EA045265B1 - METHODS FOR MODULATION OF GASTROINTESTINAL METABOLITES - Google Patents

Description

Related applications

This application claims priority to U.S. Provisional Patent Application No.

62/757446, filed November 8, 2018, US Provisional Patent Application No. 62/757471, filed November 8, 2018, and US Provisional Patent Application No. 62/757475, filed November 8, 2018, the disclosures of each of which are incorporated herein by reference in its entirety.

Prior Art

Gut microorganisms such as bacteria, viruses, fungi, molds, protozoa, etc. are responsible for converting undigested and unabsorbed components of an animal's diet into thousands of biologically active metabolites. These metabolites, in turn, interact with the local and systemic physiology of the animal.

Under normal conditions, the biochemical output of the microbiome is determined by the composition of the food consumed by the animal. In a traditional diet, especially diets containing plant fiber polysaccharides such as cellulose, lignin, hemicellulose, pectins and starch-bound protein, part of the food consumed by the animal remains undigested and not absorbed during the primary digestion process. These unabsorbed species reach the lower intestine, where they can be processed and used by the microbiota and converted into metabolites. Thus, the composition of the resulting metabolome is influenced by the structure of the unabsorbed components of its diet.

Metabolites produced in the intestine can be absorbed, for example, through the colonic or portal vein circulatory system, and transported to other organs of the animal, where they can affect the structure and/or function of those organs. These biochemicals in turn influence various biological functions such as nutrient absorption, energy regulation, mitochondrial function, systemic inflammation, stress response, liver function, kidney function, cardiometabolic function, satiety, mood, and responsiveness.

In some cases, metabolites produced by the microbiome are harmful to the host or otherwise undesirable due to their effects on the animal's environment. For example, processing indole into indoxyl sulfate and p-cresol into p-cresol sulfate places additional stress on the animal's liver and kidneys. Indoxyl sulfate is also associated with poorer cardiovascular health. These factors are especially relevant in high protein diets, which are typical for both production and domestic animals. Microbial conversion of carnitine to trimethylamine and amino acids to ammonia not only increases the burden on the liver and kidneys, but also creates negative consequences for the welfare of both food producing animals and pets. Ammonia and trimethylamine that accumulate in litter can increase the incidence of foot diseases and lead to environmental degradation due to the strong odor of these volatile nitrogenous substances. Therefore, there is a need for tailored nutritional compositions, including animal feeds, that modulate the intestinal metabolome by selectively inhibiting the production of undesirable metabolites and improving animal health.

In some cases, the metabolites produced are beneficial to the host or otherwise necessary. For example, neurotransmitters can have a positive effect on an animal's health and mood. Other metabolites can have a positive effect on the quality of animal meat, such as taste and aroma. Therefore, there is also a need for tailored nutritional compositions, including animal feeds, that modulate the intestinal metabolome, selectively promoting the production of essential metabolites and improving animal health.

Various non-medicinal feed additives to improve growth performance are considered. Unfortunately, many of these alternative feed additives are quite difficult to incorporate into the feed and deliver to the appropriate part of the animal's digestive system. For example, short-chain fatty acids (SCFAs) such as butyric acid and propionic acid are beneficial for the lower digestive tract. Butyrate nourishes colonocytes and helps reduce inflammation, but is difficult to use in feed due to its volatility and unpleasant odor. Preparation of butyrate in various salt forms (eg calcium butyrate) or encapsulation with coatings and/or oils help improve stability, however, despite these efforts, the resulting feed still exhibits reduced palatability and therefore reduced animal consumption. In addition, butyric acid is at least partially absorbed before reaching the lower digestive system, resulting in impaired delivery to the target site of the intestine. Similarly, essential oils have been studied as feed additives because many plant oils have antimicrobial and antipathogenic effects. However, essential oils degrade quickly in feed production environments and are susceptible to absorption before reaching the lower digestive system. Thus, it is difficult to deliver vital doses of essential oils to the target area of the digestive system. Thus, there is a need in the art for methods of delivering the active components of non-antibiotic feed additives to the lower digestive system of animals.

Summary of the invention

The present invention is directed to a method of modulating a metabolite in the gastrointestinal tract of an animal, comprising administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, wherein said synthetic oligosaccharide preparation contains at least n oligosaccharide fractions, each of which has different degrees of 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.

The present invention is also directed to a method of targeting a metabolite to a target compartment in the gastrointestinal tract of an animal, which is the hindgut, comprising administering to the animal a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation, wherein said 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 wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry.

According to the invention, said metabolite is butyric acid or propionic acid.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation, wherein said 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 wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% anhydro subunit-containing oligosaccharides, as determined by mass spectrometry for metabolite modulation in the animal's gastrointestinal tract.

The present invention is also directed to the use of a nutritional composition comprising a basic nutritional composition and a synthetic oligosaccharide preparation, wherein said 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 wherein each of the DP1 and DP2 fractions independently comprises from 0.5 to 15% anhydro subunit-containing oligosaccharides, as measured by mass spectrometry to target the metabolite to a target compartment in the animal's gastrointestinal tract, the hindgut.

According to the invention, said metabolite is butyric acid or propionic acid.

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 drawings

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 presents illustrative embodiments using the principles of the invention, and the following accompanying drawings (also figures and figs in this application).

Fig. 1 illustrates part of the 1H, 13C-HSQC NMR spectrum of oligosaccharide drug 9.2.

Fig. 2 illustrates the MALDI-MS spectrum of the oligosaccharide preparation from Example 9.7, which demonstrates the presence of anhydrosubunits.

Fig. 3 illustrates the 1D 3D proton NMR spectrum of the anhydro-DP1 component from the oligosaccharide preparation of Example 9.

Fig. 4 illustrates the ID APT 13C-NMR spectrum of the anhydro-DP1 component from the oligosaccharide preparation of Example 9.

Fig. 5 shows an enlargement of the GC-MS chromatogram (TIC and XIC plots (m/z 229)) for the oligosaccharide preparation of Example 2.9 after derivatization.

Fig. 6 illustrates the 1H,13C-HSQC spectrum of an oligosaccharide drug with an anhydrous caramelization subunit.

Fig. 7 illustrates a subset 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

- 2 045265 as a result of LC-MS/MS detection from FIG. 8A.

Fig. 9 shows the 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 anhydrosubunits.

Fig. 11 is a typical 1H,13C-HSQC NMR spectrum of a sample of gluco-oligosaccharides containing anhydrosubunits, with the corresponding resonances and assignments used for bond distribution.

Fig. 12 illustrates an overlay of the 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-D-cellobiose (DP2-18) and a sample of oligosaccharides containing anhydrosubunits.

Fig. 14 illustrates mass chromatograms of oligosaccharides containing anhydrosubunits (top) and digested oligosaccharides containing anhydrosubunits (bottom) under selective multiple reaction monitoring (MRM).

Fig. 15 shows a graph of relative abundance versus degree of polymerization (DP) of the oligosaccharide from Example 9. The graph shows that the oligosaccharide preparation has a monotonically decreasing DP distribution.

Fig. 16 shows a plot of relative abundance versus degree of polymerization of the oligosaccharide from Example 9. The plot shows that the oligosaccharide preparation has a non-monotonically decreasing DP distribution.

Fig. 17 shows a typical 600 MHz 1H NMR spectrum of cecal extracts obtained from chickens. The spectrum in the aromatic region (6.5-8.5 ppm) was increased approximately 10 times compared to the aliphatic region. The assignment of specific metabolite peaks is indicated.

Fig. 18 shows a plot of scores from the PLS-DA classification showing Component 1 versus Component 2 as measured by NMR. Explained deviations are shown in parentheses.

Fig. 19 shows a dose response demonstrating a non-linear effect on the selectivity of SCFA fermentation by the cecal microbiota in the presence of various synthetic oligosaccharide preparations described in the invention.

Fig. 20 is a graph showing the effect of the oligosaccharide preparation of Example 9 on the functional metagenomics of piglets.

Fig. 21 shows two anhydrosaccharide containing oligosaccharides from the DP1 fraction and one from the DP2 fraction.

Fig. 22 illustrates an oligosaccharide containing anhydrosubunits (cellotriosan).

Fig. 23A shows the MALDI-MS spectrum of the oligosaccharide preparation from Example 2, which demonstrates the presence of anhydrosubunits. Fig. 23B illustrates the MALDI-MS spectrum of the oligosaccharide preparation from Example 2, which demonstrates the presence of anhydrosubunits.

Fig. 24A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 1. FIG. 24B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 1. FIG. 24C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 1.

Fig. 25A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 3. FIG. 25B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 3. FIG. 25C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 3.

Fig. 26A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 4. FIG. 26B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 4. FIG. 26C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 4.

Fig. 27A illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 7. FIG. 27B illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydro-DP1 and DP2 species of the oligosaccharide drug from Example 7. FIG. 27C illustrates the detection by LC-MS/MS of the anhydro-DP2, anhydroDP1 and DP2 species of the oligosaccharide drug from Example 7.

Fig. 28A illustrates the GC/MS detection spectrum with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 1. FIG. 28B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 28A.

Fig. 29A illustrates the GC-MS detection spectrum with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 3. FIG. 29B illustrates the increase in DP2 and anhydro fractions

- 3 045265

DP2, as shown in FIG. 29A.

Fig. 30A illustrates the spectrum from GC-MS detection with the anhydro-DP2, anhydro-DPI and DP2 fractions of the oligosaccharide drug from Example 4. FIG. 30B illustrates the increase in DP2 and anhydro fractions

DP2, as shown in FIG. 30A.

Fig. 31A illustrates the GC-MS detection spectrum with the anhydro-DP2, anhydro-DP1 and DP2 fractions of the oligosaccharide drug from Example 7. FIG. 31B illustrates the increase in DP2 and anhydro DP2 fractions as shown in FIG. 31A.

Fig. 32 illustrates the effect of reaction temperature, water content and reaction time on the amount of oligosaccharides containing DP2 anhydrosubunits in oligosaccharide preparations compared to the oligosaccharide preparation according to Example 2.

Fig. 33 illustrates the NMR assignments of 1,6-anhydro-beta-D-glucofuranose and 1,6-anhydro-beta-Dglucopyranose.

Fig. 34 illustrates improved butyrate production by the microbiome in domestic dogs (Canis familiaris) for oligosaccharide preparations compared to conventional prebiotics.

Fig. 35 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 this application, 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 invention have the same meaning as commonly understood by one skilled in the art to which the invention relates.

The section headings used in the 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 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 forms 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 invention, all combinations and subcombinations of ranges and specific embodiments are intended to be included therein. The term is approximately

- 4 045265 when referring to a number or numerical range means that the number or numerical range stated 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 a specified 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 other day for at least 7, 14, 21, 30, 45, 60, 75, 90, or 120 days. 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) means the ratio of the mass of feed consumed (eg, consumed by an animal) to the animal product, the animal product being the target product of animal origin. 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 anhydrosubunit refers to the product of thermal dehydration of a monosaccharide (or monosaccharide subunit) or the product of caramelization of sugar. For example, the anhydrosubunit may be an anhydro-monosaccharide such as anhydroglucose. As another example, an anhydrosubunit 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 anhydrosaccharide 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 preparation refers to a preparation 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, anhydroglucose, 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 anhydroglucose subunit.

As used herein, the term galactooligosaccharide refers to galactose or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds. Essentially, a galactooligosaccharide includes galactose, anhydrogalactose, 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 anhydrogalactose subunit.

As used herein, the term glucogalacto-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-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 glucogalactose-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 glucogalactose-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 comprising 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 anhydrous subunit. The term regular disaccharide refers to a disaccharide that does not contain an anhydrous subunit. Accordingly, the term regular subunit refers to a subunit that is not an anhydrosubunit.

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

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 anhydro subunits, 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 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 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 fractions 1 to n in the oligosaccharide preparation independently comprises from 0.1 to 90% anhydrous subunit-containing oligosaccharides, as measured by relative abundance by 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 anhydrosubunits. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.5 to 15% oligosaccharides containing anhydrosubunits. In some embodiments, each fraction from DP1 and DP2 independently comprises from about 0.1 to 15% oligosaccharides containing anhydrosubunits, 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 anhydrosubunits. In some embodiments, each fraction of DP1 and DP2 independently comprises from 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% oligosaccharides containing anhydrosubunits, 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 drug 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 invention discloses oligosaccharide preparations containing anhydrosaccharide 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 galactooligosaccharides 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 by affecting the animal microbiome. 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 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 anhydrosubunits. 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 itself from other sources of carbohydrates in a nutritional composition through anhydrosubunits.

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 to DPn). For example, in some embodiments, the DP1 fraction contains one or more monosaccharides and/or one or more anhydromonosaccharides. 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 anhydrous 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,

- 8 045265

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

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 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 to 25%, about from 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%, from about 5 to 25%, from 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 up 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 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 MALDI-MS. In some embodiments, the DP2 fraction is determined using HPLC. In some embodiments, the DP2 fraction is determined using LC-MS/MS or 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 up 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%, about 1 to 10 or 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 preparation described herein has an average DP value in the range of 2 to 10. In some embodiments, the oligosaccharide preparation has an average DP value of 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 anhydrosubunits.

In some embodiments, each of the n oligosaccharide fractions in an oligosaccharide preparation described herein independently includes a level of anhydrosubunits. For example, in some embodiments, fraction DP1 contains about 10% anhydrosubunit-containing oligosaccharides by relative abundance, and fraction DP2 contains about 15% anhydrosubunit-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 anhydrosaccharide-containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fractions each contain approximately 5% anhydrosubunit-containing oligosaccharides, by relative abundance.

In some embodiments, each of fractions 1 to n in an oligosaccharide preparation described herein independently comprises from about 0.1 to 15% anhydrous subunit-containing oligosaccharides, as measured by mass spectrometry, LC-MS/MS, or GC-containing oligosaccharides. MS. In some embodiments, each of fractions 1 to n in the oligosaccharide preparation independently comprises from about 0.5 to 15% oligosaccharides containing anhydrosubunits, as measured by relative abundance by mass spectrometry, LC-MS/MS, or GC-MS. In some embodiments, LC-MS/MS is used to determine the relative abundance of oligosaccharides in the DP1, DP2 and/or DP3 fractions. In some embodiments, GC-MS 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 anhydrosubunits 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 anhydrosubunits 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 anhydrosubunits is determined, at least in part, by HPLC. In some embodiments, the level of oligosaccharides containing anhydrosubunits is determined by 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 4. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using MALDI-MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS, as shown in FIG. 24A-24C, 25A-25C, 26A-26C and 27A-27C. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS, as shown in FIG. 28A-28B, 29A-29B, 30A-30B and 31A-31B.

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%,

- 11 045265 less than 3%, less than 2% or less than 1% of oligosaccharides containing anhydrosubunits, 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 anhydrosubunits, 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 anhydrosubunits, 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% of anhydrosubunit-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%, approximately 0.9%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6% , about 7%, about 8%, about 9%, or about 10% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, at least one fraction (such as DP1, DP2, and/or DP3) of the oligosaccharide preparation 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 up to 20%, from about 0.5 to 10%, from about 0.5 to 9%, from about 0.5 to 8%, from about 0.5 to 7%, from about 0.5 to 6 from about 0 .5 to 5%, about 0.5 to 4%, about 0.5 to 3%, 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% oligosaccharides containing anhydrosubunits , by relative prevalence. In some embodiments, each of the oligosaccharide preparation fractions DP1 and DP2 independently comprises oligosaccharides containing anhydrosubunits 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% 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 anhydrosubunit-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% of oligosaccharides containing anhydrosubunits, 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% anhydrosaccharide-containing oligosaccharides, by relative abundance. In other embodiments, each fraction of the oligosaccharide preparation described herein contains more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, or 80% oligosaccharides containing anhydrosubunits, by relative abundance. In other embodiments, each fraction of an oligosaccharide preparation described herein contains greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% anhydrosaccharide-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 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% anhydrous oligosaccharides

- 12 045265 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 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9% or approximately 10% of oligosaccharides containing anhydrosubunits, 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%, about from 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%, from 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% anhydrosubunit-containing oligosaccharides, 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% of oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, the oligosaccharide preparation contains less than 10, 9, 8, 7, 6, 5, 4, 3, or 2% anhydrosubunit-containing oligosaccharides, by relative abundance. 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 anhydrosubunits, by relative abundance. In other embodiments, the oligosaccharide preparation contains greater than 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30% anhydrosaccharide-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 anhydrosubunits, 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 anhydrosubunits, 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 about 5 to 10% anhydrosubunit-containing oligosaccharides, by relative abundance.

In some embodiments, the DP1 fraction of an oligosaccharide drug 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 anhydrosubunits , by relative prevalence. In some embodiments, the DP1 fraction of an oligosaccharide drug described herein includes more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more 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 anhydrosubunits , by relative prevalence. 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 anhydrosubunits, by relative abundance. In some

- 13 045265 embodiments, the DP1 fraction of the oligosaccharide preparation described herein contains from about 0.1 to 15%, from about 0.1 to 20%, from about 0.5 to 20%, from 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 anhydrosaccharide-containing oligosaccharides, by relative abundance, or anywhere in between. In some embodiments, the DP1 fraction of an oligosaccharide preparation described herein comprises from about 0.5% to 10% anhydrosubunit-containing oligosaccharides, by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using mass spectrometry, such as MALDIMS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS.

In some embodiments, the DP2 fraction of an oligosaccharide drug 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 anhydrosubunits , by relative prevalence. In some embodiments, the DP2 fraction of an oligosaccharide drug described herein includes more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more 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 anhydrosubunits , by relative prevalence. 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 anhydrosubunits, according to relative abundance. In some embodiments, the DP2 fraction of an oligosaccharide drug 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 from 0.5 to 15%, from about 1 to 20%, from about 1 to 15%, from about 1 to 10%, from about 2 to 14%, from about 3 to 13%, from 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 therebetween. In some embodiments, the DP2 fraction of an oligosaccharide preparation described herein comprises about 5 to 10% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using mass spectrometry, such as MALDIMS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS.

In some embodiments, the DP3 fraction of an oligosaccharide drug 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 anhydrosubunits , by relative prevalence. In some embodiments, the DP3 fraction of an oligosaccharide drug described herein includes more than 0.1%, more than 0.5%, more than 0.8%, more than 1%, more than 1.5%, more than 2%, more than 3%, more 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 anhydrosubunits , by relative prevalence. 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 anhydrosubunits, by relative abundance. In some embodiments, the DP3 fraction of an oligosaccharide drug 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 from 0.5 to 15%, from about 1 to 20%, from about 1 to 15%, from about 1 to 10%, from about 2 to 14%, from about 3 to 13%, from about 4 to 12%, about 5 to 11%, about 5 to 10%, about 6 to 9%, or about 7 to 8% anhydrosaccharide-containing oligosaccharides, by relative abundance, or any range therebetween. In some variants

- 14 045265 In each embodiment, the DP3 fraction of the oligosaccharide preparation described herein contains from about 0.5% to 10% oligosaccharides containing anhydrosubunits, by relative abundance. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using mass spectrometry, such as MALDIMS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using LC-MS/MS. In some embodiments, the relative abundance of oligosaccharides containing anhydrosubunits is determined using GC-MS.

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

In some embodiments, the one or more oligosaccharides in the oligosaccharide preparation or in each fraction of the oligosaccharide preparation contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anhydrosubunits, each of which is linked through a glycosidic bond, where the glycosidic bond , binding each anhydrosubunit was selected independently. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation in or each fraction of the oligosaccharide preparation contain 1, 2, or 3 anhydrosubunits, each of which is linked by a glycosidic bond, wherein the glycosidic bond linking each anhydrosubunit 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 anhydrosubunits, each linked by a glycosidic bond, wherein the glycosidic bond linking each anhydrosubunit is independently selected . In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or in each fraction contain 1 anhydrosubunit 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 anhydrosubunit linked through a glycosidic bond.

D. Types of anhydrosubunits.

In some embodiments, the oligosaccharide preparation contains various types of anhydrosubunits. In some embodiments, exemplary oligosaccharides containing anhydrosubunits are illustrated in FIG. 33, 21 and 22. In some embodiments, the oligosaccharide preparation contains one or more anhydrosaccharide subunits, which are products of thermal dehydration of monosaccharides, that is, anhydrosaccharide 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 an anhydromonosaccharide (or anhydromonosaccharide subunit) refers to one or more types of thermal dehydration products of a monosaccharide. For example, in some embodiments, anhydro-glucose refers to 1,6-anhydro-β-D-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 variety 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, the oligosaccharide preparation as described herein contains one or more subunits of anhydro-glucose, anhydro-galactose, anhydro-mannose, 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 herein comprises one or more of 1,6-anhydro-3-O-b-O-glucopyranosyl-b-Oglucopyranose, 1,6-anhydro-3-O-a- O-glucopyranosyl-in-O-glucopyranoses, 1,6-anhydro-2-O-in-O-glucopyranosyl-in-O-glucopyranoses, 1,6-anhydro-2-O-a-O-glucopyranosyl-in- O-glucopyranoses, 1,6-anhydro-15 045265 β-O-cellobiose (cellobiosan), 1,6-anhydro-β-O-cellotriose (cellotriosan), 1,6-anhydro-β-O-cellotetraose (cellotetraosan) , 1,6-anhydro-O-cellopentaose (cellopentaosane) and 1,6-anhydro-O-maltose (maltosan).

In some embodiments, the oligosaccharide preparation contains one or more 1,6-anhydro-β-O-glucofuranose subunits. In some embodiments, the oligosaccharide preparation contains one or more 1,6-anhydro-β-D-glucopyranose (levoglucosan) subunits. For example, FIG. 33 illustrates two DP1 oligosaccharides containing anhydrosubunits (levoglucosan and 1,6-anhydro-β-O-glucofuranose) and a DP2 oligosaccharide containing anhydrosubunits (anhydrocellobiose).

The presence and level of anhydrosubunit species may vary depending on the feed Sugars used to produce the oligosaccharide. For example, in some embodiments, gluco-oligosaccharides contain anhydroglucose subunits, galacto-oligosaccharides contain anhydrogalactose subunits, and glucogalacto-oligosaccharides contain anhydroglucose and anhydrogalactose subunits.

In some embodiments, the oligosaccharide preparation contains both 1,6-anhydro-β-Oglucofuranose and 1,6-anhydro-β-D-glucopyranose anhydrosubunits. In some embodiments, at least 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 99% of the anhydro subunits are selected from the group consisting of 1,6-anhydro-β- D-glucofuranoses and 1,6-anhydro-β-Dglucopyranoses. In some embodiments, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the anhydrosubunits are 1,6-anhydro-β-O-glucofuranose anhydrosubunits. In some embodiments, at least 1, 5, 10, 20, 30, 40, 50, or 60% of the anhydro subunits are 1,6-anhydro-β-O-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, 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 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, 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 , 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, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1 :5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 in at least one fraction. In some embodiments, the ratio of 1,6-anhydro-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-O-glucofuranose to 1,6-anhydro-O-glucopyranose is about 2:1 in at least one fraction.

In some embodiments, an oligosaccharide preparation described herein includes OP2 oligosaccharides containing anhydrosaccharides. In some embodiments, the oligosaccharide preparation contains anhydrolactose, anhydrosucrose, 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 anhydrosubunits. In some embodiments, the oligosaccharide preparation described herein does not contain cellobiosan or does not contain a detectable level of cellobiosan.

In some embodiments, an oligosaccharide preparation described herein contains one or more anhydrosubunits that are caramelization products of sugar. In some embodiments, the oligosaccharide preparation contains one or more anhydrosubunits, which are sugar caramelization products selected from the group consisting of methanol; ethanol; furan; methylglyoxal; 2-methylfuran; vinyl acetate; glycol aldehyde 16 045265 hydride; acetic acid; acetol; furfural; 2-furanmethanol; 3-furanmethanol; 2-hydroxycyclopent2-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 anhydrosubunits.

In some embodiments, in the oligosaccharide preparation or at least one of the DP fractions, anhydrosubunits that are products of caramelization are less abundant than anhydrosubunits that are products of reversible thermal dehydration of the monosaccharide. In some embodiments, in the oligosaccharide preparation or at least one of the fractions, anhydrosubunits that are products of caramelization are more abundant than anhydrosubunits 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 anhydrosubunits that are products of caramelization and the anhydrosubunits 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%, about from 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 to 1%, from 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 about 10 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 anhydrosubunits 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 anhydrosubunits 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 anhydrous subunits 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%, about from 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 to 1%, from about 0.01 about from 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 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 anhydrosubunits in at least one fraction (eg, DP1, DP2 and/or DP3) of the 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 anhydrosubunits in at least one fraction (e.g., DP1, DP2, and/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% anhydrosubunits 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 anhydrous subunits in fractions DP1, DP2 and/or DP3 of the oligosaccharide preparation described herein 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%, about from 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 to 1%, from about 0.01 about from 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 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 anhydrous subunits in each fraction of the oligosaccharide preparation described in this application are caramelization products. In some embodiments, about 0.1 to 5%, about 0.1 to 2%, or about 0.1 to 1% of the anhydrous 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 anhydrous subunits in each fraction of the drug are caramelization products.

In some embodiments, each of the oligosaccharides in an oligosaccharide preparation described herein independently and optionally contains an anhydrosubunit. In some embodiments, two or more independent oligosaccharides contain the same or different anhydrosubunits. In some embodiments, two or more independent oligosaccharides contain different anhydrous subunits. For example, in some embodiments, the oligo-saccharide preparation includes a DP1 anhydrosubunit-containing oligosaccharide that includes

1,6-anhydro-β-O-glucopyranose, and an anhydro subunit-containing oligosaccharide DP2, which includes a 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 anhydrosubunits.

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 anhydrosubunit is associated with one, two, or three other regular or anhydrosubunits. In some embodiments, in the DP2-DPn fractions, at least one anhydrosubunit 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 anhydro subunits are associated with a single regular subunit. In some embodiments, in each of the DP2-DPn fractions, more than 99, 90, 80, 70, 60, 50, 40, or 30% of the anhydro subunits are associated with a single 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, more than 99, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30% of the anhydrosubunits 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 anhydrosubunits 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 anhydrosubunit-containing oligosaccharides include an anhydrosubunit 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 anhydrosubunit-containing oligosaccharides include an anhydrosubunit 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%, about 5 up to 40 mol.%, about 5 to 35 mol.%, about 5 to 30 mol.%, about 5 to 25 mol.%, about 10 to 60 mol.%, about 10 to 55 mol.% , about 10 to 50 mol.%, about 10 to 45 mol.%, about 10 to 40 mol.%, about 10 to 35 mol.%, about 15 to 60 mol.%, about 15 to 55 mol.%, about 15 to 50 mol.%, about 15 to 45 mol.%, about 15 to 40 mol.%, about 15 to 35 mol.%, about 20 to 60 mol.%, about 20 to 55 mole%, about 20 to 50 mole%, about 20 to 45 mole%, about 20 to 40 mole%, about 20 to 35 mole%, about 25 to 60 mol.%, about 25 to 55 mol.%, 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 bonds.

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%,

- 18 045265 from about 0 to 30 mol.%, from 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.%, from about 5 to 20 mol.%, from about 10 to 40 mol.%, from about 10 to 35 mol.%, from about 10 to 20 mol.% about 15 to 40 mole%, about 15 to 35 mole%, about 15 to 30 mole%, about 15 to 25 mole%, or about 15 to 20 mole% α-(1,3 ) 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%. 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 from 2 to 20 mol.%, from about 2 to 15 mol.%, from about 2 to 10 mol.%, from about 3 to 30 mol.%, from about 3 to 25 mol.%, from 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% 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.%, from 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 bonds. 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 bonds. 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.

- 19 045265

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), β-(1,4) and/or β-(1,6) glycosidic bonds in the distribution of the glycosidic bond type of the oligosaccharide preparations described in this application by at least 50 mol.%, by 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 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 30 mol.%, at least 20 mol.%, at least 15 mol.%, at least 10 mol.%, at least 5 mol.%, at least 2 mol.% or according 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 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, the 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 from 1300 to 4500 g/mol, from about 1500 to 4500 g/mol, from about 1700 to 4500 g/mol, from about 1900 to 4500 g/mol, from about 300 to 4000 g/mol, from about 500 to 4000 g/ mole, about 700 to 4000 g/mol, about 900 to 4000 g/mol, about 1100 to 4000 g/mol, about 1300 to 4000 g/mol, about 1500 to 4000 g/mol, about 1700 to 4000 g/mol, from about 1900 to 4000 g/mol, from about 300 to 3000 g/mol, from about 500 to 3000 g/mol, from about 700 to 3000 g/mol, from about 900 to 3000 g/mol, from about 1100 to 3000 g/mol, about 1300 to 3000 g/mol, about 1500 to 3000 g/mol, about 1700 to 3000 g/mol, about 1900 to 3000 g/mol, about 2100 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, from about 1500 to 2500 g/mol, from about 1700 to 2500 g/mol, from about 1900 to 2500 g/mol, from about 2100 to 2500 g/mol, from about 300 to 1500 g/mol, from about 500 to 1500 g/mol, about 700 to 1500 g/mol, about 900 to 1500 g/mol, about 1100 to 1500

- 20 045265 g/mol, from about 1300 to 1500 g/mol, from about 2000 to 2800 g/mol, from about 2100 to 2700 g/mol, from about 2200 to 2600 g/mol, from about 2300 to 2500 g/ mole 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 an oligosaccharide drug described herein is determined using HPLC according to Example 9.

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, from about 300 to 4500 g/mol, from about 500 to 4500 g/mol, from about 700 to 4500 g/mol, from about 900 to 4500 g/mol, from about 1100 to 4500 g/mol, from 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, about 700 to 4000 g/mol, about 900 to 4000 g/mol, about 1100 to 4000 g/mol, about 1300 to 4000 g/mol, about 1500 to 4000 g/mol, 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/mol, about from 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 to 3000 g /mol, from about 300 to 2500 g/mol, from about 500 to 2500 g/mol, from about 700 to 2500 g/mol, from about 900 to 2500 g/mol, from about 1100 to 2500 g/mol, from 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 mol, from about 500 to 2000 g/mol, from about 700 to 2000 g/mol, from about 900 to 2000 g/mol, from about 1100 to 2000 g/mol, from about 300 to 1500 g/mol, from about 500 up 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 using 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 glucogalactooligosaccharides, 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 oli

- 21,045,265 gosaccharides, 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 , 103, 104, 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, where 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 comprises one or more monosaccharide subunits selected from the group consisting of triose, tetrose, pentose, hexose, heptose, and any combination thereof, wherein each of said triose, tetrose, pentose, hexose, or heptose subunits independently and optionally functionalized and/or replaced by one of its corresponding anhydrosubunits. In some embodiments, the corresponding anhydrous 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 the hexose subunits is independently and optionally replaced by one of its corresponding anhydrosubunits.

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, an 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, an oligosaccharide preparation described herein comprises fructose and glucose subunits, wherein at least one fructose subunit or at least one glucose subunit is optionally replaced by one of its corresponding anhydrous 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 anhydrosubunits.

In some embodiments, the oligosaccharide drug described in the invention includes a glucogalactose-oligosaccharide drug, a gluco-oligosaccharide drug, a galacto-oligosaccharide drug, a fructo-oligosaccharide drug, a manno-oligosaccharide drug, an arabino-oligosaccharide drug, a xyloo-oligosaccharide drug, a glucofructo-oligosaccharide drug, a glucomanno-oligosaccharide drug. drug, glucoarabinooligosaccharide drug, glucoxylooligosaccharide drug, galactofructooligosaccharide drug, mannooligosaccharide drug, galactoarabinooligosaccharide drug, galactoxylooligosaccharide drug, fructomannooligosaccharide drug, fructoarabinooligosaccharide drug, fructoxylooligosaccharide drug, mannoarabinooligosaccharide drug, mannoxylooligosaccharide drug, arabinoxylooligosaccharide

- 22 045265 drug, galactoarabinoxylooligosaccharide drug, fructogalactoxylooligosaccharide drug, arabinofructomannoxylooligosaccharide drug, glucofructogalactoarabinooligosaccharide drug, fructoglucoarabinomannoxylooligosaccharide drug, glucogalactofructomannoarabinoxylooligosaccharide drug, or any of them combinations; 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 preparation described herein contains about 45 to 55% glucose subunits and 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 from about 80 to 95% glucose subunits, from 0 to 8% galactose subunits, and from 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 herein contains from about 1 to 100 wt%, about 50 to 100 wt%, about 80 to 98 wt%, or about 85 to 95 wt% glucose subunits , or any range in between. In some embodiments, the galactose subunits are present in the oligosaccharide preparation described herein in an amount of from about 0 to 90 wt%, about 1 to 50 wt%, about 2 to 20 wt%, or about 5 to 15 wt.%, or any range in between. In some embodiments, the mannose 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 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. 26.

Table 26. Approximate composition of oligosaccharide preparations

No. oligosaccharides of the 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 AND 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

- 23 045265 their natural common form, for example, in the form of 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, about 1:80, about 1:85, about 1:90, about 1:100 or about 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 (eg, -COOH), hydroxyl groups, or N-containing groups (eg, -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 of these 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 anhydrosubunit; (ii) the amount and level of anhydrosubunit; (iii) type of anhydrosubunit 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.

III. Methods for the production of oligosaccharide preparations.

In one aspect, this application provides methods for the production of oligosaccharide preparations. In some embodiments, this application 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, this application 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; 3pyridine 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; 6quinoline 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'-diyl-hydrogenphosphate; bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5xylyl)phosphinic acid; L-cysteine acid monohydrate; poly(styrenesulfonic acid co-divinylbenzene); lysine; ethanesulfonic acid; ethanesulfonic acid; isethionic acid - 24 045265 slots; homocysteine acid; HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-(N-morpholino)ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; 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 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 yet other embodiments, where one or more feed sugars include xylose, glucose and galactose, the resulting oligosaccharide preparations include glucogalactoxylooligosaccharide 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 herein is carried out on a gram scale. In some embodiments, the method of producing an oligosaccharide preparation described herein 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; 2-pyridine sulfonic acid; 3-pyridine sulfonic acid; 8-hydroxy-5-quinoline sulfonic acid hydrate; α-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)phosphine acid 26 045265; 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-(2hydroxyethyl)piperazine-N-(4-butanesulfonic acid)); HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); 2-hydroxy-3-morpholinopropanesulfonic acid; 2-N-morpholino) ethanesulfonic acid; methanesulfonic acid; metaniazide; naphthalene-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; 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 provided herein 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)-5,6diphenyl-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-1-sulfonic acid; naphthalene-2-sulfonic acid; perfluorobutanesulfonic acid; 6-sulfoquinovose; 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 invention is (+)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is 2-pyridine sulfonic acid. In some embodiments, the catalyst provided herein is 3-pyridine sulfonic acid. In some embodiments, the catalyst provided herein is 8-hydroxy-5-quinoline sulfonic acid hydrate. In some embodiments, the catalyst provided herein is αhydroxy-2-pyridinemethanesulfonic acid. In some embodiments, the catalyst provided herein is (c)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is butylphosphonic acid. In some embodiments, the catalyst provided herein is diphenylphosphinic acid. In some embodiments, the catalyst provided herein is a hexylphosphonic acid. In some embodiments, the catalyst provided herein is methylphosphonic acid. In some embodiments, the catalyst provided herein is phenylphosphinic acid. In some embodiments, the catalyst provided herein is a phenylphosphonic acid. In some embodiments, the catalyst provided herein is tert-butylphosphonic acid. In some embodiments, the catalyst provided herein is (S)-VAPOL hydrogen phosphate. In some embodiments, the catalyst provided herein is 6-quinoline sulfonic acid. In some embodiments, the catalyst provided herein is 3-(1-pyridino)-1-propanesulfonate. In some embodiments, the catalyst provided herein is 2-(2-pyridinyl)ethanesulfonic acid. In some embodiments, the catalyst provided herein is 3-(2pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate. In some embodiments, the catalyst provided herein is 1'

- 27 045265 binaphthyl-2,2'-diylhydrogen phosphate. In some embodiments, the catalyst provided herein is bis(4-methoxyphenyl)phosphinic acid. In some embodiments, the catalyst provided herein is phenyl(3,5xylyl)phosphinic acid. In some embodiments, the catalyst provided herein is L-cysteine acid monohydrate. In some embodiments, the catalyst provided herein is poly(styrenesulfonic acid-co-divinylbenzene). In some embodiments, the catalyst provided herein 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 herein 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 several feed sugars by dry matter weight. In some embodiments, the catalyst provided herein 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 provided herein is present in an amount of about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1 .1%, 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.

- 28 045265

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 weight of dry matter. In some embodiments, the catalyst provided herein is present in an amount of about 1 to 2% of the aqueous composition by dry weight. In some embodiments, the catalyst provided herein is present in an amount of about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4 %, 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 provided herein 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 herein. 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 provided herein 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, a method for producing oligosaccharide preparations described herein 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 progress, water can be produced through the reaction. For example, in some embodiments, water is produced (i) by forming a glycosidic bond, (ii) by forming 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 anhydrous subunits. Accordingly, the present invention describes a suitable water content for the production of oligosaccharide preparations.

In some embodiments, a method for producing an oligosaccharide preparation described herein 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 from 0 to 30%, from about 0 to 25%, from about 0 to 20%, from about 0 to 19%, from about 0 to 18%, from about 0 to 17%, from about 0 to 16%, from 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 2% 1% water of the total mass. 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

- 29045265 embodiments, the aqueous composition contains about 3 to 16%, about 3 to 14%, about 3 to 12%, about 3 to 10%, about 3 to 8%, about 3 to 6%, about 5 to 16%, about 5 to 14%, 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 by weight. 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 herein 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%, 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 2 to 8%, about 2 to 6%, about 4 to 16%, about 4 to 14%, 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% by 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 about 2 to 10%, about 2 to 8%, or 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 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 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 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% by 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

- 30 045265 ranged from about 2 to 10%, from about 2 to 8%, or from about 4 to 8% 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 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 anhydrosubunits 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 anhydrosubunits 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 herein. 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 up to 10%, from about 1 to 8%, from about 2 to 16%, from about 2 to 14% from about 2 to 12%, from about 2 to 10%, from about 2 to 8%, from about 2 to 6 %, about 4 to 16%, about 4 to 14%, 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 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% by 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 anhydrous subunits in the oligosaccharide preparation described herein. For example, as shown in FIG. 21, in some embodiments, higher water content correlates with lower levels of anhydrous subunits. In some embodiments, a lower reaction temperature may correlate with a lower level of anhydrous subunits.

E. Temperature.

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

F. Reaction time.

In some embodiments, a method for producing an oligosaccharide preparation described herein comprises 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, anhydrous 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 herein. 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, 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 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

- 32 045265 embodiments, the equilibrium is determined by measuring the molecular weight, viscosity or DP distribution of the 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, 2, 1 hour, 30, 20, 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, 2, 1 hour, 30, 20, 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 Km series, where

Kt ₌ Noo] where [H ₂ O] represents the molar concentration of water (mol/l), and [DP1], [DP _{m_1} ] and [DP _m ] represent the molar concentrations of oligosaccharides (mol/l) in DP1, DP _m _1 and DP _m 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 fractions DP1, DP _{m_1} and DPm 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

- 33 045265 to K11, K2 to K12, K2 to K13, K2 to K14, K2 to K15, K3 to K5, K3 to K66, 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 from about 0.1 to 100, about 0.1 to 90, about 0.1 to 80, about 0.1 to 70, about 0.1 to 60, about 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 5 to 10. In some embodiments, Km is from about 1 to 15, or 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 about 5 to 80%, about 10 to 70%, about 10 to 60%, about 10 to 50%, about 10 to 40%, about 10 to 30 %, or approximately 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.

N. Reactors.

The methods described herein may include the use of one or more reactors suitable for condensing the sugar, taking into account reaction temperature, pH, pressure and other 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, a 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.

This application provides nutritional compositions containing an oligosaccharide preparation. In some embodiments, this application 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 production 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 the anhydrosaccharide-containing oligosaccharides.

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 beans, peas and chickpeas), corn, cassava or other starchy foods (e.g. acorns, arrowroot, arracachis, 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 anhydrosubunits or oligosaccharides containing anhydrosubunits. In some embodiments, the basic nutritional composition contains less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm anhydrosubunits or oligosaccharides containing anhydrosubunits. In some embodiments, the basic nutritional composition is substantially free of anhydrous subunits.

In some embodiments, the base nutritional composition lacks detectable levels of anhydrous subunits. Depending on the detection or determination methods, levels of anhydrosubunits below a certain threshold may not be detectable. For example, in some embodiments, the detectable level of anhydrosubunits may be at least 1000 ppm, at least 500 ppm, at least 400 ppm, at least 300 ppm, at least 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 million anhydrosubunits or oligosaccharides containing anhydrosubunits in the basic 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.

- 36 045265

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. Feed composition for animals.

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 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 ppm, about 10 to 5000 ppm, about 10 to 3000 ppm, about 10 to 2000 ppm, about 10 to 1500 ppm, about 10 to 1000 ppm ppm, about 10 to 500 ppm, about 10 to 250 ppm, about 10 to 100 ppm, about 50 to 5000 ppm, about 50 to 3000 ppm 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 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 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 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 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 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 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 ppm, about 50 to 5000 ppm, about 50 to 2000 ppm, about 50 to 1000 ppm, about 50 to 500 ppm, about 50 to 250 ppm 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 5,000 ppm, about 10 to 1,000 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, more than 500 ppm, more than 600 ppm, more than 1000 ppm or more 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 application also provides a method of administering to an animal a nutritional composition containing a basic nutritional composition and the described 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 options

- 37 045265 implementation 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 regimen may vary depending on the type and age of the animal.

C. Methods for producing nutritional compositions.

Provided herein are 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 comprises from 0.1% to 90% anhydrosubunit-containing oligosaccharides, by relative prevalence 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 about 0.5 to 10%, anhydrosubunit-containing oligosaccharides, as measured by relative abundance by mass spectrometry. In some embodiments, each of the DP1 and DP2 fractions of the oligosaccharide preparation independently comprises oligosaccharides containing anhydrosubunits 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% 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 of 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 herein. 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

- 38 045265 removal of part of the oligosaccharide preparation 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.

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 feed 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 animal feed 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 drug by dry weight. In some embodiments, the animal feed premix contains at most 50, 60, 70, 80, 90, 95, or 99% oligosaccharide drug by dry matter weight.

In some embodiments, the animal food premix or carrier material further comprises other animal food 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 anhydrous 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.

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V. Methods for providing animals with oligosaccharide preparations.

In some embodiments, the methods described herein include providing oligosaccharide preparations to animals. 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 of 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. Selective stimulation or inhibition of the production of gastrointestinal metabolites.

A. Metabolites of the gastrointestinal tract.

In some embodiments, the methods described herein include selectively promoting or inhibiting the production of one or more metabolites in the gastrointestinal tract of an animal. In some embodiments, one or more metabolites are detected and quantified. Metabolites include short-chain fatty acid (SCFA), bile acid, polyphenol, amino acid, neurotransmitter (or neurotransmitter precursor), signaling factor, nitrogenous metabolite, butyric acid, propionic acid, acetic acid, lactic acid, valeric acid, isovaleric acid, amino-SCFA , thioate, terpenoid, a-terpenoid, essential oil, betazole, milk oligosaccharide, fucosylated oligosaccharide, 2'-fucosyllactose (2FL), stylated oligosaccharide, steroid, anamine, trimethylamine, ammonia, indole, indoxyl sulfate, pro-inflammatory metabolite, histamine, lipopolysaccharide, betazole, gamma-aminobutyric acid (GABA), linalool, eucalyptol, geraniol, dipeptide, fatty alcohol, p-cresol, sulfide, hydrosulfide, volatile amine, thiol, dopamine, aminoindole, fat-soluble metabolite, aliphatic aldehyde, aliphatic ketone, 2-methylthioethanol, 3 -methyl-2-butanone, 3-methylbutanal, pentanal, 3-hydroxy-2-butanone, (E)-2-pentenal, 1-pentanol, (E)-2-decenal, hexanal, (E)-2-hexenal , 1-hexanol, heptanal, styrene, oxime-, methoxyphenylbutyrolactone, (E)-2-heptenal, benzaldehyde, dimethyl trisulfide, 1-heptanol, octanal, 1-octen-3-one, 1-octen-3-ol, (E , E)-2,4-heptadienal, 2acetylthiazole, D-limonene, 4-ethylcyclohexanol, 2,4-dimethylcyclohexanol, (E)-2-octenal, benzolacetaldehyde, 1-octanol, 2-butylcyclohexanone, 4-(benzoyloxy)- (E)-2-octen-1-ol, 1-octanol, octadecanoic acid, ethenyl ester, nonanal, (E)-2-nonen-1-ol, 3-octadecine, cyclooctanemethanol, dodecanal, (E)-2 -nonenal, 2,6/3,5-dimethylbenzaldehyde, 1-nonanol, 2-n-heptylfuran, cis-4-decenal, decanal, (E,E)-2,4-nonadienal, 1,3-hexadiene, 3 -ethyl-2-methyl-2-nonenal, (E)-2-undecenal, trans-3nonen-2-one, 2,5-furandione, 3-dodecenyl-trans-2-undecen-1-ol and eicosanoic acid, but are not limited to them.

In some embodiments, one or more metabolites are beneficial to the animal

- 40 045265 (for example, beneficial for the health of the animal). Examples of beneficial metabolites include short-chain fatty acid (SCFA), amino-SCFA, neurotransmitter, neurotransmitter precursor, neurochemical, gamma-aminobutyric acid (GABA), dopamine, aminoindole, volatile fatty acids (VFA), butyric acid, propionic acid, acetic acid, lactic acid, valeric acid, isovaleric acid, essential oils, a-terpenoid, eucalyptol, geraniol, betazole, lactic oligosaccharide, fucosylated oligosaccharide, stylated oligosaccharide, 2-fucosyllactose and aminoindole.

In some embodiments, one or more metabolites promote growth of the animal. Examples of metabolites include, but are not limited to, butyric acid, propionic acid, acetic acid, lactic acid, valeric acid and isovaleric acid.

In some embodiments, one or more metabolites are harmful to the health of the animal. Examples of harmful or undesirable metabolites include, but are not limited to, nitrogenous metabolite, amino acid degradation product, ammonia, trimethylamine, indole, p-cresol, trimethylamine N-oxide (TMAO), uremic solutes, or bile acid.

In some embodiments, the metabolite is a pro-inflammatory metabolite. Examples of pro-inflammatory metabolites include, but are not limited to, histamine and LPS.

In some embodiments, the metabolite is related to the quality of the animal meat, including, for example, the taste, color, and texture of the animal meat. Examples of metabolites include 2-methylthioethanol, 3methyl-2-butanone, 3-methylbutanal, pentanal, 3-hydroxy-2-butanone, (E)-2-pentenal, 1-pentanol, (E)-2decenal, hexanal, (E) -2-hexenal, 1-hexanol, heptanal, styrene, oxime, methoxyphenyl-butyrolactone, (E)-2-heptenal, benzaldehyde, dimethyl trisulfide, 1-heptanol, octanal, 1-octen-3-one, 1-octen-3 -ol, (E,E)-2,4-heptadienal, 2-acetylthiazole, D-limonene, 4-ethylcyclohexanol, 2,4-dimethylcyclohexanol, (E)-2-octenal, benzolacetaldehyde, 1-octanol, 2 -butylcyclohexanone, 4-(benzoyloxy)-(E)-2-octen-1 ol, 1-octanol, octadecanoic acid, ethenyl ester, nonanal, (E)-2-nonen-1-ol, 3-octadecine, cyclooctanemethanol, dodecanal, (E)-2-nonenal, 2,6/3,5-dimethylbenzaldehyde, 1-nonanol, 2-n-heptylfuran, cis-4-decenal, decanal, (E,E)-2,4-nonadienal, 1,3-hexadiene, 3-ethyl-2-methyl-2-nonenal, (E)-2-undecenal, trans-3-nonen-2-one, 2,5-furandione, 3-dodecenyl-trans-2-undecen- 1-ol and eicosanoic acid, but are not limited to them.

In some embodiments, the methods described herein include promoting or inhibiting the production of one or more metabolites in the gastrointestinal tract of an animal. In some embodiments, one or more metabolites are detected and quantified. Metabolites include short-chain fatty acid (SCFA), bile acid, polyphenol, amino acid, neurotransmitter (or neurotransmitter precursor), signaling factor, nitrogenous metabolite, butyric acid, propionic acid, acetic acid, lactic acid, valeric acid, isovaleric acid, amino-SCFA , thioate, terpenoid, a-terpenoid, essential oil, betazol, milk oligosaccharide, fucosylated oligosaccharide, 2'fucosyllactose (2FL), stylated oligosaccharide, steroid, anamine, trimethylamine, ammonia, indole, indoxyl sulfate, pro-inflammatory metabolite, histamine, lipopolysaccharide, betazol , gamma-aminobutyric acid (GABA), linalool, eucalyptol, geraniol, dipeptide, fatty alcohol, p-cresol, sulfide, hydrosulfide, volatile amine, thiol, dopamine, aminoindole, fat-soluble metabolite, aliphatic aldehyde, aliphatic ketone, 2-methylthioethanol, 3- methyl 2-butanone, 3-methylbutanal, pentanal, 3-hydroxy-2-butanone, (E)-2-pentenal, 1-pentanol, (E)-2-decenal, hexanal, (E)-2hexenal, 1- hexanol, heptanal, styrene, oxime-, methoxyphenylbutyrolactone, (E)-2-heptenal, benzaldehyde, dimethyl trisulfide, 1-heptanol, octanal, 1-octen-3-one, 1-octen-3-ol, (E, E) -2,4-heptadienal, 2acetylthiazole, D-limonene, 4-ethylcyclohexanol, 2,4-dimethylcyclohexanol, (E)-2-octenal, benzolacetaldehyde, 1-octanol, 2-butylcyclohexanone, 4-(benzoyloxy)-(E) -2-octen-1-ol, 1-octanol, octadecanoic acid, ethenyl ester, nonanal, (E)-2-nonen-1-ol, 3-octadecine, cyclooctanemethanol, dodecanal, (E)-2-nonenal, 2,6/3,5-dimethylbenzaldehyde, 1-nonanol, 2-n-heptylfuran, cis-4-decenal, decanal, (E,E)-2,4-nonadienal, 1,3-hexadiene, 3-ethyl- 2-methyl-2-nonenal, (E)-2-undecenal, trans-3nonen-2-one, 2,5-furandione, 3-dodecenyl-trans-2-undecen-1-ol and eicosanoic acid, but not limited to them.

In some embodiments, the metabolite is selected from the group consisting of linalool, eucalyptol, geraniol, terpenoid, α-terpenoid, gentisic acid, lactic oligosaccharide, fucosylated oligosaccharide, 2'-fucosyllactose (2FL), sialylated oligosaccharide, α-aminoisobutyric acid, D-alpha -aminobutyric acid and 3-aminoisobutanoic acid, butyric acid, propionic acid, acetic acid, lactic acid, valeric acid, isovaleric acid, amino SCFA, thioate, essential oil, betazol, steroid, anamine, trimethylamine, ammonia, indole, indoxyl sulfate, pro-inflammatory metabolite, histamine, lipopolysaccharide, betazole, gamma-aminobutyric acid (GABA), dipeptide, fatty alcohol, p-cresol, sulfide, hydrogen sulfide, volatile amine, thiol, dopamine and aminoindole.

In some embodiments, the metabolite is associated with animal health. Examples of metabolites include, but are not limited to, linalool, eucalyptol, geraniol, terpenoid, α-terpenoid, gentisic acid, milk oligosaccharide, fucosylated oligosaccharide, 2'-fucosyllactose (2FL), and stylated oligosaccharide. Other typical metabolites include short-chain fatty acid (SCFA), amino-SCFA, thioates, terpenoids, α-terpenoids, anamines, ammonia, indole, butyric acid, histamine, betazole, GABA, 2FL, eucalyptol, and geraniol.

In some embodiments, the metabolite is associated with mood. Examples of metabolites include, but are not limited to, gamma-aminobutyric acid (GABA), aminoisobutyric acid, D-alpha-aminobutyric acid, and 3-aminoisobutanoic acid.

In some embodiments, one or more metabolites are harmful to the health of the animal. Examples of metabolites include, but are not limited to, short-chain fatty acid (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic solutes, and bile acid.

In some embodiments, the metabolite is associated with at least one quality attribute of animal meat, such as taste, color, aroma, etc. Examples of metabolites include fat-soluble metabolite, aliphatic aldehyde, aliphatic ketone, 1-methylthiopropane, 2methylthiolethanol, p-menth-1-en-4-ol and the compounds 1-nitroheptane, octanal, 2-octanone and 2,3-heptanedione, 3-methyl -2-butanone, 3-methylbutanal, pentanal, 3-hydroxy-2-butanone, (E)-2-pentenal, 1-pentanol, (E)2-decenal, hexanal, (E)-2-hexenal, 1- hexanol, heptanal, styrene, oxime, methoxyphenylbutyrolactone, (E)-2-heptenal, benzaldehyde, dimethyl trisulfide, 1-heptanol, octanal, 1-octen-3-one, 1-octen-3ol, (E,E)-2, 4-heptadienal, 2-acetylthiazole, D-limonene, 4-ethylcyclohexanol, 2,4-dimethylcyclohexanol, (E)-2-octenal, benzolacetaldehyde, 1-octanol, 2-butylcyclohexanone, 4-(benzoyloxy)-(E)- 2-octen-1-ol, 1-octanol, octadecanoic acid, ethenyl ester, nonanal, (E)-2-nonen-1-ol, 3-octadecine, cyclooctanemethanol, dodecanal, (E)-2-nonenal, 2, 6/3,5-dimethylbenzaldehyde, 1-nonanol, 2-n-heptylfuran, cis4-decenal, decanal, (E,E)-2,4-nonadienal, 1,3-hexadiene, 3-ethyl-2-methyl- 2-nonenal, (E)-2-undecenal, trans-3-nonen-2-one, 2,5-furandione, 3-dodecenyl-trans-2-undecen-1-ol and eicosanoic acid, but not limited to.

B. Sampling and detection of gastrointestinal metabolites.

In some embodiments, the methods described herein include detecting or quantifying one or more metabolites in the gastrointestinal tract of an animal. In some embodiments, the metabolite is detected or quantified in a gastrointestinal sample of the animal. 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 feces, saliva and gastrointestinal ascitic fluid are also collected. Methods for obtaining gastrointestinal tract samples are standard and known to one skilled in the art.

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, metabolites are purified from the sample prior to analysis. In some embodiments, metabolites from a single sample are purified. In some embodiments, metabolites from multiple samples from a single animal are purified and then pooled before analysis.

Metabolites that are present in gastrointestinal samples collected from animals or in fresh or spent culture media can be determined using methods described herein and known to one skilled in the art. Such methods include, for example, chromatography (eg, gas chromatography (GC) or liquid chromatography (LC)) in combination with mass spectrometry or NMR (eg, ¹ H-NMR). Measurements can be confirmed by running metabolite standards on the same analytical systems.

For gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) analysis, polar metabolites and fatty acids can be extracted using single-phase or two-phase organic solvent and aqueous sample systems and derivatized . An exemplary protocol for the derivatization of polar metabolites involves the formation of methoxime-tBDMS derivatives by incubating the metabolites with 2% methoxylamine hydrochloride in pyridine followed by the addition of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) with 1% tert-butyldimethylchlorosilane (tBDMCS). Non-polar fractions, including triacylglycerides and phospholipids, can be saponified to free fatty acids and esterified to form fatty acid methyl esters, for example by incubation with 2% H2SO4 in methanol or using Methyl-8 reagent (Thermo Scientific). The derivatized samples can then be analyzed by GC-MS using standard LC-MS methods, such as a DB-35MS column (30 m x 0.25 mm i.d.

- 42 045265 χ 0.25 µm, Agilent J&W Scientific) installed on a gas chromatograph (GC) coupled to a mass spectrometer (MS). Mass isotopomer distributions can be determined by integrating metabolite fragment ions and corrected for natural abundance using standard algorithms. For liquid chromatography-mass spectrometry (LC-MS), polar metabolites can be analyzed using a standard benchtop LCMS/MS equipped with a column such as a SeQuant ZIC-Philic polymer column (2.1 × 150 mm; EMD Millipore). Examples of mobile phases used for separation may include buffers and organic solvents adjusted to a specific pH.

In combination or alternatively, extracted samples can be analyzed using 1H nuclear magnetic resonance (1H-NMR). Samples can be combined with isotope- _enriched solvents such as _D2O , _if necessary in the presence of a buffer solution (eg _Na2HPO4 , _NaH2PO4 in D2O, pH 7.4). Samples can also be supplemented with a standard for calibration and chemical shift determination (e.g. 5 mM sodium salt of 2,2-dimethyl-2silapentane-5-sulfonate (DSS-d6, Isotec, USA)). Before analysis, the solution can be filtered or centrifuged to remove any sediment or deposits and then transferred to a suitable tube or vessel for NMR analysis (eg, a 5 mm NMR tube). ¹ H-NMR spectra can be obtained on a standard NMR spectrometer such as an Avance II+ 500 Bruker (500 MHz) spectrometer (Bruker, DE) equipped with a 5 mm QXI-Z C/N/P probe head) and analyzed with using spectrum integration software (eg, Chenomx NMR Suite 7.1; Chenomx Inc., Edmonton, Alberta). Alternatively, ¹ H-NMR can be performed in accordance with other published protocols known in the art (see, for example, Chassaing et al., Lack of soluble fiber drives diet-induced adiposity in mice, Am J Physiol Gastrointest Liver Physiol, 2015; Bai et al., Comparison of Storage Conditions for Human Vaginal Microbiome Studies, PLoS ONE, 2012:e36934).

C. Beneficial microorganisms.

In some embodiments, the methods described herein include selectively enhancing or promoting the growth of one or more species of microorganisms (eg, bacteria) in the gastrointestinal tract of an animal. In some embodiments, the microbial (eg, bacterial) species is beneficial to the animal (eg, healthy). In some embodiments, the methods described herein include selectively enhancing or promoting the growth of one or more species of microorganisms (eg, bacteria) in the gastrointestinal tract of an animal, wherein the species of microorganisms produce one or more selected metabolites. In some embodiments, the microorganism species is an archaeal species. In other embodiments, the microbial species is a virus, bacteriophage, or protozoan. In some embodiments, the microbial species is a bacterial species.

Bacteria disclosed herein include, but are not limited to, organisms classified in the genera Bacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, Barnesiella or Ruminococcus. Examples of bacteria also include, but are not limited to, organisms classified in the genera Enterococcus, Lactobacillus, Propionibacterium, Bifidobacterium and Streptococcus.

Bacterial species include, but are not limited to, Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, and Barnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiota containing at least 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of at least one bacterial species classified as genera Bacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, Barnesiella or Ruminococcus (eg, when measured in a gastrointestinal sample as disclosed herein). In some embodiments, the animal has a gastrointestinal microbiota containing at least 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of at least one bacterial species classified as genera Enterococcus, Lactobacillus, Propionibacterium, Bifidobacterium or Streptococcus (for example, when measured in a gastrointestinal sample as disclosed herein).

In some embodiments, the animal has a gastrointestinal microbiota containing at least 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of at least one of Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus or Barnesiella intestinihominis (eg, when measured in a gastrointestinal sample as described herein).

In some embodiments, the animal has a gastrointestinal microbiota of at least 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50% combination of one or more bacterial species classified as the genera Bacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, Barnesiella, or Ruminococcus (e.g., as measured in a gastrointestinal sample as described herein). In some embodiments, the animal has a gastrointestinal microbiota of at least 0.1, 1, 2, 3, 4, 5.

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6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50% combination of one or more species of bacteria classified as the genera Enterococciis, Lactobacillus, Propionibacterium, Bifidobacterium or Streptococcus (for example, when measured in the gastrointestinal sample as described in this application).

In some embodiments, the animal has a gastrointestinal microbiota of at least 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50% combination of one or more of Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus or Barnesiella intestinihominis (eg, when measured in a gastrointestinal sample as described herein).

D. Pathogenic microorganisms.

In some embodiments, the methods described herein include reducing or inhibiting the growth of one or more species of microorganisms (e.g., bacteria) in the gastrointestinal tract of an animal, and in some embodiments, quantifying the level of a species of one or more microorganisms (e.g., bacteria ). In some embodiments, the methods described herein include reducing or inhibiting the growth of one or more species of microorganisms (e.g., bacteria) in the gastrointestinal tract of an animal, wherein the microorganisms (e.g., bacteria) produce one or more metabolites that are harmful to animal health. In some embodiments, the microbial (eg, bacterial) species is pathogenic to the animal. In some embodiments, the microbial (eg, bacterial) species is pathogenic to humans but not to animals. In some embodiments, the microorganism species is an archaeal species. In other embodiments, the microbial species is a virus, bacteriophage, or protozoan. In some embodiments, the microbial species is a bacterial species.

Bacteria disclosed herein include, but are not limited to, bacteria of the phylum Proteobacteria. Bacteria also include, but are not limited to, organisms classified in the genera Helicobacter, Escherichia, Salmonella, Vibrio, or Yersinia. Examples of bacteria also include organisms classified in 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, but are not limited to. Bacterial species include, but are not limited to, Helicobacter pullorum, Proteobacteria johnsonii, Escherichia coli, Campylobacter jejuni and Lactobacillus crispatus. Bacterial species include Aeromonas hydrophila, 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, Helico bacter pylori, Klebsiellia pneumonia, hysteria Monocytogenes, Plesiomonas shigelloides, Salmonella spp., Salmonella Typhi, Salmonella Paratyphi, Shigella spp., Staphylococcus spp., Staphylococcus aureus, Vancomycin -Resistant enter ococcus spp., Vibrio spp., Vibrio Cholerae, Vibrio Parahahaemolyticus, Vibrio Vulnificus and Yersinia Enterocolitica, but are not limited to them. In some embodiments, the bacteria are resistant to one or more drugs.

In some embodiments, the animal has a gastrointestinal microbiota of less than 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.1% of bacteria classified such as the genera Helicobacter, Proteobacteria, Escherichia, Campylobacter or Lactobacillus, for example when measured in a gastrointestinal sample as described here). In some embodiments, the combination of bacteria classified as Helicobacter, Proteobacteria, Escherichia, Campylobacter, or Lactobacillus genera is less than 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2. 1 or 0.1% of the animal's gastrointestinal microbiota, for example, when measured in a gastrointestinal sample as described herein).

In some embodiments, the animal has a gastrointestinal microbiota of less than 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.1% Helicobacter pullorum, Proteobacteria johnsonii, Escherichia coli, Campylobacter jejuni or Lactobacillus crispatus, for example when measured in a gastrointestinal sample as described here). In some embodiments, the combination of Helicobacter pullorum, Proteobacteria johnsonii, Escherichia coli, Campylobacter jejuni, or Lactobacillus crispatus is less than 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.1% of the animal's gastrointestinal tract microbiota (eg, when measured in a gastrointestinal sample as described herein).

E. Sampling and detection of gastrointestinal microorganisms.

In some embodiments, the methods described herein include detecting or quantifying one or more species of microorganisms (eg, bacteria) in the gastrointestinal microbiota of an animal. In some embodiments, microbial (eg, bacterial) species are detected or quantified in a sample of the animal's gastrointestinal tract microbiota. Gastrointestinal microbiota samples can be obtained from an animal in any standard form that reflects the microbial content of the animal's gastrointestinal tract. Gastrointestinal microbiota samples include samples

- 44 045265 tissues of the gastrointestinal tract obtained, for example, using endoscopic biopsy.

Gastrointestinal tissues include, for example, tissues of the oral cavity, esophagus, stomach, intestine, ileum, cecum, colon or rectum. Samples of feces, saliva and gastrointestinal ascitic fluid are also collected. Methods for obtaining gastrointestinal microbiota samples are standard and known to one skilled in the art.

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, microorganisms (eg, bacteria, eg, all bacteria) are purified from the sample prior to analysis. In some embodiments, microorganisms (eg, bacteria) are purified from a single sample. In some embodiments, microorganisms (eg, bacteria) from multiple samples from a single animal are purified and subsequently pooled before analysis.

In some embodiments, total DNA or total RNA is isolated from the sample. Genomic DNA can be extracted from samples using standard methods known to one of ordinary skill in the art, including commercially available kits such as the Mo Bio Powersoil®-htp 96-well Soil DNA Extraction Kit (Mo Bio Laboratories, Carlsbad, CA), Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) or QIAamp Stool DNA Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RNA can be extracted from samples using standard assays known to those skilled in the art, including commercial kits such as the RNeasy PowerMicrobiome kit (QIAGEN, Valencia, Calif.) and the RiboPure Bacterial RNA Purification Kit (Life Technologies, Carlsbad, Calif.). Another method for isolating microbial (e.g., bacterial) RNA may involve enriching mRNA in purified bacterial RNA samples by removing tRNA. Alternatively, the RNA can be converted to cDNA which can be used to generate sequencing libraries using standard methods such as the Nextera XT sample preparation kit (Illumina, San Diego, CA).

Identification and determination of the relative abundance of microbial (e.g., bacterial) species in a sample can be accomplished by standard molecular biology techniques known to one of ordinary skill in the art, including, for example, genetic analysis (e.g., DNA sequencing (e.g., whole genome sequencing, whole genome sequencing shotgun (WSG)), RNA sequencing, NLR, quantitative NLR (qPCR), serology and antigen analysis, microscopy, metabolite identification, Gram staining, flow cytometry, immunological and culture-based methods such as colony-forming unit counts.

In some embodiments, the identification and relative abundance of microbial species (e.g., bacteria) is determined using whole genome shotgun sequencing (WGS), in which extracted DNA is fragmented into pieces of varying lengths (from 300 to about 40,000 nucleotides) and directly sequenced without amplification. Sequence data may be obtained using any sequencing technology, including, for example, but not limited to, Sanger, Illumina, 454 Life Sciences, Ion Torrent, ABI, Pacific Biosciences, and/or Oxford Nanopore.

Sequencing libraries for microbial (eg, bacterial) whole genome sequencing (WGS) can be generated from microbial (eg, bacterial) genomic DNA. For genomic DNA isolated from an animal sample, the DNA can be enriched with microbial (eg, bacterial) DNA if necessary using commercial kits such as the NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, Ipswich, MA) or other enrichment kit. Sequencing libraries can be prepared from genomic DNA using also commercial kits such as the Nextera Mate-Pair Sample Preparation Kit, TruSeq DNA PCR-Free or TruSeq Nano DNA, or the Nextera XT Sample Preparation Kit (Illumina, San Francisco). Diego, California) according to manufacturer's instructions.

Alternatively, libraries can be generated using other kits compatible with the Illumina sequencing platform, such as the NEBNext DNA Library Construction Kit (New England Biolabs, Ipswich, MA). The libraries can then be sequenced using standard sequencing technology including, but not limited to, a MiSeq, HiSeq, or NextSeq sequencer (Illumina, San Diego, CA).

Alternatively, a whole genome shotgun sequencing fragment library generated using standard methods in the art may be used. For example, a library of fragments from shotgun sequencing can be generated using the GS FLX Titanium Rapid Library Preparation Kit (454 Life Sciences, Branford, CT), amplified using the GS FLX Titanium emPCR kit (454 Life Sciences, Branford, CT), and sequenced according to standard 454 pyrosequencing protocols on a 454 sequencer (454 Life Sciences, Branford, CT).

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Nucleic acid sequences can be analyzed to determine taxonomic assignments using sequence similarity and phylogenetic placement methods or a combination of these two strategies. A similar approach can be used to annotate protein names, protein functions, transcription factor names, and any other nucleic acid sequence classification scheme. Methods based on sequence similarity include BLAST, BLASTx, tBLASTn, tBLASTx, RDP-classifier, DNAclust, RapSearch2, DIAMOND, USEARCH and various implementations of these algorithms such as QIIME or Mothur. These methods match the read sequence to a reference database and select the best match. Common databases include KEGG, MetaCyc, NCBI non-redundant database, Greengenes, RDP and Silva for taxonomic assignments. For functional assignment, reads are mapped to various functional databases such as COG, KEGG, BioCyc, MetaCyc and the Carbohydrate Active Enzyme (CAZy) database. Classes of microorganisms are assigned using software including MetaPhlAn.

In some embodiments, bacterial constituents are identified by characterizing the DNA sequence of the bacterial 16S small subunit ribosomal RNA gene (16S rRNA gene). The 16S rRNA gene is approximately 1500 nucleotides long and is generally highly conserved across all organisms, but contains specific variable and hypervariable regions (V1-V9) that have sufficient nucleotide diversity to differentiate taxa at the species and strain level in most organisms. These regions in bacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465, respectively, using a numbering system based on E. coli nomenclature.

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

Sequences that are at least 97% identical to each other are grouped into Operational Taxonomic Units (OTUs). OTUs that contain sequence with 97% similarity roughly correspond to species-level taxa. At least one representative sequence from each OTU is selected and used to obtain a taxonomic assignment for the OTU by comparison to a reference database of curated 16S rRNA gene sequences (eg, Greengenes or SILVA databases). The relationships among OTUs in a microbial community can be inferred by constructing a phylogenetic tree from the representative sequences of each OTU. Using known methods to determine the complete 16S sequence or the sequence of any variable region of the 16S sequence, genomic DNA is extracted from the bacterial sample, 16S rRNA (the complete region or specific variable regions) is amplified using polymerase chain reaction (PCR), the PCR products are purified, and the nucleotide sequences are delineated to determine the genetic composition of the 16S rRNA gene or variable region of the gene. If full 16S sequencing is performed, Sanger sequencing may be used, but is not limited to. If one or more variable regions are used, such as the V4 region, sequencing can be performed using the Sanger method or a next generation sequencing method such as, but not limited to, the Illumina method. Primers designed to anneal to conserved regions of the 16S rRNA genes (e.g., primers 515F and 805R to amplify the V4 region) can contain unique barcode sequences that allow the simultaneous characterization of multiple microbial communities.

In addition to the 16S rRNA gene, to assess microbial community composition, a selected set of genes known to be marker genes for a given species or taxonomic group are analyzed. Alternatively, these genes are analyzed using a PCR-based screening strategy. For example, different strains of pathogenic Eschenchia coli are distinguished using genes encoding heat-labile (LTI, LTIIa and LTIIb) and heat-stable (STI and STII) toxins, verotoxins types 1, 2 and 2e (VT1, VT2, and VT2e, respectively), cytotoxic necrotoxins factors (CNF1 and CNF2), attachment and displacement mechanisms (eaeA), enteroaggregative mechanisms (Eagg) and enteroinvasive mechanisms (Einv). The optimal genes to use to determine the taxonomic composition of a microbial community using marker genes will be familiar to one of ordinary skill in the art of sequence-based taxonomic identification.

In some embodiments, the identity of the microbial composition is characterized by the identification of nucleotide markers or genes, particularly highly conserved genes (eg, housekeeping genes) or combinations thereof. Using certain methods, DNA isolated from a bacterial sample will have specific regions of the genome amplified by PCR and sequenced to determine the nucleotide sequence of the amplified

- 46 045265 bathroom products.

VII. Targeted delivery of metabolites to the gastrointestinal tract.

A. Metabolites of the gastrointestinal tract.

In some embodiments, the methods described herein include delivering or increasing one or more gastrointestinal metabolites in the gastrointestinal tract of an animal. In some embodiments, one or more metabolites are detected and quantified. In some embodiments, the metabolites include short-chain fatty acids (SCFAs), nitrogenous metabolites, bile acids, polyphenols, amino acids, neurotransmitters, signaling factors, butyric acid, propionic acid, acetic acid, lactic acid, valeric acid, isovaleric acid, amino-SCFA, thioates, terpenoids, a-terpenoids, anamines, ammonia, indole, butyric acid, histamine, betazole, GABA, 2FL, eucalyptol, geraniol, 2-MThEtOH, 3-methyl-2-butanone, 3-methylbutanal, pentanal, 3-hydroxy -2butanone, (E)-2-pentenal, 1-pentanol, (E)-2-decenal, hexanal, (E)-2-hexenal, 1-hexanol, heptanal, styrene, oxime-, methoxyphenylbutyrolactone, (E)- 2-heptenal, benzaldehyde, dimethyl trisulfide, 1-heptanol, octanal, 1-octen-3-one, 1-octen-3-ol, (E,E)-2,4-heptadienal, 2-acetylthiazole, D-limonene, 4-ethylcyclohexanol, 2,4-dimethylcyclohexanol, (E)-2-octenal, benzolacetaldehyde, 1-octanol, 2butylcyclohexanone, 4-(benzoyloxy)-(E)-2-octen-1-ol, 1-octanol, octadecanoic acid, ethenyl ester, nonanal, (E)-2-nonen-1-ol, 3-octadecine, cyclooctanemethanol, dodecanal, (E)-2-nonenal, 2,6/3,5dimethylbenzaldehyde, 1-nonanol, 2-n-heptylfuran, cis- 4-decenal, decanal, (E,E)-2,4-nonadienal, 1,3hexadiene, 3-ethyl-2-methyl-2-nonenal, (E)-2-undecenal, trans-3-nonen-2- he, 2,5-furandione, 3dodecenyl-trans-2-undecen-1-ol, eicosanoic acid or any combination thereof.

In some embodiments used herein, butyric acid and butyrate are used interchangeably. In some embodiments of the present invention, propionic acid and propionate are used interchangeably.

In some embodiments, one or more metabolites are beneficial to the animal (eg, beneficial to the health of the animal). Examples of beneficial metabolites include short-chain fatty acid (SCFA), amino-SCFA, neurotransmitter, neurotransmitter precursor, neurochemical, gamma-aminobutyric acid (GABA), dopamine, aminoindole, volatile fatty acids (VFA), butyric acid, propionic acid, acetic acid, lactic acid, valeric acid, isovaleric acid, essential oils, a-terpenoid, eucalyptol, geraniol, betazole, milk oligosaccharide, fucosylated oligosaccharide, stylated oligosaccharide, 2-fucosyllactose and aminoindole.

In some embodiments, one or more metabolites comprise butyrate, propionate, or both. In some embodiments, one or more metabolites comprise essential oils. In some embodiments, the one or more metabolites comprise a dipeptide, fatty alcohol, or a-terpenoid. In some embodiments, one or more metabolites comprise linalool, eucalyptol, or geraniol. In some embodiments, one or more metabolites contain a neurotransmitter. In some embodiments, one or more metabolites contain ammonia.

In some embodiments, one or more metabolites promote growth of the animal. In some embodiments, the one or more metabolites promote the growth of the animal, and are selected from the group consisting of butyric acid, propionic acid, acetic acid, lactic acid, valeric acid and isovaleric acid.

In some embodiments, one or more metabolites are harmful to the health of the animal. In some embodiments, one or more metabolites are harmful to the health of the animal, and are selected from the group consisting of short chain fatty acid (SCFA), ammonia, trimethylamine (TMA), trimethylamine N-oxide (TMAO), uremic acid solute, and bile acid .

In some embodiments, the metabolite is a pro-inflammatory metabolite. Examples of pro-inflammatory metabolites include, but are not limited to, histamine and LPS.

In some embodiments, the metabolite is related to the quality of the animal meat, including, for example, the taste, color, and texture of the animal meat. In some embodiments, the one or more metabolites include 2-MThEtOH, 3-methyl-2-butanone, 3-methylbutanal, pentanal, 3-hydroxy-2butanone, (E)-2-pentenal, 1-pentanol, (E)-2 -decenal, hexanal, (E)-2-hexenal, 1-hexanol, heptanal, styrene, oxime-, methoxyphenyl-butyrolactone, (E)-2-heptenal, benzaldehyde, dimethyl trisulfide, 1-heptanol, octanal, 1-octene-3- he, 1-octen-3-ol, (E,E)-2,4-heptadienal, 2-acetylthiazole, D-limonene, 4ethylcyclohexanol, 2,4-dimethylcyclohexanol, (E)-2-octenal, benzolacetaldehyde, 1-octanol, 2butylcyclohexanone, 4-(benzoyloxy)-(E)-2-octen-1-ol, 1-octanol, octadecanoic acid, ethenyl ester, nonanal, (E)-2-nonen-1-ol, 3- octadecine, cyclooctanemethanol, dodecanal, (E)-2-nonenal, 2,6/3,5dimethylbenzaldehyde, 1-nonanol, 2-n-heptylfuran, cis-4-decenal, decanal, (E,E)-2,4- nonadienal, 1,3hexadiene, 3-ethyl-2-methyl-2-nonenal, (E)-2-undecenal, trans-3-nonen-2-one, 2,5-furandione, 3dodecenyl-trans-2-undecen- 1-ol, eicosanoic acid or any combination thereof.

- 47 045265

In some embodiments, at least one of the one or more metabolites is volatile, such as volatile fatty acids. Volatile fatty acids may refer to short-chain fatty acids such as C2-C6 carboxylic acids. In some embodiments, at least one of the one or more metabolites has a strong malodor. Examples of strong odor or malodor substances may include, but are not limited to, butyric acid and butyric anhydride. In some embodiments, at least one of the one or more metabolites results in decreased palatability and a corresponding decrease in feed intake. In some embodiments, at least one of the one or more metabolites is unstable to oxidation. For example, the iodine value may be used to measure the sensitivity of a substance to oxidation, and a metabolite that is unstable to oxidation may have an iodine value greater than 10, 20, 30, 40, 50, 60, 70, 80 or more according to the Kauffman method. In some embodiments, at least one of the one or more metabolites is unstable to oxidation under commercial animal feed manufacturing conditions.

In some embodiments, one or more metabolites are absorbed in the upper digestive tract of the animal. In some embodiments, all of one or more metabolites are absorbed in the upper digestive tract of the animal.

B. Sampling and detection of gastrointestinal metabolites.

In some embodiments, the methods described herein include detecting or quantifying one or more metabolites in the gastrointestinal tract of an animal. In some embodiments, the metabolite is detected or quantified in a gastrointestinal sample of the animal. 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 feces, saliva and gastrointestinal ascitic fluid are also collected. In some embodiments, the sample is a biopsy of gastrointestinal tissue or a stool sample. Methods for obtaining gastrointestinal tract samples are standard and known to one skilled in the art.

In some embodiments, the sample is taken from a compartment of the animal's gastrointestinal tract. In some embodiments, the sample taken represents the level of one or more metabolites in a compartment of the animal's gastrointestinal tract. In some embodiments, the compartment is part of the animal's lower digestive tract. In some embodiments, the compartment includes all or part of the small intestine and all or part of the colon.

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, metabolites are purified from the sample prior to analysis. In some embodiments, metabolites from a single sample are purified. In some embodiments, metabolites from multiple samples from a single animal are purified and then pooled before analysis.

Metabolites that are present in gastrointestinal samples taken from animals, or in fresh or spent culture media, can be determined using the methods described herein and known to one skilled in the art. Such methods include, for example, chromatography (eg, gas chromatography (GC) or liquid chromatography (LC)) in combination with mass spectrometry or NMR (eg, ¹ H-NMR). Measurements can be confirmed using metabolite standards in the same analytical systems.

In the case of gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS), polar metabolites and fatty acids can be extracted using single-phase or two-phase systems of organic solvents and an aqueous sample solution, and subjected to derivatization. An exemplary protocol for the derivatization of polar metabolites involves the formation of methoxime-tBDMS derivatives by incubating the metabolites with 2% methoxylamine hydrochloride in pyridine followed by the addition of N-tert-butyldimethylsilyl-K-methyltrifluoroacetamide (MTBSTFA) with 1% tert-butyldimethylchlorosilane (t-BDMCS). Non-polar fractions, including triacylglycerides and phospholipids, can be saponified to free fatty acids and esterified to form fatty acid methyl esters, for example by incubation with 2% H2SO4 in methanol or using Methyl-8 reagent (Thermo Scientific). The derivatized samples can then be analyzed by GCMS using standard LC-MS methods, such as a DB-35MS column (30 m x 0.25 mm i.d. x 0.25 µm, Agilent J&W Scientific) mounted on a gas chromatograph ( GC),

- 48 045265 coupled with a mass spectrometer (MS). Mass isotopomer distributions can be determined by integrating metabolite fragment ions and correcting for natural abundance using standard algorithms. For liquid chromatography-mass spectrometry (LC-MS), polar metabolites can be analyzed using a standard benchtop LC-MS/MS setup equipped with a column such as the SeQuant ZIC-Philic polymer column (2.1 × 150 mm; EMD Millipore ). Examples of mobile phases used for separation may include buffers and organic solvents adjusted to a specific pH.

In combination or alternatively, the extracted samples can be analyzed using ^1H nuclear magnetic resonance (1H-NMR). Samples can be combined with isotope- _enriched solvents such as _D2O , _if necessary in the presence of a buffer solution (eg _Na2HPO4 , _NaH2PO4 in D2O, pH 7.4). Samples can also be supplemented with a standard for calibration and chemical shift determination (e.g. 5 mM sodium salt of 2,2-dimethyl-2silapentane-5-sulfonate (DSS-d6, Isotec, USA)). Before analysis, the solution can be filtered or centrifuged to remove any sediment or deposits and then transferred to a suitable tube or vessel for NMR analysis (eg, a 5 mm NMR tube). ¹ H-NMR spectra can be obtained on a standard NMR spectrometer such as an Avance II+ 500 Bruker (500 MHz) spectrometer (Bruker, DE) equipped with a 5 mm QXI-Z C/N/P probe head) and analyzed with using spectrum integration software (eg, Chenomx NMR Suite 7.1; Chenomx Inc., Edmonton, Alberta). Alternatively, ¹ H-NMR can be performed in accordance with other published protocols known in the art (see, for example, Chassaing et al., Lack of soluble fiber drives diet-induced adiposity in mice, Am J Physiol Gastrointest Liver Physiol, 2015; Bai et al., Comparison of Storage Conditions for Human Vaginal Microbiome Studies, PLoS ONE, 2012:e36934).

C. Level of metabolites.

In some embodiments, a method of delivering or increasing one or more metabolites in the gastrointestinal tract of an animal includes determining the level of at least one of the one or more metabolites in a sample. In some embodiments, a method of delivering or increasing one or more metabolites in the gastrointestinal tract of an animal includes determining the level of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 metabolites in a sample. In some embodiments, the metabolite level is determined, in whole or in part, by LC or GC. In some embodiments, the metabolite level is determined in whole or in part by mass spectrometry. In some embodiments, the metabolite level is determined in whole or in part using NMR.

In some embodiments, the level of metabolites in a compartment of the animal's gastrointestinal tract is determined. Accordingly, in some embodiments, the level of one or more metabolites in the same compartment is compared. In some embodiments, the level of one or more metabolites in different compartments is compared.

In some embodiments, the level of one or more metabolites in the gastrointestinal tract of an animal to which a nutritional composition containing an oligosaccharide drug is administered is increased compared to the level of a metabolite in the gastrointestinal tract of an animal to which a nutritional composition without the oligosaccharide drug is administered.

For example, in some specific embodiments, the level of butyric acid in the gastrointestinal tract of an animal treated with a nutritional composition containing an oligosaccharide drug is increased compared to the level of butyric acid in the gastrointestinal tract of an animal treated with a nutritional composition not containing an oligosaccharide drug. drug. In some specific embodiments, the level of propionic acid in the gastrointestinal tract of an animal treated with a nutritional composition containing the oligosaccharide drug is increased compared to the level of propionic acid in the gastrointestinal tract of an animal treated with a nutritional composition not containing the oligosaccharide drug. In some specific embodiments, the level of one or more essential oils in the gastrointestinal tract of an animal to which a nutritional composition containing an oligosaccharide preparation is administered is increased compared to the level of one or more essential oils in the gastrointestinal tract of an animal to which a nutritional composition is administered. , which does not contain an oligosaccharide drug.

In some embodiments, each level of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more metabolites in the gastrointestinal tract of an animal to which a nutritional composition comprising an oligosaccharide drug is administered is increased relative to the level of the metabolite in gastrointestinal tract of an animal in which a nutritional composition not containing an oligosaccharide drug is used.

For example, in some specific embodiments, the level of each of butyric acid, propionic acid, and one or more essential oils in the gastrointestinal tract of an animal in which a nutritional composition containing an oligosaccharide drug is administered is elevated compared to the level of the metabolite in the gastrointestinal tract. - the intestinal tract of an animal in which a nutritional composition that does not contain an oligosaccharide drug is used.

In some embodiments, administration of a disclosed nutritional composition increases the level of one or more metabolites in a compartment of the animal's gastrointestinal tract compared to the metabolite level prior to administration of the nutritional composition. For example, in some embodiments, administration of a disclosed nutritional composition increases the level of butyric acid, propionic acid, or one or more essential oils in a compartment of the animal's gastrointestinal tract compared to the metabolite level prior to administration of the nutritional composition.

In some embodiments, the effect of the nutritional composition on the level of one or more metabolites in a compartment of the animal's gastrointestinal tract depends on the composition and characteristics of the oligosaccharide preparation. For example, some oligosaccharide drugs increase the level of butyric acid in the animal's gastrointestinal tract. As another example, a certain oligosaccharide drug increases the level of butyric acid and propionic acid in the gastrointestinal tract of an animal. As another example, a certain oligosaccharide preparation increases the level of butyric acid and one or more essential oils in the gastrointestinal tract of an animal, but does not increase the level of propionic acid.

In some embodiments, detection of the level of one or more metabolites is performed after administration of the nutritional composition. For example, in some embodiments, depending on the type and age of the animal, the level of one or more metabolites is determined at at least 10, 20, 30 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24 hours, 2 or 3 days after application of the nutritional composition. In some embodiments, the level of one or more metabolites is determined after a maximum of 10, 20, 30 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24 hours, 2 or 3 days from the moment of use of the nutritional composition.

VII. Ways to increase animal productivity.

A. Feed conversion ratio.

In some embodiments, the methods described herein 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 health, nutritional, environmental and/or community factors.

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

- 50 045265 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 effects of social interaction, such as difficulty accessing food or drinking water.

In some embodiments, an animal fed a diet not including a synthetic oligosaccharide drug described herein 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 herein 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 an animal receiving a diet that does 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.

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 herein 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 preparation, nutritional preparation, 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 a synthetic oligosaccharide drug where the FCR is adjusted to account 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 to 10%, from 5 to 10%, from 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 fed a feed composition without synthetic oligosaccharide drug.

In some embodiments, the animal is a swine and the animal feed composition is a swine feed, wherein the synthetic oligosaccharide drug, swine nutritional composition, swine feed premix, or swine feed composition reduces 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 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%, 2 to 9% or 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 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%, 2 to 6%, 2 to 7%, 2 to 8%, 2 to 9%, or 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 herein 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 the control animal that received 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 70 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 grams, at least 40 grams, or at least 50 grams per 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 grams, at least 60 grams, or at least 70 grams per 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

- 53 045265 increases the average daily gain of birds 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%, from 2 up 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 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 up 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 preparation.

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

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 average daily weight gain of pigs fed a diet that does not include a synthetic oligosaccharide preparation.

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

- 54045265 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 weight 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 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 receiving 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 net weight gain compared to a population of animals fed the diet without the synthetic oligosaccharide drug.

In some embodiments, providing an animal or animal population 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 by an individual animal 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

- 55 045265 up 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 poultry, and the poultry is between 0 and 14 days, 15 and 28 days, 29 and 35 days, 0 and 42 days, 0 and 6 weeks, or 0 and 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 start phase is from 0 to 14 days, the growth 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 a synthetic oligosaccharide preparation, a swine nutritional composition, a swine feed premix, or a porcine feed composition

- 56,045,265 pigs, has an average final weight gain of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 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 average final weight gain of pigs fed a diet that does not include a synthetic oligosaccharide drug.

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 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 per eviscerated poultry carcass is at least 6%, at least 8%, at least 10%, at least 12%, 6 to 12% . from 8 to 12%, from 10 to 18%,

- 57 045265 from 12 to 16% or from 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 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 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 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%, 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 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 for

- 58 045265

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 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 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 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%, 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 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 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%, 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 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 yield of leg meat 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%, according to 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 1%. by 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%, at least

11%, at least 12%, 1-10%, 2-8% or 3-5% more than poultry fed a diet not containing the 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 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 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 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 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%, by 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.

- 60 045265

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 vary between different types of animals and may differ between 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 vary between different types of animals and may differ between 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%, 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%, 2 to 7%, 2 to 8%, 2 to 9%, or 1 to 5% when fed to poultry compared to poultry fed the feed composition without the synthetic oligosaccharide preparation.

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 suffers from a disease or is raised in an unfavorable manner

- 61 045265 pleasant environment where the synthetic oligosaccharide preparation, swine nutritional composition, swine feed premix or swine feed composition increases average daily feed intake by about 40%, by about 35%, by about 30%, by about 25%, by about by 20%, by about 15%, by about 10%, or by 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 up 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 herein 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 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 herein 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 - bad) 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.

I. Quality of animal feces.

Gut microbiome metabolites influence fecal quality in animals. For example, volatile amines, thiols and sulfides play an important role in the formation of odors associated, for example, with animal litter (including livestock and pets). The methods described herein include methods for improving the quality of animal feces. Quality attributes include, for example, odor, consistency and pathogen levels. Each of the qualities of feces can be assessed by standard methods known to one skilled in the art.

The level of pathogens in a fecal sample can be assessed using standard methods and commercial kits. In some embodiments, total DNA or total RNA is isolated from the sample. Genomic DNA can be extracted from samples using standard methods known to those skilled in the art, including commercial kits such as the Mo Bio Powersoil®-htp 96-well Soil DNA Extraction Kit (Mo Bio Laboratories, Carlsbad, CA), Mo Bio Powersoil® DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) or QIAamp DNA Stool Mini Kit QIAamp (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RNA can be extracted from samples using standard assays known to those skilled in the art, including commercial kits such as the RNeasy PowerMicrobiome kit (QIAGEN, Valencia, Calif.) and the RiboPure Bacterial RNA Purification Kit (Life Technologies, Carlsbad, Calif.). Another method for isolating bacterial RNA may involve enriching the mRNA of purified bacterial RNA samples by removing tRNA. Alternatively, the RNA can be converted to cDNA, which can be used to generate sequencing libraries using standard methods such as the Nextera XT sample preparation kit (Illumina, San Diego, CA).

Identification and determination of the relative abundance of a pathogen in a sample can be accomplished by standard molecular biology techniques known to one of ordinary skill in the art, including, for example, genetic analysis (e.g., DNA sequencing (e.g., whole genome sequencing, whole genome shotgun sequencing (WSG)), RNA sequencing, NLR, quantitative NLR (qPCR), serological and antigen analysis, microscopy, metabolite identification, Gram staining, flow cytometry, immunological methods and

- 62 045265 culture-based methods such as colony-forming unit counting.

J. Leg disease.

Some metabolites, such as ammonia, in animal litter lead to increased moisture and elevated litter pH, which contribute to the development of foot diseases such as leg dermatitis. Ammonia production by the gut microbiome contributes to increased ammonia levels in litter. The length of time between housing successive herds or groups in commercial livestock production is often determined by the amount of time the facility must be ventilated to remove ammonia from the bedding.

The methods described herein include methods for reducing ammonia levels in the gastrointestinal tract of an animal and reducing ammonia levels in bedding to prevent foot diseases. The methods described herein further include methods for reducing ammonia production by gut microflora to reduce downtime between herds or groups, thereby improving productivity and economics of production. Foot diseases include, for example, leg dermatitis.

IX. Animals.

A. Type of 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 preruminant phase are dairy calves.

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 livestock. In some embodiments, the animal is a pet. 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 or cat. 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.

B. Digestive system of animals.

The synthetic oligosaccharide preparation, nutritional composition, animal feed premix or animal feed composition can be provided to an animal having any type of digestive system, such as the digestive system of a monogastric animal, poultry, ruminants and pseudoruminants.

In some embodiments, the animal has a monogastric digestive system. In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes the esophagus, stomach, small intestine, colon, anus, rectum, or any combination thereof. In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes an upper digestive tract, a lower digestive tract, or both.

In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes the lower digestive tract. In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes the small intestine, large intestine, or both. In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes all or part of the small intestine. In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes all or part of the colon. In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes the gastrointestinal tract below the stomach.

In some embodiments, the animal has a avian digestive system. In some embodiments, the compartment in the avian gastrointestinal tract includes the esophagus, crop, proventriculus, stomach, small intestine, colon, cloaca, or any combination thereof. In some embodiments, the compartment in the poultry's gastrointestinal tract includes an upper digestive tract, a lower digestive tract, or both.

In some embodiments, the compartment in the poultry gastrointestinal tract includes the lower digestive tract. In some embodiments, the compartment in the avian gastrointestinal tract includes the proventriculus, stomach, small intestine, colon, or any combination thereof. In some embodiments, the compartment in the poultry's gastrointestinal tract includes the stomach, small intestine, colon, or any combination thereof.

In some embodiments, the compartment in the poultry gastrointestinal tract includes all or part of the small intestine. In some embodiments, the compartment in the avian gastrointestinal tract includes all or part of the colon. In some embodiments, the compartment in the gastrointestinal tract of a monogastric animal includes the gastrointestinal tract located below the proventriculus.

In some embodiments, the animal has a ruminant digestive system. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes the esophagus, rumen, reticulum, book, abomasum, small intestine, colon, or any combination thereof. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes an upper digestive tract, a lower digestive tract, or both.

In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes the lower digestive tract. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes a rumen, mesh, book, abomasum, small intestine, colon, or any combination thereof. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes a rumen, mesh, book, abomasum, small intestine, or any combination thereof.

In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes all or part of the rumen. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes all or a portion of the mesh. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes all or part of the booklet. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes all or part of the abomasum. In some embodiments, the compartment in the gastrointestinal tract of a ruminant animal includes all or part of the small intestine.

In some embodiments, the animal has a pseudoruminant digestive system. In some embodiments, the compartment in the gastrointestinal tract of a pseudoruminant animal includes the esophagus, stomach, small intestine, colon, cecum, rectum, anus, or any combination thereof. In some embodiments, the compartment in the gastrointestinal tract of a pseudoruminant animal includes an upper digestive tract, a lower digestive tract, or both.

In some embodiments, the compartment in the gastrointestinal tract of a pseudoruminant animal includes the lower digestive tract. In some embodiments, the compartment in the gastrointestinal tract of a pseudoruminant animal includes the small intestine, colon, cecum, or any combination thereof. In some embodiments, the compartment in the gastrointestinal tract of a pseudoruminant includes all or part of the small intestine. In some embodiments, the compartment in the gastrointestinal tract of the pseudoruminant includes all or part of the colon. In some embodiments, the compartment in the gastrointestinal tract of a pseudoruminant includes all or part of the cecum.

In some embodiments, the animal may have digestive system features of more than one of the above types. In some embodiments, the animal may have digestive system features other than the above types. In some embodiments, a compartment in the animal's gastrointestinal tract includes one or more organs or sites where the animal absorbs the majority of its food. In some embodiments, a compartment in the animal's gastrointestinal tract includes one or more organs or sites where the animal digests the majority of its food. In some embodiments, a compartment in the animal's gastrointestinal tract includes an organ or site where the majority of food is digested or absorbed by the animal.

In some embodiments, a compartment in the gastrointestinal tract of an animal includes all or part of the stomach (or equivalents thereof, such as rumen, mesh, book, and abomasum), all or part of the small intestine, all or part of the large intestine, or any of these. a combination of the colon - 64 045265, or any combination thereof. In some embodiments, the compartment in the animal's gastrointestinal tract includes all or part of the small intestine and all or part of the large intestine.

In some embodiments, the gastrointestinal compartment includes all or part of the lower digestive tract. In some embodiments, the gastrointestinal compartment is all or part of the lower digestive tract. In some embodiments, the compartment in the gastrointestinal tract is the stomach, small intestine, and colon. In some embodiments, the compartment in the gastrointestinal tract is the small intestine or the colon.

X. Application.

In some embodiments, use includes providing a synthetic oligosaccharide preparation, nutritional composition, or animal feed composition described herein to an animal so that the animal can consume the synthetic oligosaccharide preparation, 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 every month.

In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal several times per day. For example, in some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day. In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day.

In some embodiments, the nutritional composition, oligosaccharide preparation of oligosaccharides, animal feed premix, or animal feed composition is administered to an animal at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times in Week. In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 times per week . In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal every day, every other day, every 3 days, every 4 days, every week, every other week, or every month.

In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal at specific times during the day. For example, in some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to an animal in the morning, afternoon, evening, or any combination thereof. In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal in the morning. In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to the animal in the afternoon. In some embodiments, the nutritional composition, oligosaccharide preparation, animal feed premix, or animal feed composition is administered to an animal in the evening.

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

- 65 045 265 days, and the final diet at the age of 29 to 35 days. 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 herein 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 glucogalacto-oligosaccharide drug.

Synthesis of the glucogalacto-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.

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 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. When the reaction mixture reached 120°C, the reflux condenser was set to 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 glucooligosaccharide 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 a glass

- 66 045265 stirring rod with 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.

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 round bottom distillation configuration. 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 a glucogalactomannooligosaccharide drug.

Synthesis of the glucogalactomannooligosaccharide 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 glucogalactomannooligosaccharide 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 glucogalactomannooligosaccharide 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 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 29/42 ground 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 viscous, dark amber colored material was collected and measured at 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° on the scale.

- 67 045265

Brix.

Example 4. Synthesis of glucomannooligosaccharide 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.

The glucomannooligosaccharide 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 glucomannooligosaccharide 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-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 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 distillation configuration with a round bottom collection flask. 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 glucomannooligosaccharide drug.

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

The glucomannooligosaccharide 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 glucomannooligosaccharide 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 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 h, 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 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 glucogalacto-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 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 µm 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 g of Dowex Marathon C resin (moisture content 0.48 g _H2O /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 µm filter to remove resin. 1113 g of gluco-oligosaccharide syrup of dark amber color with a concentration of 60° Brix were 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 0.270 g 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 220 mm 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 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. Synthesis and characterization 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. The resulting materials were analyzed by HPLC size exclusion chromatography (SEC) to characterize molecular weight distribution, LC-MS/MS analysis to quantify DP2 anhydrosaccharide content, and 2D 1H, ^13C -HSQC NMR to determine the molecular structure of the corresponding oligosaccharide drugs.

Example 9.1. Eleven batches of the oligosaccharide preparation from Example 1 were prepared and mixed in 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 g 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:

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

Example 9.12. 270 g of glucose, 30 g of lactose, 3 g of phenylphosphinic acid and 27 g of water were reacted at 130° C. for three to five hours to produce a dark brown oligosaccharide syrup, which was 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 produce 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.

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 anhydrosaccharide content, and 2D 1H, ^13C -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 drugs 9.1-9.7

Oligosaccharide MWn (g/mol) MWw (g/mol) a drug 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 content of DP2 anhydrooligosaccharides in oligosaccharide preparations was determined by LC-MS/MS using a Capcell Pak NH2 column (Shiseido; 250 χ 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 content of anhydro-BH2 in oligosaccharide preparations was first determined relative to the content in the oligosaccharide preparation from Example 9.7 as a reference composition. The absolute aHrugpo-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.

Table 2. Anhydro-DP2 content in oligosaccharide preparations with several series

Oligosaccharide drug Relative content of anhydro-OP2, in% compared to example 9.7 Anhydro-OP2 content (gram anhydro-OP2/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 by 2D ¹ H, 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 ¹ H, ¹³ C HSQC NMR spectrum of the oligosaccharide preparation from Example 9.7.

Anomeric region of the spectrum ¹ H, ¹³ C-HSQC, F2 ( ¹ H) = 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 ¹ H, ¹³ C 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% 4.50 103.30 25.9% ± 2.2% 4.45 103.56 2.4% ± 1.3%

Example 10. Determination of anhydrous sugar subunits in an oligosaccharide preparation.

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The relative abundance of anhydrous 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 sugar anhydrosubunits 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 anhydrous 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. 23A and 23B illustrate the MALDI spectra of the oligosaccharide preparation from Example 2. Anhydrous sugar subunits are observed at each DP level with relative intensities in the range of 5-10%.

Example 11. Characteristics of anhydrosubunits 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 anhydrous 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-angide glucose rho-beta-Dzuranose 1,6-Anhydro-beta-Oglucopyranose # 'N (m.d.) ¹³ C (ppm) 'N (m.d.) °C (ppm) 1 5.33 101.9 5.01 104.4 2 3.40 70.6 4.37 79.8 3 3.56(ov) ^a 73.0 4.27 78.3 4 3.56(ov) ^a 71.3 4.38 80.6 5 4.50 76.7 3.74 64.1 6 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 HP-5MS stationary phase with helium at a constant pressure of 21.57 psi as 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 per minute 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. 28A28B, 29A-29B, 30A-30B and 31A-31B 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. 28A-28B, 29A-29B, 30A-30B and 31A-31B, the anhydro-DP1 and DP1 fractions have retention times of approximately 12-17 minutes, and the anhydro-DP2 and DP2 fractions have retention times of approximately 2-25 minutes.

Fig. 35 illustrates MALDI-MS spectra comparing the oligosaccharide formulation of Example 9 at various 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 anhydrosaccharide subunits in the oligosaccharide preparation has been proven.

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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 : δ=9.39 ppm (SNO, m); 7.44 ppm (Ar-H, m); 6.68 ppm (ArH, m); 4.60 ppm (CH2, m); and ¹³ C-NMR: -180.0; 150.6; 126.2; 112.7; 159.9; 64.5. Fig. 6 presents an exemplary 2D HSQC spectrum that demonstrates the incorporation of 5-hmf into the structure of an oligosaccharide drug via a glycosidic bond.

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 NH2 column (Shiseido; 250 × 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. 24A-24C, 25A-25C, 26A-26C and 27A-27C 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 microingredient 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 weighing scale. for microingredients 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 of oligosaccharide drug. For the processed diet, the oligosaccharide drug was provided in powder form and the powder was added to a mixer using a microingredient balance 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 of the oligosaccharide formulation from Example 9.1 to control feed 15.7 (CTR). 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%, chloride sodium 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 of the oligosaccharide formulation from Example 9.3 to control feed 15.7 (CTR). 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 Da.

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 glucooligosaccharides 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 (0.2 M ammonium acetate, 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 U. /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 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.

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Table 5. List of feed samples for extraction and digestion

Matrix Extraction solvent Filtration Enzymatic digestion 1 Control food Water 0.22 µM 2 Anhydrooligomeric feed 1000 mg/kg Water 0.22 µM - 3 Control food Ethanol/Water 50/50 0.22 µM - 4 Anhydrooligomeric feed 1000 mg/kg Ethanol/Water 50/50 0.22 µM - 5 Anhydrooligomers Water 0.22 µM a + b (4 hours 60°C) 6 Control food Water 0.22 µM a + b (4 hours 60°C) 7 Anhydrooligomeric 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 Anhydrooligomeric feed 1000 mg/kg Ethanol/Water 50/50 0.22 µM - 12 Anhydrooligomeric feed 1000 Water 0.45 µM a + b (4 hours 60°C) mg/kg 13 Anhydrooligomeric 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 Anhydrooligomeric 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 Anhydrooligomeric feed 1000 mg/kg Water 0.45 µM a + b + c (4 hours 60°C) 18 Rice and spelled/ Anhydrooligomers Water 0.45 µM 19 Rice and spelled/ Anhydrooligomers Ethanol/Water 50/50 0.45 µM - 20 Control food Water 0.45 µM a + b + c (4 hours 60°C) 21 Anhydrooligomeric 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 (Anhydro oligomers 1000 mg/kg) Water 0.45 µM 26 Growth feed C (control) Water 0.45 µM a + b + c + d(4h 60°C) 27 Growth feed D (Anhydrooligomers 2000 mg/kg) Water 0.45 µM a + b + c + d(4h 60°C) 28 Pre-start food A (control) Water 0.45 µM a + b + c + d(4h 60°C) 29 Pre-starter feed D (Anhydro oligomers 1000 mg/kg) Water 0.45 µM a + b + c + d(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 + d(4h 60°C) 34 Wheat Water 0.45 µM a + b + c + d(4h 60°C) 35 Soybeans Water 0.45 µM a + b + c + d(4h 60°C) 41 Control food Water 0.45 µM 42 Anhydrooligomeric feed 1000 mg/kg Water 0.45 µM 43 Control food Water ultra 5000 MWCO a + b + c X2 (overnight at 60°C) 44 Anhydrooligomeric feed 1000 mg/kg Water ultra 5000 MWCO a + b + c X2 (overnight at 60°C)

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Anhydrooligomers refer to oligosaccharides containing anhydrosubunits.

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 raw materials 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-BG2 were compared with standard calibration curve for determining the concentration of the oligosaccharide drug in feed (Table 6).

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 glucooligosaccharides containing anhydrosubunits.

Gluco-oligosaccharide preparations containing anhydrosubunits 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 assay, approximately 50 mg of lyophilized product was dissolved in D2O 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 carried out at 298 K. ¹ H-NMR spectra were recorded and calibrated in deuterated water (4.75 ppm). ¹³ C-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 anhydrous subunits, with solvent pre-saturation. 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 glucooligosaccharides containing anhydrosubunits (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% 3(1,2) 5.7% 6.5% 3(1,3) 13.3% 6.3% 3(1.4) 17.9% 10.7% 3(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. 11 shows a typical 1H, ^13C -HSQC NMR spectrum of a sample of glucooligosaccharides containing anhydrosubunits, with the corresponding resonances and assignments used for bond assignment. 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 glucooligosaccharides containing anhydrosubunits (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 anhydrosubunits 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. 9. The collected DP1 fractions from 8 separations were pooled, dried and redissolved in 0.75 ml 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 LCMS/MS and NMR analysis.

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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 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 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 μm syringe filter and analyzed by HPLC size exclusion chromatography (SEC). SEC analysis was performed on an Agilent serine 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. The DP equilibrium constant was determined to be K = 3.3, and the DP distribution was found to decrease monotonically. Fig. 15 and 16 illustrate the DP distribution shape of the 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-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 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. When 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 μm 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 nonmonotonically decreasing. Fig. 16 shows that the content

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DP3 is greater than the content of DP2, and that the content of DP4 and DP5 is essentially 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 μm syringe filter and analyzed by HPLC size exclusion 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. The DP equilibrium constant was determined to be 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% AOAC 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 M99 x Cobb 500 chicks were obtained on hatch day from a commercial hatchery and randomly housed in 36 ft x 40 ft 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 (i.e. containing 2 lb/ton feed additive) and birds in even-numbered pens were fed a control diet.

- 80 045265 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.

The relative abundance of the identified taxa was determined for 96 sampled birds from Study Example 23. For each microbiota sample obtained in Example 23, cecal contents were thawed and DNA 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). Cecal microbiota was also assessed by 16S rRNA analysis (16Sv4 PNR/Illumina MiSeq sequencing at 2x250 bp) against a standard 16S rRNA line (USEARCH and SILVA(v4) DB) with a rarefaction cutoff of 12,230 reads per sample.

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 23.

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 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

- 81 045265 by combining 25 µl of water, 225 µl of feed hydrolysate 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 a minimum of 5 minutes, and the resulting supernatant was collected.

Example 28. Stimulation of beneficial SCFAs by oligosaccharide preparations.

The supernatant from Example 25 was analyzed by HPLC to determine its short chain fatty acid (SCFA) content. Acetic acid, lactic acid, isovaleric acid, propionic acid and butyric acid were determined by high performance liquid chromatography using an Agilent Serine 1100 HPLC instrument using a BioRad HPC-98H 300 x 7.8 mm column and isocratic elution with a mobile phase of 0.05% aqueous solution of trifluoroacetic acid. Detection was performed using refractive index and UV absorbance at 210 nm. An analytical procedure was implemented to determine the total concentrations of butyrate, propionate, acetate, lactate, and isovalerate in an analytical sample, with the mobile phase selected such that the salts or other ionic base species of the analyte conjugate were detected collectively as their corresponding acid.

For each oligosaccharide preparation, its effect on the SCFA production profile was determined by comparing the relative concentration of each SCFA species with the concentration observed in control digestion wells on the same microtiter plate as the test oligosaccharide preparation. For each well and each species, the effect was quantified by calculating log2 (FC), the base 2 logarithm of the fold change (FC), defined as the ratio of the analyte concentration to the concentration of the feed digestion control.

Significant modulation of butyric acid and propionic acid production due to oligosaccharide drugs was observed.

Up-modulation of butyrate.

The oligosaccharide preparation from Example 9.9 showed a 10-fold increase in butyrate production, log2 (FC) = 3.4, compared to the feed digestion control. The effect was found to be consistent across multiple replicates of the experiment, as a 10-fold increase was observed in more than two-thirds of the wells tested, including several digestion media preparations. Significant increases in butyrate production were also observed for the oligosaccharide preparations of Examples 9.8, 9.10 and 9.11.

In contrast, the oligosaccharide preparation from Example 9.7 showed only a small increase in butyrate production with log2(FC)=0.6. In addition, it was found that 163 other oligosaccharide formulations prepared by varying the compositions according to the procedures of Example 9 did not provide an increase in butyrate, log2(FC)~0, or a decrease in butyrate production, log2(FC)<0.

Propionate up-modulation.

The oligosaccharide preparation from Example 9.5 showed a more than 7-fold increase in propionate production, log2(FC)=2.9, compared to the feed digestion control. The effect was found to be consistent across multiple replicates of the experiment, as a 7-fold increase was observed in several wells tested, including several digestion media preparations. Significant increases in propionate production were also observed for the oligosaccharide preparations of Examples 9.3 and 9.4.

In contrast, the oligosaccharide preparation from Example 9.7 showed only a small increase in propionate production with log2 (FC)=0.8. In addition, it was found that 191 other oligosaccharide preparations prepared by varying the compositions according to the procedures of Example 9 produced neither an increase in propionate production, log2 (FC) ~ 0, nor a decrease in propionate production, log2 (FC) < 0.

Example 29. In vivo metabolomic analysis of animals treated with oligosaccharide preparations.

The effect of the oligosaccharide drug from Example 9.7 was assessed in vivo on broiler chickens from Example 23 using cecal microbiota samples from Example 24.

For each cecal sample, the contents were thawed and an aliquot of the cecal contents was added to D ₂ O to form a 0.1% w/v suspension. The resulting suspension was homogenized by vortexing and then centrifuged for at least five minutes at 14,000 rpm to obtain a precipitate. The supernatant was extracted and analyzed by 1D 1H NMR. Fig. 17 presents a typical NMR spectrum showing peak assignments for several metabolites, including various short-chain fatty acids, various nitrogenous bases such as uracil, amino acids such as tryptophan, and other biochemical species associated with the microbiome.

The integrals for the assigned peaks in the NMR spectrum were tabulated. A contrast analysis was performed by grouping the bird samples from Example 24 into two cohorts: (1) a control group consisting of only birds fed the control diet; and (2) an experimental group including only birds fed the treated diet. NMR data were compared between control and experimental groups to determine the relative shift of polar metabolites.

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The untargeted in vivo metabolomics analysis was consistent with the ex vivo SCFA results from Example 26 in that a small increase of approximately 1.5-fold in butyrate was observed in the treatment group compared to the control group.

Fig. 18 shows that PLS-DA analysis was assessed using MetaboAnalyst 4.0, which displays centroids and cluster groups for polar metabolites analyzed by NMR and shows a clear separation of the metabolomic profile of birds in the experimental group from those in the control group.

Example 30. Dose-dependent response of SCFA production to oligosaccharide preparations.

The ratio of the mass of the oligosaccharide preparation to the mass of the digested feed was changed ex vivo during the microbial conversion of Example 25 and the dose rate was measured by changing the ratio of oligosaccharides.

Aliquots of the oligosaccharide preparations to be tested were diluted to 20 wt% oligosaccharide in water, microfiltered through a 0.22 μm PES syringe, and degassed under anaerobic conditions. A hydrolyzed poultry feed was prepared and simultaneously sterilized by suspending 50 g of the control feed sample from Example 23 in 50 ml of water and then subjecting it to two cycles of autoclaving at 120° C. for 5 min followed by aqueous extraction and resuspension. The resulting feed digestion product was degassed under anaerobic conditions.

Extracted cecal content samples from Example 23 were thawed anaerobically and used to prepare a 20% suspension in phosphate buffered saline (PBS) pH 7.4 containing 15% glycerol. 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 the cecal contents in a sterile aqueous mixture: 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 sodium phosphate dibasic, 5 g/l sodium bicarbonate, 0.125 mg/l biotin, 1 mg/l pyridoxine, 1 m/l pantothenate, 75 mg/l histidine, 75 mg/l glycine, 75 mg/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 tested, aliquots of the 20 wt.% oligosaccharide solution were combined with aliquots of the digestion feed in the wells of a 96-well deep well microtiter plate (eg, Costar 3958 plates). The wells were then inoculated with an aliquot of a 1% suspension of cecal contents. For each oligosaccharide, a series of aliquots were prepared to reflect the range of oligosaccharide to digestible weight ratios, as shown in Table 1 below. 12.

Table 12. Oligosaccharide aliquots

Dose Oligosaccharide, µl Feed hydrolysate, µl Suspension, µl Oligosaccharides in extract, % Level 1 0 250 250 0% Level 2 12.5 237.5 250 5% Level 3 25 225 250 10% Level 4 50 200 250 20% Level 5 250 0 250 100%

The loaded plate was then incubated at 37°C for 45 hours under anaerobic conditions. After incubation, the contents of the wells were centrifuged and the supernatant was analyzed by HPLC for SCFAs according to the method of Example 26. FIG. 19 represents the dose-response measured as selectivity for SCFA in mol% compared to the carbon content in the medium, in mol%, provided by the oligosaccharide drug compared to the feed digestion medium. A clear nonlinear dose-dependent response is observed, indicating that the oligosaccharide drug modulates the selectivity of microbial fermentation for SCFAs depending on the choice of oligosaccharide drug provided to the microbiota. The modulation effect is particularly strong for the oligosaccharide preparation of Example 9.1 compared to the oligosaccharide preparation of Example 9.3.

Moreover, the modulation effect is observed at low doses of the oligosaccharide drug. For example, at an incorporation rate in which only 10-20% of the fermentable carbon available to the microbiota came from the oligosaccharide preparation itself (i.e., 80-90% of the fermentable carbon came from the feed digestion environment), 40-50% of the carbon was converted in SCFA for the oligosaccharide drug from example 9.1. This compares to the case where 100% of the carbon was associated with the production of the oligosaccharide from Example 9.1 (i.e., when no feed hydrolysate was present in the culture medium), only approximately 15% of the carbon was converted to SCFAs.

Surprisingly, the total mass of SCFAs observed during ex vivo growth exceeded the theoretical maximum mass of the oligosaccharide preparations themselves, providing clear evidence that the oligosaccharides acted to regulate the metabolic flow of other carbon present in the growth medium to the SCFAs.

Example 31. Constancy of the effect, despite differences in the composition of the microflora.

The ex vivo fermentations from Examples 25-26 were repeated using cecal microbiota obtained from animals raised in different locations. Analysis by 16S rRNA sequencing confirmed that the phylogenetic composition was significantly different from that of examples 25-26. In one cecal microbiota sample, the abundance of Firmicutes was approximately 65% and the abundance of Bacteroidetes was approximately 16%. In another cecal sample, the abundance of Firmicutes and Bacteroidetes was approximately 25%, with more than 5% Helicobacter.

Anaerobic growth using different cecal microbiota was performed in microtiter plates according to the procedures of Example 25, and the resulting butyrate and propionate concentrations were measured by HPLC using the methods of Example 26. The concordance scale for each oligosaccharide drug and SCFA was determined as the fraction of wells in which the concentration of the corresponding SCFA was at least 20% of the average concentration observed in all wells for a given SCFA and oligosaccharide drug analyzed (i.e., in different microbiota inocula).

High concordance between butyrate and propionate was observed, in particular, for the oligosaccharide preparation from Example 9.8, where the concordance rates were in the range of 80-90%. Other oligosaccharide preparations from Example 9 showed agreement above 70%. The oligosaccharide drug from Example 9.14 showed low compliance with a concordance rate below 50%.

Example 32. Metabolomic assessment of oligosaccharide drugs.

The metabolomic effects of the oligosaccharide drugs from Example 9 were further assessed ex vivo in the broiler chickens from Example 23 using cecal microbiota samples from Example 24.

For each oligosaccharide analyzed, approximately 10 μl of the corresponding supernatant from Example 25 was diluted with 90 μl of acetonitrile/methanol solvent containing 0.2% formic acid and isotope-labeled internal standards. The resulting dilutions were analyzed by LC/MS using reversed-phase HPLC (Waters 150x2 mm Atlantis HILIC column) with gradient elution and time-of-flight mass spectrometry (TOFMS) detection using electrospray ionization at 3.5 kV. The resulting LC/MS data were processed to obtain a series of retention time and parent ion molecular weight pairs. Molecular weight data were searched in the Human Metabolome Database (HMDB) [Wishart D.S., Tzur D., Knox S., et al., HMDB: the Human Metabolome Database. Nucleic Acids Res., 35(Database issue), D521-6 (2007)] to generate a list of metabolites with corresponding MS peak intensities measured by TIC.

Example 33: Down-regulation of inflammation-associated metabolites.

Metabolome effect contrast analysis from Example 29 was performed by grouping supernatant samples into cohorts by oligosaccharide drug, determining the fold change (FC) and log2 (FC) for each analyte relative to the control, and then screening for a statistically significant reduction (log2(FC) ) <0 and P <0.05 by ANOVA) due to the oligosaccharide drug.

The oligosaccharide preparation from Example 9.3 was found to provide significant histamine reduction. Oligosaccharide preparations from examples 9.6 and 9.7 did not reduce histamine.

Example 34 Down-regulation of microbial ammonia production.

The effect of oligosaccharide preparations on microbial ammonia production was assessed ex vivo using the methods of Examples 25 and 27. Cecal microbiota were grown anaerobically on the medium of Example 25 without the addition of digestible growth feed. After growth, cultures were transferred to 1.5 ml Eppendorf tubes and centrifuged for 10 minutes at 4000xg. 10 μL of the resulting supernatant was transferred back to the wells of a 96-well plate for ammonia determination using an enzymatic assay (Biovision K370-100 Ammonia Assay Kit). Specifically, each 10 μL aliquot was diluted to 50 μL with assay buffer. The assay, developer, and conversion enzyme mixture was prepared by resuspending in 200 μL of assay buffer with vortex mixing. The assay master mixture was prepared by adding 42 μL assay buffer, 2 μL Oxired probe, 2 μL enzyme mixture, 2 μL developer, and 2 μL converting enzyme. Then 50 μl of the master mixture was added to each well. The plate was covered with foil and incubated for 60 min at 37°C in the dark. The absorbance was read at 570 nm and the ammonia concentration was determined by comparison with a standard curve constructed using known concentrations of ammonium chloride. A significant reduction in ammonia content was observed for the oligosaccharide preparation from Example 9.7.

Example 35. Rearing and sampling of piglets treated with an oligosaccharide preparation.

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Weaned piglets were raised on the diets of Example 15 to determine the effect of the oligosaccharide formulation on the functional metagenomics of porcine ileal, cecal, and fecal microbiomes.

One hundred and forty-four Redon x Large-White weaned pigs with an initial body weight of 8.54 ± 1.70 kg were reared for 42 days in a single-tier battery cage in an environmentally controlled facility. The animals were divided into 4 equal groups of 36 piglets in 9 pens with 4 animals per pen. Each pen had a plastic-coated welded wire bottom and two nipple drinkers and two individual stainless steel feeders. Room humidity was maintained at 50% and temperature was initially maintained at 27°C and decreased weekly by approximately 2°C per week to 21–22°C. The animals were fed in accordance with a two-phase dietary program compiled in accordance with the NRC Nutritional Guidelines (2012) using the diet composition given in Table. 13.

Table 13. Composition of diets for weaned piglets

Ingredient Pre-start feed (wt.%) Starter feed (wt.%) Corn 52.20 62.25 Soy flour 26.00 26.00 Corn starch 6.00 - Soy protein concentrate 5.00 3.00 Calcium carbonate 0.60 0.55 Dicalcium phosphate 1.7 1.50 Sodium bicarbonate 0.45 0.40 Sodium chloride 0.25 0.20 L-lysine 0.80 0.65 L-threonine 0.30 0.25 L-methionine 0.20 0.20 Soybean oil 3.00 2.00 Vitamin and mineral premix 3.50 3.00

The pre-starter diet was given from days 0 to 14, and the starter diet was given from days 15 to 42. All rations were given without restriction in the form of mash. The piglets were divided into experimental groups in accordance with Table 14.

Table 14. Experimental groups of weaned piglets

Experimental group Pre-start diet Starter diet Control Ration 15.3 (CTR) Example 15.5 (CTR) Experimental group A Diet 15.3 (CTR) supplemented with 125 ppm oligosaccharide preparation 9.7 Diet 15.5 (CTR) supplemented with 125 ppm oligosaccharide preparation 9.7 Experimental group B Diet 15.3 (CTR) supplemented with 250 ppm oligosaccharide preparation 9.7 Diet 15.5 (CTR) supplemented with 250 ppm oligosaccharide preparation 9.7 Treatment Diet 15.3 (TRT) Diet 15.6 (TRT)

The average daily weight gain (ADWG) of piglets in the experimental group was determined to be 508.1 g/day compared to the control group, whose weight gain was 494.9 g/day. The feed conversion ratio for the control group was 1.781 kg/kg, and for the experimental group - 1.748.

Example 36. Effect of oligosaccharide drug on functional metagenomics.

Weaned piglets were raised in pens in accordance with the general protocol from Example 33. The piglets were divided into two experimental groups: the control group received diets 15.3 (CTR) and 15.5 (CTR); and the experimental group received diets 15.3 (CTR) and 15.5 (TRT). For each experimental group, 13 animals were randomly selected, sacrificed, and dissected to obtain ileal, cecal, and fecal (colon) microbiota samples.

microbiota samples were prepared by SAMBO DNA extraction and sequenced using MetaQuant (MGP). On average, 21.6 million cleaned reads were obtained per sample. Mapping and counting were performed using METEOR. Mapping with Bowtie2 (95% identity) was performed to remove host contaminants (pig genome), and the resulting sequences were profiled against a custom gene catalog containing 9M genes (ileal, cecal, and fecal microbiomes). Biostatistical analysis was performed using MetaOMineR. More than 2 x 10 ⁷ high quality reads were obtained. To avoid bias due to mapping rate variability, all samples were downsampled to 10M mapped reads and gene abundance was normalized using FPKM according to gene length and sequencing depth.

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Gene richness and functional metagenomic analysis.

Samples were grouped into cohorts according to experimental group and microbiome type (ileum, cecum, feces), and contrast analyzes were performed to determine statistically significant changes in gene richness, measured as the number of individual genes. Animals in the experimental group showed a 2-5-fold increase in gene richness, significant at P < 0.006 for cecal microbiota and P < 0.06 for fecal microbiota (Wilcoxon test). There was also a numerical increase in ileal gene richness.

Genes were annotated using the KEGG database (v82) and grouped according to gut metabolic modules (GMM) [Darzi, Y. et al., The ISME Journal, 10, 1025-1028 (2015)] using an internal line. Contrast analysis was performed on GMM prevalence data to determine the metabolic pathways and functions that were modulated in the experimental group compared to the control group.

Fig. 20 illustrates the effect of an oligosaccharide drug on the functional metagenomics of the piglet microbiome. A statistically significant increase in pathways associated with acetogenesis was observed in the ileal microflora. Statistically significant increases in pathways related to pectin degradation and SCFA production, among others, were observed in the cecum. In addition, SCFA pathways for butyrate and propionate production were enriched in the cecal microbiota for the experimental group. In particular, animals in the experimental group demonstrated a statistically significant decrease in the metabolic pathways of the cecal microbiota associated with hydrogen metabolism and putrescine degradation. Pathways in the fecal microbiome associated with anaerobic fatty acid beta-oxidation were also significantly reduced.

Example 37: Up-regulation of health-related metabolites.

Contrasting metabolomic effects analysis from Example 31 was performed by grouping supernatant samples into groups by oligosaccharide drug, determining the fold change (FC) and log2 (FC) for each analyte relative to the control, and then screening for statistically significant increases (P < 0 .05 by ANOVA) concentration due to the oligosaccharide drug.

The oligosaccharide formulation of Example 9.7 was found to provide significant increases in multiple target metabolites including gentisic acid, alpha-terpineol, D-linalool, eucalyptol, geraniol. In contrast, the oligosaccharide preparation from Example 9.6 did not produce a significant increase in these metabolites.

The oligosaccharide preparation of Example 9.3 provides a significant increase in the fucosylated milk oligosaccharide 2-fucosyllactose. In contrast, the oligosaccharide preparation from Example 9.7 did not produce a significant increase in these metabolites.

Example 38: Up-regulation of mood-related metabolites.

Contrast analysis of metabolome effects from Example 31 was performed by grouping supernatant samples into cohorts by oligosaccharide drug, determining the fold change (FC) and log2 (FC) for each sample analyzed relative to the control, and then screening for a statistically significant increase (P < 0.05 by ANOVA) concentration due to the oligosaccharide drug.

The oligosaccharide formulation of Example 9.3 provided a significant increase in the neurochemical gamma-aminobutyric acid (GABA), as well as increases in 2-aminoisobutyric acid, D-alpha-aminobutyric acid, and 3-aminoisobutanoic acid. In contrast, the oligosaccharide preparations from Examples 9.6 and 9.7 did not produce a significant increase in GABA.

The oligosaccharide preparation of Example 9.7 provided a significant increase in the neurochemical dopamine, while the oligosaccharide preparations of Examples 9.3 and 9.6 did not increase dopamine levels.

Example 39: Up-regulation of metabolites associated with meat flavor and quality.

Contrast analysis of metabolome effects from Example 31 was performed by grouping supernatant samples into cohorts by oligosaccharide drug, determining the fold change (FC) and log2 (FC) for each sample analyzed relative to the control, and then screening for statistically significant increases (P < 0. 05 by ANOVA) concentration due to the oligosaccharide drug.

The oligosaccharide preparation from Example 9.3 provided a significant increase in the flavoring compounds 1-methylthiopropane and 2-methylthiopropane. The oligosaccharide preparation of Example 9.7 provided significant increases in the aromatic compound p-menth-1-en-4-ol and the compounds 1-nitroheptane, octanal, 2-octanone, and 2,3-heptanedione.

The oligosaccharide preparation from Example 9.6 did not provide a significant increase in any of the metabolites associated with taste, aroma or meat quality.

Example 40. Method for targeted delivery of butyrate to the lower intestine of animals.

The results from Examples 23-27 demonstrate a method for targeting butyrate to the hindgut, such as the cecum and lower digestive tract of broiler chickens. Oligosa-86 045265 charide preparations, such as those from Example 9, were added to the feed and given to the animals. Feed additives containing butyrate were not included in the feed itself. After ingestion, the oligosaccharide preparation was processed by the intestinal microflora in the presence of other undigested feed components (for example, hydrolysate from the cecum) and converted into butyrate.

Example 41. Method for targeted delivery of essential oils to the hindgut of animals.

The results from Example 41 demonstrate a method for targeted delivery of essential oils to the hindgut, such as the cecum and lower digestive tract of broiler chickens. The oligosaccharide preparation from Example 9 was added to the feed and given to the animals. Feed additives based on essential oils were not included in the feed itself. After ingestion, the oligosaccharide drug is processed by the intestinal microflora in the presence of other undigested feed components (for example, hydrolyzate from the cecum) and is converted into essential oils, including D-linalool, eucalyptol and geraniol, as well as p-menth-1-en-4-ol .

Example 42. Live growth performance of broiler chickens under various conditions.

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. Table 17 provides detailed protocol information for each study included in the analysis.

Table 15. Protocols

Study A country Season Duration Type of diet Replication/experiment Quantity/repetition Genetics Floor Litter Program against coccidiosis Etc. 42.1 USA Spring 35 Corn/Soybeans 6 14 Cobb 500 M/F Use Saccox Etc. 42.2 USA Winter 49 Corn/Soybeans 12 60 Cobb 500 M/F Use Maxiban Etc. 42.3 Canada Winter 35 Corn/Soybeans 8 60 Ross 708 M Use Saccox Etc. 42.4 USA Winter 49 Corn/Soybeans 12 60 Cobb 500 M/F Use Maxiban Etc. 42.5 Conn. Kingdom N/A N/A Wheat/Soybean N/A N/A N/A n/a Clean No Etc. 42.6 USA Winter 33 Corn/Soybeans 12 100 Cobb 500 M/F Use Amprol Etc. 42.7 USA Summer 49 Corn/Soybeans 12 18 Hubbard M99 M Use No Etc. 42.8 Canada Winter 42 Corn/Soybeans 12 17 Ross308 M Use Vaccine Etc. 42.9 Great Britain Autumn 42 Wheat/Soybean 16 35 Ross308 M Fresh No Etc. 42.10 France Autumn 42 Wheat/Soybean 17 thirty Ross308 M Fresh No Etc. 42.11 USA Autumn 42 Corn/Soybeans 21 40 Cobb500 M Use Vaccine Etc. 42.12 France Summer 36 Corn/Soybeans 12 18 Cobb500 M Fresh Vaccine Etc. 42.13 Canada Spring 42 Corn/Soybeans 10 20 Ross708 M Use Vaccine Etc. 42.14 USA Spring 42 Corn/Soybeans 14 40 Cobb500 M Use Vaccine Etc. 42.15 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 percentage (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.

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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. 16 (NA = data not available from the site).

Table 16. Starter control diets

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. 42.1 63.5 N/A 27.4 N/A 22.1 N/A 2988 1.35 N/A Etc. 42.2 0 N/A 0 N/A N/A N/A N/A N/A n/a Etc. 42.3 63.4 n/a 28.3 n/a 20.9 n/a 2940 1.14 n/a Etc. 42.4 N/A n/a N/A n/a N/A n/a N/A N/A n/a Etc. 42.5 N/A n/a n/a n/a N/A n/a N/A N/A n/a Etc. 42.6 0 n/a 0 n/a N/A n/a ZON N/A n/a Etc. 42.7 0 n/a 0 n/a N/A n/a N/A n/a n/a Etc. 42.8 54.52 0 34.38 5 22.23 2.81 2900 1.24 0.63 Etc. 42.9 0 51.78 30.5 0 21.31 5.74 2899 1.251 0.622 Etc. 42.10 0 55.1 28 0 22.49 5.42 2899 1.237 N/A Etc. 42.11 58,353 2,377 29,992 5 20.3 N/A 2900 1.33 n/a Etc. 42.12 N/A N/A N/A N/A 22 N/A ZON n/a n/a Etc. 42.13 56.08 0 34.1 5 22.23 2.31 2900 1.24 0.63 Etc. 42.14 58,353 0 29,992 5 20.3 N/A 2900 1.33 n/a Etc. 42.15 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. 17 (N/A - data not available from the site).

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Table 17. Diets for the growth phase

Study Corn flour, % Wheat flour, % Soy flour, % corn fermentation,% Unpurified protein Unrefined fat metabolic value (kcal/kg) Lysine (SID) Methionine (SID) Ex.42.1 68.6 N/A 22 N/A 19.95 N/A 3059 1.2 N/A Ex.42.2 N/A N/A N/A N/A N/A N/A N/A N/A n/a Ex.42.3 65.6 n/a 26.3 n/a 19.9 n/a 2988 1.06 n/a Ex.42.4 N/A n/a N/A n/a N/A n/a N/A N/A n/a Project 42.5 N/A n/a n/a n/a N/A n/a N/A N/A n/a Project 42.6 N/A n/a n/a n/a N/A n/a 3102 N/A n/a Project 42.7 N/A n/a n/a n/a N/A n/a N/A N/A n/a Project 42.8 54.95 0 28.31 10 20.8 3.88 3000 1.11 0.56 Project 42.9 0 57.135 26 0 19.01 6.55 2997 1.08 0.533 Project 42.10 0 55.77 24 0 20.94 7.4 2998 1.11 N/A Project 42.11 65,383 0.344 20,404 10 17.5 N/A 3040 1.33 n/a Project 42.12 N/A N/A N/A n/a 19 N/A 3035 n/a n/a Project 42.13 56.53 0 28.02 10 20.8 3.39 3000 1.11 0.56 Project 42.14 65,383 0 20,404 5 17.5 N/A 3040 1.14 N/A Project 42.15 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. 18 (N/A=data not available from the site).

Table 18. Final phase rations

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. 42.1 74.3 N/A 27.4 N/A N/A N/A 3155 1.06 N/A Etc. 42.2 N/A N/A N/A N/A n/a N/A N/A n/a n/a Etc. 42.3 70.8 n/a 28.3 n/a n/a n/a 3059 0.94 n/a Etc. 42.4 N/A n/a N/A n/a n/a n/a N/A N/A n/a Etc. 42.5 n/a n/a n/a n/a n/a n/a N/A N/A n/a Etc. 42.6 n/a n/a n/a n/a n/a n/a 3203 N/A n/a Etc. 42.7 n/a n/a n/a n/a n/a n/a N/A N/A n/a Etc. 42.8 57.37 0 24.85 10 19.4 5.16 3100 1.03 0.55 Etc. 42.9 0 59.94 23 0 17.53 7.71 3097 1.003 0.503 Etc. 42.10 0 87.67 21 0 19.53 8.88 3099 0.994 N/A Etc. 42.11 69.41 0.12 16,879 10 16 N/A 3084 1.01 N/A Etc. 42.12 N/A N/A N/A n/a N/A N/A N/A N/A N/A Etc. 42.13 58.95 0 24.56 10 19.4 4.66 3100 1.03 0.55 Etc. 42.14 69.41 0 16,879 5 16 N/A 3084 1.01 N/A Etc. 42.15 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 oly

- 89 045265 gosaccharide preparations 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 19.

Table 19. 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. 42.1 Control Etc. 9.7 (500 ppm) Etc. 42.2 Control Etc. 9.7 (500 ppm) Etc. 42.3 Control Etc. 9.7 (500 ppm) Etc. 42.4 Control Etc. 9.7 (500 ppm) Etc. 42.5 Control Etc. 9.7 (100 ppm) Etc. 9.7 (500 ppm) Comp. Project 36 (1500 ppm) Etc. 42.6 Control Etc. 9.7 (500 ppm) Etc. 42.7 Control Etc. 9.7 (500 ppm) Etc. 42.8 Control Etc. 9.7 (500 ppm) Etc. 9.3 (500 ppm) Etc. 42.9 Control Etc. 9.7 (500 ppm) Etc. 42.10 Control Etc. 9.7 (500 ppm) Etc. 9.2 (500 ppm) Etc. 9.3 (500 ppm) Comp. Etc. 36 (1250 ppm) Etc. 42.11 Control Etc. 9.7 (500 ppm) Etc. 9.2 (500 ppm) Etc. 9.3 (500 ppm) Etc. 9.4 (500 ppm) Etc. 9.5 (500 ppm) Comp. Etc. 36 (1250 ppm) Etc. 42.12 Control Etc. 9.7 (500 ppm) Etc. 9.3 (500 ppm) Etc. 42.13 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. 42.14 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) (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. 42.15 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.

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, feed conversion ratio (FCR) was calculated by dividing the total feed intake for the interval by 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 43. Meta-analysis.

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 meta-analysis results for cFCR are presented in Table. 20.

Table 20. 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 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. The results are presented in table. 21.

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Table 21. Consistency of application effect on cFCR

Experimental group 1 improvement unit 2 upgrade units 3 upgrade units 4 upgrade units 5 upgrade units 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 relationship was observed between cFCR and the degree of oligosaccharide inclusion in the diet. For the oligosaccharide from Example 9.2, a 2.4 unit improvement in cFCR was observed with 100 ppm inclusion (P>0.05), a 3.7 unit improvement in cFCR was observed with 250 ppm inclusion (P<0.05) , a 6.4 unit improvement in cFCR was observed with 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 g compared to 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. 22.

Table 22. Meta-analysis of poultry weight

Experimental group BW (Ismean) S.E. df Grouping by Tyoki ABW (g vs control) Control 2.755 113 14 A 0 Etc. 9.2 (500 ppm) 2.817 113 14 b 62.6 Etc. 9.3 (500 ppm) 2,807 113 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) 2,794 113 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 44. Metabolic shifts in the rumen microbiome.

The modulating effect of the oligosaccharide drugs from Example 9 on the metabolic output of the rumen microbiome of dairy cows was demonstrated in vitro. Rumen fluid samples were collected from commercial dairy cows before morning feeding and immediately after milking. In each animal from which a sample was taken, rumen fluid was removed using a gastric tube [Ramos-Morales E, A Arco-Perez, AI Martin-Garcia, DR Yanez-Ruiz. Animal Feed Science and Technology 198, 57-66 (2014)] and transferred to thermoses. The liquid was filtered before use, and inoculation for in vitro experiments was performed within 30 minutes of sample collection.

Batch incubations of in vitro cultures were performed at 39°C for 72 h in 125 ml serum bottles. Bottles were prepared by adding 500 mg of nutritious oat-hay concentrate 50:50[“. eg Yanez-Ruiz DR, et al Animal Feed Science and Technology, 216, 1-18 (2016)] _K 50 _mL incubation culture containing 16.7 ml rumen fluid and 33.3 ml anaerobic buffer solution. The initial pH of each bottle was adjusted to approximately 6.2. Empty vials were prepared in duplicate for each individual rumen sample, excluding the nutritional concentrate.

The effect of oligosaccharides on incubation was assessed by adding a certain mass of oligosaccharide to the vial before incubation. The mass of added oligosaccharide was selected to obtain the target dose, measured in micromoles per liter of inoculum. Molar amounts were determined based on monomeric sugar. For each of n = 3 individual rumen samples, each oligosaccharide treatment was performed in triplicate, and the control (containing no added oligosaccharides) was performed in duplicate. Oligosaccharide treatment was analyzed at 6 dose levels: 0 (control), 10, 50, 100, 250, and 500 μmol/L.

After inoculation, the vials were placed on trays in a thermostatic incubator and kept at 39°C for 72 hours. The volumes of gas fermentation were recorded at 2, 4, 6, 8, 12, 24, 36, 48 and 72 hours after the start of incubation. The bottles were stirred gently after each reading of the gas content. After 24 h, 2 ml of liquid was collected from the culture to measure microbiome metabolites and ammonia nitrogen. pH was measured using a calibrated pH probe. Volatile fatty acids (VFAs) were analyzed by gas chromatography with a flame ionization detector (AutoSystem; Perkin-Elmer Corp., Shelton, CT, USA) and ammonia nitrogen by colorimetric methods. Total VFA concentration was determined in mmol/L, and the molar fractions of acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate were calculated as the ratio of the molar concentration of individual species to the molar concentration of total VFA. The acetate/propionate ratio was calculated from the measured acetate and propionate concentrations.

The effects of oligosaccharide treatments compared with controls were determined by statistical analysis using a model that included fixed effects for oligosaccharide treatment groups. The animal was the experimental unit. When significant effects were found, means were compared using Fisher's protected least significant difference test using SPSS version 21 (IBM Corp., Armonk, NY, USA). P <0.05 was considered statistically significant, with a trend toward differences at P <0.10.

VFA metabolites of the microbiome.

For the oligosaccharide from Example 9.2, a numerical increase in total VFA production was observed at all dose levels compared to the control. The maximum VFA yield was observed at a dose of 250 μmol/L. A clear effect of the oligosaccharide on increasing acetate production (P < 0.01) was observed at each dose level with a 15% increase over control at the 250 μM dose level. The mole fraction of acetic acid was also increased compared to the control. Similarly, the total molar concentration of propionic acid was significantly increased (P < 0.05) compared to control, but the molar fraction of propionic acid was decreased. A detailed breakdown of specific volatile fatty acids is presented in Table. 23.

Table 23. VFA metabolites of the microbiome

Treatment Total VFA (mM) Acetate, mol% Propione, mol% Isobutyrate, mol% Butyrate, mol% Isovalerate, mol% Valerat, mol% Control 75.28 64.67 20.52 1.52 9.25 2.22 1.80 Treatment (10 µM) 81.39 65.33 20.17 1.40 9.00 2.25 1.82 Treatment (50 µM) 83.44 65.63 20.02 1.38 8.97 2.25 1.77 Treatment (100 µM) 84.09 65.58 20.07 1.43 8.92 2.27 1.78 Treatment (250 µM) 85.54 65.57 20.28 1.35 8.95 2.13 1.70 Treatment (500 µM) 81.90 65.85 20.03 1.33 8.87 2.17 1.77

The catalytic effect of oligosaccharides on the metabolomic yield of the microbiome was observed. For example, at a concentration of 10 μM, the oligosaccharide from Example 9.2 increased acetate production by the rumen microbiome to 53.2 mM from 48.7 mM, suggesting an increase of 450 mol of acetate per mole of oligosaccharide.

pH, ammonia nitrogen and total gas production.

A numerical decrease in total gas production was observed with oligosaccharide treatment compared to control. The pH value decreased by approximately 0.1 pH unit in the dose range from 10 to 500 μmol of oligosaccharides with a statistical trend (P = 0.057). There was no increase in ammonia nitrogen observed as a result of treatment with the oligosaccharide drugs, indicating that exposure to the oligosaccharides from Example 9.2 did not result in an increase in protein deamination.

Example 45. Method for improving milk quality.

Dairy cows were fed a diet containing one or more of the oligosaccharide preparations from Example 9.2. There was an increase in acetate and propionate production in the rumen, with a relative decrease in the ratio of acetate and propionate concentrations in the rumen. Milk taken from oligosaccharide-fed animals contains a higher percentage of milk solids compared to milk

- 93 045265 obtained from animals fed a similar diet, but without oligosaccharide preparations.

Example 46. Method for improving the quality of beef.

Beef cattle were fed a diet containing one or more of the oligosaccharide drugs from Example 9.2 through a feedlot. There was an increase in ruminal acetate and propionate production with a relative decrease in ruminal acetate and propionate concentrations. Beef from animals fed oligosaccharides contained a higher percentage of marbling compared to beef from animals fed a similar diet without oligosaccharides.

Example 47: Method for reducing gas emissions due to fermentation in ruminants.

Cows, cattle or other ruminants were fed diets containing one or more of the oligosaccharide preparations from Example 9.2. Total rumen fermentation-related gas production and resulting emissions were reduced compared to animals fed a similar diet without oligosaccharide supplements.

Example 48: 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.

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. 24, the following batch characterization data were obtained (N/R = not reported).

Table 24. Characteristics of oligosaccharide preparations

The consignment Sol. solid, substance, wt.% pH Residual catalyst DPI, wt.% MWn MWw AnhydroDP2 content (g anhydro-OP2/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%

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Example 49: Improved butyrate production in dogs compared to prebiotics.

The effect of oligosaccharide drugs on gut microbiome metabolites was assessed in domestic dogs (Canis familiaris). Improved butyrate production was observed relative to comparative examples provided by two common prebiotics.

Fecal microbiome samples were obtained from six dogs of different breeds, ages and sexes. Collected samples were immediately frozen and assessed for SCFA production using the methods of Examples 27 and 28. Microbiome samples were assessed ex vivo for beneficial SCFA production using the oligosaccharide preparations of Examples 9.2 and 9.3 and the two prebiotics of Comparative Examples:

1. Comparative Example 2. Short-chain fructo-oligosaccharide (scFOS) prebiotics (Profeed®, Beghin Meiji); And

2. Comparative Example 3. Prebiotics from corn cob xyloo-oligosaccharides (XOS) (Longlive Bio-Technology, Shandong).

Fig. 34 shows the concentrations of butyrate and propionate obtained for each of the four experimental groups, demonstrating superior production of key beneficial short-chain fatty acids for the microbiota of dogs fed the oligosaccharide preparations of Example 9. Statistical analysis was performed to confirm the improvement. For example, increased butyrate formation was significant at P < 0.05 for the oligosaccharide drug from Example 9.2, as shown in Table. 25.

Table 25. Statistical analysis of butyrate production in dogs

Experimental group Average concentration DF Paired comparisons (P<0.05) Comparative example 2 0.0939 5 A Comparative example 3 0.1633 5 ab Example 9.2 0.2961 5 With Example 9.3 0.2622 5 bc

Example 50. Regulation of the 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, 6.53 ml of a 1.0 M aqueous solution of sodium hydroxide was added. The resulting mixture was vigorously stirred until a homogeneous syrup was obtained with the pH adjusted. 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 carried out 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 51. 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% of the powder mass having a particle size of less than 196 μm. 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

- 95 045265 to 0°C at a rate of -5°C/min, followed by final reheating 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 of 5°C/min. 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 52. 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 53 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 grits 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, from 90% to mass less than 2 mm in size. The resulting powder was free-flowing and stable.

Example 54. Preparation of stable powder compositions.

Solid compositions, including those of Examples 51-53, were evaluated to determine their stability and hygroscopicity. The powders of Examples 52 and 53 were resistant to segregation and agglomeration, while the powder from Example 51 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 52 was found to be unstable when exposed to moisture.

Example 55. 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 51) 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 injection, 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 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 preparation from Example 9.2 was analyzed and the residual catalyst concentration was determined to be 0.62 mg per gram of 70 wt.% syrup.

Example 56. Quantitative analysis of residual catalyst concentration for batch acceptance.

The catalyst residual determination in Example 55 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 57. Preparation of the product in the form of syrup.

In the oligosaccharide preparation from example 9.7, the pH value was adjusted to 4.2 with sodium hydroxide

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Claims (2)

food grade according to the procedure in Example 50. 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. The total solids content of the syrup has been 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 counts were confirmed to be less than 10 MPN/r using the method described in Chapter 4 of the FDA BAM. E. coli was confirmed to be less than 3 MPN/r 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 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 58. Preparation of treated drinking water. Drinking water containing 250 ppm of the 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 herein, which depend on a variety of conditions and variables. It should be understood that various alternatives to the embodiments described herein 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 for modulating a metabolite in the gastrointestinal tract of an animal, including administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where 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 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 anhydrosubunits, as determined by relative abundance by mass spectrometry. 2. A method of directing a metabolite to a target compartment in the gastrointestinal tract of an animal, which is the hindgut, including administering to the animal a nutritional composition containing a basic nutritional composition and a synthetic oligosaccharide preparation, where 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% anhydro subunit-containing oligosaccharides as measured by mass spectrometry. -