethyl)-1-piperazinethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6sulfoquinovose; triflic 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; polymeric acid; carbon-based acid; or any combination thereof.

In some embodiments, the catalyst of the present invention includes: (+)-camphor-10-sulfonic acid; 2-pyridine sulfonic acid; 3-pyridine sulfonic acid; 8-hydroxy-5-quinoline sulfonic acid hydrate; a-hydroxy-2-pyridinemethanesulfonic acid; (c)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; (S)-VAPOL-hydrogen phosphate; 6-quinoline sulfonic acid, 3-(1-pyridino)-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'diylhydrogen phosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid; L-cysteine acid monohydrate; poly(styrenesulfonic acid-co-divinylbenzene); lysine; ethanesulfonic acid; ethanesulfonic acid; isethionic acid; homocysteine acid; HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(Nmorpholino)ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6sulfoquinovose; triflic 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.

In some embodiments, the catalyst of the present invention is (+)-camphor-10-sulfonic acid. In some embodiments, the catalyst of the present invention is 2-pyridine sulfonic acid. In some embodiments, the catalyst of the present invention is 3-pyridine sulfonic acid. In some embodiments, the catalyst of the present invention is 8-hydroxy-5-quinoline sulfonic acid hydrate. In some embodiments, the catalyst of the present invention is a-hydroxy-2-pyridinemethanesulfonic acid. In some embodiments, the catalyst of the present invention is (c)-camphor-10sulfonic acid. In some embodiments, the catalyst of the present invention is butylphosphonic acid. In some embodiments, the catalyst of the present invention is diphenylphosphinic acid. In some embodiments, the catalyst of the present invention is hexylphosphonic acid. In some embodiments, the catalyst of the present invention is methylphosphonic acid. In some embodiments, the catalyst of the present invention is phenylphosphinic acid. In some embodiments, the catalyst of the present invention is phenylphosphonic acid. In some embodiments, the catalyst of the present invention is tert-butylphosphonic acid. In some embodiments, the catalyst of the present invention is (S)-VAPOL hydrogen phosphate. In some embodiments, the catalyst of the present invention is 6-quinoline sulfonic acid. In some embodiments, the catalyst of the present invention is 3(1-pyridino)-1-propanesulfonate. In some embodiments, the catalyst of the present invention is 2-(2-pyridinyl)ethanesulfonic acid. In some embodiments, the catalyst of the present invention is 3(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate. In some embodiments, the catalyst of the present invention is G-binaphthyl-2,2'-diyl hydrogen phosphate. In some embodiments, the catalyst of the present invention is bis(4-methoxyphenyl)phosphinic acid. In some embodiments, the catalyst of the present invention is phenyl(3,5-xylyl)phosphinic acid. In some embodiments, the catalyst of the present invention is L-cysteine acid monohydrate. In some embodiments, the catalyst of the present invention is poly(styrenesulfonic acid-co-divinylbenzene). In some embodiments, the catalyst of the present invention is lysine.

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 homocysteine acid. In some embodiments, the catalyst is HEPBS (K-(2-hydroxyethyl)piperazine-K'-(4butanesulfonic 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-(K-morpholino)ethanesulfonic acid. In some embodiments, the catalyst is methanesulfonic acid. In embodiments, the catalyst is naphthalene-1-sulfonic acid. In some embodiments, the catalyst is a metathaniazide. In some embodiments, the catalyst 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 triflic 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 an 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.

In some embodiments, the catalyst provided by the present invention is a polymer or carbon-supported catalyst disclosed in WO 2016122887, which is incorporated herein by reference in its entirety and for its disclosure.

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

In some embodiments, the catalyst provided herein is present in an amount of about 0.01% to 5%, about 0.02% to 4%, about 0.03% to 3%, or about 0.05% to 2% % of the aqueous composition by dry matter weight. In some variants

- 29 044964 implementation of the proposed in the present invention, the catalyst is present in an amount of from about 1% to 2% of the aqueous composition by weight of dry matter. 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 %, approximately 1.5%, approximately 1.6%, approximately 1.7%, approximately 1.8%, approximately 1.9%, approximately 2.0%, approximately 2.1%, approximately 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 weight of dry matter.

In some embodiments, the catalyst of the present invention is a combination of two or more different catalysts. In some embodiments, the catalyst includes a recyclable catalyst, such as resins and polymer catalysts, and a non-recyclable catalyst. In some embodiments, when the catalyst includes at least two different catalysts, each of the catalysts is present in the amount provided in the present invention. In other embodiments, where the catalyst includes at least two different catalysts, the at least two different catalysts are present together in the amount specified herein.

In some embodiments, the catalyst is added to the aqueous composition in dry form. In other embodiments, the catalyst is added to the aqueous composition in wet form, for example, in 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 it is combined with one or more feed sugars. In some embodiments, the method of the present invention includes preparing an aqueous composition by combining one or more feed sugars in dehydrated form and a catalyst in wet form (eg, as an aqueous solution).

C. Adding water.

In some embodiments, the method of producing oligosaccharide preparations described in the present invention includes adding water to form an aqueous composition. In some embodiments, all or a portion 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, such as saccharide mono- 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 some embodiments, all or a portion of the water in the aqueous composition is added with the catalyst, that is, through a catalyst solution.

D. Water content.

As methods for producing oligosaccharide preparations develop, water can be produced through the reaction. For example, in some embodiments, water is produced (i) through the formation of a glycosidic bond, (ii) through the formation of an anhydro subunit, or (iii) through other mechanisms or sources. Because both sugar condensation and dehydration reactions involve water, in some embodiments, water content affects the composition of the oligosaccharide preparation.

Additionally, in some embodiments, water content affects the viscosity of the aqueous composition, which in turn may affect the mixing efficiency of the aqueous composition. For example, in some embodiments, an excessively viscous aqueous composition may result in undesirable heterogeneous distribution of the catalyst in the aqueous composition. Moreover, in some embodiments, very low water content may cause the aqueous composition to harden, preventing effective mixing. On the other hand, in some other embodiments, extremely high water content may inhibit the sugar condensation reaction and lower the level of anhydro subunits. Accordingly, the present invention describes a suitable water content for the production of oligosaccharide preparations.

In some embodiments, a method of producing an oligosaccharide preparation described in the present invention includes forming and/or heating an aqueous composition. In some embodiments, the aqueous composition contains from about 0% to 80%, about 0% to 70%, about 0% to 60%, about 0% to 50%, about 0% to 40%, about 0 % to 35%, about 0% to 30%, about 0% to 25%, about 0% to 20%, about 0% to 19%, about 0% to 18%, about 0% to 17%, about 0% to 16%, about 0% to 15%, about 0% to 14%, about 0% to 13%, about 0% to 12%, about 0% to 11% , about 0% to 10%, about 0% to 9%, about 0% to 8%, about 0% to 7%, about 0% to 6%, about 0% to 5%, about 0% to 4%, about 0% to 3%, about 0% to 2%, or about 0% to 1% water by weight. In some embodiments, the aqueous composition contains from about 1% to 20%, about 1% to 18%, about 1% to 16%, about 1% to 14%, about 1% to 12%, about 1% % to 10%, about 1% to 8%, about 1% to 6%, or about 1% to 4% water by weight. In some embodiments, the aqueous composition contains from about 3% to 16%, from about 3% to 14%, from about 3%

- 30 044964 up to 12%, from about 3% to 10%, from about 3% to 8%, from about 3% to 6%, from about 5% to 16%, from about 5% to 14%, from about 5 % to 12%, about 5% to 10%, about 7% to 16%, about 7% to 14%, about 7% to 12%, about 7% to 10%, or about 8% to 10% water of the total mass. In some embodiments, the aqueous composition contains 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 weight. In some embodiments, the aqueous composition contains about 9% water by weight. However, it should be understood that the amount of water in the aqueous composition can be adjusted depending on the reaction conditions and the particular catalyst used. In some embodiments, the water content of the aqueous composition, as described above, is measured at the beginning of the reaction, for example, before heating the feed sugars. In some embodiments, the water content of the aqueous composition described above is measured at the end of the polymerization or condensation reaction. In some embodiments, the water content of the aqueous composition described above is measured as the average water content at the beginning of the reaction and at the end of the reaction.

In some embodiments, the method described in the present invention may further include monitoring the content of water present in the aqueous composition and/or the ratio of water to sugars or catalyst over a period of time. In some embodiments, the method further includes removing at least a portion of the water from the aqueous composition, such as by distillation. Any method known in the art can be used to remove water from the aqueous composition, including, for example, vacuum filtration, vacuum distillation, heat, steam, hot air and/or evaporation.

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

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

In some embodiments, the method described herein includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is from about 1% to 20%, about 1% to 18%, about 1% to 16% , about 1% to 14%, about 1% to 12%, about 1% to 10%, about 1% to 8%, about 2% to 16%, about 2% to 14%, about from 2% to 12%, from about 2% to 10%, from about 2% to 8%, from about 2% to 6%, from about 4% to 16%, from about 4% to 14%, from about 4 % to 12%, about 4% to 10%, about 4% to 8%, about 6% to 16%, about 6% to 12%, about 6% to 10%, or about 6% to 8% of the total mass. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 2% to 10%, about 2% to 8%, or

- 31 044964 measured from 4% to 8% of the total mass. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9% or about 10% of the total weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is from about 4% to 8% by weight. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that, at the end of the polymerization and/or condensation reaction, the water content of the aqueous composition is as described above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that, at the start of the polymerization and/or condensation reaction, the water content of the aqueous composition is as described above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that the average water content of the aqueous composition at the beginning and end of the polymerization and/or condensation reaction is within the range described above. In some embodiments, the method includes adding at least a portion of water to the aqueous composition such that, throughout the polymerization and/or condensation reaction, the water content of the aqueous composition remains within the range described above.

In some embodiments, the degree of polymerization of the oligosaccharides and/or the amount and type of anhydro subunits in the oligosaccharide preparation can be controlled by adjusting or controlling the water content present in the aqueous composition throughout the manufacturing process. For example, in some embodiments, the degree of oligosaccharide polymerization and the number of anhydro subunits are increased by decreasing the water content.

Accordingly, in some embodiments, the method described herein includes in-process control (IPC) of water content, which may include monitoring water content, maintaining water content, increasing water content, decreasing 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 temperature described in the present invention. In some embodiments, the method includes maintaining a water content for a time sufficient to induce polymerization. In some embodiments, the method includes maintaining the water content within a disclosed range by either adding water or removing water from the aqueous composition, or both. In some embodiments, the method includes maintaining the water content within the disclosed range by distillation. In some embodiments, the method includes maintaining the water content within a disclosed range using vacuum distillation. In some embodiments, the method includes maintaining the water content within the disclosed range by distillation at atmospheric pressure.

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

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

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

Without wishing to be bound by theory, it is believed that the water content during the sugar polymerization and/or condensation reaction may influence the level of anhydro subunits in the oligosaccharide preparation described herein. For example, as shown in FIG. 30, in some embodiments, higher water content correlates with lower levels of anhydro subunits. In some embodiments, a lower reaction temperature may correlate with a lower level of anhydro subunits.

E. Temperature.

In some embodiments, the degree of polymerization of the oligosaccharides and/or the number and type of anhydro subunits in the oligosaccharide preparation can be controlled by adjusting the temperature to which the aqueous composition is heated. In some embodiments, a method of producing an oligosaccharide preparation described herein comprises heating the aqueous composition to a temperature of about 80°C to 250°C, about 90°C to 200°C, about 100°C to 200°C, about 100°C to 180°C, about 110°C to 170°C, about 120°C to 160°C, about 130°C to 150°C, or about 135°C to 145°C. In some embodiments, a method for producing an oligosaccharide preparation includes heating the aqueous composition to a temperature of about 100°C to 200°C, about 100°C to 180°C, about 110°C to 170°C, about 120°C to 160°C, about 130°C to 150°C, or about 135°C to 145°C. In some embodiments, a method for producing an oligosaccharide preparation includes heating the aqueous composition to a temperature of from about 135°C to 145°C. In other embodiments, a method for producing an oligosaccharide preparation includes heating the aqueous composition to a temperature of from about 125°C to 135°C.

F. Reaction time.

In some embodiments, the method of producing an oligosaccharide preparation described in the present invention includes heating the aqueous composition for a sufficient time. In some embodiments, the degree of polymerization of oligosaccharides prepared in accordance with the methods described herein can be adjusted by reaction time.

In some embodiments, sufficient time is described in terms of hours. For example, in some embodiments, a 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, a sufficient time is about 1 to 24 hours, about 1 to 16 hours, about 1 to 8 hours, about 1 to 4 hours, about 1 to 3 hours, about 1 to 2 hours, about 2 to 12 hours, about 2 to 10 hours, about 2 to 8 hours, about 2 to 6 hours, about 2 to 4 hours, about 3 to 8 hours, about 3 to 6 hours, about 3 to 5 hours, or about 3 to 4 hours.

In other 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.

In some embodiments, the molecular weight of the oligosaccharide drug is controlled during polymerization. In some embodiments, the method includes heating the aqueous composition for a time sufficient to cause the aqueous composition to reach a number average molecular weight or weight average molecular weight, as described in the present invention. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve a number average molecular weight of the aqueous composition in the range of about 300 to 5000 g/mol, about 500 to 5000 g/mol, about 700 to 5000 g/mol, about 500 to 2000 g/mol, about 700 to 2000 g/mol, about 700 to 1500 g/mol, about 300 to 1500 g/mol, about 300 to 2000 g/mol, about 400 to 1000 g/mol, about 400 to 900 g/mol, about 400 to 800 g/mol, about 500 to 900 g/mol, or about 500 to 800 g/mol. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve a number average molecular weight of the aqueous composition from about 500 to 2000 g/mol. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve a weight average molecular weight of the aqueous composition in the range of about 300 to 5000 g/mol, about 500 to 5000 g/mol, about 700 to 5000 g/mol , approximately from 500 to

2000 g/mol, about 700 to 2000 g/mol, about 700 to 1500 g/mol, about 300 to

1500 g/mol, about 300 to 2000 g/mol, about 400 to 1300 g/mol, about 400 to

1200 g/mol, about 400 to 1100 g/mol, about 500 to 1300 g/mol, about 500 to

1200 g/mol, about 500 to 1100 g/mol, about 600 to 1300 g/mol, about 600 to

1200 g/mol or about 600 to 1100 g/mol. In some embodiments, the method includes heating the aqueous composition for a time sufficient to achieve a weight average molecular weight of the aqueous composition from about 700 to 3000 g/mol.

In some embodiments, sufficient time is the time required for the aqueous composition to reach reaction equilibrium at the appropriate reaction temperature. Accordingly, 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 molecular weight, viscosity, or

- 33 044964 DP distribution of aqueous composition.

In some embodiments, equilibrium is determined by measuring the number average or weight average molecular weight of the aqueous composition. In some embodiments, the equilibrium is determined by the number-average or weight-average molecular weight of the aqueous composition, which remains substantially unchanged over time. In some embodiments, equilibrium is determined by a change in the number-average or weight-average molecular weight of the aqueous composition that is less than a certain percentage over a period of time. In some embodiments, the molecular weight of the aqueous composition is measured using HPLC or SEC.

In some embodiments, equilibrium is determined by a change in the number average or weight average molecular weight of the aqueous composition of less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a period of time. In some embodiments, equilibrium is determined by changing the number average 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 a change in the weight average molecular weight of the aqueous composition by less than 15% over a period of 1 hour.

In some 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, which remains substantially unchanged over time. In some embodiments, equilibrium is determined by a change in viscosity of the aqueous composition that is less than a certain percentage over a period of time. In some embodiments, the viscosity of the aqueous composition is measured with a viscometer or rheometer.

In some embodiments, equilibrium is determined by a change in viscosity of the aqueous composition of less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a period of time. In some embodiments, equilibrium is determined by the change in viscosity 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 a change in the viscosity of the aqueous composition of less than 15% over a period of 1 hour.

In some embodiments, equilibrium is determined by measuring the DP distribution of the aqueous composition. In some embodiments, equilibrium is determined by the DP distribution of the aqueous composition, which remains substantially unchanged over time. In some embodiments, the change in DP distribution of an aqueous composition is determined by calculating the [D^][H ₂ O] series Km, where Km=[ ^Dp mi][0Fi]' where [H ₂ O] is the molar concentration of water (mol/L ), and [DP1], [DPm-1] and [DPm] are the molar concentrations of oligosaccharides (mol/L) in DP1, DPm-1 and DPm fractions, respectively. For example, K2 is equal to [DP2][H ₂ O]/[DP1][DP1] according to the above formula. In some embodiments, m is an integer greater than 1 and less than n. In other embodiments, m is equal to n. In some embodiments, m is an integer greater than 1 and equal to or less than n. In some embodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the concentration of oligosaccharides in the DP1, DPm-1, and DPm fractions is determined using SEC, HPLC, PFP, A4F, mass spectrometry, or any other suitable method. In some embodiments, the concentration of oligosaccharides in the DP1, DPm-1, and DPm fractions is determined using SEC, such as GPC. In some embodiments, the concentration of oligosaccharides 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 oligosaccharides in the DP1, DPm-1, and DPm fractions is determined by HPLC. In some embodiments, the water concentration is determined by evaporation method (eg, loss on drying method), distillation method, or chemical reaction method (eg, Karl Fischer titration). In some embodiments, the water concentration is determined by any suitable analytical instrument, such as a moisture analyzer.

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 to K4, K2 to K5, K2 to K6, K2 to K7, K2 to K8, K2 to K9, K2 to K10, K2 to K11, K2 to K12, K2 to K13, K2 to K14, K2 to K15, K3 to K5, K3 to K6, K3 to K7, K3 to K8, K3 to K9, K3 to K10, K3 to K11, K3 to K12, K3 to K13, K3 to K14, or K3 to K15. In some embodiments, the method includes calculating K2 to K4 or K3 to K5.

In some embodiments, the Km value depends on temperature, water concentration, and/or the amount and type of feed sugars. In some embodiments, Km is at

- 34 044964 measured from 0.1 to 100, from approximately 0.1 to 90, from approximately 0.1 to 80, from approximately 0.1 to 70, from approximately 0.1 to 60, from approximately 0.1 to 50 , about 0.1 to 40, about 0.1 to 30, about 0.1 to 25, about 0.1 to 20, or about 0.1 to 15. In some embodiments, Km is about 1 to 100, about 1 to 90, about 1 to 80, about 1 to 70, about 1 to 60, about 1 to 50, about 1 to 40, about 1 to 30, about 1 to 25 , about 1 to 20, about 1 to 15, about 1 to 10, about 5 to 50, about 5 to 40, about 5 to 30, about 5 to 20, about 5 to 15 or about from 5 to 10. In some embodiments, Km is from about 1 to 15, or from about 5 to 15.

In some embodiments, the mean, standard deviation, and/or relative standard deviation are determined for a series of calculated Km. In this context, 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.

In some embodiments, equilibrium is defined by a relative standard deviation of the series Km 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 a relative standard deviation of the Km series of less than 15%, less than 10%, or less than 5%.

G. Post-reaction steps.

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

In some embodiments, the method includes a dilution step in which water is added to the oligosaccharide preparation to produce a syrup of the oligosaccharide preparation. In some embodiments, the concentration of oligosaccharide drug in the syrup is from about 5% to 80%, about 10% to 70%, about 10% to 60%, about 10% to 50%, about 10% to 40%, from about 10% to 30%, or from about 15% to 25%. In other embodiments, the method does not include a dilution step, but rather the oligosaccharide preparation is allowed to solidify. In some embodiments, the method includes a filtration step. In some embodiments, the method includes recycling the catalyst by filtration.

In some embodiments, the described method includes a decolorization step. In some embodiments, the oligosaccharide preparation may be subjected to a decolorization step using any method known in the art, including, for example, treatment with an absorbent, activated carbon, chromatography (eg, using an ion exchange resin), hydrogenation, and/or filtration (eg, microfiltration ).

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

In some embodiments, the method includes a concentration step. In some embodiments, the concentration step produces an increased concentration of the oligosaccharide drug. For example, in some embodiments, the concentration step includes evaporation (eg, vacuum evaporation), drying (eg, lyophilization and spray drying), or any combination thereof.

In some embodiments, the method includes a separation step in which at least a portion of the oligosaccharide drug is separated. In some embodiments, the isolation step includes crystallization, precipitation, filtration (eg, vacuum filtration), and centrifugation, or any combination thereof.

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, at least a portion of the unreacted feed sugars, or both. In some embodiments, the separation step includes filtration, chromatography, differential solubility, precipitation, extraction, or centrifugation.

H. Reactors.

The methods described in the present invention may include the use of one or more reactors suitable for condensing sugar, taking into account reaction temperature, pH, pressure, etc.

- 35 044964 different factors. In some embodiments, one or more suitable reactors include a fed-batch reactor, a stirred batch reactor, a continuous stirred flow reactor, a continuous plug flow reactor, an attrition reactor, or an electromagnetic field induced stirred reactor. In some embodiments, the one or more suitable reactors include the reactor 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).

In some embodiments, one or more suitable reactors include fluidized bed, upflow blanket, immobilization, or extruder type reactors for hydrolysis and/or fermentation. In some embodiments, the one or more suitable reactors include an open reactor, a closed reactor, or both. In some embodiments, when the method involves a continuous process, one or more suitable reactors may include a continuous mixer, such as a screw mixer.

I. Process.

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

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

In some embodiments, the method of producing an oligosaccharide drug is a continuous process. For example, in some embodiments of a continuous process, the reactor contents flow continuously through the reactor. In some embodiments, the combination of feed sugars with a catalyst and removal of at least a portion of the oligosaccharide drug are performed simultaneously.

In some embodiments, a method for producing an oligosaccharide drug includes a one-pot and a multi-pot process. For example, in some embodiments of a one-pot process, the polymerization is carried out in a single reactor. As another example, in some embodiments of a multi-reactor process, polymerization is carried out in more than one reactor. In some embodiments of a multi-reactor process, the method includes 2, 3, or more reactors. In some embodiments of a multi-reactor process, the method includes a combining step in which polymerization products from two or more reactors are combined.

IV. Nutritional compositions including oligosaccharide preparations.

The present invention provides nutritional compositions containing an oligosaccharide preparation. In some embodiments, the present invention provides nutritional compositions containing a disclosed oligosaccharide drug, wherein the presence and/or concentration of the oligosaccharide drug in the nutritional compositions can be selectively determined and/or detected. Oligosaccharide drugs that exhibit complex functional modulation of the microbial community can be important components of nutritional formulations. Thus, the presence and/or concentration of the oligosaccharide drug in nutritional compositions may be one of the factors that needs to be measured during quality control and production of nutritional compositions. Accordingly, the nutritional compositions of the invention are advantageous for quality control and manufacturing purposes because the presence and/or concentration of the oligosaccharide drug can be selectively determined and/or detected. For example, in some embodiments, the presence and concentration of an oligosaccharide drug can be determined and/or detected by measuring the signal associated with oligosaccharides containing anhydro subunits.

In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the nutritional composition includes a base nutritional composition.

A. Basic nutritional compositions.

In some embodiments, the primary nutritional composition contains a carbohydrate source other than the oligosaccharide drug. For example, in some embodiments, the base nutritional composition includes a natural carbohydrate source such as starch and plant fiber. In some embodiments, the base nutritional composition comprises starch. In some embodiments, the base nutritional composition comprises plant fibers.

In some embodiments, the base nutritional composition contains one or more carbohydrate sources that are derived from seeds, roots, tubers, corn, tapioca, arrowroot, wheat, rice, potatoes, sweet potatoes, sago, beans (e.g., broad beans, lentils, beans mung, peas and chickpeas), corn, cassava or other starchy foods (e.g. acorns, arrowroot, arracachi, bananas, barley, breadfruit, buckwheat, canna, taro, japonica, pueraria, xanthosoma, millet, oats, oxalis) , Polynesian arrowroot, sorghum, rye, taro, chestnut, water chestnut and yam).

In some embodiments, the base nutritional composition contains one or more carbohydrate sources that are derived from legumes (e.g., peas, soybeans, lupine, green beans, and other beans), oats, rye, chia, barley, fruits (e.g., figs, avocado , plums, prunes, berries, bananas, apple, quince and pear peels), vegetables (such as broccoli, carrots, cauliflower, zucchini, celery, nopal and Jerusalem artichoke), root tubers, root vegetables (such as sweet potatoes and onions), psyllium husks, seeds (eg flax seeds), nuts (eg almonds), whole grains, wheat, corn bran, lignans, or any combination thereof. In some embodiments, the composition includes one or more plant fibers derived from wheat bran, sugar beet pulp, cottonseed, soybean husk, or any combination thereof.

In some embodiments, the base nutritional composition contains less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm or less than 1 ppm anhydro subunits or oligosaccharides containing anhydro subunits. In some embodiments, the base nutritional composition contains less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm anhydro subunits or oligosaccharides containing anhydro subunits. In some embodiments, the base nutritional composition is substantially free of anhydro subunits.

In some embodiments, the base nutritional composition lacks detectable levels of anhydro subunits. Depending on the detection or determination methods, the level of anhydro subunits below a certain threshold may not be detectable. For example, in some embodiments, the detectable level of anhydro subunits may be 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 anhydro subunits or oligosaccharides containing anhydro subunits, in the main nutritional composition.

In some embodiments, the base nutritional composition contains a plurality of oligosaccharides. In some embodiments, the base nutritional composition includes a glycosidic linkage type distribution that is different from the oligosaccharide preparation. For example, in some embodiments, the base nutritional composition contains a higher percentage of α-(1,4) glycosidic linkages than the oligosaccharide preparation. In some embodiments, glycosidic linkages, such as α-(1,4) glycosidic linkages in basic nutritional compositions, are cleaved by one or more enzymes. In some embodiments, the glycosidic bonds in the base nutritional composition are more easily cleaved and/or hydrolyzed than the glycosidic bonds 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 main nutritional composition by at least 2%, at least 3%, at least 4%, according to at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least at least 12%, at least 13%, at least 14%, or at least 15% below the level of the corresponding 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) a glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond in the base nutritional composition that is at least 10% lower than the level of the corresponding glycosidic bond in the oligosaccharide preparation.

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

B. Animal feed composition.

Depending on the type and age of the animal, the nutritional composition may contain the oligosaccharide preparation and the main nutritional composition in different ratios. For example, the oligosaccharide preparation can be combined with the basic nutritional composition in various ratios suitable for the type and age of the animal. In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of about 1 to 10,000 ppm, about 1 to 5,000 ppm, about 1 to 3,000 ppm, about 1 to 2,000 ppm, about 1 to 1500 ppm, about 1 to 1000 ppm, about 1 to 500 ppm, about 1 to 250 ppm, about 1 to 100 ppm, about 10 to 5000 ppm ppm, about 10 to 3000 ppm, about 10 to 2000 ppm, about 10 to 1500 ppm, about 10 to 1000 ppm, about 10 to 500 ppm, about 10 up to 250 ppm, about 10 to 100 ppm, about 50 to 5000 ppm, about 50 to 3000 ppm, about 50 to 2000 ppm, about 50 to 1500 ppm , about 50 to 1000 ppm, about 50 to 500 ppm, about 50 to 250 ppm, about 50 to 100 ppm, about 100 to 5000 ppm, about 100 to 3000 ppm, about 100 to 2000 ppm, about 100 to 1500 ppm, about 100 to 1000 ppm, about 100 to 500 ppm, about 100 to 400 ppm, about 100 to 300 ppm, about 100 to 200 ppm, about 200 to 5000 ppm, about 200 to 3000 ppm, about 200 to 2500 ppm, about 200 to 2000 ppm, about 200 to 1500 ppm, about 200 to 1000 ppm, about 200 to 500 ppm, about 500 to 5000 ppm, about 500 to 3000 ppm, about 500 to 2500 ppm, about 500 to 2000 ppm, about 500 to 1500 ppm, or about 500 to 1000 ppm. In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of about 1 to 5000 ppm, about 1 to 1000 ppm, about 1 to 500 ppm, about 10 to 5000 ppm, about 10 to 2000 ppm, about 10 to 1000 ppm, about 10 to 500 ppm, about 10 to 250 ppm, about 10 to 100 ppm, about 50 to 5000 ppm ppm, about 50 to 2000 ppm, about 50 to 1000 ppm, about 50 to 500 ppm, about 50 to 250 ppm, or about 50 to 100 ppm. In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of about 1 to 5000 ppm, about 10 to 1000 ppm, about 10 to 500 ppm, or about 50 to 500 ppm.

In some embodiments, the oligosaccharide drug is present in the nutritional composition at a concentration of 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 drug is present in the nutritional composition at a concentration of greater than 10 ppm, greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, or greater than 500 ppm.

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

The present invention also provides a method of administering to an animal a nutritional composition comprising a basic nutritional composition and a disclosed oligosaccharide preparation. In some embodiments, the animal is selected from cattle (eg, beef and dairy cattle), swine, aquatic animals, poultry, and humans. In some embodiments, the animal is a pig, such as sows, piglets, and hogs. In other embodiments, the animal is a poultry such as chicken, duck, turkey, goose, quail, and chicken. In some embodiments, the poultry is a broiler, breeder, or layer. In some embodiments, the animal is an aquatic animal such as salmon, catfish, grouper, eel, tilapia, flounder, shrimp, and crab. In some embodiments, the nutritional composition is provided to the animal in dry form, liquid form, paste form, or a combination thereof. In some embodiments, the form of application, feeding rate, and feeding schedule may vary depending on the type and

- 38 044964 age of the animal.

C. Methods for producing nutritional compositions.

The present invention provides methods for producing a nutritional composition comprising combining an oligosaccharide preparation with a base nutritional composition. In some embodiments, the oligosaccharide preparation includes oligosaccharides containing anhydrosubunits. In some embodiments, the oligosaccharide preparation includes a glycosidic linkage type distribution that is different from that of the base nutritional composition.

In some embodiments, the oligosaccharide drug is a synthetic oligosaccharide drug. In some embodiments, the synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each having a different degree of polymerization selected from 1 to n (fractions DP1 to DPn). In some embodiments, n is an integer equal to 2 or more. In some embodiments, n is an integer greater than 2. In some embodiments, n is an integer equal to 3 or greater. In some embodiments, n is an integer ranging from 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 DP1DPn fractions includes from 0.1% to 90% oligosaccharides containing anhydro subunits, by relative abundance measured by mass spectrometry. In some embodiments, each of the oligosaccharide preparation fractions DP1 and DP2 independently comprises from about 0.1 to 15%, or from about 0.5% to 10%, anhydro subunit-containing oligosaccharides, as measured by relative abundance by mass spectrometry. In some embodiments, each of the oligosaccharide preparation fractions DP1 and DP2 independently comprises oligosaccharides containing anhydro subunits 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 8%, 9%, 10%, 11%, 12%, 15% or 20% by relative abundance measured by mass spectrometry. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with the degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, 10, 20, or 30 DP fractions decreases monotonically with degree of polymerization.

In some embodiments, a method for producing a nutritional composition includes mixing an oligosaccharide preparation with a base nutritional composition. For example, in some embodiments, mixing may be accomplished using a commercial mixer and/or a mixer such as a drum mixer, a double cone mixer, a ribbon mixer, a V mixer, a shear mixer, and a paddle mixer.

In some embodiments, the method of producing a nutritional composition further includes a quality control step described in the present invention. In some embodiments, the quality control step described herein includes determining a signal level in a sample of the nutritional composition and calculating the concentration of an oligosaccharide drug in the nutritional composition based on the signal level. In some embodiments, the quality control step described herein includes detecting a signal in a sample of the nutritional composition using analytical instruments, and accepting or rejecting a batch of the nutritional composition based on the presence or absence of the signal. In some embodiments, the quality control step described herein includes detecting by analytical instruments 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, and comparing the first signal and the second signal. In some embodiments, the signal, the first signal and/or the second signal (i) indicates oligosaccharides containing one or more anhydrosubunits; (ii) is associated with the distribution of oligosaccharides according to the degree of polymerization (DP); or (iii) associated with α-(1,2) glycosidic bonds, α-(1,3) glycosidic bonds, α-(1,6) glycosidic bonds, β-(1,2) glycosidic bonds, β-(1 ,3) glycosidic bonds, β-(1,4) glycosidic bonds or β-(1,6) glycosidic bonds of oligosaccharides.

Additionally, in some embodiments, a method for producing a nutritional composition includes, after performing a quality control step, further mixing the oligosaccharide drug with the base nutritional composition, adjusting the level of the oligosaccharide drug, or a combination thereof. In some embodiments, adjusting the level of the oligosaccharide drug includes adding an additional oligosaccharide drug to the nutritional composition or removing a portion of the oligosaccharide drug from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide drug includes adding an additional primary nutritional composition to the nutritional composition or removing a portion of the primary nutritional composition from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide drug includes adding an additional oligosaccharide drug to the nutritional composition.

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D. Animal feed premix.

In some embodiments, the nutritional composition includes an animal feed premix containing a disclosed oligosaccharide preparation.

In some embodiments, the animal food premix comprises a carrier material that can be combined with an oligosaccharide drug to form the animal food premix. In some embodiments, the carrier material can be any material in dry or liquid form that is suitable for combination with an oligosaccharide drug in a nutritional composition. In some embodiments, the carrier material includes grain fermentation solids, clay, vermiculite, diatomaceous earth, hulls such as milled rice hulls and milled oat hulls, silica such as feed grade silica gel and feed grade colloidal silica, corn, such as corn gluten, 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 milled rice hulls or milled oat hulls.

In some embodiments, the animal feed premix is prepared by combining a carrier material with an oligosaccharide drug, both in dry form. In some embodiments, the animal feed premix is prepared by combining a carrier material with an oligosaccharide preparation; one of which is in dry form. In some embodiments, the animal feed premix is prepared by combining a carrier material with an oligosaccharide drug, both of which are in liquid form. For example, in some embodiments, an oligosaccharide preparation in liquid form refers to an oligosaccharide in solution, such as an aqueous solution of oligosaccharides, such as a syrup.

In some embodiments, the animal feed premix is prepared by combining a carrier material with a syrup containing an oligosaccharide drug. In some embodiments, the concentration of oligosaccharide drug in the syrup is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70 %, at least 75% or at least 80% by weight. In some embodiments, the concentration of oligosaccharide drug in the syrup is from about 40 to 80%, from 50 to 75%, or from 60 to 70% by weight.

In some embodiments, the pet food premix is in the form of a powder (eg, a pourable powder), slurry, slurry, granules, or liquid. In some embodiments, the animal food premix has a moisture content of less than 40%, 30%, 20%, 15%, 10%, or 5% by weight. In some embodiments, the pet food premix has a moisture content of less than 10% or 5% by weight. In some embodiments, the pet food premix has a moisture content of greater than 5%, 10%, 15%, 20%, 25%, or 30% by weight. In additional embodiments, the moisture content of the animal feed premix is adjusted to any of the described ranges. For example, in some embodiments, the pet food premix is dried to increase the moisture content within the described range.

In some embodiments, depending on the specific application, the animal feed premix contains varying levels of the oligosaccharide drug. In some embodiments, the animal food premix contains at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95% or 99% oligosaccharide preparation by dry matter weight. In some embodiments, the animal food premix contains at most 50%, 60%, 70%, 80%, 90%, 95%, or 99% oligosaccharide drug by dry matter weight.

In some embodiments, the animal feed premix or carrier material further comprises other animal feed such as minerals, fats and proteins. In some embodiments, the animal feed carrier material or premix contains copper, zinc, or both. In some embodiments, the carrier material or animal feed premix contains an ionophore or other coccidiostat. In some embodiments, the animal feed carrier material or premix contains an antibiotic. In some embodiments, the carrier material includes a source of carbohydrates. In some embodiments, the carbohydrate source in the carrier material does not contain an anhydro subunit. In some embodiments, the carbohydrate source in the carrier material includes a glycosidic linkage type distribution that is different from the glycosidic linkage type distribution of the oligosaccharide drug.

Accordingly, in some embodiments, a method of producing a nutritional composition includes combining an animal feed premix with a base nutritional composition.

V. Methods for providing animals with oligosaccharide preparations.

In some embodiments, the methods described in the present invention include providing animals with oligosaccharide preparations. In some embodiments, the animals are treated by feeding or providing an oligosaccharide drug. In some embodiments, the oligosaccharide drug is administered to animals at a specific targeted dose. A specific dose may be quantified, for example, as the mass of the oligosaccharide drug consumed by the animal per unit of time (e.g., grams per day), or as the mass of the oligosaccharide drug consumed by the animal per unit of time per unit of animal body weight (e.g., mg oligosaccharide per kg body weight per day). In some embodiments, the specific dose of the oligosaccharide drug is 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000 or 10,000 mg/kg/day. In some embodiments, the weight of the oligosaccharide preparation is measured as the DP1+ content on a dry solids basis. In some embodiments, the weight of the oligosaccharide preparation is measured as the DP2+ content on a dry solids basis.

In some embodiments, the animals are provided with oligosaccharide preparations by oral administration in the form of nutritional compositions. In some embodiments, the nutritional composition includes an oligosaccharide preparation at a fixed concentration or inclusion level. The concentration or level of inclusion of the oligosaccharide in the nutritional composition can be quantified, for example, by the mass fraction of the oligosaccharide preparation per total weight of the final feed or nutritional composition. In some embodiments, the concentration or inclusion level is measured in parts per million (ppm) of the oligosaccharide on a solids basis for the final nutritional composition in its original state. In some embodiments, the concentration of the oligosaccharide drug is measured as the mass fraction of DP1+ particles on a dry solids basis. In some embodiments, the concentration of the oligosaccharide drug is measured as the mass fraction of DP2+ particles on a dry solids basis.

One of ordinary skill in the art is aware of various methods and techniques for determining the concentration of an oligosaccharide drug in a nutritional composition or final feed to achieve a given specific dose. For example, average daily feed intake by age has been established for various types of broiler chickens and can be used by a nutritionist or veterinarian to determine the appropriate level of inclusion in the final feed.

In some embodiments, oligosaccharide preparations are administered to animals by oral administration through ingested fluids. In some embodiments, the oligosaccharide preparations are provided in drinking water. In some embodiments, the concentration of the oligosaccharide drug in the drinking water is selected to provide the animal with a given, specific dose of the oligosaccharide drug.

VI. Dysfunction of the gastrointestinal barrier.

In some embodiments, the methods described in the present invention include preventing or treating dysfunction of the gastrointestinal barrier of an animal. Animal gastrointestinal barriers consist of a layer of mucus and a layer of intestinal epithelial cells (including, for example, goblet cells). The intestinal epithelium acts as a barrier to prevent the entry of harmful intraluminal particles, including foreign antigens, microorganisms and their toxins, and other intestinal contents (eg, food particles). The intestinal epithelium also acts as a selective filter, allowing the movement of essential dietary nutrients, electrolytes, and water from the intestinal lumen into the bloodstream.

The gastrointestinal epithelium provides selective permeability through two main pathways: transepithelial/transcellular and paracellular. Transcellular permeability is typically associated with the transport of solutes across epithelial cells and is predominantly regulated by selective transporters of amino acids, electrolytes, short-chain fatty acids, and sugars. Paracellular permeability is associated with transport in the space between epithelial cells and is regulated by intercellular complexes localized at the junction of the apical-lateral membrane and along the lateral membrane. Contact between intestinal epithelial cells includes three main components: desmosomes, adhesions, and tight junctions.

The permeability of gastrointestinal barriers is regulated by a variety of factors, including, for example, exogenous factors, epithelial apoptosis, cytokines, immune cells, and gastrointestinal microbes. Microbiome bacteria and intestinal pathogens can disrupt the intestinal barrier directly by binding to cell surface molecules and causing changes in the expression of tight junction proteins. Microbiome bacteria and gut pathogens can also generate toxins and proteases that can promote cell damage and apoptosis, alter epithelial ion transport, and disrupt tight junctions and the cytoskeleton.

Dysfunction of the gastrointestinal barrier allows the movement of intestinal contents into the bloodstream, including, for example, pathogens and toxins, which can adversely affect the health of the animal and cause disease. Inflammation and/or damage to epithelial cells can also reduce the animal's ability to absorb nutrients. Consequently, animals with a compromised gastrointestinal barrier exhibit malnutrition and poor health, resulting in decreased weight gain, loss of body weight, decreased nutritional efficiency, decreased meat production, increased feed conversion ratio, decreased meat quality, and higher mortality.

A. Ways to prevent gastrointestinal barrier dysfunction.

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In some embodiments, the methods described in the present invention include methods for preventing gastrointestinal barrier dysfunction in an animal. In some embodiments, gastrointestinal barrier dysfunction is an increase in the permeability of the gastrointestinal barrier compared to a healthy state, which allows the movement of intestinal contents, including, for example, partially digested food, pathogens and toxins, into the bloodstream. In some embodiments, gastrointestinal barrier dysfunction is increased gastrointestinal barrier permeability. Hyperpermeability is also known in the art as leaky gut. As described above, leaky gut can allow toxins, bacteria, and food particles to pass through the lining of the gastrointestinal tract and enter the body's bloodstream. Leaky gut may indicate an acute or chronic condition. Leaky gut can lead to infection, sepsis, or damage to the intestinal lining.

Symptoms of increased intestinal permeability may include decreased mucus synthesis, decreased mucin synthesis, decreased mucus secretion, decreased mucin secretion, decreased nutrient absorption, increased inflammation, decreased resistance to infection, decreased intestinal epithelial proliferation, decreased intestinal epithelial cell maturation, malnutrition, decreased weight gain, increased feed conversion ratio, decreased feed efficiency, increased mortality, but not limited to. In some embodiments, the methods described herein prevent one or more symptoms of gastrointestinal barrier dysfunction, such as a leaky gut symptom.

In some embodiments, dysfunction is prevented by reducing the permeability of the animal's gastrointestinal barrier. In some embodiments, gastrointestinal barrier permeability is reduced by about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% compared to the permeability of the gastrointestinal barrier of the specified animal before the use of the specified synthetic oligosaccharide drug. 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- 4%, 0.5-3%, 0.5-3%, 0.5-2%, 0.5-1%, 1-40%, 1-30%, 1-20%, 1-10% , 1-5%, 14%, 1-3%, 1-2%, 5-40%, 5-30%, 5-20%, 5-10%, 10-40%, 10-30% or on 10-20% less than the permeability of the gastrointestinal barrier of the specified animal before the use of the specified synthetic oligosaccharide drug.

In some embodiments, dysfunction is prevented by reducing the permeability of the gastrointestinal barrier in the animal. In some embodiments, the permeability of the gastrointestinal barrier is about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50 % less than the permeability of the gastrointestinal barrier of an animal in which a nutritional composition that does not contain a synthetic oligosaccharide drug was used. In some embodiments, the gastrointestinal barrier permeability is about 0.540%, 0.5-30%, 0.5-20%, 0.5-10%, 0.5-5%, 0.5-4%, 0 ,5-3%, 0.5-2%, 0.5-1%, 1-40%, 1-30%, 1-20%, 1-10%, 15%, 1-4%, 1- 3%, 1-2%, 5-40%, 5-30%, 5-20%, 5-10%, 10-40%, 10-30% or 10-20% less than gastrointestinal barrier permeability animal in which a nutritional composition without a synthetic oligosaccharide preparation was used.

B. Methods for treating gastrointestinal barrier dysfunction.

In some embodiments, the methods described in the present invention include methods for treating gastrointestinal barrier dysfunction in an animal. In some embodiments, gastrointestinal barrier dysfunction is an increase in the permeability of the gastrointestinal barrier, compared with a healthy state, allowing intestinal contents, including, for example, partially digested food, pathogens and toxins, to move into the bloodstream.

In some embodiments, gastrointestinal barrier dysfunction is increased gastrointestinal barrier permeability. Hyperpermeability is also known in the art as leaky gut. As described above, leaky gut allows toxins, bacteria, and food particles to pass through the lining of the gastrointestinal tract and enter the body's bloodstream. Leaky gut may indicate an acute or chronic condition. Leaky gut can lead to infection, sepsis, or damage to the intestinal lining.

Symptoms of increased intestinal permeability may include, but are not limited to: decreased mucus synthesis, decreased mucin synthesis, decreased mucus secretion, decreased mucin secretion, decreased nutrient absorption, increased inflammation, decreased resistance to infection, decreased intestinal epithelial cell proliferation, decreased intestinal epithelial cell maturation , malnutrition, decreased weight gain, increased feed conversion ratio, decreased nutrition efficiency, increased mortality. In some embodiments, one or more symptoms of leaky gut are improved.

In some embodiments, dysfunction is prevented by reducing the permeability of the animal's gastrointestinal barrier. In some embodiments, the implementation

- 42 044964 the gastrointestinal barrier is reduced by approximately 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40% or 50 %, compared with the permeability of the gastrointestinal barrier of the specified animal before the use of the specified synthetic oligosaccharide drug. 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- 4%, 0.5-3%, 0.5-2%, 0.5-1%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 1 -4%, 1-3%, 1-2%, 5-40%, 5-30%, 5-20%, 5-10%, 10-40%, 10-30% or 10-20% less, than the permeability of the gastrointestinal barrier of the specified animal before the use of the specified synthetic oligosaccharide drug.

In some embodiments, the dysfunction is treated by reducing the permeability of the gastrointestinal barrier in the animal. In some embodiments, the gastrointestinal barrier permeability is about 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% less than the gastrointestinal permeability - the intestinal barrier of the animal before the use of a nutritional composition that does not contain a synthetic oligosaccharide drug. In some embodiments, the permeability of the gastrointestinal barrier is about 0.5-40%, 0.5-30%, 0.5-20%, 0.5-10%, 0.5-5%, 0.5- 1%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 1-2%, 5-40%, 5-30%, 5-20%, 5- 10%, 10-40%, 10-30%, or 10-20% less than the permeability of the animal's gastrointestinal barrier before administration of a nutritional composition that does not contain a synthetic oligosaccharide drug.

C. Methods for determining gastrointestinal barrier permeability and nutrient absorption.

The permeability of the gastrointestinal barrier can be demonstrated by methods known in the art, such as the lactulose/mannitol test, where the animal is given a solution containing mannitol and lactulose and the urine is collected for six hours and examined. Lactulose (a disaccharide) and mannitol (a monosaccharide) are two water-soluble, non-metabolizable sugar molecules. Mannitol is easily absorbed, passively entering cells, while lactulose, with its larger molecular weight and size, is only partially absorbed by the healthy gastrointestinal barrier. If the levels of mannitol and lactulose in the collected urine sample are high, this indicates that the gastrointestinal tract is too permeable. Low levels of both molecules indicate malabsorption of nutrients. High levels of mannitol and low levels of lactulose indicate that the animal has normal gastrointestinal barrier function.

The permeability of the gastrointestinal barrier can also be assessed using post-digestive stool analysis, which includes testing fecal material for digestive function, the extent to which lipids, proteins, carbohydrates and other nutrients are absorbed in the small intestine and colon, and the presence of Candida or other bacterial infections, dysbiosis (imbalance of intestinal bacteria), parasitic infections and other indicators of digestive dysfunction. The improvement in intestinal function caused by the use of the synthetic oligosaccharide preparations described in the present invention can also be measured by a decrease in diarrhea, a decrease in one or more symptoms of leaky gut, an increase in hard stools, an increase in the production of 5-hydroxytryptophan and 5-hydroxytryptamine, and 2-methyl -5hydroxytryptamine and other neurotransmitter precursors that can be produced by bacterial populations (serotonin precursors). Assessment of gastrointestinal barrier permeability can also be performed using fluorescent tracers (e.g., as described in Droshow et al., Measurement of gut permeability using fluorescent tracer agent technology, Scientific Reports, 7:108888 (2017)). Other methods include, for example, those disclosed in Wang et al., Methods to determine intestinal permeability and bacterial translocation during liver disease, J Immunol Methods, 421:44-53 (2015); Galipeau et al., Neurogastroenterology and Motility, 28:7 (2016); and are described in the present invention in examples. The permeability of the gastrointestinal barrier can also be assessed using a variety of non-digestible sugars, labeled dextrans and radioisotopes known in the art.

The permeability of the gastrointestinal barrier can also be assessed by histological analysis of a tissue sample or a sample of cells of the specified gastrointestinal barrier. In some embodiments, such an assay uses one or more dyes or labels that are known in the art. These include, for example, hematoxylin and eosin staining, immunofluorescent staining, the use of immunofluorescent antibodies or other targeting agents. Microscopy methods include all methods known in the art, for example, light microscopy, confocal microscopy, electron microscopy, etc. In some embodiments, the methods described in the present invention include assessing the mucosal morphology of said gastrointestinal barrier. In some embodiments, said assessment includes determining villous length, crypt length, level of inflammatory cell infiltration, or any combination thereof.

Methods similar to those described above can be used to assess the ability of the gastrointestinal tract to absorb nutrients. For example, the level of certain macro- or micronutrients can be assessed in a stool sample, such as fat. Sweat samples can be analyzed

- 43 044964 to determine the level of enzymes necessary for the correct distribution of nutrients. To determine the amount of hydrogen in the air exhaled by an animal after ingesting a milk sugar solution (for example, lactose), a lactose hydrogen breath test can be performed. A biopsy of the gastrointestinal barrier may be taken to assess nutrient absorption. Numerous tests for measuring the ability to absorb nutrients from the gastrointestinal tract are known in the art and are included herein.

In some embodiments, the level of at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10) nutrient transporters expressed by the gastrointestinal barrier is assayed to determine the ability of the gastrointestinal barrier to absorption of nutrients in general or of a specific macro- or micronutrient. Methods for such analysis are known in the art and are incorporated herein by reference. Transporters include the expressed proteins SI (sucrose isomaltase),

SLC5A10,

SLC34A1, SLC2A2, SLC34A2, SLC23A1, SLC23A2, SLC5A8, SLC16A3, SLC4, SLC4A9, SLC4A2, SLC4A3, NPC1L1, C6orf58, DDC, MCT1, MCT4, NaSl, DTDST, PAT1, DRA, CLD, SAT1, SUT2, SGLT 1, GLUT2, B0AT1, ATB0+, SITI, TAUT, EAAC1, ASCT2, SN1, SN2, PEPT1, SNAT2, GLYT1, y+LATl, y+LAT2, CD36, LFABP, NPC1L1, ABCG5, ABCG8, SVCT1, SVCT2, SMVT, GIF, AMN, CUBL, MRP1, FOLT, PCFT, FOLRI, OAT10, RFVT1, RFVT2, THTR1, THTR2, VDR, DCYTB, DMT1, HCP1, FPN1, HEPH, HAMP, ZIP4, ZIP11, ZIP8, ZIP14A, ZIP14B, ZnTl, ZnT2, CTR1, but not limited to SLC3A1, SLC1A4, ALPI, C17orf78, MUC17, DEFA5, RBP2, DEFA6, MLN, MEP1B, LCT, TM4SF20 or FABP6.

The permeability of the gastrointestinal barrier can be assessed using a variety of samples, for example, as described in the present invention. For example, the samples include a gastrointestinal barrier tissue sample, a gastrointestinal barrier biopsy, a gastrointestinal barrier cell sample, a blood sample, a serum sample, a urine sample, a stool sample, a cecal sample (e.g., its contents), a sweat sample, or saliva sample, but not limited to.

Gastrointestinal barriers include any barrier of the gastrointestinal tract, for example, ileum, cecum, small intestine, colon, stomach, duodenum, jejunum.

D. Goblet cell proliferation and mucus production.

In some embodiments, the methods described in the present invention include increasing the proliferation of goblet cells in the gastrointestinal tract of the animal. Goblet cells are simple columnar epithelial cells that form part of the epithelial lining of the gastrointestinal barrier. Goblet cells secrete mucus that contains large glycoproteins called mucins. The mucous layer of the gastrointestinal barrier protects the epithelial cells and prevents damage to the epithelial cells by microorganisms, toxins, etc.

Standard assays known to one skilled in the art can be used to assess goblet cell proliferation. For example, in vitro assays can be used, as described in the examples in the present invention. Various molecular or biochemical markers can also be used to assess goblet cell proliferation, including but not limited to gene expression or expression of goblet cell-specific proteins, such as mucins.

Certain mucin genes, such as MUC5B, are expressed in the gastrointestinal tract and contain a gene product that is widely represented in mucus. Therefore, mucin gene expression is a suitable marker for determining mucus production.

Expression of mucin genes can be assessed using conventional technologies such as Northern blot, polymerase chain reaction (PCR), mucin gene promoter assay, or in situ analysis. Alternatively, mucin proteins are assessed using conventional methodology such as Western blotting, ELISA, immunochemistry, etc., using detectable labeled antibodies. Morphological criteria can also be used to determine the presence or absence of goblet cells in culture; such as detection of mucins using Alcian blue/PAS staining (Lou et al. (1998) Am. J. Respir. Crit. Care Med. 157: 1927-1934). Antibodies to mucins can be tested using ELISA tests.

In vivo assays can also be used to assess goblet cell proliferation in animals. The animal's gastrointestinal tract tissue can be assessed for the prevalence of goblet cells. Tissue samples from the gastrointestinal tract of animals can also be assessed.

- 44 044964 test for the same molecular and biochemical markers described above for in vitro analyses.

E. Susceptibility to infection.

In some embodiments, the methods described in the present invention include reducing the animal's susceptibility to infection. The methods also include preventing infection in an animal or animal population. As described above, dysfunction of the gastrointestinal barrier, resulting in increased permeability of the gastrointestinal barrier, allows the movement of pathogenic microorganisms from the intraluminal space into the bloodstream. The methods described here prevent infection of the animal by reducing the permeability of the gastrointestinal barrier. The methods described here also reduce the animal's susceptibility to infection by reducing the permeability of the gastrointestinal barrier. Infection can result from any pathogenic microorganism present in the lumen of the gastrointestinal tract. The pathogen can enter the gastrointestinal tract through contaminated food. In some embodiments, the pathogen is a microorganism. In some embodiments, the pathogen is a bacterium, virus, parasite, or fungus. In some embodiments, the pathogen is a member of the phylum Proteobacteria. In some embodiments, the pathogen is a bacterium and is classified as a member of the genera Helicobacter, Escherichia, Salmonella, Vibrio, or Yersinia.

In some embodiments, the pathogen is a bacterium and is classified as a member of the genera

Treponema, Streptococcus, Staphylococcus,

Shigella, Rickettsia, Orientia, Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira, Legionella, Klebsiella, Haemophilus, Francisella, Ehrlichia, Enterococcus, Coxiella, Corynebacterium, Clostridium, Chlamydia, Chlamydophila, Campylobacter, Burkholderia, Brucella, Borrelia, Bordetella, Bifidobacterium and Bacillus.

Bacterial species include

Helicobacter pylori, Proteobacteria johnsonii, Escherichia coli, Campylobacter jejuni and

Lactobacillus crispatus, but not limited to them. In some embodiments, the pathogen is

Aeromonas hy dr ophila, Campylobacter fetus, Plesiomonas shigelloides, Bacillus cereus, Campylobacter jejuni, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, enteroaggregative Escherichia coli, enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli, enterotoxigenic Escherichia coli, Helicobacter pylori, Klebsiellia pneumonia, Lysteria monocytogenes , Plesiomonas shigelloides, Salmonella spp., Salmonella typhi, Salmonella paratyphi, Shigella spp., Staphylococcus spp., Staphylococcus aureus, vancomycin-resistant enterococcus spp., Vibrio spp., Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Yersinia enterocolitica.

In some embodiments, the pathogen is resistant to one or more drugs. Infection of an animal with one or more pathogens can be assessed by standard methods known to one skilled in the art.

In some embodiments, the pathogen is a parasite. In some embodiments, said parasite is an Eimeria parasite. In some embodiments, said parasite is

Eimeria acervuline,

Eimeria maxima, Eimeria mitis, Eimeria tenella, Toxoplasma gondii, Hammondia spp. Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Isospora canis, Isospora ohioensis, Isospora burrosi, Isospora fells.

In some embodiments, said pathogen is a coccidia protozoan. In some embodiments, said protozoan coccidia are

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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.

In some embodiments, the level of at least one pro-inflammatory cytokine or chemokine in the gastrointestinal tract of the animal is increased. Examples of cytokines and chemokines include tumor necrosis factors (TNF) such as TNF-α and TNF-β, TNF superfamily molecules such as FasL, CD27L, CD30L, CD40L, Ox40L, 4-1BBL, TRAIL, TWEAK and Apo3L, interleukin- 1 (IL-1), including IL-1a and IL-1e, IL-2, interferon-γ (IFN-γ), IFN-α/β, IL-6, IL-8, IL-12, IL-15 , IL-17, IL-18, IL-21, LIF, CCL5, GROa, MCP-1, MIP-1a, MIP-1e, RANTES, macrophage colony-stimulating factor (MCSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) , CXCL2, CCL2, lymphotactin, fractalkine or GRO/KC, but are not limited to.

In some embodiments, the level of at least one anti-inflammatory cytokine or chemokine in the animal's gastrointestinal tract is reduced. Examples of anti-inflammatory cytokines and chemokines include, but are not limited to, interleukin (IL)-1 receptor antagonist, IL-4, IL-6, IL-10, IL-11, IL-13 and TGFe.

In some embodiments, the level of at least one immune cell is increased in the gastrointestinal tract of the animal. In other embodiments, the level of at least one immune cell is increased in the lymphatic system of the animal. In some embodiments, the immune cell is an innate immune cell. In some embodiments, the immune cell is an adaptive immune cell. Examples of immune cells include, but are not limited to, T cells, B cells, natural killer cells, macrophages, dendritic cells, granulocytes (including, for example, basophils, eosinophils and neutrophils), mast cells and monocytes. In some embodiments, the immune cell is a phagocytic cell. Phagocytic immune cells include, but are not limited to, neutrophils, monocytes, macrophages, mast cells, and dendritic cells. In some embodiments, the phagocytic activity of the immune cell is increased. In some embodiments, immune cell differentiation occurs in Peyer's patches.

F. Gastrointestinal specimens.

In some embodiments, the methods described in the present invention include detecting a property (including, for example, mucus level, immune cell level, cytokine level, chemokine level, etc.) in the gastrointestinal tract of the animal. In some embodiments, this property is detected or quantified in a sample of the animal's gastrointestinal tract. Gastrointestinal tract samples can be obtained from the animal in any standard form that reflects the metabolic content of the animal's gastrointestinal tract. Gastrointestinal samples include samples of gastrointestinal tissue obtained, for example, by endoscopic biopsy. Tissues of the gastrointestinal tract include, for example, tissue of the oral cavity, esophagus, stomach, intestine, ileum, cecum, colon or rectum. Samples of stool, saliva, urine and gastrointestinal ascitic fluid are also taken. Methods for obtaining gastrointestinal tract samples are standard and known to one skilled in the art. In some embodiments, said sample is a blood or serum sample.

In some embodiments, the sample is a single sample from a single animal. In some embodiments, the sample is a combination of multiple samples from a single animal. In some embodiments, cells or proteins are purified from the sample prior to analysis. In some embodiments, cells or proteins are purified from a single sample. In some embodiments, cells or proteins from multiple samples from a single animal are purified and then pooled before analysis.

VI I. Methods for increasing animal productivity.

A. Feed conversion ratio.

In some embodiments, the methods described in the present invention include reducing the animal's feed conversion ratio. In some embodiments, an animal fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein has a lower feed conversion ratio compared to an animal fed a diet that does not include the synthetic oligosaccharide. a drug. As used herein, the term feed conversion ratio (FCR) refers to the ratio of the input mass of feed (eg, consumed by an animal) to the animal product, the animal product being the target animal product. For example, the animal product for dairy animals is milk, and the animal product for animals raised for meat is body weight.

In some embodiments, the animal is raised for meat and the target animal product is body weight. Thus, in some embodiments, FCR refers to the ratio of the weight of feed consumed to the final body weight of the animal before processing. In some embodiments, FCR refers to the ratio of the weight of feed consumed to the final weight gain of the animal before processing. It should be understood that FCR can be measured for an animal or population of animals over different time periods. For example, in some embodiments, the FCR is an FCR throughout the life of the animal. In other embodiments, the FCR is a daily FCR, or a weekly FCR, or a cumulative FCR measured up to a specific point in time (eg, a specific day).

One skilled in the art will appreciate that the target minimum FCR (optimal FCR) may be different for different types of animals and can be different for different breeds of the same type of animal (eg, different breeds of broiler chickens, or different breeds of pigs). The minimum FCR of target production may also vary depending on the age of the animal (eg, chicks or pigs in the first finishing period versus the second finishing period) or the sex of the animal. It should be clear that the optimal FCR may differ depending on any combination of these factors.

The minimum target usually refers to the lowest nutritional efficiency observed for a given animal and breed under ideal rearing conditions, ideal animal health and ideal dietary nutrition. Those skilled in the art are well aware that under normal rearing conditions an animal may not achieve the minimum target FCR. An animal may not achieve its minimum target FCR due to various factors related to health, nutrition, environment and/or community. An animal may not achieve its minimum target FCR when raised in a challenging environment, which may include, for example, pathogenic environmental stress, excessive environmental temperature (heat stress), excessive environmental humidity, crowding, or other social interaction effects such as difficulty with access to food or drinking water. In some embodiments, the animal may not achieve its minimum target FCR due to illness or pathogenic environmental stress. In other embodiments, the animal may not achieve its minimum target FCR due to excessive environmental temperature (heat stress) or excessive environmental humidity. In other embodiments, the animal may not achieve its minimum target FCR due to crowding or other social interaction effects, such as difficulty accessing food or drinking water.

In some embodiments, an animal fed a diet not including a synthetic oligosaccharide drug described in the present invention has an FCR that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% %, 9% or 10% higher than the minimum planned FCR. In some embodiments, an animal fed a diet that does not include a synthetic oligosaccharide drug described in the present invention has an FCR that is 1-10% above the minimum target, 2-10% above the minimum target, or 5-10% above the minimum planned indicator.

In some embodiments, an animal receiving a nutritional composition comprising a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein has an FCR that is closer to the minimum target compared to the animal who received a diet that did not include a synthetic oligosaccharide drug. In specific embodiments, an animal fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein has an FCR that is 0-10% higher than the minimum target, 0-5% higher the minimum planned indicator or 0-2% above the minimum planned indicator.

In some embodiments, an animal fed a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition as described herein has a lower feed conversion ratio compared to an animal fed a diet that does not include the synthetic oligosaccharide drug . For example, in some embodiments, an animal fed a diet including a synthetic oligosaccharide drug consumes less feed but produces the same animal product as an animal fed a diet not including the synthetic oligosaccharide drug. In other embodiments, an animal fed a diet including a synthetic oligosaccharide drug consumes the same amount of feed but produces a higher animal product compared to an animal fed a diet not including the synthetic oligosaccharide drug. In other embodiments, an animal fed a diet including a synthetic oligosaccharide drug consumes less food and produces a higher animal product compared to an animal fed a diet not including the synthetic oligosaccharide drug.

- 47 044964

In some embodiments, the FCR of an animal fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is reduced by at least 1%, by at least 2%, by at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, 1-10%, 4-10%, 1-8%, at 4-8%, 1-6% or 4-6% compared to an animal fed a diet that did not include a synthetic oligosaccharide drug. In some embodiments, the animal is a poultry. In some embodiments, the poultry FCR is reduced at 0 to 14 days of age, 15 to 28 days, 29 to 35 days, greater than 35 days, greater than 42 days, greater than 6 weeks, greater than 6.5 weeks, 0 to 35 days, 0 to 42 days, 0 to 6 weeks, 0 to 6.5 weeks, 15 to 35 days, 36 to 42 days, 15 to 39 days, or 40 to 46 days.

In one embodiment, the 35 day FCR for poultry fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition as described herein is reduced by 4-6% compared to poultry fed the diet , which does not include a synthetic oligosaccharide drug. For example, in a certain embodiment, the FCR per 35 days for poultry fed a nutritional composition containing a synthetic oligosaccharide drug, nutritional composition, animal feed premix or animal feed composition as described in the present invention is 1.53, FCR per 35 days for poultry fed a diet without a synthetic oligosaccharide drug is 1.61, and the FCR of birds fed a nutritional composition containing an oligosaccharide drug, nutritional composition, animal feed premix or animal feed composition is reduced by approximately 5% compared to birds fed a diet without a synthetic oligosaccharide drug. In some embodiments, the FCR of 42 days, greater than 6 weeks, or greater than 6.5 weeks for poultry fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is reduced by 4- 6% compared to poultry fed a diet that did not include a synthetic oligosaccharide drug.

In some embodiments, a population of animals fed a synthetic oligosaccharide drug, nutritional product, animal feed premix, or animal feed composition as described herein has a lower FCR compared to a population of animals fed a diet that does not include the synthetic oligosaccharide drug. where FCR is adjusted for mortality in the animal population.

In some embodiments, an animal fed a synthetic oligosaccharide drug, animal feed premix, or animal feed composition has a lower FCR than an animal fed a diet that does not include the synthetic oligosaccharide drug but includes one or more antibiotics, one or more ionophores, soluble corn fiber, modified wheat starch or yeast mannan, or any combination thereof.

Those skilled in the art will know that when determining FCR, it can be adjusted for mortality to reduce errors due to small statistical data. Methods for adjusting FCR for mortality are well known to those skilled in the art.

In some embodiments, which may be combined with any of the above embodiments, poultry is a single poultry bird, while in other embodiments, poultry is a population of poultry birds.

In some embodiments, the animal is poultry and the animal feed composition is a poultry feed, wherein the synthetic oligosaccharide drug, poultry nutritional composition, poultry feed premix, or poultry feed composition reduces the feed conversion ratio (FCR). ) by about 10%, or by about 5%, or by 1-10%, by 2-10%, by 3-10%, by 4-10%, by 5-10%, by 2-5%, by 2-6%, by 2-7%, by 2-8%, by 2-9% or by 1-5% when fed to poultry, compared to poultry receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the poultry suffers from a disease or disorder, or is raised in an unfavorable environment, where the synthetic oligosaccharide drug, poultry nutritional composition, poultry feed premix, or poultry feed composition reduces the feed conversion ratio (FCR) by approximately 30% , about 25%, about 20%, about 15%, about 10% or about 5%, or from 1% to 30%, from 5% to 30%, from 10% to 30%, from 5 % to 20%, from 10% to 20%, from 1% to 20%, from 1% to 15%, from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% up to 10%, from 5% to 10%, from 2% to 5%, from 2% to 6%, from 2% to 7%, from 2% to 8%, from 2% to 9% or from 1% to 5% when fed to poultry compared to poultry receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the animal is a pig and the animal feed composition is a pig feed, wherein the synthetic oligosaccharide preparation, pig nutrition composition, pig feed premix, or pig feed composition

- 48 044964 reduce the feed conversion ratio (FCR) by about 15%, by about 10% or by about 5%, or from 1% to 15%, from 2% to 15%, from 3% to 15%, from 4% up to 15%, from 5% to 15%, from 10% to 15%, from 1% to

10%, from 2% to 10%, from 3% to 10%, from 4% to 10%, from 5% to 10%, from 2% to 5%, from 2% to 6%, from 2% to 7 %, from 2% to 8%, from 2% to 9% or from 1% to 5% when fed to pigs, compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the pig suffers from a disease or disorder, or is raised in an unfavorable environment, where the synthetic oligosaccharide drug, swine nutritional composition, swine feed premix, or swine feed composition reduces the feed conversion ratio (FCR) by about 40%, by about 35 %, about 30%, about 25%, about 20%, about 15%, about 10% or about 5%, or from 1% to 40%, from 5% to 40%, from 10% up to 40%, from 15% to 40%, from 20% to 40%, from 25% to 40%, from 30% to 40%, from 1% to 30%, from 5% to 30%, from 10% to 30%, from 5% to 20%, from 10% to 20%, from 1% to 20%, from 1% to 15%, from 1% to 10%, from 2% to 10%, from 3% to 10 %, from 4% to 10%, from 5% to 10%, from 2% to 5%, from 2% to 6%, from 2% to 7%, from 2% to 8%, from 2% to 9% or from 1% to 5% when fed to pigs, compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation.

B. Body weight.

In some embodiments, an animal in accordance with the invention that receives a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition described in the present invention may exhibit an increase in body weight compared to a control animal not receiving the oligosaccharide drug. a nutritional composition, an animal feed premix or an animal feed composition. In some embodiments, both the animal of the invention and the control animal consume the same amount of food on a body weight basis, but the animal of the invention fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition exhibits increase in weight gain compared to control animals receiving a diet that did not include a synthetic oligosaccharide drug.

An animal's weight gain can be determined by any suitable methods known in the art. For example, to determine the weight gain of an animal that is subjected to a feeding regimen of a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition, one skilled in the art may measure the animal's body weight before the feeding regimen, measure the animal's weight after the animal will receive a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition, and determine the difference between these two measurements.

In some embodiments, the gain may be average daily weight gain (also called average daily weight gain (ADG)), average weekly weight gain (AWG), or total weight gain (BWG).

C. Average daily weight gain.

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in increased average daily weight gain compared to an animal fed the diet without the synthetic oligosaccharide drug. In some embodiments, providing a population of animals with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or feed composition results in an increase in average daily weight gain, compared to a group of animals receiving the diet without the synthetic oligosaccharide drug.

In one embodiment, an animal's average daily weight gain is the weight gained each day by an individual animal, averaged over a given period of time. In some embodiments, the average daily gain for a population of animals is the average daily gain for each individual animal averaged over the population; where average daily weight gain is the weight gained each day by an individual animal, averaged over a given period of time. In other embodiments, the average daily weight gain for a population of animals is the total weight gained by the population each day, divided by the number of individual animals in the population, averaged over a given period of time. It should be understood that daily gain or average daily gain can be further averaged, for example, to provide average daily gain among animal populations.

In some embodiments, the animal is a poultry and the poultry receiving the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average daily weight gain of at least 20 grams per day, at least 30 grams per day , at least 40 grams per day, at least 50 grams per day, at least 60 grams per day, at least 70 grams per day, at least 80 grams per day, at least 90 grams per day, from 20 to 100 grams per day, 20 to 80 grams per day, 30 to 50 grams per day, 40 to 60 grams per day, 50 to

- 49,044,964 grams per day or 70 to 90 grams per day. In one embodiment, the animal is a poultry, and the poultry receiving the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average daily weight gain of at least 50 grams per day. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average daily weight gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least 11%, at least at least 12%, 1-10%, 2-8%, or 35% more than the average daily weight gain of poultry fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, the animal is a poultry, and the poultry is between 0 and 14 days old and the average daily weight gain is at least 30 g, at least 40 g, or at least 50 g/day.

In other embodiments, the animal is a poultry, the poultry is between 14 and 28 days old, and the average daily weight gain is at least 70 grams, at least 80 grams, or at least 90 grams per day.

In yet other embodiments, the animal is a poultry, the poultry is between 29 and 35 days old, and the average daily weight gain is at least 50 g, at least 60 g, or at least 70 g/day.

In some embodiments, which may be combined with the foregoing, the animal is a poultry and the animal feed composition is a poultry feed, wherein the synthetic oligosaccharide preparation, poultry nutritional composition, poultry feed premix, or poultry feed composition poultry increases the average daily gain of poultry to approximately 10%, or approximately 5%, or from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% to 10%, from 5% to 10 %, 2% to 5%, 2% to 6%, 2% to 7%, 2% to 8%, 2% to 9% or 1% to 5% when fed to poultry, compared with poultry receiving a feed composition without a synthetic oligosaccharide drug.

In some embodiments, the poultry is suffering from a disease or disorder or is being raised in an unfavorable environment where the synthetic oligosaccharide preparation, poultry nutritional composition, poultry feed premix, or poultry feed composition increases the average daily weight gain of the poultry by about 30%, by about 30%. 25%, about 20%, about 15%, about 10% or about 5%, or from 1% to 30%, from 5% to 30%, from 10% to 30%, from 5% to 20 %, from 10% to 20%, from 1% to 20%, from 1% to 15%, from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% to 10% , from 5% to 10%, from 2% to 5%, from 2% to 6%, from 2% to 7%, from 2% to 8%, from 2% to 9%, or from 1% to 5% when fed to poultry, compared to poultry receiving a feed composition without a synthetic oligosaccharide drug.

In some embodiments, which may be combined with the foregoing, the animal is a pig and the animal feed composition is a pig feed, wherein the synthetic oligosaccharide preparation, swine nutritional preparation, swine feed premix, or swine feed composition increases average daily weight gain pigs by about 15%, by about 10% or by about 5%, or from 1% to 15%, from 2% to 15%, from 3% to 15%, from 4% to 15%, from 5% to 15 %, from 10% to 15%, from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% to 10%, from 5% to 10%, from 2% to 5% , from 2% to 6%, from 2% to 7%, from 2% to 8%, from 2% to 9% or from 1% to 5% when fed to pigs compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation .

In some embodiments, the pig suffers from a disease or disorder, or is raised in an unfavorable environment, wherein the oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition increases the pig's average daily growth rate by about 40%, by about 35%, by about 30%, about 25%, about 20%, about 15%, about 10% or about 5%, or from 1% to 40%, from 5% to 40%, from 10% to 40%, from 15% to 40%, from 20% to 40%, from 25% to 40%, from 30% to 40%, from 1% to 30%, from 5% to 30%, from 10% to 30%, from 5% to 20%, from 10% to 20%, from 1% to 20%, from 1% to 15%, from 1% to 10%, from 2% to 10%, from 3% and 10%, from 4 % to 10%, from 5% to 10%, from 2% to 5%, from 2% to 6%, from 2% to 7%, from 2% to 8%, from 2% and 9%, or from 1 % up to 5%, when fed to pigs, compared to pigs receiving a feed composition without a synthetic oligosaccharide drug.

In some embodiments, the animal is a pig and the pig fed the synthetic oligosaccharide preparation, swine nutritional preparation, swine feed premix, or swine feed composition has an average daily gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1 to 10%, 2 to 8%, or 3 to 5% more than the daily average

- 50 044964 weight gain of pigs fed a diet that does not include a synthetic oligosaccharide drug.

D. Average weekly weight gain.

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in an increase in average weekly weight gain compared to an animal fed a diet without the synthetic oligosaccharide drug. In some embodiments, providing a population of animals with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or feed composition results in increased average weekly weight gain compared to a population of animals fed the diet without the synthetic oligosaccharide drug.

In one embodiment, the average weekly weight gain of an animal is the weight gained each week by an individual animal, averaged over a given period of time. In some embodiments, the average weekly weight gain for a population of animals is the average weekly weight gain for each individual animal averaged over the population; where average weekly weight gain is the weight gained each week by an individual animal, averaged over a given period of time. In other embodiments, the average weekly weight gain for a population of animals is the total weight gained by the population each week divided by the number of individual animals in the population averaged over a given period of time. It should be understood that the average weekly gain may be further averaged, for example, to provide an average weekly gain across animal populations.

In some embodiments, the animal is a poultry and the poultry receiving the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average weekly weight gain of at least 100 grams per week, at least 200 grams per week , at least 300 grams per week, at least 400 grams per week, at least 500 grams per week, at least 600 grams per week, at least 700 grams per week, at least 800 grams per week, from 100 to 800 grams per week, 100 to 400 grams per week, 300 to 600 grams per week, 500 to 800 grams per week, or 350 to 550 grams per week. In one embodiment, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average weekly gain of at least 400 grams per week. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average weekly weight gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least 11%, at least at least 12%, 1-10%, 2-8% or 35% more than the average weekly gain of poultry fed a diet that does not include an oligosaccharide drug.

In some embodiments, the animal is a pig and the pig fed the synthetic oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition has an average weekly weight gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than the average weekly weight gain of pigs fed a diet that does not include a synthetic oligosaccharide preparation.

E. Final weight gain.

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in an increase in final weight gain, compared to an animal fed a diet without the synthetic oligosaccharide drug. In some embodiments, providing a population of animals with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or feed composition results in an increase in average net weight gain compared to a population of animals fed the diet without the synthetic oligosaccharide drug.

In some embodiments, providing an animal or population of animals with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition results in a final weight gain or average final weight gain that is closer to the maximum performance targets than the animals or animal population receiving food without synthetic oligosaccharide preparation. The maximum performance target usually refers to the highest practical gain observed for a given animal type and breed under ideal rearing conditions, ideal animal health and ideal dietary nutrition.

In one embodiment, the final weight gain is the amount of weight gained

- 51 044964 to individual animals over a period of time. For example, in one embodiment, total weight gain is the amount of weight that an individual animal gains from 0 days of age to the last weight taken before the animal was processed, or the final weight taken on the day the animal was processed. For example, in one embodiment, the total weight gain from days 0 to 28 for an animal is the amount of weight that an individual animal gains from 0 days of age to 28 days of age.

In another embodiment, average total weight gain is the amount of weight that an individual animal gains over a period of time, averaged over a population of animals. For example, in one embodiment, the average total weight gain is the amount of weight that an individual animal gains from 0 days of age to the final weight taken before the animal was processed, or the final weight taken on the day the animal was processed, the animal averaged over the animal population. In yet another embodiment, average total weight gain is the amount of weight that a population of animals gains over a period of time divided by the number of individual animals in the population. For example, in one embodiment, the average total weight gain is the amount of weight gained by a population of animals from 0 days of age to the final weight taken before the animal population was processed, or the final weight taken on the day the animal was processed, divided by the number of individual animals in the population.

It should be understood that the total weight gain and average total weight gain may be further averaged. For example, the average weight gain for different populations of the same animal type can be averaged to obtain the average overall weight gain for the populations.

In some embodiments, the animal is a poultry and the poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has a final weight gain of at least 3 kg, at least 2.5 kg, at least 2 kg, at least 1.5 kg, at least 1 kg, from 1 to 3 kg or from 1.5 to 2.5 kg. In one embodiment, poultry fed the synthetic oligosaccharide preparation, animal feed premix, or animal feed composition have a final weight gain of at least 2 kg. In some embodiments, poultry fed the synthetic oligosaccharide preparation, animal feed premix, or animal feed composition have a final weight gain of at least 1%, at least 2%, at least 3%, at least by 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, by at least 11%, by at least 12%, 1-10%, 2-8% or 3-5% more than the final weight gain of birds fed a diet that did not include a synthetic oligosaccharide drug. In some embodiments, poultry fed the synthetic oligosaccharide preparation, animal feed premix, or animal feed composition have a final weight gain of at least 0.01 kg, at least 0.02 kg, or at least 0.03 kg, at least 0.04 kg, at least 0.05 kg, at least 0.06 kg, at least 0.07 kg, at least 0.08 kg, at least 0.09 kg, at least 0.1 kg, 0.01-0.1 kg, 0.03-0.07 kg or 0.04-0.06 kg more than the final weight gain of the bird , who received a diet that did not include a synthetic oligosaccharide drug.

In some embodiments, the animal is a poultry and the poultry receiving the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition has an average final weight gain of at least 3 kg, at least 2.5 kg, at least 2 kg, at least 1.5 kg, at least 1 kg, from 1 to 3 kg or from 1.5 to 2.5 kg. In one embodiment, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average final weight gain of at least 2 kg. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average final weight gain of at least 1%, at least 2%, at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, by at least 11%, according to at least 12%, 1-10%, 2-8%, or 3-5% more than the average final weight gain of poultry fed a diet that does not include a synthetic oligosaccharide preparation. In some embodiments, poultry fed the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition have an average final weight gain of at least 0.01 kg, at least 0.02 kg, at least by 0.03 kg, by at least 0.04 kg, by at least 0.05 kg, by at least 0.06 kg, by at least 0.07 kg, by at least 0.08 kg , at least 0.09 kg, at least 0.1 kg, 0.01-0.1 kg, 0.03-0.07 kg or 0.04-0.06 kg more than average the final weight gain of birds fed a diet that did not include a synthetic oligosaccharide drug.

In some embodiments, the animal is a domestic bird

- 52 044964 tsu, and poultry is aged from 0 to 14 days, from 15 to 28 days, from 29 to 35 days, from 0 to 42 days, from 0 to 6 weeks or from 0 to 6.5 weeks. In some embodiments, the start phase is from 0 to 14 days, the growth phase is from 15 to 28 days, and the finishing phase is from 29 to 35 days. In other embodiments, the starting phase is from 0 to 14 days, the growing phase is from 15 to 35 days, and the finishing phase is from 36 to 42 days. In other embodiments, the start phase is from 0 to 14 days, the growth phase is from 15 to 39 days, and the finishing phase is from 40 to 46 days. It should be understood that the duration of the starter phase, rearing phase and finishing phase for poultry may vary depending on the intended use of the poultry or poultry product. For example, in some embodiments, the duration of the starter phase, grower phase, and finisher phase may be different if the poultry is being processed for broiler chicken production versus processing for tray-packed chicken meat.

In some embodiments, which may be combined with any of the above embodiments, poultry is a single poultry bird, while in other embodiments, poultry is a population of poultry birds.

In some embodiments, a pig fed the synthetic oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition has a final weight gain of at least 1%, at least 2%, at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, by at least 11%, according to at least 12%, 1-10%, 2-8% or 3-5% more than the final weight gain of pigs fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, a pig fed the synthetic oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition has an average final weight gain of at least 1%, at least 2%, or at least 3% , at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than the average final weight gain of pigs fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, which may be combined with any of the above embodiments, the pig is an individual pig, while in other embodiments, the pig is a population of pigs.

F. Animal Product Yield.

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition as described herein results in increased yield of the animal product compared to animals fed a diet that does not include the synthetic oligosaccharide drug. In some embodiments, an animal receiving a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition provides at least 1%, at least 2%, at least 3%, at least an increase in 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, 1-10% , 4-10%, 6-10% or 2-8% more animal product, compared to an animal fed a diet that does not include a synthetic oligosaccharide drug. For example, in some embodiments, the animal product is the meat of an animal, and an animal treated with a synthetic oligosaccharide drug as described herein produces a greater amount of meat compared to an animal not treated with the oligosaccharide drug. In some embodiments, providing an animal population with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition results in an increase in the average yield of the animal product compared to an animal population fed a feed that does not include the synthetic oligosaccharide drug. In some embodiments, the average animal product yield is the amount of animal product obtained from each individual animal averaged over a population of animals.

In some embodiments, the animal product is meat from an animal (eg, which may be sold to consumers, processed into a food product, or consumed by humans). In some embodiments, the animal is poultry and the animal product is a poultry eviscerated carcass, poultry eviscerated leg meat, poultry eviscerated breast meat, poultry eviscerated drumstick meat, poultry eviscerated fat. , breast fillet from a poultry carcass or chicken leg fillet from a poultry carcass. In other embodiments, the animal is poultry and the animal product is white meat, breast fillet, and breast meat. In another embodiment, the animal is poultry and the product is tray-packaged chicken. In yet another embodiment, the animal is poultry and the product is a whole bird without giblets (WOG).

In some embodiments, the yield of the animal product is the yield of the product from an individual animal. In some embodiments, the average yield of an animal product is the yield obtained from each individual animal in a population of animals, averaged over the population. In yet another embodiment, the average animal product yield is the total animal product yield obtained from a population of animals divided by the number of individual animals in the animal population.

In some embodiments, the animal is poultry, the leg meat yield from the eviscerated poultry carcass is at least 6%, at least 8%, at least 10%, at least 12%, 6 to 12%, 8 up to 12%, 10 to 18%, 12 to 16% or 12 to 14% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the yield of leg meat from an eviscerated poultry carcass treated with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 35% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average leg meat yield per eviscerated poultry carcass is at least 6%, at least 8%, at least 10%, at least 12%, 6 to 12% . 8 to 12%, 10 to 18%, 12 to 16%, or 12 to 14% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition. In some embodiments, the average yield of leg meat from an eviscerated poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the breast meat yield from the poultry is at least 10%, at least 12%, at least 15%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 28%, 10 to 18%, 12 to 16%, 18 to 29%, 20 to 27% or 20 up to 25% of the live weight of birds receiving a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition. In some embodiments, the yield of breast meat from an eviscerated poultry carcass treated with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average breast meat yield from a gutted poultry carcass is at least 10%, at least 12%, at least 15%, at least 16%, at least 18% , at least 20%, at least 22%, at least 24%, at least 28%, from 10 to 18%, from 12 to 16%, from 18 to 29%, from 20 to 27% or from 20 to 25% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average breast meat yield from an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than in poultry fed a diet that does not include a synthetic oligosaccharide preparation .

In some embodiments, the animal is poultry and the leg meat yield from the eviscerated poultry carcass is at least 5%, at least 7%, at least 8%, at least 9%, at least 10%, at at least 11%, at least 12%, from 5 to 14%, from 7 to 10%, from 7 to 15%, from 9 to 13% or from 9 to 11% of the live weight of birds receiving synthetic oligosaccharide a preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the yield of drumstick meat from an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average leg meat yield from a gutted poultry carcass is at least 5%, at least 7%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 5 to 14%, 7 to 10%, 7 to 15%, 9 to 13% or 9 to 11% of the live weight of poultry treated with synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average drumstick meat yield from an eviscerated poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% %, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, the animal is poultry and the breast fillet yield from the boneless poultry carcass is at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at at least 24%, 14 to 16%, 18 to 30%, 20 to 28% or 20 to 26% of live weight for poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or feed composition for animals. In some embodiments, the yield of breast fillet from a boneless poultry carcass fed with an oligosaccharide preparation, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 35% more than poultry fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, the animal is poultry and the average breast fillet yield from a boneless poultry carcass is at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, 14 to 16%, 18 to 30%, 20 to 28% or 20 to 26% of live weight for poultry fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or feed composition for animals. In some embodiments, the average yield of breast fillet from a boneless poultry carcass fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1% by at least 1%. 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10 %, at least 11%, at least 12%, 110%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide drug.

In some embodiments, the animal is poultry and the boneless poultry leg meat yield is at least 6%, at least 8%, at least 10%, at least 12%, at least 14% , at least 16%, at least 18%, 6 to 18%, 8 to 16%, 12 to 21%, 14 to 19% or 14 to 17% of the live weight of poultry treated with synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the yield of leg meat from a boneless poultry carcass treated with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%,

- 55 044964 by at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9 %, at least 10%, at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than the poultry fed the diet, not including a synthetic oligosaccharide drug.

In some embodiments, the animal is poultry and the average leg meat yield from a boneless poultry carcass is at least 6%, at least 8%, at least 10%, at least 12%, at least 14 %, at least 16%, at least 18%, 6 to 18%, 8 to 16%, 12 to 21%, 14 to 19% or 14 to 17% of the live weight of poultry fed a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average leg meat yield from a boneless poultry carcass treated with an oligosaccharide preparation, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the poultry eviscerated carcass fat yield is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4% , at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, at least 1.6%, 0.1 to 2%, 0.2 to 1%, 0.5 to 2% or 0.3 to 0.7% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the fat yield from an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, by at least 3%, by at least 4%, by at least 5%, by at least 6%, by at least 8%, by at least 9%, by at least 10%, according to at least 11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average fat yield from the eviscerated poultry carcass is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4 %, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at at least 1.2%, at least 1.4%, at least 1.6%, from 0.1 to 2%, from 0.2 to 1%, from 0.5 to 2% or from 0, 3 to 0.7% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average fat yield from an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2% , by at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10% , at least 11%, at least 12%, 1-10%, 2-8% or 35% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the poultry eviscerated yield is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least 80%, at least 85%, at least 90%, from 50 to 95%, from 60 to 85% or from 65 to 75% of the live weight of poultry treated with a synthetic oligosaccharide preparation, a nutritional composition, an animal feed premix or an animal feed composition. In some embodiments, the yield of an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry and the average poultry evisceration yield is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least at least 75%, at least

- 56 044964 at least 80%, at least 85%, at least 90%, from 50 to 95%, from 60 to 85% or from 65 to 75% of the live weight of poultry fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition. In some embodiments, the average yield of an eviscerated poultry carcass fed with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition as described herein is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least at least 11%, at least 12%, 1-10%, 2-8%, or 3-5% more than poultry fed a diet that does not include a synthetic oligosaccharide preparation.

Methods for deboning poultry carcasses are well known to those skilled in the field of poultry processing. It should be understood that meat obtained from poultry can be measured, for example, as the ratio of the mass of meat extracted to the final weight of the bird before processing. In some embodiments, the animal is a poultry, and the poultry is at least 35 days, at least 42 days, at least 6 weeks, at least 6.5 weeks old before the poultry is processed for evisceration production poultry carcasses, boneless poultry carcasses, white meat, breast fillets and breast meat, tray-packed chicken, whole poultry without giblets (WOG) or meat as described above.

In other embodiments, the animal is poultry and the animal product is eggs. In some embodiments, the animal is a pig and the pig product is pig meat (eg, which may be sold to consumers, processed into food, or consumed by humans). In some embodiments, the yield of the pork product is the yield obtained from an individual pig. In some embodiments, the average yield of a pork product is the yield obtained from each individual pig in a population of pigs, averaged across the population. In yet another embodiment, the average yield of a swine product is the total yield of a swine population divided by the number of individuals in the swine population.

In some embodiments, an animal or population of animals fed a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition has a higher average daily gain, a higher average weekly gain, a higher total gain, a higher average total gain, or more a higher average yield of the animal product, or any combination thereof, than that of an animal or population of animals fed a diet that does not include a synthetic oligosaccharide preparation, but includes one or more antibiotics, one or more ionophores, soluble corn fiber, modified wheat starch or yeast mannan, or any combinations thereof.

One skilled in the art will appreciate that the maximum theoretical weight gain may be different for different types of animals, and may be different for different breeds of the same type of animal (eg, different types of broiler chickens or different types of pigs).

One skilled in the art will appreciate that the maximum theoretical weight gain may be different for different types of animals, and may be different for different breeds of the same type of animal (eg, different types of broiler chickens or different types of pigs).

In some embodiments, the animal is a poultry. In some embodiments, which may be combined with any of the above embodiments, poultry is a single poultry bird, while in other embodiments, poultry is a population of poultry birds. In other embodiments, the animal is a pig. In some embodiments, which may be combined with any of the above embodiments, the pig is an individual pig, while in other embodiments, the pig is a population of pigs.

G. Feed consumption.

In some embodiments, providing an animal with a synthetic oligosaccharide drug, nutritional composition, animal feed premix, or animal feed composition as described herein results in increased average daily feed intake compared to an animal food that does not include the synthetic oligosaccharide drug.

Average daily feed intake (ADFI) refers to the average weight of feed consumed by an animal over a given period of time. In some embodiments, average daily feed intake is measured by distributing a known mass of feed to a group of a fixed number of animals, allowing the animals in the group to freely consume the distributed feed without restriction for a specified number of days, weighing the mass of unconsumed feed at the end of the time period, and calculating the average daily feed intake (ADFI) as the difference between the distributed feed mass minus the residual feed mass divided by the number of animals in the group and divided by the number of days in the period. In other embodiments, the average daily feed intake may be adjusted for any animals that die or are removed from the group using methods known to one of ordinary skill in the art.

In some embodiments, the animal is poultry and the animal feed composition is poultry feed, wherein the synthetic oligosaccharide drug, poultry feed premix, or poultry feed composition increases average daily feed intake by about 10%, or about 5 %, or from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% to 10%, from 5% to 10%, from 2% to 5%, from 2% to 6 %, from 2% to 7%, from 2% to 8%, from 2% to 9% or from 1% to 5% when fed to poultry compared to poultry receiving a feed composition without a synthetic oligosaccharide preparation.

In some embodiments, the poultry is suffering from a disease or is being raised in an unfavorable environment where the synthetic oligosaccharide drug, poultry nutritional composition, poultry feed premix, or poultry feed composition increases average daily feed intake by about 30%, about 25%, by about 20%, by about 15%, by about 10%, or by about 5%, or from 1% to 30%, from 5% to 30%, from 10% to 30%, from 5% to 20%, from 10% to 20%, from 1% to 20%, from 1% to 15%, from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% to 10%, from 5 % to 10%, 2% to 5%, 2% to 6%, 2% to 7%, 2% to 8%, 2% to 9% or 1% to 5% when fed to poultry compared to poultry receiving a feed composition without a synthetic oligosaccharide drug.

In some embodiments, which may be combined with the foregoing, the animal is a pig and the animal feed composition is a pig feed, wherein the oligosaccharide preparation, swine nutritional composition, swine feed premix, or swine feed composition increases average daily feed intake by about 15%, by about 10% or by about 5%, or from 1% to 15%, from 2% to 15%, from 3% to 15%, from 4% to 15%, from 5% and 15% , from 10% to 15%, from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% to 10%, from 5% to 10%, from 2% to 5%, from 2% to 6%, from 2% to 7%, from 2% to 8%, from 2% to 9% or from 1% to 5% when fed to pigs, compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation .

In some embodiments, the pig has a disease or is raised in an unfavorable environment where the synthetic oligosaccharide drug, swine nutritional composition, swine feed premix, or swine feed composition increases average daily feed intake by about 40%, about 35%, about 30% , about 25%, about 20%, about 15%, about 10% or about 5%, or from 1% to 40%, from 5% to 40%, from 10% to 40%, from 15 % to 40%, from 20% to 40%, from 25% to 40%, from 30% to 40%, from 1% to 30%, from 5% to 30%, from 10% to 30%, from 5% up to 20%, from 10% to 20%, from 1% to 20%, from 1% to 15%, from 1% to 10%, from 2% to 10%, from 3% to 10%, from 4% to 10%, from 5% to 10%, from 2% to 5%, from 2% to 6%, from 2% to 7%, from 2% to 8%, from 2% to 9%, or from 1% to 5% when fed to pigs compared to pigs receiving a feed composition without a synthetic oligosaccharide preparation.

Methods for enhancing the growth of an animal or animal population described in the present invention include providing an oligosaccharide preparation, an animal feed premix, or an animal feed to an animal or animal population. The oligosaccharide preparation, animal feed premix, or animal feed may be provided in any suitable form to any suitable type of animal using any suitable feeding regimen to promote growth of the animal or population of animals.

H. Quality of the animal product.

In some embodiments, the animal product, such as animal meat, is of improved quality. Animal products described herein include non-meat products such as milk and eggs. Animal meat qualities include, for example, color, integrity, texture, taste, mouthfeel, aroma and tenderness. It is clear to a qualified professional that the quality of animal meat will depend on the type of animal. Standard tests known to one skilled in the art can be used to evaluate the quality of animal meat, including, for example, color, taste, tenderness, and aroma. The animal meats described in the present invention can be evaluated by trained experts. Evaluation may include inspection, touch, chewing and tasting of the product to determine the product's appearance, color, integrity, texture, aroma, mouthfeel, etc. Samples can be presented to experts under red or white light. Samples can be assigned random three-digit numbers and rotated in selected positions to prevent bias. Sensory evaluations can be divided into acceptance or attractiveness, or specific terminology may be used. For example, you can use letter scales (A - excellent, B - good, C - poor) or numerical scales (1=unacceptable, 2=satisfactory, 3=good; 4=very good; 5=excellent). The scale can be used to evaluate the overall acceptability or quality of animal meat, or specific quality characteristics such as texture and taste. Panelists may be asked to rinse their mouths with water between samples and given the opportunity to comment on each sample.

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VIII. Animals.

The synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition may be provided to any suitable animal. In some embodiments, the animal is monogastric. It is generally believed that a monogastric animal has a single-chamber stomach. In other embodiments, the animal is a ruminant. It is generally accepted that ruminants have a multi-chambered stomach. In some embodiments, the animal is a ruminant in the preruminant phase. Examples of such ruminant animals in the pre-ruminant phase are dairy calves.

In some embodiments, the animal is livestock. In some embodiments, the animal is a pet. In some embodiments, the animal is a fish (e.g., salmon, tilapia, tropical fish), poultry (e.g., chicken, turkey), marine organism (e.g., shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g. growing piglets, piglets in the first/second phase of fattening, cat, dog, rabbit, goat, guinea pig, donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird or human.

In some embodiments, the animal is a poultry. Examples of poultry include chicken, duck, turkey, goose, quail or fighting cornish. In one embodiment, the animal is a chicken. In some embodiments, the poultry is a laying hen, broiler chicken, or turkey. In other embodiments, the animal is a mammal, including, for example, cow, pig, goat, sheep, deer, bison, rabbit, alpaca, llama, mule, horse, reindeer, buffalo, yak, guinea pig, rat, mouse, alpaca , dog, cat or person. In one embodiment, the animal is a cow. In another embodiment, the animal is a pig. The animal feed composition can also be used in aquaculture. In some embodiments, the animal is an aquatic animal. Examples of aquatic animals may include trout, salmon, grouper, tilapia, shrimp, oyster, mussel, clam, lobster or crayfish. In one embodiment, the animal is a fish.

IX. Application.

In some embodiments, use includes providing the synthetic oligosaccharide drug, nutritional composition, or animal feed composition described herein to an animal so that the animal can consume the synthetic oligosaccharide drug, nutritional composition, or animal feed composition as desired. In such embodiments, the animal consumes a portion of the synthetic oligosaccharide drug, nutritional composition, or animal feed composition.

The synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition may be provided to the animal on any suitable schedule. In some embodiments, the animal is provided with a synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition daily, weekly, monthly, every other day, for at least three days a week, or at least seven days a month. In some embodiments, the oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal during certain phases of the diet.

For example, some animals are given a starter diet between 0 and 14 days of age. In other embodiments, the animal is given a growth diet at 15 to 28 days, 15 to 35 days, or 15 to 39 days of age. In other embodiments, the animal is given a finishing diet at 29 to 35 days, 36 to 42 days, or 40 to 46 days of age.

In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition is provided to the animal during the starter phase of the diet, the growth phase of the diet, or the finishing phase of the diet, or any combinations thereof.

In some embodiments, the animal is a poultry and the poultry is fed a starter diet from 0 to 15 days of age, a grower diet from 16 to 28 days of age, and a finisher diet from 29 to 35 days of age. In other embodiments, the animal is a poultry, and the poultry is fed a starter diet from 0 to 14 days of age, a grower diet from 15 to 35 days of age, and a finisher diet from 36 to 42 days of age. In other embodiments, the animal is a poultry and the poultry is fed a starter diet from 0 to 14 days of age, a grower diet from 15 to 39 days of age, and a finisher diet from 20 to 46 days of age.

In some embodiments, the synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition is provided to poultry during the starter phase of the diet, the growth phase of the diet, or the finishing phase of the diet, or any combination thereof.

The oligosaccharide preparations described in the present invention can be fed to individual animals or populations of animals. For example, in one embodiment, where the animal is poultry, the oligosaccharide preparations can be fed to an individual poultry or group of poultry.

The synthetic oligosaccharide preparation, nutritional composition, animal feed premix, or animal feed composition may be provided to the animal in any suitable form, including, for example, solid form, liquid form, or a combination thereof. In some embodiments, the oligosaccharide preparation or animal feed composition is a liquid, such as a syrup or solution. In other embodiments, the oligosaccharide preparation, animal feed premix, or animal feed composition is a solid, such as a granule or powder. In still other embodiments, the oligosaccharide preparation, animal feed premix, or animal feed composition may be provided to the animal in both liquid and solid components, such as a mash.

Examples

Example 1. Synthesis of a gluco-galacto-oligosaccharide drug.

Synthesis of the gluco-galacto-oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time and reaction temperature that were chosen to ensure suitable production on a kilogram scale.

D-glucose monohydrate (825.16 g), D-lactose monohydrate (263.48 g) and 2-pyridine sulfonic acid (1.0079 g, Sigma-Aldrich, St. Louis, USA) were added to a three-liter three-neck round bottom flask and a central neck made of ground glass 29/42 and two side necks made of ground glass 24/40. A 133 mm Teflon stirring paddle was attached to the glass stirring rod using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control unit through a J-type thermocouple rod inserted through a rubber baffle in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction mixture reached 120°C, the reflux condenser was placed in distillation configuration, with the distilled product collected in a 250 mL round bottom flask placed in an ice bath. The mixture was kept at 130°C with continuous stirring for 6 hours, after which the thermocouple unit was turned off. The distillation apparatus was removed, and 390 g of distilled water at a temperature of 60°C was gradually added to the three-neck flask. The resulting mixture was left to stir at 40 rpm for 10 hours. Approximately 1250 g of a viscous light amber colored material was collected and its concentration was 71.6°Brix based on the refractive index.

The final water content of the reactor product was measured by Karl Fischer titration of a representative aliquot of the reactor contents taken at the end of the reaction. At a reaction temperature of 130°C, the water content in the reaction product was determined to be 5.8 wt.% water without conversion to dry matter.

Example 2. Synthesis of a gluco-oligosaccharide drug.

Synthesis of the gluco-oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time, and reaction temperature that were selected to allow suitable kilogram-scale production.

D-glucose monohydrate (1150 g) was added to a three-liter, three-neck round bottom flask with one 29/42 ground glass center neck and two 24/40 ground glass side necks. A 133 mm Teflon stirring paddle was attached to the glass stirring rod using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device, through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. A secondary temperature probe connected to the secondary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

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The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction temperature increased to 120-130°C, (+)-camphor-10-sulfonic acid (1.16 g, Sigma-Aldrich, St. Louis) was added to the three-neck flask and the apparatus was switched from reflux to the distillation configuration with round bottom collection flask placed in an ice bath. This setting was maintained for 1.5 hours, after which the thermocouple unit was turned off, the distillation apparatus was removed, and 390 g of distilled water at a temperature of 23°C was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 1300 g of dark amber viscous material was collected and the concentration was 72.6° Brix.

Example 3. Synthesis of gluco-galacto-manno-oligosaccharide drug.

The synthesis of the gluco-galacto-manno-oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time and reaction temperature that were chosen to ensure suitable production on a kilogram scale.

The gluco-galacto-manno-oligosaccharide preparation was prepared as two separate components synthesized in separate reaction vessels that were assembled independently. Each synthesis used different starting reagents but followed the same procedures and methods until completion. The final gluco-galacto-manno-oligosaccharide preparation was a homogeneous syrup obtained by mixing both synthesis products.

To synthesize the first component, 990.54 g of glucose monohydrate, 105.58 g of lactose monohydrate and 1.00 g of 2-pyridine sulfonic acid were added to a three-liter three-neck round-bottom flask with a central ground neck 29/42 and side ground necks 24/40. A 133mm Teflon stirring paddle was attached to a 440mm diameter glass stirrer using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was placed in a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum into one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control chamber reached 120°C to 130°C, the unit was switched from reflux to a distillation configuration with a round-bottom collection flask placed in an ice bath. This setting was maintained for approximately 6 hours and 10 minutes, after which the heating mantle was turned off, the distillation apparatus was removed, and 390 g of distilled water at 60° C. was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 1250 g of a viscous, light amber colored material was collected and measured at 73.1° Brix by refractive index.

To synthesize the second component, 825.04 g of glucose monohydrate, 251.16 g of pure mannose from wood, 25.10 g of distilled water and 1.00 g of 2-pyridine sulfonic acid were added to a three-liter three-neck round bottom flask with one central ground 29/42 neck. on the sides of which there are two polished necks 24/40. The remainder of the synthesis of the second component followed the same procedure and methods as for the first component until collection. Approximately 1250 g of dark amber viscous material was collected and the concentration was 72.3° Brix.

The first and second components were completely transferred into a suitable size HDPE container and thoroughly mixed by hand until homogeneous. The final syrup mixture weighed approximately 2.5 kg, was dark amber in color, was viscous and had a concentration of approximately 72° Brix.

Example 4. Synthesis of a gluco-manno-oligosaccharide drug.

Synthesis of the gluco-oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loading, reaction time, and reaction temperature that were chosen to ensure suitable kilogram-scale production.

The gluco-manno-oligosaccharide preparation was prepared as two separate components, synthesized in separate reaction vessels, which were collected independently. Each synthesis used different starting reagents but used the same procedures and methods until completion. The final gluco-manno-oligosaccharide preparation was a homogeneous syrup obtained by mixing both synthesis products.

To synthesize the first component, 1264.80 g of glucose monohydrate was added to a three-liter three-liter

- 61 044964 neck round-bottom flask with one central 29/42 ground neck surrounded by two 24/40 ground necks. A 133 mm Teflon stirring paddle was attached to a 440 mm diameter glass stirrer using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was placed in a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control chamber reached 120°C to 130°C, 1.15 g of (+)-camphor-10-sulfonic acid was added to the three-neck flask and the unit was switched from reflux to the round-bottom flask distillation configuration for collection, placed in an ice bath. This setting was maintained for approximately 1 hour, after which the thermocouple unit was turned off, the distillation apparatus was removed, and 390 g of distilled water at 23°C was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 1350 g of viscous, light amber colored material was collected and the concentration was 71.8° Brix.

To synthesize the second component, 949.00 g of glucose monohydrate, 288.00 g of pure mannose from wood, 27.94 g of distilled water and 1.15 g of 2-pyridine sulfonic acid were added to a three-liter three-neck flask with one central polished neck 29/42, according to on the sides of which there are two polished necks 24/40. The remainder of the synthesis of the second component followed the same procedure and methods as for the first component until collection, except that (+)-camphor-10-sulfonic acid was not added since the reflux condenser was switched to the distillation configuration, and The resulting setup was maintained for approximately 6 hours. Approximately 1350 g of a viscous dark amber colored material was collected and its concentration was 72.0° Brix.

The first and second components were completely transferred into a suitable size HDPE container and thoroughly mixed by hand until homogeneous. The final syrup mixture weighed approximately 2.7 kg, was dark amber in color, was viscous and, as determined by refractive index, had a concentration of approximately 72° Brix.

Example 5. Synthesis of a gluco-manno-oligosaccharide drug.

Kilogram scale production of the oligosaccharide drug was carried out in a three liter reaction vessel using a catalyst loading, reaction time and reaction temperature found to be suitable for 1 kg scale production.

The gluco-manno-oligosaccharide preparation was prepared as two separate components, synthesized in separate reaction vessels, which were collected independently. Each synthesis used different starting reagents but followed the same procedures and methods until completion. The final gluco-manno-oligosaccharide preparation was a homogeneous syrup obtained by mixing both synthesis products.

To synthesize the first component, 1261.00 g of glucose monohydrate and 1.15 g of 2-pyridine sulfonic acid were added to a three-liter, three-neck round bottom flask with one central 29/42 ground neck surrounded by two 24/40 ground necks. A 133 mm Teflon stirring paddle was attached to a 440 mm diameter glass stirrer using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control unit reading reached 120°C to 130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round-bottom collection flask placed in an ice bath. This setting was maintained for approximately 6 hours, after which the thermocouple unit was turned off, the distillation apparatus was removed, and 390 g of distilled water at 23°C was gradually added to the three-neck flask. The resulting mixture

- 62 044964 was left to stir at 40 rpm for 10 hours until collection. Approximately 1250 g of viscous light yellow material was collected and the concentration was 73.5° Brix.

To synthesize the second component, 949.00 g of glucose monohydrate, 288.00 g of pure mannose from wood, 28.94 g of distilled water and 1.15 g of 2-pyridine sulfonic acid were added to a three-liter three-neck flask with one central polished neck 29/42, according to on the sides of which there are two polished necks 24/40. The remainder of the synthesis of the second component followed the same procedure and methods as for the first component until collection. Approximately 1250 g of viscous, dark amber colored material was collected and measured at 73.3° Brix.

The first and second components were completely transferred into a suitable size HDPE container and thoroughly mixed by hand until homogeneous. The final syrup mixture weighed approximately 2.5 kg, was dark amber in color, viscous and had a concentration of approximately 73° Brix.

Example 6. Synthesis of a gluco-galacto-oligosaccharide drug.

Kilogram-scale production of the oligosaccharide drug was carried out in a three-liter reaction vessel using catalyst loadings, reaction times, and reaction temperatures that were found to be suitable for 1-kg scale production.

A 3-L three-neck flask was equipped with an overhead mixer connected through a 10 mm diameter glass stirrer to a 14 cm crescent-shaped mixing element. The mixing element was located at a distance of approximately 5 mm from the walls of the flask. The flask was heated by a hemispherical electric heating mantle controlled by a temperature control unit connected to a rod-shaped thermocouple probe inserted into the reaction flask. The thermocouple probe was placed so that there was a gap of 5-10 mm above the mixing element. The flask was charged with 576 g of food grade dextrose monohydrate and 577 g of food grade D-galactose monohydrate and heated to approximately 115° C. to obtain a molten sugar syrup. After obtaining the syrup, the flask was equipped with a jacketed reflux condenser, cooled to 4°C by circulating cooled glycol/water and temperature. 31 g of Dowex Marathon C resin (moisture content 0.48 g H ₂ O/g resin) was added to the mixture to form a stirred slurry. The condenser was placed in distillation configuration and the slurry was heated to 145°C.

The stirring speed of approximately 80 rpm and the temperature of 145°C were maintained for 3.8 hours, after which the temperature on the temperature control unit was reduced to 80°C and 119 ml of deionized water at 60°C was gradually added to the flask to obtain dark amber syrup containing residual Dowex resin. The resulting suspension was further diluted to 60° Brix, cooled to room temperature and vacuum filtered through a 0.45 micron filter to remove resin. 1200 g of light amber syrup with a concentration of 60° Brix were obtained.

Example 7. Synthesis of a gluco-oligosaccharide drug.

Kilogram-scale production of the oligosaccharide drug was accomplished in a three-liter reaction vessel using catalyst loadings, reaction times, and reaction temperatures that were found to be suitable for 1-kg scale production.

A three-neck flask with a capacity of 3 L was equipped with an overhead mixer connected through a glass stirrer with a diameter of 10 mm to a crescent-shaped mixing element measuring 14 cm. The mixing element was placed at a distance of approximately 5 mm from the walls of the flask. The flask was heated by a hemispherical electric heating mantle controlled by a temperature control unit connected to a rod-shaped thermocouple probe inserted into the reaction flask. The thermocouple probe was placed so that there was a gap of 5-10 mm above the mixing element. The flask was gradually charged with 1148 g of food grade dextrose monohydrate and heated to approximately 115° C. to obtain a molten sugar syrup. After obtaining the syrup, the flask was equipped with a jacketed distillation condenser, cooled to 4°C by circulating cooled glycol/water. The reaction temperature was gradually increased to 145°C. Once the temperature had been reached and stabilized, 31 grams of Dowex Marathon C resin (moisture content 0.48 g H ₂ O/g resin) was added to the mixture and the stirring speed was maintained at approximately 80 rpm and the temperature at 145°C for 3.8 hours.

After 3.8 hours, the temperature control unit was lowered to 80°C and 119 mL of 60°C deionized water was gradually added to the flask to produce a dark amber syrup containing residual Dowex resin. The resulting suspension was further diluted to 60° Brix, cooled to room temperature and vacuum filtered through a 0.45 micron filter to remove resin. 1113 g of dark amber-colored gluco-oligosaccharide syrup with a concentration of 60° Brix was obtained.

Example 8. One-pot synthesis of oligosaccharide drugs.

A one-pot (one-component) synthesis of the oligosaccharide from Example 3 was demonstrated on a 300 g scale in a one-liter reaction vessel using catalyst loadings, reaction times and reaction temperatures that were found to be suitable for a one-pot reaction.

g food grade D-glucose monohydrate from corn, 37.50 g food grade D-mannose from wood, 15.60 g food grade D-lactose monohydrate, 3.96 g distilled water and

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0.270 g of 2-pyridine sulfonic acid (Sigma-Aldrich, St. Louis) was added to a one-liter, three-neck round-bottom flask with one central 29/42 ground neck surrounded by two 24/40 ground necks. A Teflon stirring paddle was attached to a 220mm glass stirring shaft using PTFE tape. The stirrer rod was secured through the center point using a Teflon carrier adapter and attached to an overhead high torque mechanical mixer via a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a J-type thermocouple rod inserted through a rubber septum in one of the side ports. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The secondary temperature probe connected to the auxiliary temperature sensor was also inserted and secured in the same manner. The second side port of the flask was equipped with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. Once the temperature control chamber reached 120°C to 130°C, the unit was switched from reflux to a distillation configuration with a round-bottom collection flask placed in an ice bath. The mixture was maintained at 130° C. with continuous stirring for approximately 5 hours 40 minutes, after which the heating mantle and distillation apparatus were removed. Approximately 40 g of distilled water at a temperature of 23°C was gradually added to a three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 h until collection. Approximately 389 g of dark amber viscous material was collected and the concentration was 67.0° Brix. Compliance with the oligosaccharide drug from Example 3 was confirmed by SEC chromatography and 2D HSQC NMR spectroscopy.

Example 9. Characteristics of oligosaccharide preparations.

The methods and procedures from Examples 1-8 were used to prepare repeat batches and mixtures of oligosaccharides from Examples 1-7. When preparing various batches from examples 9.1-9.7, both multi-reactor (multicomponent) and single-reactor versions of the corresponding synthesis schemes were used. The resulting materials were analyzed by HPLC size exclusion chromatography (SEC) to characterize molecular weight distribution, LC-MS/MS analysis to quantify DP2 anhydro-sugar content, and 2D 1H,13C-HSQC NMR to determine the molecular structure of the corresponding oligosaccharide preparations.

The materials were prepared as follows.

Example 9.1: Eleven batches of the oligosaccharide preparation from Example 1 were prepared and mixed into four separate batches to obtain oligosaccharide preparation 9.1.

Example 9.2: Seven batches of the oligosaccharide preparation from Example 2 were prepared and mixed in two separate batches to obtain oligosaccharide preparation 9.2.

Example 9.3: Twelve batches of the oligosaccharide preparation from Example 3 were prepared and mixed in five separate batches to obtain oligosaccharide preparation 9.3.

Example 9.4: Four batches of the oligosaccharide preparation from Example 4 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.4.

Example 9.5: Four batches of the oligosaccharide preparation from Example 5 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.5.

Example 9.6: Two batches of the oligosaccharide preparation from Example 6 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.6.

Example 9.7: Two batches of the oligosaccharide preparation from Example 7 were prepared and mixed into one batch to obtain oligosaccharide preparation 9.7.

Additional structural variants of the oligosaccharide drugs from Examples 1-7 were synthesized on a 300 gram scale using the methods from Examples 1-7, but with variations in the starting sugar compositions, acid, acid load, reaction time and temperature.

Oligosaccharide preparations were synthesized as follows.

Example 9.8: 300 g of sucrose, 3 g of phosphoric acid and 27 g of water were reacted at 125° C. for approximately one hour to obtain a dark brown oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.9: React 270 g of glucose, 30 g of sucrose, 0.3 g of phenylphosphonic acid and 27 g of water at 130°C for one to four hours to produce a dark brown oligosaccharide syrup, which is then diluted to 60° Brix with distilled water. water.

Example 9.10: 225 g glucose, 75 g lactose, 3 g butylphosphonic acid and 27 g water were reacted at 130°C for one to four hours to produce a dark amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water .

Example 9.11: 225 g glucose, 75 g lactose, 3 g phenylphosphonic acid and 27 g water were reacted at 130°C for one to five hours to produce a dark amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

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Example 9.12: React 270 g of glucose, 30 g of lactose, 3 g of phenylphosphinic acid and 27 g of water at 130° C. for three to five hours to produce a dark brown oligosaccharide syrup, which is then diluted to 60° Brix with distilled water.

Example 9.13: 300 g of glucose, 3 g of phenylphosphinic acid and 27 g of water were reacted at 130°C for one to three hours to produce a dark amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.14: 300 g of glucose, 2 g of propionic acid and 27 g of water were reacted at 130°C for one to four hours to obtain an amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.15: 300 g of glucose, 0.15 g of 8-hydroxy-5-quinoline sulfonic acid hydrate and 27 g of water were reacted at 130°C for two to four hours to obtain an amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

In the above reactions, all masses refer to the masses of the pure components, and the total mass of reacting water included any additional water provided by moisture content and/or water from hydration of the reacting sugars.

Example 9.16: 10 g of glucose, 0.01 g of 8-hydroxy-5-quinoline sulfonic acid hydrate and 1 g of water were reacted at 130°C to obtain an succinic oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Example 9.17: About 8 g of glucose, about 2 g of lactose, 0.01 g of 2-pyridine sulfonic acid and 1 g of water were reacted at 130° C. to produce an amber oligosaccharide syrup, which was then diluted to 60° Brix with distilled water.

Characteristics of oligosaccharide preparations.

The resulting materials were analyzed by HPLC size exclusion chromatography (SEC) to characterize molecular weight distribution, LC-MS/MS analysis to quantify DP2 anhydro-sugar content, and 2D ¹ H, ¹³ C-HSQC NMR to determine the molecular structure of the corresponding oligosaccharide drugs.

Analysis of molecular weight of polymers using HPLC.

The number average molecular weight (MWn) and weight average molecular weight (MWw) of the oligosaccharide preparations from Examples 9.1-9.7 were determined using HPLC. SEC analysis was performed on an Agilent 1100 series HPLC with refractive index determination using an Agilent PL aquagel-OH 20 column at 40°C with distilled water as the mobile phase at 0.45 mL/min. Retention time calibration to MW was performed using standard solutions of known molecular weight, and standard prior art methods were used to determine various distribution properties from the SEC chromatogram. The MWn and MWw values of oligosaccharide preparations from many series are shown in the table below. 1.

Table 1

MWn and MWw of oligosaccharide preparations

Oligosaccharide drug MWn (g/mol) MWw (g/mol) 9.1 719 ± I 1.063 ±23 9.2 808 ±30 1.336 ± 122 9.3 757 ± 15 1.186 ±49 9.4 761 1,196 9.5 755 1.177 9.6 505 709 9.7 762 ± 12 1.154 ± 14

Analysis of anhydro-DP2 content by LC-MS/MS.

The DP2 anhydro-oligosaccharide content of the oligosaccharide preparations was determined by LC-MS/MS using a Capcell Pak NH2 column (Shiseido; 250 x 4.6 mm, 5 μm) at a flow rate of 1 mL/min under isocratic water/acetonitrile 35/65 conditions. Before MS, the flow was split 1:4 and a feed flow of 50 μL of 0.05% NH4OH was added to enhance ionization. An ESI probe in negative mode was used for MS detection, and the multiple reaction monitoring (MRM) method allowed for targeted analysis.

The anhydro-DP2 content of the oligosaccharide preparations was first determined relative to the content of the oligosaccharide preparation from Example 9.7 as a reference composition. The absolute anhydro-DP2 content of the reference oligosaccharide preparation from Example 9.7 was then determined by HPLC-MS/MS to be approximately 10%, and the anhydro-DP2 content of the oligosaccharide preparations from Examples 9.1-9.6 was then calculated. The relative and absolute content of DP2 was determined as described in Table. 2.

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table 2

Anhydro-DP2 content in oligosaccharide preparations with several series

Oligosaccharide drug Relative content of anhydro-PR2, in% compared to Example 9.7 Anhydro-PR2 content (gram anhydro-PR2/gram total DP2) 9.1 53% 5.3% 9.2 14% 1.4% 9.3 57% 5.7% 9.4 53% 5.3% 9.5 33% 3.3% 9.6 50% 5.0% 9.7 100% 10.0%

Molecular fingerprinting using 2D 1H, ¹³ C-HSQC NMR.

The molecular structures of the oligosaccharide preparations from Example 9 were characterized using 2D 1H, ¹³ C-HSQC NMR spectroscopy. Samples were prepared by drying 125 mg (dry matter basis) of the oligosaccharide preparation at 40°C and redissolving in D2O containing 0.1% acetone. NMR spectra were obtained at 300 K on either a Bruker Avance 400 MHz NMR spectrometer or a Bruker Avance III 600 MHz NMR spectrometer equipped with a cryogenically cooled 5 mm TCI probe. In fig. 1 shows an illustrative 2D 1H, ^13C HSQC NMR spectrum of an oligosaccharide preparation from oligosaccharide preparation 9.7.

Anomeric region of the spectrum 1H, ¹³ C-HSQC, F2 (1H)=4.2-6.0 ppm. and F1 ( ¹³ C) = 90-120 ppm. was used to fingerprint the bond distribution of oligosaccharide drugs. Each peak in the anomeric region was integrated and its relative abundance was determined relative to that of the entire anomeric region. 2D 1H, ^13C HSQC fingerprinting was carried out on four series of oligosaccharide preparation 9.1.

Table 3

Relative abundance of peaks F1 and F2 from oligosaccharide preparation 9.1

F2 (ppm) F1 (ppm) AUC (Mean ± SEM) 5.43 92.42 0.4% ± 0.3% 5.44 102.07 0.4% ± 0.1% 5.43 90.05 0.5% ± 0.2% 5.40 100.22 1.6% ± 0.4% 5.37 98.33 0.7% ± 0.4% 5.35 99.70 2.7% ± 0.6% 5.33 96.53 0.3% ± 0.2% 5.24 100.86 0.5% ± 0.2% 5.22 92.71 20.2% ± 3.9% 5.21 102.45 0.5% ± 0.4% 5.18 93.86 0.9% ± 0.4% 5.17 96.01 0.4% ± 0.1% 5.09 96.88 0.6% ± 0.3% 5.03 108.49 0.4% ± 0.2% 5.02 109.16 0.4% ± 0.4% 4.98 99.19 0.6% ± 0.3% 4.95 98.51 30.6% ±4.1% 4.86 98.53 0.7% ± 0.5% 4.79 96.84 0.6% ± 0.3% 4.71 103.48 2.5% ± 0.7% 4.64 103.56 0.8% ± 0.4% 4.63 102.49 0.7% ± 0.5% 4.62 104.56 1.4% ± 0.4% 4.57 97.07 1.6% ± 0.3%

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4.50 103.30 25.9% ± 2.2% 4.45 103.56 2.4% ± 1.3%

Example 10. Determination of anhydro-sugar subunits in an oligosaccharide preparation.

The relative abundance of anhydro sugar subunits in the oligosaccharide preparations of Example 9 was determined using MALDI-MS on a Bruker Ultraflex instrument. Samples were dissolved in water to a concentration of 10 mg/ml, of which 5 μl was mixed with matrix solution (30 mg/ml DHB in 80% ethanol and water in a ratio of 1:10). The plates were prepared by applying 1 μL of analyte solution to the target plate and allowed to dry in ambient air. In some cases, samples were recrystallized using 1 μL ethanol before MS analysis.

Fig. 2 presents an exemplary MALDI spectrum of the oligosaccharide preparation from Example 9. The anhydro sugar subunits are clearly observed as shifted peaks, shifted by -18 g/mol relative to its corresponding major parent DP. Shifted peaks are observed at all DP values, indicating that anhydro sugar subunits are found at all oligosaccharide sizes. The relative peak intensity of the anhydro subunits as determined was approximately 10% of the total peak intensity for each DP value, from which the relative abundance of all anhydro subunits was determined to be approximately 10%. Fig. 32A and FIG. 32B illustrates the MALDI spectra of the oligosaccharide preparation from Example 2. Anhydro sugar subunits are observed at each DP level with relative intensities in the range of 5-10%.

Example 11. Characteristics of anhydro-subunits of an oligosaccharide drug.

The anhydrosaccharide subunits of the oligosaccharide preparations from Example 9 were characterized using a combination of LC-MS, GC-MS, LC-MS/MS and NMR methods.

Characteristics of anhydro-DP1 components.

The anhydro component DP1 from the oligosaccharide preparation of Example 9 was isolated using preparative liquid chromatography. The isolated anhydro-DP1 component was obtained for NMR by dissolving it in 0.75 ml of D2O. In fig. 3 shows an exemplary 1D ¹ H-NMR spectrum of the anhydro DP1 fraction isolated from the oligosaccharide of Example 9, and FIG. 4 shows an illustrative APT ¹³ C-NMR spectrum of the same isolated fraction of anhydro DP1.

Using the following peak assignments in the table. 4, the ratio of 1,6-anhydro-beta-D-glucofuranose to 1,6-anhydro-beta-D-glucopyranose as determined by NMR was 2:1.

Table 4

NMR peak assignments

1,6-anhydride aphids rho-beta-Dzuranose 1, b-anhydro-beta-Dglucopyranose # Ή (ppm) ¹³ C (ppm) Ή (ppm) ¹³ C (ppm) 1 5.33 101.9 5.01 104.4 3.40 70.6 4.37 79.8 3.56(ov) ^a 73.0 4.27 78.3 3.56(ov) ^a 71.3 4.38 80.6 4.50 76.7 3.74 64.1 3.97; 3.64 65.7 4.14; 3.72 66.7

^a Ov means overlapping signal

Characteristics of anhydro-DP2 components.

The anhydro-DP2 content of the oligosaccharide preparations from Example 9 was determined using a combination of LC-MS, GC-MS, LC-MS/MS and NMR methods. The content of anhydro-DP2 in the oligosaccharide preparations from Example 9 was determined using GC-MS and LC-MS/MS analyses. Gas chromatography was performed using a 30 m x 0.25 mm fused silica column containing an HP-5MS stationary phase with helium at a constant pressure of 21.57 psi as the carrier gas. Aliquots were pre-derivatized by acetylation by dissolving 20 mg of sample in 0.5 ml of pyridine with 0.5 ml of acetic anhydride for 30 min at 60°C. 1 μL samples were injected at 300°C with an oven temperature program starting at 70°C and gradually increasing by 10°C/min to 315°C. Detection was performed on an Agilent 5975C MSD with an electron energy of 70 eV.

Fig. Figure 5 shows an enlargement of the GC-MS chromatogram for oligosaccharide drug 9.7. TIC and XIC plots (m/z 229) demonstrate that the DP2 and anhydro-DP2 components are clearly separated. Fig. 37A37B, 38A-38B, 39A-39B, and 40A-4B illustrate the presence of the DP1, anhydro-DP1, DP2, and anhydro-DP2 fractions as determined by GC-MS in the oligosaccharide preparation of Example 1, Example 3, Example 4, and Example 7, respectively. As shown in FIG. 37A-37B, 38A-38B, 39A-39B and 40A-40B, the anhydro-DP1 and DP1 fractions have a retention time of approximately 12-17 min, and the anhydro-DP2 and

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DP2 have a retention time of approximately 2-25 min.

Fig. 43 illustrates MALDI-MS spectra comparing the oligosaccharide preparation of the example at different laser energies. The relative abundance of the signals remained virtually unchanged, indicating that laser ionization does not result in water loss. Consequently, the presence of anhydro-sugar subunits in the oligosaccharide preparation has been proven.

Example 12. Observation of subunits from caramelization in an oligosaccharide preparation.

An oligosaccharide preparation comprising the 5-hydroxymethylfurfural subunit from caramelization was demonstrated by a combination of HPLC and 2D 1H, ^13C HSQC NMR analyses. A 50 mg aliquot of the oligosaccharide drug from Example 9 was dissolved in 0.8 ml D ₂ O. The resulting 2D 1H, ¹³ C HSQC spectrum was analyzed for the presence of a glycosidic bond between 5-hmf and the anomeric carbon of glucose with the following peak assignments: ¹ H-NMR : 8=9.39 ppm (SNO, m); 7.44 ppm (Ar-H, m); 6.68 ppm (ArH, m); 4.60 ppm (CH2, m); and 13C-NMR: -180.0; 150.6; 126.2; 112.7; 159.9; 64.5. The absence of free 5hmf was confirmed by HPLC using an Agilent 1100 HPLC system equipped with a 3000 mm Bio-Rad Aminex 87H using 25 mM aqueous sulfuric acid at an isocratic elution of 0.65 mL/min and UV detection. 5-hmf was quantified in comparison with an authentic sample. The 5-hmf peak was not observed in the oligosaccharide preparation of Example 9, but was observed after low-temperature acid hydrolysis of the oligosaccharide preparation under conditions not allowing for the formation of free 5-hmf in the hydrolysate. Fig. 6 shows an exemplary 2D 1H, ¹³ CHSQC NMR spectrum of an oligosaccharide preparation that includes the caramelization subunit.

Example 13. Comparative example.

Commercial 5 kDa dextran was analyzed by MALDI-MS for the presence of anhydrosugar subunits. Fig. 7 illustrates the clear presence of a shifted peak with a -18 g/mol shift from the main DP peak (Na+ adduct at 851.268 g/mol). In contrast, the dextran sample was found to contain virtually no anhydrous-sugar subunits.

Example 14 Quantitative determination of the anhydro-DP component using LC-MS/MS.

The DP2 fraction of the oligosaccharide preparation was isolated by liquid chromatography. Samples were dissolved in water and separated using a Capcell Pak NH ₂ column (Shiseido; 250 x 4.6 mm, 5 μm) at a flow rate of 1 ml/min under isocratic conditions of water/acetonitrile 35/65. In some cases, 50 μl of 0.05% NH ₄ OH was added after chromatographic separation to enhance ionization. The content of anhydro-DP2 was determined by MS/MS detection. An ESI probe in negative mode was used for MS detection, and the MRM method allowed for targeted analysis. Fig. 8A illustrates the detection of the oligosaccharide drug of Example 9 over the concentration range of 1-80 μg/mL in water with a linear calibration curve (shown in FIG. 8B) versus area under the LCMS/MS chromatogram by concentration.

Fig. 33A-33C, 34A-34C, 35A-35C and 36A-36C illustrate the presence of anhydroDP2, anhydro-DP1 and DP2 species detected by LC-MS/MS in the oligosaccharide preparation of Example 1, Example 3, Example 4 and Example 7. respectively.

Example 15. Preparation of feed containing oligosaccharide preparations.

Poultry and pig diets were prepared to demonstrate the inclusion of oligosaccharide drugs in the diet. Control foods showing different ingredient compositions and corresponding treated foods obtained by fortifying the corresponding control foods with the oligosaccharide preparations of Example 9 were prepared as follows.

Sample food 15.1. Control food 15.1 (CTR) was prepared using 62% corn meal and 32% soybean meal. Treated diet 15.1 (TRT) was prepared by adding 500 mg/kg of the oligosaccharide drug from Example 9 to control food 15.1 (CTR). For the treated diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on ground rice husks as a carrier and adding the powder into a mixer using a micro-ingredient scale before granulation.

Sample food 15.2. Control food 15.2 (CTR) was formulated using 62% corn meal, 3% soybean concentrate and 26% soybean meal. Treated diet 15.2 (TRT) was prepared by adding 500 mg/kg of the oligosaccharide drug from Example 9 to control food 15.2 (CTR). For the treated diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on ground rice husks as a carrier and adding the powder into a mixer using a micro-ingredient scale before granulation.

Sample food 15.3. Control 15.3 (CTR) was formulated using 52% corn meal, 6% corn starch, 5% soybean concentrate, 26% soybean meal and titanium oxide microindicator. Treated feed 15.3 (TRT) was prepared by adding 500 mg/kg of oligosaccharide drug to control feed 15.3 (CTR). For the processed diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on crushed rice husk as a carrier and adding the powder to a mixer using a micro-ingredient balance before pelleting.

Sample food 15.4. Control 15.4 (CTR) was prepared using 55% corn meal and 39% soybean meal. Treated feed 15.4 (TRT) was prepared by adding 1000 mg/kg of oligosaccharide drug to control feed 15.4 (CTR). For the processed diet, the oligosaccharide drug was provided in powder form by drying the oligosaccharide on crushed rice husk as a carrier and adding the powder to a mixer using a microingredient balance before pelleting.

Approximate feed 15.5. Control 15.5 (CTR) was formulated using 62% corn meal, 3% soybean concentrate and 26% soybean meal. Treated diet 15.5 (TRT) was prepared by adding 500 mg/kg of the oligosaccharide drug from Example 9 to control food 15.5 (CTR). For the treated diet, the oligosaccharide drug was provided in powder form, with the powder added to a mixer using a micro-ingredient scale before granulation.

Sample feed 15.6. Control feed 15.6 (CTR) was a commercial corn soy poultry starter feed from the USA. Treated Feed 15.6 (TRT) was a commercial poultry starter feed made from corn and soybeans in the United States containing 500 ppm. oligosaccharide drug. For the processed diet, the oligosaccharide drug was provided in powder form and the powder was added to a mixer using a micro-ingredient scale before pelleting.

Approximate feed 15.7. Control Feed 15.7 (CTR) was a test corn soy poultry feed with the following diet composition: corn meal 62.39%, soybean meal 31.80%, calcium carbonate 0.15%, dicalcium phosphate 2.2%, sodium chloride 0 .15%, DL-methionine 0.15%, L-lysine 0.10%, soybean oil 2.00%, vitamin-mineral premix 1.00% and coccidiostat 0.06%. Treated feed 15.7 (TRT) was prepared by adding 1000 ppm to control feed 15.7 (CTR). oligosaccharide drug from example 9.1. For the treated diet, the oligosaccharide drug was provided as a 60% syrup in water and applied by spraying the syrup onto the feed after pelleting.

Approximate feed 15.8. Control Feed 15.8 (CTR) was a test corn soy poultry feed with the following composition: wheat flour 48.45%, soybean flour 32.00%, rye 12%, calcium carbonate 0.20%, bicalcium phosphate 2.00% , sodium chloride 0.20%, DLmethionine 0.15%, soybean oil 4.00%, vitamin-mineral premix 1.00%. Treated feed 15.7 (TRT) was prepared by adding 1000 ppm to control feed 15.7 (CTR). oligosaccharide drug from example 9.3. For the treated diet, the oligosaccharide drug was provided as a 60% syrup in water and applied by spraying the syrup onto the feed after pelleting.

As one skilled in the art will appreciate, 15.1-15.6 also contain industry standard levels of fats, vitamins, minerals, amino acids, other micronutrients and feed enzymes. In some cases, the feed contained a coccidiostat, but in all cases it did not contain antibiotic growth promoters. In accordance with standard practice, feed was provided in the form of purees, pellets or crumbs.

Example 16 Extraction of feed samples.

Diets prepared according to the procedures of Example 15 were processed by extraction. Feed samples were ground in a mill. A sample of five grams of the resulting crushed food was prepared in a 50 ml volumetric flask and hot water (approximately 80°C) was added. After shaking, the mixture was incubated in an ultrasonic water bath at 80°C for 30 min. After cooling, the solution was centrifuged for 20 min at 3000 g, and the supernatant was filtered through a 1.2 μm filter and then a 0.45 μm filter (and in some cases a 0.22 μm filter). The resulting filtered solutions were evaporated to dryness on a rotary evaporator.

In some cases, extraction was performed using 50 wt.% ethanol in water as an alternative extraction solvent. In some cases, the filtration step was performed using a membrane filter with a molecular weight cutoff of 5000 Daltons.

Example 17 Enzymatic treatment of feed extracts.

The feed extracts from Example 16 were subjected to one or more enzymatic hydrolysis steps to break down the oligosaccharides naturally present in the feed. A mixture of α-amylase and amyloglycosidases was used to cleave the α-(1,4) bonds of gluco-oligosaccharides and starch. Invertase and α-galactosidase were used to remove sucrose, raffinose, and other common fiber saccharides.

Enzyme solutions were prepared as follows: amyloglucosidase (A. niger) 36000 U/g, solution 800 U/ml in ammonium acetate buffer (ammonium acetate 0.2 M, pH 5, containing 0.5 mM MgCl ₂ and 200 mM CaCl ₂ ), α-amylase (from porcine pancreas) 100,000 U/g, Megazyme: solution 800 U/ml in ammonium acetate, invertase from baker's yeast (S. cerevisiae) 300 U/mg, Sigma: solution 600 Units/ml in ammonium acetate buffer; α-galactosidase from A. Niger, Megazyme 1000 U/ml.

The dry feed extracts from Example 16 were resuspended in 10 ml of ammonium acetate buffer. 50 μl of α-amylase (final concentration 4 U/ml), 50 μl of amyloglucosidase (final concentration 4 U/ml), 50 μl of invertase (final concentration 3 U/ml) were added. If necessary, 20 μl of α-galactosidase was added (final concentration 2 U/ml). The solution was incubated for 4 hours at 60°C. The digested extract was then filtered through an ultrafiltration filter (Vivaspin Turbo 4, 5000

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MWCO, Sartorius) before evaporation to dryness in a nitrogen evaporation system. In enzymatic digestion embodiments, one or more of the above enzymes were used in combinations and the digestion period varied from 4 hours to overnight digestion. Enzyme concentrations were changed to double the above loadings, and the complete enzymatic digestion procedure was repeated several times in a row on the same feed.

Table 5

List of feed samples for extraction and digestion

Matrix Extraction solvent Filtration Enzymatic digestion 1 Control food Water 0.22 µM 2 Anhydro-oligomeric feed 1000 mg/kg Water 0.22 µM - 3 Control food Ethanol/Water 50/50 0.22 µM - Anhydro-oligomeric feed 1000 mg/kg Ethanol/Water 50/50 0.22 µM - Anhydro-oligomers Water 0.22 µM a + b (4 hours 60°C) Control food Water 0.22 µM a + b (4 hours 60°C) Anhydro-oligomeric feed 1000 mg/kg Water 0.22 µM a + b (4 hours 60°C) 10 Control food Ethanol/Water 50/50 0.22 µM - eleven Anhydro-oligomeric feed 1000 mg/kg Ethanol/Water 50/50 0.22 µM - 12 Anhydro-oligomeric feed 1000 mg/kg Water 0.45 µM a + b (4 hours 60°C) 13 Anhydro-oligomeric feed 1000 mg/kg Water 0.22 µM a + b (4 hours 60°C) 14 Control starter feed A Water 0.45 µM a + b (4 hours 60°C) 15 Anhydro-oligomeric starter feed B 2000 mg/kg Water 0.45 µM a + b (4 hours 60°C) 16 Control food Water 0.45 µM a + b + c (4 hours 60°C) 17 Anhydro-oligomeric feed 1000 mg/kg Water 0.45 µM a + b + c (4 hours 60°C) 18 Rice and spelled/ Anhydro-oligomers Water 0.45 µM 19 Rice and spelled/ Anhydro-oligomers Ethanol/Water 50/50 0.45 µM -

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20 Control food Water 0.45 µM a + b + c (4 hours 60°C) 21 Anhydro-oligomeric feed 1000 mg/kg Water 0.45 µM a + b + c (4 hours 60°C) 22 Growth feed C (control) Water 0.45 µM 23 Growth feed D (anhydro-oligomers 2000 mg/kg) Water 0.45 µM 24 Pre-start food A (control) Water 0.45 µM 25 Pre-starter feed D (Anhydrooligomers 1000 mg/kg) Water 0.45 µM 26 Growth feed C (control) Water 0.45 µM a + b + c + c! (4h 60°C) 27 Growth feed D (Anhydrooligomers 2000 mg/kg) Water 0.45 µM a + b + c + c! (4h 60°C) 28 Pre-start food A (control) Water 0.45 µM a + b + c + c! (4h 60°C) 29 Pre-starter feed D (Anhydrooligomers 1000 mg/kg) Water 0.45 µM a + b + c + c! (4h 60°C) thirty Corn Water 0.45 µM 31 Wheat Water 0.45 µM 32 Soybeans Water 0.45 µM 33 Corn Water 0.45 µM a + b + c + c! (4h 60°C) 34 Wheat Water 0.45 µM a + b + c + c! (4h 60°C) 35 Soybeans Water 0.45 µM a + b + c + c! (4h 60°C) 41 Control food Water 0.45 µM 42 Anhydro-oligomeric feed 1000 mg/kg Water 0.45 µM 43 Control food Water ultra 5000 MWCO a + b + c X2 (overnight at 60°C) 44 Anhydro-oligomeric feed 1000 mg/kg Water ultra 5000 MWCO a + b + c X2 (overnight at 60°C)

Anhydro-oligomers refer to oligosaccharides containing anhydro-subunits.

Control foods refer to nutritional compositions without added anhydro-oligomers.

Enzyme a=amyloglucosidase (A. niger) 36000 U/g Megazyme.

Enzyme b=α-amylase (from porcine pancreas) 100,000 U/g Megazyme.

Enzyme c=invertase from baker's yeast (S. cerevisiae) 300 U/mg Sigma.

Enzyme d=α-galactosidase from A. niger 620 U/g Megazyme.

Example 18. Detection of oligosaccharide drugs in feed.

Control and treated diets according to Example 15 were analyzed to detect the absence or presence of oligosaccharide drugs. After production of the feed, 1 kg samples were taken from the final feed. The extractable solids content of the feed was obtained by aqueous extraction using the procedure of Example 16. The resulting extracts were analyzed for the presence of anhydro-DP using LC-MS/MS according to the procedure of Example 14.

Fig. 9 shows the absence of anhydro-DP2 species in the control diet of Examples 15.1 (CTR) to 15.6 (CTR) compared to the presence of anhydro-DP2 species in the treated diet of Examples 15.1 (TRT) - 15.6 (TRT). Integration of the resulting LC-MS/MS chromatograms was used to determine the presence of the oligosaccharide compositions from Example 9 in the final feedstock. Specifically, feeds were determined to contain the appropriate oligosaccharide drug if the area under the anhydrPO-DP2 peak exceeded the detection limit (or any other suitable threshold specified in the method).

Example 19. Quantitative assessment of oligosaccharide drugs in feed.

The control and treated diets according to Example 15 were analyzed to determine the concentration of oligosaccharide drugs in the final feed. After production of the feed, 1 kg samples were taken from the final feed. The extractable solids content of the feedstock was obtained by aqueous extraction using the procedure of Example 16. The resulting extracts were analyzed for the presence of anhydro-DP by LC-MS/MS according to the procedure of Example 14, and the peak areas of anhydro-DP2 were compared with standard calibration curve for determining the concentration of the oligosaccharide drug in feed (Table 6).

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Table 6

Concentration of oligosaccharide drug in feed

Feed Oligosaccharide content (ppm) in control feed Oligosaccharide content (ppm) in processed feed Example 15.1 Not detected 1642 Example 15.2 Not detected 953 Example 15.3 Not detected 1912 Example 15.4 Not detected 549 Example 15.5 Not detected 406 Example 15.6 Trace amount 401

Example 20. NMR characterization of gluco-oligosaccharides containing anhydro subunits.

Gluco-oligosaccharide preparations containing anhydro subunits were characterized by (i) degree of polymerization and (ii) distribution of glycosidic bonds from glucose units.

Relative molar abundance of α-(1,1)-α, α-(1,1)-β, β-(1,1)-β, α-(1,2), β-(1,2), α (1,3), β-(1,3), α-(1,4), β-(1,4), α-(1,6), and β-(1,6) bonds were identified by NMR - spectroscopy. Chemical shifts ¹ H-NMR for anomeric protons were determined as follows: the region d=4.5-5.5 ppm was considered. with resonances from C-2 to C-6 of covalently bound clusters at ~d 3.2 and 3.9 ppm. Due to the bond with the H atom in C-2, the anomeric proton appears as a doublet, and the axial position appears at a higher field than the equatorial position. The conformation of the sugar was inferred from the binding constant of neighboring protons: equatorial-equatorial, equatorial-axial (small binding constants), or axial-axial (large binding constants).

The identification of glycosidic bonds was also carried out using ¹³ C NMR. Primary, secondary, tertiary and quaternary carbon atoms differ in proton non-resonance decoupling or polarization transfer. Carbons attached to methoxy groups resonate at a lower field than the corresponding carbon atoms with free hydroxy groups, and ring carbon atoms with axial hydroxy groups generally absorb at a higher field than the corresponding carbon atoms with equatorial hydroxy groups. Thus, following these recommendations and comparing with the chemical shifts described in the literature for 1H and ^13C of similar sugars, the purpose of most of the signals was determined.

J-RES and 1H,13C-HSQC were used to determine the binding distribution. For some samples, the HSQC method showed superior performance. For each analysis, approximately 50 mg of the lyophilized product was dissolved in D ₂ O and transferred to a 5 mm NMR tube. Any residual catalyst or solids were removed by filtration. NMR experiments were performed on a Bruker Avance III NMR spectrometer operating at 600 MHz protons, corresponding to the carbon Larmor frequency of 150 MHz. The instrument was equipped with a cryogenically cooled 5 mm TCI probe. All experiments were performed at 298 K. ¹ H-NMR spectra were recorded and calibrated in deuterated water (4.75 ppm). 13C-NMR spectra were calibrated with acetone (30.9 ppm). Data were acquired using TopSpin 3.5 and processed using ACD/Labs on a personal computer.

Fig. 10 shows a typical 2D-1H JRES NMR spectrum of a sample of gluco-oligosaccharides containing anhydro subunits, pre-saturated with solvent. The assignments of various glycosidic bonds are made in accordance with the table. 7.

Table 7

Relative molar abundance of glycosidic bonds in a sample of gluco-oligosaccharides containing anhydro subunits (2D-1H JRES NMR method)

Connection Relative molar abundance tested in laboratory I Relative molar abundance tested in laboratory II a(1,2) 10.1% 9.2% a(1.4) 2.0% 17.0% a(1,3) 4.5% 1.3% a(1.6) 28.9% 33.6% β(1,2) 5.7% 6.5% β(1,3) 13.3% 6.3% β(1,4) 17.9% 10.7% β(1.6) 18.9% 14.5%

As shown in table. 7, for some samples, discrepancies were observed between experiments performed in different laboratories using different 2D-1H JRES NMR analysis instruments.

Fig. Figure 11 shows a typical 1H, 13C-HSQC NMR spectrum of a sample of glucooligosaccharides containing anhydro subunits, with the corresponding resonances and assignments used to assign bonds. In contrast, determination by 1H, ^13C -HSQC NMR was found to be consistent between two different laboratories and instruments, as shown in Table 1. 8.

Table 8

Relative molar abundance of glycosidic bonds in four samples of gluco-oligosaccharides containing anhydro subunits (1H method, ¹³ C-HSQC NMR)

Connection Sample 1 Sample 2 Sample 3 Sample 4 Lab I Lab II Lab I Lab II Lab I Lab II Lab I Lab II a(1,2) 9.2% 9.2% 9.0% 9.5% 9.1% 9.9% 9.1% - a(1.4) 1.4% 1.3% 1.2% 1.3% 1.3% 1.3% 0.0% - a(1,3) 17.7% 17.0% 17% 17.7% 17.5% 16.7% 21.9% - a(1.6) 33.9% 33.6% 33.6% 30.9% 36.0% 31.6% 34.4% - 3(1,2) 5.7% 6.5% 5.7% 7.6% 5.2% 7.6% 5.2% - 3(1,3) 4.1% 6.3% 4.2% 6.2% 4.4% 6.1% 5.6% - 3(1.4) 8.5% 10.7% 8.9% 10.7% 7.6% 10.7% 8.3% - 3(1.6) 12.3% 14.5% 12.6% 11.6% 11.7% 11.6% 11.0% -

Diffusion-ordered NMR spectroscopy (DOSY) was performed to separate NMR signals of different species according to diffusion coefficient and hence MW. Signals in the upper part of the DOSY spectra in Fig. 12 corresponds to species with high DP value, and species with lower DP value are shown below. Fig. 12 illustrates the superposition of the 1H DOSY spectra of three oligosaccharides containing anhydro subunits in table. 8.

Example 21. Semi-preparative isolation of fractions DP1 and DP2.

Preparative isolation of the DP1 fraction was carried out by preparative HPLC using a Waters BEN Amide 19x150 mm column. For the mobile phase, water was used as solvent A and acetonitrile as solvent B, where each solvent contained 0.1% ammonia. The applied gradient is shown in table. 19. Collected DP1 fractions from 8 separations were pooled, dried and redissolved in D2O for NMR analysis as described above.

To characterize the DP2 fraction, a two-step purification was carried out. The first step was carried out in a flash chromatography system using an ELSD (evaporative light scattering detector). 2 ml (2.65 g) of the oligosaccharide preparation was diluted in 1 ml DMSO, 0.5 ml water and 0.5 ml acetonitrile. The solution was stirred and treated with ultrasound for 15 min. 1 ml of solution was injected onto a column YMC DispoPackAT, NH2, spherical beads, 25 μm, 120 g. The oligosaccharide preparation was separated using an isocratic gradient method with 75% acetonitrile in water at a speed of 40 ml/min. The resulting DP2 fraction was dried with nitrogen and redissolved in DMS/water (80:20, v/v).

For the 2nd stage of purification, a UHPLC analytical system with a YMC NH2 4.6x250 mm (5 μm) column at 40°C was used. Fraction DP2 was purified using an isocratic gradient (Table 10) and a flow rate of 1 ml/min. A 1:5 split downstream of the column was used to trigger collection by ELSD. Fraction DP2 from 12 chromatography sessions was collected, acetonitrile was removed with heated nitrogen, and residual water was removed by lyophilization. The dry fraction was redissolved for subsequent LC-MS/MS and NMR analysis.

Table 9

Gradient method

Time (min) Flow rate(ml/min) Solvent A Solvent B 0 25 10 90 2.5 25 10 90 23 25 25 75 23.1 25 10 90 47 25 10 90

Table 10

Isocratic method

Time (min) Flow rate(ml/min) Water (%) Acetonitrile (%) 0 1 25 75 15 1 25 75

Example 22. Synthesis of an oligosaccharide drug with a monotonically decreasing DP distribution.

330 g of D-glucose monohydrate and 0.3 g of (+)-camphor-10-sulfonic acid were added to a 1 L three-neck flask with mechanical stirring provided by an overhead high-torque mechanical mixer connected through a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a thermocouple rod inserted into the reaction mixture. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of several mm above the mixing element. The flask was equipped with a reflux condenser cooled by the water-glycol mixture, the temperature of which was maintained below 4°C using a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction temperature increased to 120-130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round bottom receiving flask placed in an ice bath. The reaction was maintained at 130°C with a stirring speed of 120 rpm for 60 min, and the mass of condensate collected in the collection flask was recorded over time at 10-min intervals. The reaction was stopped by adding distilled water and stopping heating. After cooling the product mixture to room temperature, an aliquot of the product syrup was diluted to approximately 1° Brix, as determined by refractive index. The diluted aliquot was microfiltered using a 0.2 micron syringe filter and analyzed by HPLC size exclusion chromatography (SEC). SEC analysis was performed on an Agilent 1100 series HPLC with refractive index determination using an Agilent PL aquagelOH 20 column at 40°C with distilled water as the mobile phase at a flow rate of 0.45 mL/min. MW retention time calibration was performed using standard solutions of known molecular weight. The DP equilibrium constant was determined to be K=3.3, and the DP distribution was found to decrease monotonically. Fig. 15 and fig. 16 illustrates the DP distribution shape of various oligosaccharide preparations from Example 9, as determined by HPLC-SEC.

Example 23. Synthesis of an oligosaccharide drug with a non-monotonic DP distribution.

330 g of D-glucose monohydrate and 0.3 g of (+)-camphor-10-sulfonic acid were added to a 1 L three-neck flask with mechanical stirring provided by an overhead high-torque mechanical mixer attached through a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a thermocouple rod inserted into the reaction mixture. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The flask was equipped with a reflux condenser cooled by the water-glycol mixture, the temperature of which was maintained below 4°C using a recirculating cooling bath.

The reaction mixture was gradually heated to 135°C with continuous stirring at a stirring speed of 80-100 rpm. As the reaction temperature increased to 130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round bottom collection flask placed in an ice bath. The reaction was maintained at 135°C with stirring at 120 rpm for 35 minutes. The reaction was stopped by adding distilled water and stopping heating. After cooling the product mixture to room temperature, an aliquot of the product syrup was diluted to approximately 1°Brix, as determined by the refractive index. The diluted aliquot was microfiltered using a 0.2 micron syringe filter and analyzed by HPLC size exclusion chromatography (SEC). SEC analysis was performed on an Agilent 1100 series HPLC with refractive index determination using an Agilent PL aquagel-OH 20 column at 40°C with distilled water as the mobile phase at a flow rate of 0.45 ml/min. MW retention time calibration was performed using standard solutions of known molecular weight. The DP distribution turned out to be non-monotonically decreasing. Fig. 16 shows that the content of DP3 is greater than that of DP2, and that the content of DP4 and DP5 is substantially equal.

Example 24. Fed-batch synthesis of an oligosaccharide drug.

330 g of D-glucose monohydrate and 0.3 g of 2-pyridine sulfonic acid were added to a liter three-neck flask with mechanical stirring provided by an overhead high-torque mechanical mixer attached through a flexible connector. The flask was secured inside a hemispherical electric heating mantle, controlled by a temperature control device through a thermocouple rod inserted into the reaction mixture. The thermocouple tip was adjusted so that it was in the reaction mixture with a gap of a few mm above the mixing element. The flask was equipped with a reflux condenser cooled by a water-glycol mixture, the temperature of which was maintained below 4°C using a recirculating cooling bath.

The reaction mixture was gradually heated to 130°C with continuous stirring at a stirring speed of 80-100 rpm. When the reaction temperature increased to 120-130°C, the apparatus was switched from a reflux condenser to a distillation configuration with a round bottom collection flask placed in an ice bath. The reaction was maintained at 130°C at 120 rpm, and the mass of condensate collected in the receiving flask was recorded over time at 20-minute intervals. After 210 minutes, another 110 g of D-glucose monohydrate was added to the reaction mixture. After 420 min, the reaction was stopped by adding distilled water and stopping heating. After cooling the product mixture to room temperature, an aliquot of the product syrup was diluted to approximately 1° Brix, as determined by refractive index. The diluted aliquot was microfiltered using a 0.2 micron syringe filter and analyzed using size exclusion

- 74 044964 HPLC chromatography (SEC). SEC analysis was performed on an Agilent 1100 HPLC with refractive index determination using an Agilent PL aquagel-OH 20 column at 40°C with distilled water as the mobile phase at a flow rate of 0.45 ml/min. MW retention time calibration was performed using standard solutions of known molecular weight. Equilibrium constant

DP was defined as K=0.8, and the DP distribution was found to decrease monotonically.

Example 25: Growth performance of commercial broiler chickens treated with an oligosaccharide preparation.

Broiler chickens were raised on the diets from Example 15.6 to determine the effect of the oligosaccharide drug on animal growth performance. Specifically, commercial corn and soybean meal poultry feeds containing a grain fermentation residue solution (DDGS), a coccidiostatic agent, and a standard micronutrient blend were produced according to industry practices and a three-phase feeding program. Through preliminary analysis, feed compositions were determined as shown in Table. eleven.

Table 11

Nutritional composition

Component Starter feed Growth feed Weaning food Method Humidity 13.0% 13.0% 12.9% AOAS 930.15 (drawer oven) Crude protein (CP) 24.1% 21.5% 19.6% AOAC 992.15; AOAC 990.03 Fat (EE) 3.2% 3.8% 3.9% AOAC 920.39 (ether extraction) Crude fibers (CF) 2.7% 2.4% 2.4% AOAS 962.09 (hydrolysis) Mineral Residue (AR) 5.2% 4.3% 4.3% AOAS 942.05 (muffle furnace) NFE, by difference 51.9% 55.1% 56.9% Calculated: 1-CP-EECF-AR Total 100.0% 100.0% 100.0%

Control (CTR) and treated (TRT) diets were prepared for each phase as described in Example 15.6, where the treated diets were prepared by fortifying the control diet with one pound per US ton treated using the oligosaccharide preparation from Example 9.7. In total, approximately 50 US tons of each ration were produced.

Hubbard M99xCobb 500 chicks were obtained at hatch day from a commercial hatchery and randomly housed in 36' x 40' pens constructed in a tunnel-ventilated house with a dirt floor. A total of approximately 30,000 birds were housed, with each pen containing the same number of birds. The bedding in the room consisted of loose bedding covered with fresh wood shavings. A standard commercial environmental and lighting program was used. Animals and living quarters were checked daily, including recording of general health status, feed intake, water supply and room temperature. Fatalities were recorded daily.

Birds in odd-numbered pens were fed a treated diet (ie, containing 2 lb/ton of feed additive), and birds in even-numbered pens received a control diet. All diets were provided ad libitum through automatic feeders in each pen and feeding trays from days 1 to 7. Water was supplied without restriction from the drinking nipple line.

The start phase ran from day 0 to day 13, the rearing phase from day 14 to day 27, and the stop phase from day 28 to the end of the study, day 31. The weight of the birds in the pen was recorded on days 0 and 31. For each pen, the total weight of the birds consumed was recorded. stern. Weight gain and FCR were then determined for each pen according to standard industry practice.

On day 31, six male birds were randomly selected from each pen for blood and cecal sampling. The live weight of each selected bird was recorded. The blood sample was collected by wing puncture into Vacutainer tubes and frozen after coagulation and serum separation. Each selected bird was then sacrificed by cervical dissection followed by cecal removal using standard veterinary techniques. After dissection, the cecal contents were transferred into 15 ml conical tubes, the weight of the cecal contents was recorded, and the contents were snap frozen to -80°C.

Based on the weight of the selected birds, the experimental group showed an increase of 11 points in body weight, significant at P < 0.05 (by ANOVA).

Example 26: Shotgun sequencing of poultry cecal microbiota.

Relative abundance of identified taxa was determined for 96 sampled birds taken from Case Study 25. For each microbiota sample obtained from Case Study 25,

- 75 044964 the contents of the cecum were thawed and DNA was extracted using standard methods. Extracted DNA was analyzed on an Illumina HiSeq-X instrument with 2x150 bp reads. Standard analyzes were performed to process the raw sequencing data, including: trimming (adapter, BBDuk), entropy filtering (k=5, window=20, min=50, BBDuk), qualitative filtering (average Q20, BBDuk), filtering Gallus (Bowtie2). Taxonomic assignments were determined from the MetaPhlAn2 database (db_v20).

Example 27. Microbial conversion of undigested feed and oligosaccharide preparations.

The effect of oligosaccharide preparations on the profile of metabolites produced by microbial fermentation of undigested feed was assessed ex vivo on poultry cecal microbiota obtained from selected birds from Example 25.

Aliquots of the test oligosaccharide preparations were diluted to 20 wt.% oligosaccharide in water, microfiltered through a syringe filter with a pore size of 0.22 μm, and degassed under anaerobic conditions. A poultry feed hydrolysate (simulating cecal digestion) was prepared and simultaneously sterilized by suspending a 10 g sample of commercial corn and soy broiler feed in 50 ml water, followed by two cycles of autoclaving at 120°C for 5 min, s subsequent aqueous extraction and resuspension. The resulting feed hydrolyzate was degassed under anaerobic conditions.

Extracted cecal content samples were thawed anaerobically and used to prepare a 20% suspension in phosphate-buffered saline (PBS), pH 7.4, containing 15% glycerol. The resulting suspension of cecal contents was analyzed to confirm that its phylogenetic composition closely matched that of the originally sampled microbiota (sequenced in Example 24). Cecal content suspension was assessed using 16S rRNA analysis (16Sv4 PCR/Illumina MiSeq sequencing at 2s250 bp) according to a standard 16S rRNA pipeline (USEARCH and SILVA(v4) DB) with a rarefaction cutoff of 12,230 reads per sample. By number of phyla, the cecal content suspension included approximately 70% Firmicutes, 20% Bacteroidetes, 7% Tenericutes, and the remainder as Proteobacteria, Cyanobacteria, Actinobacteria, and Verrucomicrobia.

When working under anaerobic conditions, an aliquot of the suspension was centrifuged at 2000 g, the supernatant was pipetted and the pellet was resuspended to form a 1% suspension of cecal contents in minimal growth medium consisting of a sterile aqueous mixture of 900 mg/l sodium chloride, 26 mg/l calcium chloride dihydrate, 20 mg/l magnesium chloride hexahydrate, 10 mg/l manganese chloride tetrahydrate, 40 mg/l ammonium sulfate, 4 mg/l iron sulfate heptahydrate, 1 mg/l cobalt chloride hexahydrate, 300 mg/l dibasic potassium phosphate, 1.5 g/l dibasic sodium phosphate, 5 g/l sodium bicarbonate, 0.125 mg/l biotin, 1 mg/l pyridoxine, 1 mg/l pantothenate, 75 mg/l histidine, 75 mg/l glycine, 75 mg/l l tryptophan, 150 mg/l arginine, 150 mg/l methionine, 150 mg/l threonine, 225 mg/l valine, 225 mg/l isoleucine, 300 mg/l leucine, 400 mg/l cysteine and 450 mg/l proline .

For each oligosaccharide preparation in Example 9, a 25 μl aliquot of a 20 wt% oligosaccharide solution, a 225 μl aliquot of the feed hydrolysate, and 250 μl of a 1% cecal contents suspension were loaded in triplicate into wells of a 96 well deep well microtiter plate (e.g., Costar plates 3958). Each plate contained a set of empty wells prepared by combining 25 μl of water, 225 μl of digested food and 250 μl of a 1% suspension of cecal contents. The loaded plate was then incubated at 37°C for 45 hours under anaerobic conditions. After incubation, the contents of the wells were transferred into 1.5 ml Eppendorf tubes, microcentrifuged at 2000 g for at least 5 min, and the resulting supernatant was collected.

Example 28. Stimulation of goblet cell proliferation with oligosaccharide preparations.

Proliferation of goblet cells in the presence of oligosaccharide preparations from example 9 was assessed in vitro. Wells in a 96-well Falcon plate were seeded with 200 μl of growth medium and 40,000 goblet cells and grown for three days. Oligosaccharide preparations from Example 9 were retested (n=6 wells per oligosaccharide) at a concentration of 0.1 mg/ml compared to control (nutrient medium only). The cells were kept for differentiation for four days with complete replacement of the culture medium after two days. The plates were then washed with phosphate-buffered saline (PBS) and fixed by treating the cells with 4% paraformaldehyde (Cellomics #28906, diluted 1:4 in PBS) for 30 min. The plates were then washed twice with PBS and stained with the mucin dye Alcian blue (Abcam ab150662 pH 2.5). Staining was performed by incubating the cells in an acetic acid solution for 3 min, followed by incubating the cells in an Alcian blue solution for 30 min at room temperature, followed by a 2-min wash with water and two final washes with distilled water.

Nuclei were quantified by Hoechst staining and automated microscopy (10x objective, 49 fields) with a field size of 641 x 641 microns. At a concentration of 0.1 mg/ml, various oligosaccharide preparations provided an increase in goblet cell proliferation of approximately 1% to 5% (Table 12).

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Table 12

Number of goblet cells

Oligosaccharide drug Increase in cell number compared to control P-value Example 9.1 3.2% 0.003 *** Example 9.3 2.3% 0.007 *** Example 9.4 3.1% 0.002 *** Example 9.5 3.6% 0.001 *** Example 9.6 2.7% 0.007 *** Example 9.7 1.2% 0.115 Example 9.16 3.5% 0.001 *** Example 9.17 4.9% 0.001 ***

Example 29. Stimulation of mucus production with oligosaccharide preparations.

The effect of oligosaccharide drugs on mucus production by goblet cells was assessed in vitro. Wells in a 96-well Falcon plate were seeded with 200 μl of growth medium and 40,000 goblet cells and grown for three days. Oligosaccharide preparations were tested repeatedly (n=6 wells per oligosaccharide) by adding to the cell growth medium the supernatant obtained by ex vivo fermentation of the oligosaccharide preparations in Example 27. For each oligosaccharide preparation, the corresponding supernatant from Example 27 was diluted 1:20 with culture medium and subjected to microfiltration. The control was obtained by diluting the fermentation medium 1:20. The cells were kept for differentiation for four days with complete replacement of the culture medium after two days. The plates were then washed with phosphate-buffered saline (PBS) and fixed by treatment with 4% paraformaldehyde (Cellomics #28906, diluted 1:4 in PBS) for 30 min.

The plates were then washed twice with PBS and stained with the mucin dye Alcian blue (Abcam ab150662 pH 2.5). Staining was performed by incubating the cells in an acetic acid solution for 3 minutes, followed by incubating the cells in an Alcian blue solution for 30 minutes at room temperature, followed by a 2-minute wash with water and two final washes with distilled water. Nuclei were quantified by Hoechst staining and automated microscopy (10x objective, 49 fields) with a field size of 641 x 641 microns.

Mucin production was quantified by absorbance at 600 nm on a Spectramax plate reader. To account for differences in cell numbers between wells, optical density was normalized to OD600 per 100,000 cells. There was a clear and statistically significant effect on mucus production by goblet cells, as shown in Table 1. 13.

Table 13

Mucus production

Oligosaccharide drug Increased mucin production S.E.M. P-value Example 9.2 7.9% 0.2% 7.21E-05 * * * Example 9.1 11.1% 0.4% 2.4E-05 * * and Example 9.6 9.1% 0.3% 0.00013 * * * Example 9.4 -0.8% 0.0% 0.561442 Example 9.7 9.7% 0.4% 0.000192 * * * Example 9.17 9.0% 0.4% 0.00112 * * * Example 9.5 9.1% 0.3% 2.34E-05 * * and Example 9.3 -4.1% 0.1% 0.002309 * * *

Example 30: Stimulate the integrity of a healthy intestinal barrier.

The effects of oligosaccharide preparations on healthy intestinal barrier function were assessed in vitro. Caco-2 cells were cocultured with goblet cells on a semipermeable membrane, differentiated, and then treated with the microbiome supernatant of Example 27 prepared using the cecal microbiota samples of Example 25. Transendothelial electrical resistance (TEER) changes were performed relative to control. Specifically, Caco-2 and goblet cells were seeded onto a semi-permeable insert and placed in a chamber containing a suitable growth medium, as shown in FIG. 18. Cells were grown for 13 days to obtain a differentiated monolayer with tight junctions, as confirmed by performing a basic TEES measurement.

Multiple oligosaccharide preparations from Example 9 were analyzed with n=6 replicates. For each oligosaccharide preparation, the corresponding supernatant from Example 27 was diluted 1:20 with growth medium, microfiltered through a 0.2 micron filter, and applied

- 77 044964 on the apical side of the chamber. Control was provided by using a diluted nutrient medium

1:20. After 24 h, TEES measurements were performed for each oligosaccharide preparation. For each drug, the change in TEES after treatment was determined compared to the value before treatment.

The change in TEES for the control was normalized to 100%.

Statistically significant improvements in healthy intestinal barrier function measured by TEES were observed for several oligosaccharide drugs. The improvement in TEES for different oligosaccharide preparations varied from approximately 0% to 15% relative to control. In particular, the oligosaccharide preparation from example 9.2 provided an increase in TEES by 14% compared to the control. The results are presented in table. 14.

Table 14

Increase in TEES compared to control for synthetic oligosaccharide drugs

Oligosaccharide drug Increased TEES compared to control Example 9.1 8%±2% Example 9.2 14% ± 1% Example 9.3 2% ± 1% Example 9.4 9% ± 2% Example 9.6 15%±1% Example 9.7 2% ± 1%

Example 31. Stimulation of nonspecific immune function.

The effect of oligosaccharide preparations on nonspecific phagocytosis was determined in vitro. Piglet granulocytes were analyzed by flow cytometry for the uptake of opsonized E. coli cells in the presence of the oligosaccharide drug from Example 9.3 at two differential concentrations: 1 mg/ml and 2 mg/ml. The number of granulocytes with engulfed pathogens was compared with the number of granulocytes that engulfed the pathogen to determine the phagocytic index.

In the presence of 1 mg/ml of the oligosaccharide drug from Example 9.3, the phagocytic index was determined to be 82, while at a concentration of 2 mg/ml the phagocytic index was determined to be 106, reflecting a 29% increase in pathogen uptake. Fig. 19 is an exemplary flow cytogram illustrating the identification of granulocytes with and without engulfed pathogens.

The effects of oligosaccharide drugs on the immune and inflammatory response were determined in vivo in broiler chickens exposed to common pathogens.

Birds were fed in experimental groups using two types of background diet, as described in Table. 15. Diets containing oligosaccharide preparations were given to the experimental groups described in Table. 16.

Table 15

Types of background rations

Ingredient Type 1 (mass%) Type 2 (mass%) Corn 62.39 - Soy flour 31.80 32.00 Wheat - 48.45 Calcium carbonate - 12 Calcium carbonate 0.15 0.20 Dicalcium phosphate 2.2 2.00 NaCl 0.15 0.20 DL-methionine 0.15 0.15 L-lysine 0.10 - Soybean oil 2.00 4.00 Vitamin and mineral premix 1.00 1.00 Coccidiostatic 0.06 -

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Table 16

Experimental groups

Experimental groups Background diet Oligosaccharide drug Dose (ppm) Control 1 Type 1 No Control 2 Type 2 No Experience 1 Type 2 Example 9.17 1,000 Experience 2 Type 2 Example 9.6 1,000 Experience 3 Type 2 Example 9.1 1,000 Experience 4 Type 2 Example 9.4 1,000 Experience 5 Type 2 Example 9.8 1,000 Experience 6 Type 2 Example 9.3 1,000

Day-old Cobb 500 broiler chicks were obtained from a commercial hatchery. On the day of arrival (day -8), chicks were housed in groups of 50 for an induction period of 8 days. During the run-in period, animals received a control diet. On day 0, animals were divided by weight into groups of 8 birds each and received a 10-fold dose of the attenuated oral coccidial vaccine Paracox-5 (MSD Animal Health) (a mixture of E. acervulina, E. maxima, E. mitis and E. tenella ) by gavage to all birds on diet type 2. Each group was then placed in a cage battery and randomly assigned to treatment groups, each with two replicates per group.

The animals were kept in a room with a controlled environment and temperature adapted to the age of the birds. Water and all rations were provided without restrictions. On days 7 and 14, birds from each replicate were randomly selected and killed. The selected birds were immediately dissected. Standard techniques were used to obtain samples of ileal contents, cecal contents, ileal tissue, and Peyer's patch tissue. Tissue samples were analyzed by PCR array for the expression of genes relevant to inflammation and immune system cytokines and chemokines.

At day 7, the expression of ΓΕ12β, IL18, IFNy, IL10, IL22, IL1β, IL21 and IL6 in Peyer's patches of birds in control group 2 (type 2 diet) increased by at least 1.5-fold compared with birds in the control group 1 (diet type 1), as shown in FIG. 20. Ileal expression of IL12e, IFNy, IL17a, IL16, IL21, IL8L1 and TNFSF13e was also increased by at least 1.5-fold in control group 2. Cytokine and chemokine expression data suggested an inflammatory and immune response in birds fed the diet type 2 exposed to Eimeria pathogens.

An improved immune response was observed in birds in experimental groups 3 and 6, which were given the oligosaccharide preparations from examples 9.1 and 9.3, respectively. According to the expression of cytokines and chemokines, on day 7, increased activity of the adaptive immune system was observed, corresponding to T-helper type 2, as well as stimulation of granulocytes and stem cells. Expression data were consistent with increased differentiation of multipotent hematopoietic stem cells into myeloid progenitor cells, increased granulocyte proliferation, and general activation of T cells, B cells, and mast cells.

By day 14, interleukin expression data suggested regulation of the immune system and anti-inflammatory pathways, including IL9 signaling from regulatory T cells and anti-inflammatory myokine activity of IL6. Expression data indicated an enhanced adaptive immune response to pathogens associated with the type 2 diet, followed by a more rapid return to homeostasis compared to control group 2 birds that were exposed to the pathogenic stimulus but did not receive oligosaccharide drugs.

Example 32. Restoring the damaged integrity of the intestinal barrier.

Multiple oligosaccharide preparations from Example 9 were analyzed in n=6 replicates. For each oligosaccharide preparation, the corresponding supernatant from Example 27 was diluted 1:20 with growth medium, microfiltered through a 0.2 micron filter, and applied to the apical side of the chamber. Control was provided by using a nutrient medium at a dilution of 1:20. After 24 h, TEES measurements were performed for each oligosaccharide preparation. For each, the change in TEES after drug use was determined compared to TEES before drug use. The change in TEES for the control was normalized to 100%.

The effect of oligosaccharides on the barrier function of leaky gut was assessed in vitro. Both the direct effect of the oligosaccharide preparations themselves and the indirect effect of microbiome products derived from the oligosaccharide preparations were assessed. Caco-2 and goblet cells were plated on a semipermeable insert and inserted into a chamber containing a suitable growth medium and treated with the various oligosaccharide preparations from Example 2 or the appropriate supernatant from Example 3. Barrier integrity was assessed using transendothelial electrical resistance and by measuring fluorescent permeability dye.

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Caco-2 and goblet cells were seeded onto a semi-permeable insert and placed in a chamber containing a suitable growth medium as shown in FIG. 21. Oligosaccharide preparations, negative control containing medium only, and positive control containing medium only, were evaluated in n=6 replicates. For each of them, cells were grown for 13 days until a differentiated monolayer was obtained. Cells were washed and TEES measurements were performed to establish a baseline value. Control was normalized to 100%.

Multiple oligosaccharide preparations from Example 9 were analyzed in n=6 replicates. For each oligosaccharide preparation tested, the corresponding syrup from Example 9 was diluted to 5% with distilled water, then further diluted 1:20 with distilled water, microfiltered through a 0.2 micron filter, and applied to the apical side of the chamber. To evaluate the indirect effect for each oligosaccharide drug tested, the corresponding supernatant from Example 25 was diluted 1:20 with distilled water, microfiltered through a 0.2 micron filter, and applied to the apical side of the chamber. Control was provided by using a nutrient medium at a dilution of 1:20.

After determining the basal value of TEES, the rhamnolipid apical stressor was applied to the apical chamber for each of the oligosaccharide drugs and the positive control. Lucifer Yellow fluorescent dye was applied to the apical chamber for each of the oligosaccharide preparations, positive control and negative control. After three hours in the presence of the apical stressor and Lucifer Yellow dye, a second TEES measurement was performed to assess the effect of the stressor on barrier integrity. Barrier permeability was assessed by measuring the extent of Lucifer Yellow penetration from the apical to basolateral side of the cell layer and membrane.

Statistically significant improvements in impaired intestinal barrier function, measured by TEES, and dye permeability were observed for several oligosaccharide drugs. The improvement in TEES for different oligosaccharide preparations varied from approximately 0% to 30% relative to control. Both direct and indirect effects of oligosaccharide drugs were observed.

Specifically, the oligosaccharide formulation from Example 9.3 provided a significant direct reduction in dye permeability compared to the control, while the oligosaccharide from Example 9.2 provided a significant indirect reduction in dye permeability compared to the control. The oligosaccharide from Example 9.7 provided neither a direct nor indirect reduction in dye permeability, and direct application of the oligosaccharide from Example 9.6 exacerbated the disruption of barrier function due to the apical stressor.

Example 33: Meta-analysis of studies on live growth performance of broiler chickens.

The effects of oligosaccharide formulations on live growth performance of commercial broiler chickens were assessed in vivo through a series of independent studies conducted across different regions, seasons, basal diet types, avian genetics and management practices, including litter treatments and coccidiosis control programs. In each study, birds were assigned to experimental groups, including one control group and one or more experimental groups. The control group was fed only the background diet. The experimental groups were given a background diet supplemented with a certain dose of oligosaccharide preparations from Example 9. In some studies, a commercial feed additive used in poultry farming was included as a comparative example.

For each study, birds were housed in pens located in a typical broiler house with a specified number (Hd/Rep) of birds in each pen. Statistical replicates were performed by randomly assigning pens to experimental groups with a specified number (Reps/Trt) of replicates per experiment. In table Table 17 provides detailed protocol information for each study included in the analysis.

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Table 17

Protocol details for each study

Study A country Season Duration Type of diet Replication/experiment Quantity/repetition Genetics Floor Litter Program against coccidiosis Etc. 36.1 USA Spring 35 Corn/Soybeans 6 14 Cobb 500 M/F Use Saccox Etc. 36.2 USA Winter 49 Corn/Soybeans 12 60 Cobb 500 M/F Use Maxiban Etc. 36.3 Canada Winter 35 Corn/Soybeans 8 60 Ross 708 M Use Saccox Etc. 36.4 USA Winter 49 Corn/Soybeans 12 60 Cobb 500 M/F Use Maxiban Etc. 36.5 Conn. Kingdom N/A N/A Wheat/Soybean N/A N/A N/A N/A Clean No Etc. 36.6 USA Winter 33 Ku ku ru for / Soya 12 100 Cobb 500 M/F Use Amprol Etc. 36.7 USA Summer 49 Corn/Soybeans 12 18 Hubbard M99 M Use No Etc. 36.8 Canada Winter 42 Ku ku ru for / Soya 12 17 Ross308 M Use Vaccine Etc. 36.9 Great Britain Autumn 42 Wheat/Soybean 16 35 Ross308 M Fresh No Etc. 36.10 France Autumn 42 Wheat/Soybean 17 thirty Ross308 M Fresh No Etc. 36.11 USA Autumn 42 Corn/Soybeans 21 40 Cobb500 M Use Vaccine Etc. 36.12 France Summer 36 Ku ku ru for / Soya 12 18 Cobb500 M Fresh Vaccine Etc. 36.13 Canada Spring 42 Ku ku ru for / Soya 10 20 Ross708 M Use Vaccine Etc. 36.14 USA Spring 42 Ku ku ru for / Soya 14 40 Cobb500 M Use Vaccine Etc. 36.15 New Zealand Spring 35 Wheat/Soybean 12 20 Ross308 M Fresh Vaccine

Study results included bird weight (BW), feed intake (FI), feed conversion ratio (FCR), mortality rate (per head) and mortality rate. The pen was a statistical unit. Spatial blocking was used whenever possible, and experimental groups were randomly assigned to blocks.

Background rations.

Birds were fed phased diets according to local commercial practices for a total study duration of 35 to 49 days. Starter phase diets were typically provided in kibble form from the time birds were housed until day 15 of the study. All diets did not contain growth promoting antibiotics. The designs of the starter control diet are described in Table. 18 (NA=data not available from the site).

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Table 18

Compositions of the starter control diet

Study Corn flour, % Wheat flour, % Soy flour, % Residue solution after corn fermentation, % Unpurified protein Unrefined fat True Metabolic Value (kcal/kg) D NN And S S h Methionine (SID) Etc. 36.1 63.5 N/A 27.4 N/A 22.1 N/A 2988 1.35 N/A Etc. 36.2 0 N/A 0 N/A N/A N/A N/A N/A n/a Etc. 36.3 63.4 N/A 28.3 n/a 20.9 n/a 2940 1D4 n/a Etc. 36.4 N/A n/a N/A n/a N/A n/a N/A N/A n/a Etc. 36.5 N/A N/A n/a n/a N/A n/a N/A N/A n/a Etc. 36.6 0 N/A 0 n/a N/A n/a ZON N/A n/a Etc. 36.7 0 N/A 0 n/a N/A n/a N/A N/A n/a Etc. 36.8 54.52 0 34.38 5 22.23 2.81 2900 1.24 0.63 Etc. 36.9 0 51.78 30.5 0 21.31 5.74 2899 1.251 0.622 Etc. 36.10 0 55.1 28 0 22.49 5.42 2899 1.237 N/A Etc. 36.11 58,353 2,377 29,992 5 20.3 N/A 2900 1.33 N/A Etc. 36.12 N/A N/A N/A N/A 22 N/A ZON n/a n/a Etc. 36.13 56.08 0 34.1 5 22.23 2.31 2900 1.24 0.63 Etc. 36.14 58,353 0 29,992 5 20.3 N/A 2900 1.33 n/a Etc. 36.15 0 54.92 28.31 5 N/A 6.9 2900 1.24 n/a

Growth phase diets were provided in pellet form from days 16 to 24. The compositions of control growth diets are described in detail in Table. 19 (N/A=data not available from the site).

Table 19

Control diet compositions for growth phase

Study Corn flour, % Wheat flour, % Soy flour, % Residue solution after corn fermentation, % Unpurified protein Unrefined fat True Metabolic Value (kcal/kg) Lysine (SID) Methionine (SID) Ex.36.1 68.6 N/A 22 N/A 19.95 N/A 3059 1.2 N/A Ex.36.2 N/A N/A N/A N/A N/A N/A N/A N/A n/a Ex.36.3 65.6 n/a 26.3 n/a 19.9 N/A 2988 1.06 n/a Ex.36.4 N/A n/a N/A n/a N/A N/A N/A N/A n/a

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Project 36.5 N/A N/A N/A N/A n/a n/a N/A N/A n/a Project 36.6 n/a N/A n/a n/a n/a n/a 3102 N/A n/a Project 36.7 n/a N/A n/a n/a n/a n/a N/A N/A n/a Project 36.8 54.95 0 28.31 10 20.8 3.88 3000 1.11 0.56 Project 36.9 0 57.135 26 0 19.01 6.55 2997 1.08 0.533 Project 36.10 0 55.77 24 0 20.94 7.4 2998 1.11 N/A Project 36.11 65,383 0.344 20,404 10 17.5 N/A 3040 1.33 n/a Project 36.12 N/A N/A N/A n/a 19 N/A 3035 n/a n/a Project 36.13 56.53 0 28.02 10 20.8 3.39 3000 1.11 0.56 Project 36.14 65,383 0 20,404 5 17.5 N/A 3040 1.14 N/A Project 36.15 0 57.3 23.05 6 N/A 6.27 3000 1.11 N/A

Terminal phase diets were provided in pellet form from days 16 to 24. The compositions of control diets of the final phase are described in detail in Table. 20 (N/A=data not available from the site).

Table 20

Compositions of the control diet of the final phase

Study Corn flour, % Wheat flour, % Soy flour, % Residue solution after corn fermentation, % Unpurified protein Unrefined fat True Metabolic Value (kcal/kg) Lysine (SID) Methionine (SID) Etc. 36.1 74.3 N/A 27.4 N/A N/A N/A 3155 1.06 N/A Etc. 36.2 N/A N/A N/A N/A n/a N/A N/A n/a n/a Etc. 36.3 70.8 n/a 28.3 n/a n/a n/a 3059 0.94 n/a Etc. 36.4 N/A n/a N/A n/a n/a n/a N/A N/A n/a Etc. 36.5 N/A n/a n/a n/a n/a n/a N/A N/A n/a Etc. 36.6 N/A n/a n/a n/a n/a n/a 3203 N/A n/a Etc. 36.7 N/A n/a n/a n/a n/a n/a N/A N/A n/a Etc. 36.8 57.37 0 24.85 10 19.4 5.16 3100 1.03 0.55 Etc. 36.9 0 59.94 23 0 17.53 7.71 3097 1.003 0.503 Etc. 36.10 0 87.67 21 0 19.53 8.88 3099 0.994 N/A Etc. 36.11 69.41 0.12 16,879 10 16 N/A 3084 1.01 N/A Etc. 36.12 N/A N/A N/A n/a N/A N/A N/A N/A N/A Etc. 36.13 58.95 0 24.56 10 19.4 4.66 3100 1.03 0.55 Etc. 69.41 0 16,879 5 16 N/A 3084 1.01 N/A 36.14 Etc. 36.15 0 62.02 17.62 6 N/A 7.25 3100 0.99 n/a

Experimental groups.

For each study, experimental groups were created to compare the effect of oligosaccharide drugs with a control diet. For selected studies, experimental groups were designed to evaluate the dose-response curve for oligosaccharide drugs. Treated diets were prepared by mixing a sufficient amount of the appropriate oligosaccharide preparation from Example 9 such that the final oligosaccharide content reached the target dose (units ppm on a dry solids basis). In separate studies, a comparative example (ref. 36) was provided by a commercial whole yeast product (Diamond V XPC Original). The experiment was carried out in accordance with table. 21.

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Table 21

Distribution of experimental groups

Study Experimental group 1 Experimental group 2 Experimental group 3 Experimental group 4 Experimental group 5 Experimental group 6 Experimental group 7 Etc. 36.1 Control Etc. 9.7 (500 ppm) Etc. 36.2 Control Etc. 9.7 (500 ppm) Etc. 36.3 Control Etc. 9.7 (500 ppm) Etc. 36.4 Control Etc. 9.7 (500 ppm) Etc. 36.5 Control Etc. 9.7 (100 ppm) Etc. 9.7 (500 ppm) Comp. Project 36 (1500 ppm) Etc. 36.6 Control Etc. 9.7 (500 ppm) Etc. 36.7 Control Etc. 9.7 (500 ppm) Etc. 36.8 Control Etc. 9.7 (500 ppm) Etc. 9.3 (500 ppm) Etc. 36.9 Control Etc. 9.7 (500 ppm) Etc. 36.10 Control Etc. 9.7 (500 ppm) Etc. 9.2 (500 ppm) Etc. 9.3 (500 ppm) Comp. Etc. 36 (1250 ppm) Etc. 36.11 Control Etc. 9.7 (500 ppm) Etc. 9.2 (500 Etc. 9.3 (500 ppm) Etc. 9.4 (500 Etc. 9.5 (500 Comp. Etc. 36 ppm) ppm) ppm) (1250 ppm) Etc. 36.12 Control Etc. 9.7 (500 ppm) Etc. 9.3 (500 ppm) Etc. 36.13 Control Etc. 9.2 (100 ppm) Etc. 9.2 (250 ppm) Etc. 9.2 (500 ppm) Etc. 9.2 (750 ppm) Etc. 9.2 (1000 ppm) Etc. Control Etc. 9.3 (100 ppm) Etc. 9.3 (250 ppm) Etc. 9.3 (500 ppm) Etc. 9.3 (750 ppm) Etc. 9.3 (1000 ppm) Etc. 9.7 (500 ppm) 36.14 (continued, 8-13) Etc. 9.2 (100 ppm) Etc. 9.2 (250 ppm) Etc. 9.2 (500 ppm) Etc. 9.2 (750 ppm) Etc. 9.2 (1000 ppm) Comp. Etc. 36 (1250 ppm) Etc. 36.15 Control Etc. 9.2 (100 ppm) Etc. 9.2 (250 ppm) Etc. 9.2 (500 ppm) Etc. 9.3 (100 ppm) Etc. 9.3 (250 ppm) Etc. 9.3 (500 ppm)

Standard equipment and methods known in the art were used to prepare both the background and treated diets. For the treated diets, oligosaccharide preparations and comparison products were prepared on top of the background diet and added to the pre-pellet mixer. Inclusion of oligosaccharides was confirmed by feeding assay.

Living growth phase and sampling.

Food and water were provided without restrictions. Commercial lighting and temperature programs were implemented in each study in accordance with local industry practices in the respective region. Pens were checked daily and the number and percentage of any deaths recorded in the study log. No veterinary intervention was required.

For each diet phase, total pen weight gain, initial and final bird numbers, and total feed intake for each pen were measured. For each pen, average bird weight (BW) was calculated by dividing the total weight in the pen by the number of birds in the pen at the time of weighing. For each paddock, the feed conversion ratio (FCR) was calculated by dividing the total intake

- 84 044964 feed per interval for the total weight gain of the corresponding paddock. FCRs were adjusted for mortality (FCRma) by adding the overall percentage of deaths for the period. To account for differences in feather weight, FCRma was adjusted for total body weight to obtain a adjusted FCR (cFCR) for each pen using methods known in the art. The correction factor was determined for different avian genetics using published growth day-specific BW and FCR targets for the respective genetics.

In the selected studies, one bird was randomly selected from each pen for sampling on either day 15 or the last day of the study. For each sampled bird, 5 ml of blood was collected from a wing vein into vacuum serum containers. After coagulation, the serum was separated by centrifugation, recovered, and frozen on dry ice for later processing. Each selected bird was then euthanized according to local ethical procedures and necropsied. Cecal contents were collected into 5 mL conical tubes and immediately frozen for whole microbiome genome sequencing and cecal metabolomics. Minor ileal tissue resection was performed, and the tissue was processed to deactivate RNA and frozen for subsequent gene expression analysis.

Example 34: Meta-analysis of a study.

A statistical meta-analysis of the in vivo studies from Example 36 was performed to evaluate the effects of oligosaccharide feed additives and comparator products on poultry performance compared to birds fed control diets. The analysis used a linear mixed model with treatment group as a fixed effect and random effects for study nested within block. Statistical analysis was performed in R version 3.4.4 (2018-03-15). The results were assessed using the least squares method with statistical significance P<0.05. Pairwise comparisons were performed using Tukey's method, assigning letter designations: a, b, c, d,... Treatments without a common letter in their Tukey grouping were significantly different in pairwise comparisons at P < 0.05.

Feed conversion ratio.

Study effects for cFCR were significant at P < 0.05. The oligosaccharide treatment provided a cFCR improvement of at least 2.7% at 500 ppm versus the control diet, compared to the comparative example, which provided a 2.2 unit improvement in cFCR at 1250 ppm. The oligosaccharide from Example 9.4 provided a 6.4 unit improvement in cFCR when included at 500 ppm. The results of the meta-analysis for cFCR are presented in Table. 22.

Table 22

Meta-analysis of cFCR

Experimental group cFCR (Is average) S.E. df Tukey grouping D cFCR (compared to control) Control 1.6511 0.043 14 d Etc. 9.2 (500 ppm) 1.6019 0.044 14 A -4.9 Etc. 9.3 (500 ppm) 1.6106 0.044 14 abc -4.1 Etc. 9.4 (500 ppm) 1.5876 0.044 14 A -6.4 Etc. 9.5 (500 ppm) 1.5948 0.044 14 ab -5.6 Etc. 9.7 (500 ppm) 1.6246 0.043 14 bc -2.7 Comp. Etc. 36 (1250 ppm) 1.6293 0.044 14 With -2.2

The oligosaccharide treatment groups showed greater consistency of effect compared to Comparative Example 36. For each oligosaccharide included across multiple studies, the consistency of its effect on cFCR was assessed by determining the proportion of studies that observed a given value of cFCR improvement compared to control. For example, the oligosaccharide from Example 9.2, when included at 500 ppm, provided at least 3 units of cFCR improvement in 80% of studies, at least 4 units of cFCR improvement in 60% of studies, at least 5 units of cFCR improvement in 40% of studies, and at least 6 units of cFCR improvement in 40% of studies. The comparator at inclusion of 1250 ppm provided a cFCR improvement of 3 units in only 25% of the studies and did not provide a cFCR improvement of 4 units or greater in any of the studies.

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Table 23

Consistency of the effect of experimental group on cFCR

Experimental group 1 improvement unit 2 upgrade units 3 upgrade units 4 upgrade units 5 units of improvement 6 upgrade units Etc. 9.2 (500 ppm) 100% 80% 80% 60% 40% 40% Etc. 9.3 (500 ppm) 100% 83% 67% 67% 50% 33% Etc. 9.7 (500 ppm) 85% 85% 38% 23% 15% 0% Comp. Etc. 36 (1250 ppm) 100% 100% 25% 0% 0% 0%

A clear dose-dependent response was observed between cFCR and the degree of oligosaccharide inclusion in the diet. For the oligosaccharide of Example 9.2, a 2.4 unit improvement in cFCR was observed at 100 ppm inclusion (P>0.05), a 3.7 unit improvement in cFCR was observed at 250 ppm inclusion (P<0.05), an improvement A cFCR of 6.4 units was observed at 1000 ppm inclusion (P<0.05).

Bird weight.

Study effects for BW were significant at P < 0.05. The oligosaccharides provided an increase in body weight of at least 48.9 grams over the control diet at 500 ppm, compared to the comparative example which provided a 39.6 g increase in body weight at 1250 ppm. The oligosaccharide from Example 9.5 provided an increase in body weight of 81.8 g compared to the control when included at 500 ppm. The results of the meta-analysis of poultry weight are presented in Table. 24.

Table 24

Meta-analysis of poultry weight

Experimental group BW (Ismean) S.E. df Tukey Grouping ABW (g vs control) Control 2.755 FROM 14 A 0 Etc. 9.2 (500 ppm) 2.817 from 14 b 62.6 Etc. 9.3 (500 ppm) 2,807 from 14 b 52.6 Etc. 9.4 (500 ppm) 2.838 114 14 b 83.3 Etc. 9.5 (500 ppm) 2.837 114 14 b 81.8 Etc. 9.7 (500 ppm) 2,804 FROM 14 b 48.9 Comp. Etc. 36 (1250 ppm) 2,794 FROM 14 b 39.6

Group homogeneity.

Birds fed the 500 ppm oligosaccharide formulations showed improved group uniformity compared to birds fed the control diet. For each treatment group, group homogeneity was assessed by calculating the proportion of bird weight that was within ±5% of the mean bird weight for the corresponding study. On average across all studies 36.1-36.15, 81.7% of birds fed the control diet had a weight within ±5% of the average bird weight, while 91.3% of birds fed the oligosaccharide-supplemented diets from Example 9.2 is within ±5% of the average bird weight. The effect of homogeneity was significant at P < 0.01, as measured by the nonparametric Ansari-Bradley test.

Example 35: Reduced inflammation and improved nutrient absorption in broilers fed oligosaccharides.

The effects of oligosaccharides on the inflammatory response and nutrient absorption were assessed in vivo in commercial broiler chickens. Day-old broiler chicks (Cobb 500) were obtained from a commercial hatchery (Joseph Grelier S.A., Elevage avicole de la Bohadiere, F-49290 Saint-Laurent de la Plaine, France).

Treatment and diets.

Birds were fed standard commercial corn/soybean meal diets containing solids from grain fermentation but no growth promoting antibiotics or coccidiostats. The diets were fed in two feeding phases: starter phase diets were fed from days 1 to 22 and were formulated to provide 210 g/kg crude protein and 12.5 MJ/kg ME. Growth phase diets were fed from days 23 to 36 and were designed to provide 190 g/kg crude protein and 12.9 MJ/kg ME. All diets were supplemented with feed enzymes phytase (Ronozyme HiPhos) in

- 86 044964 standard dose of 100 mg per kg of feed.

On the day of study acceptance (day 1), birds were housed in floor pens with 18 birds per pen and wood shaving bedding. Pens were randomly assigned to one of four treatment groups with 12 replicates per treatment. The control group was fed diets containing no added oligosaccharides, while the experimental groups were fed diets produced by adding oligosaccharides to the control diet. The different experimental groups are presented in Table. 25.

Table 25

Description of experimental groups

Experimental group Application of oligosaccharides A (Control) No IN Example 9.1 (500 ppm) WITH Example 9.3 (500 ppm) D Example 9.7 (500 ppm)

Cultivation and sampling.

The birds were kept in a controlled environment with temperature and lighting adjusted to the age of the birds. During the first few days of the study, an infrared electric lamp was placed in each pen. Food and water were provided ad libitum, with food provided as crushed pellets for the first ten days of the study and as pelleted food for the remainder of the rearing period. Pen weight and feed intake were recorded on days 1, 22 and 26. Mortality was recorded daily.

On day 36, one bird was randomly selected from each pen, sacrificed, and necropsied. Ileal and cecal contents were collected and immediately frozen for analysis. An area of approximately 1 cm ² of ileal tissue approximately 10 cm below the origin of the jejunum was processed to preserve RNA and genetic material for gene expression analysis.

Gene expression analysis.

Ileal tissue was analyzed using the Gut Health Array to quantify the expression levels of 18 genes associated with inflammatory response and immunity, 5 genes associated with intestinal integrity, and 4 genes associated with nutrient absorption. Gene expression levels were compared between birds fed diets containing oligosaccharides and birds fed a control diet. Statistically significant effects were observed for four genes in birds fed diets containing 500 ppm of the oligosaccharide from Example 9.3, as shown in FIG. 22. A 1.5-2-fold increase in the expression of immunity genes and anti-inflammatory genes IL1B, IL4, IL10 was observed. A twofold increase in the carbohydrate transporter gene SLC5A10 was observed. These data indicate reduced ileal inflammation and improved nutrient absorption in birds fed the oligosaccharide from Example 9.3 compared to birds fed the control diet. In contrast, birds fed diets containing the oligosaccharide from Example 9.1 showed increased expression of the IFNG and IL17B genes, which are associated with a pro-inflammatory response.

Example 36 Effect of oligosaccharides on birds exposed to an inflammatory stimulus.

The effects of oligosaccharides on local and systemic immune responses, inflammatory responses, organ health, and intestinal morphology were assessed in vivo in broiler chickens.

Treatment, diets and inflammatory stimulus.

Day-old male birds (Cobb 500) were housed in battery cages of ten birds per cage. For the first eight days of the study, all birds were fed the same control diet, consisting of a conventional poultry corn-soy diet supplemented with phytase (Ronozyme HiPhos) at a standard dose of 100 mg per kg feed, but without growth promoting antibiotics or coccidiostats. .

Starting on day 9, cells were distributed into four experimental groups, two cells per experimental group. Detailed information about each experimental group is presented in Table. 26.

Table 26

Composition of experimental groups

Experimental group Diet (days 1-8) Diet (days 9-28) Inflammatory stimulus Application of oligosaccharides A (Control) Control Control No No B (Stimulus) Control With incentive cocci-vac (Yuh) No C(Treatment 1) Control With incentive cocci-vac (Yuh) Example 9.3 (500 ppm) D (Treatment 2) Control With incentive cocci-vac (Yuh) Example 9.2 (500 ppm)

Experimental group A (control group) continued to receive the same diet as before day 8. Experimental group B (stimulus group) received the control diet with the addition of

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1% potato protein to cause mild nutritional stress on the digestive tract.

Experimental group C received the stimulus diet supplemented with 500 ppm of the oligosaccharide from Example 9.3.

Experimental group D received the stimulus diet supplemented with 500 ppm of the oligosaccharide from Example 9.2.

The birds were kept in an environmentally controlled facility where food and water were provided ad libitum. On day 14, all birds in experimental groups B, C and D received a 10-dose of coccidial vaccine (Paracox®-5, MSD Animal Health) to induce an inflammatory gut response to sporulated oocysts of attenuated Eimeria acervulina, E. maxima, E. mitis and E. tenella.

Selection of biological samples.

On each of days 21 and 28, one cell was removed from each experimental group. Birds from each cage were euthanized and dissected for biological sampling and analysis. Ileal tissue and Peyer's patches were collected and processed for gene expression and histological analysis. Blood samples were collected to analyze the local immune response by phenotyping lymphocytes (Peyer's patches) and the systemic immune and inflammatory response by measuring immunoglobulin (IgA) and alpha-glycoprotein (an acute phase response protein produced by the liver). Blood was also analyzed for diamine oxidase (DAO) levels, and liver tissue was collected for histology.

Performing the analysis both one and two weeks after application of the inflammatory stimulus provided an assessment of the effect of oligosaccharides on both the amplitude and time course of the animal's immune and inflammatory response.

Absorption of nutrients by the ileum.

Ileal gene expression analysis showed that the inflammatory stimulus in Experimental Group B significantly impaired the ileal absorption of carbohydrates, proteins, and phosphorus-containing nutrients. A 2-3-fold decrease in the expression of transport genes SI, SLC34A2 and SCL15A1 was observed in Experimental Group B with an inflammatory stimulus, compared to control group A on day 21.

In contrast, experimental groups C and D, receiving the oligosaccharides from Examples 9.3 and 9.2, respectively, showed a decrease in ileal absorption of less than 0.5. At day 28, phosphorus absorption as indicated by SLC34A2 expression for Experimental Groups C and D was statistically similar to control and improved as shown in FIG. 23.

Ileal villi quality and goblet cell number.

Images of ileal tissue collected from birds in Experimental Group D compared with those in Experimental Group B are shown in FIG. 24. Birds in experimental group D showed significantly increased villous length and less inflammatory damage as shown by ileal histology. Thus, birds fed certain oligosaccharides from Example 9 exhibited increased resistance to ileal tissue damage resulting from an acute inflammatory stimulus compared to birds not fed the oligosaccharides from Example 9.

A significant (P<0.05) increase in the number of ileal goblet cells was observed in group D compared to inflammatory stimulus-treated birds in group B (FIG. 25). Goblet cells are responsible for the production of mucin among other key functions of the ileum. Therefore, oligosaccharide-fed birds receiving the specific oligosaccharides from Example 9 exhibited an improved ability to maintain healthy ileal function under acute inflammatory stimulus compared to birds not receiving the oligosaccharides from Example 9.

T cell differentiation.

As shown in FIG. 26, birds in experimental groups C and D showed increased proliferation of T helper cells compared to birds in group B receiving an inflammatory stimulus. Birds in group D exhibited a level of T helper cells, measured as a percentage of total Peyer's patch lymphocytes, that was statistically indistinguishable from the percentage for unstimulated birds in group A. In addition, birds in groups C and D showed a lower percentage of lymphocytes, differentiated as T-cytotoxic cells, compared with birds in group B receiving an inflammatory stimulus.

Liver histology and acute phase liver proteins.

Liver histology and blood analysis showed that birds in experimental group C exhibited less inflammatory damage compared to birds in group B, indicating that the oligosaccharide from Example 9.3 provides protection against liver damage (Fig. 27A). Histological evaluation was performed as shown in FIG. 27V. Birds in group C showed approximately 50% less increase in liver damage than birds in group B, compared with the control group A without the inflammatory stimulus. Birds in group B showed a significant increase in circulating

- 88 044964 acute phase hepatic alpha-glycoprotein (AGP) (Fig. 27C). By day 28 of the study, birds receiving the oligosaccharide from Example 9.3 exhibited blood levels of AGP that were statistically indistinguishable from control birds in Group A that did not receive the inflammatory stimulus.

Increased immunoglobulin yield and diamine oxidase levels. Serum immunoglobulin A (IgA) levels were measured to observe the effect of oligosaccharide drugs on the immune response and recovery rate of birds exposed to inflammatory conditions. IgA is an antibody involved in immune function and the inflammatory response, especially of membranes. At day 21, one week after application of the inflammatory stimulus, birds in group B showed an increase in serum IgA concentration of approximately 2.25 mg/ml compared to approximately 1.85 mg/ml in unstimulated control birds in group A. In contrast, , birds in group D, treated with the oligosaccharide preparation from Example 9.2, showed an increased IgA response of approximately 4.4 mg/ml. On day 28, two weeks after application of the inflammatory stimulus, birds in group B showed a serum IgA concentration of approximately 4.35 mg/ml, whereas birds in group D showed a serum IgA concentration that had decreased to approximately 2.1 mg/ml. It was concluded that birds treated with the oligosaccharide preparation from Example 9.2 exhibited a faster immune response and faster recovery from an inflammatory stimulus. Serum IgA levels increased more rapidly and fell back to baseline control levels in Group A more rapidly than in stimulus-exposed birds in Group B that were not given the oligosaccharide drug.

Serum diamine oxidase (DAO) levels were measured to observe the effect of oligosaccharide drugs on the inflammatory response and recovery rate of birds exposed to an inflammatory stimulus. DAO is involved in histamine regulation and inflammatory pathways. At day 21, one week after application of the inflammatory stimulus, birds in groups B and D showed a decrease in serum DAO concentration of approximately 25-30% compared to a concentration of approximately 28.5 μg/ml for unstimulated control birds in the group A. The decrease in DAO concentration in group D birds receiving the oligosaccharide from Example 9.2 was more pronounced than in group B birds not receiving the oligosaccharide preparation. On day 28, two weeks after application of the inflammatory stimulus, up to experimental error, birds in group D showed a return to baseline DAO concentrations, as observed in unstimulated birds in group A. It was concluded that birds treated with the oligosaccharide drug from Example 9.2, exhibited a similar inflammatory response and faster restoration of homeostasis compared to birds not receiving the oligosaccharide.

Example 37 Microarray analysis of gene expression.

The expression of all known genes in the ileum of broiler chickens was performed using microarray analysis. Ileal tissue from broilers from Example 36.4 was analyzed to determine the effect of oligosaccharide drugs and doses on gene expression in the ileum compared to control birds not given the oligosaccharide drug. Tissue samples were prepared according to the procedures of Examples 33 and 36.

To prepare for microarray analysis, RNA samples were amplified and labeled in a T7 RNA polymerase-based reaction. The Agilent Low Input Quick Amp Labeling Kit, Single Color was used to generate fluorescent cRNA (complementary RNA) with a sample RNA input of 100 ng of total RNA for single color processing. The method used a mixture of T7 RNA polymerase, which simultaneously amplified the target material and included cyanine 3-CTP. Typically, amplification was at least 100-fold from total RNA to cRNA.

Double-stranded cDNA was produced by reverse transcription performed in the presence of labeled ribonucleotides, yielding microgram quantities of labeled RNA for array hybridization. Labeled cRNA was hybridized onto microarray slides overnight at a concentration of 1.65 μg per sample. After hybridization (at least 17 hours), the slides were washed and scanned on an Agilent SureScan Mircroarray scanner. Data were analyzed using the Partek Genomics package. Mechanistic and ontological grouping was done as shown in Table. 27.

Table 27

Mechanistic and ontological grouping

Mode of action Gene association Gut Integrity CLDN1, CLDN2, CLDN3, CLDN5, ZO1, ZO2, MUC2, FOXP1, MUC, OCLN, TFF2, TJP1 Inflammation/Immunity ALPI, C5, CCL1, CCL20, CRP, CSF1, CSF2, FASLG, GATA3, IFNG, IL10, IL12A, IL16, IL17A, IL17B, IL18, IL1B, IL2, IL21, IL22, PD IL4, IL4, IL5, IL6, IL8L1 , LYZ, NoDl, NoS2, ТВХ21, TGFB2, TLR21, TLR4, TNFSF10, TNFSF13B Absorption nutrients SCL15A1, SI, SLC34A2, SLC5A10, MGAM, SLC6A19, SLC6A14, SLC7A9, SLC6A20, SLC6A6, SLC1A1, SLC1A5, SLC38A3, SLC36A1, SLC3A1, SLC38A5

In addition, untargeted analysis was performed using the following multistep approach. Ten annotated genes with the largest fold change observed between oligosaccharide-fed animals compared to controls were identified. Ten annotated genes with the lowest fold change observed between oligosaccharide-treated animals compared to controls were also identified. Finally, the ten annotated genes with the highest statistical significance of differences between oligosaccharide-treated and control animals were analyzed. Pathway analysis was performed against Gallus gallus KEGG pathways using Genome Analysis and Gene Expression software (Partek, St. Louis).

Example 38. Method for stimulating recovery from coccidiosis.

Coccidiosis is a parasitic disease of the intestinal tract of animals caused by exposure to coccidia protozoa, including, for example, Eimeria acervuline, Eimeria maxima, Eimeria mitis, Eimeria tenella, Toxoplasma gondii, Hammondia spp. Cryptosporidium parvum, Cryptosporidium muris, Cryptosporidium hominis, Isospora canis, Isospora ohioensis, Isospora burrosi, Isospora felis and others.

Broiler chickens from Example 36 were exposed to the following levels of sporulated oocysts derived from early coccidia lines: 5000-6000 Eimeria acervuline HP oocysts, 2000-2300 Eimeria maxima CP oocysts, 1000-1300 Eimeria maxima MFP oocysts, 10,000-13,000 E oocysts imeria mitis HP and 5,000 - 6,500 Eimeria tenella HP oocysts. Exposed birds exhibited a strong inflammatory response as evidenced by serum IgA production, serum DAO, liver histology, and intestinal luminal histology from Example 36.

Birds in Group D from Example 36 that were given 500 ppm of the oligosaccharide formulation from Example 9.2 showed significantly less intestinal and liver damage and faster restoration of homeostasis compared to birds not receiving the oligosaccharide. Oligosaccharide-fed birds also showed improved nutrient absorption and improved T-cell differentiation compared to non-oligosaccharide-fed group B birds with inflammatory stimulation.

Example 39. Method for increasing tolerance to vaccines against coccidiosis.

Birds from Example 35 demonstrated reduced liver damage, improved intestinal histology, improved nutrient absorption, and faster recovery from inflammation when exposed to a 10-fold dose of a commercial coccidiosis vaccine used in broiler production.

Example 40: Scaling up repeat batches for production.

The production scale of oligosaccharide preparations was increased to a 720 L tank reactor with an overhead stirrer. Twelve batch reactions using the scaled procedure derived from Example 9.2 were performed on a 720 L scale. The resulting oligosaccharide formulations were characterized according to predefined quality control acceptance criteria to perform batch validation and evaluate process stability. Residual catalyst in the final product was determined on a batch basis using the procedure from Example 26.

For twelve batches, process conditions such as temperature, reaction time, and reaction pressure were deliberately varied within a range from the nominal conditions of Example 9.2 to evaluate the sensitivity of the resulting product to reasonable variations in process conditions that might be expected in a typical manufacturing environment. For selected batches, an in situ viscosity probe was used to monitor the viscosity of the reactor contents as a function of time. In some batches, reaction stop times were used for in-process control (IPC) based on continuous viscosity measurement. Quantities of materials, including reactant metering, distillation water, and condensate released, were measured either by mass using load cells on the reactor and auxiliary tanks, or by volumetric flow and time.

The final water content of the reactor product was measured by Karl Fischer titration of a typical aliquot of the reactor contents taken at the end of the reaction, i.e. before pH neutralization and dilution. At a reaction temperature of 120°C, the water content in the reaction product was determined to be 8 and 9 wt.% water, without conversion to dry matter. At a reaction temperature of 130°C, the water content in the reaction product was determined to be from 5 to 7 wt.% water without conversion to dry matter.

The appearance of the resulting oligosaccharide syrup of all batches was determined by visual inspection to be caramel syrup. Total dissolved solids were determined by Karl Fischer titration, residual monomer content, MWn and MWw were determined by HPLC/GPC chromatography, pH was determined by a calibrated pH meter, and anhydro-DP2 content was determined by LC-MS/MS. As shown in table. 28, the following batch characterization data were obtained (N/R=not shown).

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Table 28

Characteristics of oligosaccharide preparations

[arty Sol. pH Residual DPI MWn MWw Content of anhydro- solid, substance, wt.% catalyst wt.% DP2 (g anhydro-DP2/ g total DP2) 27.1 66.4 N/D N/A 17.5 777 1218 0.84% 27.2 68.8 z, z N/R 17.9 735 1091 0.91% 27.3 69.4 3.1 0.095 14.8 807 1276 N/R 27.4 71.0 N/D 0.068 15.5 793 1241 1.04% 27.5 70.9 3.2 0.057 15.8 777 1196 1.15% 27.6 70.9 z, z N/A 16.3 773 1170 N/A 27.7 70.7 3.0 n/a 15.7 783 1226 1.13% 27.8 70.5 3.9 n/a 16.1 785 1182 1.09% 27.9 71.1 4.1 n/a 17.1 761 1169 1.09% 27.10 70.4 4.1 n/a 16.3 778 1193 1.15% 27.11 70.5 4.7 n/a 18.6 696 995 1.33% 27.12 70.9 3.9 n/a 16.7 769 1194 1.12%

Example 41. Regulation of pH of an oligosaccharide preparation.

The pH value of the oligosaccharide preparation from Example 9.2 at a solids content of 50 wt.% was determined in triplicate by diluting 5.00 ± 0.05 g aliquots of the oligosaccharide preparation in 1.80 ± 0.02 ml of deionized water and vortexing to obtain uniform concentration. The pH value of each aliquot was measured using a calibrated pH meter (VWR, Symphony B30PCI) to obtain an average pH value of 2.4.

To 1.2 kg of the oligosaccharide preparation from Example 9.2 was added 6.53 ml of a 1.0 M aqueous solution of sodium hydroxide. The resulting mixture was stirred vigorously until a homogeneous syrup was obtained with an adjusted pH. The pH of the resulting adjusted syrup was then determined at 50 wt% solids in triplicate as described above to obtain an average pH of 4.1.

The pH adjustment procedure was repeated for replicate batch syntheses at different scales, but with some variations in procedure to provide a basis for the composition of the oligosaccharide product. For one batch, pH adjustment was performed as the final reaction step before diluting the reaction water. In another batch, pH adjustment was performed simultaneously with the dilution step by first dissolving the required amount of base in the dilution water; thus, the base and diluent water were added together to stop the reaction in one step to obtain the final syrup with the desired pH. In another batch, the base came in the form of food grade sodium hydroxide granules. In another batch, 10 ppm food grade silicone emulsion (Dow Xiameter AFE-0100) was added to the reaction mixture before dilution and pH adjustment.

Example 42. Preparation of a glass-like powder composition from an oligosaccharide preparation.

Approximately 50 g of the oligosaccharide preparation from Example 9.1 was spread on a drying tray and placed in a forced convection heater at 60° C. to produce a brittle, caramel-colored glass. The glass was removed from the drying tray and ground using a rotary shear mill to obtain a flowable light orange powder. The particle size of the powder was determined by sieving and ranged from 100 to 2000 microns, with 90% of the mass less than 1350 microns. The true density of the coarse crushed powder, determined by a helium pinkometer, was 1.3063 g/ml. The resulting powder was free-flowing.

The forming procedure was repeated using a hammer mill to obtain a fine powder with 90% by weight of the powder having a particle size of less than 196 microns. The true density of the finely ground powder was 1.5263 g/ml. The resulting powder was neither stable nor free-flowing.

DSC measurements were carried out on powders using two temperature cycle programs. In the first program, the temperature was shifted to 160°C from 0°C at a rate of 5°C/min, then annealed again to 0°C at a rate of -5°C/min, followed by a final return to 160°C. In the second program, the temperature was shifted to 50°C from -50°C at a rate of 5°C/min, annealed to -60°C at a rate of -5°C/min, and then heated to 60°C at a rate 5°C/min. The powder was found to have a glass transition temperature of 20 to 40° C., depending on the residual water content of the solid from 5 to 10 wt.% moisture.

The process of grinding the composition was repeated for each of the oligosaccharide preparations from example 9.2, example 9.3, example 9.4 and example 9.5. The powders were easily redissolved in water and water-alcohol mixtures, but did not dissolve in acetone, methanol, and anhydrous ethanol.

Example 43. Preparation of a powder composition loaded onto a carrier.

Equal weights of 70 wt.% aqueous syrup of the oligosaccharide preparation from Example 9.2 and diatomaceous earth were combined at room temperature to obtain a stable free-flowing powder. The resulting powder contained about 35 wt.% adsorbed oligosaccharide (based on dry solids) and about 50 wt.% carrier. The particle size distribution of the powder was measured by sieving. 10% by weight of the powder had a particle size of less than 290 microns, 50% by weight of the powder had a particle size of less than 511 microns, and 90% by weight of the powder had a particle size of less than 886 microns. The powder was resistant to segregation and cohesion as determined by standard aeration and compressibility tests. The true density of the resulting powder, measured with a helium pycnometer, was 1.8541 g/ml.

Formation with carrier loading was repeated using feed grade silica to obtain a stable free-flowing powder having an oligosaccharide formulation content of at least 50 wt.% (on a dry solids basis) relative to the final powder. The true density of the resulting powder was 1.5562 g/ml.

Example 44 Preparation of an extruded solid form.

A solid extruded product was prepared by mixing 20% of the oligosaccharide preparation from Example 9.2 with coarse wheat flour, and preparing the mixture through a twin screw jacketed dry matter extruder to obtain a free-flowing powder with a particle size of 0.2 to 3.0 mm, with 90% by weight less than 2 mm in size. The resulting powder was free-flowing and stable.

Example 45. Preparation of stable powder compositions.

Solid compositions, including those of Examples 42-44, were evaluated to determine their stability and hygroscopicity. The powders of Examples 43 and 44 were resistant to segregation and agglomeration, while the powder from Example 42 was unstable to segregation.

Samples of each powder composition studied were placed in sealed climate chambers at 50% relative humidity and 65% relative humidity for up to two weeks at 25°C. Of the molds tested, some showed little or no weight gain when exposed to humidity, and remained free-flowing after a two-week exposure period. The fine powder of Example 23 was found to be unstable when exposed to moisture.

Example 46. Determination of residual catalyst in oligosaccharide preparations.

The residual content of the acid catalyst in the oligosaccharide preparations was determined by ion chromatography. 80 to 100 mg of the powdered oligosaccharide formulation (prepared, for example, as described in Example 42) was dissolved in exactly 1.00 ml and centrifuged to remove particles, if necessary. The resulting solution was analyzed by ion chromatography at 30°C using a Thermo Dionex ICS-3000 system equipped with a conductivity detector, an Ion Pac AS19A 4x250 mm column, an Ion Pac AS19G 50 4x50 mm precolumn and a continuously regenerated CR-ATC anion precolumn using KOH in water as eluent. Elution was performed at 10 mM KOH for the first ten minutes after administration, followed by a gradient elution, increasing linearly to 55 mM KOH after 25 min, then decreasing to 10 mM KOH after 26 min, and maintaining the level of 10 mM KOH until the end of the program.

For the oligosaccharide preparation of Example 9.2, the concentration of residual catalyst was determined from a standard calibration curve constructed using an authentic sample of (+)camphor-10-sulfonic acid. A typical batch of the oligosaccharide formulation from Example 9.2 was analyzed and the residual catalyst concentration was determined to be 0.62 mg/g of 70 wt.% syrup.

Example 47. Quantitative analysis of residual catalyst concentration for batch acceptance.

The catalyst residual determination in Example 46 was compared to the lot acceptance criterion to determine the suitability of the lot for further use. The acceptance limit for the concentration of residual catalyst in the oligosaccharide product formulation was previously established to be <1.0 mg per gram of syrup product. The catalyst residual was measured to be 0.62 mg per gram of syrup product. Thus, the acceptance criterion was met for the tested lot and the lot was accepted for further use.

Example 48. Preparation of the product in the form of syrup.

The oligosaccharide preparation of Example 9.7 was adjusted to pH 4.2 with food grade sodium hydroxide according to the procedure of Example 41. The resulting syrup was packaged in a 20-liter bottle with a tamper-resistant cap. Immediately before sealing the container, a 500 g sample was taken and subjected to quality control. Was

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Claims (3)

The total solids content of the syrup is verified to be greater than 70% by weight according to FCC methods APPENDIX X: Carbohydrates (starches, sugars and related substances): Total solids content. Reducing sugars have been confirmed to be less than 50% as D-glucose on a dry matter basis according to the FCC method APPENDIX X: Carbohydrates (starches, sugars and related substances): Analysis of reducing sugars. The sulphated ash content has been verified to be less than 1% of the dry matter weight using the FCC Method, APPENDIX II: Physical Tests and Determinations: C. OTHER: Mineralization Residue (Sulphated Ash), Method II (for liquids). The sulfur dioxide content was less than 40 mg/kg using the optimized Monier-Williams method. The lead content was less than 1 mg/kg using the AOAC Official International Method 2013.06. The total aerobic microbial count by plate assay was confirmed to be below 1000 CFU/g using the methods described in Chapter 7 of the CMMEF. The total number of yeasts and molds was less than 100 CFU/g using the international method approved by AACC 42-50. Coliform levels were confirmed to be less than 10 MPN/g using the method described in Chapter 4 of the FDA BAM. E. coli was confirmed to be less than 3 MPN/g using the FDA BAM Chapter 4 method. Salmonella was confirmed to be undetectable in the 25-gram sample according to the method specified in Chapter 5 of the FDA BAM. Staphylococcus aureus was found to be below 10 CFU/g using the FDA BAM method, Chapter 12. The color was determined to be caramel by visual analysis. The container was sealed, the remaining sample was frozen and stored for future use, and a certificate of analysis was issued for the resulting batch. Example 49. Preparation of treated drinking water. Drinking water containing 250 ppm. oligosaccharide drug from example 9.7 was prepared as follows. 37 ml of the oligosaccharide syrup from Example 33 and 40 g of potassium sorbate were gradually added to 50 gallons of drinking tap water in a 55 gallon blue polymer drum. The solution was stirred manually using a paddle mixer for 10 min at room temperature. The method was repeated without the inclusion of potassium sorbate. Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are presented by way of example only. The invention is not intended to be limited to the specific examples presented in the specification. Although the invention has been described with reference to the above specification, the descriptions and illustrations of embodiments herein are not intended to be construed in a limiting sense. Numerous variations, changes and substitutions will be apparent to those skilled in the art without departing from the invention. Moreover, it should be understood that all aspects of the invention are not limited to the specific images, configurations or relative proportions set forth in the present invention, which depend on a variety of conditions and variables. It should be understood that various alternatives to the embodiments described in the present invention may be used in the practice of the invention. Accordingly, the invention is also intended to cover any such alternatives, modifications, variations or equivalents. CLAIM 1. A method of treating or preventing dysfunction of the gastrointestinal barrier in an animal in need thereof, comprising administering a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation to said animal, wherein said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree of polymerization, selected from 1 to n (fractions from DP1 to DPn), where n is an integer greater than 3; and wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% oligosaccharides containing anhydro subunits, as determined by relative abundance by mass spectrometry. 2. A method of treating or preventing an infection in an animal in need thereof, comprising administering a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation to said animal, wherein said synthetic oligosaccharide preparation contains at least n fractions of oligosaccharides, each of which has a different degree polymerization selected from 1 to n (fractions DP1 to DPn), where n is an integer greater than 3; and wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% oligosaccharides containing anhydro subunits, as determined by relative abundance by mass spectrometry. 3. The use of a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation in an animal, wherein said synthetic oligosaccharide pre- -