Method for quantifying oligosaccharide preparations

Abstract

The present disclosure relates to selective analytical methods for the detection and / or quantification of oligosaccharide preparations in nutritional compositions, such as animal feed. Also disclosed are methods for producing nutritional compositions containing oligosaccharide preparations, which can selectively detect or determine the presence or concentration of the oligosaccharide preparations.

Description

Detailed Description of the Invention

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 757,231, filed November 8, 2018, the disclosure of which is incorporated herein by reference in its entirety.

[0002] [background]

[0002] Oligosaccharide preparations (which may generally contain monosaccharides, oligosaccharides, polysaccharides, functional oligosaccharides, or combinations thereof) are used as additives in nutritional compositions such as animal feed. The addition of oligosaccharide preparations can improve the health and performance of animals. However, because nutritional compositions typically contain other carbohydrate sources that may have structural similarity to the oligosaccharide preparations, it is difficult to detect or quantify the oligosaccharide preparation additives in nutritional compositions. As a result, there is a need for a method for selectively detecting or quantifying the oligosaccharide preparations in nutritional compositions.

[0003] [overview]

[0003] Disclosed herein are simple, selective, and sensitive analytical methods for the detection and / or quantification of oligosaccharide preparations or compositions containing oligosaccharide preparations in nutritional compositions. Also disclosed are methods for producing nutritional compositions containing oligosaccharide preparations, which allow for the selective and sensitive detection or determination of the presence or concentration of oligosaccharide preparations.

[0004] In one aspect, described herein is a method for correlating a synthetic oligosaccharide preparation in a nutritional composition, wherein the nutritional composition comprises a synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition, the method comprising the steps of: (a) providing a sample of the nutritional composition; (b) detecting a signal of at least a portion of the oligosaccharides in the sample of the nutritional composition; and (c) correlating the concentration of the synthetic oligosaccharide preparation in the nutritional composition, wherein the signal is determined by: (i) detecting one or more oligosaccharides present in the sample; or (ii) exhibits an α-(1,2) glycosidic, α-(1,3) glycosidic, α-(1,6) glycosidic, β-(1,2) glycosidic, β-(1,3) glycosidic, β-(1,4) glycosidic, β-(1,6) glycosidic, α-(1,1)-α glycosidic, α-(1,1)-β glycosidic, β-(1,1)-α glycosidic, or β-(1,1)-β glycosidic linkages in the oligosaccharide. In one aspect, a method for performing quality control of a nutritional composition is described herein, comprising the steps of: (a) providing a batch of a nutritional composition, the nutritional composition comprising a synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition; (b) obtaining a sample of the nutritional composition from the batch; (c) detecting a signal of at least a portion of the oligosaccharides in the sample of the nutritional composition using an analytical device; and (d) passing or failing the batch of nutritional composition, wherein the signal is In some embodiments, the signal indicates one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the signal indicates one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the one or more anhydro-subunit-containing oligosaccharides have a degree of polymerization (DP2) of 2.In one aspect, described herein is a method for performing quality control in a nutritional composition, comprising: (a) providing a sample of the nutritional composition, wherein the nutritional composition comprises a naturally occurring oligosaccharide composition; and (b) detecting, using an analytical device, a signal of at least a portion of the oligosaccharides in the sample of the nutritional composition, wherein the signal is indicative of one or more anhydrosubunit-containing oligosaccharides having a degree of polymerization of 2 (DP2). In some embodiments, the nutritional composition comprises a synthetic oligosaccharide preparation. In some embodiments, the method comprises correlating the concentration of the synthetic oligosaccharide preparation in the nutritional composition. In some embodiments, the signal is determined by high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, size exclusion chromatography (SEC), field-flow fractionation (FFF), asymmetric field-flow fractionation (A4F), weight determination of fractions by preparative chromatography, or any combination thereof. In some embodiments, the nutritional composition comprises a basal nutritional composition. In some embodiments, the basal nutritional composition comprises a naturally occurring oligosaccharide composition. In some embodiments, the basal nutritional composition does not contain detectable levels of anhydro-subunit-containing oligosaccharides. In some embodiments, the basal nutritional composition is substantially free of anhydro-subunit-containing oligosaccharides. In some embodiments, one or more anhydro-subunit-containing oligosaccharides are derived from a synthetic oligosaccharide preparation. In some embodiments, the signal is attributed to an anhydro-subunit-containing oligosaccharide having a degree of polymerization of 1 (DP1). In some embodiments, the signal is attributed to levoglucosan, 1,6-anhydro-β-D-glucofuranose, or a combination thereof. In some embodiments, the signal is attributed to an anhydro-subunit-containing oligosaccharide having a degree of polymerization of 3 (DP3). In some embodiments, the signal is attributed to DP1, DP2, or DP3 anhydro-subunit-containing oligosaccharides, or a combination thereof. In some embodiments, the signal is attributed to DP2 anhydro-subunit-containing oligosaccharides.In some embodiments, the signal is attributable to anhydro-cellobiose. In some embodiments, the detecting step comprises a gravimetric determination of one or more degree of polymerization (DP) fractions of oligosaccharides. In some embodiments, the detecting step comprises a gravimetric determination of at least a portion of the anhydro-subunit-containing oligosaccharides from the sample. In some embodiments, at least a portion of the anhydro-subunit-containing oligosaccharides have a degree of polymerization of 1, 2, or 3. In some embodiments, one or more DP fractions of oligosaccharides or at least a portion of the anhydro-subunit-containing oligosaccharides are isolated by preparative chromatography. In some embodiments, the signal is detected, at least in part, by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). In some embodiments, the signal is detected, at least in part, by liquid chromatography-mass spectrometry (LC-MS) / MS. In some embodiments, the signal is detected, at least in part, by GC-flame ionization detection (GC-FID) or GC-MS. In some embodiments, the signal is detected, at least in part, by NMR spectroscopy. In some embodiments, the signal is associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond of the oligosaccharide. In some embodiments, the detecting step comprises obtaining NMR spectroscopy. In some embodiments, the signal is associated with an α-(1,2) glycosidic bond. In some embodiments, the signal is associated with an α-(1,3) glycosidic bond. In some embodiments, the signal is associated with an α-(1,6) glycosidic bond. In some embodiments, the signal is associated with a β-(1,2) glycosidic bond. In some embodiments, the signal is associated with a β-(1,3) glycosidic bond. In some embodiments, the signal is associated with a β-(1,4) glycosidic bond. In some embodiments, the signal is associated with a β-(1,6) glycosidic bond.In some embodiments, the detecting step comprises determining the presence or absence of a signal. In some embodiments, the detecting step comprises determining the presence or absence of DP1 or DP2 anhydrosubunit-containing oligosaccharides, or both. In some embodiments, the detecting step comprises determining or correlating the level of a signal. In some embodiments, the detecting step comprises correlating the level of DP1 anhydrosubunit-containing oligosaccharides in the nutritional composition. In some embodiments, the detecting step comprises correlating the level of DP2 anhydrosubunit-containing oligosaccharides in the nutritional composition.

[0005] In one aspect, described herein is a method for performing quality control of a nutritional composition, including a synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition, the method comprising the steps of: (a) providing a first sample of the nutritional composition; (b) providing a second sample of the nutritional composition; (c) detecting a first signal of at least a portion of the oligosaccharides in the first sample; (d) detecting a second signal of at least a portion of the oligosaccharides in the second sample; and (e) comparing the first signal with the second signal, The first signal and the second signal are independently (i) indicative of one or more anhydrosubunit-containing oligosaccharides, or (ii) associated with an α-(1,2) glycosidic linkage, an α-(1,3) glycosidic linkage, an α-(1,6) glycosidic linkage, a β-(1,2) glycosidic linkage, a β-(1,3) glycosidic linkage, a β-(1,4) glycosidic linkage, a β-(1,6) glycosidic linkage, an α-(1,1)-α glycosidic linkage, an α-(1,1)-β glycosidic linkage, a β-(1,1)-α glycosidic linkage, or a β-(1,1)-β glycosidic linkage of the oligosaccharide. In some embodiments, the method includes correlating the concentration of the synthetic oligosaccharide preparation in the nutritional composition of the first sample, the concentration of the synthetic oligosaccharide preparation in the nutritional composition of the second sample, or both. In some embodiments, the first signal and the second signal each independently represent one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the first signal and the second signal are attributable to the same species of anhydro-subunit-containing oligosaccharides. In some embodiments, the first signal and the second signal are attributable to different species of anhydro-subunit-containing oligosaccharides. In some embodiments, the first sample and the second sample are taken from different batches of the nutritional composition. In some embodiments, the first signal and the second signal are each independently determined by high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, size-exclusion chromatography (SEC), field-flow fractionation (FFF), asymmetric field-flow fractionation (A4F), gravimetric determination of fractions by preparative chromatography, or a combination thereof.In some embodiments, the nutritional composition comprises a basal nutritional composition. In some embodiments, the basal nutritional composition comprises a naturally occurring oligosaccharide composition. In some embodiments, the basal nutritional composition does not contain detectable levels of anhydro-subunit-containing oligosaccharides. In some embodiments, the basal nutritional composition is substantially free of anhydro-subunit-containing oligosaccharides. In some embodiments, one or more anhydro-subunit-containing oligosaccharides are derived from a synthetic oligosaccharide preparation. In some embodiments, the first signal, the second signal, or both, are independently attributable to anhydro-subunit-containing oligosaccharides having a degree of polymerization of 1 (DP1). In some embodiments, the first signal, the second signal, or both, are independently attributable to levoglucosan, 1,6-anhydro-β-D-glucofuranose, or a combination thereof. In some embodiments, the first signal, the second signal, or both, are independently attributable to anhydro-subunit-containing oligosaccharides having a degree of polymerization of 2 (DP2). In some embodiments, the signals are attributable to anhydro-cellobiose. In some embodiments, the first signal, the second signal, or both, are independently attributable to anhydro-subunit-containing oligosaccharides having a degree of polymerization of 3 (DP3). In some embodiments, the first signal, the second signal, or both, are independently attributable to DP1, DP2, or DP3 anhydro-subunit-containing oligosaccharides, or combinations thereof. In some embodiments, the detecting step comprises a gravimetric determination of one or more degree of polymerization (DP) fractions from the sample or the second sample. In some embodiments, the detecting step comprises a gravimetric determination of at least a portion of the anhydro-subunit-containing oligosaccharides from the first sample or the second sample. In some embodiments, at least a portion of the anhydro-subunit-containing oligosaccharides have a degree of polymerization of 1, 2, or 3. In some embodiments, one or more DP fractions of oligosaccharides or at least a portion of the anhydro-subunit-containing oligosaccharides are isolated by preparative chromatography.In some embodiments, the first signal, the second signal, or both, are independently, at least in part, detected by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). In some embodiments, the first signal, the second signal, or both, are independently, at least in part, detected by liquid chromatography-mass spectrometry (LC-MS) / MS. In some embodiments, the first signal, the second signal, or both, are independently, at least in part, detected by GC-flame ionization detector (GC-FID) or GC-MS. In some embodiments, the first signal, the second signal, or both, are independently, at least in part, detected by NMR spectroscopy. In some embodiments, the first signal, the second signal, or both, independently, are associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond of the oligosaccharide. In some embodiments, the detecting step comprises obtaining NMR spectroscopy. In some embodiments, the first signal, the second signal, or both, are associated with an α-(1,2) glycosidic bond. In some embodiments, the first signal, the second signal, or both, are associated with an α-(1,3) glycosidic bond. In some embodiments, the first signal, the second signal, or both, are associated with an α-(1,6) glycosidic bond. In some embodiments, the first signal, the second signal, or both, are associated with a β-(1,2) glycosidic bond. In some embodiments, the first signal, the second signal, or both, are associated with a β-(1,3) glycosidic bond. In some embodiments, the first signal, the second signal, or both, are associated with a β-(1,4) glycosidic bond. In some embodiments, the first signal, the second signal, or both, are associated with a β-(1,6) glycosidic bond.In some embodiments, the first signal is indicative of one or more anhydrosubunit-containing oligosaccharides, and the second signal is associated with an α-(1,2) glycosidic linkage, an α-(1,3) glycosidic linkage, an α-(1,6) glycosidic linkage, a β-(1,2) glycosidic linkage, a β-(1,3) glycosidic linkage, a β-(1,4) glycosidic linkage, a β-(1,6) glycosidic linkage, an α-(1,1)-α glycosidic linkage, an α-(1,1)-β glycosidic linkage, a β-(1,1)-α glycosidic linkage, or a β-(1,1)-β glycosidic linkage of the oligosaccharide. In some embodiments, the detecting step comprises determining the presence or absence of the first signal and the second signal. In some embodiments, the detecting step comprises determining the presence or absence of DP1 or DP2 anhydrosubunit-containing oligosaccharides, or both. In some embodiments, the detecting step comprises determining or correlating the levels of the first signal and the second signal. In some embodiments, the detecting step comprises correlating the levels of DP1 anhydrosubunit-containing oligosaccharides in the nutritional composition. In some embodiments, the detecting step comprises correlating the levels of DP2 anhydrosubunit-containing oligosaccharides in the nutritional composition.

[0006] In some embodiments, the methods described herein include a step of passing or failing a batch of a nutritional composition. In some embodiments, the method includes a step of adjusting the level of the synthetic oligosaccharide preparation in the nutritional composition after detection. In some embodiments, the basal nutritional composition includes a plurality of oligosaccharides. In some embodiments, the basal nutritional composition includes starch or plant fiber. In some embodiments, the level of α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, or β-(1,4) glycosidic linkages in the basal nutritional composition is at least 10% lower than the level of the same glycosidic linkages in the synthetic oligosaccharide preparation. In some embodiments, the method includes a derivatization step prior to the detection step. In some embodiments, the method includes a step of extracting oligosaccharides from a sample of the nutritional composition. In some embodiments, the method includes a step of filtering or clarifying the extracted oligosaccharides. In some embodiments, the method includes a step of concentrating the extracted oligosaccharides. In some embodiments, the concentrating step comprises lyophilization. In some embodiments, the concentrating step comprises nanofiltration. In some embodiments, the method comprises introducing an internal standard into the extracted or enriched oligosaccharides. In some embodiments, the method comprises reducing the extracted or enriched oligosaccharides. In some embodiments, the method comprises digesting the extracted or enriched oligosaccharides with one or more hydrolases. In some embodiments, the one or more hydrolases comprise a carbohydratase, a protease, a lipase, or any combination thereof. In some embodiments, the one or more hydrolases comprise α-amylase, amyloglycosidase, invertase, α-galactosidase, or a combination thereof. In some embodiments, the one or more hydrolases cleave one or more naturally occurring glycosidic bonds. In some embodiments, the method comprises isolating undigested oligosaccharides. In some embodiments, the method comprises separating the extracted, enriched, digested, or reduced oligosaccharides. In some embodiments, the oligosaccharides are separated by chromatography.In some embodiments, the method comprises isolating the separated oligosaccharides. In some embodiments, oligosaccharides are separated or isolated by their degree of polymerization. In some embodiments, the method comprises isolating or separating oligosaccharides having a degree of polymerization of 1, 2, 3, 4, or 5. In some embodiments, DP1 oligosaccharides are isolated or separated. In some embodiments, DP2 oligosaccharides are isolated or separated. In some embodiments, DP3 oligosaccharides are isolated or separated. In some embodiments, the method comprises isolating at least a portion of DP1 anhydrosubunit-containing oligosaccharides from the sample. In some embodiments, the method comprises isolating at least a portion of DP2 anhydrosubunit-containing oligosaccharides from the sample. In some embodiments, the method comprises isolating at least a portion of DP3 anhydrosubunit-containing oligosaccharides from the sample. In some embodiments, the isolated or separated oligosaccharides are quantified. In some embodiments, the majority of the quantified oligosaccharides are derived from a synthetic oligosaccharide preparation. In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99% by weight of the quantified oligosaccharides are derived from synthetic oligosaccharide preparations.

[0007]

[0007] In one aspect, a method for correlating a synthetic oligosaccharide preparation in a nutritional composition is described herein, wherein the nutritional composition comprises (i) a synthetic oligosaccharide preparation comprising anhydro-subunit-containing oligosaccharides and (ii) a naturally occurring oligosaccharide composition, the method comprising: (a) providing a sample of the nutritional composition; (b) isolating one or more anhydro-subunit-containing oligosaccharides from the sample; (c) detecting a signal indicative of one or more anhydro-subunit-containing oligosaccharides, the signal comprising: (i) determining the weight of at least a portion of the anhydro-subunit-containing oligosaccharides from the sample; or (ii) analyzing at least a portion of the anhydro-subunit-containing oligosaccharides from the sample by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), liquid chromatography-mass spectrometry (LC-MS) / MS, or gas chromatography (GC)-MS; and (d) correlating the concentration of the synthetic oligosaccharide preparation in the nutritional composition. In some embodiments, the detecting step comprises determining the weight of at least a portion of anhydro-subunit-containing oligosaccharides having a degree of polymerization of 1 (DP1) from the sample. In some embodiments, the detecting step comprises determining the weight of at least a portion of anhydro-subunit-containing oligosaccharides having a degree of polymerization of 2 (DP2) from the sample. In some embodiments, the detecting step comprises analyzing at least a portion of DP1 or DP2 anhydro-subunit-containing oligosaccharides from the sample by MALDI-MS. In some embodiments, the detecting step comprises analyzing at least a portion of DP1 or DP2 anhydro-subunit-containing oligosaccharides from the sample by LC-MS / MS. In some embodiments, the detecting step comprises analyzing at least a portion of DP1 or DP2 anhydro-subunit-containing oligosaccharides from the sample by GC / MS. In some embodiments, the detecting step comprises analyzing at least a portion of DP3 anhydro-subunit-containing oligosaccharides from the sample. In some embodiments, the isolating step comprises separating one or more anhydro-subunit-containing oligosaccharides by preparative chromatography.In some embodiments, the synthetic oligosaccharide preparation is present in the nutritional composition at a concentration of about 1 to about 5000 ppm, about 1 to about 1000 ppm, about 1 to about 500 ppm, about 10 to about 5000 ppm, about 10 to about 2000 ppm, about 10 to about 1000 ppm, about 10 to about 500 ppm, about 10 to about 250 ppm, about 10 to about 100 ppm, about 50 to about 5000 ppm, about 50 to about 2000 ppm, about 50 to about 1000 ppm, about 50 to about 500 ppm, about 50 to about 250 ppm, or about 50 to about 100 ppm. In some embodiments, the synthetic oligosaccharide preparation is present in the nutritional composition at a concentration of greater than 10 ppm, greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, greater than 300 ppm, greater than 400 ppm, greater than 500 ppm, greater than 600 ppm, greater than 1000 ppm, or greater than 2000 ppm, hi some embodiments, the nutritional composition is an animal feed composition.

[0008] In one aspect, a method of producing a nutritional composition is described herein, comprising: (a) combining a basal nutritional composition with a synthetic oligosaccharide preparation comprising anhydro-subunit-containing oligosaccharides; and (b) performing a quality control method (e.g., a method of correlating the synthetic oligosaccharide preparation in the nutritional composition) as described herein. In some embodiments, the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a different degree of polymerization selected from 1 to n (DP1 to DPn fractions), where n is an integer greater than or equal to 3; the DP1 and DP2 fractions each independently comprise about 0.5% to about 15% anhydro-subunit-containing oligosaccharides by relative abundance as measured by mass spectrometry. In some embodiments, the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides each having a different degree of polymerization selected from 1 to n (DP1 to DPn fractions), where n is an integer greater than or equal to 2; the DP1 and DP2 fractions each independently comprise anhydro-subunit-containing oligosaccharides at a relative abundance of about 0.1% to about 15% as measured by mass spectrometry. In some embodiments, the relative abundance is determined by LC-MS / MS. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with their degree of polymerization.

[0009] In one aspect, provided herein is a method for quantifying an oligosaccharide preparation in a nutritional composition, comprising: (a) determining a level of a signal in a sample of the nutritional composition; and (b) calculating the concentration of the oligosaccharide preparation in the nutritional composition based on the level of the signal, wherein the signal (i) is indicative of one or more anhydrosubunit-containing oligosaccharides; or (ii) is associated with a degree of polymerization (DP) distribution of the oligosaccharides. or (iii) associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, an α-(1,1)-α glycosidic bond, an α-(1,1)-β glycosidic bond, a β-(1,1)-α glycosidic bond, or a β-(1,1)-β glycosidic bond in an oligosaccharide. In one aspect, provided herein is a method for performing quality control of a nutritional composition, comprising: (a) detecting a signal in a sample of the nutritional composition using an analytical device; and (b) passing or rejecting a batch of the nutritional composition based on the presence or absence of the signal, wherein the signal is either (i) indicative of one or more anhydro-subunit-containing oligosaccharides or (ii) associated with a degree of polymerization (DP) distribution of the oligosaccharides. or (iii) associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, an α-(1,1)-α glycosidic bond, an α-(1,1)-β glycosidic bond, a β-(1,1)-α glycosidic bond, or a β-(1,1)-β glycosidic bond in an oligosaccharide.In another aspect, provided herein is a method for performing quality control of a nutritional composition, comprising: (a) detecting, with an analytical device, 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 (b) comparing the first signal and the second signal, wherein the first signal and the second signal are indicative of either (i) one or more anhydro-subunit-containing oligosaccharides or (ii) a degree of polymerization (DP) of the oligosaccharides. ) distribution of oligosaccharides, or (iii) associated with α-(1,2) glycosidic, α-(1,3) glycosidic, α-(1,6) glycosidic, β-(1,2) glycosidic, β-(1,3) glycosidic, β-(1,4) glycosidic, β-(1,6) glycosidic, α-(1,1)-α glycosidic, α-(1,1)-β glycosidic, β-(1,1)-α glycosidic, or β-(1,1)-β glycosidic linkages of oligosaccharides. In some embodiments, the signal, the level of the signal, the first signal, and / or the second signal are determined or detected by high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, size exclusion chromatography (SEC), field-flow fractionation (FFF), asymmetric flow field-flow fractionation (A4F), or a combination thereof. In some embodiments, the nutritional composition comprises a basal nutritional composition. In some embodiments, the signal, the first signal, and / or the second signal are indicative of one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the one or more anhydro-subunit-containing oligosaccharides are derived from an oligosaccharide preparation. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP1 fraction. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to levoglucosan, 1,6-anhydro-β-D-glucofuranose, or a combination thereof.In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to one or more anhydro-subunit-containing oligosaccharides in the DP2 fraction. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to cellobiosan. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP3 fraction. In some embodiments, the first signal and the second signal are attributable to the same species of anhydro-subunit-containing oligosaccharides. In some embodiments, the first signal and the second signal are attributable to different species of anhydro-subunit-containing oligosaccharides. In some embodiments, the signal, the level of the signal, the first signal, and / or the second signal are determined or detected by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS). In some embodiments, the signal, the level of the signal, the first signal, and / or the second signal are determined or detected by liquid chromatography-mass spectrometry (LC-MS) / MS. In some embodiments, the signal, the level of the signal, the first signal, and / or the second signal are determined or detected by GC-flame ionization detection (GC-FID) or GC-MS. In some embodiments, the signal, the level of the signal, the first signal, and / or the second signal are detected by NMR. In some embodiments, a derivatization step is performed prior to detection. In some embodiments, the signal, the level of the signal, the first signal, and / or the second signal are determined by weight of isolated and / or purified fractions from preparative chromatography. In some embodiments, the basal nutritional composition lacks detectable levels of anhydro subunits. In some embodiments, the basal nutritional composition is substantially free of anhydro subunits.In certain embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, an α-(1,1)-α glycosidic bond, an α-(1,1)-β glycosidic bond, a β-(1,1)-α glycosidic bond, or a β-(1,1)-β glycosidic bond of an oligosaccharide. In some embodiments, the signal, the first signal, and the second signal are detected or determined by NMR. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,2) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,3) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,6) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,2) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,3) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,4) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,6) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,1)-α glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,1)-β glycosidic bond or a β-(1,1)-α glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,1)-β glycosidic bond.In certain embodiments, the signal, the first signal, and / or the second signal are associated with the DP distribution of oligosaccharides. In some embodiments, one or more of the signals, the first signal, and the second signal are attributable to oligosaccharides in the DP2 fraction. In some embodiments, the method further comprises passing or rejecting the batch of nutritional composition. In some embodiments, the method further comprises adjusting the level of the oligosaccharide preparation after the determination or detection. In some embodiments, the basal nutritional composition comprises a plurality of oligosaccharides. In some embodiments, the basal nutritional composition comprises starch and / or plant fiber. In some embodiments, the level of an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, or a β-(1,4) glycosidic bond in the basal nutritional composition is at least 10% lower than the level of the same glycosidic bond in the oligosaccharide preparation. In some embodiments, the level of α-(1,1)-α glycosidic linkages, α-(1,1)-β glycosidic linkages, β-(1,1)-α glycosidic linkages, or β-(1,1)-β glycosidic linkages in the basal nutritional composition is at least 10% lower than the level of the same glycosidic linkages in the oligosaccharide preparation. In some embodiments, the method further comprises extracting oligosaccharides from a sample of the nutritional composition. In some embodiments, the method further comprises filtering or clarifying the extracted oligosaccharides. In some embodiments, the method further comprises concentrating the extracted oligosaccharides. In some embodiments, concentrating the extracted oligosaccharides comprises lyophilization. In some embodiments, the method further comprises incorporating an internal standard into the extracted or enriched oligosaccharides. In some embodiments, the method further comprises reducing the extracted or enriched oligosaccharides. In some embodiments, the method further comprises digesting the extracted or enriched oligosaccharides with one or more hydrolases. In some embodiments, the one or more hydrolytic enzymes comprise a carbohydratase, a protease, a lipase, or any combination thereof.In some embodiments, the one or more hydrolases comprise α-amylase, amyloglycosidase, invertase, α-galactosidase, or a combination thereof. In some embodiments, the one or more hydrolases cleave one or more naturally occurring glycosidic bonds. In some embodiments, the method further comprises isolating undigested oligosaccharides. In some embodiments, the method further comprises separating the extracted, concentrated, digested, or reduced oligosaccharides. In some embodiments, the oligosaccharides are separated by chromatography. In some embodiments, the method further comprises isolating the separated oligosaccharides. In some embodiments, the oligosaccharides are separated or isolated by their degree of polymerization. In some embodiments, the method comprises isolating or separating oligosaccharides having a degree of polymerization of 1, 2, 3, 4, or 5. In some embodiments, the oligosaccharides in the DP1 fraction are isolated or separated. In some embodiments, the oligosaccharides in the DP2 fraction are isolated or separated. In some embodiments, the oligosaccharides in the DP3 fraction are isolated or separated. In some embodiments, oligosaccharides in the DP4 fraction are isolated or separated. In some embodiments, oligosaccharides in the DP5 fraction are isolated or separated. In some embodiments, the isolated or separated oligosaccharides are quantified. In some embodiments, anhydro-subunit-containing oligosaccharides in the isolated or separated oligosaccharides are quantified. In some embodiments, the majority of the oligosaccharides quantified. In some embodiments, greater than 50, 60, 70, 80, 90, 95, or 99% by weight of the quantified oligosaccharides are derived from the oligosaccharide preparation. In some embodiments, the method comprises analyzing a glycosidic bond of an oligosaccharide (wherein the glycosidic bond is an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, an α-(1,1)-α glycosidic bond, an α-(1,1)-β glycosidic bond, a β-(1,1)-α glycosidic bond, or a β-(1,1)-β glycosidic bond) by NMR, thereby determining or detecting a signal, a level of a signal, a first signal, or a second signal associated with the corresponding glycosidic bond. In some embodiments, the methods involve analyzing the anhydro-subunit-containing oligosaccharides by mass spectrometry to determine or detect a signal, signal level, first signal, or second signal indicative of one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the methods involve analyzing the anhydro-subunit-containing oligosaccharides by HPLC to determine or detect a signal, signal level, first signal, or second signal indicative of one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the methods involve analyzing the anhydro-subunit-containing oligosaccharides by FFF or A4F to determine or detect a signal, signal level, first signal, or second signal indicative of one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the methods involve analyzing the DP distribution of the oligosaccharides by SEC, GC, or HPLC to determine or detect a signal, signal level, first signal, or second signal associated with the DP distribution. In some embodiments, the method comprises quantifying the oligosaccharides in the DP2 fraction by SEC, GC or HPLC, thereby determining or detecting a signal, a level of a signal, a first signal or a second signal associated with the DP distribution.In some embodiments, the oligosaccharide preparation is present at 1-5000 ppm, 1-1000 ppm, 1-500 ppm, 10-5000 ppm, 10-2000 ppm, 10-1000 ppm, 10-500 ppm, 10-250 ppm, 10-100 ppm, 50-5000 ppm, 50-2000 ppm, 50-1000 ppm, 50-500 ppm, 50-250 ppm, or 50-100 ppm relative to the nutritional composition. In some embodiments, the oligosaccharide preparation is present at more than 10 ppm, more than 50 ppm, more than 100 ppm, more than 200 ppm, more than 300 ppm, more than 400 ppm, more than 500 ppm, more than 600 ppm, more than 1000 ppm, or more than 2000 ppm relative to the nutritional composition. In some embodiments, the nutritional composition is an animal feed composition.

[0010] In one aspect, a method of producing a nutritional composition is described herein, comprising: (a) combining a synthetic oligosaccharide preparation comprising anhydro-subunit-containing oligosaccharides with a basal nutritional composition; and (b) performing a quality control process as described herein. In some embodiments, the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1-DPn fractions), each having a different degree of polymerization selected from 1 to n, where n is an integer greater than or equal to 2; each fraction comprising about 0.1% to about 15% anhydro-subunit-containing oligosaccharides in relative abundance as measured by mass spectrometry. In some embodiments, the synthetic oligosaccharide preparation comprises n fractions of oligosaccharides (DP1-DPn fractions), each having a different degree of polymerization selected from 1 to n, where n is an integer greater than or equal to 3; each fraction comprising 0.5% to 15% anhydro-subunit-containing oligosaccharides in relative abundance as measured by mass spectrometry. In some embodiments, the relative abundance of an oligosaccharide in each of the n fractions decreases monotonically with its degree of polymerization.

[0011]

[0011] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0012] [Incorporated by reference]

[0012] All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification supersedes and / or is intended to take precedence over such conflicting material.

[0013] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figures" and "FIG."). [Brief explanation of the drawings]

[0014] [Figure 1] 1 shows the 1H,13C-HSQC NMR spectrum of the oligosaccharide preparation of Example 9.7. [Figure 2] 1 shows a MALDI-MS spectrum of the oligosaccharide preparation from Example 9 showing the presence of anhydro subunits. [Figure 3] 1D 1H-proton NMR spectrum of the anhydro-DP1 fraction isolated from the oligosaccharides of Example 9. [Figure 4] 1D APT 13C-NMR spectrum of the anhydro-DP1 fraction isolated from the oligo of Example 9. [Figure 5]The structures of two anhydro DP1 compounds (1,6-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranose) and their NMR assignments are shown. [Figure 6] Figure 9 shows an enlargement of the GC-MS chromatogram (TIC and XIC (m / z 229) plot) for the oligosaccharide preparation of Example 9.7 after derivatization. [Figure 7] 1 shows MALDI-MS spectra comparing the oligosaccharide preparation from Example 9 with conventional dextran. [Figure 8] LC-MS / MS detection of two anhydroDP species from oligosaccharide preparations in water at concentrations ranging from 1 to 80 μg / mL is shown. [Figure 9] Figure 8 shows the linear calibration curve obtained from LC-MS / MS. [Figure 10A] Quantification of the anhydro-DP2 content of various control and treated dietary compositions is shown. [Figure 10B] Quantification of the anhydro-DP2 content of various control and treated dietary compositions is shown. [Figure 11] 2D-1H JRES NMR spectra of anhydro-subunit-containing gluco-oligosaccharide samples. [Figure 12] 1 is a representative 1H, 13C-HSQC NMR spectrum of an anhydro subunit-containing glucooligosaccharide sample with relevant resonances and assignments used for bond distribution. [Figure 13] 1 shows an overlay of 1H DOSY spectra of three anhydrosubunit-containing oligosaccharides. [Figure 14] A comparison of 1,6-anhydro-β-D-glucose (DP1-18), 1,6-anhydro-β-D-cellobiose (DP2-18), and an anhydro-subunit-containing oligosaccharide sample is shown. [Figure 15] Selected multiple reaction monitoring (MRM) mass chromatograms of anhydro-subunit-containing oligosaccharides (top) and digested anhydro-subunit-containing oligosaccharides (bottom) are shown. [Figure 16]Selected MRMs are shown for mass chromatograms of (1) feed containing anhydro subunit-containing oligosaccharides, (2) digested feed containing anhydro subunit-containing oligosaccharides, and (3) blank digested feed. [Figure 17] A representative workflow for the analysis of oligosaccharide preparations in animal feed is shown. [Figure 18] Two DP1 and one DP2 anhydrosubunit-containing oligosaccharides are shown. [Figure 19] An anhydrosubunit-containing oligosaccharide (cellotriosan) is shown. [Figure 20A] 1 shows a MALDI-MS spectrum of the oligosaccharide preparation from Example 2 showing the presence of anhydro subunits. [Figure 20B] FIG. 20B shows an enlarged view of a portion of the MALDI-MS spectrum shown in FIG. 20A. [Figure 21A] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 1. [Figure 21B] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 1. [Figure 21C] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 1. [Figure 22A] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 3. [Figure 22B] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 3. [Figure 22C] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 3. [Figure 23A] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 4. [Figure 23B] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 4. [Figure 23C] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 4. [Figure 24A]1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 7. [Figure 24B] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 7. [Figure 24C] 1 shows LC-MS / MS detection of two anhydro DP species in the oligosaccharide preparation of Example 7. [Figure 25A] 1 shows GC-MS spectral detection of DP1, anhydroDP1, DP2 and anhydroDP2 fractions of the oligosaccharide preparation of Example 1. [Figure 25B] 25A shows a close-up of the DP2 and anhydroDP2 fractions shown in FIG. 25A. [Figure 26A] 1 shows GC-MS spectral detection of DP1, anhydroDP1, DP2 and anhydroDP2 fractions of the oligosaccharide preparation of Example 3. [Figure 26B] 26A shows a close-up of the DP2 and anhydroDP2 fractions shown in FIG. 26A. [Figure 27A] 1 shows GC-MS spectral detection of DP1, anhydroDP1, DP2 and anhydroDP2 fractions of the oligosaccharide preparation of Example 4. [Figure 27B] A magnified view of the DP2 and anhydroDP2 fractions shown in Figure 27A is shown. [Figure 28A] 1 shows GC-MS spectral detection of DP1, anhydroDP1, DP2 and anhydroDP2 fractions of the oligosaccharide preparation of Example 7. [Figure 28B] A magnified view of the DP2 and anhydroDP2 fractions shown in Figure 28A is shown. [Figure 29] 1 shows the effect of reaction time, water content and reaction time on the content of DP2 anhydrosubunit-containing oligosaccharides in oligosaccharide preparations compared to oligosaccharide preparations according to Example 2. [Figure 30] 1 shows MALDI-MS spectra comparing oligosaccharide preparations from Example 9 at different laser energies.

[0015] [Detailed explanation]

[0044] The following description and examples will explain in detail the embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may vary as such. Those skilled in the art will appreciate that there are many variations and modifications of the present disclosure that are within its scope.

[0016]

[0045] All terms are intended to be understood as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0017]

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0018]

[0047] While various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be practiced in a single embodiment.

[0019]

[0048] The following definitions supplement those in the art and are directed to this application and are not attributable to, for example, commonly owned patents or applications, whether related or unrelated. Although any methods and materials similar or equivalent to those described herein can be used to carry out the testing of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is merely for the purpose of describing particular embodiments and is not intended to be limiting.

[0020] [I. Definition]

[0049] As used herein, "administering" includes providing a synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition described herein to an animal such that the animal has free access to the synthetic oligosaccharide preparation, nutritional composition, liquid, or animal feed composition. In such embodiments, the animal ingests a portion of the synthetic oligosaccharide preparation, nutritional composition, or animal feed composition. In some embodiments, the animal ingests a portion of the synthetic oligosaccharide preparation, 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 preparation can be dissolved in water or another liquid, and the animal ingests a portion of the oligosaccharide preparation by drinking the liquid. In certain embodiments, the oligosaccharides are provided to the animal via its drinking water. In certain embodiments, the oligosaccharide preparation, nutritional composition, liquid, or animal feed composition is consumed ad libitum.

[0021]

[0050] As used herein, the term "inclusion level" or "dosage" refers to the concentration of an oligosaccharide preparation in a nutritional composition, liquid, diet, or animal feed composition fed to an animal. In some embodiments, the inclusion level is measured as the mass concentration of the oligosaccharide preparation in the final nutritional composition, liquid, diet, or animal feed. For example, the inclusion level may be measured in parts per million (ppm) of oligosaccharides based on dry solids weight per total weight of the final nutritional composition, liquid, diet, or animal feed. In certain embodiments, the dry solids weight of the oligosaccharide preparation is measured as the dry basis weight of the DP1+ species. In other embodiments, the dry solids weight of the oligosaccharide preparation is measured as the dry basis weight of the DP2+ species.

[0022]

[0051] As used herein, the term "specific dose" refers to the amount of oligosaccharide preparation consumed by an animal per unit of time and relative to its body mass. In some embodiments, the specific dose can be measured in mg of oligosaccharide preparation (on a dry solids basis) per kg of animal body weight per day (i.e., mg / kg / day).

[0023]

[0052] As used herein, the term "anhydro subunit" refers to a monosaccharide (or monosaccharide subunit) or a thermal dehydration product of a sugar caramelization product. For example, an "anhydro subunit" can be an anhydro-monosaccharide, such as anhydro-glucose. As another example, an "anhydro subunit" can be linked to one or more regular or anhydro-monosaccharide subunits via glycosidic bonds.

[0024]

[0053] As used herein, the terms "anhydroDPn oligosaccharide," "anhydroDPn species," or "DPn anhydrosubunit-containing oligosaccharide" refer to an oligosaccharide having a degree of polymerization of n and containing one or more anhydrosubunits. Thus, anhydroglucose is an oligosaccharide containing a DP1 anhydrosubunit, and cellotriosan is an oligosaccharide containing a DP3 anhydrosubunit.

[0025]

[0054] As used herein, the term "feed conversion ratio (FCR)" refers to the ratio of feed mass input (e.g., consumed by an animal) to animal output, where the animal output is the animal product of interest. For example, the animal output of dairy animals is milk, and the animal output of livestock raised for meat is body mass.

[0026]

[0055] The term "oligosaccharide" refers to a monosaccharide or a compound containing two or more monosaccharide subunits linked by glycosidic bonds. As such, oligosaccharides include regular monosaccharides; anhydro-monosaccharides; or compounds containing two or more monosaccharide subunits, where one or more monosaccharide subunits are optionally independently replaced with one or more anhydro-subunits. Oligosaccharides can be functionalized. As used herein, the term oligosaccharide encompasses all species of oligosaccharides, where each monosaccharide subunit in an oligosaccharide is independently and optionally functionalized and / or replaced with its corresponding anhydro-monosaccharide subunit.

[0027]

[0056] As used herein, the term "oligosaccharide preparation" refers to a preparation that includes at least one oligosaccharide.

[0028]

[0057] As used herein, the term "gluco-oligosaccharide" refers to a compound containing glucose or two or more glucose monosaccharide subunits linked by glycosidic bonds. As such, gluco-oligosaccharides include compounds containing glucose; anhydro-glucose; or two or more glucose monosaccharide subunits linked by glycosidic bonds, wherein one or more of the glucose monosaccharide subunits are each optionally and independently replaced with an anhydro-glucose subunit.

[0029]

[0058] As used herein, the term "galacto-oligosaccharide" refers to a compound containing galactose or two or more galactose monosaccharide subunits linked by glycosidic bonds. As such, galacto-oligosaccharides include galactose; anhydro-galactose; or a compound containing two or more galactose monosaccharide subunits linked by glycosidic bonds, wherein one or more of the galactose monosaccharide subunits are each optionally and independently replaced with an anhydro-galactose subunit.

[0030]

[0059] As used herein, the term "gluco-galactose-oligosaccharide preparation" refers to a composition produced from a complete or incomplete glycocondensation reaction of glucose and galactose. Thus, in some embodiments, gluco-galactose-oligosaccharide preparations include gluco-oligosaccharides, galacto-oligosaccharides, compounds containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by glycosidic bonds, or combinations thereof. In some embodiments, gluco-galactose-oligosaccharide preparations include 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, gluco-galactose-oligosaccharide preparations include 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 gluco-galactose-oligosaccharide preparation comprises a compound containing one or more glucose monosaccharide subunits and one or more galactose monosaccharide subunits linked by a glycosidic bond.

[0031]

[0060] As used herein, the terms "monosaccharide unit" and "monosaccharide subunit" are used interchangeably. A "monosaccharide subunit" refers to a monosaccharide monomer of an oligosaccharide. For oligosaccharides having a degree of polymerization of 1, the oligosaccharide may be referred to as a monosaccharide subunit or a monosaccharide. For oligosaccharides having a degree of polymerization of 2 or higher, the monosaccharide subunits are linked via glycosidic bonds.

[0032]

[0061] As used herein, the term "normal monosaccharide" refers to a monosaccharide that does not contain an anhydro subunit. The term "normal disaccharide" refers to a disaccharide that does not contain an anhydro subunit. Thus, the term "normal subunit" refers to a subunit that is not an anhydro subunit.

[0033]

[0062] The terms "relative abundance" or "abundance," as used herein, refer to the abundance of a species relative to how common or rare the species is. For example, a DP1 fraction containing 10% anhydrosubunit-containing oligosaccharides in relative abundance refers to a plurality of DP1 oligosaccharides, where 10% of the DP1 oligosaccharides are anhydromonosaccharides. For example, for a particular DP fraction of oligosaccharides, the relative abundance can be determined by suitable analytical equipment, e.g., mass spectrometry and liquid chromatography, such as LC-MS / MS, GC-MS, HPLC-MS, and MALDI-MS. In some embodiments, the relative abundance is determined by integrating the area under the peak in a chromatograph (e.g., LC-MS / MS, GC-MS, and HPLC-MS) corresponding to the fraction of interest. In some embodiments, the relative abundance is determined by peak intensity (e.g., MALDI-MS). In some embodiments, the relative abundance is determined by a combination of analytical methods, such as gravimetric determination after liquid chromatographic separation.

[0034]

[0063] As used herein, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, and reference to "an oligosaccharide" includes one or more oligosaccharides (or oligosaccharides) and equivalents thereof known to those skilled in the art.

[0035]

[0064] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formula, all combinations and subcombinations of the ranges and specific embodiments described herein are intended to be encompassed. The term "about" when referring to a numerical value or numerical range means that the stated numerical value or numerical range is an approximation within experimental variability (or within statistical experimental error); thus, the numerical value or numerical range may, in some cases, vary by 1% to 15% of the stated numerical value or numerical range. In some embodiments, the term "about" means within 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of the given numerical value or range.

[0036]

[0065] "Comprising" (and related terms such as "comprise" or "comprises" or "having" or "including") is intended to include, but not necessarily be limited to, the items so designated.

[0037] II. Application of the Method

[0066] Oligosaccharide preparations are used as additives in nutritional compositions such as complete animal feeds. The addition of such oligosaccharide preparations improves the health and performance (weight, growth rate, feed conversion efficiency) of animals such as poultry (broilers, layers, broiler breeders, turkeys) and pigs (pigs, breeder / finisher pigs, sows, etc., as well as other species).

[0038]

[0067] The effect of oligosaccharide preparations on animal health and performance depends on the chemical and physicochemical properties of the oligosaccharides. In particular, the chemical composition of an oligosaccharide preparation can affect how it is used by an animal's microflora, particularly the intestinal microflora. In turn, the animal's microflora, particularly the intestinal microflora, affects the overall health and performance of the animal. Such effects include, for example, physiological and morphological effects on the gastrointestinal tract, immune system activation, stimulation of mucus production, improvement of intestinal barrier function, improvement of nutrient release and absorption, regulation of the abundance of various members of the intestinal microflora (e.g., suppression of pathogenic species, increase of beneficial symbiotic species), and regulation of biochemical species produced by the intestinal microflora. Therefore, oligosaccharide preparations can be added to nutritional compositions such as animal feed to act as prebiotics.

[0039]

[0068] Provided herein are methods for producing oligosaccharide preparations suitable for use as additives in nutritional compositions. Oligosaccharide preparations can also be produced by other means, including enzymatic or acidic hydrolysis of plant fiber, extraction of bacterial and yeast cell wall glycans and glycopeptides, enzyme enrichment from sugars, and fermentation by wild-type or recombinant microorganisms.

[0040]

[0069] Due to the structural complexity of both nutritional compositions and oligosaccharide preparations, there are currently no simple, selective, and sensitive techniques for analyzing the presence and concentration of oligosaccharide preparations in feed materials. Nutritional compositions contain a large amount and a variety of carbohydrate structures (e.g., starch, vegetable fiber, and pectin). Therefore, it is particularly difficult to distinguish small amounts of oligosaccharide-based feed additives from the vast number of other carbohydrates present as the matrix of nutritional compositions. Therefore, analytical methods with the required sensitivity and selectivity are needed to distinguish these oligosaccharide feed additives from natural oligomers.

[0041]

[0070] Assaying for the presence and / or concentration of feed additives is commercially useful. Such assays can be performed for quality control purposes to determine whether the additive has been consistently blended with the base nutritional composition to provide a final nutritional composition containing the additive at the intended dose or level of content. In certain embodiments, the manufacturing process can include either passing or failing a given amount (e.g., lot or batch) of the final feed composition based on a determination that the additive was contained within a predetermined range of values.

[0042]

[0071] Such oligosaccharide preparations contain a wide variety of glycosidic linkages between the various monomers in the oligosaccharide preparation. In certain embodiments, the relative abundance of glycosidic linkages, e.g., α-(1,4) linkages, which are readily hydrolyzed by the digestive acids and enzymes of an animal's upper gastrointestinal tract, is low. Thus, the oligosaccharides survive primary digestion by the animal and pass through the gastrointestinal tract to the lower gastrointestinal tract, where they interact with the gut microbiota. In some embodiments, the relative abundance of glycosidic linkages commonly found in plant fiber and pectin is also low. In some embodiments, the oligosaccharide preparations contain linkages that are present in low relative abundance in the base carbohydrates (i.e., background carbohydrates) in the nutritional composition. In certain embodiments, the oligosaccharide preparations contain anhydrosubunit-containing oligosaccharides. In certain embodiments, the oligosaccharide preparations contain chemically synthesized anhydrosubunit-containing oligosaccharides.

[0043]

[0072] Despite the need to detect and / or quantify the relative abundance of oligosaccharide preparations in nutritional compositions, existing analytical methods for quantifying various glycosidic linkages in complex carbohydrates may lack sensitivity. Certain existing analytical methods have minimum thresholds that resolve glycosidic linkages at the percentage level. However, commercially relevant inclusion levels in feed additives are typically in the range of 1-5000 ppm, 10-1000 ppm, 10-500 ppm, or 50-500 ppm. Therefore, glycosidic linkages in feed additives are undetectable by some existing methods.

[0044]

[0073] Surprisingly, the inventors have found that it is possible to analyze and quantify oligosaccharide preparations in complex nutritional compositions, such as complete animal feeds, as described herein.

[0045] III. Oligosaccharide Preparations [Oligosaccharides by manufacturing method]

[0074] Provided herein are oligosaccharide preparations suitable for use in nutritional compositions. In some embodiments, the oligosaccharide preparations provided herein can comprise monosaccharides, oligosaccharides, polysaccharides, or any combination thereof, where one or more monosaccharide subunits in either the monosaccharides, oligosaccharides, or polysaccharides can be independently functionalized. In some embodiments, the oligosaccharide preparations include oligosaccharides produced by hydrolysis or pyrolysis of polysaccharides such as cellulose and starch, by condensation or polymerization of monosaccharides or oligosaccharides, by enzymatic or acid hydrolysis of polysaccharides such as plant fibers, by extraction of bacterial and yeast cell wall glycans and glycopeptides, by enzymatic condensation from sugars, by fermentation with wild-type or recombinant microorganisms, or any combination thereof. In some embodiments, the oligosaccharide preparations can include any oligosaccharide known in the art. In some embodiments, the oligosaccharide preparations are produced chemically, naturally, or enzymatically.

[0046]

[0075] In some embodiments, the oligosaccharide preparation is a synthetic oligosaccharide preparation. In some embodiments, a synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a process that does not require a living organism. In some embodiments, a synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a process that does not require an enzyme. In some embodiments, a synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by a chemical process. In certain embodiments, a synthetic oligosaccharide preparation refers to a plurality of oligosaccharides produced by condensation of sugars.

[0047] [Polymerization degree (DP) distribution]

[0076] In some embodiments, the oligosaccharide preparations described herein comprise at least n fractions of oligosaccharides (DP1-DPn fractions), each having a different degree of polymerization selected from 1 to n. In some embodiments, the oligosaccharide preparations comprise n fractions of oligosaccharides, each fraction having a different degree of polymerization selected from 1 to n (DP1-DPn fractions). In some embodiments, the DP1 fraction comprises one or more monosaccharides and / or one or more anhydro-monosaccharides. As another example, in some embodiments, the DP1 fraction comprises glucose, galactose, fructose, 1,6-anhydro-β-D-glucofuranose, 1,6-anhydro-β-D-glucopyranose, or any combination thereof. For example, in some embodiments, the DP2 fraction comprises one or more normal disaccharides and one or more anhydro-subunit-containing disaccharides. In some embodiments, the DP2 fraction comprises lactose.

[0048]

[0077] 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 at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99 or at least 100.In some embodiments, n is about 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 2 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100. In some embodiments, n is less than 10, less than 11, less than 12, less than 13, less than 14, less than 15, less than 16, less than 17, less than 18, less than 19, less than 20, less than 21, less than 22, less than 23, less than 24, less than 25, less than 26, less than 27, less than 28, less than 29, less than 30, less than 31, less than 32, less than 33, less than 34, less than 35, less than 36, less than 37, less than 38, less than 39, less than 40, less than 41, less than 42, less than 43, less than 44, less than 45, less than 46, less than 47, less than 48, less than 49, less than 50, less than 51, less than 52, less than 53, less than 54 less than 55, less than 56, less than 57, less than 58, less than 59, less than 60, less than 61, less than 62, less than 63, less than 64, less than 65, less than 66, less than 67, less than 68, less than 69, less than 70, less than 71, less than 72, less than 73, less than 74, less than 75, less than 76, less than 77, less than 78, less than 79, less than 80, less than 81, less than 82, less than 83, less than 84, less than 85, less than 86, less than 87, less than 88, less than 89, less than 90, less than 91, less than 92, less than 93, less than 94, less than 95, less than 96, less than 97, less than 98, less than 99 or less than 100. In some embodiments, n is 2 to 100, 5 to 90, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 15 to 60, 15 to 50, 15 to 45, 15 to 40, 15 to 35, or 15 to 30.

[0049]

[0078] The degree of polymerization distribution of an oligosaccharide preparation can be determined by any suitable analytical method and instrument, including, but not limited to, end-group analysis, osmotic pressure (osmometry), ultracentrifugation, viscometry, light scattering, size-exclusion chromatography (SEC), SEC-MALLS, field-flow fractionation (FFF), asymmetric flow field-flow fractionation (A4F), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). For example, the degree of polymerization distribution can be determined and / or detected by mass spectrometry, such as matrix-assisted laser desorption / ionization (MALDI)-MS, liquid chromatography (LC)-MS, or gas chromatography (GC)-MS. As another example, the degree of polymerization distribution can be determined and / or detected by SEC, such as gel permeation chromatography (GPC). As yet another example, the degree of polymerization distribution can be determined and / or detected by HPLC, FFF, or A4F. In some embodiments, the degree of polymerization distribution is determined and / or detected by MALDI-MS. In some embodiments, the degree of polymerization distribution is determined and / or detected by GC-MS or LC-MS. In some embodiments, the degree of polymerization distribution is determined and / or detected by SEC. In some embodiments, the degree of polymerization distribution is determined and / or detected by HPLC. In some embodiments, the degree of polymerization distribution is determined and / or detected by a combination of analytical instruments, such as MALDI-MS and SEC. In some embodiments, the degree of polymerization of an oligosaccharide preparation can be determined based on its molecular weight and molecular weight distribution. For example, Figure 2 shows a MALDI-MS spectrum showing the degree of polymerization of various fractions and the presence of anhydrosubunit-containing oligosaccharides (-18 g / mol MW offset peak) in all observed fractions.

[0050]

[0079] In some embodiments, the relative abundance of oligosaccharides in the majority of the fractions decreases monotonically with their degree of polymerization, hi some embodiments, the relative abundance of oligosaccharides in less than 6, less than 5, less than 4, or less than 2 fractions of an oligosaccharide preparation does not decrease monotonically with their degree of polymerization.

[0051]

[0080] In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 consecutive DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 consecutive DP fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in at least 5, at least 10, at least 20, or at least 30 consecutive DP fractions decreases monotonically with their degree of polymerization.

[0052]

[0081] In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with their degree of polymerization. For example, Figure 10 provides an example of a DP distribution in which the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with their DP. For example, in some embodiments, only the relative abundance of oligosaccharides in the DP3 fraction does not decrease monotonically with their degree of polymerization, i.e., the relative abundance of oligosaccharides in the DP3 fraction is lower than the relative abundance of oligosaccharides in the DP4 fraction. In some embodiments, the relative abundance of oligosaccharides in the DP2 fraction is lower than the relative abundance of oligosaccharides in the DP3 fraction. For example, Figure 16 shows a degree of polymerization distribution in which the relative abundance of oligosaccharides in the DP2 fraction does not decrease monotonically with their degree of polymerization.

[0053]

[0082] In some embodiments, the oligosaccharide preparations described herein have a DP1 fraction content by weight or relative abundance of about 1% to about 50%, about 1% to about 40%, about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 5% to about 50%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 10% to about 50%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, or about 10% to about 15%. In some embodiments, the oligosaccharide preparation has a DP1 fraction content of about 10% to about 35%, about 10% to about 20%, or about 10% to about 15% by weight or relative abundance. In some embodiments, the DP1 fraction content is determined by mass spectrometry. In some embodiments, the DP1 fraction content is determined by HPLC. In some embodiments, the DP1 fraction content is determined by LC-MS / MS or GC-MS.

[0054]

[0083] In some embodiments, the oligosaccharide preparations described herein have a DP2 fraction content of about 1% to about 35%, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% by weight or relative abundance. In some embodiments, the oligosaccharide preparations have a DP2 fraction content of about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% by weight or relative abundance. In some embodiments, the DP2 fraction content is determined by mass spectrometry. In some embodiments, the DP2 fraction content is determined by HPLC. In some embodiments, the DP2 fraction content is determined by LC-MS / MS or GC-MS.

[0055]

[0084] In some embodiments, the oligosaccharide preparations described herein have a DP3 fraction content of about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, or about 5% to about 10% by weight or relative abundance. In some embodiments, the oligosaccharide preparations have a DP3 fraction content of about 1% to about 15%, about 1% to about 10%, about 5% to about 15%, or about 5% to about 10% by weight or relative abundance. In some embodiments, the DP3 fraction content is determined by MALDI-MS. In some embodiments, the DP3 fraction content is determined by HPLC. In some embodiments, the DP3 fraction content is determined by LC-MS / MS or GC-MS.

[0056]

[0085] In some embodiments, the oligosaccharide preparations described herein have a DP4 fraction content of about 0.1% to about 20%, about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% by weight or relative abundance. In some embodiments, the oligosaccharide preparations described herein have a DP4 fraction content of about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% by weight or relative abundance. In some embodiments, the oligosaccharide preparations described herein have a DP5 fraction content of about 0.1% to about 15%, about 0.1% to about 10%, about 0.1% to about 5%, about 1% to about 15%, about 1% to about 10%, or about 1% to about 5% by weight or relative abundance. In some embodiments, the oligosaccharide preparation has a DP5 fraction content of about 1% to about 10% or about 1% to about 5% by weight or relative abundance. In some embodiments, the DP4 and / or DP5 fraction content is determined by MALDI-MS. In some embodiments, the DP4 and / or DP5 fraction content is determined by HPLC. In some embodiments, the DP4 and / or DP5 fraction content is determined by LC-MS / MS or GC-MS.

[0057]

[0086] In some embodiments, the ratio of the DP2 fraction to the DP1 fraction in the oligosaccharide preparation is about 0.01 to about 0.8, about 0.02 to about 0.7, about 0.02 to about 0.6, about 0.02 to about 0.5, about 0.02 to about 0.4, about 0.02 to about 0.3, about 0.02 to about 0.2, about 0.1 to about 0.6, about 0.1 to about 0.5, about 0.1 to about 0.4, or about 0.1 to about 0.3, in terms of weight or relative abundance. In some embodiments, the ratio of the DP2 fraction to the DP1 fraction in the oligosaccharide preparation is about 0.02 to about 0.4, in terms of weight or relative abundance.

[0058]

[0087] In some embodiments, the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation, in terms of weight or relative abundance, is about 0.01 to about 0.7, about 0.01 to about 0.6, about 0.01 to about 0.5, about 0.01 to about 0.4, about 0.01 to about 0.3, or about 0.01 to about 0.2. In some embodiments, the ratio of the DP3 fraction to the DP2 fraction in the oligosaccharide preparation, in terms of weight or relative abundance, is about 0.01 to about 0.3.

[0059]

[0088] In some embodiments, the aggregate content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% by weight or relative abundance. In some embodiments, the aggregate content of the DP1 and DP2 fractions in the oligosaccharide preparation is less than 50%, less than 30%, or less than 10% by weight or relative abundance.

[0060]

[0089] In some embodiments, the oligosaccharide preparations described herein have an average DP value in the range of 2 to 10. In some embodiments, the oligosaccharide preparations have an average DP value of about 2 to about 8, about 2 to about 5, or about 2 to about 4. In some embodiments, the oligosaccharide preparations have an average DP value of about 3.5. The average DP value can be determined by SEC or elemental analysis.

[0061] [Anhydro subunit level]

[0090] In some embodiments, the oligosaccharide preparations described herein contain one or more anhydrosubunits. That is, the oligosaccharide preparations contain one or more anhydrosubunit-containing oligosaccharides. In some embodiments, each of the n fractions of oligosaccharides in the oligosaccharide preparations described herein independently contains a particular anhydrosubunit level. For example, in some embodiments, the DP1 fraction contains an anhydrosubunit-containing oligosaccharide at a relative abundance of about 10%, and the DP2 fraction contains an anhydrosubunit-containing oligosaccharide at a relative abundance of about 15%. For another example, in some embodiments, the DP1, DP2, and DP3 fractions contain anhydrosubunit-containing oligosaccharides at a relative abundance of about 5%, about 10%, and about 2%, respectively. In some embodiments, two or more fractions of oligosaccharides contain the same level of anhydrosubunit-containing oligosaccharides. For example, in some embodiments, the DP1 and DP3 fractions each contain anhydrosubunit-containing oligosaccharides at a relative abundance of about 5%. In some embodiments, the oligosaccharide preparations described herein do not contain any anhydro subunits, i.e., the oligosaccharide preparations do not contain any anhydro subunit-containing oligosaccharides.

[0062]

[0091] In some embodiments, each of the 1-n fractions in an oligosaccharide preparation described herein independently comprises about 0.1% to 15% anhydro-subunit-containing oligosaccharides in relative abundance as measured by mass spectrometry, LC-MS / MS, or GC-MS. In some embodiments, each of the 1-n fractions in an oligosaccharide preparation independently comprises about 0.5% to 15% anhydro-subunit-containing oligosaccharides in relative abundance as measured by mass spectrometry, LC-MS / MS, or GC-MS. In some embodiments, LC-MS / MS is used to determine the relative abundance of oligosaccharides in the DP1, DP2, and / or DP3 fractions.

[0063]

[0092] In some embodiments, the presence, species type, and / or level of anhydro subunits can be determined and / or detected by any suitable analytical method, such as nuclear magnetic resonance (NMR) spectroscopy, HPLC, FFF, A4F, or any combination thereof. In some embodiments, the presence and level of anhydro subunit-containing oligosaccharides is determined and / or detected by MALDI-MS, as exemplified by the -18 g / mol MW offset peak in Figure 2. In some embodiments, the presence and species type of anhydro subunits is determined and / or detected by NMR, as exemplified in Example 11, Figures 3, and 4. In some embodiments, the presence, species type, and / or level of anhydro subunits or anhydro subunit-containing oligosaccharides is determined and / or detected, at least in part, by mass spectrometry, such as MALDI-MS. In some embodiments, the presence, species type, and / or level of anhydro subunits or anhydro subunit-containing oligosaccharides is determined and / or detected, at least in part, by NMR. In some embodiments, the presence, species type, and / or levels of anhydro-subunits or anhydro-subunit-containing oligosaccharides are determined and / or detected, at least in part, by HPLC. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by MALDI-MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by LC-MS / MS, as illustrated in Figures 21A-21C, 22A-22C, 23A-23C, and 24A-24C. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by GC-MS, as illustrated in Figures 25A-25B, 26A-26B, 27A-27B, and 28A-28D. 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 fraction and higher DP fractions.In some embodiments, the relative abundance of a particular fraction is determined by integrating the area under the peak in an LC-MS / MS chromatogram designated as corresponding to that fraction. In some embodiments, the relative abundance of a particular fraction is determined by integrating the area under the peak in a GC-MS chromatogram designated as corresponding to that fraction.

[0064]

[0093] In some embodiments, at least one fraction of an oligosaccharide preparation described herein comprises anhydro-subunit-containing oligosaccharides at a relative abundance of less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, at least one fraction of an oligosaccharide preparation described herein comprises anhydro-subunit-containing oligosaccharides at a relative abundance of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. In other embodiments, at least one fraction of the oligosaccharide preparations described herein comprises anhydro-subunit-containing oligosaccharides at a relative abundance of greater than 0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, greater than 15%, greater than 16%, greater than 17%, greater than 18%, greater than 19%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80%. In other embodiments, at least one fraction of the oligosaccharide preparations described herein comprises an anhydro-subunit-containing oligosaccharides at a relative abundance of greater than 20%, greater than 21%, greater than 22%, greater than 23%, greater than 24%, greater than 25%, greater than 26%, greater than 27%, greater than 28%, greater than 29%, or greater than 30%. In some embodiments, at least one fraction (such as DP1, DP2, and / or DP3) of the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides at a relative abundance of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30%.In some embodiments, at least one fraction (such as DP1, DP2, and / or DP3) of the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides at a relative abundance of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, at least one fraction of the oligosaccharide preparation (e.g., DP1, DP2, and / or DP3) has a relative abundance of between about 0.1% and about 90%, between about 0.5% and about 90%, between about 0.5% and about 80%, between about 0.5% and about 70%, between about 0.5% and about 60%, between about 0.5% and about 50%, between about 0.5% and about 40%, between about 0.5% and about 30%, between about 0.5% and about 20%, between about 0.5% and about 10%. , about 0.5% to about 9%, about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, or about 5% to about 10% anhydro-subunit-containing oligosaccharides. In some embodiments, the relative abundance is measured by mass spectrometry, LC-MS / MS, or LC-MS / MS. In some embodiments, the DP1 and DP2 fractions each independently comprise about 0.5% to about 15% anhydro-subunit-containing oligosaccharides in relative abundance as measured by mass spectrometry, LC-MS / MS, or GC-MS.

[0065]

[0094] In some embodiments, each fraction of the oligosaccharide preparations described herein comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, each fraction of the oligosaccharide preparations described herein comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of anhydrosubunit-containing oligosaccharides by relative abundance. In other embodiments, each fraction of the oligosaccharide preparations described herein comprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydrosubunit-containing oligosaccharides by relative abundance. In other embodiments, each fraction of the oligosaccharide preparations described herein comprises greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% anhydrosubunit-containing oligosaccharides by relative abundance. In some embodiments, each fraction of the oligosaccharide preparations described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30% anhydro-subunit-containing oligosaccharides by relative abundance. In some embodiments, each fraction of the oligosaccharide preparations described herein comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% anhydrosubunit-containing oligosaccharides in relative abundance.In some embodiments, each fraction of the oligosaccharide preparations described herein has a relative abundance of between about 0.1% and about 90%, between about 0.1% and about 15%, between about 0.5% and about 90%, between about 0.5% and about 80%, between about 0.5% and about 70%, between about 0.5% and about 60%, between about 0.5% and about 50%, between about 0.5% and about 40%, between about 0.5% and about 30%, between about 0.5% and about 20%, between about 0.5% and about 10%, between about 0.5% and about 9%, about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 2% to about 9%, about 1% to about 10%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, or about 5% to about 10% anhydrosubunit-containing oligosaccharides.

[0066]

[0095] In some embodiments, the oligosaccharide preparations described herein comprise anhydro-subunit-containing oligosaccharides at a relative abundance of less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the oligosaccharide preparations comprise anhydro-subunit-containing oligosaccharides at a relative abundance of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In other embodiments, the oligosaccharide preparation comprises an anhydro subunit-containing oligosaccharides at a relative abundance of greater than 0.5%, 0.8%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%. In other embodiments, the oligosaccharide preparation comprises an anhydro subunit-containing oligosaccharides at a relative abundance of greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%. In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides at a relative abundance of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, or about 30%. In some embodiments, the oligosaccharide preparation comprises an anhydrosubunit-containing oligosaccharides at a relative abundance of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%.In some embodiments, the oligosaccharide preparation comprises, in relative abundance, about 0.1% to about 90%, about 0.1% to about 15%, about 0.5% to about 90%, about 0.5% to about 80%, about 0.5% to about 70%, about 0.5% to about 60%, about 0.5% to about 50%, about 0.5% to about 40%, about 0.5% to about 30%, about 0.5% to about 20%, about 0.5% to about 10%, about 0.5% to about 9% , about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, or about 5% to about 10% anhydrosubunit-containing oligosaccharides.

[0067]

[0096] In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises anhydro-subunit-containing oligosaccharides at a relative abundance of less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises anhydro-subunit-containing oligosaccharides at a relative abundance of greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15%. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises an anhydro-subunit-containing oligosaccharides at a relative abundance of about 0.1% to about 15%, about 0.1% to about 20%, about 0.5% to about 20%, 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the DP1 fraction of the oligosaccharide preparations described herein comprises an anhydro-subunit-containing oligosaccharides at a relative abundance of about 5% to about 10%. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by mass spectrometry. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by LC-MS / MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by GC-MS.

[0068]

[0097] In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides at a relative abundance of less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises anhydro-subunit-containing oligosaccharides at a relative abundance of greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15%. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises an anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.1% to about 15%, about 0.1% to about 20%, about 0.5% to about 20%, 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the DP2 fraction of the oligosaccharide preparations described herein comprises an anhydro-subunit-containing oligosaccharides in a relative abundance of about 5% to about 10%. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by mass spectrometry, such as MALDI-MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by LC-MS / MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by GC-MS.

[0069]

[0098] In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides at a relative abundance of less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises anhydro-subunit-containing oligosaccharides at a relative abundance of greater than 0.1%, greater than 0.5%, greater than 0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%, greater than 10%, greater than 11%, greater than 12%, greater than 13%, greater than 14%, or greater than 15%. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises anhydrosubunit-containing oligosaccharides at a relative abundance of about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises an anhydro-subunit-containing oligosaccharides in a relative abundance of about 0.1% to about 15%, about 0.1% to about 20%, about 0.5% to about 20%, 0.5% to about 10%, about 0.5% to about 15%, about 1% to about 20%, about 1% to about 15%, about 1% to about 10%, about 2% to about 14%, about 3% to about 13%, about 4% to about 12%, about 5% to about 11%, about 5% to about 10%, about 6% to about 9%, or about 7% to about 8%, or any range therebetween. In some embodiments, the DP3 fraction of the oligosaccharide preparations described herein comprises an anhydro-subunit-containing oligosaccharides in a relative abundance of about 5% to about 10%. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by mass spectrometry, such as MALDI-MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by LC-MS / MS. In some embodiments, the relative abundance of anhydro-subunit-containing oligosaccharides is determined by GC-MS.

[0070]

[0099] In some embodiments, anhydrosubunit-containing oligosaccharides contain one or more anhydrosubunits. For example, DP1 anhydrosubunit-containing oligosaccharides contain one anhydrosubunit. In some embodiments, DPn anhydrosubunit-containing oligosaccharides can contain 1 to n anhydrosubunits. For example, in some embodiments, DP2 anhydrosubunit-containing oligosaccharides contain 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 anhydrosubunit-containing oligosaccharides have only one anhydrosubunit. In some embodiments, greater than 99%, 95%, 90%, 85%, or 80% of the anhydro-subunit-containing oligosaccharides have only one anhydro-subunit.

[0071]

[0100] In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or each fraction of the oligosaccharide preparation comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anhydro subunits, each linked via a glycosidic bond, where the glycosidic bond linking each anhydro subunit is independently selected. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation or each fraction of the oligosaccharide preparation comprise 1, 2, or 3 anhydro subunits, each linked via a glycosidic bond, where the glycosidic bond linking each anhydro subunit is independently selected. In some embodiments, more than 50%, 60%, 70%, 80%, 90%, or 99% of the oligosaccharides in the oligosaccharide preparation or each fraction comprise 1, 2, or 3 anhydro subunits, each linked via a glycosidic bond, where the glycosidic bond linking each anhydro subunit is independently selected. In some embodiments, one or more of the oligosaccharides in the oligosaccharide preparation or each fraction contain one anhydro subunit linked via a glycosidic bond, hi 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 each fraction contain one anhydro subunit linked via a glycosidic bond.

[0072] [Anhydro subunit species]

[0101] In some embodiments, the oligosaccharide preparation comprises different species of anhydro subunits. In some embodiments, exemplary anhydro subunit-containing oligosaccharides are shown in Figures 5, 18, and 19. In some embodiments, the oligosaccharide preparation comprises one or more anhydro subunits that are the thermal dehydration products of monosaccharides, i.e., anhydro-monosaccharide subunits. In some embodiments, the oligosaccharide preparation comprises one or more anhydro subunits that are the reversible thermal dehydration products of monosaccharides.

[0073]

[0102] An anhydro-monosaccharide (or anhydro-monosaccharide subunit) should be understood to refer to the thermal dehydration product of one or more species of monosaccharide. For example, in some embodiments, an anhydro-glucose refers to 1,6-anhydro-β-D-glucopyranose (levoglucosan) or 1,6-anhydro-β-D-glucofuranose. In some embodiments, a plurality of anhydro-glucoses refers to a plurality of 1,6-anhydro-β-D-glucopyranoses (levoglucosan), a plurality of 1,6-anhydro-β-D-glucofuranoses, a plurality of other thermal dehydration products of glucose, or any combination thereof. Similarly, in some embodiments, a plurality of anhydro-galactoses refers to a plurality of any thermal dehydration products of galactose, or any combination thereof.

[0074]

[0103] In some embodiments, the oligosaccharide preparations described herein comprise one or more anhydroglucose, anhydrogalactose, anhydromannose, anhydroallose, anhydroaltrose, anhydrogulose, anhydroindose, anhydrotalose, anhydrofructose, anhydroribose, anhydroarabinose, anhydrorhamnose, anhydrolyxose, anhydroxylose, or any combination of these subunits. In some embodiments, the oligosaccharide preparations comprise one or more anhydroglucose, anhydrogalactose, anhydromannose, or anhydrofructose subunits. In some embodiments, the oligosaccharide preparations described herein include 1,6-anhydro-3-O-β-D-glucopyranosyl-β-D-glucopyranose, 1,6-anhydro-3-O-α-D-glucopyranosyl-β-D-glucopyranose, 1,6-anhydro-2-O-β-D-glucopyranosyl-β-D-glucopyranose, 1,6-anhydro-2-O-α-D-glucopyranosyl-β-D-glucopyranose, 1,6-anhydro- β-D-cellobiose (cellobiosan), 1,6-anhydro-β-D-cellotriose (cellotriosan), 1,6-anhydro-β-D-cellotetraose (cellotetraosan), 1,6-anhydro-β-D-cellopentaose (cellopentaosan), and 1,6-anhydro-β-D-maltose (maltosan).

[0075]

[0104] In some embodiments, the oligosaccharide preparation comprises one or more 1,6-anhydro-β-D-glucofuranose subunits. In some embodiments, the oligosaccharide preparation comprises one or more 1,6-anhydro-β-D-glucopyranose (levoglucosan) subunits. For example, Figure 18 shows two DP1 anhydrosubunit-containing oligosaccharides (levoglucosan and 1,6-anhydro-β-D-glucofuranose) and one DP2 anhydrosubunit-containing oligosaccharide (anhydro-cellobiose).

[0076]

[0105] The presence and level of anhydro subunit species can vary based on the feed sugar used to produce the oligosaccharides, for example, in some embodiments, gluco-oligosaccharides contain anhydro-glucose subunits, galacto-oligosaccharides contain anhydro-galactose subunits, and gluco-galacto-oligosaccharides contain anhydro-glucose and anhydro-galactose subunits.

[0077]

[0106] In some embodiments, the oligosaccharide preparation comprises both 1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranose anhydrous subunits. In some embodiments, at least 0.1%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the anhydrous subunits are selected from the group consisting of 1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranose. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the anhydrous subunits are 1,6-anhydro-β-D-glucofuranose. In some embodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the anhydro subunits are 1,6-anhydro-β-D-glucopyranose.

[0078]

[0107] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose in the preparation is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose in the preparation is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose in the preparation is about 2:1.

[0079]

[0108] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose in each fraction is 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 in each fraction. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in each fraction.

[0080]

[0109] In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose in at least one fraction is about 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 in at least one fraction. In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about 2:1 in at least one fraction.

[0081]

[0110] In some embodiments, the oligosaccharide preparations described herein comprise anhydro-subunit-containing DP2 oligosaccharides. In some embodiments, the oligosaccharide preparations comprise anhydro-lactose, anhydro-sucrose, anhydro-cellobiose, or a combination thereof. In some embodiments, the oligosaccharide preparations comprise about 2-20, 2-15, 5-20, 5-15, or 5-10 DP2 anhydro-subunit-containing oligosaccharides. In some embodiments, the oligosaccharide preparations described herein are free of cellobiosan or do not contain detectable levels of cellobiosan.

[0082]

[0111] In some embodiments, the oligosaccharide preparations described herein comprise one or more anhydrous subunits that are sugar caramelization products. In some embodiments, the oligosaccharide preparations comprising one or more anhydrous subunits are sugar caramelization products selected from the group consisting of methanol, ethanol, furan, methylglyoxal, 2-methylfuran, vinyl acetate, glycolaldehyde, acetic acid, acetol, furfural, 2-furanmethanol, 3-furanmethanol, 2-hydroxycyclopent-2-en-1-one, 5-methylfurfural, 2(5H)-furanone, 2-methylcyclopentenolone, levoglucosenone, cyclic hydroxylactones, 1,4,3,6-dianhydro-α-D-glucopyranose, dianhydroglucopyranose, and 5-hydroxymethylfurfural (5-hmf).

[0083]

[0112] In some embodiments, in at least one of the oligosaccharide preparations or DP fractions, anhydro subunits that are caramelization products are less abundant than anhydro subunits that are reversible thermal dehydration products of monosaccharides. In some embodiments, in at least one of the oligosaccharide preparations or fractions, anhydro subunits that are caramelization products are more abundant than anhydro subunits that are reversible thermal dehydration products of monosaccharides. In some embodiments, in at least one of the oligosaccharide preparations or fractions, anhydro subunits that are caramelization products and anhydro subunits that are reversible thermal dehydration products of monosaccharides have similar abundance ratios.

[0084]

[0113] In some embodiments, the anhydro subunits in the oligosaccharide preparations described herein are from about 0.01% to about 50%, from about 0.01% to about 40%, from about 0.01% to about 30%, from about 0.01% to about 20%, from about 0.01% to about 10%, from about 0.01% to about 5%, from about 0.01% to about 4%, from about 0.01% to about 3%, from about 0.01% to about 2%, or from about 0.01% to about 10%. Between about 0.01% and about 1%, between about 0.01% and about 0.5%, between about 0.1% and about 50%, between about 0.1% and about 40%, between about 0.1% and about 30%, between about 0.1% and about 20%, between about 0.1% and about 10%, between about 0.1% and about 5%, between about 0.1% and about 4%, between about 0.1% and about 3%, between about 0.1% and about 2%, between about 0.1% and about 1%, or between about 0.1% and about 0.5% of the anhydro subunits in the oligosaccharide preparation are caramelization products. In some embodiments, between about 0.1% and about 5%, between about 0.1% and about 2%, or between about 0.1% and about 1% of the anhydro subunits in the oligosaccharide preparation are caramelization products. In some embodiments, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the anhydro subunits in the oligosaccharide preparation are caramelized products.

[0085]

[0114] In some embodiments, about 0.01% to about 50%, about 0.01% to about 40%, about 0.01% to about 30%, about 0.01% to about 20%, about 0.01% to about 10%, about 0.01% to about 5%, about 0.01% to about 4%, about 0.01% to about 3% of the anhydro subunits in at least one fraction (e.g., DP1, DP2, and / or DP3 fraction) of the preparations described herein. , about 0.01% to about 2%, about 0.01% to about 1%, about 0.01% to about 0.5%, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 20%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 4%, about 0.1% to about 3%, about 0.1% to about 2%, about 0.1% to about 1%, or about 0.1% to about 0.5% of the anhydro subunits in at least one fraction (e.g., DP1, DP2, and / or DP3) of the preparation is a caramelization product. In some embodiments, about 0.1% to about 5%, about 0.1% to about 2%, or about 0.1% to about 1% of the anhydro subunits in at least one fraction (e.g., DP1, DP2, and / or DP3) of the preparation is a caramelization product. In some embodiments, less than 50%, 40%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the anhydro subunits in at least one fraction of the preparation are caramelization products. In some embodiments, less than 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the anhydro subunits in the DP1, DP2, and / or DP3 fractions of the oligosaccharide preparations described herein are caramelization products.

[0086]

[0115] In some embodiments, the anhydro subunits in each fraction of the oligosaccharide preparations described herein are between about 0.01% and about 50%, between about 0.01% and about 40%, between about 0.01% and about 30%, between about 0.01% and about 20%, between about 0.01% and about 10%, between about 0.01% and about 5%, between about 0.01% and about 4%, between about 0.01% and about 3%, between about 0.01% and about 2%, between about 0.01% and about 3%, between about 0.01% and about 4%, between about 0.01% and about 5%, between about 0.01% and about 6%, between about 0.01% and about 6%, between about 0.01% and about 7%, between about 0.01% and about 8%, between about 0.01% and about 9%, between about 0.01 Between 0.01% and about 1%, between 0.01% and about 0.5%, between 0.1% and about 50%, between 0.1% and about 40%, between 0.1% and about 30%, between 0.1% and about 20%, between 0.1% and about 10%, between 0.1% and about 5%, between 0.1% and about 4%, between 0.1% and about 3%, between 0.1% and about 2%, between 0.1% and about 1%, or between 0.1% and about 0.5% of the anhydro subunits in each fraction of the preparation are caramelization products. In some embodiments, between 0.1% and about 5%, between 0.1% and about 2%, or between 0.1% and about 1% of the anhydro subunits in each fraction of the preparation 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 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the anhydro subunits in each fraction of the preparation are caramelized products.

[0087]

[0116] In some embodiments, each of the oligosaccharides in the oligosaccharide preparations described herein independently and optionally comprises an anhydrosubunit. In some embodiments, two or more independent oligosaccharides comprise the same or different anhydrosubunits. In some embodiments, two or more independent oligosaccharides comprise different anhydrosubunits. For example, in some embodiments, the oligosaccharide preparation comprises an oligosaccharide containing a DP1 anhydrosubunit comprising a 1,6-anhydro-β-D-glucopyranose subunit and an oligosaccharide containing a DP2 anhydrosubunit comprising a 1,6-anhydro-β-D-glucofuranose subunit. In some embodiments, one or more oligosaccharides in the oligosaccharide preparation comprise two or more of the same or different anhydrosubunits.

[0088]

[0117] In some embodiments, in any fraction of the oligosaccharide preparation having a degree of polymerization of 2 or greater (i.e., the DP2-DPn fractions), an anhydro subunit may be linked to one or more regular or anhydro subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydro subunit is linked to one, two, or three other regular or anhydro subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydro subunit is linked to one or two regular subunits. In some embodiments, in the DP2-DPn fractions, at least one anhydro subunit is linked to 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 linked to one regular subunit. In some embodiments, in each of the DP2 through DPn fractions, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% of the anhydro subunits are bound to one regular subunit.

[0089]

[0118] In some embodiments, in any fraction of an oligosaccharide preparation having a degree of polymerization of 2 or greater (i.e., DP2-DPn fractions), anhydro subunits can be located at the chain ends of the oligosaccharides. In some embodiments, in any fraction of an oligosaccharide preparation having a degree of polymerization of 3 or greater (i.e., DP3-DPn fractions), anhydro subunits can be located at positions other than the chain ends of the oligosaccharides. In some embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% of the anhydro subunits in the DP2-DPn fractions are located at the chain ends of the oligosaccharides. In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the anhydro subunit-containing oligosaccharides comprise chain-terminal anhydro subunits. In some embodiments, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anhydro subunit-containing oligosaccharides comprise chain-terminating anhydro subunits.

[0090]

[0119] In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the anhydro subunit-containing oligosaccharides comprise chain-terminal anhydro subunits, hi some embodiments, greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anhydro subunit-containing oligosaccharides comprise chain-terminal anhydro subunits.

[0091] [Glycosidic bond]

[0120] In some embodiments, the oligosaccharide preparations described herein used in the methods described herein comprise a variety of glycosidic linkages. The type and distribution of glycosidic linkages may depend on the source and production method of the oligosaccharide preparation. In some embodiments, the type and distribution of glycosidic linkages may be determined and / or detected by any suitable method known in the art, such as NMR. For example, in some embodiments, the glycosidic linkages are 1 H NMR, 13In some embodiments, the glycosidic bonds are determined and / or detected, at least in part, by proton NMR. In some embodiments, the glycosidic bonds are determined and / or detected, at least in part, by C NMR, 2D NMR such as 2D JRES, HSQC, HMBC, DOSY, COSY, ECOSY, TOCSY, NOESY, or ROESY, or any combination thereof. In some embodiments, the glycosidic bonds are determined and / or detected, at least in part, by proton NMR. In some embodiments, the glycosidic bonds are determined and / or detected, at least in part, by 13 In some embodiments, the glycosidic bond is at least partially determined and / or detected by 2D C NMR. 1 H, 13 Determined and / or detected by C-HSQC NMR.

[0092]

[0121] In some embodiments, the oligosaccharide preparations described herein comprise one or more α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,4) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, β-(1,6) glycosidic linkages, α-(1,1)-α glycosidic linkages, α-(1,1)-β glycosidic linkages, β-(1,1)-β glycosidic linkages, or combinations thereof.

[0093]

[0122] In some embodiments, the oligosaccharide preparation has from about 0 to about 60 mol%, about 5% to about 55 mol%, about 5% to about 50 mol%, about 5% to about 45 mol%, about 5% to about 40 mol%, about 5% to about 35 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 10% to about 60 mol%, about 10% to about 55 mol%, about 10% to about 50 mol%, about 10% to about 45 mol%, about 10% to about 40 mol%, about 10% to about 35 mol%, about 15% to about 60 mol%, or about 15% to about The glycoside bond distribution is about 55 mol%, about 15% to about 50 mol%, about 15% to about 45 mol%, about 15% to about 40 mol%, about 15% to about 35 mol%, about 20% to about 60 mol%, about 20% to about 55 mol%, about 20% to about 50 mol%, about 20% to about 45 mol%, about 20% to about 40 mol%, about 20% to about 35 mol%, about 25% to about 60 mol%, about 25% to about 55 mol%, about 25% to about 50 mol%, about 25% to about 45 mol%, about 25% to about 40 mol%, or about 25% to about 35 mol%.

[0094]

[0123] In some embodiments, the oligosaccharide preparation has a glycosidic bond distribution of about 0 to about 50 mol%, about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 5% to about 40 mol%, about 5% to about 35 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 10% to about 40 mol%, about 10% to about 35 mol%, about 10% to about 20 mol%, about 15% to about 40 mol%, about 15% to about 35 mol%, about 15% to about 30 mol%, about 15% to about 25 mol%, or about 15% to about 20 mol% α-(1,3) glycosidic linkages.

[0095]

[0124] In some embodiments, the oligosaccharide preparation has a glycosidic bond distribution of about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 2% to about 30 mol%, about 2% to about 25 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, about 3% to about 30 mol%, about 3% to about 25 mol%, about 3% to about 20 mol%, about 3% to about 15 mol%, about 3% to about 10 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 5% to about 15 mol%, or about 5% to about 10 mol%.

[0096]

[0125] In some embodiments, the oligosaccharide preparation has a glycosidic linkage type distribution of about 0 to about 40 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, or about 0 to about 5 mol% of α-(1,4) glycosidic linkages. In some embodiments, the oligosaccharide preparation has 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% of α-(1,4) glycosidic linkages.

[0097]

[0126] In some embodiments, the oligosaccharide preparation comprises about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 2% to about 30 mol%, about 2% to about 25 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, about 5% The glycosidic bond type distribution of β-(1,6) glycosidic bonds is about 8% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 5% to about 15 mol%, about 5% to about 10 mol%, about 8% to about 30 mol%, about 8% to about 25 mol%, about 8% to about 20 mol%, about 8% to about 15 mol%, about 8% to about 10 mol%, or about 10% to about 15 mol%.

[0098]

[0127] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution of β-(1,4) glycosidic linkages of about 0 to about 40 mol%, about 0 to about 35 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 2% to about 30 mol%, about 2% to about 25 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, about 3% to about 30 mol%, about 3% to about 25 mol%, about 3% to about 20 mol%, about 3% to about 15 mol%, about 3% to about 10 mol%, about 5% to about 30 mol%, about 5% to about 25 mol%, about 5% to about 20 mol%, about 5% to about 15 mol%, or about 5% to about 10 mol%.

[0099]

[0128] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution of β-(1,2) glycosidic linkages of about 0 to about 40 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 0 to about 5 mol%, about 1% to about 20 mol%, about 1% to about 15 mol%, about 1% to about 10 mol%, about 1% to about 5 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, about 2% to about 10 mol%, or about 2% to about 5 mol%. In some embodiments, the oligosaccharide preparation has a glycosidic linkage 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 linkages.

[0100]

[0129] In some embodiments, the oligosaccharide preparation has a glycosidic bond type distribution of β-(1,3) glycosidic linkages of about 0 to about 40 mol%, about 0 to about 30 mol%, about 0 to about 25 mol%, about 0 to about 20 mol%, about 0 to about 15 mol%, about 0 to about 10 mol%, about 0 to about 5 mol%, about 1% to about 20 mol%, about 1% to about 15 mol%, about 1% to about 10 mol%, about 1% to about 5 mol%, about 2% to about 20 mol%, about 2% to about 15 mol%, or about 2% to about 10 mol%, or about 2% to about 5 mol%. In some embodiments, the oligosaccharide preparation has 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 linkages.

[0101]

[0130] In some embodiments, the oligosaccharide preparation has a glycosidic linkage distribution that differs from that of a non-synthetic oligosaccharide preparation. For example, in some embodiments, the oligosaccharide preparation has a glycosidic linkage distribution that differs from that of a basal nutritional composition. In some embodiments, the basal nutritional composition includes a natural carbohydrate source, such as starch and plant fiber. Some natural carbohydrate sources have a high proportion of α-(1,4), α-(1,6), and / or β-(1,6) glycosidic linkages. Thus, in some embodiments, the oligosaccharide preparation has a lower proportion of α-(1,4) glycosidic linkages than the basal nutritional composition. In some embodiments, the oligosaccharide preparation has a lower proportion of α-(1,6) glycosidic linkages than the basal nutritional composition. In other embodiments, the oligosaccharide preparation has a higher proportion of α-(1,6) glycosidic linkages than the basal nutritional composition. In some embodiments, the oligosaccharide preparation has a lower percentage of β-(1,6) glycosidic linkages than the basal nutritional composition, hi some embodiments, the oligosaccharide preparation comprises glycosidic linkages that are not readily digested or hydrolyzed by enzymes.

[0102]

[0131] Specifically, in some embodiments, the glycosidic linkage distribution of the oligosaccharide preparations described herein has at least 50 mol%, at least 40 mol%, at least 30 mol%, at least 20 mol%, at least 15 mol%, at least 10 mol%, at least 5 mol%, at least 2 mol%, or at least 1 mol% lower than that of the basal nutritional composition. In some embodiments, the glycosidic linkage distribution of the oligosaccharide preparation is at least 50 mol%, at least 40 mol%, at least 30 mol%, at least 20 mol%, at least 15 mol%, at least 10 mol%, at least 5 mol%, at least 2 mol%, or at least 1 mol% higher than that of the basal nutritional composition.

[0103]

[0132] It should be understood by those skilled in the art that a particular type of glycosidic bond may not be applicable to an oligosaccharide containing a particular type of monosaccharide. For example, in some embodiments, the oligosaccharide preparation comprises an α-(1,2) glycosidic bond and an α-(1,6) glycosidic bond. In other embodiments, the oligosaccharide preparation comprises an α-(1,2) glycosidic bond and a β-(1,3) glycosidic bond. In some embodiments, the oligosaccharide preparation comprises an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, and a β-(1,6) glycosidic bond. In some embodiments, the oligosaccharide preparation comprises α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,4) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, and β-(1,6) glycosidic linkages.

[0104] [Molecular weight]

[0133] The molecular weight and molecular weight distribution of the oligosaccharide preparations described herein can be determined by any suitable analytical means and instrumentation, such as end-group methods, osmotic pressure (osmometry), ultracentrifugation, viscometry, light scattering, SEC, SEC-MALLS, FFF, A4F, HPLC, and mass spectrometry. In some embodiments, the molecular weight and molecular weight distribution are determined by mass spectrometry, such as MALDI-MS, LC-MS, or GC-MS. In some embodiments, the molecular weight and molecular weight distribution are determined by size exclusion chromatography (SEC), such as gel permeation chromatography (GPC). In other embodiments, the molecular weight and molecular weight distribution are determined by HPLC. In some embodiments, the molecular weight and molecular weight distribution are determined by MALDI-MS.

[0105]

[0134] In some embodiments, the oligosaccharide preparations described herein comprise from about 100 to about 10,000 g / mol, from about 200 to about 8,000 g / mol, from about 300 to about 5,000 g / mol, from about 500 to about 5,000 g / mol, from about 700 to about 5,000 g / mol, from about 900 to about 5,000 g / mol, from about 1100 to about 5,000 g / mol, from about 1300 to about 5,000 g / mol, from about 1500 to about 5,000 g / mol, from about 1700 to about 5,000 g / mol, from about 300 to about 4,500 g / mol, from about 500 to about 4,500 g / mol, from about 700 to about 4,500 g / mol, from about 900 to about 4,500 g / mol, / mol, about 1100 to about 4500 g / mol, about 1300 to about 4500 g / mol, about 1500 to about 4500 g / mol, about 1700 to about 4500 g / mol, about 1900 to about 4500 g / mol, about 300 to about 4000 g / mol, about 500 to about 4000 g / mol, about 700 to about 4000 g / mol, about 900 to about 4000 g / mol, about 1100 to about 4000 g / mol, about 1300 to about 4000 g / mol, about 1500 to about 4000 g / mol, about 1700 to about 4000 g / mol, about 1900 to about 4000 g / mol, about 300 to about 3000 g / mol, about 500 to about 3000 g / mol, about 700 to about 3000 g / mol, about 900 to about 3000 g / mol, about 1100 to about 3000 g / mol, about 1300 to about 3000 g / mol, about 1500 to about 3000 g / mol, about 1700 to about 3000 g / mol, about 1900 to about 3000 g / mol, about 2100 to about 3000 g / mol, about 300 to about 2500 g / mol, about 500 to about 2500 g / mol, about 700 to about 2500 g / mol, about 900 to about 2500 g / mol, about 1100 to about 2500 g / mol, about 1300 to about 2500 g / mol, about 1500 to about 2 The weight average molecular weight is about 500 g / mol, about 1700 to about 2500 g / mol, about 1900 to about 2500 g / mol, about 2100 to about 2500 g / mol, about 300 to about 1500 g / mol, about 500 to about 1500 g / mol, about 700 to about 1500 g / mol, about 900 to about 1500 g / mol, about 1100 to about 1500 g / mol, about 1300 to about 1500 g / mol, about 2000 to about 2800 g / mol, about 2100 to about 2700 g / mol, about 2200 to about 2600 g / mol, about 2300 to about 2500 g / mol, or about 2320 to about 2420 g / mol.In some embodiments, the weight-average molecular weight of the oligosaccharide preparation is about 2000 to about 2800 g / mol, about 2100 to about 2700 g / mol, about 2200 to about 2600 g / mol, about 2300 to about 2500 g / mol, or about 2320 to about 2420 g / mol. In some embodiments, the oligosaccharide preparation has a weight-average molecular weight ranging from at least 500 g / mol, 750 g / mol, 1000 g / mol, or 1500 g / mol to a maximum of 1750 g / mol, 2000 g / mol, 2250 g / mol, 2500 g / mol, or 3000 g / mol. In some embodiments, the weight-average molecular weight of the oligosaccharide preparations described herein is determined by HPLC according to Example 9.

[0106]

[0135] In some embodiments, the oligosaccharide preparations described herein comprise from about 100 to about 10,000 g / mol, about 200 to about 8,000 g / mol, about 300 to about 5,000 g / mol, about 500 to about 5,000 g / mol, about 700 to about 5,000 g / mol, about 900 to about 5,000 g / mol, about 1,100 to about 5,000 g / mol, about 1,300 to about 5,000 g / mol, about 1,500 to about 5,000 g / mol, about 1,700 to about 5,000 g / mol, about 300 to about 4,500 g / mol, about 500 to about 4,500 g / mol, about 700 to about 4,500 g / mol, about 900 to about 4,500 g / mol, about 1,100 to about 4500 g / mol, about 1300 to about 4500 g / mol, about 1500 to about 4500 g / mol, about 1700 to about 4500 g / mol, about 1900 to about 4500 g / mol, about 300 to about 4000 g / mol, about 500 to about 4000 g / mol, about 700 to about 4000 g / mol, about 900 to about 4000 g / mol, about 1100 to about 4000 g / mol, about 1300 to about 4000 g / mol, about 1500 to about 4000 g / mol, about 1700 to about 4000 g / mol, about 1900 to about 4000 g / mol, about 300 to about 3000 g / mol, about 500 to about 3000 g / mol, about 700 to about 3000 g / mol, about 900 to about 3000 g / mol, about 1100 to about 3000 g / mol, about 1300 to about 3000 g / mol, about 1500 to about 3000 g / mol, about 1700 to about 3000 g / mol, about 1900 to about 3000 g / mol, about 2100 to about 3000 g / mol, about 300 to about 2500 g / mol, about 500 to about 2500 g / mol, about 700 to about 2500 g / mol, about 900 to about 2500 g / mol, about 1100 to about 2500 g / mol, about 1300 to about 2500 g / mol, about 1500 to about 2500 g / mol, about 1700 to about 2500 g / mol, about 19 00 to about 2500 g / mol, about 2100 to about 2500 g / mol, about 300 to about 2000 g / mol, about 500 to about 300 to 2000 g / mol, about 700 to about 2000 g / mol, about 900 to about 2000 g / mol, about 1100 to about 2000 g / mol, about 300 to about 1500 g / mol, about 500 to about 1500 g / mol, about 700 to about 1500 g / mol, about 900 to about 1500 g / mol, about 1100 to about 1500 g / mol, about 1300 to about 1500 g / mol, about 1000 to about 2000 g / mol, about 1100 to about 1900 g / mol, about 1200 to about 1800 g / mol,The oligosaccharide preparation has a number average molecular weight of about 1300 to about 1700 g / mol, about 1400 to about 1600 g / mol, or about 1450 to about 1550 g / mol. In some embodiments, the number average molecular weight of the oligosaccharide preparation is about 1000 to about 2000 g / mol, about 1100 to about 1900 g / mol, about 1200 to about 1800 g / mol, about 1300 to about 1700 g / mol, 1400 to 1600 g / mol, or 1450 to 1550 g / mol. In some embodiments, the oligosaccharide preparation has a number average molecular weight ranging from at least 500 g / mol, 750 g / mol, 1000 g / mol, or 1500 g / mol to a maximum of 1750 g / mol, 2000 g / mol, 2250 g / mol, 2500 g / mol, or 3000 g / mol. In some embodiments, the number average molecular weight of the oligosaccharide preparations described herein is determined by HPLC according to Example 9.

[0107] [Types of oligosaccharides]

[0136] In some embodiments, the oligosaccharide preparations described herein comprise one or more species of monosaccharide subunits, hi some embodiments, the oligosaccharide preparations may comprise oligosaccharides having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more different species of monosaccharide subunits.

[0108]

[0137] In some embodiments, the oligosaccharide preparations described herein comprise one or more monosaccharide subunits selected from the group consisting of triose, tetrose, pentose, hexose, heptose, and any combination thereof, wherein each of the triose, tetrose, pentose, hexose, or heptose subunits is independently and optionally functionalized and / or substituted with one of its corresponding anhydrous subunits. In some embodiments, the corresponding anhydrous subunit is the product of reversible thermal dehydration of the monosaccharide subunit. In some embodiments, the corresponding anhydrous subunit is the product of caramelization of the monosaccharide subunit.

[0109]

[0138] In some embodiments, the oligosaccharide preparations described herein comprise pentose subunits, hexose subunits, or any combination thereof, wherein each of the pentose or hexose subunits is independently and optionally functionalized and / or substituted with one of its corresponding anhydro subunits. In some embodiments, the oligosaccharide preparations comprise hexose subunits, wherein each of the hexose subunits is independently and optionally functionalized and / or substituted with one of its corresponding anhydro subunits.

[0110]

[0139] As used herein, tetrose refers to a monosaccharide having four carbon atoms, such as erythrose, threose, and erythrulose. As used herein, pentose refers to a monosaccharide having five carbon atoms, such as arabinose, lyxose, ribose, and xylose. As used herein, hexose refers to a monosaccharide having six carbon atoms, such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, and tagatose. As used herein, heptose refers to a monosaccharide having seven carbon atoms, such as sedoheptulose and mannoheptulose.

[0111]

[0140] In some embodiments, the oligosaccharide preparations described herein comprise glucose subunits, wherein at least one glucose subunit is optionally replaced with an anhydro-glucose subunit. In some embodiments, the oligosaccharide preparations described herein comprise galactose subunits, wherein at least one galactose subunit is optionally replaced with an anhydro-galactose subunit. In some embodiments, the oligosaccharide preparations described herein comprise galactose and glucose subunits, wherein at least one galactose subunit or at least one glucose subunit is optionally replaced with one of its corresponding anhydro-subunits. In some embodiments, the oligosaccharide preparations described herein comprise fructose and glucose subunits, wherein at least one fructose subunit or at least one glucose subunit is optionally replaced with one of its corresponding anhydro-subunits. In some embodiments, the oligosaccharide preparations described herein comprise mannose and glucose subunits, wherein at least one mannose subunit or at least one glucose subunit is optionally substituted with one of its corresponding anhydro subunits.

[0112]

[0141] In some embodiments, the oligosaccharide preparations described herein are selected from the group consisting of gluco-galactose-oligosaccharide preparations, gluco-oligosaccharide preparations, galacto-oligosaccharide preparations, fructo-oligosaccharide preparations, manno-oligosaccharide preparations, arabino-oligosaccharide preparations, xylo-oligosaccharide preparations, gluco-fructo-oligosaccharide preparations, gluco-manno-oligosaccharide preparations, gluco-arabino-oligosaccharide preparations, gluco-xylo-oligosaccharide preparations, galacto-fructo-oligosaccharide preparations, galacto-manno-oligosaccharide preparations, galacto-arabino-oligosaccharide preparations, galacto-xylo-oligosaccharide preparations, fructo-manno-oligosaccharide preparations, fructo-arabino-oligosaccharide preparations, fructo-xylo-oligosaccharide preparations. and / or any combination thereof; wherein each of the monosaccharide subunits within the preparation is independently and optionally functionalized and / or substituted with one of its corresponding anhydro subunits.

[0113]

[0142] In certain embodiments, the oligosaccharide preparations described herein comprise greater than 99% glucose subunits by weight, hi some embodiments, the oligosaccharide preparations comprise only glucose subunits.

[0114]

[0143] In some embodiments, the oligosaccharide preparations described herein contain about 45%-55% glucose subunits and about 55%-45% galactose subunits by weight, hi some embodiments, the oligosaccharide preparations contain about 50% glucose and 50% galactose subunits by weight.

[0115]

[0144] In some embodiments, the oligosaccharide preparations described herein comprise, by weight, about 80%-95% glucose subunits and about 20%-5% mannose subunits, hi some embodiments, the oligosaccharide preparations comprise, by weight, about 85%-90% glucose subunits and about 15%-10% mannose subunits.

[0116]

[0145] In some embodiments, the oligosaccharide preparations described herein comprise, by weight, about 80%-95% glucose subunits and about 20%-5% galactose subunits, hi some embodiments, the oligosaccharide preparations comprise, by weight, about 85%-90% glucose subunits and about 15%-10% galactose subunits.

[0117]

[0146] In some embodiments, the oligosaccharide preparations described herein comprise, by weight, about 80%-95% glucose subunits, 0%-8% galactose subunits, and 5%-20% mannose subunits. In some embodiments, the oligosaccharide preparations comprise, by weight, about 80%-90% glucose subunits, 1%-5% galactose subunits, and 10%-15% mannose subunits.

[0118]

[0147] In some embodiments, the oligosaccharide preparations described herein contain about 1% to about 100%, about 50% to about 100%, about 80% to about 98%, or about 85% to about 95% glucose subunits by weight, or any range therebetween. In some embodiments, galactose subunits are present in the oligosaccharide preparations described herein in an amount of about 0% to about 90%, about 1% to about 50%, about 2% to about 20%, or about 5% to about 15% by weight, or any range therebetween. In some embodiments, mannose subunits are present in the oligosaccharide preparations described herein in an amount of about 0% to about 90%, about 1% to about 50%, about 2% to about 20%, or about 5% to about 15% by weight, or any range therebetween.

[0119] [D-type vs. L-type]

[0148] In some embodiments, at least one monosaccharide subunit in the oligosaccharide is in the L-form. In some embodiments, at least one monosaccharide subunit in the oligosaccharide is in the D-form. In some embodiments, the monosaccharide subunits in the oligosaccharide preparations described herein are in their naturally abundant form, such as D-glucose, D-xylose, and L-arabinose.

[0120]

[0149] In some embodiments, the oligosaccharide preparations described herein comprise a mixture of L- and D-type monosaccharide subunits. In some embodiments, the ratio of L- to D- or D- to L-type monosaccharide subunits is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:14, about 1:16, about 1:18, about 1:20, about 1:25, about 1:30, about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60, about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90, about 1:100, or about 1:150.

[0121] [Functionalized Oligosaccharides]

[0150] In some embodiments, one or more oligosaccharides in the oligosaccharide preparation described herein are independently functionalized.Functionalized oligosaccharides can be produced by combining one or more sugars with one or more functionalizing compounds in the presence of a catalyst.Methods for producing functionalized oligosaccharides are described in International Publication Nos. 2012 / 118767, 2014 / 031956, and 2016 / 122887 (their entire disclosures are incorporated herein by reference).

[0122]

[0151] In some embodiments, the functionalized compound contains one or more acidic groups (e.g., —COOH), hydroxyl groups, N-containing groups (e.g., —CN, —NO, and —N(R a)2 (wherein Ra is a hydrogen group, an alkyl group, an alkenyl group, an alkynyl group, a haloalkyl group, a heteroalkyl group, a cycloalkyl group, an aryl group, a heterocycloalkyl group, or a heteroaryl group), an S-containing group (e.g., thiol and sulfate), a halide (e.g., -Cl), a P-containing group (e.g., phosphate), or any combination thereof. In some embodiments, the functionalizing compound is attached to at least one monosaccharide subunit through an ether bond, an ester bond, an oxygen-sulfur bond, an amine bond, or an oxygen-phosphorus bond. In some embodiments, one or more functionalizing compounds are attached to the monosaccharide subunit through a single bond. In some embodiments, at least one functionalizing compound is attached to one or two oligosaccharides through two or more bonds.

[0123]

[0152] For each oligosaccharide in the oligosaccharide preparation, each of the described embodiments is independent and can be combined as if each combination were described separately; therefore, it should be understood that any combination of embodiments is included in the present disclosure. For example, various embodiments can be grouped into several categories, including, but not limited to, (i) the presence or absence of anhydro subunits; (ii) the number and level of anhydro subunits; (iii) the type of anhydro subunit species; (iv) the position of anhydro subunits; (v) the degree of polymerization; (vi) molecular weight; (vii) the presence or absence of any functional groups; (viii) the type of oligosaccharide; (ix) the type of glycosidic bond; and (x) L-type vs. D-type. Thus, the described oligosaccharide preparations contain multiple oligosaccharides of different species. In some embodiments, the oligosaccharide preparations described herein contain at least 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 or 10 10 In some embodiments, the preparation comprises at least 10 different oligosaccharide species. 3, 10 4 , 10 5 , 10 6 or 10 9 In some embodiments, the preparation comprises at least 10 different oligosaccharide species. 3 It contains different oligosaccharide species.

[0124] IV. Methods for Producing Oligosaccharide Preparations

[0153] In one aspect, provided herein are methods for producing an oligosaccharide preparation. In some embodiments, provided herein are methods for producing an oligosaccharide preparation suitable for use in nutritional compositions, such as animal feed compositions, or fed directly to an animal. In one aspect, provided herein are methods for producing an oligosaccharide preparation, comprising heating an aqueous composition comprising one or more feed sugars and a catalyst to a temperature and for a time sufficient to induce polymerization, wherein the catalyst is selected from the group consisting of (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate; α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexyl Phosphonic acid;Methylphosphonic acid;Phenylphosphinic acid;Phenylphosphonic acid;tert-Butylphosphonic acid;(SS)-VAPOL hydrogen phosphate;6-Quinolinesulfonic acid, 3-(1-pyridinio)-1-propanesulfonate;2-(2-Pyridinyl)ethanesulfonic acid;3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate;1,1'-Binaphthyl-2,2'-diyl hydrogen phosphate;Bis(4-methoxyphenyl)phosphinic acid ;Phenyl(3,5-xylyl)phosphinic acid;L-Cysteic acid monohydrate;Poly(styrenesulfonic acid-co-divinylbenzene);Lysine;Ethanedisulfonic acid;Ethanesulfonic acid;Isethionic acid;Fomicysteic acid;HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid));HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);2-Hydroxy-3-morpholinopropanesulfonic acid;2-(N-morpholino)ethanesulfonic acid Sulfonic acid;Methanesulfonic acid;Methaniazid;Naphthalene-1-sulfonic acid;Naphthalene-2-sulfonic acid;Perfluorobutanesulfonic acid;6-sulfoquinovose;Trifluoromethanesulfonic acid;2-aminoethanesulfonic acid;Benzoic acid;Chloroacetic acid;Trifluoroacetic acid;Caproic acid;Enanthic acid;Caprylic acid;Pelargonic acid;Lauric acid;Palmitic acid;Stearic acid;Arachidic acid;Aspartic acid;Glutamic acid;Serine;Threonine;Glutamine;Cysteine;Glycine;Proline;The oligosaccharide preparation may comprise at least n fractions of oligosaccharides, each having a distinct degree of polymerization selected from the group consisting of alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; and tryptophan, wherein the oligosaccharide preparation comprises at least n fractions of oligosaccharides, each having a distinct degree of polymerization selected from 1 (DP1 fraction) to n (DPn fraction), where n is an integer equal to or greater than 2. In some embodiments, n is an integer equal to or greater than 3. In some embodiments, n is an integer in the range of 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, polymerization of the feed sugars is achieved by step-growth polymerization. In some embodiments, polymerization of the feed sugars is achieved by polycondensation.

[0125] [Sugar supplied]

[0154] In some embodiments, the methods of producing an oligosaccharide preparation described herein include heating one or more types of feed sugars, in some embodiments, the one or more types of feed sugars include monosaccharides, disaccharides, trisaccharides, tetrasaccharides, or any mixture thereof.

[0126]

[0155] 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.

[0127]

[0156] 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 certain embodiments, the one or more feed sugars comprise glucose, mannose, galactose, xylose, maltodextrin, arabinose, or galactose, or any combination thereof. In certain 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.

[0128]

[0157] In some embodiments, the type of feed sugar can affect the resulting oligosaccharide preparation produced. For example, in some variations where one or more feed sugars are all glucose, the resulting oligosaccharide preparation comprises a gluco-oligosaccharide preparation. In other embodiments, where one or more feed sugars are all mannose, the resulting oligosaccharide preparation comprises a manno-oligosaccharide preparation. In some embodiments, where one or more feed sugars comprise glucose and galactose, the resulting oligosaccharide preparation comprises a gluco-galacto-oligosaccharide preparation. In yet other embodiments, where one or more feed sugars comprise xylose, glucose, and galactose, the resulting oligosaccharide preparation comprises a gluco-galacto-xylo-oligosaccharide preparation.

[0129]

[0158] In some embodiments, each of the one or more feed sugars may be independently 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 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 sugar dihydrate, such as trehalose dihydrate. In some embodiments, the one or more feed sugars comprise at least one sugar in its dehydrate form and at least one sugar in its hydrate form.

[0130]

[0159] In some embodiments, one or more feed sugars can be provided as a sugar solution in which the sugars are combined with water and fed to the reactor. In some embodiments, the sugars can be fed 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 adding water. In some embodiments, one or more feed sugars are combined with water and then mixed.

[0131]

[0160] In some embodiments, the method comprises combining two or more feed sugars with a catalyst to produce an oligosaccharide preparation. In some embodiments, the two or more feed sugars comprise glucose, galactose, fructose, mannose, lactose, or any combination thereof. In some embodiments, the method comprises combining a mixture of sugars (e.g., monosaccharides, disaccharides, and / or trisaccharides) with a catalyst to produce the oligosaccharide preparation. In other embodiments, the method comprises combining a mixture of sugars and sugar alcohols with a catalyst to produce the oligosaccharide preparation.

[0132]

[0161] In some embodiments, one or more of the source sugars comprises a functionalized or modified sugar. The functionalized or modified sugar may comprise an amino sugar, a sugar acid, a sugar alcohol, a sugar amide, a sugar ether, or any combination thereof. In some embodiments, an amino sugar refers to a sugar molecule in which a hydroxyl group has been replaced with an amine group. Exemplary amino sugars include, but are not limited to, N-acetyl-d-glucosamine, mannosamine, neuraminic acid, muramic acid, N-acetyl-neuramine, N-acetyl-muramine, N-acetyl-galactosamine, N-acetyl-mannosamine, N-glycolylneuramine, acarviosin, D-glucosamine, and D-galactosamine.

[0133]

[0162] In some embodiments, sugar acid refers to a sugar having a carboxyl group. Exemplary sugar acids include, but are not limited to, aldonic acids (such as glyceric acid, xylonic acid, gluconic acid, and ascorbic acid), ulosonic acids (such as neuraminic acid and ketodeoxyoctulosonic acid), uronic acids (such as glucuronic acid, galacturonic acid, and iduronic acid), and aldaric acids (such as tartaric acid, mucic acid, and saccharinic acid).

[0134]

[0163] In some embodiments, sugar alcohol refers to a polyol derived from a sugar. Exemplary sugar alcohols include, but are not limited to, ethylene glycol, arabitol, glycerol, erythritol, threitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, and volemitol.

[0135]

[0164] In embodiments, sugar amide refers to a sugar molecule containing a -C(=O)-N- group. In embodiments, sugar ether refers to a sugar molecule containing an ether bond, such as a glucoside.

[0136]

[0165] In some embodiments, the functionalized or modified sugar acid comprises glucosamine, N-acetylglucosamine, glucuronic acid, galacturonic acid, glucitol, xylitol, mannitol, sorbitol, hi some embodiments, the one or more feed sugars comprise a deoxy sugar, such as fucose, rhamnose, deoxyribose, or fuculose.

[0137]

[0166] In some embodiments, the methods for producing oligosaccharide preparations described herein are carried out on a gram scale. In some embodiments, the methods for producing oligosaccharide preparations described herein are carried out on a kilogram or larger scale. Thus, in some embodiments, the methods involve heating an aqueous composition comprising one or more feed sugars in an amount greater than 0.5 kg, greater than 1 kg, greater than 2 kg, greater than 3 kg, greater than 4 kg, greater than 5 kg, greater than 6 kg, greater than 7 kg, greater than 9 kg, greater than 10 kg, greater than 100 kg, or greater than 1000 kg. In some embodiments, the methods involve heating an aqueous composition comprising one or more feed sugars in an amount equal to or less than 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10, 100, 1000, or 1500 kg. In some embodiments, the methods involve heating an aqueous composition comprising one or more feed sugars in an amount equal to or less than 1 kg.

[0138] [catalyst]

[0167] In some embodiments, the method for producing an oligosaccharide preparation described herein includes the addition of one or more 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; an amino acid; a sulfonic acid; a boronic acid; a phosphonic acid; a phosphinic acid; a sulfuric acid; a phosphoric acid; a poly(styrenesulfonic acid-co-vinylbenzyl-imidazolium sulfate-co-divinylbenzene); a poly(styrenesulfonic acid-co-divinylbenzene); (+)-camphor-10-sulfonic acid; a 2-pyridine sulfonic acid; a 3-pyridine sulfonic acid; a 8-hydroxy-5-quinolinesulfonic acid hydrate; a α-hydroxy-2-pyridine Methanesulfonic acid;(β)-Camphor-10-sulfonic acid;Butylphosphonic acid;Diphenylphosphinic acid;Hexylphosphonic acid;Methylphosphonic acid;Phenylphosphinic acid;Phenylphosphonic acid;tert-Butylphosphonic acid;(SS)-VAPOL hydrogen phosphate;6-Quinolinesulfonic acid;3-(1-Pyridinio)-1-propanesulfonate;2-(2-Pyridinyl)ethanesulfonic acid;3-(2-Pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate ;1,1'-Binaphthyl-2,2'-diyl hydrogen phosphate;Bis(4-methoxyphenyl)phosphinic acid;Phenyl(3,5-xylyl)phosphinic acid;L-Cysteic acid monohydrate;Acetic acid;Propionic acid;Butanoic acid;Glutamic acid;Lysine;Ethanedisulfonic acid;Ethanesulfonic acid;Isethionic acid;Fomycysteic acid;HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid));HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);2-Hydroxy 3-Morpholinopropanesulfonic acid;2-(N-morpholino)ethanesulfonic acid;Methanesulfonic acid;Methaniazid;Naphthalene-1-sulfonic acid;Naphthalene-2-sulfonic acid;Perfluorobutanesulfonic acid;6-Sulfoquinovose;Trifluoromethanesulfonic acid;2-Aminoethanesulfonic acid;Benzoic acid;Chloroacetic acid;Trifluoroacetic acid;Caproic acid;Enanthic acid;Caprylic acid;Pelargonic acid;Lauric acid;Palmitic acid;Stearic acid;Arachidic acid;Aspartic acid;Glutamic acid;Serinethreonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan; polymeric acids; carbon-bearing acids; or any combination thereof;

[0139]

[0168] In some embodiments, the catalyst provided herein is selected from the group consisting of (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid; 3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate; α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid; butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid; methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid; tert-butylphosphonic acid; (SS)-VAPOL hydrogen phosphate 6-Quinolinesulfonic acid, 3-(1-pyridinio)-1-propanesulfonate; 2-(2-pyridinyl)ethanesulfonic acid; 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate; 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate; Bis(4-methoxyphenyl)phosphinic acid; Phenyl(3,5-xylyl)phosphinic acid; L-Cysteic acid monohydrate; Poly(styrenesulfonic acid-co-divinylbenzene); Lysine; Ethanedisulfonic acid;Ethanesulfonic acid;Isethionic acid;Fomicysteic acid;HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid));HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);2-Hydroxy-3-morpholinopropanesulfonic acid;2-(N-morpholino)ethanesulfonic acid;Methanesulfonic acid;Methaniazid;Naphthalene-1-sulfonic acid;Naphthalene-2-sulfonic acid;Perfluorobutanesulfonic acid;6-sulfonic acid sulfoquinovose; trifluoromethanesulfonic acid; 2-aminoethanesulfonic acid; benzoic acid; chloroacetic acid; trifluoroacetic acid; caproic acid; enanthic acid; caprylic acid; pelargonic acid; lauric acid; palmitic acid; stearic acid; arachidic acid; aspartic acid; glutamic acid; serine; threonine; glutamine; cysteine; glycine; proline; alanine; valine; isoleucine; leucine; methionine; phenylalanine; tyrosine; tryptophan; or any combination thereof.

[0140]

[0169] In some embodiments, the catalyst provided herein is (+)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is 2-pyridinesulfonic acid. In some embodiments, the catalyst provided herein is 3-pyridinesulfonic acid. In some embodiments, the catalyst provided herein is 8-hydroxy-5-quinolinesulfonic acid hydrate. In some embodiments, the catalyst provided herein is α-hydroxy-2-pyridinemethanesulfonic acid. In some embodiments, the catalyst provided herein is (β)-camphor-10-sulfonic acid. In some embodiments, the catalyst provided herein is butylphosphonic acid. In some embodiments, the catalyst provided herein is diphenylphosphinic acid. In some embodiments, the catalyst provided herein is hexylphosphonic acid. In some embodiments, the catalyst provided herein is methylphosphonic acid. In some embodiments, the catalyst provided herein is phenylphosphinic acid. In some embodiments, the catalyst provided herein is phenylphosphonic acid. In some embodiments, the catalyst provided herein is tert-butylphosphonic acid. In some embodiments, the catalyst provided herein is (SS)-VAPOL hydrogen phosphate. In some embodiments, the catalyst provided herein is 6-quinolinesulfonic acid. In some embodiments, the catalyst provided herein is 3-(1-pyridinio)-1-propanesulfonate. In some embodiments, the catalyst provided herein is 2-(2-pyridinyl)ethanesulfonic acid. In some embodiments, the catalyst provided herein is 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate. In some embodiments, the catalyst provided herein is 1,1'-binaphthyl-2,2'-diyl hydrogen phosphate. In some embodiments, the catalyst provided herein is bis(4-methoxyphenyl)phosphinic acid. In some embodiments, the catalyst provided herein is phenyl(3,5-xylyl)phosphinic acid.In some embodiments, the catalyst provided herein is L-cysteic acid monohydrate. In some embodiments, the catalyst provided herein is poly(styrenesulfonic acid-co-divinylbenzene). In some embodiments, the catalyst provided herein is lysine.

[0141]

[0170] In some embodiments, the catalyst is ethanedisulfonic acid. In some embodiments, the catalyst is ethanesulfonic acid. In some embodiments, the catalyst is isethionic acid. In some embodiments, the catalyst is homocysteic acid. In some embodiments, the catalyst is HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)). In some embodiments, the catalyst is HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the catalyst is 2-hydroxy-3-morpholinopropanesulfonic acid. In some embodiments, the catalyst is 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the catalyst is methanesulfonic acid. In some embodiments, the catalyst is naphthalene-1-sulfonic acid. In some embodiments, the catalyst is methanazid. 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 trifluoromethanesulfonic acid. In some embodiments, the catalyst is 2-aminoethanesulfonic acid. In some embodiments, the catalyst is benzoic acid. In some embodiments, the catalyst is chloroacetic acid. In some embodiments, the catalyst is trifluoroacetic acid. In some embodiments, the catalyst is caproic acid. In some embodiments, the catalyst is enanthic acid. In some embodiments, the catalyst is caprylic acid. In some embodiments, the catalyst is pelargonic acid. In some embodiments, the catalyst is lauric acid. In some embodiments, the catalyst is palmitic acid. In some embodiments, the catalyst is stearic acid. In some embodiments, the catalyst is arachidic acid. In some embodiments, the catalyst is aspartic acid. In some embodiments, the catalyst is glutamic acid. In some embodiments, the catalyst is serine. In some embodiments, the catalyst is threonine. In some embodiments, the catalyst is glutamine. In some embodiments, the catalyst is cysteine. In some embodiments, the catalyst is glycine.In some embodiments, the catalyst is proline. In some embodiments, the catalyst is alanine. In some embodiments, the catalyst is valine. In some embodiments, the catalyst is isoleucine. In some embodiments, the catalyst is leucine. In some embodiments, the catalyst is methionine. In some embodiments, the catalyst is phenylalanine. In some embodiments, the catalyst is tyrosine. In some embodiments, the catalyst is tryptophan. In some embodiments, the catalysts provided herein are polymeric catalysts or carbon-supported catalysts disclosed in WO2016122887, which is incorporated herein by reference in its entirety and for its disclosure.

[0142]

[0171] In some embodiments, the catalysts provided herein are present in an amount of about 0.01% to about 5%, about 0.02% to about 4%, about 0.03% to about 3%, or about 0.05% to about 2% by dry weight of one or more feed sugars. In some embodiments, the catalysts provided herein are present in an amount of about 1% to 2% by dry weight of one or more feed sugars. In some embodiments, the catalyst provided herein is present in an amount of about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0% by dry weight of one or more feed sugars.

[0143]

[0172] In some embodiments, the catalysts provided herein are present in an amount of about 0.01% to about 5%, about 0.02% to about 4%, about 0.03% to about 3%, or about 0.05% to about 2% by dry weight of the aqueous composition. In some embodiments, the catalysts provided herein are present in an amount of about 1% to 2% by dry weight of the aqueous composition. In some embodiments, the catalysts provided herein are present in an amount of about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0% by dry weight of the aqueous composition.

[0144]

[0173] In some embodiments, the catalysts provided herein are combinations of two or more different catalysts. In some embodiments, the catalysts include recyclable catalysts, such as resin and polymer catalysts, and non-recyclable catalysts. In some embodiments, when the catalyst includes at least two different catalysts, each of the catalysts is present in the amounts provided herein. In other embodiments, when the catalyst includes at least two different catalysts, the at least two different catalysts are present in an aggregate in the amounts provided herein.

[0145]

[0174] In some embodiments, the catalyst is added to the aqueous composition in a dry form. In other embodiments, the catalyst is added to the aqueous composition in a wet form, such as an aqueous solution. In some embodiments, the catalyst is combined with one or more feed sugars before adding water. In other embodiments, the catalyst is dissolved in water before combining with the one or more feed sugars. In some embodiments, the methods provided herein include producing an aqueous composition by combining one or more feed sugars in a dehydrated form and a catalyst in a wet form (e.g., an aqueous solution).

[0146] [Addition of water]

[0175] In some embodiments, the method of producing an oligosaccharide preparation 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, e.g., in a sugar monohydrate or dihydrate. In some embodiments, all of the water in the aqueous composition is added as bound water, e.g., in a monosaccharide monohydrate, such as glucose monohydrate. In certain embodiments, all or a portion of the water in the aqueous composition is added with a catalyst, i.e., via a catalyst solution.

[0147] [Water content]

[0176] As the process for producing an oligosaccharide preparation progresses, water can be generated by reactions. For example, in some embodiments, water is generated (i) with the formation of glycosidic bonds, (ii) with the formation of anhydro subunits, or (iii) by other mechanisms or sources. Because both sugar condensation and dehydration reactions involve water, in some embodiments, the water content affects the composition of the oligosaccharide preparation.

[0148]

[0177] Furthermore, in some embodiments, the water content affects the viscosity of the aqueous composition, which in turn can affect the effectiveness of mixing of the aqueous composition. For example, in some embodiments, an overly viscous aqueous composition may result in undesirable uneven catalyst distribution in the aqueous composition. Moreover, in some embodiments, a very low water content may cause the aqueous composition to solidify, preventing effective mixing. On the other hand, in other embodiments, an extremely high water content may hinder the sugar condensation reaction and reduce the level of anhydrous subunits. Therefore, the present disclosure describes a water content suitable for producing oligosaccharide preparations.

[0149]

[0178] In some embodiments, the method of producing the oligosaccharide preparations described herein comprises forming and / or heating an aqueous composition. In some embodiments, the aqueous composition comprises from about 0% to about 80%, from about 0% to about 70%, from about 0% to about 60%, from about 0% to about 50%, from about 0% to about 40%, from about 0% to about 35%, from about 0% to about 30%, from about 0% to about 25%, from about 0% to about 20%, from about 0% to about 19%, from about 0% to about 18%, from about 0% to about 17%, from about 0% to about In some embodiments, the aqueous composition comprises about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 15%, about 0% to about 14%, about 0% to about 13%, about 0% to about 12%, about 0% to about 11%, about 0% to about 10%, about 0% to about 9%, about 0% to about 8%, about 0% to about 7%, about 0% to about 6%, about 0% to about 5%, about 0% to about 4%, about 0% to about 3%, about 0% to about 2%, or about 0% to about 1% water by total weight. In some embodiments, the aqueous composition comprises about 3% to about 16%, about 3% to about 14%, about 3% to about 12%, about 3% to about 10%, about 3% to about 8%, about 3% to about 6%, about 5% to about 16%, about 5% to about 14%, about 5% to about 12%, about 5% to about 10%, about 7% to about 16%, about 7% to about 14%, about 7% to about 12%, about 7% to about 10%, or about 8% to about 10% water by total weight. In some embodiments, the aqueous composition comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% water by total weight. In some embodiments, the aqueous composition comprises about 9% water by total weight. However, it should be understood that the amount of water in the aqueous composition can be adjusted based on the reaction conditions and the particular catalyst used. In some embodiments, the water content in the aqueous composition as disclosed above is measured at the beginning of the reaction, for example, before heating the sugar feed. In some embodiments, the water content in the aqueous composition as disclosed above is measured at the end of the polymerization or condensation reaction. In some embodiments, the water content in the aqueous composition as disclosed above is measured as the average water content at the beginning and end of the reaction.

[0150]

[0179] In certain embodiments, the methods described herein can further include monitoring the water content and / or the ratio of water to sugar or catalyst present in the aqueous composition over a period of time. In some embodiments, the method further includes removing at least a portion of the water in the aqueous composition, for example, by distillation. Water can be removed from the aqueous composition using any method known in the art, including, for example, vacuum filtration, vacuum distillation, heating, steam, hot air, and / or evaporation.

[0151]

[0180] In some embodiments, the oligosaccharide preparations described herein are hygroscopic. Thus, in some embodiments, the hygroscopicity of the feed sugars and oligosaccharides formed in the polymerization can affect the rate at which water can be removed from the aqueous composition.

[0152]

[0181] In some embodiments, the methods described herein include removing at least a portion of the water in the aqueous composition such that the water content in the aqueous composition is about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 6% to about 16%, about 6% to about 12%, about 6% to about 10%, or about 6% to about 8%, by total weight. In some embodiments, the method comprises 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 about 10%, about 2% to about 8%, or about 4% to about 8% by total weight. In some embodiments, the method comprises 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% by total weight. In some embodiments, the method comprises removing at least a portion of the water from the aqueous composition such that the water content of the aqueous composition is about 4% to about 8% by total weight. In some embodiments, the method comprises removing at least a portion of the water in the aqueous composition such that the water content of the aqueous composition at the end of the polymerization and / or condensation reaction is as disclosed above. In some embodiments, the method comprises removing at least a portion of the water in the aqueous composition such that the water content of the aqueous composition at the start of the polymerization and / or condensation reaction is as disclosed above. In some embodiments, the method comprises removing at least a portion of the water in the aqueous composition such that the average water content in the aqueous composition at the beginning and end of the polymerization and / or condensation reaction is within the ranges disclosed above. In some embodiments, the method comprises removing at least a portion of the water in the aqueous composition throughout the polymerization and / or condensation reaction such that the water content in the aqueous composition remains within the ranges disclosed above.

[0153]

[0182] In some embodiments, the methods described herein comprise adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 6% to about 16%, about 6% to about 12%, about 6% to about 10%, or about 6% to about 8%, by total weight. In some embodiments, the method comprises adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 2% to about 10%, about 2% to about 8%, or about 4% to about 8% by total weight. In some embodiments, the method comprises 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% by total weight. In some embodiments, the method comprises adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition is about 4% to about 8% by total weight. In some embodiments, the method comprises adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition at the end of the polymerization and / or condensation reaction is as disclosed above. In some embodiments, the method comprises adding at least a portion of water to the aqueous composition such that the water content of the aqueous composition at the start of the polymerization and / or condensation reaction is as disclosed above. In some embodiments, the method comprises adding at least a portion of the water to the aqueous composition such that the average water content in the aqueous composition at the beginning and end of the polymerization and / or condensation reaction is within the ranges disclosed above. In some embodiments, the method comprises adding at least a portion of the water to the aqueous composition throughout the polymerization and / or condensation reaction such that the water content in the aqueous composition remains within the ranges disclosed above.

[0154]

[0183] In some embodiments, the degree of polymerization of the oligosaccharides and / or the amount and type of anhydro subunits in the oligosaccharide preparation can be adjusted by adjusting or controlling the water content present in the aqueous composition throughout the manufacturing process. For example, in some embodiments, the degree of polymerization of the oligosaccharides and / or the amount and type of anhydro subunits is increased by decreasing the water content.

[0155]

[0184] Thus, in some embodiments, the methods described herein include in-process control (IPC) of water content, which may include monitoring the water content, maintaining the water content, increasing the water content, decreasing the water content, or any combination thereof. In some embodiments, the IPC process includes maintaining the water content while the aqueous composition is heated to a temperature described herein. In some embodiments, the method includes maintaining the water content for a time sufficient to induce polymerization. In some embodiments, the method includes maintaining the water content within the disclosed ranges by adding water or removing water from the aqueous composition, or both. In some embodiments, the method includes maintaining the water content within the disclosed ranges by distillation. In some embodiments, the method includes maintaining the water content within the disclosed ranges by vacuum distillation. In some embodiments, the method includes maintaining the water content within the disclosed ranges by distillation at atmospheric pressure.

[0156]

[0185] In some embodiments, the water content of the aqueous composition is maintained within the range of about 1% to about 20%, about 1% to about 18%, about 1% to about 16%, about 1% to about 14%, about 1% to about 12%, about 1% to about 10%, about 1% to about 8%, about 2% to about 16%, about 2% to about 14%, about 2% to about 12%, about 2% to about 10%, about 2% to about 8%, about 2% to about 6%, about 4% to about 16%, about 4% to about 14%, about 4% to about 12%, about 4% to about 10%, about 4% to about 8%, about 6% to about 16%, about 6% to about 12%, about 6% to about 10%, or about 6% to about 8% by total weight. In some embodiments, the water content of the aqueous composition is maintained within a range of about 2% to about 10%, about 2% to about 8%, or about 4% to about 8% by total weight. In some embodiments, the water content of the aqueous composition is maintained within a range of about 2% to about 8% by total weight.

[0157]

[0186] The water content of an aqueous composition can be determined by various analytical methods and instruments. In some embodiments, the water content is determined by evaporation (e.g., loss on drying), distillation, or chemical reaction (e.g., Karl Fischer titration). In some embodiments, the water content is determined by an analytical instrument, such as a moisture meter. In some embodiments, the water content is determined by Karl Fischer titration.

[0158]

[0187] In some embodiments, the water content of the aqueous composition is measured during the reaction and used to implement in-process control (IPC) of the water content. In certain embodiments, the water content of the reaction is measured by Karl Fischer titration, infrared spectroscopy, near-infrared spectroscopy, conductivity, viscosity, density, torque mixing, or energy mixing. In some embodiments, the measurement of the water content of the reaction is used to control devices that actively regulate the water content of the reaction, such as a water addition pump or a flow valve.

[0159]

[0188] Without being bound by theory, it is believed that water content during the sugar polymerization and / or condensation reaction can affect the level of anhydro subunits in the oligosaccharide preparations described herein. For example, as shown in Figure 29, in some embodiments, higher water content correlates with lower levels of anhydro subunits. In some embodiments, lower reaction temperatures can correlate with lower levels of anhydro subunit content.

[0160] [temperature]

[0189] In some embodiments, the degree of polymerization of the oligosaccharides and / or the amount and type of anhydro subunits in the oligosaccharide preparation can be controlled by adjusting the temperature to which the aqueous composition is heated. In some embodiments, methods for producing the oligosaccharide preparations described herein include heating the aqueous composition to a temperature of about 80°C to about 250°C, about 90°C to about 200°C, about 100°C to about 200°C, about 100°C to about 180°C, about 110°C to about 170°C, about 120°C to about 160°C, about 130°C to about 150°C, or about 135°C to about 145°C. In some embodiments, methods for producing the oligosaccharide preparation include heating the aqueous composition to a temperature of about 100°C to about 200°C, about 100°C to about 180°C, about 110°C to about 170°C, about 120°C to about 160°C, about 130°C to about 150°C, or about 135°C to about 145°C. In some embodiments, the method of producing an oligosaccharide preparation comprises heating an aqueous composition to a temperature of about 135° C. to about 145° C. In other embodiments, the method of producing an oligosaccharide preparation comprises heating an aqueous composition to a temperature of about 125° C. to about 135° C.

[0161] [Reaction time]

[0190] In some embodiments, the methods for producing the oligosaccharide preparations described herein comprise heating an aqueous composition for a sufficient period of time, hi some embodiments, the degree of polymerization of the oligosaccharides produced according to the methods described herein can be controlled by the reaction time.

[0162]

[0191] In some embodiments, a sufficient time is defined as a substantial number 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 about 24 hours, about 1 to about 16 hours, about 1 to about 8 hours, about 1 to about 4 hours, about 1 to about 3 hours, about 1 to about 2 hours, about 2 to about 12 hours, about 2 to about 10 hours, about 2 to about 8 hours, about 2 to about 6 hours, about 2 to about 4 hours, about 3 to about 8 hours, about 3 to about 6 hours, about 3 to about 5 hours, or about 3 to about 4 hours.

[0163]

[0192] In some embodiments, a sufficient time is determined by measuring one or more chemical or physical properties of the oligosaccharide preparation, such as water content, viscosity, molecular weight, anhydro subunit content, and / or degree of polymerization distribution.

[0164]

[0193] In some embodiments, the molecular weight of the oligosaccharide preparation is monitored during polymerization. In some embodiments, the method includes heating the aqueous composition for a time sufficient for the aqueous composition to achieve a number average molecular weight or a weight average molecular weight described herein. In certain embodiments, the method comprises heating the aqueous composition for a sufficient time such that the aqueous composition achieves a number average molecular weight within the range of about 300 to about 5,000 g / mol, about 500 to about 5,000 g / mol, about 700 to about 5,000 g / mol, about 500 to about 2,000 g / mol, about 700 to about 2,000 g / mol, about 700 to about 1,500 g / mol, about 300 to about 1,500 g / mol, about 300 to about 2,000 g / mol, about 400 to about 1,000 g / mol, about 400 to about 900 g / mol, about 400 to about 800 g / mol, about 500 to about 900 g / mol, or about 500 to about 800 g / mol. In certain embodiments, the method comprises heating the aqueous composition for a sufficient time such that the aqueous composition achieves a number average molecular weight within the range of about 500 to about 2,000 g / mol. In certain embodiments, the method comprises providing an aqueous composition having a solubility of about 300 to about 5000 g / mol, about 500 to about 5000 g / mol, about 700 to about 5000 g / mol, about 500 to about 2000 g / mol, about 700 to about 2000 g / mol, about 700 to about 1500 g / mol, about 300 to about 1500 g / mol, about 300 to about 2000 g / mol, about 400 to about 1300 g / mol, The method includes heating the aqueous composition for a sufficient time to achieve a weight average molecular weight of about 400 to about 1200 g / mol, about 400 to about 1100 g / mol, about 500 to about 1300 g / mol, about 500 to about 1200 g / mol, about 500 to about 1100 g / mol, about 600 to about 1300 g / mol, about 600 to about 1200 g / mol, or about 600 to about 1100 g / mol. In certain embodiments, the method includes heating the aqueous composition for a sufficient time to achieve a weight average molecular weight of about 700 to about 3000 g / mol.

[0165]

[0194] In some embodiments, the sufficient time is the time required for the aqueous composition to reach reaction equilibrium at the respective reaction temperature. Thus, in some embodiments, the method comprises heating the aqueous composition for a sufficient time for the aqueous composition to reach equilibrium. For example, in some embodiments, the equilibrium is determined by measuring the molecular weight, viscosity, or DP distribution of the aqueous composition.

[0166]

[0195] In certain embodiments, equilibrium is determined by measuring the number-average or weight-average molecular weight of the aqueous composition. In some embodiments, equilibrium is determined by the number- or weight-average molecular weight of the aqueous composition remaining essentially unchanged over time. In some embodiments, equilibrium is determined by a change in the number- or weight-average molecular weight of the aqueous composition being less than a certain percentage over a period of time. In some embodiments, the molecular weight of the aqueous composition is measured by HPLC or SEC.

[0167]

[0196] In some embodiments, equilibrium is determined by a change in the number or weight average molecular weight of the aqueous composition of less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% over a period of time. In some embodiments, equilibrium is determined by a change in the number or weight average molecular weight of the aqueous composition of less than 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, equilibrium is determined by a change in the weight average molecular weight of the aqueous composition of less than 15% over 1 hour.

[0168]

[0197] In certain embodiments, equilibrium is determined by measuring the viscosity of the aqueous composition. In some embodiments, equilibrium is determined by the viscosity of the aqueous composition remaining essentially unchanged over time. In some embodiments, equilibrium is determined by a change in the viscosity of the aqueous composition being less than a certain percentage over a period of time. In some embodiments, the viscosity of the aqueous composition is measured by a viscometer or rheometer.

[0169]

[0198] 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 a change in viscosity of the aqueous composition of less than 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, equilibrium is determined by a change in viscosity of the aqueous composition of less than 15% over 1 hour.

[0170]

[0199] In certain embodiments, the equilibrium is determined by measuring the DP distribution of the aqueous composition. In some embodiments, the equilibrium is determined by the DP distribution of the aqueous composition remaining essentially unchanged over time. In some embodiments, the change in the DP distribution of the aqueous composition is determined by calculating a series of Km, wherein

number

[0171]

[0200] In some embodiments, the concentrations of oligosaccharides in the DP1, DPm-1, and DPm fractions are determined by SEC, HPLC, FFF, A4F, mass spectrometry, or any other suitable method. In some embodiments, the concentrations of oligosaccharides in the DP1, DPm-1, and DPm fractions are determined by SEC, such as GPC. In some embodiments, the concentrations of oligosaccharides in the DP1, DPm-1, and DPm fractions are determined by mass spectrometry, such as GC-MS, LC-MS / MS, and MALDI-MS. In some embodiments, the concentrations of oligosaccharides in the DP1, DPm-1, and DPm fractions are determined by HPLC. In some embodiments, the water concentration is determined by evaporation (e.g., loss on drying), distillation, or chemical reaction (e.g., Karl Fischer titration). In some embodiments, the water concentration is determined by any suitable analytical instrument, such as a moisture meter.

[0172]

[0201] In some embodiments, the method comprises calculating a set of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 Km numbers. In some embodiments, the method comprises calculating a set of at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 15 Km numbers. In some embodiments, the method comprises calculating about 3, 4, 5, 6, 7, 8, 9, 10, or 15 Km numbers. In some embodiments, the method includes calculating K2-K4, K2-K5, K2-K6, K2-K7, K2-K8, K2-K9, K2-K10, K2-K11, K2-K12, K2-K13, K2-K14, K2-K15, K3-K5, K3-K6, K3-K7, K3-K8, K3-K9, K3-K10, K3-K11, K3-K12, K3-K13, K3-K14, or K3-K15. In certain embodiments, the method includes calculating K2-K4 or K3-K5.

[0173]

[0202] In some embodiments, the Km value depends on temperature, water concentration, and / or the amount and type of sugar supplied. In some embodiments, Km is about 0.1 to about 100, about 0.1 to about 90, about 0.1 to about 80, about 0.1 to about 70, about 0.1 to about 60, about 0.1 to about 50, about 0.1 to about 40, about 0.1 to about 30, about 0.1 to about 25, about 0.1 to about 20, or about 0.1 to about 15. In some embodiments, Km is about 1 to about 100, about 1 to about 90, about 1 to about 80, about 1 to about 70, about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, about 5 to about 50, about 5 to about 40, about 5 to about 30, about 5 to about 20, about 5 to about 15, or about 5 to about 10. In some embodiments, Km is about 1 to about 15 or about 5 to about 15.

[0174]

[0203] In some embodiments, the mean, standard deviation, and / or relative standard deviation are determined for a set of calculated Km. As used herein, the 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.

[0175]

[0204] In some embodiments, equilibrium is determined by a relative standard deviation of a series of Km's of less than 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, equilibrium is determined by a relative standard deviation of a series of Km's of less than 15%, 10%, or 5%.

[0176] [Post-reaction process]

[0205] In some embodiments, the methods for producing an oligosaccharide preparation described herein further comprise one or more additional processing steps after heating the aqueous composition at a temperature for a sufficient period of time. In some embodiments, the additional processing steps include, for example, separation (such as chromatographic separation), dilution, concentration, drying, filtration, desalting, extraction, decolorization, or any combination thereof. For example, in some embodiments, the method includes a dilution step and a decolorization step. In some embodiments, the method includes a filtration step and a drying step.

[0177]

[0206] In some embodiments, the method includes a dilution step in which water is added to the oligosaccharide preparation to form a syrup of the oligosaccharide preparation. In some embodiments, the concentration of the oligosaccharide preparation in the syrup is about 5% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, or about 15% to about 25%. In other embodiments, the method does not include a dilution step, but rather solidifies the oligosaccharide preparation. In some embodiments, the method includes a filtration step. In some embodiments, the method includes recycling the catalyst by filtration.

[0178]

[0207] In some embodiments, the described methods include a decolorization step, which can be accomplished using any method known in the art, such as treatment with absorbents, activated carbon, chromatography (e.g., using ion exchange resins), hydrogenation, and / or filtration (e.g., microfiltration).

[0179]

[0208] In some embodiments, the oligosaccharide preparation is contacted with a material to remove salts, minerals, and / or other ionic species. In certain embodiments, the oligosaccharide preparation is passed through an anion / cation exchange column pair. In one embodiment, the anion exchange column contains a weak base exchange resin in its hydroxide form, and the cation exchange column contains a strong acid exchange resin in its protonated form.

[0180]

[0209] In some embodiments, the method includes a concentration step. In some embodiments, the concentration step produces an increased concentration of the oligosaccharide preparation. For example, in some embodiments, the concentration step includes evaporation (e.g., vacuum evaporation), drying (e.g., freeze drying and spray drying), or any combination thereof.

[0181]

[0210] In some embodiments, the isolation step comprises separating at least a portion of the oligosaccharide preparation, hi some embodiments, the isolation step comprises crystallization, precipitation, filtration (e.g., vacuum filtration), and centrifugation, or any combination thereof.

[0182]

[0211] In some embodiments, the method includes a separation step. In some embodiments, the separation step includes separating at least a portion of the oligosaccharide preparation from at least a portion of the catalyst, from at least a portion of the unreacted feed sugars, or both. In some embodiments, the separation step includes filtration, chromatography, differential solubility, precipitation, extraction, or centrifugation.

[0183] [Reactor]

[0212] The methods described herein can include the use of one or more reactors suitable for sugar condensation, taking into consideration reaction temperature, pH, pressure, and other factors. In some embodiments, the one or more suitable reactors include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor, a continuous plug flow column reactor, a friction reactor, or a reactor using stirring induced by an electromagnetic field. In some embodiments, one or more suitable reactors include those 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 reactors for the cellobiose hydrolysis, Acta Scientiarum. Technology, 25:33-38 (2003).

[0184]

[0213] In some embodiments, the one or more suitable reactors include a fluidized bed reactor, an upflow blanket reactor, a fixed bed reactor, or an extruder reactor 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 comprises a continuous process, the one or more suitable reactors may include a continuous mixer, such as a screw mixer.

[0185] [process]

[0214] In some embodiments, the methods for producing an oligosaccharide preparation described herein include a batch process, a continuous process, or both. In some embodiments, the methods for producing an oligosaccharide preparation include a batch process. For example, in some embodiments of a batch process, production of a subsequent batch of oligosaccharide preparation does not begin until the current batch is completed. In some embodiments, during a batch process, all or a substantial amount of the oligosaccharide preparation is removed from the reactor. In some embodiments, during a batch process, all of the feed sugar and catalyst are combined in the reactor before the aqueous composition is heated to the specified temperature or before polymerization is induced. In some embodiments, during a batch process, the feed sugar is added before, after, or simultaneously with the addition of the catalyst.

[0186]

[0215] In some embodiments, the batch process is a fed-batch process, where not all of the feed sugar is added to the reactor at the same time. In some embodiments of a fed-batch process, at least a portion of the feed sugar is added to the reactor during polymerization or after the aqueous composition has been heated to a recited temperature. In some embodiments of a fed-batch process, at least 10%, 20%, 30%, 40%, 50%, or 60% by weight of the feed sugar is added to the reactor during polymerization or after the aqueous composition has been heated to a recited temperature.

[0187]

[0216] In some embodiments, the method for producing an oligosaccharide preparation comprises a continuous process. For example, in some embodiments of a continuous process, the contents of the reactor flow continuously throughout the reactor. In some embodiments, combining the feed sugar with the catalyst and removing at least a portion of the oligosaccharide preparation are performed simultaneously.

[0188]

[0217] In some embodiments, the method for producing an oligosaccharide preparation comprises a single-container or multi-container process. For example, in some embodiments of a single-container process, polymerization is carried out in a single reaction vessel. For another example, in some embodiments of a multi-container process, polymerization is carried out in two or more reaction vessels. In some embodiments of a multi-container process, the method comprises two, three, or more reaction vessels. In some embodiments of a multi-container process, the method comprises a combining step in which polymerization products from two or more reactors are combined.

[0189] V. Nutritional Compositions Comprising Anhydro Subunits

[0218] Provided herein are nutritional compositions comprising an oligosaccharide preparation. In certain embodiments, provided herein are nutritional compositions comprising the described oligosaccharide preparation, wherein the presence and / or concentration of the oligosaccharide preparation within the nutritional composition can be selectively determined and / or detected. Oligosaccharide preparations, which exhibit complex functional regulation of microbial communities, can be important components of nutritional compositions. Thus, the presence and / or concentration of the oligosaccharide preparation within a nutritional composition can be one of the factors that need to be measured in the quality control and manufacturing process of nutritional compositions. Thus, the provided nutritional compositions are advantageous from the perspective of quality control and manufacturing purposes, since the presence and / or concentration of the oligosaccharide preparation can be selectively determined and / or detected. For example, in some embodiments, the presence and concentration of the oligosaccharide preparation can be determined and / or detected by measuring a signal associated with anhydrosubunit-containing oligosaccharides.

[0190]

[0219] In some embodiments, the nutritional composition is an animal feed composition. In some embodiments, the nutritional composition comprises a basal nutritional composition.

[0191] [Basic nutritional composition]

[0220] In some embodiments, the nutritional compositions described herein comprise a basal nutritional composition and a disclosed oligosaccharide preparation. In some embodiments, the basal nutritional composition comprises a carbohydrate source different from the oligosaccharide preparation. For example, in some embodiments, the basal nutritional composition comprises a naturally occurring carbohydrate source (or a naturally occurring oligosaccharide composition) such as starch and plant fiber. In some embodiments, the basal nutritional composition comprises starch. In some embodiments, the basal nutritional composition comprises plant fiber.

[0192]

[0221] In some embodiments, the basal nutritional composition comprises one or more carbohydrate sources derived from seeds, roots, tubers, corn, tapioca, arrowroot, wheat, rice, potato, sweet potato, sago, beans (e.g., fava beans, lentils, mung beans, peas, and chickpeas), maize, cassava, or other starchy foods (e.g., acorns, arrowroot, arracacha, bananas, barley, breadfruit, buckwheat, canna, colacasia, dogtooth violet, kudzu, malanga, millet, oats, okara, tsorghum, sorghum, rye, taro, chestnut, water chestnut, and yam).

[0193]

[0222] In some embodiments, the basal nutritional composition comprises one or more carbohydrate sources derived from legumes (e.g., peas, soybeans, lupins, green beans, and other beans), oats, rye, chia, barley, fruits (e.g., figs, avocados, plums, prunes, berries, bananas, apple peels, quince, and pears), vegetables (e.g., broccoli, carrots, cauliflower, zucchini, celery, nopal cactus, and Jerusalem artichoke), root tubers, root vegetables (e.g., sweet potato and onion), psyllium husks, seeds (e.g., flaxseed), nuts (e.g., almonds), whole grain foods, wheat, corn bran, lignans, or any combination thereof. In some embodiments, the basal nutritional composition comprises one or more vegetable fibers derived from wheat bran, beet pulp, fuzzy cottonseed, soybean hulls, or any combination thereof.

[0194]

[0223] In some embodiments, the basal nutritional composition comprises less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm of anhydro subunits or anhydro subunit-containing oligosaccharides. In some embodiments, the basal nutritional composition comprises less than 50 ppm, less than 10 ppm, less than 5 ppm, or less than 1 ppm of anhydro subunits or anhydro subunit-containing oligosaccharides. In some embodiments, the basal nutritional composition is essentially free of anhydro subunits.

[0195]

[0224] In some embodiments, the basal nutritional composition does not contain detectable levels of anhydro subunits. Depending on the detection or determination method, anhydro subunit levels below a certain threshold may not be detectable. For example, in some embodiments, a detectable level of anhydro subunits refers to at least 1000 ppm, at least 500 ppm, at least 400 ppm, at least 300 ppm, at least 200 ppm, at least 100 ppm, at least 50 ppm, at least 10 ppm, at least 5 ppm, or at least 1 ppm of anhydro subunits or anhydro subunit-containing oligosaccharides in the basal nutritional composition.

[0196]

[0225] In some embodiments, the basal nutritional composition comprises a plurality of oligosaccharides. In some embodiments, the basal nutritional composition comprises a different distribution of glycosidic linkage types than the oligosaccharide preparation. For example, in some embodiments, the basal nutritional composition comprises a higher proportion of α-(1,4) glycosidic linkages than the oligosaccharide preparation. In some embodiments, the glycosidic linkages, such as α-(1,4) glycosidic linkages, in the basal nutritional composition are digestible by one or more enzymes. In some embodiments, the glycosidic linkages in the basal nutritional composition are more readily digestible and / or hydrolyzable than the glycosidic linkages in the oligosaccharide preparation.

[0197]

[0226] In some embodiments, the level of α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, or β-(1,6) glycosidic linkages in the basal nutritional composition is at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% lower than the level of the respective glycosidic linkages in the oligosaccharide preparation. In some embodiments, the level of an α-(1,2) glycosidic linkage, an α-(1,3) glycosidic linkage, an α-(1,6) glycosidic linkage, a β-(1,2) glycosidic linkage, a β-(1,3) glycosidic linkage, a β-(1,4) glycosidic linkage, or a β-(1,6) glycosidic linkage in the basal nutritional composition is at least 10% lower than the level of the respective glycosidic linkage in the oligosaccharide preparation.

[0198]

[0227] In some embodiments, the level of α-(1,4) glycosidic linkages in the basal nutritional composition is at least 50%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5%, or at least 2% higher than the level of α-(1,4) glycosidic linkages in the oligosaccharide preparation. In some embodiments, the level of α-(1,4) glycosidic linkages in the basal nutritional composition is at least 10% higher than the level of α-(1,4) glycosidic linkages in the oligosaccharide preparation.

[0199] [Animal feed composition]

[0228] Depending on the type and age of the animal, the nutritional composition may contain different ratios of the oligosaccharide preparation and the basal nutritional composition. For example, the oligosaccharide preparation may be combined with the basal nutritional composition in various ratios appropriate for the type and age of the animal. In some embodiments, the oligosaccharide preparation may be present in an amount of about 1 to about 10,000 ppm, about 1 to about 5,000 ppm, about 1 to about 3,000 ppm, about 1 to about 2,000 ppm, about 1 to about 1,500 ppm, about 1 to about 1,000 ppm, about 1 to about 500 ppm, about 1 to about 250 ppm, about 1 to about 100 ppm, about 10 to about 5,000 ppm, about 10 to about 3,000 ppm, about 10 to about 2,000 ppm, or about 10 to about 3,000 ppm. ppm, about 10 to about 1500ppm, about 10 to about 1000ppm, about 10 to about 500ppm, about 10 to about 250ppm, about 10 to about 100ppm, about 50 to about 5000ppm, about 5 0 to about 3000ppm, about 50 to about 2000ppm, about 50 to about 1500ppm, about 50 to about 1000ppm, about 50 to about 500ppm, about 50 to about 250ppm, about 50 to about 10 0 ppm, about 100 to about 5000 ppm, about 100 to about 3000 ppm, about 100 to about 2000 ppm, about 100 to about 1500 ppm, about 100 to about 1000 ppm, about 100 to about 500 ppm, about 100 to about 400 ppm, about 100 to about 300 ppm, about 100 to about 200 ppm, about 200 to about 5000 ppm, about 200 to about 3000 ppm, about 200 to about 2 The nutrient composition is present at a concentration of about 500 ppm, about 200 to about 2000 ppm, about 200 to about 1500 ppm, about 200 to about 1000 ppm, about 200 to about 500 ppm, about 500 to about 5000 ppm, about 500 to about 3000 ppm, about 500 to about 2500 ppm, about 500 to about 2000 ppm, about 500 to about 1500 ppm, or about 500 to about 1000 ppm. In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration of about 1 to about 5000 ppm, about 1 to about 1000 ppm, about 1 to about 500 ppm, about 10 to about 5000 ppm, about 10 to about 2000 ppm, about 10 to about 1000 ppm, about 10 to about 500 ppm, about 10 to about 250 ppm, about 10 to about 100 ppm, about 50 to about 5000 ppm, about 50 to about 2000 ppm, about 50 to about 1000 ppm, about 50 to about 500 ppm, about 50 to about 250 ppm, or about 50 to about 100 ppm.In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration of about 1 to about 5000 ppm, about 10 to about 1000 ppm, about 10 to about 500 ppm, or about 50 to about 500 ppm.

[0200]

[0229] In some embodiments, the oligosaccharide preparation is present in the nutritional composition at a concentration of greater than 10 ppm, greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, greater than 300 ppm, greater than 400 ppm, greater than 500 ppm, greater than 600 ppm, greater than 1000 ppm, or greater than 2000 ppm. In some embodiments, the oligosaccharide preparation 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.

[0201]

[0230] In some embodiments, depending on the species and age of the animal, the nutritional composition may further comprise protein, minerals (such as copper, calcium and zinc), salts, essential amino acids, vitamins and / or antibiotics.

[0202]

[0231] In some embodiments, methods for producing nutritional compositions for animal feed are described herein. In some embodiments, the animal is selected from cattle (e.g., beef and dairy cattle), pigs, aquatic animals, poultry, and humans. In some embodiments, the animal is a pig, such as a sow, a piglet, or a barrow. In other embodiments, the animal is a poultry, such as a chicken, a duck, a turkey, a goose, a quail, or a hen. In embodiments, the poultry is a broiler, a breeder, or a layer. In some embodiments, the animal is an aquatic animal, such as salmon, catfish, bass, eel, tilapia, flounder, shrimp, and crab. In some embodiments, the nutritional composition is administered to the animal in a dry form, a liquid form, a paste, or a combination thereof. In some embodiments, the form of administration, feeding rate, and feeding schedule can vary depending on the type and age of the animal.

[0203] Methods for Producing Nutritional Compositions

[0232] Provided herein are methods for producing a nutritional composition, comprising combining an oligosaccharide preparation with a basal nutritional composition. In some embodiments, the oligosaccharide preparation comprises anhydro-subunit-containing oligosaccharides. In some embodiments, the oligosaccharide preparation comprises a glycosidic linkage distribution that differs from the glycosidic linkage distribution of the basal nutritional composition.

[0204]

[0233] In some embodiments, the oligosaccharide preparation is a synthetic oligosaccharide preparation. In some embodiments, the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1-DPn fractions), each having a distinct degree of polymerization selected from 1 to n. In some embodiments, n is an integer greater than or equal to 2. In some embodiments, n is an integer greater than or equal to 3. In some embodiments, n is an integer in the range of 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, each of the DP1-DPn fractions comprises between 0.1% and 90% anhydro-subunit-containing oligosaccharides by relative abundance as measured by mass spectrometry. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently comprise anhydro-subunit-containing oligosaccharides at a relative abundance of about 0.1% to about 15% or about 0.5% to about 10%, as measured by mass spectrometry. In some embodiments, the DP1 and DP2 fractions of the oligosaccharide preparation each independently comprise anhydro-subunit-containing oligosaccharides at a relative abundance ranging from about 0.1%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5% to about 8%, 9%, 10%, 11%, 12%, 15%, or 20%, as measured by mass spectrometry. In some embodiments, the relative abundance of oligosaccharides in each of the n fractions decreases monotonically with their degree of polymerization. In some embodiments, the relative abundance of oligosaccharides in DP fractions of at least 5, 10, 20, or 30 decreases monotonically with their degree of polymerization.

[0205]

[0234] In some embodiments, the method of producing the nutritional composition comprises mixing the oligosaccharide preparation with the basal nutritional composition. For example, in some embodiments, the mixing can be performed with an industrial blender and / or mixer, such as a drum blender, a double cone blender, a ribbon blender, a V-blender, a shear mixer, and a paddle mixer.

[0206]

[0235] In some embodiments, the method of producing a nutritive composition further comprises a quality control step as described herein. In some embodiments, the quality control step as described herein comprises determining a signal level in a sample of the nutritive composition and calculating the concentration of the oligosaccharide preparation in the nutritive composition based on the signal level. In some embodiments, the quality control step as described herein comprises detecting a signal in a sample of the nutritive composition with an analytical instrument and accepting or rejecting a batch of the nutritive composition based on the presence or absence of the signal. In some embodiments, the quality control step as described herein comprises detecting the presence or absence of a first signal in a first sample of the nutritive composition and a second signal in a second sample of the nutritive composition with an analytical instrument and comparing the first signal with the second signal. In some embodiments, the signal, the first signal, and / or the second signal (i) are indicative of one or more anhydrosubunit-containing oligosaccharides, (ii) are related to the degree of polymerization (DP) distribution of the oligosaccharides, or (iii) are related to an α-(1,2) glycosidic linkage, an α-(1,3) glycosidic linkage, an α-(1,6) glycosidic linkage, a β-(1,2) glycosidic linkage, a β-(1,3) glycosidic linkage, a β-(1,4) glycosidic linkage, a β-(1,6) glycosidic linkage, an α-(1,1)-α glycosidic linkage, an α-(1,1)-β glycosidic linkage, a β-(1,1)-α glycosidic linkage, or a β-(1,1)-β glycosidic linkage of the oligosaccharides.

[0207]

[0236] Furthermore, in some embodiments, the method of producing a nutritional composition, after performing the quality control step, further comprises blending the oligosaccharide preparation with the basal nutritional composition, adjusting the level of the oligosaccharide preparation, or a combination thereof. In some embodiments, adjusting the level of the oligosaccharide preparation comprises adding additional oligosaccharide preparation to the nutritional composition or removing a portion of the oligosaccharide preparation from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide preparation comprises adding additional basal nutritional composition to the nutritional composition or removing a portion of the basal nutritional composition from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide preparation comprises adding additional oligosaccharide preparation to the nutritional composition.

[0208] VI. Methods for Correlating Oligosaccharide Preparations

[0237] Provided herein are methods for correlating the presence, absence, and / or concentration of a described oligosaccharide preparation in a nutritional composition. Provided herein are methods for implementing quality control methods during the manufacturing process of a nutritional composition, including an oligosaccharide preparation and a basal nutritional composition. Provided herein are methods for determining the quality of a nutritional composition, including an oligosaccharide preparation and a basal nutritional composition. In some embodiments, provided herein are methods for correlating a synthetic oligosaccharide preparation in a nutritional composition. In some embodiments, a correlation method can refer to establishing a relationship, relating, and / or linking between two things, and can also refer to comparing the presence and amount of two things and evaluating the ratio of the two things. In some embodiments, provided herein are methods for detecting the concentration, presence, and / or absence of a synthetic oligosaccharide preparation in a nutritional composition. In some embodiments, the nutritional composition comprises a described synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition (e.g., a basal nutritional composition).

[0209]

[0238] In some embodiments, the term "quality," as used herein, can refer to the level of an oligosaccharide preparation (e.g., a synthetic oligosaccharide preparation) in a nutritional composition, e.g., whether the level is within a specified range, such as 1-5000 ppm, 10-1000 ppm, 10-500 ppm, or 50-500 ppm. In some embodiments, the term "quality" can refer to the level of an oligosaccharide preparation in a nutritional composition as evidenced by a signal provided by an analytical instrument; e.g., whether a specified peak is present in an NMR, GC-MS, LC-MS / MS, MALDI-MS, or HPLC chromatogram or spectrum and / or weight determination.

[0210]

[0239] In some embodiments, the term "quality" can refer to the distribution of an oligosaccharide preparation (e.g., a synthetic oligosaccharide preparation) in a nutritional composition, for example, whether the oligosaccharide preparation is consistently and homogeneously distributed in the nutritional composition. Thus, in some embodiments, the quality of a batch of nutritional composition can be determined by comparing the levels and / or signals of the oligosaccharide preparation in two or more samples of the nutritional composition taken from the same batch. In some embodiments, the quality of a batch of nutritional composition can be determined by comparing the levels and / or signals of the oligosaccharide preparation in two or more samples of the nutritional composition taken from different batches.

[0211]

[0240] Thus, provided herein is a method for quantifying an oligosaccharide preparation in a nutritional composition, comprising determining the level of a signal in a sample of the nutritional composition and calculating the concentration of the oligosaccharide preparation in the nutritional composition based on the level of the signal. Provided herein is a method for performing quality control of a nutritional composition, comprising using an analytical device to detect a signal in a sample of the nutritional composition and passing or rejecting a batch of the nutritional composition based on the presence or absence of the signal. Further provided herein is a method for performing quality control of a nutritional composition, comprising using an analytical device to detect 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 and second signals. In some embodiments, the signal, the first signal, and / or the second signal (i) are indicative of one or more anhydrosubunit-containing oligosaccharides, (ii) are associated with the degree of polymerization (DP) distribution of the oligosaccharides, or (iii) are associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, an α-(1,1)-α glycosidic bond, an α-(1,1)-β glycosidic bond, a β-(1,1)-α glycosidic bond, or a β-(1,1)-β glycosidic bond of the oligosaccharides.

[0212]

[0241] In some embodiments, provided herein are methods for quantifying a synthetic oligosaccharide preparation in a nutritional composition, wherein the nutritional composition comprises a synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition, the method comprising: (a) determining a level of a signal in a sample of the nutritional composition; and (b) correlating a concentration of the oligosaccharide preparation in the nutritional composition based on the level of the signal, wherein the signal is either (i) indicative of one or more anhydro-subunit-containing oligosaccharides or (ii) or (iii) the degree of polymerization (DP) distribution of the oligosaccharides; or (iv) the α-(1,2) glycosidic, α-(1,3) glycosidic, α-(1,6) glycosidic, β-(1,2) glycosidic, β-(1,3) glycosidic, β-(1,4) glycosidic, β-(1,6) glycosidic, α-(1,1)-α glycosidic, α-(1,1)-β glycosidic, β-(1,1)-α glycosidic, or β-(1,1)-β glycosidic linkages of the oligosaccharides. In some embodiments, described herein are methods for performing quality control in a nutritional composition, the methods comprising: (a) providing a batch of the nutritional composition, the nutritional composition comprising a synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition; (b) obtaining a sample of the nutritional composition from the batch; (c) detecting a signal of at least a portion of the oligosaccharides in the sample of the nutritional composition using an analytical device; and (d) passing or failing the batch of the nutritional composition, the signal being determined by: (i) one or more (ii) they represent anhydrosubunit-containing oligosaccharides, or (iii) they relate to the degree of polymerization (DP) distribution of the oligosaccharides, or (iv) they relate to the α-(1,2) glycosidic, α-(1,3) glycosidic, α-(1,6) glycosidic, β-(1,2) glycosidic, β-(1,3) glycosidic, β-(1,4) glycosidic, β-(1,6) glycosidic, α-(1,1)-α glycosidic, α-(1,1)-β glycosidic, β-(1,1)-α glycosidic, or β-(1,1)-β glycosidic linkages of the oligosaccharides.In some embodiments, described herein is a method for performing quality control in a nutritional composition, the method comprising: (a) providing a sample of the nutritional composition, wherein the nutritional composition comprises a naturally occurring oligosaccharide composition; (b) detecting, with an analytical device, a signal of at least a portion of the oligosaccharides in the sample of the nutritional composition, the signal being indicative of one or more anhydro-subunit-containing oligosaccharides; or (ii) detecting a degree of polymerization (DP) of the oligosaccharides. ) distribution of oligosaccharides, or (iii) associated with α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, β-(1,6) glycosidic linkages, α-(1,1)-α glycosidic linkages, α-(1,1)-β glycosidic linkages, β-(1,1)-α glycosidic linkages, or β-(1,1)-β glycosidic linkages of oligosaccharides. In some embodiments, the signal is indicative of one or more anhydrosubunit-containing oligosaccharides. In some embodiments, described herein are methods for performing quality control in nutritional compositions, including synthetic oligosaccharide preparations and naturally occurring oligosaccharide compositions, the methods comprising: (a) detecting, with an analytical device, 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 (b) comparing the first signal and the second signal, wherein the first signal and the second signal are indicative of (i) one or more anhydro-subunit-containing oligosaccharides. or (ii) associated with the degree of polymerization (DP) distribution of the oligosaccharides, or (iii) associated with the α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, β-(1,6) glycosidic linkages, α-(1,1)-α glycosidic linkages, α-(1,1)-β glycosidic linkages, β-(1,1)-α glycosidic linkages, or β-(1,1)-β glycosidic linkages of the oligosaccharides. In some embodiments, the first signal and / or the second signal is indicative of one or more anhydrosubunit-containing oligosaccharides.

[0213]

[0242] Depending on the type, the signal (e.g., level of the signal), the first signal, and / or the second signal can be determined or detected by any suitable analytical method, including, but not limited to, NMR, HPLC, SEC, FFF, A4F, GC, LC, GC-MS, LC-MS / MS, MALDI-MS, IR, or CD spectroscopy. The level of the signal, the first signal, and / or the second signal can be determined or detected by purifying the sample using LC and weighing the sample. In some embodiments, the signals of the described oligosaccharide preparations are determined or detected by MALDI-MS. In some embodiments, the signals of the described oligosaccharide preparations are determined or detected by LC-MS / MS. In some embodiments, the signals of the described oligosaccharide preparations are determined or detected by GC-MS. In some embodiments, the signals of the described oligosaccharide preparations are determined or detected by NMR, such as 2D HSQC NMR. In some embodiments, the relative abundance or concentration, or both, of the described oligosaccharide preparations is calculated based on the level of signal of the oligosaccharide preparation, e.g., the relative abundance of one or more peaks in a mass spectrum or the intensity of one or more peaks in an NMR spectrum.

[0214] [anhydro subunit signal]

[0243] In some embodiments, the methods described herein involve detecting a signal from an oligosaccharide preparation. In some embodiments, the signal from an oligosaccharide preparation described herein is indicative of one or more anhydrosubunit-containing oligosaccharides. In some embodiments, the methods described herein involve detecting two or more signals, i.e., a first signal, a second signal, etc. In some embodiments, at least one of the first signal and the second signal is indicative of one or more anhydrosubunit-containing oligosaccharides. In some embodiments, both the first signal and the second signal are indicative of one or more anhydrosubunit-containing oligosaccharides.

[0215]

[0244] In some embodiments, signals indicative of one or more anhydro-subunit-containing oligosaccharides can be determined or detected by any suitable analytical device, including, but not limited to, liquid chromatography (e.g., HPLC), FFF, A4F, NMR, SEC, mass spectrometry, such as LC-MS / MS, GC-MS, and MALDI-MS. As used herein, in some embodiments, signals indicative of one or more anhydro-subunit-containing oligosaccharides can include signals attributable to one or more anhydro-subunit-containing oligosaccharides.

[0216]

[0245] In some embodiments, the signal representing one or more anhydro-subunit-containing oligosaccharides comprises one or more peaks attributable to anhydro-subunit-containing oligosaccharides by mass spectrometry, NMR, or liquid chromatography (e.g., HPLC), and / or weight determination. In some embodiments, the one or more peaks attributable to anhydro-subunit-containing oligosaccharides from the oligosaccharide preparation. In some embodiments, the one or more peaks attributable to mass spectrometry, NMR, or liquid chromatography (e.g., HPLC), and / or weight determination are attributable to anhydro-subunit-containing oligosaccharides from any of the DP1 to DPn fractions in the oligosaccharide preparation. In some embodiments, the one or more peaks attributable to mass spectrometry, NMR, or liquid chromatography (e.g., HPLC), and / or weight determination are attributable to anhydro-subunit-containing oligosaccharides in the DP1 fraction. In some embodiments, one or more peaks on mass spectrum, NMR or liquid chromatography (eg, HPLC) and / or weight determination are assigned to levoglucosan, 1,6-anhydro-β-D-glucofuranose, or a combination thereof.

[0217]

[0246] In some embodiments, the signal representing one or more anhydro-subunit-containing oligosaccharides comprises one or more peaks attributable to anhydro-subunit-containing oligosaccharides in the DP2 fraction by mass spectrometry, NMR, or liquid chromatography (e.g., HPLC), and / or weight determination. In some embodiments, the one or more peaks attributable to anhydro-cellobiose by mass spectrometry, NMR, or liquid chromatography (e.g., HPLC), and / or weight determination. In some embodiments, the one or more peaks attributable to anhydro-subunit-containing oligosaccharides in the DP3, DP4, DP5, or DP6 fractions.

[0218]

[0247] In some embodiments, one or more peaks on mass spectrum, NMR, or liquid chromatography (e.g., HPLC) and / or weight determination are attributable to anhydro-subunit-containing oligosaccharides from one or more fractions. For example, in some embodiments, signals are attributable to anhydro-subunit-containing oligosaccharides from the DP1 and DP2 fractions. In some embodiments, signals are attributable to anhydro-subunit-containing oligosaccharides from the DP1, DP2, and DP3 fractions.

[0219]

[0248] For example, in some embodiments, the signal representing one or more anhydro-subunit-containing oligosaccharides comprises one or more mass spectral peaks attributable to anhydro-subunit-containing oligosaccharides in the DP2 fraction. In some embodiments, the signal representing one or more anhydro-subunit-containing oligosaccharides comprises one or more mass spectral peaks attributable to anhydro-subunit-containing oligosaccharides in the DP1 fraction. In some embodiments, the signal representing one or more anhydro-subunit-containing oligosaccharides comprises a weight determination of DP2 anhydro-subunit-containing oligosaccharides. In some embodiments, the weight determination is performed on at least a portion of the DP2 anhydro-subunit-containing oligosaccharides isolated and / or purified by preparative chromatography.

[0220]

[0249] In some embodiments, the methods described herein involve detecting two or more signals, i.e., a first signal, a second signal, a third signal, etc. In some embodiments, the first signal and the second signal are attributable to anhydrosubunit-containing oligosaccharides in the same fraction. For example, in some embodiments, the first signal is attributable to levoglucosan and the second signal is attributable to 1,6-anhydro-β-D-glucofuranose. In some embodiments, the first signal and the second signal are attributable to the same species of anhydrosubunit-containing oligosaccharides. For example, in some embodiments, the first signal and the second signal are attributable to 1,6-anhydro-β-D-glucofuranose. In some embodiments, the first signal and the second signal are attributable to anhydrosubunit-containing oligosaccharides from different fractions. In some embodiments, the first signal and the second signal are attributable to different species of anhydrosubunit-containing oligosaccharides. For example, in some embodiments, the first signal is attributed to levoglycosan and the second signal is attributed to anhydro-cellobiose. In some embodiments, at least a portion of the first and second signals representing one or more anhydro-subunit-containing oligosaccharides comprise one or more mass spectral peaks attributable to anhydro-subunit-containing oligosaccharides in the DP2 fraction. In some embodiments, at least a portion of the first and second signals representing one or more anhydro-subunit-containing oligosaccharides comprise one or more mass spectral peaks attributable to anhydro-subunit-containing oligosaccharides in the DP1 fraction. In some embodiments, at least a portion of the first and second signals representing one or more anhydro-subunit-containing oligosaccharides comprise a weight determination of DP1 and / or DP2 anhydro-subunit-containing oligosaccharides. In some embodiments, both the first and second signals representing one or more anhydro-subunit-containing oligosaccharides comprise one or more mass spectral peaks attributable to anhydro-subunit-containing oligosaccharides in the DP2 fraction, such as anhydro-cellobiose.In some embodiments, both the first signal and the second signal indicative of one or more anhydro-subunit-containing oligosaccharides comprise one or more mass spectral peaks attributable to anhydro-subunit-containing oligosaccharides in the DP1 fraction, such as levoglucosan, 1,6-anhydro-β-D-glucofuranose, or a combination thereof.

[0221]

[0250] In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP1 fraction. In some embodiments, all of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP1 fraction. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to levoglucosan, 1,6-anhydro-β-D-glucofuranose, or a combination thereof. In some embodiments, all of the signal, the first signal, and the second signal are attributable to levoglucosan, 1,6-anhydro-β-D-glucofuranose, or a combination thereof. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP2 fraction. In some embodiments, all of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP2 fraction. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to anhydro-cellobiose. In some embodiments, all of the signal, the first signal, and the second signal are attributable to anhydro-cellobiose. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP3 fraction. In some embodiments, all of the signal, the first signal, and the second signal are attributable to anhydro-subunit-containing oligosaccharides in the DP3 fraction.

[0222]

[0251] In some embodiments, the described signal level can be determined by any suitable analytical device, including, but not limited to, equilibrium, liquid chromatography (e.g., HPLC), NMR, SEC, mass spectrometry, such as LC-MS / MS, GC-FID, GC-MS, and MALDI-MS. For example, in some embodiments, the signal level is the relative abundance of one or more anhydrosubunit-containing oligosaccharides represented by the signal. In some embodiments, the signal level is the concentration of one or more anhydrosubunit-containing oligosaccharides represented by the signal. In some embodiments, the signal level is the weight of at least a portion of an isolated fraction (e.g., DP2 anhydrosubunit-containing oligosaccharides) of the described oligosaccharide preparation.

[0223] [Glycosidic bond signal]

[0252] In some embodiments, the signals described herein are associated with one or more glycosidic bonds. For example, in some embodiments, the signals are associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,4) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, or a combination thereof. In some embodiments, the signals are associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond.

[0224]

[0253] In some embodiments, the methods described herein involve detecting two or more signals, i.e., a first signal, a second signal, a third signal, etc. In some embodiments, at least one of the first signal and the second signal is associated with one or more glycosidic bonds. For example, in some embodiments, at least one of the first signal and the second signal is associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,4) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, or a combination thereof. In some embodiments, at least one of the first signal and the second signal is associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond.

[0225]

[0254] In some embodiments, both the first signal and the second signal are associated with one or more glycosidic bonds. For example, in some embodiments, both the first signal and the second signal are associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,4) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, a β-(1,6) glycosidic bond, or a combination thereof. In some embodiments, both the first signal and the second signal are associated with an α-(1,2) glycosidic bond, an α-(1,3) glycosidic bond, an α-(1,6) glycosidic bond, a β-(1,2) glycosidic bond, a β-(1,3) glycosidic bond, a β-(1,4) glycosidic bond, or a β-(1,6) glycosidic bond.

[0226]

[0255] In some embodiments, signals associated with one or more glycosidic bonds can be analyzed by any suitable analytical device, including but not limited to, 1D 1 H NMR, 1D 13The signal can be determined or detected by NMR, including C NMR, 2D NMR such as 2D JRES, HSQC, DOSY, HMBC, COSY, ECOSY, TOCSY, NOESY, or ROESY, or a combination thereof. In some embodiments, a signal associated with one or more glycosidic bonds can be attributed to one or more glycosidic bonds.

[0227]

[0256] In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,2) glycosidic bond. In some embodiments, all of the signal, the first signal, and the second signal are associated with an α-(1,2) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,3) glycosidic bond. In some embodiments, all of the signal, the first signal, and the second signal are associated with an α-(1,3) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with an α-(1,6) glycosidic bond. In some embodiments, all of the signal, the first signal, and the second signal are associated with an α-(1,6) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,2) glycosidic bond. In some embodiments, all of the signal, the first signal, and the second signal are associated with a β-(1,2) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,3) glycosidic bond. In some embodiments, all of the signal, the first signal, and the second signal are associated with a β-(1,3) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,4) glycosidic bond. In some embodiments, all of the signal, the first signal, and the second signal are associated with a β-(1,4) glycosidic bond. In some embodiments, one or more of the signal, the first signal, and the second signal are associated with a β-(1,6) glycosidic bond. In some embodiments, all of the signal, the first signal, and the second signal are associated with a β-(1,6) glycosidic bond.

[0228]

[0257] In some embodiments, the signal associated with one or more glycosidic bonds comprises one or more peaks in the NMR spectrum attributable to one or more glycosidic bonds. In some embodiments, the position, intensity, shape, and other characteristics of the signal may vary depending on the type of NMR spectrum and the conditions under which the NMR is performed. In some embodiments, the presence or absence of a signal associated with one or more glycosidic bonds refers to the presence or absence of one or more peaks in the NMR spectrum attributable to one or more glycosidic bonds. In some embodiments, the level of a signal refers to the intensity of one or more peaks in the NMR spectrum attributable to one or more glycosidic bonds, wherein the relative abundance and / or concentration of one or more glycosidic bonds may or may not be calculated based on the intensity.

[0229] [DP distribution signal]

[0258] In some embodiments, the signals described herein are related to the DP distribution of oligosaccharides. In some embodiments, the methods described herein involve detecting two or more signals, i.e., a first signal, a second signal, a third signal, etc. In some embodiments, at least one of the first signal and the second signal is related to the DP distribution of oligosaccharides. In some embodiments, both the first signal and the second signal are related to the DP distribution of oligosaccharides. In some embodiments, the DP distribution of oligosaccharides is primarily attributable to the oligosaccharide preparation.

[0230]

[0259] In some embodiments, signals related to the DP distribution of oligosaccharides can be determined or detected by any suitable analytical method, including but not limited to, HPLC, SEC, FFF, A4F, mass spectrometry, such as LC-MS / MS, GC-MS, and MALDI-MS. In some embodiments, signals related to the DP distribution of oligosaccharides can be determined based on the molecular weight distribution of the oligosaccharides.

[0231]

[0260] In some embodiments, the signal associated with the DP distribution of oligosaccharides may refer to a signal attributable to any one or more of the DP1 to DPn fractions, as acquired by a suitable analytical device. In some embodiments, the signal associated with the DP distribution of oligosaccharides is attributable to oligosaccharides in the DP1, DP2, DP3, DP4, or DP5 fraction.

[0232]

[0261] In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to oligosaccharides in the DP1 fraction. In some embodiments, all of the signal, the first signal, and the second signal are attributable to oligosaccharides in the DP1 fraction. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to oligosaccharides in the DP2 fraction. In some embodiments, all of the signal, the first signal, and the second signal are attributable to oligosaccharides in the DP2 fraction. In some embodiments, one or more of the signal, the first signal, and the second signal are attributable to oligosaccharides in the DP3 fraction. In some embodiments, all of the signal, the first signal, and the second signal are attributable to oligosaccharides in the DP3 fraction.

[0233]

[0262] Depending on the type of analytical instrument, in some embodiments, the concentration and / or relative abundance of the oligosaccharide associated with the signal can be determined based on the level of the signal. In some embodiments, the signal associated with the DP distribution of the oligosaccharides is attributed to the DP2 fraction and determined or detected by SEC, where the amount of the DP2 fraction in the oligosaccharides can be determined by SEC. In some embodiments, the signal associated with the DP distribution of the oligosaccharides is attributed to the DP2 fraction and determined or detected by HPLC, where the amount of the DP2 fraction in the oligosaccharides can be determined by HPLC. In some embodiments, the signal associated with the DP distribution of the oligosaccharides is attributed to the DP2 fraction and determined or detected by LC-MS / MS, where the amount of the DP2 fraction in the oligosaccharides can be determined by LC-MS / MS.

[0234]

[0263] It should be understood that each of the signals (e.g., the first signal and the second signal) can be independently selected, determined, and / or detected. For example, in some embodiments, the first signal is associated with the DP distribution and is determined by SEC, while the second signal is indicative of anhydrosubunit-containing oligosaccharides in the DP2 fraction and is determined by LC-MS / MS. In some embodiments, the first signal is associated with an α-(1,6) glycosidic bond and is determined by 2D 1 H, 13 The first signal is detected by C-HSQC, while the second signal represents anhydro-subunit-containing oligosaccharides in the DP1 fraction and is determined or detected by LC-MS / MS. In some embodiments, the first signal and the second signal both represent and are attributed to anhydro-subunit-containing oligosaccharides in the DP1 fraction and are determined or detected by mass spectrometry. In some embodiments, the first signal and the second signal both represent and are attributed to anhydro-subunit-containing oligosaccharides in the DP2 fraction and are determined or detected by mass spectrometry. In some embodiments, the first signal and the second signal both represent and are attributed to anhydro-subunit-containing oligosaccharides in the DP2 fraction and are determined or detected by weight determination after isolation by preparative chromatography.

[0235] [extraction]

[0264] In some embodiments, the methods described herein include extracting at least a portion of the oligosaccharides from a sample of a nutritional composition. In some embodiments, the extraction solvent comprises one or more solvents capable of dissolving or partially dissolving the oligosaccharides. In some embodiments, the extraction solvent comprises water, an alcohol (e.g., ethanol, methanol, or propanol), a buffer, one or more organic solvents, or any combination thereof. In some embodiments, a buffer suitable for extraction may comprise citric acid, acetic acid, phosphate, CHES, borate, diethylbarbituric acid, carbonic acid, bicarbonate, hydrochloric acid, sodium hydroxide, sodium acetate, imidazole, sodium carbonate, any combination thereof, or other buffers known in the art. In some embodiments, the buffer has a pH less than 7. In other embodiments, the buffer has a pH greater than or equal to 7. In some embodiments, the extraction solvent comprises water and ethanol in a weight or volume ratio of about 5:95, about 10:90, about 20:80, about 30:70, about 40:60, about 50:50, about 60:40, about 70:30, about 80:20, about 90:10, or about 95:5. In some embodiments, the extraction solvent comprises water and ethanol in a weight or volume ratio of about 50:50. In some embodiments, the extraction solvent comprises water and ethanol in a volume ratio of about 50:50. In some embodiments, the extraction solvent comprises more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, or more than 99% water by weight. In some embodiments, the extraction solvent is water.

[0236]

[0265] In some embodiments, the methods described herein involve varying extraction times and temperatures. One of skill in the art can select an appropriate extraction solvent, extraction time, and extraction temperature based on the chemical and physical properties of the oligosaccharides and nutritional composition. For example, in some embodiments, the extraction temperature is about 20 to about 100°C, about 30 to about 95°C, about 40 to about 95°C, about 50 to about 95°C, about 60 to about 95°C, about 70 to about 95°C, about 80 to about 895°C, about 60 to about 90°C, about 70 to about 90°C, or about 75 to about 95°C. In some embodiments, the extraction temperature is about 60 to about 90°C. In some embodiments, the extraction temperature is about 80°C. For example, in certain embodiments, the extraction solvent is heated to the desired extraction temperature.

[0237]

[0266] Depending on the dissolution rate, some extraction solvents and / or oligosaccharides may require longer extraction times. In some embodiments, the extraction time is about 5 minutes to 24 hours, 5 minutes to 10 hours, 5 minutes to 5 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 5 minutes to 1 hour, 10 minutes to 10 hours, 10 minutes to 5 hours, 10 minutes to 2 hours, or 10 minutes to 1 hour. In some embodiments, the extraction time is approximately 5 minutes to 1 hour. In some embodiments, the extraction time is about 30 minutes.

[0238]

[0267] In some embodiments, the extraction comprises physically breaking down a sample of the nutritional composition into fine fragments, for example, by grinding in a mill. In some embodiments, the method comprises filtering the extracted oligosaccharides, for example, to remove solids from the solution. In some embodiments, the filtering step can be performed by paper filter, ultrafiltration, microfiltration, centrifugation, sedimentation, or any other separation technique. In some embodiments, the method comprises clarifying the extracted oligosaccharides. In certain embodiments, clarifying the extracted oligosaccharides comprises contacting the extracted oligosaccharide solution with a dye absorber, such as activated carbon or an ion exchange resin. In some embodiments, the method comprises multiple extraction steps. For example, the method can include a solid-liquid extraction step and one or more liquid-liquid and / or solid-liquid extraction steps.

[0239] [concentration]

[0268] In some embodiments, the methods described herein include concentrating at least a portion of the extracted oligosaccharides. In some embodiments, the concentration of the extracted oligosaccharides increases by more than 1000-fold, more than 500-fold, more than 100-fold, more than 10-fold, or more than 5-fold after the concentration step. In some embodiments, the concentration of the extracted oligosaccharides increases by more than 100-fold after the concentration step.

[0240]

[0269] In some embodiments, the concentrating step comprises lyophilization, vacuum distillation, membrane separation, solvent extraction, evaporation of the solvent in the oligosaccharide solution at room temperature or elevated temperature, or any combination thereof. In some embodiments, the concentrating step comprises lyophilization. In some embodiments, the concentrating step comprises evaporation of the solvent in the oligosaccharide solution under vacuum and / or elevated temperature. In some embodiments, the concentrating step comprises nanofiltration. In some embodiments, the concentrating step comprises concentrating DP1 anhydrosubunit-containing oligosaccharides using nanofiltration. In some embodiments, the concentrating step comprises concentrating DP2 anhydrosubunit-containing oligosaccharides using nanofiltration. In some embodiments, the concentrating step comprises concentrating DP3 anhydrosubunit-containing oligosaccharides using nanofiltration.

[0241]

[0270] In some embodiments, the extracted and enriched oligosaccharides have enriched DP1, DP2, DP3, DP4, or DP5 fractions. In some embodiments, the extracted and enriched oligosaccharides have enriched anhydro-subunit-containing oligosaccharides.

[0242]

[0271] In some embodiments, the methods described herein include introducing an internal standard into the extracted or enriched oligosaccharides. For example, the internal standard is introduced for MS quantitation purposes. In some embodiments, the internal standard is 13 In some embodiments, the internal standard is an isotopically labeled substance, such as a synthetic oligosaccharide preparation containing C. 13C is a gluco-oligosaccharide synthesized from glucose.

[0243] [digestion]

[0272] In some embodiments, the methods described herein include selectively degrading a carbohydrate source in a basal nutritional composition. For example, naturally occurring carbohydrate sources in a basal nutritional composition may be more susceptible to enzymatic hydrolysis than synthetic oligosaccharide preparations. Thus, in some embodiments, the methods described herein include digesting at least a portion of the extracted or enriched oligosaccharides with one or more hydrolases. In some embodiments, the one or more hydrolases can include any enzyme that promotes hydrolysis of naturally occurring polysaccharides. In some embodiments, the one or more hydrolases can include any enzyme that cleaves one or more naturally occurring glycosidic bonds. In some embodiments, the one or more hydrolases selectively cleave α-(1,4) glycosidic bonds or naturally occurring α-(1,4) glycosidic bonds. In some embodiments, the one or more hydrolytic enzymes comprise a carbohydratase, a protease, a lipase, an amylase (e.g., α-amylase and β-amylase), amyloglycosidase, an invertase, α-galactosidase, a cellulase, a xylanase, a chitinase, a lysozyme, a glucoamylase, a pullulanase, or any combination thereof. In some embodiments, the one or more hydrolytic enzymes comprise a carbohydratase, a protease, a lipase, or any combination thereof. In some embodiments, the one or more hydrolytic enzymes comprise an α-amylase, amyloglycosidase, an invertase, α-galactosidase, or any combination thereof.

[0244]

[0273] In some embodiments, the concentration of the one or more hydrolases is about 0.1-40 U / mL, 0.1-20 U / mL, 0.5-20 U / mL, 0.5-15 U / mL, 0.5-10 U / mL, 0.5-9 U / mL, 0.5-8 U / mL, 0.5-7 U / mL, 0.5-6 U / mL, 0.5-5 U / mL, 0.5-4 U / mL, 1-10 U / mL, 1-9 U / mL, 1-8 U / mL, 1-7 U / mL, 1-6 U / mL, 1-5 U / mL, 2-5 U / mL, or 3-4 U / mL for each enzyme. In some embodiments, the concentration of the one or more hydrolases is about 1-10 U / mL for each enzyme. In some embodiments, the concentration of the one or more hydrolases is about 3-4 U / mL for each enzyme.

[0245]

[0274] Depending on the type and concentration of enzyme and other digestion conditions, the digestion time can vary. In some embodiments, the digestion time is about 10 minutes to 24 hours, 30 minutes to 12 hours, 1 hour to 12 hours, 2 hours to 11 hours, 3 hours to 10 hours, 4 hours to 12 hours, 4 hours to 11 hours, 4 hours to 10 hours, 4 hours to 9 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours to 6 hours, 3 hours to 8 hours, or 3 hours to 5 hours. In some embodiments, the digestion time is about 4 hours to 12 hours. In some embodiments, the digestion time is about 4 hours.

[0246]

[0275] In certain embodiments, changes in digestion temperature can affect the digestion rate of the enzyme. In some embodiments, the digestion temperature is about 20-100°C, 30-90°C, 40-80°C, 50-70°C, or 55-65°C. In some embodiments, the digestion temperature is about 40-80°C. In some embodiments, the digestion temperature is about 60°C.

[0247]

[0276] In some embodiments, the method includes multiple digestion steps, for example, the digestion steps can be repeated multiple times until all or substantially all of the carbohydrate source from the basal nutritional composition is hydrolyzed.

[0248]

[0277] In certain embodiments, the method includes reducing the extracted or enriched oligosaccharides. In some embodiments, reducing the oligosaccharides to their respective alditols can result in a simpler chromatogram, i.e., sharper peaks, and therefore more sensitive detection. In some embodiments, the extracted or enriched oligosaccharides are reduced with one or more reducing agents, such as sodium borohydride (NaBH), ferrous sulfate, sulfur dioxide, dithionate, thiosulfate, iodide, hydrazine, and ascorbic acid. In some embodiments, the extracted or enriched oligosaccharides are reduced with sodium borohydride.

[0249] [Separation, isolation and quantification]

[0278] In some embodiments, the methods described herein include a separation and / or isolation step. In some embodiments, the methods described herein include a step of separating at least a portion of the extracted, enriched, digested, or reduced oligosaccharides. In some embodiments, the methods described herein include a step of separating extracted oligosaccharides. In some embodiments, the methods described herein include a step of separating enriched oligosaccharides. In some embodiments, the methods described herein include a step of separating digested oligosaccharides. In some embodiments, the methods described herein include a step of separating reduced oligosaccharides.

[0250]

[0279] Oligosaccharides can be separated by any suitable means. For example, in some embodiments, oligosaccharides are separated by flash chromatography, low, medium, or high pressure liquid chromatography, ultrafiltration, membrane filtration, reverse osmosis, or any combination thereof. In some embodiments, oligosaccharides are separated by flash chromatography. In some embodiments, oligosaccharides are separated by chromatography. In some embodiments, oligosaccharides are separated by precipitation. In some embodiments, oligosaccharides are separated by nanofiltration. In some embodiments, DP1 anhydrosubunit-containing oligosaccharides or DP1 oligosaccharides are separated by nanofiltration. In some embodiments, DP2 anhydrosubunit-containing oligosaccharides or DP1 oligosaccharides are separated by nanofiltration. In some embodiments, DP3 anhydrosubunit-containing oligosaccharides or DP1 oligosaccharides are separated by nanofiltration.

[0251]

[0280] In some embodiments, the methods described herein include isolating at least a portion of the extracted, concentrated, undigested, reduced, or separated oligosaccharides. In some embodiments, the methods described herein include isolating undigested oligosaccharides. In some embodiments, the methods described herein include isolating separated oligosaccharides. In some embodiments, the methods described herein include isolating each fraction of the separated oligosaccharides. In some embodiments, the methods include isolating anhydrosubunit-containing oligosaccharides. In some embodiments, the methods include isolating a specific fraction of anhydrosubunit-containing oligosaccharides, such as DP1 or DP2 anhydrosubunit-containing oligosaccharides. In some embodiments, isolating oligosaccharides can include enriching and / or combining oligosaccharides with similar characteristics, e.g., degree of polymerization.

[0252]

[0281] In certain embodiments, oligosaccharides are separated and / or isolated by degree of polymerization. In some embodiments, the method comprises isolating and / or separating oligosaccharides having a degree of polymerization of 1, 2, 3, 4, or 5. In some embodiments, at least a portion of the oligosaccharides in the DP1 fraction are isolated and / or separated. In some embodiments, at least a portion of the oligosaccharides in the DP2 fraction are isolated and / or separated. In some embodiments, at least a portion of the oligosaccharides in the DP3 fraction are isolated and / or separated. In some embodiments, at least a portion of the oligosaccharides in the DP4 fraction are isolated and / or separated. In some embodiments, at least a portion of the oligosaccharides in the DP5 fraction are isolated and / or separated. In some embodiments, two or more fractions of oligosaccharides are isolated and / or separated. In some embodiments, oligosaccharides in the DP1 and DP2 fractions are isolated and / or separated. In some embodiments, oligosaccharides in the DP1, DP2, and DP3 fractions are isolated and / or separated.

[0253]

[0282] In certain embodiments, oligosaccharides are separated and / or isolated based on whether they contain anhydro subunits. In some embodiments, the method comprises isolating and / or separating anhydro subunit-containing oligosaccharides having a degree of polymerization of 1, 2, 3, 4, or 5. In some embodiments, at least a portion of the anhydro subunit-containing oligosaccharides in the DP1 fraction are isolated and / or separated. In some embodiments, at least a portion of the anhydro subunit-containing oligosaccharides in the DP2 fraction are isolated and / or separated. In some embodiments, at least a portion of the anhydro subunit-containing oligosaccharides in the DP3 fraction are isolated and / or separated. In some embodiments, at least a portion of the anhydro subunit-containing oligosaccharides in the DP4 fraction are isolated and / or separated. In some embodiments, at least a portion of the anhydro subunit-containing oligosaccharides in the DP5 fraction are isolated and / or separated. In some embodiments, two or more fractions of anhydro subunit-containing oligosaccharides are isolated and / or separated.

[0254]

[0283] In certain embodiments, the methods described herein include a quantification step. In some embodiments, at least a portion of the digested, undigested, extracted, enriched, separated, and / or isolated oligosaccharides are quantified. In some embodiments, the separated and / or isolated oligosaccharides are quantified. In some embodiments, the isolated oligosaccharides are quantified.

[0255]

[0284] In some embodiments, oligosaccharides are quantified by weight, concentration, and / or relative abundance. In some embodiments, oligosaccharides are quantified by weight. In some embodiments, at least a portion of the oligosaccharides in the DP2 fraction are isolated and then quantified by weight. In some embodiments, at least a portion of the anhydro-subunit-containing oligosaccharides in the DP2 fraction are isolated and then quantified by weight. In some embodiments, the anhydro-subunit-containing oligosaccharides within the isolated oligosaccharides are quantified by relative abundance, for example, using mass spectrometry. For example, the anhydro-subunit DP2-containing oligosaccharides in the isolated DP2 fraction are quantified by relative abundance as indicated by mass spectrometry.

[0256]

[0285] In some embodiments, the majority of the quantified oligosaccharides are derived from the oligosaccharide preparation. In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the oligosaccharides quantified by weight are derived from the oligosaccharide preparation. In some embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the oligosaccharides quantified by relative abundance are derived from the oligosaccharide preparation.

[0257] [Signal Analysis]

[0286] In some embodiments, the methods described herein include analyzing at least a portion of the extracted, digested, undigested, separated, or enriched oligosaccharides to obtain a signal. In some embodiments, the methods described herein include analyzing the separated oligosaccharides using an analytical device. In some embodiments, the methods described herein include analyzing the isolated oligosaccharides using an analytical device. In some embodiments, the methods described herein include analyzing the digested oligosaccharides using an analytical device. In some embodiments, the methods described herein include analyzing the quantified oligosaccharides using an analytical device.

[0258]

[0287] In some cases, a derivatization step of oligosaccharides is performed either before or during their analysis by various instruments. The derivatization step can be performed to modify the chemical and / or physical properties of oligosaccharides to facilitate their quantification or separation. During derivatization, one or more functional groups or tagging groups can be attached to the oligosaccharide. The derivatization step can include chemical reactions that add polar or non-polar groups to the oligosaccharide; for example, chemical reactions such as silylation, acylation, alkylation, esterification, and transesterification can be performed. Derivatization methods are described in the art, for example, in Ruiz-Matute et al., J. Chromatography B, vol. 879(17-18), 1226-1240 and S. Ahuja, J. Pharmaceutical Sciences, vol. 65(2), February 1976, 163-182, which are incorporated herein by reference in their entireties. In some embodiments, the derivatization step is performed before signal detection.

[0259]

[0288] In some embodiments, the methods described herein comprise analyzing glycosidic linkages of oligosaccharides by NMR, thereby determining or detecting one or more described signals (e.g., levels of a plurality of signals, a first signal and a second signal) associated with the corresponding glycosidic linkages. 1H NMR, 1D 13 The method includes analyzing glycosidic linkages by C NMR, 2D NMR, such as 2D JRES, HSQC, HMBC, COSY, ECOSY, TOCSY, NOESY, and ROESY, or a combination thereof. In some embodiments, the glycosidic linkages analyzed are α-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, α-(1,6) glycosidic linkages, β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4) glycosidic linkages, β-(1,6) glycosidic linkages, α-(1,1)-α glycosidic linkages, α-(1,1)-β glycosidic linkages, β-(1,1)-α glycosidic linkages, or β-(1,1)-β glycosidic linkages of oligosaccharides.

[0260]

[0289] In some embodiments, the method comprises analyzing the α-(1,2) glycosidic linkages of the oligosaccharide by NMR, thereby determining or detecting the described signals associated with the α-(1,2) glycosidic linkages. In some embodiments, the method comprises analyzing the α-(1,3) glycosidic linkages of the oligosaccharide by NMR, thereby determining or detecting the described signals associated with the α-(1,3) glycosidic linkages. In some embodiments, the method comprises analyzing the α-(1,6) glycosidic linkages of the oligosaccharide by NMR, thereby determining or detecting the described signals associated with the α-(1,6) glycosidic linkages. In some embodiments, the method comprises analyzing the β-(1,2) glycosidic linkages of the oligosaccharide by NMR, thereby determining or detecting the described signals associated with the β-(1,2) glycosidic linkages. In some embodiments, the method comprises analyzing the β-(1,3) glycosidic linkages of the oligosaccharide by NMR, thereby determining or detecting the described signals associated with the β-(1,3) glycosidic linkages. In some embodiments, the method comprises analyzing the β-(1,4) glycosidic linkages of the oligosaccharide by NMR, thereby determining or detecting the described signals associated with the β-(1,4) glycosidic linkages. In some embodiments, the method comprises analyzing the β-(1,6) glycosidic linkages of the oligosaccharide by NMR, thereby determining or detecting the described signals associated with the β-(1,6) glycosidic linkages.

[0261]

[0290] In some embodiments, the methods described herein comprise analyzing anhydro-subunit-containing oligosaccharides to thereby determine or detect a signal (e.g., a plurality of signals, a first signal, and a second signal level) indicative of one or more anhydro-subunit-containing oligosaccharides. In some embodiments, the anhydro-subunit-containing oligosaccharides are analyzed by gravimetric determination, HPLC, NMR, SEC, mass spectrometry, such as LC-MS / MS, GC-MS, and MALDI-MS, or a combination thereof. In some embodiments, the anhydro-subunit-containing oligosaccharides are analyzed by LC-MS / MS. In some embodiments, the anhydro-subunit-containing oligosaccharides are analyzed by GC-MS. In some embodiments, the anhydro-subunit-containing oligosaccharides are analyzed by MALDI-MS. In some embodiments, the anhydro-subunit-containing oligosaccharides are analyzed by HPLC. In some embodiments, the anhydro-subunit-containing oligosaccharides are analyzed by gravimetric determination of fractions isolated and / or purified by liquid chromatography.

[0262]

[0291] In some embodiments, the methods described herein comprise analyzing anhydro-subunit-containing oligosaccharides in any one or more of the DP1 through DPn fractions. In some embodiments, the methods comprise analyzing anhydro-subunit-containing oligosaccharides in any one of the DP1 through DP5 fractions. In some embodiments, the methods comprise analyzing anhydro-subunit-containing oligosaccharides in the DP1 fraction. In some embodiments, the methods comprise analyzing anhydro-subunit-containing oligosaccharides in the DP2 fraction. In some embodiments, the methods comprise analyzing anhydro-subunit-containing oligosaccharides in the DP3 fraction. In some embodiments, the methods comprise analyzing anhydro-subunit-containing oligosaccharides in the DP4 fraction. In some embodiments, the methods comprise analyzing anhydro-subunit-containing oligosaccharides in the DP5 fraction.

[0263]

[0292] In some embodiments, the methods described herein comprise analyzing the DP distribution of oligosaccharides, thereby determining or detecting a described signal (e.g., the level of a plurality of signals, a first signal, and a second signal) associated with the DP distribution. In some embodiments, the described signal is provided by HPLC or SEC.

[0264]

[0293] In some embodiments, analyzing the DP distribution of oligosaccharides comprises determining, detecting, or quantifying oligosaccharides in one or more of the DP1-DPn fractions. In some embodiments, analyzing the DP distribution of oligosaccharides comprises determining, detecting, or quantifying oligosaccharides in one of the DP1-DP5 fractions. In some embodiments, analyzing the DP distribution of oligosaccharides comprises determining, detecting, or quantifying oligosaccharides in the DP1 fraction. In some embodiments, analyzing the DP distribution of oligosaccharides comprises determining, detecting, or quantifying oligosaccharides in the DP2 fraction. In some embodiments, analyzing the DP distribution of oligosaccharides comprises determining, detecting, or quantifying oligosaccharides in the DP3 fraction.

[0265]

[0294] In some embodiments, the method comprises quantifying at least a portion of the oligosaccharides in any one of the DP1-DPn fractions. In some embodiments, the method comprises quantifying at least a portion of the oligosaccharides in any one of the DP1-DP5 fractions. In some embodiments, the method comprises quantifying at least a portion of the oligosaccharides in the DP1 fraction. In some embodiments, the method comprises quantifying at least a portion of the oligosaccharides in the DP2 fraction. In some embodiments, the method comprises quantifying at least a portion of the oligosaccharides in the DP3 fraction.

[0266]

[0295] Depending on the analytical device, the methods described herein may include various variations. In some variations, the method includes detecting the presence or absence of a signal. In some embodiments, the detecting step can be performed manually, automatically, e.g., by a machine, or any combination thereof. In some variations, the method includes determining a characteristic of the signal, including, but not limited to, the intensity, strength, shape, and / or area of ​​the signal. In some embodiments, determining a characteristic of the signal includes determining the presence or absence of a signal. In some embodiments, determining a characteristic of the signal includes determining the level of the signal.

[0267]

[0296] For example, in some embodiments, the absence of a DP2 anhydrosubunit-containing oligosaccharide signal in mass spectrometry indicates that a sample of a nutritional composition does not contain an oligosaccharide preparation or does not contain detectable levels of an oligosaccharide preparation. As another example, in some embodiments, the presence and / or level of a DP2 anhydrosubunit-containing oligosaccharide signal in mass spectrometry indicates that a sample of a nutritional composition may contain a level of an oligosaccharide preparation corresponding to the level of the signal. As yet another example, in some embodiments, the presence and / or level of an α-(1,6) glycosidic bond signal in an NMR spectrum indicates that a sample of a nutritional composition may contain a level of an oligosaccharide preparation corresponding to the level of the signal.

[0268]

[0297] In some embodiments, the method includes correlating (e.g., calculating or estimating) the concentration of an oligosaccharide preparation in a nutritional composition based on the described signals (e.g., the level of a particular signal, a first signal, and a second signal). For example, the relative abundance of oligosaccharides can be correlated based on mass spectrometry signals. In some embodiments, the concentration and / or relative abundance of oligosaccharides can be correlated based on SEC, HLPC, and / or NMR signals.

[0269]

[0298] In some embodiments, the oligosaccharide preparation can be analyzed to calculate or estimate its concentration in the nutritional composition. For example, in some embodiments, the level of anhydro-subunit-containing oligosaccharides in the oligosaccharide preparation is calculated when the quality control step determines or detects signals indicative of anhydro-subunit-containing oligosaccharides. As another example, in some embodiments, the DP distribution of the oligosaccharide preparation is determined when the quality control step determines or detects signals associated with the DP distribution of the oligosaccharides. As yet another example, in some embodiments, the relative abundance of α-(1,6) glycosidic linkages in the oligosaccharide preparation is determined when the quality control step determines or detects signals associated with α-(1,6) glycosidic linkages.

[0270]

[0299] In some embodiments, the method includes comparing the signals, where any characteristic of the signals can be compared. For example, in some embodiments, the method includes comparing a first signal to a second signal. In some embodiments, the method includes comparing the level of the first signal to the level of the second signal. In some embodiments, a difference between the first signal and the second signal indicates uneven distribution of the oligosaccharide preparation in the nutritional composition. In other embodiments, a similarity between the first signal and the second signal indicates consistent distribution of the oligosaccharide preparation in the nutritional composition.

[0271]

[0300] It should be understood that the quality control methods provided herein can include any combination of the steps and embodiments described above. Furthermore, in some embodiments, the quality control methods are repeated. In certain embodiments, the quality control methods are performed on multiple samples taken from the same or different batches of nutritional composition.

[0272]

[0301] In certain embodiments, the first and second samples are taken from the same batch of nutritional composition. In certain embodiments, the first and second samples are taken from different batches of nutritional composition. In some embodiments, the first and second samples are taken from the same or different manufacturing facilities. In some embodiments, the first and second samples are taken at the same or different time points. In certain embodiments, the second sample is taken more than one day, more than one week, more than one month, more than six months, or more than one year after the first sample is taken. In certain embodiments, the first and second samples are taken within a time period of one day, one week, one month, six months, or one year. In some embodiments, the first and second samples are taken from different locations in the same mixer that combines the oligosaccharide preparation and the basal nutritional composition. In some embodiments, the first sample is taken during the mixing process of the oligosaccharide preparation and the nutritional composition, and the second sample is taken after mixing.

[0273]

[0302] In some embodiments, a detection limit exists for the methods described herein, where levels of the oligosaccharide preparation above the detection limit cannot be detected by the method. In some embodiments, the detection limit of the oligosaccharide preparation is greater than 1 ppm, 5 ppm, 10 ppm, 50 ppm, 100 ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, or 1000 ppm of the nutritional composition. In some embodiments, the detection limit of the oligosaccharide preparation is greater than 10 ppm, 50 ppm, 100 ppm, or 500 ppm of the nutritional composition.

[0274] [Additional Steps]

[0303] In some embodiments, the methods described herein include a step of passing or rejecting a batch of nutritional composition. In some embodiments, after a quality control step (or after performing a method described herein), the batch of nutritional composition is passed or rejected. In some embodiments, the passing or rejecting step is performed manually, automatically, such as by a machine, or a combination thereof.

[0275]

[0304] In some embodiments, the passing or failing step is based in whole or in part on the presence or absence of a described signal. In other embodiments, the passing or failing step is based in whole or in part on the level of a described signal, and the concentration of the oligosaccharide preparation in the nutritional composition is calculated based on this signal. In some embodiments, the passing or failing step is based in whole or in part on a comparison of the first signal and the second signal.

[0276]

[0305] In further embodiments, the passing or rejecting step is based in whole or in part on a predetermined concentration range of the oligosaccharide preparation in the nutritional composition. For example, the predetermined concentration range may vary depending on the particular animal feed composition. By way of example, in some embodiments, a batch of nutritional composition may be rejected if the signal level indicates that the oligosaccharide preparation is not within 10-1000 ppm, 10-500 ppm, or 50-500 ppm of the nutritional composition.

[0277]

[0306] In some embodiments, the methods described herein include adjusting the level of the oligosaccharide preparation after the determination or detection, e.g., after a quality control step. In some embodiments, adjusting the level of the oligosaccharide preparation includes adjusting the level of a basal nutritional composition, adjusting the level of the oligosaccharide preparation, or a combination thereof. In some embodiments, adjusting the level of the oligosaccharide preparation includes adding an additional oligosaccharide preparation to the nutritional composition or removing a portion of the oligosaccharide preparation from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide preparation includes adding an additional basal nutritional composition to the nutritional composition or removing a portion of the basal nutritional composition from the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide preparation includes adding an additional oligosaccharide preparation to the nutritional composition. In some embodiments, adjusting the level of the oligosaccharide preparation includes mixing the nutritional composition to increase consistency. In certain embodiments, the method includes adjusting the level of the oligosaccharide preparation to a predetermined range, which may vary depending on the specific animal feed composition.

[0278]

[0307] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the invention, as set forth in the appended claims.

[0279]

[0308] The present invention will be further described in the following examples, which are given for illustrative purposes only and are not intended to limit the invention in any way.

[0280] [Example] Example 1: Synthesis of gluco-galacto-oligosaccharide preparations

[0309] The synthesis of the gluco-galacto-oligosaccharide preparations was carried out in a 3 liter reactor using catalyst loading, reaction time and reaction temperature selected to allow suitable production on a kg scale.

[0281]

[0310] D-Glucose monohydrate (825.16 g), D-lactose monohydrate (263.48 g), and 2-pyridinesulfonic acid (1.0079 g, Sigma-Aldrich, St. Louis, US) were added to a 3-liter, three-neck round-bottom flask equipped with a 29 / 42 center ground joint and two 24 / 40 side ground joints. A 133 mm Teflon stirring blade was secured to a glass stirring shaft using PTFE tape. The stirring rod was secured via its center point using a Teflon bearing adapter and attached to an overhead high-torque mechanical mixer via a flexible coupler. The flask was secured within a hemispherical electric heating mantle operated by a temperature control unit via a J-type wand thermocouple inserted through a rubber septum in one of the side ports. The tip of the thermocouple was adjusted so that it was positioned within the reaction mixture with a gap of several mm above the mixing element. A second temperature probe connected to an auxiliary temperature monitor was inserted and secured in the same manner. A second side port of the flask was fitted with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating bath refrigeration unit.

[0282]

[0311] The reaction mixture was gradually heated to 130°C with continuous mixing at a stirring rate of 80-100 rpm. When the reaction mixture reached 120°C, the reflux condenser was replaced in the distillation setup, and the distillate was collected in a 250 mL round-bottom flask placed in an ice bath. The mixture was maintained at 130°C with continuous mixing for 6 hours, after which the thermocouple box was turned off. The distillation setup was removed, and 390 g of 60°C distilled water was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 RPM for 10 hours. Approximately 1,250 g of a viscous, light amber material was collected, with a refractive index of 71.6 Brix.

[0283] Example 2: Synthesis of gluco-oligosaccharide preparations

[0312] The synthesis of gluco-oligosaccharide preparations was carried out in a 3 liter reactor using catalyst loading, reaction time and reaction temperature selected to allow suitable production on a kg scale.

[0284]

[0313] D-glucose monohydrate (1,150 g) was added to a 3-liter, three-necked round-bottom flask equipped with one central 29 / 42 ground joint and two side 24 / 40 ground joints. A 133 mm Teflon stirring blade was secured to a glass stirring shaft using PTFE tape. The stirring rod was secured through the flask's center point using a Teflon bearing adapter and attached to an overhead high-torque mechanical mixer via a flex coupling. The flask was secured within a hemispherical electric heating mantle operated by a temperature control unit via a J-type wand thermocouple inserted into a rubber septum in one of the side ports. The tip of the thermocouple was adjusted so that it was positioned within the reaction mixture with a gap of several mm above the mixing element. A second temperature probe connected to an auxiliary temperature monitor was inserted and secured in the same manner. The second side port of the flask was fitted with a reflux condenser cooled by a water-glycol mixture maintained at less than 4°C using a recirculating bath chiller.

[0285]

[0314] The reaction mixture was gradually heated to 130 °C with continuous mixing at a stirring rate of 80-100 rpm. When the reaction temperature rose to between 120 °C and 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 a reflux condenser to a distillation setup with a round-bottom collection flask placed in an ice bath. After maintaining this setup for 1 hour and 30 minutes, the thermocouple box was turned off, the distillation apparatus was removed, and 390 g of 23 °C distilled water was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 hours until collection. Approximately 1300 g of a viscous, dark amber material was collected and measured to have a density of 72.6 Brix.

[0286] Example 3: Synthesis of gluco-galacto-manno-oligosaccharide preparations

[0315] The synthesis of the gluco-galacto-manno-oligosaccharide preparations was carried out in a 3 liter reactor using catalyst loading, reaction time and reaction temperature selected to allow suitable production on a kg scale. MH47-32-A / MH46-35-B: 8 / 10 / 18

[0287]

[0316] The gluco-galacto-manno-oligosaccharide preparation was prepared as two separate components synthesized in separate, independently harvested reaction vessels. Each synthesis used different starting reactants but followed the same procedures and methods until completion. The final gluco-galacto-manno-oligosaccharide preparation was a homogenous syrup formed from the mixture of the products of both syntheses.

[0288]

[0317] For the synthesis of the first component, 990.54 g of glucose monohydrate, 105.58 g of lactose monohydrate, and 1.00 g of 2-pyridinesulfonic acid were added to a 3-liter, three-necked round-bottom flask equipped with a central 29 / 42 ground joint flanked by two 24 / 40 ground joints. A 133 mm Teflon stirring blade was secured to a 440 mm glass stirring shaft using PTFE tape. The stirring rod was secured via its center point using a Teflon bearing adapter and attached to an overhead high-torque mechanical mixer via a flexible coupler. The flask was placed in a hemispherical electric heating mantle operated by a temperature control unit via a J-type wand thermocouple inserted through a rubber septum in one of the side ports. The tip of the thermocouple was adjusted so that it was positioned within the reaction mixture with a gap of a few millimeters above the mixing element. A second temperature probe connected to an auxiliary temperature monitor was inserted and secured in the same manner. A second side port of the flask was fitted with a reflux condenser cooled by a water-glycol mixture maintained below 4°C by a recirculating bath refrigeration unit.

[0289]

[0318] The reaction mixture was gradually heated to 130°C with continuous mixing at a stirring speed of 80-100 rpm. When a temperature control box reading between 120°C and 130°C was observed, the apparatus was switched from the reflux condenser to a distillation setup with a round-bottom collection flask placed in an ice bath. This setup 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 60°C distilled water was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 hours until collection. Approximately 1250 g of a viscous, light amber material was collected, with a refractive index of 73.1 Brix.

[0290]

[0319] For the synthesis of the second component, 825.04 g of glucose monohydrate, 251.16 g of pure wood-derived mannose, 25.10 g of distilled water, and 1.00 g of 2-pyridinesulfonic acid were added to a 3-liter, three-necked round-bottom flask equipped with a central 29 / 42 grinding joint flanked by two 24 / 40 grinding joints. The remainder of the synthesis of the second component followed the same procedures and methods as the first, up to the moment of collection. Approximately 1250 g of a viscous, dark amber material was collected and measured to have a density of 72.3 Brix.

[0291]

[0320] The entire first and second components were transferred to an appropriately sized 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 measured to have a consistency of approximately 72 Brix.

[0292] Example 4: Synthesis of gluco-manno-oligosaccharide preparations

[0321] The synthesis of gluco-oligosaccharide preparations was carried out in a 3 liter reactor using catalyst loading, reaction time and reaction temperature selected to allow suitable production on a kg scale.

[0293]

[0322] The gluco-manno-oligosaccharide preparation was prepared as two separate components synthesized in separate, independently harvested reaction vessels. Each synthesis used different starting reactants but followed the same procedures and methods until completion. The final gluco-manno-oligosaccharide preparation was a homogenous syrup formed from the mixture of the products of both syntheses.

[0294]

[0323] For the synthesis of the first component, 1264.80 g of glucose monohydrate was added to a 3-liter, three-neck round-bottom flask equipped with a central 29 / 42 ground joint flanked by two 24 / 40 ground joints. A 133 mm Teflon stirring blade was secured to a 440 mm glass stirring shaft using PTFE tape. The stirring rod was secured via a center point using a Teflon bearing adapter and attached to an overhead high-torque mechanical mixer via a flexible coupler. The flask was placed in a hemispherical electric heating mantle operated by a temperature control unit via a J-type wand thermocouple inserted into a rubber septum in one of the side ports. The tip of the thermocouple was adjusted so that it was positioned within the reaction mixture with a gap of a few mm above the mixing element. A second temperature probe connected to an auxiliary temperature monitor was inserted and secured in the same manner. The second side port of the flask was fitted with a reflux condenser cooled by a water-glycol mixture maintained at below 4 °C by a recirculating bath chiller.

[0295]

[0324] The reaction mixture was gradually heated to 130 °C with continuous mixing at a stirring speed of 80-100 rpm. When a temperature control box reading between 120 °C and 130 °C was observed, 1.15 g of (+)-camphor-10-sulfonic acid was added to the three-neck flask, and the apparatus was switched from a reflux condenser to a distillation setup with a round-bottom collection flask placed in an ice bath. After maintaining this setup for approximately 1 hour, the thermocouple box was turned off, the distillation apparatus was removed, and 390 g of 23 °C distilled water was gradually added to the three-neck flask. The resulting mixture was left stirring at 40 rpm for 10 hours until collection. Approximately 1350 g of a viscous, light amber material was collected and measured to have a density of 71.8 Brix.

[0296]

[0325] For the synthesis of the second component, 949.00 g of glucose monohydrate, 288.00 g of pure wood-derived mannose, 27.94 g of distilled water, and 1.15 g of 2-pyridinesulfonic acid were added to a 3-liter, three-necked round-bottom flask equipped with a central 29 / 42 ground joint flanked by two 24 / 40 ground joints. The remainder of the synthesis of the second component followed the same procedure and method as the first, up to the moment of collection, except that no (+)-camphor-10-sulfonic acid was added, as the reflux condenser was switched to a distillation configuration and the resulting setup was maintained for approximately 6 hours. Approximately 1350 g of a viscous, dark amber material was collected and measured to have a density of 72.0 Brix.

[0297]

[0326] The entire first and second components were transferred to an appropriately sized HDPE container and thoroughly mixed by hand until homogeneous. The final syrup mixture weighed approximately 2.7 kg, was dark amber in color, viscous, and measured to have a refractive index of approximately 72 Brix.

[0298] Example 5: Synthesis of gluco-manno-oligosaccharide preparations

[0327] Kilogram-scale production of oligosaccharide preparations was carried ou...

Claims

1. 1. A method for correlating anhydro-subunit-containing oligosaccharides in a synthetic oligosaccharide preparation in a nutritional composition, comprising: the nutritional composition comprises the synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition, wherein the naturally occurring oligosaccharide composition has an anhydro-subunit-containing oligosaccharide content of less than 1 ppm; The method comprises: a. providing a sample of the nutritional composition; b. detecting a signal of at least a portion of an oligosaccharide in a sample of said nutritional composition; and c) correlating the level of the signal with the concentration of the anhydro-subunit-containing oligosaccharide in the nutritional composition by comparing the level of the signal with a standard calibration curve. Including, The signal is indicative of one or more anhydrosubunit-containing oligosaccharides.

2. 1. A method for performing quality control of a nutritional composition, comprising: a) providing a batch of the nutritional composition, the nutritional composition comprising a synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition, wherein the naturally occurring oligosaccharide composition contains less than 1 ppm of anhydro-subunit-containing oligosaccharides; b. Obtaining a sample of said nutritional composition from said batch; c. detecting a signal of at least a portion of the oligosaccharides in the sample of the nutritional composition using an analytical device; and d. Passing or failing the batch of nutritional composition based on the presence or absence of the signal or the level of the signal. Including, The signal is indicative of one or more anhydrosubunit-containing oligosaccharides.

3. 3. The method of claim 1 or 2, wherein the one or more anhydro-subunit-containing oligosaccharides have a degree of polymerization of 2 (DP2).

4. 4. The method of any one of claims 1 to 3, wherein the signal is determined by high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), nuclear magnetic resonance (NMR) spectroscopy, size exclusion chromatography (SEC), field-flow fractionation (FFF), asymmetric flow field-flow fractionation (A4F), gravimetric determination of fractions by preparative chromatography, or any combination thereof.

5. The method of any one of claims 1 to 4, wherein the nutritional composition comprises a basal nutritional composition.

6. 1. A method for performing quality control of a nutritional composition comprising a synthetic oligosaccharide preparation and a naturally occurring oligosaccharide composition, the method comprising: a. providing a first sample of the nutritional composition; b. providing a second sample of said nutritional composition; c. detecting a first signal of at least a portion of the oligosaccharides in the first sample; d. detecting a second signal of at least a portion of the oligosaccharides in the second sample; and e. Comparing the first signal with the second signal. Including, The method, wherein the first signal and the second signal each represent one or more anhydro-subunit-containing oligosaccharides, and the first signal and the second signal belong to the same species of anhydro-subunit-containing oligosaccharides.

7. The method described in claim 6, comprising a step of correlating the level of a first signal with the concentration of the anhydro subunit-containing oligosaccharide in the nutritional composition of the first sample, a step of correlating the level of a second signal with the concentration of the anhydro subunit-containing oligosaccharide in the nutritional composition of the second sample, or both.

8. 1. A method for correlating anhydro-subunit-containing oligosaccharides in a synthetic oligosaccharide preparation in a nutritional composition, comprising: The nutritional composition comprises: (i) a synthetic oligosaccharide preparation comprising anhydro-subunit-containing oligosaccharides; and (ii) a naturally occurring oligosaccharide composition having an anhydro-subunit-containing oligosaccharide content of less than 1 ppm; The method comprises: a. providing a sample of said nutritional composition; b. isolating one or more anhydro-subunit-containing oligosaccharides from the sample; c) detecting a signal indicative of the one or more anhydro-subunit-containing oligosaccharides, comprising (i) determining the weight of at least a portion of the anhydro-subunit-containing oligosaccharides from the sample, or (ii) analyzing at least a portion of the anhydro-subunit-containing oligosaccharides from the sample by matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), liquid chromatography-mass spectrometry (LC-MS) / MS, or gas chromatography (GC)-MS; d. Correlating the level of said signal with the concentration of anhydro-subunit-containing oligosaccharides in said nutritional composition. A method comprising:

9. 9. The method of claim 8, wherein the detecting step comprises determining the weight of at least a portion of the anhydro-subunit-containing oligosaccharides having a degree of polymerization of 1 (DP1) from the sample.

10. 9. The method of claim 8, wherein the detecting step comprises determining the weight of at least a portion of the anhydro-subunit-containing oligosaccharides having a degree of polymerization of 2 (DP2) from the sample.

11. 11. The method of any one of claims 1 to 10, wherein the synthetic oligosaccharide preparation is present in the nutritional composition at a concentration of 1 to 5000 ppm, 11 to 1000 ppm, 1 to 500 ppm, 10 to 5000 ppm, 10 to 2000 ppm, 10 to 1000 ppm, 10 to 500 ppm, 10 to 250 ppm, 10 to 100 ppm, 50 to 5000 ppm, 50 to 2000 ppm, 50 to 1000 ppm, 50 to 500 ppm, 50 to 250 ppm or 50 to 100 ppm.

12. 1. A method for producing a nutritional composition, comprising: a. combining a basal nutritional composition comprising a naturally occurring oligosaccharide composition with a synthetic oligosaccharide preparation comprising anhydro-subunit-containing oligosaccharides; and b) A step of carrying out the quality control method according to any one of claims 2 to 7 and 11. A method comprising:

13. 13. The method of claim 12, wherein the synthetic oligosaccharide preparation comprises at least n fractions of oligosaccharides (DP1 to DPn fractions), each having a different degree of polymerization selected from 1 to n, where n is an integer greater than or equal to 3; and the DP1 and DP2 fractions each independently comprise anhydro-subunit-containing oligosaccharides in a relative abundance of 0.1% to 15% as measured by mass spectrometry (MS).

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