High yield peroxide quenching controlled polysaccharide depolymerization and compositions thereof

Abstract

In one embodiment, a method is provided for cleaving a polysaccharide comprising reacting a polysaccharide with a Fenton reagent, and cleaving the treated polysaccharide with a nitrogen-based cleaving reagent, preferably, the nitrogen-based cleaving reagent is also a peroxide quencher. Synthetic oligosaccharide compositions produced by such polysaccharide cleavage methods are also disclosed. Such oligosaccharide compositions exhibit utility in a variety of aspects including modulation of microbial growth and / or microbial metabolism or host metabolism.

Description

This application is a divisional application of patent application 202080079305.X (based on PCT application No. PCT / US2020 / 060297) entitled "High-yield peroxide quenching-controlled polysaccharide depolymerization and its compositions" filed by the applicant on November 12, 2020. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 935,583, filed on November 14, 2019, the entirety of which is incorporated herein by reference. Technical Field

[0002] The present invention relates generally to polysaccharide depolymerization, and more particularly to polysaccharide depolymerization for producing oligosaccharides, and more particularly to polysaccharide depolymerization using chemical oxidation and cleavage, and even more particularly to a controlled oligosaccharide generation ("COG") process for polysaccharide depolymerization and production of oligosaccharides by oxidizing a polysaccharide material using a Fenton reagent to provide a polysaccharide treated with hydrogen peroxide radicals, followed by peroxide elimination (peroxide quenching) and controlled cleavage of the treated polysaccharide using a compatible peroxide quenching / cleavage reagent (PQC-reagent) to eliminate residual hydrogen peroxide and induce high-yield polysaccharide cleavage while minimizing or eliminating undesirable side reactions. Background Art

[0003] Oligosaccharides are short chains of carbohydrates, usually ranging from 3 to 20 monomers in length. Oligosaccharides have been shown to have a variety of functions (e.g., biologically active functions, etc.), which are affected by many structural properties, such as stereochemistry, branching, degree of polymerization, monosaccharide composition, and glycosidic bond position (Amicucci, Nandita et al. 2019). For example, oligosaccharides (HMOs) from human milk can promote the growth of certain microorganisms in the intestines of infants, while also regulating the immune system, reducing the occurrence of diarrhea, and protecting the host from pathogen adhesion (Morrow, Ruiz-Palacios et al. 2004, LoCascio, Niñuevo et al. 2007, Smilowitz, Lebrilla et al. 2014).

[0004] Currently, biosynthesis is the main tool for large-scale production of human milk oligosaccharides (Merighi et al. 2016, Yu et al. 2018). In addition, apart from HMO and two other common oligosaccharides (galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS)), little work has been done to expand the field of oligosaccharide manufacturing (Gosling et al. 2010, Dominguez et al. 2014). Polysaccharides (e.g., homopolymer polysaccharides or heteropolymer polysaccharides) can contain, for example, up to 100,000 monomer building blocks and are ubiquitous in all organisms, including, for example, plants, mammals, fungi, bacteria, diatoms and algae (Bar-On et al. 2018). Polysaccharides are often used for their rheological properties, but their prebiotic and immunomodulatory potential have recently been explored, however, these properties are limited by their low solubility and intercellular transport (Hamaker and Tuncil 2014). Therefore, soluble and easily transportable oligosaccharides with epitopes similar to their parent polysaccharides may provide a more effective avenue for microbiome modulation and immune regulation.

[0005] The polymerization of oligosaccharides from large polysaccharides can provide the opportunity to produce a large number of oligosaccharides with diverse structures from natural starting materials. Enzymatic methods have been used to produce oligosaccharides from polysaccharides, however, their inherent specificity limits each enzyme to depolymerize only a single type of glycosidic bond, thereby only depolymerizing a very limited number of polysaccharides (Pauly et al. 1999, Bauer et al. 2006). Although chemical methods for depolymerizing polysaccharides are known in the art and are not routinely used, they can provide a more robust and extensive polysaccharide depolymerization pathway.

[0006] For example, oxidation chemistry is often used to modify the molecular weight and functional groups of carbohydrates ( et al. 2015, Sun et al. 2015). Fenton and Fenton-like reactions rely on transition metals and hydrogen peroxide to produce peroxide radicals that can drive many oxidation reactions (Wardman and Candeias 1996). Currently, Fenton reactions have been used on a large scale for wastewater treatment (Wang et al. 2016). Recently, a method has been described that uses Fenton reactions for polysaccharide depolymerization followed by the use of a strong Arrhenius base (Na + OH - , K + OH - or Ca 2+ (OH - ) 2) for cleavage (Amicucci, Park et al. 2018). However, polysaccharide depolymerization using such strong Arrhenius bases as cleavage agents not only requires the use of large-scale dialysis to remove residual post-reaction salts (after neutralization of the strong Arrhenius base), but also "peeling" (Cancilla et al. 1998) and off-target side reactions (e.g., C-6 oxidation produces oligosaccharide-containing uronic acid and other potentially unwanted substances) during the strong Arrhenius base cleavage step and post-cleavage (e.g., dialysis) steps. This is because while the strong Arrhenius base cleavage agent "quenches" the Fenton reaction (i.e., by flocculating the metal ion reactants), such bases do not (as disclosed below) quench / eliminate residual peroxides or peroxide radicals themselves. In addition, such peeling and off-target reactions may become problematic at the optimal peroxide and pH concentrations / conditions used for the peroxidation reaction and cleavage steps, while oligosaccharide yields may be affected if lower concentrations or suboptimal conditions are used. Summary of the invention

[0007] Various aspects of the present disclosure can be described from the following aspects:

[0008] 1. A method for cleaving polysaccharides, comprising: reacting the polysaccharide with a Fenton reagent containing a peroxide agent and a metal ion in a reaction mixture to provide a treated polysaccharide; and The treated polysaccharide is cleaved with a nitrogen-based cleavage reagent to produce at least one polysaccharide cleavage product and / or oligosaccharide having the characteristics of the polysaccharide.

[0009] 2. The method of aspect 1, wherein the cleavage produces a polysaccharide cleavage product mixture and / or an oligosaccharide mixture having the characteristics of the polysaccharide.

[0010] 3. The method of aspect 1 or 2, wherein the Fenton reagent comprises hydrogen peroxide and one or more metals selected from the following: transition metals Fe(II), Fe(III), Cu(I), Cu(II), Mn(II), Zn(II), Ni(II) and Co(II); alkaline earth metals Ca(II) and Mg(II); and lanthanide element Ce(IV).

[0011] 4. The method according to any one of aspects 1 to 3, wherein the nitrogen-based cleavage reagent is one or more selected from ammonium hydroxide, ammonium bicarbonate, ammonia, urea, sodium amide, dimethylamine, trimethylamine, pyridine and N,N-diisopropylethylamine.

[0012] 5. The method according to aspect 4, wherein the nitrogen-based cleavage reagent is one or more selected from ammonium hydroxide, ammonium bicarbonate and ammonia.

[0013] 6. The method of any one of aspects 1-5, wherein the nitrogen-based cleavage reagent is also a peroxide quencher, and the initiation of polysaccharide cleavage is simultaneous or substantially simultaneous with the initiation of peroxide quenching.

[0014] 7. The method of any one of aspects 1-6, wherein the nitrogen-based cleavage agent is not a peroxide quencher, and the method further comprises initiating peroxide quenching with an additional peroxide quencher.

[0015] 8. The method of aspect 7, wherein the additional peroxide quencher comprises a peroxide quenching enzyme or one or more peroxide quenchers listed in Table 1.

[0016] 9. The method of any one of aspects 6-8, wherein the additional peroxide quencher is also an additional polysaccharide cleaving reagent that can cleave the treated polysaccharide.

[0017] 10. The method of any one of Aspects 1-5 and Aspects 7-9, wherein the additional peroxide quencher is introduced before, during, or after the polysaccharide cleavage is initiated with the nitrogen-based cleavage reagent.

[0018] 11. The method of any one of aspects 1-10, further comprising removing the nitrogen-based cleavage reagent and / or quencher, or one or more of their reactive components, by evaporation.

[0019] 12. The method of any one of aspects 1-11, wherein the yield of the oligosaccharide is increased and / or wherein off-target side reactions are reduced and / or stripping is reduced relative to cleavage of the treated polysaccharide with a strong Arrhenius base.

[0020] 13. The method of any of aspects 1-12, wherein the polysaccharide is derived from or is present in the form of at least one material selected from the group consisting of plants, bacteria, yeast, algae, animals, fungi, and waste stream materials.

[0021] 14. The method of aspect 13, wherein the polysaccharide comprises one or more selected from the group consisting of amylose, amylopectin, betaglucan, pullulan, xyloglucan, arabinogalactan I and arabinogalactan II, rhamnogalacturonan I, rhamnogalacturonan II, polygalacturonic acid, polyglucose, galactan, arabinan, arabinoxylan, xylan (e.g., beech xylan), glycogen, mannan, glucomannan, curdlan, galactomannan, lichenan, and inulin.

[0022] 15. The method of any one of aspects 1-14, wherein the reacting and the cleaving change at least one structural property and / or chemical property of the material comprising the polysaccharide, wherein the property is selected from solubility, texture, porosity, permeability, elasticity, rheological properties and chemical reactivity.

[0023] 16. A composition comprising one or more polysaccharide cleavage products, oligosaccharides, or a mixture of polysaccharide cleavage products and / or oligosaccharides produced by the method of any one of aspects 1-15.

[0024] 17. A method of regulating microbial growth and / or microbial metabolism or host metabolism, comprising contacting a microorganism with a composition according to aspect 16 in vitro or in vivo.

[0025] 18. A method for cleaving polysaccharides, comprising:

[0026] reacting the polysaccharide with a Fenton reagent comprising a peroxidizing agent and a metal ion in a reaction mixture to provide a treated polysaccharide; and

[0027] The treated polysaccharide is cleaved with a polysaccharide cleaving agent in the presence of a peroxide quencher to produce at least one polysaccharide cleavage product and / or oligosaccharide having the characteristics of the polysaccharide.

[0028] 19. The method of aspect 18, wherein the cleavage produces a mixture of polysaccharide cleavage products and / or a mixture of oligosaccharides having polysaccharide characteristics.

[0029] 20. The method of aspect 18 or 19, wherein the Fenton reagent comprises one or more metals selected from the following: transition metals Fe(II), Fe(III), Cu(I), Cu(II), Mn(II), Zn(II), Ni(II) and Co(II); alkaline earth metals Ca(II) and Mg(II); and lanthanide element Ce(IV).

[0030] 21. The method of any one of aspects 18-20, wherein the polysaccharide lysing agent comprises one or more strong Arrhenius bases, weak Arrhenius bases, or non-Arrhenius bases.

[0031] 22. The method of any one of aspects 18-21, wherein the polysaccharide cleaving agent comprises one or more nitrogen-based cleaving agents selected from the group consisting of ammonium hydroxide, ammonium bicarbonate, ammonia, urea, sodium amide, dimethylamine, trimethylamine, pyridine and N,N-diisopropylethylamine.

[0032] 23. The method of aspect 22, wherein the nitrogen-based cleavage reagent is one or more selected from ammonium hydroxide, ammonium bicarbonate and ammonia.

[0033] 24. The method of any one of aspects 18-23, wherein the polysaccharide cleaving agent is also the peroxide quencher, and the initiation of polysaccharide cleavage is simultaneous or substantially simultaneous with the initiation of peroxide quenching.

[0034] 25. The method of any one of aspects 18-23, wherein the polysaccharide cleaving agent is not the peroxide quencher.

[0035] 26. The method of aspect 24 or 25, wherein the peroxide quencher comprises a peroxide quenching enzyme or one or more peroxide quenchers listed in Table 1.

[0036] 27. The method of aspect 26, wherein the peroxide quencher is also an additional polysaccharide lysing reagent that lyses the treated polysaccharide.

[0037] 28. The method of any one of Aspects 18-23 and Aspects 25-27, wherein the peroxide quencher is introduced before initiating polysaccharide lysis with the polysaccharide lysis reagent, while initiating polysaccharide lysis with the polysaccharide lysis reagent, or after initiating polysaccharide lysis with the polysaccharide lysis reagent.

[0038] 29. The method of any one of aspects 18-28, further comprising removing the polysaccharide cleaving agent and / or quenching agent, or one or more of their reactive components, by evaporation (eg, as a gas).

[0039] 30. The method of any one of aspects 18-29, wherein the oligosaccharide yield is increased and / or wherein off-target side reactions are reduced and / or stripping is reduced relative to cleavage of the treated polysaccharide with a strong Arrhenius base.

[0040] 31. The method of any of aspects 18-30, wherein the polysaccharide is derived from or is present in the form of at least one material selected from the group consisting of plants, bacteria, yeast, algae, animals, fungi, and waste stream materials.

[0041] 32. The method of aspect 30, wherein the polysaccharide comprises one or more selected from the group consisting of amylose, amylopectin, β-glucan, pullulan, xyloglucan, arabinogalactan I and arabinogalactan II, rhamnogalacturonan I, rhamnogalacturonan II, polygalacturonic acid, polyglucose, galactan, arabinan, arabinoxylan, xylan (e.g., beech xylan), glycogen, mannan, glucomannan, curdlan, galactomannans, galactans, lichenin, and inulin.

[0042] 33. The method of any one of aspects 18-32, wherein the reacting and the cleaving change at least one structural property and / or chemical property of the material comprising the polysaccharide, wherein the property is selected from solubility, texture, porosity, permeability, elasticity, rheological properties and chemical reactivity.

[0043] 34. A composition comprising one or more polysaccharide cleavage products, oligosaccharides, or a mixture of polysaccharide cleavage products and / or oligosaccharides produced by the method of any one of aspects 18-33.

[0044] 35. A method of modulating microbial growth and / or microbial metabolism or host metabolism, comprising contacting a microorganism with a composition according to aspect 34 in vitro or in vivo.

[0045] 36. An oligosaccharide mixture produced by a method comprising:

[0046] a) contacting one or more polysaccharides with a Fenton's reagent comprising a peroxidizing agent and a metal ion to form a mixture;

[0047] b) allowing the Fenton reagent to react with the polysaccharide for a specified reaction time; and

[0048] c) after the specified reaction time of step b, adding a lysing agent, which may also be a peroxide quenching agent, to the mixture,

[0049] A mixture of oligosaccharides is produced.

[0050] 37. The oligosaccharide mixture of aspect 36, wherein the Fenton reagent comprises hydrogen peroxide and one or more metals selected from the group consisting of transition metals Fe(II), Fe(III), Cu(I), Cu(II), Mn(II), Zn(II), Ni(II) and Co(II); alkaline earth metals Ca(II) and Mg(II); and lanthanide element Ce(IV).

[0051] 38. The oligosaccharide mixture of aspect 36 or 37, wherein the cleavage agent which is also a peroxide quenching agent is a nitrogen-based cleavage agent.

[0052] 39. The oligosaccharide mixture of aspect 38, wherein the nitrogen-based cleavage reagent is one or more selected from ammonium hydroxide, ammonium bicarbonate, ammonia, urea, sodium amide, dimethylamine, trimethylamine, pyridine and N,N-diisopropylethylamine.

[0053] 40. The oligosaccharide mixture of aspect 38 or 39, wherein the nitrogen-based cleavage reagent is one or more selected from ammonium hydroxide, ammonium bicarbonate and ammonia.

[0054] 41. The oligosaccharide mixture of any one of aspects 36 to 40, wherein the cleavage agent that may also be a peroxide quenching agent is both a cleavage agent and a peroxide quenching agent, and the initiation of polysaccharide cleavage is simultaneous or substantially simultaneous with the initiation of peroxide quenching.

[0055] 42. The oligosaccharide mixture of any one of aspects 36 to 41, wherein the cleavage agent that may also be a peroxide quenching agent is not a peroxide quenching agent, and the method further comprises initiating peroxide quenching with an additional peroxide quenching agent.

[0056] 43. The oligosaccharide mixture of aspect 42, wherein the additional peroxide quencher comprises a peroxide quenching enzyme or one or more peroxide quenchers listed in Table 1.

[0057] 44. The oligosaccharide mixture of aspect 42 or 43, wherein the additional peroxide quencher is also an additional polysaccharide cleaving reagent that cleaves the treated polysaccharide.

[0058] 45. The oligosaccharide mixture of any one of aspects 42 to 44, wherein the additional peroxide quenching reagent is introduced before initiating polysaccharide cleavage with the cleaving reagent that is also a peroxide quenching reagent, while initiating polysaccharide cleavage with the cleaving reagent that is also a peroxide quenching reagent, or after initiating polysaccharide cleavage with the cleaving reagent that is also a peroxide quenching reagent.

[0059] 46. ​​The oligosaccharide mixture of any one of aspects 36 to 45, wherein after step (c), the cleavage agent which may also be a peroxide quenching agent and any additional polysaccharide cleavage agent, or one or more reactive components thereof, are removed by evaporation.

[0060] 47. The oligosaccharide mixture of any one of aspects 36 to 46, wherein the oligosaccharide mixture consists of different combinations of oligosaccharides, except that a strong Arrhenius base is used as a cleavage agent in step (c).

[0061] 48. The oligosaccharide mixture of any one of aspects 36 to 47, wherein the one or more polysaccharides in step (a) are derived from or are in the form of at least one material selected from the group consisting of plants, bacteria, yeast, algae, animals, fungi, and waste stream materials.

[0062] 49. The oligosaccharide mixture of any one of aspects 36 to 48, wherein the one or more polysaccharides of step (a) comprise one or more selected from the group consisting of amylose, amylopectin, β-glucan, pullulan, xyloglucan, arabinogalactan I and arabinogalactan II, rhamnogalacturonan I, rhamnogalacturonan II, polygalacturonic acid, polyglucose, galactan, arabinan, arabinoxylan, xylan (e.g., beech xylan), glycogen, mannan, glucomannan, curdlan, galactomannan, lichenin, and inulin.

[0063] 50. A method for cleaving a polysaccharide, comprising:

[0064] a) contacting one or more polysaccharides with a Fenton's reagent comprising a peroxide reagent and a metal ion to form a mixture;

[0065] b) allowing the Fenton reagent to react with the polysaccharide for a specified reaction time; and

[0066] c) After the specified reaction time of step b, adding a lysing agent, which may also be a peroxide quenching agent, to the mixture.

[0067] 51. The method of aspect 50, wherein steps (a) and (b) are performed at a pH of pH 4 to pH 7.

[0068] 52. The method of aspect 50 or 51, wherein steps (a) and (b) are performed at a pH of pH 4.5 to pH 6.5.

[0069] 53. The method of any one of aspects 50 to 52, wherein steps (a) and (b) are performed at a pH of pH 5 to pH 6.

[0070] 54. The method of any one of aspects 50 to 53, wherein step (c) is performed at a pH of 6 to 11.

[0071] 55. The method of any one of aspects 50 to 54, wherein step (c) is performed at a pH of 6.5 to 9.5.

[0072] 56. The method of any one of aspects 50 to 55, wherein step (c) is performed at a pH of 7 to 9.

[0073] 57. The method of any one of aspects 50 to 56, wherein step (c) is performed at a pH of 7 to 8.

[0074] 58. The method of any one of aspects 50 to 57, wherein steps (a) and (b) are performed at a temperature of 10 degrees Celsius to 70 degrees Celsius.

[0075] 59. The method of any one of aspects 50 to 58, wherein steps (a) and (b) are performed at a temperature of 20 degrees Celsius to 60 degrees Celsius.

[0076] 60. The method of any one of aspects 50 to 59, wherein steps (a) and (b) are performed at a temperature of 25 degrees Celsius to 55 degrees Celsius.

[0077] 61. The method of any one of aspects 50 to 60, wherein step (c) is carried out at a temperature of 10 degrees Celsius to 70 degrees Celsius.

[0078] 62. The method of any one of aspects 50 to 61, wherein step (c) is carried out at a temperature of 20 degrees Celsius to 60 degrees Celsius.

[0079] 63. The method of any one of aspects 50 to 62, wherein step (c) is carried out at a temperature of 25 degrees Celsius to 55 degrees Celsius.

[0080] 64. The method of any one of Aspects 50 to 63, wherein the Fenton reagent comprises hydrogen peroxide and one or more metals selected from the group consisting of transition metals Fe(II), Fe(III), Cu(I), Cu(II), Mn(II), Zn(II), Ni(II) and Co(II); alkaline earth metals Ca(II) and Mg(II); and the lanthanide element Ce(IV).

[0081] 65. The method of aspect 64, wherein the Fenton reagent comprises hydrogen peroxide and one or more metals selected from Fe(II), Fe(III), Cu(I) and Cu(II).

[0082] 66. The method of aspect 65, wherein the Fenton reagent comprises hydrogen peroxide and Fe(III).

[0083] 67. The method of any one of aspects 50 to 66, wherein the lysing agent that can also be a peroxide quenching agent is both a peroxide quenching agent and a lysing agent.

[0084] 68. The method of any one of aspects 50 to 67, wherein the cleavage agent, which may also be a peroxide quenching agent, is selected from one or more of ammonium hydroxide, ammonium bicarbonate, ammonia, urea, sodium amide, dimethylamine, trimethylamine, pyridine and N,N-diisopropylethylamine.

[0085] 69. The method of aspect 68, wherein the cleavage agent, which may also be a peroxide quenching agent, is selected from one or more of ammonium hydroxide, ammonium bicarbonate and ammonia.

[0086] 70. The method of any one of aspects 50 to 69, wherein the cleavage agent that can also be a peroxide quenching agent is a cleavage agent other than a peroxide quenching agent, and the method further comprises initiating peroxide quenching with an additional agent that is a peroxide quenching agent.

[0087] 71. The method of aspect 70, wherein the additional peroxide quencher comprises a peroxide quenching enzyme or one or more peroxide quenchers listed in Table 1.

[0088] 72. The method of aspect 70 or 71, wherein the additional peroxide quencher is also an additional polysaccharide lysing reagent that lyses the treated polysaccharide.

[0089] 73. The method of any one of aspects 70 to 72, wherein the additional peroxide quenching reagent is introduced before initiating polysaccharide cleavage with the cleaving reagent that is also a peroxide quenching reagent, while initiating polysaccharide cleavage with the cleaving reagent that is also a peroxide quenching reagent, or after initiating polysaccharide cleavage with the cleaving reagent that is also a peroxide quenching reagent.

[0090] 74. The method of any one of Aspects 50 to 73, further comprising removing the cleavage agent, which may also be a peroxide quenching agent, and / or the quenching agent, or one or more of their reaction components by evaporation.

[0091] 75. The method of any one of aspects 50 to 74, wherein the oligosaccharide yield is increased and / or wherein off-target side reactions are reduced and / or stripping is reduced relative to cleavage of the treated polysaccharide with a strong Arrhenius base in step (c).

[0092] 76. The method of any one of Aspects 50 to 75, wherein the one or more polysaccharides are derived from or are present in the form of at least one material selected from the group consisting of plants, bacteria, yeast, algae, animals, fungi, and waste stream materials.

[0093] 77. The method of any one of aspects 50 to 76, wherein the one or more polysaccharides comprise one or more selected from the group consisting of amylose, amylopectin, β-glucan, pullulan, xyloglucan, arabinogalactan I and arabinogalactan II, rhamnogalacturonan I, rhamnogalacturonan II, polygalacturonic acid, polyglucose, galactan, arabinan, arabinoxylan, xylan (e.g., beech xylan), glycogen, mannan, glucomannan, curdlan, galactomannans, lichenin, and inulin.

[0094] 78. The method of any one of Aspects 50 to 77, wherein the reacting and the cleaving alter at least one structural and / or chemical property of the material comprising the one or more polysaccharides, wherein the property is selected from solubility, texture, porosity, permeability, elasticity, rheological properties, and chemical reactivity.

[0095] 79. The method of any one of aspects 50 to 78, wherein the specified reaction time in step (b) is 1 hour to 3 hours.

[0096] 80. The method of any one of aspects 50 to 79, wherein the specified reaction time in step (b) is 1.5 to 2.5 hours.

[0097] 81. The method of any one of Aspects 50 to 80, wherein step (c) is performed such that step (c) is terminated by evaporating the lysing agent, which may also be a peroxide quenching agent.

[0098] 82. A composition comprising one or more polysaccharide cleavage products, oligosaccharides, or a mixture of polysaccharide cleavage products and / or oligosaccharides produced by the method of any one of aspects 50-81.

[0099] 83. A method of modulating microbial growth and / or microbial metabolism or host metabolism, comprising contacting a microorganism with a composition according to aspect 82 in vitro or in vivo.

[0100] 84. A synthetic oligosaccharide comprising an alpha-1,4 glucose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0101] 85. The synthetic oligosaccharide of aspect 84, wherein the synthetic oligosaccharide may comprise α-1,4,6 glucose branches that may terminate or extend in an α-1,4 manner.

[0102] 86. The synthetic oligosaccharide of aspect 84 or 85, wherein the oligosaccharide is described by the mass and retention time identifiers in Table 6.

[0103] 87. The synthetic oligosaccharide of aspect 86, wherein the sum of Compound 1, Compound 7, Compound 10, Compound 12, Compound 14, Compound 16, Compound 17, Compound 18, Compound 22, Compound 24, Compound 26, and Compound 28 constitutes at least 94% of the peak volume shown in Table 6.

[0104] 88. The synthetic oligosaccharide of aspect 86, wherein the sum of compound 1, compound 7, compound 10, compound 12, compound 14, compound 16, compound 17, compound 18, compound 22, compound 24, compound 26, and compound 28 constitutes 80% to 95% of the peak volume shown in Table 6.

[0105] 89. The synthetic oligosaccharide of any one of aspects 84 to 88, wherein the synthetic oligosaccharide comprises 1H-13C2D-NMR (HSQC) peaks within 10% of those described as amylopectin in Table 5.

[0106] 90. The synthetic oligosaccharide of any one of aspects 84 to 89, wherein the synthetic oligosaccharide comprises 20% to 40% terminal α-1,4 glycosidic bonds and terminal α-1,4,6 glycosidic bonds.

[0107] 91. The synthetic oligosaccharide of any one of aspects 84 to 89, wherein the synthetic oligosaccharide comprises 40% to 60% terminal α-1,4 glycosidic bonds and terminal α-1,4,6 glycosidic bonds.

[0108] 92. The synthetic oligosaccharide of any one of aspects 84 to 89, wherein the synthetic oligosaccharide comprises 60% to 80% terminal α-1,4 glycosidic bonds and terminal α-1,4,6 glycosidic bonds.

[0109] 93. The synthetic oligosaccharide of any one of aspects 84 to 89, wherein the oligosaccharide comprises at least 80% terminal α-1,4 glycosidic bonds and terminal α-1,4,6 glycosidic bonds.

[0110] 94. A synthetic oligosaccharide comprising a β-1,4 xylose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0111] 95. The synthetic oligosaccharide of aspect 94, wherein the synthetic oligosaccharide may comprise α-1,3 arabinose branches and / or α-1,2 arabinose branches.

[0112] 96. The synthetic oligosaccharide of aspect 94 or 95, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 7.

[0113] 97. The synthetic oligosaccharide of aspect 96, wherein the sum of Compound 3, Compound 4, Compound 5, Compound 7, Compound 11, Compound 12, Compound 13, Compound 20, and Compound 22 constitutes at least 55% of the peak volume shown in Table 7.

[0114] 98. The synthetic oligosaccharide of aspect 96, wherein the sum of compound 3, compound 4, compound 5, compound 7, compound 11, compound 12, compound 13, compound 20, and compound 22 constitutes 40% to 60% of the peak volume shown in Table 7.

[0115] 99. The synthetic oligosaccharide of aspect 96, wherein the sum of Compound 7, Compound 12, Compound 13, Compound 20, and Compound 22 constitutes at least 35% of the peak volume shown in Table 7.

[0116] 100. The synthetic oligosaccharide of aspect 96, wherein the sum of compound 7, compound 12, compound 13, compound 20, and compound 22 constitutes 20% to 40% of the peak volume shown in Table 7.

[0117] 101. The synthetic oligosaccharide of any one of Aspects 94 to 100, wherein the synthetic oligosaccharide comprises 1H-13C 2D-NMR (HSQC) peaks within 10% of those described as arabinoxylan in Table 5.

[0118] 102. The synthetic oligosaccharide of any one of aspects 94 to 101, wherein the synthetic oligosaccharide comprises 20% to 40% terminal xylose, terminal arabinose, β-1,4 xylose, α-1,3 xylose, α-1,2 xylose and trisecting α-1,2,3 xylose.

[0119] 103. The synthetic oligosaccharide of any one of aspects 94 to 101, wherein the synthetic oligosaccharide comprises 40% to 60% terminal xylose, terminal arabinose, β-1,4 xylose, α-1,3 xylose, α-1,2 xylose and trisprouted α-1,2,3 xylose.

[0120] 104. The synthetic oligosaccharide of any one of aspects 94 to 101, wherein the synthetic oligosaccharide comprises 60% to 80% terminal xylose, terminal arabinose, β-1,4 xylose, α-1,3 xylose, α-1,2 xylose and tri-partite α-1,2,3 xylose.

[0121] 105. The synthetic oligosaccharide of any one of aspects 94 to 101, wherein the synthetic oligosaccharide comprises at least 80% terminal xylose, terminal arabinose, β-1,4 xylose, α-1,3 xylose, α-1,2 xylose, and tri-partite α-1,2,3 xylose.

[0122] 106. A synthetic oligosaccharide comprising a β-1,4 glucose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0123] 107. The synthetic oligosaccharide of aspect 106, wherein the synthetic oligosaccharide comprises α-1,6 xylose branches that may be extended by β-2,1 galactose.

[0124] 108. The synthetic oligosaccharide of aspect 106 or 107, wherein the oligosaccharide is described by the mass and retention time identifiers in Table 8.

[0125] 109. The synthetic oligosaccharide of aspect 108, wherein the sum of Compound 1, Compound 3, Compound 6, Compound 7, Compound 9, Compound 16, Compound 18, Compound 20, Compound 21, Compound 22, Compound 24, and Compound 26 constitutes at least 58% of the peak volume shown in Table 8.

[0126] 110. The synthetic oligosaccharide of aspect 108, wherein the sum of Compound 1, Compound 3, Compound 6, Compound 7, Compound 9, Compound 16, Compound 18, Compound 20, Compound 21, Compound 22, Compound 24, and Compound 26 constitutes 45% to 65% of the peak volume shown in Table 8.

[0127] 111. The synthetic oligosaccharide of aspect 108, wherein the sum of Compound 1, Compound 3, Compound 7, Compound 9, and Compound 18 constitutes at least 36% of the peak volume shown in Table 8.

[0128] 112. The synthetic oligosaccharide of aspect 108, wherein the sum of Compound 1, Compound 3, Compound 7, Compound 9, and Compound 18 constitutes 30% to 45% of the peak volume shown in Table 8.

[0129] 113. The synthetic oligosaccharide of any one of aspects 106 to 112, wherein the synthetic oligosaccharide comprises 1H-13C2D-NMR (HSQC) peaks within 10% of those described as xyloglucan in Table 5.

[0130] 114. The synthetic oligosaccharide of any one of aspects 106 to 112, wherein the synthetic oligosaccharide comprises 20% to 40% terminal xylose, terminal glucose, (β-1,4, β-1,4,6 and β-1,6) glucose linkages, β-2,1 xylose linkages and terminal galactose linkages.

[0131] 115. The synthetic oligosaccharide of any one of aspects 106 to 112, wherein the synthetic oligosaccharide comprises 40% to 60% terminal xylose, terminal glucose, (β-1,4, β-1,4,6 and β-1,6) glucose linkages, β-2,1 xylose linkages and terminal galactose linkages.

[0132] 116. The synthetic oligosaccharide of any one of aspects 106 to 112, wherein the synthetic oligosaccharide comprises 60% to 80% terminal xylose, terminal glucose, (β-1,4, β-1,4,6 and β-1,6) glucose linkages, β-2,1 xylose linkages and terminal galactose linkages.

[0133] 117. The synthetic oligosaccharide of any one of aspects 106 to 112, wherein the synthetic oligosaccharide comprises at least 80% terminal xylose, terminal glucose, (β-1,4, β-1,4,6 and β-1,6) glucose linkages, β-2,1 xylose linkages and terminal galactose linkages.

[0134] 118. A synthetic oligosaccharide comprising a combined β-1,4 glucose and β-1,3 glucose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0135] 119. The synthetic oligosaccharide of aspect 118, wherein the synthetic oligosaccharide comprises β-1,4 glucose and β-1,3 glucose alternating in a repeating manner.

[0136] 120. The synthetic oligosaccharide of aspect 118 or 119, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 9 and Table 13.

[0137] 121. The synthetic oligosaccharide of aspect 120, wherein the sum of Compound 2, Compound 4, Compound 12, and Compound 14 constitutes at least 42% of the peak volume shown in Table 13.

[0138] 122. The synthetic oligosaccharide of aspect 120, wherein the sum of Compound 2, Compound 4, Compound 12, and Compound 14 constitutes 35% to 50% of the peak volume shown in Table 13.

[0139] 123. The synthetic oligosaccharide of aspect 120, wherein the sum of Compound 1, Compound 2, Compound 4, Compound 6, Compound 7, and Compound 12 constitutes at least 62% of the peak volume shown in Table 13.

[0140] 124. The synthetic oligosaccharide of aspect 120, wherein the sum of Compound 1, Compound 2, Compound 4, Compound 6, Compound 7, and Compound 12 constitutes 55% to 75% of the peak volume shown in Table 13.

[0141] 125. The synthetic oligosaccharide of aspect 120, wherein the sum of Compound 5, Compound 11, Compound 14, Compound 16, Compound 20, Compound 22, Compound 27, Compound 31, Compound 32, and Compound 33 constitutes at least 73% of the peak volume shown in Table 9.

[0142] 126. The synthetic oligosaccharide of aspect 120, wherein the sum of compound 5, compound 11, compound 14, compound 16, compound 20, compound 22, compound 27, compound 31, compound 32, and compound 33 constitutes 65% to 85% of the peak volume shown in Table 9.

[0143] 127. The synthetic oligosaccharide of aspect 120, wherein the sum of Compound 1, Compound 5, Compound 6, Compound 14, Compound 16, Compound 21, Compound 27, Compound 33, Compound 38, and Compound 40 constitutes at least 51% of the peak volume shown in Table 9.

[0144] 128. The synthetic oligosaccharide of aspect 120, wherein the sum of Compound 1, Compound 5, Compound 6, Compound 14, Compound 16, Compound 21, Compound 27, Compound 33, Compound 38, and Compound 40 constitutes 40% to 60% of the peak volume shown in Table 9.

[0145] 129. The synthetic oligosaccharide of any of aspects 120 to 128, wherein the synthetic oligosaccharide comprises 1H-13C 2D-NMR (HSQC) peaks within 10% of those described as lichenin or beta glucan in Table 5.

[0146] 130. The synthetic oligosaccharide of any one of aspects 120 to 129, wherein the synthetic oligosaccharide comprises 20% to 40% terminal glucose, β-1,4 glucose linkages and β-1,3 glucose linkages.

[0147] 131. The synthetic oligosaccharide of any one of aspects 120 to 129, wherein the synthetic oligosaccharide comprises 40% to 60% terminal glucose, β-1,4 glucose linkages and β-1,3 glucose linkages.

[0148] 132. The synthetic oligosaccharide of any one of aspects 120 to 129, wherein the synthetic oligosaccharide comprises 60% to 80% terminal glucose, β-1,4 glucose linkages, and β-1,3 glucose linkages.

[0149] 133. The synthetic oligosaccharide of any one of aspects 120 to 129, wherein the synthetic oligosaccharide comprises at least 80% terminal glucose, β-1,4 glucose linkages, and β-1,3 glucose linkages.

[0150] 134. A synthetic oligosaccharide comprising a β-1,4 galactose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0151] 135. The synthetic oligosaccharide of aspect 134, wherein the synthetic oligosaccharide comprises α-1,6 mannose branches at a probability of 22-4%.

[0152] 136. The synthetic oligosaccharide of aspect 134 or 135, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 10 and Table 18.

[0153] 137. The synthetic oligosaccharide of aspect 136, wherein the sum of Compound 4, Compound 7, Compound 11, Compound 20, Compound 26, Compound 38, Compound 41, and Compound 44 constitutes at least 38% of the peak volume shown in Table 10.

[0154] 138. The synthetic oligosaccharide of aspect 136, wherein the sum of Compound 4, Compound 7, Compound 11, Compound 20, Compound 26, Compound 38, Compound 41, and Compound 44 constitutes at least 30% to 50% of the peak volume shown in Table 10.

[0155] 139. The synthetic oligosaccharide of aspect 136, wherein the sum of compound 4, compound 5, compound 6, compound 7, compound 10, compound 11, compound 12, compound 20, compound 26, and compound 37 constitutes at least 55% of the peak volume shown in Table 10.

[0156] 140. The synthetic oligosaccharide of aspect 136, wherein the sum of compound 4, compound 5, compound 6, compound 7, compound 10, compound 11, compound 12, compound 20, compound 26, and compound 37 constitutes 45% to 65% of the peak volume shown in Table 10.

[0157] 141. The synthetic oligosaccharide of aspect 136, wherein the sum of compound 4, compound 5, compound 8, compound 9, compound 10, compound 13, compound 18, compound 20, compound 24, and compound 31 constitutes at least 51% of the peak volume shown in Table 18.

[0158] 142. The synthetic oligosaccharide of aspect 136, wherein the sum of compound 4, compound 5, compound 8, compound 9, compound 10, compound 13, compound 18, compound 20, compound 24, and compound 31 constitutes 40% to 60% of the peak volume shown in Table 18.

[0159] 143. The synthetic oligosaccharide of aspect 136, wherein the sum of Compound 5, Compound 8, Compound 13, Compound 18, Compound 20, Compound 24, Compound 31, Compound 35, and Compound 39 constitutes at least 33% of the peak volume shown in Table 18.

[0160] 144. The synthetic oligosaccharide of aspect 136, wherein the sum of compound 5, compound 8, compound 13, compound 18, compound 20, compound 24, compound 31, compound 35, and compound 39 constitutes 25% to 40% of the peak volume shown in Table 18.

[0161] 145. The synthetic oligosaccharide of any of aspects 136 to 144, wherein the synthetic oligosaccharide comprises 1H-13C 2D-NMR (HSQC) peaks within 10% of those described in Table 5 as galactomannan and locust bean gum.

[0162] 146. The synthetic oligosaccharide of any one of aspects 136 to 145, wherein the synthetic oligosaccharide comprises 20% to 40% terminal galactose, terminal mannose, β-1,4 mannose linkages and β-1,4,6 mannose linkages.

[0163] 147. The synthetic oligosaccharide of any one of aspects 136 to 145, wherein the synthetic oligosaccharide comprises 40% to 60% terminal galactose, terminal mannose, β-1,4 mannose linkages and β-1,4,6 mannose linkages.

[0164] 148. The synthetic oligosaccharide of any one of aspects 136 to 145, wherein the synthetic oligosaccharide comprises 60% to 80% terminal galactose, terminal mannose, β-1,4 mannose linkages and β-1,4,6 mannose linkages.

[0165] 149. The synthetic oligosaccharide of any one of aspects 136 to 145, wherein the synthetic oligosaccharide comprises at least 80% terminal galactose, terminal mannose, β-1,4 mannose linkages and β-1,4,6 mannose linkages.

[0166] 150. A synthetic oligosaccharide comprising a β-1,3 galactose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0167] 151. The synthetic oligosaccharide of aspect 150, wherein the synthetic oligosaccharide comprises β-1,6 galactose branches, β-1,3 galactose branches and β-1,3,6 galactose branches of length 1-4 and a terminal arabinose cap.

[0168] 152. The synthetic oligosaccharide of aspect 150 or 151, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 11.

[0169] 153. The synthetic oligosaccharide of aspect 152, wherein the sum of compound 7, compound 9, compound 11, compound 19, compound 25, compound 27, compound 30, compound 32, compound 36, compound 37, compound 41, compound 44, compound 47, compound 54, and compound 59 constitutes at least 35% of the peak volume shown in Table 11.

[0170] 154. The synthetic oligosaccharide of aspect 152, wherein the sum of compound 7, compound 9, compound 11, compound 19, compound 25, compound 27, compound 30, compound 32, compound 36, compound 37, compound 41, compound 44, compound 47, compound 54, and compound 59 constitutes 28% to 42% of the peak volume shown in Table 11.

[0171] 155. The synthetic oligosaccharide of aspect 152, wherein the sum of compound 5, compound 9, compound 10, compound 12, compound 14, compound 18, compound 25, compound 32, compound 37, and compound 53 constitutes at least 50% of the peak volume shown in Table 11.

[0172] 156. The synthetic oligosaccharide of aspect 152, wherein the sum of compound 5, compound 9, compound 10, compound 12, compound 14, compound 18, compound 25, compound 32, compound 37, and compound 53 constitutes 40% to 60% of the peak volume shown in Table 11.

[0173] 157. The synthetic oligosaccharide of any one of aspects 152 to 156, wherein the synthetic oligosaccharide comprises 1H-13C 2D-NMR (HSQC) peaks within 10% of those described as arabinogalactan in Table 5.

[0174] 158. The synthetic oligosaccharide of any one of aspects 152 to 157, wherein the oligosaccharide comprises 20% to 40% terminal galactose, terminal arabinose, β-1,3 galactose, β-1,3,6 galactose.

[0175] 159. The synthetic oligosaccharide of any one of aspects 152 to 157, wherein the oligosaccharide comprises 40% to 60% terminal galactose, terminal arabinose, β-1,3 galactose, β-1,3,6 galactose.

[0176] 160. The synthetic oligosaccharide of any one of aspects 152 to 157, wherein the oligosaccharide comprises 60% to 80% terminal galactose, terminal arabinose, β-1,3 galactose, β-1,3,6 galactose.

[0177] 161. The synthetic oligosaccharide of any one of aspects 152 to 157, wherein the oligosaccharide comprises at least 80% terminal galactose, terminal arabinose, β-1,3 galactose, β-1,3,6 galactose.

[0178] 162. A synthetic oligosaccharide comprising a β-1,3 glucose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0179] 163. The synthetic oligosaccharide of aspect 162, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 12.

[0180] 164. The synthetic oligosaccharide of aspect 162 or 163, wherein the sum of Compound 1, Compound 4, Compound 7, Compound 9, and Compound 10 constitutes at least 91% of the peak volume shown in Table 12.

[0181] 165. The synthetic oligosaccharide of aspect 162 or 163, wherein the sum of Compound 1, Compound 4, Compound 7, Compound 9, and Compound 10 constitutes at least 80% to 98% of the peak volume shown in Table 12.

[0182] 166. The synthetic oligosaccharide of aspect 162 or 163, wherein the sum of Compound 2, Compound 3, Compound 5, Compound 6, and Compound 8 constitutes at least 8% of the peak volume shown in Table 12.

[0183] 167. The synthetic oligosaccharide of aspect 162 or 163, wherein the sum of Compound 2, Compound 3, Compound 5, Compound 6, and Compound 8 constitutes at least 1% to 15% of the peak volume shown in Table 12.

[0184] 168. The synthetic oligosaccharide of any one of Aspects 162 to 167, wherein the synthetic oligosaccharide comprises 1H-13C2D-NMR (HSQC) peaks within 10% of those described as curdlan in Table 5.

[0185] 169. The synthetic oligosaccharide of any one of aspects 162 to 168, wherein the oligosaccharide comprises 20% to 40% terminal glucose and β-1,3 glucose linkages.

[0186] 170. The synthetic oligosaccharide of any one of aspects 162 to 168, wherein the oligosaccharide comprises 40% to 60% terminal glucose and β-1,3 glucose linkages.

[0187] 171. The synthetic oligosaccharide of any one of aspects 162 to 168, wherein the oligosaccharide comprises 60% to 80% terminal glucose and β-1,3 glucose linkages.

[0188] 172. The synthetic oligosaccharide of any one of aspects 162 to 168, wherein the oligosaccharide comprises at least 80% terminal glucose and beta-1,3 glucose.

[0189] 173. A synthetic oligosaccharide comprising a backbone of repeating linear β-1,4 mannose, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0190] 174. The synthetic oligosaccharide of aspect 173, wherein the oligosaccharide is described by the mass and retention time identifiers in Table 14.

[0191] 175. The synthetic oligosaccharide of aspect 173 or 174, wherein the sum of Compound 2, Compound 6, Compound 10, Compound 11, Compound 14, Compound 19, Compound 20, Compound 21, Compound 25, and Compound 27 constitutes at least 58% of the peak volume shown in Table 14.

[0192] 176. The synthetic oligosaccharide of aspect 173 or 174, wherein the sum of Compound 2, Compound 6, Compound 10, Compound 11, Compound 14, Compound 19, Compound 20, Compound 21, Compound 25, and Compound 27 constitutes at least 50% to 70% of the peak volume shown in Table 14.

[0193] 177. The synthetic oligosaccharide of any one of Aspects 173 to 176, wherein the synthetic oligosaccharide comprises 1H-13C2D-NMR (HSQC) peaks within 10% of those described as mannans in Table 5.

[0194] 178. The synthetic oligosaccharide of any one of aspects 173 to 177, wherein the synthetic oligosaccharide comprises 20% to 40% terminal mannose and β-1,4 mannose linkages.

[0195] 179. The synthetic oligosaccharide of any one of aspects 173 to 177, wherein the synthetic oligosaccharide comprises 40% to 60% terminal mannose and β-1,4 mannose linkages.

[0196] 180. The synthetic oligosaccharide of any one of aspects 173 to 177, wherein the synthetic oligosaccharide comprises 60% to 80% terminal mannose and β-1,4 mannose linkages.

[0197] 181. The synthetic oligosaccharide of any one of aspects 173 to 177, wherein the synthetic oligosaccharide comprises at least 80% terminal mannose and β-1,4 mannose linkages.

[0198] 182. A synthetic oligosaccharide comprising a β-1,4 xylose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0199] 183. The synthetic oligosaccharide of aspect 182, wherein the synthetic oligosaccharide comprises α-1,2 glucuronic acid-4-OMe branches on about 13% of the backbone units.

[0200] 184. The synthetic oligosaccharide of aspect 182 or 183, wherein the oligosaccharide is described by the mass and retention time identifiers in Table 15.

[0201] 185. The synthetic oligosaccharide of any one of Aspects 182 to 184, wherein the sum of Compound 3, Compound 4, Compound 10, Compound 14, and Compound 15 constitutes at least 66% of the peak volume shown in Table 15.

[0202] 186. The synthetic oligosaccharide of any one of Aspects 182 to 184, wherein the sum of Compound 3, Compound 4, Compound 10, Compound 14, and Compound 15 constitutes 55% to 75% of the peak volume shown in Table 15.

[0203] 187. The synthetic oligosaccharide of any one of Aspects 182 to 184, wherein the sum of Compound 2, Compound 6, Compound 7, Compound 8, Compound 9, Compound 11, Compound 12, and Compound 13 constitutes at least 31% of the peak volume shown in Table 15.

[0204] 188. The synthetic oligosaccharide of any one of Aspects 182 to 184, wherein the sum of Compound 2, Compound 6, Compound 7, Compound 8, Compound 9, Compound 11, Compound 12, and Compound 13 constitutes 20% to 40% of the peak volume shown in Table 15.

[0205] 189. The synthetic oligosaccharide of any one of Aspects 182 to 188, wherein the synthetic oligosaccharide comprises 1H-13C2D-NMR (HSQC) peaks within 10% of those described as xylans in Table 5.

[0206] 190. The synthetic oligosaccharide of any one of aspects 182 to 189, wherein the synthetic oligosaccharide comprises 20% to 40% terminal xylose, β-1,4 xylose linkages, and terminal glucuronic acid-4-OMe.

[0207] 191. The synthetic oligosaccharide of any one of aspects 182 to 189, wherein the synthetic oligosaccharide comprises 40% to 60% terminal xylose, β-1,4 xylose linkages, and terminal glucuronic acid-4-OMe.

[0208] 192. The synthetic oligosaccharide of any one of aspects 182 to 189, wherein the synthetic oligosaccharide comprises 60% to 80% terminal xylose, β-1,4 xylose linkages, and terminal glucuronic acid-4-OMe.

[0209] 193. The synthetic oligosaccharide of any one of aspects 182 to 189, wherein the synthetic oligosaccharide comprises at least 80% terminal xylose, β-1,4 xylose linkages, and terminal glucuronic acid-4-OMe.

[0210] 194. A synthetic oligosaccharide comprising a β-1,4 galactose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0211] 195. The synthetic oligosaccharide of aspect 195, wherein the synthetic oligosaccharide comprises β-1,4 linked galactose in a linear repeating chain.

[0212] 196. The synthetic oligosaccharide of aspect 194 or 195, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 16.

[0213] 197. The synthetic oligosaccharide of Aspect 196, wherein the sum of Compound 2, Compound 5, Compound 9, Compound 11, and Compound 13 constitutes at least 37% of the peak volume shown in Table 16.

[0214] 198. The synthetic oligosaccharide of aspect 196, wherein the sum of Compound 2, Compound 5, Compound 9, Compound 11, and Compound 13 constitutes 30% to 45% of the peak volume shown in Table 16.

[0215] 199. The synthetic oligosaccharide of Aspect 196, wherein the sum of Compound 2, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 12, and Compound 15 constitutes at least 77% of the peak volume shown in Table 16.

[0216] 200. The synthetic oligosaccharide of aspect 196, wherein the sum of Compound 2, Compound 5, Compound 6, Compound 7, Compound 9, Compound 10, Compound 12, and Compound 15 constitutes 65% to 85% of the peak volume shown in Table 16.

[0217] 201. The synthetic oligosaccharide of any one of aspects 194 to 200, wherein the synthetic oligosaccharide comprises 20% to 40% terminal galactose and β-1,4 galactose linkages.

[0218] 202. The synthetic oligosaccharide of any one of aspects 194 to 200, wherein the synthetic oligosaccharide comprises 40% to 60% terminal galactose and β-1,4 galactose linkages.

[0219] 203. The synthetic oligosaccharide of any one of aspects 194 to 200, wherein the synthetic oligosaccharide comprises 60% to 80% terminal xylose and β-1,4 xylose linkages.

[0220] 204. The synthetic oligosaccharide of any one of aspects 194 to 200, wherein the synthetic oligosaccharide comprises at least 80% terminal xylose and β-1,4 xylose linkages.

[0221] 205. A synthetic oligosaccharide comprising a backbone having β-1,4 mannose and β-1,4 glucose, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0222] 206. The synthetic oligosaccharide of aspect 205, wherein the synthetic oligosaccharide comprises β-1,4 linked mannose in a linear repeating chain with a β-1,4 glucose present approximately every 3 units in the linear repeating chain.

[0223] 207. The synthetic oligosaccharide of aspect 205 or 206, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 17.

[0224] 208. The synthetic oligosaccharide of aspect 207, wherein the sum of compound 7, compound 8, compound 13, compound 15, compound 18, compound 33, compound 36, compound 39, compound 64, compound 68, compound 71, compound 72, compound 73, and compound 74 constitutes at least 39% of the peak volume shown in Table 17.

[0225] 209. The synthetic oligosaccharide of aspect 207, wherein the sum of compound 7, compound 8, compound 13, compound 15, compound 18, compound 33, compound 36, compound 39, compound 64, compound 68, compound 71, compound 72, compound 73, and compound 74 constitutes 30% to 50% of the peak volume shown in Table 17.

[0226] 210. The synthetic oligosaccharide of aspect 207, wherein the sum of Compound 4, Compound 7, Compound 8, Compound 13, Compound 16, Compound 18, Compound 33, Compound 36, Compound 39, and Compound 74 constitutes at least 37% of the peak volume shown in Table 17.

[0227] 211. The synthetic oligosaccharide of aspect 207, wherein the sum of Compound 4, Compound 7, Compound 8, Compound 13, Compound 16, Compound 18, Compound 33, Compound 36, Compound 39, and Compound 74 constitutes at least 30% to 50% of the peak volume shown in Table 17.

[0228] 212. The synthetic oligosaccharide of any one of Aspects 205 to 211, wherein the synthetic oligosaccharide comprises 1H-13C2D-NMR (HSQC) peaks within 10% of those described as glucomannan in Table 5.

[0229] 213. The synthetic oligosaccharide of any one of aspects 205 to 212, wherein the synthetic oligosaccharide comprises 20% to 40% terminal mannose, terminal glucose, β-1,4 mannose linkages and β-1,4 glucose linkages.

[0230] 214. The synthetic oligosaccharide of any one of aspects 205 to 212, wherein the oligosaccharide comprises 40% to 60% terminal mannose, terminal glucose, β-1,4 mannose linkages and β-1,4 glucose linkages.

[0231] 215. The synthetic oligosaccharide of any one of aspects 205 to 212, wherein the oligosaccharide comprises 60% to 80% terminal mannose, terminal glucose, β-1,4 mannose linkages and β-1,4 glucose linkages.

[0232] 216. The synthetic oligosaccharide of any one of aspects 205 to 212, wherein the oligosaccharide comprises at least 80% terminal mannose, terminal glucose, β-1,4 mannose linkages and β-1,4 glucose linkages.

[0233] 217. A synthetic oligosaccharide produced from corn fiber.

[0234] 218. The synthetic oligosaccharide of aspect 217, wherein the synthetic oligosaccharide comprises a β-1,4 xylose backbone, wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30.

[0235] 219. The synthetic oligosaccharide of aspect 218, wherein the synthetic oligosaccharide further comprises α-1,3 arabinose branches and / or α-1,2 arabinose branches.

[0236] 220. The synthetic oligosaccharide of any one of Aspects 217 to 219, wherein the synthetic oligosaccharide is described by the mass and retention time identifiers in Table 19.

[0237] 221. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 1, Compound 4, Compound 8, Compound 9, Compound 10, and Compound 16 constitutes at least 44% of the peak volume shown in Table 19.

[0238] 222. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 1, Compound 4, Compound 8, Compound 9, Compound 10, and Compound 16 constitutes 35% to 55% of the peak volume shown in Table 19.

[0239] 223. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 9, Compound 10, Compound 11, Compound 13, Compound 14, Compound 15, and Compound 17 constitutes at least 54% of the peak volume shown in Table 19.

[0240] 224. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 9, Compound 10, Compound 11, Compound 13, Compound 14, Compound 15, and Compound 17 constitutes 45% to 65% of the peak volume shown in Table 19.

[0241] 225. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 7 constitutes at least 23% of the peak volume shown in Table 19.

[0242] 226. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, and Compound 7 constitutes 15% to 35% of the peak volume shown in Table 19.

[0243] 227. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 8, Compound 12, and Compound 16 constitutes at least 12% of the peak volume shown in Table 19.

[0244] 228. The synthetic oligosaccharide of aspect 220, wherein the sum of Compound 8, Compound 12, and Compound 16 constitutes at least 5% to 20% of the peak volume shown in Table 19.

[0245] 229. The synthetic oligosaccharide of any one of Aspects 217 to 228, wherein the synthetic oligosaccharide comprises 1H-13C2D-NMR (HSQC) peaks within 10% of those described in Table 5 as corn fiber.

[0246] 230. The synthetic oligosaccharide of any one of aspects 217 to 228, wherein the synthetic oligosaccharide comprises 20% to 40% terminal xylose, terminal arabinose, β-1,4 arabinose linkages, and α-1,3 arabinose linkages and α-1,2 arabinose linkages.

[0247] 231. The synthetic oligosaccharide of any one of aspects 217 to 228, wherein the synthetic oligosaccharide comprises 40% to 60% terminal xylose, terminal arabinose, β-1,4 arabinose linkages, and α-1,3 arabinose linkages and α-1,2 arabinose linkages.

[0248] 232. The synthetic oligosaccharide of any one of aspects 217 to 228, wherein the synthetic oligosaccharide comprises 60% to 80% terminal xylose, terminal arabinose, β-1,4 arabinose linkages, and α-1,3 arabinose linkages and α-1,2 arabinose linkages.

[0249] 233. The synthetic oligosaccharide of any one of aspects 217 to 228, wherein the synthetic oligosaccharide comprises at least 80% terminal xylose, terminal arabinose, β-1,4 arabinose linkages, and α-1,3 arabinose linkages and α-1,2 arabinose linkages.

[0250] 234. An oligosaccharide library produced by the method of any one of aspects 1 to 33 or 50 to 81, which does not include one or more oligosaccharides unique to the depolymerization process called FITDOG shown in Table 20.

[0251] 235. The synthetic oligosaccharide of any one of Aspects 84 to 233, wherein the synthetic oligosaccharide does not include one or more oligosaccharides unique to the depolymerization process known as FITDOG shown in Table 20. BRIEF DESCRIPTION OF THE DRAWINGS

[0252] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication in color will be provided by the Office upon request and payment of the necessary fee.

[0253] Figure 1 By way of non-limiting example of the present invention a comparison of the total yield of oligosaccharides from locust bean gum by different lysis reagents and at different temperatures is shown.

[0254] Figure 2 By way of non-limiting example of the present invention, the oligosaccharide profiles of locust bean gum for different cleavage reagents reacted at 45°C are shown.

[0255] Figure 3 By way of non-limiting example of the present invention, residual hydrogen peroxide after one hour incubation at 27°C with three exemplary lysis reagents is shown.

[0256] Figure 4 By way of non-limiting example of the present invention, the hydrogen peroxide concentration and pH after one hour incubation with ammonium bicarbonate at different temperatures are shown.

[0257] Figure 5A and Figure 5B By way of non-limiting example of the present invention, liquid chromatography-mass spectra of two spent distiller's grain fractions are shown.

[0258] Figure 6 HPLC / Q-TOF chromatograms of oligosaccharides produced from pullulan are shown. Oligosaccharides are produced by an alkaline lysis step using either ammonium hydroxide or sodium hydroxide.

[0259] Figure 7 The monosaccharide composition of oligosaccharides produced from pullulan is shown. Oligosaccharides were produced by an alkaline lysis step using ammonium hydroxide or sodium hydroxide. Monosaccharide abundance was normalized to control abundance.

[0260] Figure 8Oligosaccharide analysis of pullulan oligosaccharides produced by an alkaline lysis step using different strong Arrhenius bases and nitrogenous bases is shown.

[0261] Fig. 9 Bacterial growth of oligosaccharides produced from pullulan is shown. Oligosaccharides are produced by an alkaline lysis step using either ammonium hydroxide or sodium hydroxide.

[0262] Fig.10 The monosaccharide composition of locust bean gum polysaccharides and locust bean gum oligosaccharides is shown.

[0263] Fig.11 An HPLC / Q-TOF chromatogram showing locust bean gum oligosaccharides produced by COG is shown.

[0264] Fig.12 A comparison of corn fiber oligosaccharide yields using different catalysts and different conditions is shown.

[0265] Fig.13 1H-13C HSQC NMR spectra of COG-produced oligosaccharides are shown.

[0266] Fig.14 Annotated extracted ion chromatograms are shown, with the most abundant oligosaccharides labeled.

[0267] Fig.15 Linkage analysis chromatograms of annotated corn fiber are shown. DETAILED DESCRIPTION

[0268] A high-yield peroxide-quenched controlled method (controlled oligosaccharide generation ("COG") method) for producing oligosaccharides from polysaccharides (PS) is provided, which includes a multi-step reaction (e.g., a two-step, three-step, etc. reaction), wherein the multi-step reaction includes an initial oxidation step using a Fenton system / reagent and a subsequent peroxide quenching / PS cleavage step using either: a PS cleavage agent that also acts as a peroxide quencher; or a PS cleavage agent is used in combination with a compatible peroxide quenching agent that does not interfere with the PS cleavage reaction. In the method, the PS cleavage agent can be, for example, a weak Arrhenius base or a non-Arrhenius base. In the method, preferably, the PS cleavage initiator is also used as a peroxide quencher to quench (e.g., substantially reduce or eliminate) residual hydrogen peroxide and / or its free radicals to minimize or eliminate off-target side reactions. For example, the methods of the invention comprise reacting a polysaccharide with hydrogen peroxide and a suitable metal or metal ion as described herein (e.g., Fe(II), Fe(III), Cu(I), Cu(II), Ca(II), Mg(II), Mn(II), Zn(II), Ni(II), Ce(IV), Co(II) or other metal ions), followed by cleaving glycosidic bonds in the hydroperoxide-treated polysaccharide with a high-yield peroxide quencher / cleavage agent such as ammonium bicarbonate, ammonium hydroxide, ammonia, urea, sodium amide or other ammonium-based reagents, thereby producing high yields of oligosaccharides and lower molecular weight polysaccharides (which are still polysaccharide cleavage products) from the parent (starting material) polysaccharide while reducing or eliminating stripping and unwanted side reactions.

[0269] In the methods described herein, the cleavage agent (cleavage initiator) may also be, and preferably is, a peroxide quencher agent, and in either case may be used in combination with an additional compatible peroxide quencher, which may or may not be a cleavage agent. Exemplary cleavage agents and / or peroxide quenchers are listed in Table 1.

[0270] In the disclosed COG method, a peroxide quencher is used to quench (eg, substantially reduce or eliminate) residual hydrogen peroxide and / or its free radicals themselves, minimizing or eliminating off-target side reactions.

[0271] In the disclosed COG method, the use of specific weak Arrhenius bases and / or non-Arrhenius bases (e.g., ammonium peroxide quenchers / PS cleavage reagents, etc.; for example, see Table 1) not only provides improved high-yield oligosaccharide production (relative to the strong Arrhenius bases used in the art), but also eliminates the need for expensive and time-consuming post-reaction concentration and desalting steps. Table 1. Exemplary polysaccharide (PS) cleaving agents and / or peroxide quenchers.

[0272] In the method, the cleavage initiator can and preferably also be used as a peroxide quencher to quench (fully reduce or eliminate) residual peroxide and / or its free radical to reduce or eliminate stripping and unwanted side reactions. Alternatively, a high-yield cracking agent can be added to the reaction after or simultaneously adding a compatible peroxide quencher (which can also be a cracking agent). In the method, the peroxide quencher / cracking agent can be and preferably be selected from one or more nitrogen-based reagents as described herein (e.g., referring to Table 1 above), and it not only provides high-yield cracking and the quenching of residual peroxide, but also provides cracking specific tailoring (e.g., by replacing the hydrogen bound by nitrogen with a larger moiety to sterically hinder or otherwise change the entry or activity of the cracking agent).

[0273] The method, sometimes referred to herein as the "COG" method, can effectively produce bioactive oligosaccharides and low molecular weight polysaccharides by digesting polysaccharides from any source (including but not limited to plants, bacteria, animals, algae and fungi). In some aspects, the oligosaccharides are produced in a degree of polymerization (DP) range of 3 to 20. In some aspects, the polysaccharides are broken down into smaller polysaccharides. In some aspects, the described method will produce oligosaccharides for analysis and for use in bioactive foods, which are prebiotics, anticancer, antipathogens or have other functions (enhancing biofuel production, extractability of other compounds, etc.). The COG method can be used to convert polysaccharides (e.g., from plants, bacteria or yeast, algae, animals, fungi and waste product streams) into bioactive oligosaccharides or smaller polysaccharides.

[0274] In some aspects, the resulting oligosaccharides can be characterized (structure and / or activity / properties). In some aspects, high performance liquid chromatography-mass spectrometry (LC-MS) analysis of the product mixture shows that the size range of the structures of many oligosaccharides is from 3 to 20 (or, for example, from 3 to as many as 200) DP, depending on the source of the polysaccharide and the reaction conditions. The structure and composition of the oligosaccharides will depend on the source of the polysaccharide.

[0275] In some aspects, oligosaccharides consisting of a DP from 3 to 20 (or, for example, 3 to as many as 200) are provided from natural polysaccharide sources. The polysaccharide may include, for example, those from plants, algae, bacteria, animals, fungi, and waste product streams. In some aspects, the polysaccharide may be from food, agricultural or biofuel waste and from sources that are not usually considered as food. In some aspects, the source of the polysaccharide is processed food and plant products.

[0276] In some aspects, the COG methods provide for the production of oligosaccharides (eg, having a DP of 3 to 20 (or, eg, 3 to as high as 200)) from bacterial cell wall polysaccharides.

[0277] In some aspects, the COG methods provide for the production of oligosaccharides (eg, having a DP of 3 to 20 (or such as 3 to as high as 200)) from yeast cell wall polysaccharides.

[0278] In some aspects, the COG methods provide for the production of oligosaccharides (eg, having a DP of 3 to 20 (or, eg, 3 to as high as 200)) from algal polysaccharides.

[0279] In some aspects, the oligosaccharide is a bioactive oligosaccharide (for example, a bioactive oligosaccharide consumed by bacteria that are beneficial to human intestinal tract). In some aspects, the oligosaccharide is consumed by bacteria that are beneficial to vaginal microbiome, respiratory tract or skin. In some aspects, the oligosaccharide is consumed by bacteria that are beneficial to soil microbiome. In some aspects, the bioactive oligosaccharide can regulate the immune system (for example, for insufficient or excessive response to known or unknown stimuli). In some aspects, the bioactive oligosaccharide plays a function of blocking pathogens. In some aspects, the oligosaccharide is used as a raw material for producing biofuels. In some aspects, the oligosaccharide can be used to regulate microbial metabolite output.

[0280] In some aspects, the oligosaccharide is a selective carbon substrate for stimulating the growth of the microbial community of the soil. In some aspects, after fumigating or sterilizing the soil, the oligosaccharide is added to the soil. If not controlled, the available organic carbon can guide the soil ecology to the direction of pathogenicity. By providing a specific oligosaccharide that selectively stimulates the growth of beneficial soil microbial community (or provides growth advantage for beneficial soil microbial community), the soil pathogen group in the soil can be reduced. In some aspects, the combination of one or more oligosaccharides prepared as described herein can be added to the soil together with one or more microorganisms (for example, beneficial soil microorganisms) to reach the required microbial supplement or microbial balance in the soil, or to reduce or eliminate pathogens or unwanted microorganisms. In some aspects, the oligosaccharide can selectively promote the growth and colonization of bacteria that can repair soil by metabolizing impurities (contaminant) or pollutants (pollutant) (such as chemicals, heavy metals, etc.) in the soil. In some aspects, bacteria can be designed by recombinant methods to consume specific oligosaccharide structures. In some aspects, the oligosaccharide can selectively promote the growth of bacteria that can produce insecticidal compounds naturally or recombinantly. In some aspects, the oligosaccharides can selectively promote the growth of bacteria that naturally or recombinantly produce the herbicidal compound.

[0281] In some aspects, the oligosaccharide can be formulated into a product for oral hygiene. In some aspects, the oral hygiene product can be a toothpaste, a mouthwash, a chewing gum, a mint, a candy, a lozenge, and dental floss. In some aspects, the oligosaccharide can be configured at a dosage of about 10 mg / time. In some aspects, the oligosaccharide can be configured at a dosage of about 100 mg / time. In some aspects, the oligosaccharide can be configured at a dosage of about 200 mg / time or more / time.

[0282] In some aspects, the oligosaccharide can be in the form of an enteral composition, a topical composition, an intravaginal composition, or a disposable absorbent article, such as a diaper, a pant, an adult incontinence product, an absorbent liner for a diaper or pant, a tissue, or a feminine hygiene product such as a sanitary napkin, a tampon, and a panty liner.

[0283] In some aspects, the enteral composition comprises 0.5g to 15g of oligosaccharides, more preferably 1g to 10g. For example, the enteral composition may comprise 2g to 7.5g of oligosaccharides. Preferably, the topical composition and the intravaginal composition comprise 0.1g to 10g of oligosaccharides, more preferably 0.2g to 7.5g. For example, the topical or intravaginal composition may comprise 0.5g to 5g of oligosaccharides. When in the form of a disposable absorbent article, at least a portion of the article may be coated or impregnated with an amount of 0.2g to 200g per square meter, preferably 5.0g to 100g per square meter, more preferably 8.0g to 50g per square meter. In the case where women need to improve urogenital health or treatment, the women may first apply a higher dose and then a lower dose. Preferably, the higher dose is applied for up to 14 days, for example, up to 7 days. The lower dose can be applied for a long time. In the case where a woman is in need of treatment to reduce the risk of bacterial vaginosis, recurrence of bacterial vaginosis, urinary tract infection, or recurrence of urinary tract infection, the woman can be administered a lower maintenance dose for a longer period of time.

[0284] In some aspects, one or more oligosaccharides prepared by the COG method as described herein can be used to produce prebiotics for food supplementation. In some aspects, the oligosaccharides can be used to regulate appetite control and / or energy (calorie) intake control in subjects in need thereof (e.g., overweight and obese children or other subjects).

[0285] In some aspects, a method is provided for producing soluble fibers from insoluble fibers containing polysaccharides using COG reaction conditions as described herein. By reacting only to a certain extent (e.g., partial depolymerization of polysaccharide materials), compositions (e.g., gels or ointments) with desired characteristics can be produced. The COG method can be used to soften or change the texture, porosity or reaction characteristics of polysaccharide-containing materials that are exposed to (e.g., impregnated or infiltrated to a certain extent) reaction components. In some aspects, the COG method can be used to soften (e.g., by partial depolymerization) the cell walls of plants and / or plant materials, animals, bacteria and fungi before industrial processing. In some aspects, softening the cell walls of plants can result in greater extractability of valuable components. In some aspects, softening the cell walls of plants or plant materials can result in easier physical removal or separation of desired parts and / or unwanted parts (e.g., shells, skins, peels, seeds). In some aspects, the present invention can be used to "soften" the cell walls of plants, bacteria, animals and fungi to produce permeable membranes prior to cell modification (e.g., nucleic acid (e.g., DNA and / or RNA) transfection and / or modification). In some aspects, the COG method can be used to modify the rheological properties of gums, gels and other carbohydrate-derived tissue / sensory modifiers. In some aspects, the COG method can be used to produce smaller molecular weight carbohydrates and / or polysaccharides and / or oligosaccharides for the production of bioethanol, biofuels or other downstream compounds.

[0286] Soluble fiber products can be used for many purposes, including but not limited to medical products and devices, foods (i.e., thickeners, nutrient amendments, flavors and / or seasonings), soil amendments (to design, balance or enrich specific beneficial soil microbiome components), and fiber products (e.g., new textiles, ropes, biodegradable packaging, etc.). In some aspects, for example, the insoluble fiber is cotton, which can be treated or partially treated using the COG methods described herein to obtain one or more desired characteristics (e.g., softness, strength, elasticity, absorbency, etc.). In some aspects, the COG methods described herein can modify insoluble fibers to make them soluble.

[0287] In preferred aspects, the COG method is used to produce oligosaccharides from polysaccharides. In some aspects, the COG method comprises reacting the polysaccharides in the reaction mixture with hydrogen peroxide and a suitable transition metal, alkaline earth metal or lanthanide (e.g., Fe(II), Fe(III), Cu(I), Cu(II), Ca(II), Mg(II), Mn(II), Zn(II), Ni(II), Ce(IV), CO(II)); followed by cleaving the glycosidic bonds in the polysaccharides treated with oxidized hydroxyl groups with a high yield peroxide quenching / cleavage reagent such as ammonium bicarbonate, ammonium hydroxide, ammonia, urea, sodium amide or other nitrogen-based reagents, and / or other weak Arrhenius bases or non-Arrhenius bases (e.g., see Table 1 above), thereby producing high yields of oligosaccharides from the polysaccharides while reducing or eliminating stripping and unwanted side reactions. In the methods disclosed herein, the reaction mixture comprises a transition metal or an alkaline earth metal. In some aspects, the transition metal is selected from Fe(II), Fe(III), Cu(I), Cu(II), Mn(II), Zn(II), Ni(II), CO(II). In some aspects, the reaction mixture comprises an alkaline earth metal selected from calcium or magnesium (e.g., Ca(II), Mg(II)). In some aspects, the metal can be selected from lanthanides (e.g., Ce(IV)).

[0288] In some aspects, the cleavage reagent may include at least one reagent selected from ammonium hydroxide, ammonia, ammonium bicarbonate, urea, etc., or a combination thereof (e.g., see Table 1). In some aspects, the cleavage reagent may include the conjugate base of an alcohol or an amine. In some aspects, the cleavage reagent may include sodium methoxide, sodium ethoxide, sodium tert-butoxide, or other deprotonated alcohols. In some aspects, the cleavage reagent may be one or more relative "bulky bases" or include one or more relative "bulky bases", such as tert-butanol, triethylamine, or other sterically hindered bases. In some aspects, the use of such bulky cleavage reagents / bases results in the selective cleavage of accessible glycosidic bonds to provide a unique / specific oligosaccharide profile of the cleavage reagent / base. In some aspects, the cleavage reagent itself is not a base, but is composed of or includes one or more reactants, and the reactants react to produce alkaline conditions and / or decomposition products. In all methods described herein, the cleavage reagent (cleavage initiator) may also be, and preferably is, a peroxide quencher reagent, and in either case may be used in combination with another compatible peroxide quencher, which may or may not be a cleavage agent.

[0289] In some aspects, the concentration of the transition metal or alkaline earth metal in the reaction mixture is at least about 0.65nM (for example, at least a value in the range of 0.5nM to 0.7nM). In some aspects, the concentration of the transition metal or alkaline earth metal in the reaction mixture is 0.65nM to 500nM. In some aspects, the concentration of the peroxide agent (for example, hydrogen peroxide) in the reaction mixture is at least about 0.02M (for example, at least a value in the range of 0.015M to 0.025M). In some aspects, the concentration of the peroxide agent (for example, hydrogen peroxide) in the reaction mixture is 0.02M to 1M. In some aspects, the cleavage reagent / base is ammonium hydroxide, ammonia, ammonium bicarbonate, weak Arrhenius base, non-Arrhenius base, Lewis base and / or Bronsted-Lowry base or includes ammonium hydroxide, ammonia, ammonium bicarbonate, weak Arrhenius base, non-Arrhenius base, Lewis base and / or Bronsted-Lowry base. In addition, a combination of two or more cleavage agents / bases (e.g., cleavage agents / bases as discussed herein) can be used. In some aspects, a strong Arrhenius base (e.g., Na + OH - , K + OH - or Ca +2 (OH - ) 2) can be used in combination with the cleavage reagents / bases discussed herein. In some aspects, ammonia can be contacted with the solution by bubbling or as an atmospheric component to act as a cleavage reagent and / or quenching reagent. In some aspects, the concentration of the cleavage reagent is at least about 0.1M (+ / -20%). In some aspects, the concentration of the cleavage reagent is 0.1M to 5.0M. In some aspects, the cleavage reagent exists as a saturated solution or an insoluble substance. In some aspects, the cleavage reagent brings the solution pH to 7.5, 8, 9, 10, 12 or higher. In all methods described herein, the cleavage reagent (cleavage initiator) can also be and preferably is a peroxide quenching reagent, and in either case can be used in combination with another compatible peroxide quencher, which can be a cleavage agent or not a cleavage agent. In some aspects, the polysaccharide includes one or more of amylose, amylopectin, beta-glucan, pullulan, xyloglucan, arabinogalactan I and arabinogalactan II, rhamnogalacturonic acid polysaccharide I, rhamnogalacturonic acid polysaccharide II, polygalacturonic acid, polyglucose, galactan, arabinan, arabinoxylan, xylan (e.g., beech xylan), glycogen, mannan, glucomannan, curdlan, galactomannan, galactan, lichenan and inulin. In some aspects, the polysaccharide is from plant origin or animal origin. In some aspects, the polysaccharide is from bacterial origin, yeast origin or algae origin. In some aspects, the polysaccharide is in the form of (optionally freeze-dried) plant material. In some aspects, the plant material is locust bean gum, fenugreek seeds, distillers grains or spent distillers grains or some fraction or extract thereof. In some aspects, the method further comprises purifying one or more oligosaccharides from the oligosaccharide mixture.

[0290] In some aspects, prior to the reaction, the method comprises contacting the polysaccharide with one or more polysaccharide degrading enzymes. In some aspects, the one or more polysaccharide degrading enzymes comprise, for example, amylase, isoamylase, cellulase, maltase, glucanase, xylanase, lactase or a combination thereof.

[0291] In some aspects, the polysaccharide material can be pretreated with acids, bases, and / or oxidizing and reducing agents prior to reaction.

[0292] Also provided are compositions comprising a mixture of oligosaccharides produced using the COG methods disclosed above or elsewhere herein, or one or more purified oligosaccharides produced using the COG methods disclosed above or elsewhere herein.

[0293] In some aspects, the COG method includes contacting one or more microorganisms (e.g., bacteria, fungi, yeast) with a composition comprising an oligosaccharide or oligosaccharide mixture to selectively stimulate the growth of the one or more microorganisms. In some aspects, the microorganism includes a probiotic microorganism. In some aspects, the one or more microorganisms are in the intestinal tract of an animal, and the composition is applied to the animal. In some aspects, the one or more microorganisms (prebiotic microorganisms) and the composition are applied to the animal respectively (e.g., sequentially), or applied to the animal simultaneously with the composition (e.g., applying a composition comprising the probiotic microorganism and an oligosaccharide or oligosaccharide mixture). In some aspects, the one or more microorganisms are located at or introduced into a specific position or lumen (e.g., vagina) of an animal or a human. In some aspects, the probiotic microorganism is a pseudocatenulatum bifidobacterium. In some aspects, the probiotic microorganism is a crisp lactobacillus (Lactobacillus Crispatus). In some aspects, the one or more microorganisms are soil microorganisms, oral microorganisms (e.g., bacteria) or skin microorganisms. In some aspects, the one or more oligosaccharides can be used together with antibiotic treatment. In some aspects, the one or more oligosaccharides can be used together with antibiotic treatment and one or more probiotics. In some aspects, the one or more oligosaccharides can be used together with determined bacterial groups or undetermined bacterial groups. In some aspects, the one or more oligosaccharides can be used as excipients. definition

[0294] As used herein, the term "polysaccharide" refers to a polysaccharide or a material comprising a polysaccharide, in either case, wherein at least the polysaccharide component can be cleaved by the COG method disclosed herein. In addition, as used herein, the term "polysaccharide" refers to any carbohydrate polymer (e.g., disaccharides, oligosaccharides, polysaccharides) and may also be linked to other non-carbohydrate moieties (e.g., glycoproteins, proteoglycans, glycopeptides, glycolipids, glycoconjugates, glycosides).

[0295] As used herein, the term "peroxidase" refers to a compound containing an oxygen-oxygen bond that can naturally generate RO and / or ROO species under the action of light, temperature, or a catalyst (e.g., metals and enzymes), wherein "R" refers to a hydrogen or carbon group attached to the rest of the molecule. In one aspect, the peroxidase is hydrogen peroxide.

[0296] The "degree of polymerization" or "DP" of an oligosaccharide refers to the total number of sugar monomer units that are part of a particular carbohydrate. For example, tetragalacto-oligosaccharide has a DP of 4, with 3 galactose moieties and one glucose moiety.

[0297] The term "Bifidobacterium" and its synonyms refer to a genus of anaerobic bacteria with properties that are beneficial to humans. Bifidobacterium is one of the major bacterial strains that make up the intestinal flora, which resides in the gastrointestinal tract and contributes to the health of its host (Guarner and Malagelada 2003).

[0298] "Prebiotics" or "prebiotic nutrients" are generally non-digestible food ingredients that, when ingested, produce a beneficial effect on the host by selectively stimulating the growth and / or activity of one or a limited number of microorganisms in the gastrointestinal tract. As used herein, the term "prebiotic" refers to the above non-digestible food ingredients in their non-naturally occurring state (e.g., after purification, chemical synthesis or enzymatic synthesis, other than, for example, in whole human milk).

[0299] "Probiotic" refers to live microorganisms that when administered in adequate amounts confer a health benefit on the host.

[0300] As used herein, "stripping reaction" or "stripping" as applied to the disclosed methods refers to the sequential alkaline degradation of carbohydrates by a mechanism that releases monomer units from the reducing end of the polymer.

[0301] As used herein, preferably, "cleavage agent" or "cleavage reagent" as applied to the disclosed method refers to a single or a group of non-Arrhenius bases and / or weak Arrhenius bases used for cleaving polysaccharides after oxidation of the peroxyhydroxyl group of the polysaccharide. In certain aspects, the cleavage agent or cleavage reagent destroys glycosidic bonds in the polysaccharide, which can be present between any two sugars of the polysaccharide. In the methods described herein, the cleavage agent (cleavage initiator) can also be and preferably is a peroxide quenching agent, and in either case can be used in combination with another compatible peroxide quencher, which can also be a cleavage agent or not. In some aspects, the cleavage agent can be an enzyme. In some aspects, the cleavage agent enzyme can be a glycosyl hydrolase, a soluble polysaccharide monooxygenase, a glycosyltransferase, a transglycosidase, a polysaccharide lyase, a carbohydrate binding component, a glycosyltransferase, a carbohydrate esterase, a mixture comprising two or more of the foregoing enzymes, or any enzyme having carbohydrate activity. In some aspects, the cleavage reagent can be a solid phase acid catalyst or a solid phase base catalyst.

[0302] As used herein, "base" refers to a compound or collection of compounds that can accept hydrogen ions from carbohydrates, water, or non-aqueous solvents oxidized by hydrogen peroxide. The term "base" can include Lewis bases, non-Arrhenius bases, weak Arrhenius bases, other molecules that produce hydroxide ions, Lewis bases, non-Arrhenius bases, or weak Arrhenius bases by decomposition, or other compounds that can accept hydrogen ions from carbohydrates oxidized by hydrogen peroxide. As used herein, unless otherwise specified, "base" explicitly does not refer to strong Arrhenius bases (e.g., Na + OH - , K + OH - or Ca +2 (OH - ) 2 ).

[0303] As used herein, "ammonium bicarbonate" as applied to the disclosed methods refers to solid ammonium bicarbonate, and / or an aqueous solution comprising: ammonium and bicarbonate; ammonium, OH - and CO 2 ; Ammonia, H 2 O and CO 2 ; or any of the above products and their equilibrium products.

[0304] As used herein, "ammonium hydroxide" as applied to the disclosed methods refers to aqueous ammonium hydroxide solutions, and / or solutions comprising: ammonia and H 2 O; ammonium and OH - ; Ammonia and OH - ; or any of the above products and their equilibrium products.

[0305] As used herein, a "strong Arrhenius base" as applied to the disclosed methods refers to a compound that completely dissociates in water to release one or more hydroxide ions into solution. As used herein, a "strong Arrhenius base" as applied to the disclosed methods specifically refers to KOH, NaOH, Ba(OH) 2 、CsOH、Sr(OH) 2 , Ca(OH) 2 , LiOH and RbOH.

[0306] As used herein, a "weak Arrhenius base" as applied to the disclosed methods refers to a compound that does not completely dissociate in water to release one or more hydroxide ions into solution, such as ammonium hydroxide, H 2 O, etc. As "weak Arrhenius base" is used herein, there are no compounds that meet the definitions of both a strong Arrhenius base and a weak Arrhenius base.

[0307] As used herein, a "non-Arrhenius base" as applied to the disclosed methods refers to a base that can donate electrons (e.g., a Lewis base), accept protons (e.g., a Bronstead-Lowry base), or release hydroxide ions (NH 4 HCO 3 ), but compounds or atoms that are not explicitly considered to be Arrhenius bases.

[0308] As used herein, a "Lewis base" as applied to the disclosed methods refers to a compound or atom (e.g., F) that can donate an electron pair. - , benzene, H - , pyridine, acetonitrile, acetone, urea, etc.).

[0309] As used herein, a "Bronsted-Lowry base" as applied to the disclosed methods refers to a compound or atom (eg, methanol, formaldehyde, ammonia, etc.) that can accept or bind a hydrogen ion.

[0310] As used herein, a "peroxide quenching agent" applied to the disclosed methods refers to a compound or atom (e.g., ammonium hydroxide, ammonium bicarbonate, ammonia, etc.) that is not a strong Arrhenius base and can convert hydrogen peroxide, peroxy radicals, and peroxyhydroxyl radicals into a weaker reactive state or a non-reactive state. In some aspects, the peroxide quenching agent defined herein converts hydrogen peroxide and free radicals generated by hydrogen peroxide into a weaker reactive substance (e.g., water). In some aspects, the peroxide quenching agent can reduce the concentration of hydrogen peroxide to zero, less than 5 mg / L, less than 10 mg / L, less than 25 mg / L, or less than 50 mg / L. In some aspects, the peroxide quenching agent can form water, hydroxide ions, or oxygen. In some aspects, enzymes can be used to quench peroxidized substances. In some aspects, these enzymes can include catalase. In some aspects, these enzymes can be from animal sources. In some aspects, these enzymes can be from bovine liver. In some aspects, the enzyme can be from a microbial source. In some aspects, the enzyme can be recombinant. In certain aspects, different enzymes can be mixed to quench peroxidative species.

[0311] As used herein, "nitrogen-based" as applied to the disclosed methods refers to a compound comprising at least one nitrogen atom and four substituents, which may comprise any combination of lone electron pairs, hydrogen or carbon atoms (e.g., ammonia, sodium amide, trimethylamine, diethylamine, N,N-diisopropylethylamine, urea, pyridine, ammonium hydroxide, ammonium bicarbonate, etc.). Exemplary nitrogen-based peroxide quenchers and nitrogen-based PS cleavage agents are listed in Table 1. The nitrogen-based reagent may have an unsubstituted ammonium group or a substituted ammonium group, and may exist in a neutral and / or ionic form.

[0312] As used herein, a "reaction mixture" refers to a mixture comprising reagents that can chemically react to form products other than the reagents.

[0313] As used herein, "treated polysaccharide" refers to a polysaccharide that has been contacted with at least one reagent capable of reacting with the polysaccharide (eg, an enzyme or Fenton's reagent).

[0314] As used herein, "polysaccharide cleavage products" are products formed by chemical and / or enzymatic cleavage of polysaccharides.

[0315] As used herein, "oligosaccharide" refers to a low molecular weight polysaccharide, a polymer of 3 to 30 monosaccharide units. The oligosaccharide can be a linear polymer, a branched polymer, a main linear polymer with side chain sugar monomers, or any combination thereof.

[0316] As used herein, "polysaccharide" refers to a polymer of monosaccharide units greater than 30 monosaccharide units. The polysaccharide can be a linear polymer, a branched polymer, a primarily linear polymer with pendant sugar monomers, or any combination thereof.

[0317] As used herein, "Fenton's reagent" refers to a reagent comprising a peroxidizing agent and a metal. In certain aspects, the peroxidizing agent is hydrogen peroxide. In certain aspects, the metal is Fe(II), Fe(III), Cu(I), Cu(II), Mn(II), Zn(II), Ni(II), and Co(II); alkaline earth metals Ca(II) and Mg(II); lanthanide Ce(IV), or any combination thereof.

[0318] As used herein, the phrase "substantially simultaneous with initiation of peroxide quenching" refers to the relationship between the timing of the cleavage reaction and the timing of the peroxide quenching reaction, indicating that initiation of the cleavage reaction and initiation of the peroxide quenching reaction occur within a short time of each other (e.g., about a few seconds, or about a few minutes, but not more than a day).

[0319] As used herein, "specified reaction time" or "reaction time" refers to providing time to allow a reaction to proceed to a state of equilibrium between the added reagents and the products produced by the reaction of the reagents. In some aspects, the specified reaction time allows sufficient time to reach equilibrium. In certain other aspects, the specified reaction time, while allowing the reaction time to proceed to equilibrium, does not provide the time required to reach equilibrium.

[0320] As used herein, the term "synthetic oligosaccharide" refers to an oligosaccharide produced by the depolymerization of a polysaccharide. According to the methods described herein, by depolymerizing heteropolymer polysaccharides and homopolymer polysaccharides, a synthetic oligosaccharide according to the present invention can be obtained. In some aspects, the term synthetic oligosaccharide refers to an oligosaccharide pool produced by the methods disclosed herein.

[0321] As used herein, the term "heteropolysaccharide" refers to a polysaccharide comprising two or more monosaccharide subunits linked together by the same type of glycosidic bonds or different types of glycosidic bonds; heteropolysaccharides also include polysaccharides comprising repeating monosaccharide subunits of the same type linked together by different types of glycosidic bonds. The glycosidic bonds in the heteropolysaccharide can be β1-2 bonds, β1-3 bonds, β1-4 bonds, β1-6 bonds, α1-3 bonds, α1-4 bonds, α1-6 bonds, or combinations thereof. Examples of heteropolysaccharides include, but are not limited to, xyloglucan, lichenin, β-glucan, glucomannan, galactomannan, arabinan, xylan, and arabinoxylan.

[0322] As used herein, when the terms "about" and "approximately" are used to modify a numerical value or an amount specified in a range, it indicates that the numerical value and reasonable deviations from the value known to those skilled in the art, such as ±20%, ±10%, or ±5%, are within the intended meaning of the listed value.

[0323] As disclosed herein, controlled oligosaccharide generation ("COG") is a method for controlled degradation of polysaccharides into oligosaccharides. In some aspects, crude polysaccharides are first subjected to an initial oxidation treatment with hydrogen peroxide and a transition metal, alkaline earth metal or lanthanide catalyst to make the glycosidic bond more unstable. Ammonium hydroxide, ammonium bicarbonate, ammonia, urea, etc., or other weak Arrhenius or non-Arrhenius bases are then used for cleavage to produce a variety of unique oligosaccharides (unique oligosaccharide spectrum) or smaller polysaccharides. In some aspects, peroxide quenching and / or neutralization are immediately performed to reduce unwanted oxidation or stripping, respectively. In some aspects, a treated sample (e.g., a polysaccharide raw material after Fenton's reagent treatment) is allowed to react with a cleavage reagent at a reduced temperature, ambient temperature or room temperature to promote the production of oligosaccharides. In some aspects, the cleavage reaction is performed at 4°C to 100°C, 20°C to 80°C, 30°C to 60°C or 40°C. In some aspects, cleavage and peroxide quenching are instant. In some aspects, the cleavage step is carried out for 10 minutes to 30 minutes, 20 minutes to 60 minutes, 30 minutes to 120 minutes. In some aspects, the cleavage step is carried out for 2 hours to 6 hours, 3 hours to 12 hours, 6 hours to 24 hours or longer. In some aspects, the cleavage step is carried out overnight. In all methods described herein, the cleavage reagent (cleavage initiator) can also be and preferably is a peroxide quenching reagent, and in either case can be used in combination with other compatible peroxide quenchers, and the other compatible peroxide quenchers can be cleavage agents or not. The disclosed COG method has the ability to produce a large amount of bioactive oligosaccharides from a variety of carbohydrate sources (e.g., raw materials containing polysaccharides).

[0324] In certain aspects, the method for cleaving polysaccharides comprises a plurality of steps. For example, the method may comprise: a) contacting one or more polysaccharides with a Fenton reagent comprising a peroxide reagent and a metal ion to form a mixture; b) allowing the Fenton reagent to react with the polysaccharide for a specified reaction time; c) after step b, adding a cleaving agent, which may also be a peroxide quenching agent, to the mixture. In this regard, the steps of contacting the polysaccharide with the Fenton reagent (step a) and allowing a specified reaction time (step b) to pass can be performed at the same pH or at different pHs, wherein the pH is selected from the range of pH 3 to pH 8, pH 4 to pH 7, pH 4.5 to pH 6.5, and pH 5 to pH 6. The pH may be any possible value within the specified pH range. The step of adding a cleaving agent, which may also be a peroxide quenching agent (step c), may be performed at a pH selected from the range of pH 6 to pH 11, pH 6.5 to pH 9.5, pH 7 to pH 9, and pH 7 to pH 8. The pH may be any possible value within the specified pH range. In this regard, the step of contacting the polysaccharide with the Fenton reagent (step a) and the step of allowing the specified reaction time (step b) can be performed at the same temperature or at different temperatures, wherein the temperature is selected from the temperature range of 10 degrees Celsius to 70 degrees Celsius, 20 degrees Celsius to 60 degrees Celsius, and 25 degrees Celsius to 55 degrees Celsius. The temperature can be any possible value in the specified temperature value range. The step of adding a lysing agent, which can also be a peroxide quenching agent (step c), can be performed at a temperature selected from the temperature range of 10 to 70 degrees Celsius, 20 to 60 degrees Celsius, and 25 to 55 degrees Celsius. The temperature can be any possible value in the specified temperature value range.

[0325] In some aspects, the oligosaccharide material can be treated with a suitable resin material. The suitable resin material may include anion exchange, cation exchange, decolorization, chelating properties. For example, suitable resins may include but are not limited to Ionac NM-60, MBD-10ULTRA, Thermax Tulsion MB, Cole-Parmer RR-1400, Amberlite MB20, DOWEX Monosphere MR-450. Two or more resins can be combined to produce a mixed bed resin. The sample can be treated with carbon. The carbon can be activated carbon, charcoal, graphitized carbon, porous graphitized carbon or any carbon-based material added for the purpose of purification.

[0326] If desired, before the obtained polysaccharide is treated with the COG method, one or more polysaccharide degrading enzymes may be optionally used to treat the polysaccharide to reduce the average size or complexity of the polysaccharide. Non-limiting examples of polysaccharidase include, for example, amylase, isoamylase, cellulase, maltase, glucanase, lactase, xylanase, arabinase, pectinase, mannanase or a combination thereof. In some aspects, carbohydrate-active enzymes may be used to modify the resulting product to prepare a new product by adding or removing monomeric units.

[0327] In the COG method, the initial oxidation treatment may include hydrogen peroxide and a transition metal, an alkaline earth metal or a lanthanide metal, wherein these metals may be used alone or in combination. In the COG method, different metals may be used to produce oligosaccharides or oligosaccharide profiles with a characteristic degree of polymerization (DP) or a preferred degree of polymerization. In the COG method, different metals may be used to produce different oligosaccharide profiles from similar raw materials. The oxidation treatment of the method is followed by a peroxide quenching / cracking treatment. The COG method is capable of generating oligosaccharides from polysaccharides having different degrees of branching and having a variety of monosaccharide compositions (including natural polysaccharides and modified polysaccharides). The COG method is applicable to polysaccharides from any source. Exemplary polysaccharide substrates include, but are not limited to, one or more of amylose, amylopectin, beta-glucan, pullulan, xyloglucan, arabinogalactan I and arabinogalactan II, rhamnogalacturonan I, rhamnogalacturonan II, polygalacturonic acid, polyglucose, galactan, arabinan, arabinoxylan, xylan (e.g., beech xylan), glycogen, mannan, glucomannan, curdlan, galactomannan, galactan, lichenin, and inulin. The original or natural source and form of the material containing the polysaccharide can be used. The material containing the polysaccharide can be in natural form, or can be infiltrated, ground, chopped, cavitated, or otherwise separated or altered before contacting with the reactant.

[0328] The one or more oligosaccharides (e.g., mixture) produced by the COG process may have an average DP ranging from 2 to 200, such as 2 to 100 or 3 to 20 or 5 to 50, or any DP lower than that of the native polysaccharide, or any value in any sub-range of the foregoing exemplary ranges.

[0329] One or more oligosaccharides (e.g., mixtures) obtained by the COG method can have a variety of uses. In some aspects, the one or more oligosaccharides can be used as prebiotics to selectively stimulate the growth of one or more probiotics. In some aspects, the oligosaccharide composition can be used as a prebiotic preparation (i.e., without bacteria) or as a probiotic preparation (i.e., with one or more required bacteria, such as bifidobacteria described herein). Generally, any food or beverage that is edible or otherwise appropriately administered by humans or animals can be used to prepare a preparation containing a composition containing prebiotic and probiotic oligosaccharides. Exemplary foods include those foods that have a semi-liquid viscosity to allow the prebiotic and probiotic compositions described herein to be easily and evenly dispersed. However, other viscosities (e.g., powders, liquids, etc.) can also be used without restriction. Therefore, such foods include, but are not limited to, dairy-based products such as cheese, cottage cheese, yogurt, and ice cream. Processed fruits and vegetables, including those for infants / young children, such as applesauce or pea puree and carrot puree, are also suitable for use in combination with the oligosaccharides of the present invention. Infant cereals (such as rice-based or oat-based cereals) and baby foods such as Cream of Wheat TM Adult cereal foods such as , are also suitable for use in combination with the oligosaccharides. COG products can also be used in medical foods, such as Pedialyte TM 、Ensure TM etc. In addition to foods intended for human consumption, animal feeds can also be supplemented with compositions containing prebiotic and probiotic oligosaccharides.

[0330] Alternatively, the polysaccharide-containing material and / or oligosaccharide-containing composition (e.g., a composition containing prebiotic and probiotic oligosaccharides) treated by the COG method can be used to supplement beverages. Examples of such beverages include, but are not limited to, infant formula, follow-on formula, toddler beverages, milk, fermented milk, fruit juice, fruit drinks, and sports drinks. Many infant and toddler formulas are known in the art and are commercially available, including, for example, Carnation Good Start TM (Nestle Nutrition Division; Glendale, Calif.) and Nutrish AB produced by Mayfield Dairy Farms (Athens, Tenn.) TM Other examples of infant or baby formulas include those disclosed in US Pat. No. 5,902,617. Other beneficial formulations of the composition include supplementation with animal milk, such as cow's milk.

[0331] Alternatively, the composition containing prebiotics and probiotic oligosaccharides can be formulated into pills or tablets or encapsulated in capsules, such as gelatin capsules. Tablet forms can optionally include, for example, lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, one or more, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavorings, dyes, disintegrants and pharmaceutically compatible carriers. Lozenges or candy forms can include the composition in flavorings (such as sucrose), as well as pastilles (pastilles) containing the composition in an inert matrix, such as gelatin and glycerol or sucrose and gum arabic emulsions, gels, etc., which are known in the art, in addition to the active ingredients. Preparations containing prebiotics or probiotic oligosaccharides can also include fillers and bulking agents of conventional food supplements, such as rice flour. This product may also be used to aid in the absorption of other nutrients and minerals.

[0332] In some aspects, the composition containing prebiotic or probiotic oligosaccharides will include or further include non-human proteins, non-human lipids, non-human carbohydrates or other non-human components. For example, in some aspects, the composition can include bovine (or other non-human) milk protein, soy protein, rice protein, beta-lactoglobulin, whey, soybean oil or starch. In some aspects, the oligosaccharide is combined with a polysaccharide. In some aspects, the oligosaccharide is combined with its parent polysaccharide.

[0333] The dosage of the composition containing prebiotics and probiotic oligosaccharides will vary according to the needs of the individual, and / or will take into account factors such as age (infants and adults), weight, and the cause of loss of beneficial intestinal bacteria (e.g., antibiotic treatment, chemotherapy, radiation therapy, disease or age). In the context of the present disclosure, the administration regimen and the individual administration amount or individual consumption should preferably be sufficient to allow the colonization of beneficial bacteria in the intestine over time. The administration regimen and / or dosage size will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of the composition containing prebiotics or probiotic oligosaccharides provided. In some administration aspects, the dosage range will be effective as a food supplement and used to rebuild beneficial bacteria in the intestine. In some administration aspects, the dosage range of the oligosaccharide composition of the present invention is about 1 microgram / L to about 25 grams / L of oligosaccharides. In some aspects, the dosage of the oligosaccharide composition is about 100 micrograms / L to about 15 grams / L of oligosaccharides. In some aspects, the dosage of the oligosaccharide composition is about 1 g / L to 10 g / L, 5 g / L to 15 g / L, 10 g / L to 50 g / L, or up to 200 g / L. In some aspects, the dosage is 50 g / day to 70 g / day. In some aspects, the dosage is 10 g / day. In some aspects, the dosage is 1 g / day to 10 g / day. In some aspects, the dosage exceeds 100 g / day. In some aspects, the dosage is 0.25 g / day to 3 g / day. Exemplary Bifidobacterium dosages include, but are not limited to, about 10 g / day per dose. 4 to about 10 12 A more favorable range is about 10 6 CFU is about 10 10 Other bacteria may be administered at similar concentrations, but are not limited to approximately 10 CFU per dose. 4 to about 10 12 colony forming units (CFU) or about 10 per dose 6 CFU is about 10 10 CFU.

[0334] The disclosed preparations containing prebiotics or probiotic oligosaccharides can be applied to any subject / individual in need thereof. In some aspects, the individual is an infant or a toddler. For example, in some aspects, the individual is less than, for example, 3 months, 6 months, 9 months, 1 year old, 2 years old or 3 years old. In some aspects, the individual is 3 to 18 years old. In some aspects, the individual is an adult (e.g., 18 years old or older). In some aspects, the individual is more than 50 years old, 55 years old, 60 years old, 65 years old, 70 years old or 75 years old. In some aspects, the individual has an immunodeficiency (e.g., the individual suffers from AIDS or is receiving chemotherapy, immunotherapy or radiotherapy).

[0335] Exemplary bifidobacteria that may be included in the probiotic composition of the present invention include, but are not limited to, Bifidobacterium longum subsp. Infantis, B. longum subsp. Longum, Bifidobacterium breve, Bifidobacterium adolescentis, and B. pseudocatenulatum. The bifidobacterium used will depend in part on the target consumer.

[0336] It should be understood that it may be advantageous to include other bifidobacterial factors in the formulations described herein for some applications. Such additional components may include, but are not limited to, oligofructose such as Raffinose (Rhone-Poulenc, Cranbury, New Jersey), inulin (Imperial Holly Corp., Sugar Land, Texas) and Nutraflora (Golden Technologies, Westminister, Colorado), as well as lactose, oligoxylose, soy oligosaccharides, lactulose / lactitol and oligogalactose, etc. In some applications, the formulations may include other beneficial bacteria, such as Lactobacillus, Rumminococcus, Akkermansia, Bacteroides, Faecalibacterium. COG products described herein can be used to stimulate yeast.

[0337] Oligosaccharides as described herein can be used to stimulate any kind of microorganism. Examples of microorganisms that can be stimulated by the oligosaccharides include, for example, soil microorganisms (e.g., mycorrhizal fungi and bacteria and other microorganisms used as soil inoculants, such as Azospirillum sp.), oral bacteria (e.g., Streptococcus mutans, Streptococcus gordonii, Streptococcus sanguis, and S.oralis) and skin bacteria (e.g., Propionibacterium acnes, and ammonia oxidizing bacteria, including but not limited to Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosocvstis, Nitrosolobus, and Nitrosovibrio.

[0338] In some aspects, the disclosed oligosaccharide composition is applied to a person or animal in need. For example, in some aspects, the oligosaccharide composition is applied to a person or animal with at least one of the following conditions: inflammatory bowel syndrome, constipation, diarrhea, colitis, Crohn's disease, colon cancer, functional bowel disorder (FBD), irritable bowel syndrome (IBS), excessive sulfate-reducing bacteria, inflammatory bowel disease (IBD) and ulcerative colitis. Irritable bowel syndrome (IBS) is characterized by abdominal pain and discomfort, bloating, and changes in intestinal function, constipation and / or diarrhea. There are three types of IBS: mainly constipation-type IBS (C-IB), alternating IBS (A-IBS) and mainly diarrhea-type IBS (D-IBS). The oligosaccharide composition is useful, for example, for suppressing or prolonging the recurrence period of ulcerative patients. The oligosaccharide composition can be administered to treat or prevent any form of functional bowel disorder (FBD), in particular irritable bowel syndrome (MS), such as constipation-predominant IBS (C-IBS), alternating IBS (A-IBS) and diarrhea-predominant IBS (D-IBS); functional constipation and functional diarrhea. FBD is a general term for a series of chronic or semi-chronic gastrointestinal diseases that are associated with intestinal pain, intestinal dysfunction and social disturbance.

[0339] In some aspects, the oligosaccharide composition can be used as a bulking-agent. In some aspects, the oligosaccharide composition can be used as a bulking-agent in low-sugar food applications. In some aspects, these oligosaccharides can be used as a bulking-agent that does not affect local flavor, smell, rheological properties, and texture properties.

[0340] In another aspect, the oligosaccharide composition is administered to a human in need of stimulating the immune system and / or promoting resistance to bacterial or yeast infections (eg, candidiasis or diseases caused by sulfate-reducing bacteria).

[0341] Some aspects of the present disclosure provide synthetic oligosaccharides comprising a main chain containing glucose monomers, wherein each glucose monomer is optionally bonded to a xylose monomer of a side chain, and wherein the total number of monomers of the synthetic oligosaccharide ranges from 3 to 30. Such synthetic oligosaccharides can be obtained by, for example, depolymerizing xyloglucan according to the methods described herein. It is known that xyloglucan comprises a glucose main chain with a single unit of xylose branches, wherein the xylose branches can be modified with a galactose endcap or an arabinose endcap. For example, tamarind xyloglucan comprises a β1,4-linked glucose main chain and has a single unit branch of frequently occurring α1,6-linked xylose, which can sometimes be further connected to a single β1,2-linked galactose endcap. In other sources of xyloglucan, arabinose can be α1,2-linked to a xylose residue. Xyloglucan from other sources may contain a single fucose residue linked to galactose in α1,2.

[0342] In some aspects, the oligosaccharide comprises 2, 3, 4, 5 or 6 hexose residues. In some aspects, the oligosaccharide comprises 1, 2, 3 or more pentose residues. In some aspects, the oligosaccharide comprises the same number of hexose residues and pentose residues. In some aspects, the oligosaccharide comprises pentose residues less than hexose residues.

[0343] In some aspects, the glucose monomers in the backbone of the synthetic oligosaccharide are β1-4 linked glucose monomers. In some aspects, each side chain xylose monomer is bonded to the glucose monomer in the backbone through an α1-6 bond.

[0344] In some aspects, the synthetic oligosaccharide further comprises a galactose monomer bonded to one or more side chain xylose monomers. In some aspects, each galactose monomer is bonded to the side chain xylose monomer via a β1-2 connection. In some aspects, the synthetic oligosaccharide further comprises a fucose monomer bonded to one or more galactose monomers. In some aspects, each fucose monomer is bonded to the galactose monomer via an α1-2 bond.

[0345] In some aspects, the synthetic oligosaccharide further comprises an arabinose monomer bonded to one or more side chain xylose monomers. In some aspects, the arabinose monomer is bonded to the side chain xylose monomer via an α1-2 bond.

[0346] In some aspects, the synthetic oligosaccharide comprises 2 to 4 glucose monomers in the backbone, 1 to 2 side chain xylose monomers bonded to different glucose monomers in the backbone, and 0 to 2 galactose monomers bonded to different xylose monomers.

[0347] Some aspects of the present disclosure provide synthetic oligosaccharides having a main chain containing a mannose monomer, wherein each mannose monomer is optionally bonded to a side chain galactose monomer, and wherein the total number of monomers in the synthetic oligosaccharide is from 3 to 30. Such synthetic oligosaccharides can be obtained by depolymerizing galactomannan, for example, according to the methods described herein. Galactomannan produced by sources such as Aspergillus contains a β1-4 mannose main chain with frequent occurrences of α1-6 galactose side chains containing a single unit.

[0348] Some aspects of the present disclosure provide synthetic oligosaccharides comprising mannose monomers and glucose monomers, wherein the total number of monomers in the synthetic oligosaccharide is 3 to 30. Such synthetic oligosaccharides can be obtained, for example, by depolymerizing glucomannan according to the methods described herein. Glucomannan is a polysaccharide mainly found in konjac roots. The polymer comprises glucose residues and mannose residues linked in β1-4, which are believed to be randomly distributed in a non-repeating pattern.

[0349] Some aspects of the present disclosure provide synthetic oligosaccharides having a main chain containing arabinose monomers, wherein each arabinose monomer is optionally bonded to a side chain arabinose monomer, and wherein the total number of monomers in the synthetic oligosaccharide is 3 to 30. Such synthetic oligosaccharides can be obtained, for example, by depolymerizing arabinan according to the methods described herein. Arabinan is present as a side chain in the pectic polysaccharide rhamnogalacturonan I and is also present in the cell walls of some mycobacteria. Arabinan contains an α1-5 arabinose main chain and has short α1-3 arabinose branches.

[0350] In some aspects of the present disclosure, synthetic oligosaccharides derived from β-glucans found in cereals (e.g., rice, wheat, oats, bran, barley and malt) are provided, for example, consisting of a β1-4 connected glucose backbone and a single β1-3 glucose residue dispersed between every 2 to 3 β1-4 connected glucose residues. In some aspects of the present disclosure, synthetic oligosaccharides derived from lichen polysaccharides are provided, the lichen polysaccharides are polysaccharides found in lichens, having a structure similar to β-glucans, whose connections consist of β1-4 glucose residues and β1-3 glucose residues. However, unlike β-glucans, lichen polysaccharides have more frequent β1-3 bonds. In some aspects, oligosaccharides similar to β-glucans can be derived from waste distillers grains or other corn products. In some aspects, oligosaccharides similar to β-glucans can be derived from oats and oat agricultural wastes. In some aspects, oligosaccharides similar to β-glucans can be derived from waste beer grains or other malt products.

[0351] Some aspects of the present disclosure provide synthetic oligosaccharides having a main chain containing a xylose monomer, wherein each xylose monomer is optionally bonded to a side chain arabinose monomer or a side chain gluronic acid (gluronic acid) (e.g., 4-O methylated GlcA), and wherein the total number of monomers in the synthetic oligosaccharide is 3 to 30. Such synthetic oligosaccharides can be obtained by, for example, depolymerizing xylan and / or arabinoxylan according to the methods described herein. Xylan is a polysaccharide commonly found in the secondary cell walls of dicotyledonous plants and in the cell walls of most grasses. The structure contains a β1-4 xylose main chain and typically contains α1-2 glucuronic acid branches, which may contain a single methyl group. In some embodiments, beechwood xylan, which is known to contain a large number of 4-O-methyl-glucuronic acid branches, can be used. Arabinoxylan is a polysaccharide commonly found in cereals, which contains a β1-4 xylose main chain and has α1-2 arabinose branches and α1-3 arabinose branches. Some aspects of the present disclosure provide synthetic oligosaccharides similar to arabinoxylan. Some aspects of the present disclosure provide arabinoxylan-like oligosaccharides from the synthesis of spent distiller's grains, corn fiber or other corn-based streams. Some aspects of the present disclosure provide arabinoxylan-like oligosaccharides from the synthesis of spent distiller's grains, corn fiber or other corn-based streams. Some aspects of the present disclosure provide arabinoxylan-like oligosaccharides from the synthesis of spent beer grains or other grain-based streams.

[0352] In some aspects, synthetic oligosaccharides can also be obtained by depolymerizing homopolymer polysaccharides according to the methods described herein. As used herein, the term "homopolymer polysaccharide" refers to a polysaccharide containing repeating monosaccharide subunits of the same type, which are linked together by the same type of glycosidic bonds, including but not limited to β1-3 bonds, β1-4 bonds, β1-6 bonds, α1-3 bonds, α1-4 bonds, and α1-6 bonds. Examples of homopolymers include, but are not limited to, curdlan, galactan, and mannan. Homopolymers include, but are not limited to, curdlan (a linear polymer of glucose linked to β1-3, which is an exopolysaccharide of Agrobacterium), galactan (a linear polymer of galactose linked to β1-4, which is separated as arabinogalactan before subsequent arabinofuranosidase treatment to remove arabinose units) and mannan (a linear polymer of glucose linked to β1-3, which is an exopolysaccharide of Agrobacterium and some nuts).

[0353] In some respects, the synthetic oligosaccharide can be prepared by any suitable method, including but not limited to controlled oligosaccharide generation (COG), which is a method for controlling the degradation of polysaccharides into oligosaccharides. In some respects, firstly, crude polysaccharides are subjected to initial oxidation treatment with hydrogen peroxide and transition metal or alkaline earth metal (e.g., iron sulfate (III)) catalyst, so that glycosidic bonds are more unstable. Then weak Arrhenius base or non-Arrhenius base are used to carry out alkali-induced cracking, thereby producing a variety of oligosaccharides. Can be immediately neutralized to reduce any stripping reaction. The method can produce a large number of biologically active oligosaccharides from a variety of carbohydrate sources.

[0354] If desired, before the obtained polysaccharide is treated with oxidation and a metal catalyst, the polysaccharide may be optionally treated with one or more polysaccharide degrading enzymes to reduce the average size or complexity of the polysaccharide. Non-limiting examples of polysaccharidase include, for example, amylase, isoamylase, cellulase, maltase, glucanase or a combination thereof.

[0355] The initial oxidation treatment may include hydrogen peroxide and a transition metal or alkaline earth metal. Metals with different oxidation states, sizes, periodic table groups, and coordination numbers have been tested to understand the application of the COG process. Each different metal is active in the COG reaction. Although these metals can be used with any polysaccharide, different metals can be used to produce oligosaccharides with a preferred degree of polymerization. The oxidation treatment is followed by an alkali treatment. The method is capable of producing oligosaccharides from polysaccharides (including natural polysaccharides and modified polysaccharides) with different degrees of branching and having a variety of monosaccharide compositions.

[0356] Also provided is a mixture comprising two or more different synthetic oligosaccharides as described herein. Unpurified or semi-purified depolymerization products can be used to prepare oligosaccharide mixtures, or oligosaccharides can be purified to produce a specially formulated library. For example, the synthetic oligosaccharides in the mixture can be obtained by depolymerizing polysaccharide homopolymers, polysaccharide heteropolymers or a combination thereof. In some aspects, at least one synthetic oligosaccharide in the mixture is obtained by depolymerizing xyloglucan, curdlan, galactan, mannan, lichenin, β-glucan, glucomannan, galactomannan, arabinan, xylan, arabinoxylan, other polymers described herein or a combination thereof. In some aspects, based on the total amount of oligosaccharides in the mixture, the amount of at least one synthetic oligosaccharide in the mixture is at least 1%. The synthetic oligosaccharide can, for example, be present in an amount of about 1% to about 99%, or about 5% to about 95%, or about 10% to about 90%, or about 20% to about 80%, or about 30% to about 70%. The synthetic oligosaccharide can, for example, exist with an amount of about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99%. The percentage can be the mole % based on the total moles of oligosaccharides in the mixture, or the weight % based on the gross weight of oligosaccharides in the mixture. In some aspects, the amount of at least one synthetic oligosaccharide is at least 5mol%.

[0357] The synthetic oligosaccharides and compositions described herein can be used as synbiotics, prebiotics, immunomodulators, digestive aids, food additives, pharmaceutical excipients or analytical standards. The synthetic oligosaccharides can be combined with other ingredients to produce foods and supplements, including infant formula, elderly supplements, baked flours and snack foods. The synthetic oligosaccharides can be combined with beneficial bacteria to form synbiotics. The synthetic oligosaccharides can also be used as pharmaceutical products.

[0358] The synthetic oligosaccharides can be used for the growth or maintenance of specific microorganisms in humans, other mammals or plant rhizospheres. The synthetic oligosaccharides can contain specific glycosidic bonds that cannot be digested by a specific host (e.g., humans, livestock or pets), but can be metabolized by a specific symbiotic microbial community or probiotic community. Therefore, the synthetic oligosaccharides can be used as carriers to transport exogenous microorganisms (probiotic microorganisms or biotherapeutic microorganisms) to specific ecological niches, or as a source of nutrition for microorganisms already present in the host.

[0359] Xyloglucan can be used for the selective growth of specific Bacteroides species, such as B. ovatus (Larsbrink et al. 2014). It has been demonstrated that the xylan utilization locus with glycoside hydrolase genes belongs to the GH5 and GH31 families that can be found in B. ovatus. The presence of these genes allows the growth of this species when used as the sole carbon source. Other major Bacteroides species in the intestine, such as B. thetaiotaomicron, B. caccae or B. fragilis, lack this locus or part of this locus in their genome and are therefore unable to metabolize xyloglucan.

[0360] When the genome of a Bacteroides species encodes a specific type of glycoside hydrolase belonging to the GH16 family, curdlan can be used for the selective growth of a specific Bacteroides species (e.g., B. thetaiotaomicron or B. distasonis). Orthologs of this gene are absent in the genomes of other Bacteroides species (e.g., B. faecalis or B. ovatus) and are unable to grow on curdlan (Salyerss et al. 1997).

[0361] β-Glucan or lichenin can be used for the selective growth of specific Bacteroides species, such as Bacteroides ovatus. This species encodes a specific type of GH16 in its genome, which has β1-3,4 glucan activity (Tamura et al. 2017). This polysaccharide has been shown to promote the growth of Firmicutes species, such as Enterococcus faecium, Clostridium perfingens, Roseburia inulinivorans, and R. faecis (Beckmann et al. 2006, Sheridan et al. 2016).

[0362] Galactans can selectively enable the growth of specific Bacteroides species, such as B. thetaiotaomicron, B.dorei and B. ovatus. Different types of endogalactanases can lead to this selective growth, which belong to the GH53 and GH147 families (Lammerts van Bueren et al. 2017, Luis et al. 2018). The ability to consume galactans has also been described in some Bifidobacterium species (Bif. Breve, Bif. Longum, Biflongsubsp. Infantis) (Hinz et al. 2005).

[0363] Mannans can selectively enable the growth of specific Bacteroides species, such as Bacteroides fragilis or Bacteroides ovatus, which encode GH26 endo-β1-4-mannosidase (Kawaguchi et al. 2014). This gene is not present in the genomes of major intestinal species such as Bacteroides thetaiotaomicron that cannot grow on mannan or glucomannan. R. intestinalis and R. faecis can deplete mannan bonds (Leanti La Rosa et al. 2019), as can members of Clostridium cluster XIVa, whose genomes encode GH26 (Desai et al. 2016, Sheridan et al. 2016). In addition, GH26 has been characterized in specific species of Bifidobacteria, such as Bif. Adolescentis (Kulcinskaja et al. 2013), confirming the ability of this species to grow on mannan. Galactomannans are consumed only by microorganisms that encode endo-β1-4-mannosidase GH26 and α-galactosidase GH27 in their genome, such as Bacteroides ovatus, B. xylanisolvens (Reddy et al. 2016) or Roseburia intestinalis (Desai et al. 2016, Leanti La Rosa et al. 2019).

[0364] Xylans, arabinans and arabinoxylans can be used to selectively grow specific species of Bacteroides. Xylans can be metabolized by Bacteroides ovatus and Bacteroides uniformis, while Bacteroides thetaiotaomicron or Bacteroides faecalis cannot grow in this medium. Arabinans promote the growth of Bacteroides thetaiotaomicron and Bacteroides ovatus, while arabinoxylans show a high degree of selectivity for the growth of Bacteroides ovatus (Martens et al. 2011, Desai et al. 2016). It has been shown that strains of R.intestinalis, E.rectale and R.faecis can consume xylan or arabinoxylan as the sole carbon source (Desai et al. 2016, Sheridan et al. 2016). Certain bifidobacteria have the ability to ferment xylans or arabinofuranosyl oligosaccharides. B.adolescentis has shown selective growth on xylose and arabinoxylan-derived polysaccharides in vitro (Van Laere et al. 1999). Furthermore, additional experiments demonstrated that B. longum subsp. Longum is also able to metabolize arabinoxylan (Margolles and De Los Reyes-Gavilán 2003). Example 1 (Ammonium hydroxide and ammonium bicarbonate were used as exemplary polysaccharide (PS) cleavage reagents)

[0365] Locust bean gum is known to have a high content of galactomannan polysaccharides. Galactomannan is a polysaccharide that contains a β1-4 linked mannose backbone with α1-6 linked galactose branches. Galactomannan (or oligosaccharides that can be obtained using the disclosed method) can be used to selectively promote the growth of bacteria that can depolymerize one or both of these glycosidic bonds.

[0366] Oligosaccharide production. In the first exemplary aspect, locust bean gum (500mg) was dissolved in 20ml of HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55°C and 85RPM for 10 minutes. The pH of the solution was adjusted to 5.2 with ammonium bicarbonate (0.5M). Hydrogen peroxide (5ml) and iron (III) sulfate (2.75mg in 50μL water) were added to the reaction mixture and mixed thoroughly. The reaction in the capped reaction vessel was carried out in a shaking incubator at 55°C and 75RPM for 2 hours. The closed reaction was cooled to 20°C. Four cleavage conditions were implemented: ammonium hydroxide (1ml, 28% v / v, to pH 10), sodium hydroxide (65μl, 10.45M NaOH, to pH 10) and two concentrations of ammonium bicarbonate (1.125g and 5g, both to pH 7.5). All four conditions were reacted at 70 RPM for 1 hour in a shaking incubator at both 27°C and 45°C, with the lid loosened to release oxygen, ammonia and carbon dioxide gases. Ammonium hydroxide and ammonium bicarbonate were removed, and the solution was neutralized by evaporation. Sodium hydroxide was neutralized by adding HCl to pH 7. Samples were stored at -20°C before clean-up and subsequent mass spectrometry analysis.

[0367] Separation of oligosaccharides. The oligosaccharide samples after cleavage were reconstituted in water and subjected to C18 solid phase extraction. Before loading and collecting the samples, the solid phase column was washed with three volumes of acetonitrile and two volumes of water as direct flushing liquid (flow-through). The samples extracted by the C18 column were then subjected to non-porous graphitized carbon (NPGC) solid phase extraction. The NPGC column was pre-washed with two volumes of water, two volumes of 80% acetonitrile containing 0.01% (v / v) TFA aqueous solution, and another two volumes of water. The sample extracted by the C18 column was then loaded and washed with five volumes of water, and then eluted with 40% acetonitrile containing 0.05% (v / v) TFA. Finally, the samples after NPGC were completely dried by evaporative centrifugation and stored at -20°C until analysis.

[0368] Instrumental analysis. The dried post-NPGC samples were reconstituted in nanopure water prior to UHPLC-QqQ analysis. Analytical separation was performed using an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6495 QqQ MS. The samples were chromatographed on a 50 mm × 1 mm Waters AcquityTM BEH-AMIDE column with a particle size of 1.9 μm. A binary gradient consisting of solvent A: (3% (v / v) acetonitrile / water + 0.1% formic acid) and solvent B: (95% acetonitrile / water) was used. Chromatographic separation was performed using a 4.5-minute gradient with a flow rate of 0.6 ml / min: 70-67% B, 0 to 3 minutes; 67-25% B, 3 to 3.01 minutes; 25-25% B, 3.01 to 3.5 minutes; 25-70% B, 3.5 to 3.51 minutes; 70-70% B, 3.51 to 4.5 minutes. Electrospray ionization was used as the ion source, and data were collected in positive ion mode, and single ion monitoring (SIM) was used. The capillary voltage and fragmentor voltage were 1800 V and 280 V, respectively. The quadrupole was set to scan the mass corresponding to the oligosaccharides from 2 to 10 hexoses, with a residence time of 50 ms. All ions were observed to be their proton adducts.

[0369] Ammonium hydroxide and ammonium bicarbonate were used as exemplary PS cleavage reagents. Among the three cleavage reagents, ammonium hydroxide produced the highest amount of total oligosaccharides from locust bean gum at 45°C and 27°C, followed by two ammonium bicarbonate concentrations at 45°C, sodium hydroxide at two temperatures, and finally ammonium bicarbonate at 27°C ( Figure 1 ). Unexpectedly, ammonium hydroxide produced the highest total oligosaccharide abundance without sacrificing oligosaccharide structural diversity ( Figure 2 ), and produced twice as many total oligosaccharides as compared to sodium hydroxide and ammonium bicarbonate. The results were even more unexpected because it was expected that the concentration of hydroxide ions, which are readily present in ammonium hydroxide and sodium hydroxide, would be correlated with the production of oligosaccharides in the PS cleavage step in the COG reaction; however, both the ammonium hydroxide and sodium hydroxide reactions reached pH 10, indicating that the concentration of hydroxide ions was the same. In addition, considering that the pH at 27°C was only 7.5, it was unexpected that ammonium bicarbonate produced a large amount of oligosaccharides, although it was almost consistent with the oligosaccharide yield when NaOH was used as the cleavage reagent. According to specific aspects of the present invention, these results indicate that the non-hydroxide-mediated mechanism is cleavage mediated by ammonium bicarbonate. In addition, the increased efficiency of ammonium hydroxide as a PS cleavage reagent may indicate that a combination of hydroxide-driven and ammonia-driven mechanisms are involved.

[0370] Mechanistic differences in the specificity of the reaction were also observed regarding the size distribution of the oligosaccharides produced ( Figure 2). Oligosaccharides ranging from 3 to 10 monomers in length were observed from the reactions at 45 °C. Significant differences in the relative concentrations of trisaccharides were observed. The two ammonium bicarbonate-mediated PS cleavage reactions produced the highest relative abundance of trisaccharides (15.0% and 12.7%), while the sodium hydroxide-mediated PS cleavage reaction produced the lowest relative abundance of trisaccharides (9.52%), and ammonium hydroxide-mediated PS cleavage was intermediate (11.2%). The differential yields of trisaccharides between sodium hydroxide-mediated PS cleavage reactions and ammonium bicarbonate-mediated PS cleavage reactions provided additional evidence for different reaction mechanisms, and the intermediate amounts produced by ammonium hydroxide-mediated trisaccharides further supported a combined PS cleavage mechanism. Other significant differences included that sodium hydroxide produced the greatest relative abundance of tetrasaccharides and pentasaccharides, while the hexasaccharide remained the most abundant oligosaccharide under all four conditions. In addition, all three nitrogen-based reagent-mediated cleavage reactions produced higher amounts of decasaccharides than sodium hydroxide-mediated cleavage reactions.

[0371] Without being bound by mechanism, applicants' data are consistent with a mechanism in which cleavage of polysaccharides treated with peroxyhydroxyl radicals, such as with nitrogen-based cleavage agents (rather than with strong Arrhenius bases used in the art), is carried out via a unique β-elimination mechanism involving deprotonation of the hydroxyl moiety, such as with ammonia (or a decomposition product of a cleavage agent, such as a nitrogen-based cleavage agent), located β-relative to the glycosidic bond, because the adjacent ketone (from the peroxidation step) pulls electron density away from the hydrogen, making it a better leaving group. As suggested, when, for example, ammonia deprotonates the carbohydrate, the electrons form carbon-carbon double bonds that promote cleavage of the glycosidic bond, thereby depolymerizing the polysaccharide. Example 2 (When using ammonium bicarbonate or ammonium hydroxide as the polysaccharide (PS) lysis reagent, the need for desalting is reduced or eliminated)

[0372] Desalting of oligosaccharides is an expensive and time-consuming process, which, prior to the present invention, was a major limitation in producing oligosaccharides using existing Fenton reagent-based methods. Dialysis and chromatography desalting is a process that is performed by neutralizing traditional strong Arrhenius bases (e.g., NaOH, KOH, Ca(OH) 2) are two common methods for separating oligosaccharides from salts (e.g., sodium chloride, sodium acetate, and potassium chloride) produced when the reaction is carried out. Both methods have proven difficult because low molecular weight salts such as sodium chloride (58.44 g / mol) and oligosaccharides such as maltotriose (504.44 g / mol) are close enough in mass to be difficult to separate. Both methods also require that the volume of the sample be first reduced before separation, which further increases the cost and processing time required. According to specific aspects, the use of a currently disclosed high-yield nitrogen-based peroxide quencher / PS cleavage agent (e.g., ammonium bicarbonate, ammonium hydroxide, ammonia, etc.) eliminates the need for desalting by dialysis or other size-based methods because these reagents or their reaction products can evaporate from the solution when the reaction is complete. For example, depending on the reaction mechanism, ammonium bicarbonate can be evaporated in the form of CO 2 NH 3 and H 2 O form effectively removes: Taking ammonium hydroxide as an example, according to the reaction mechanism, NH 3 and H 2 O form removal: Example 3 (Ammonium-based PS cleavage reagents are shown as peroxide quenching reagents that eliminate hydrogen peroxide and off-target oxidation, and thus represent exemplary, preferred peroxide quenching / PS cleavage reagents)

[0373] Although hydrogen peroxide is a component of the initial oxidation step in the oligosaccharide production disclosed herein (as well as in prior art methods), there is a risk of unwanted off-target oxidation in the subsequent cleavage reaction step and any subsequent downstream processing steps due to any residual presence of hydrogen peroxide and / or its free radicals. As is understood in the art, hydrogen peroxide cannot be easily removed by standard evaporation processes due to its high boiling point (150.2°C). In addition, its presence can hinder chromatographic work for downstream glycan purification and enrichment, as many stationary phases are unstable in relatively high reduction / oxidation states. Strategies for its removal can include dialysis, use of enzymes such as horseradish peroxidase, and prolonged exposure to the open atmosphere. Enzymatic methods have the advantage of rapid quenching of hydrogen peroxide, but also require removal downstream. Both dialysis and exposure to the open atmosphere bring hydrogen peroxide (and / or any residual free radicals thereof) into contact with the produced oligosaccharides, which can produce side reactions, including C-6 oxidation to produce oligosaccharides containing uronic acid and other unwanted substances. According to specific aspects, the COG method currently disclosed solves this substantial problem.

[0374] To determine the effect of different PS cleavage reagents on hydrogen peroxide concentration, the samples were incubated with different cleavage reagents at different temperatures and then tested with test paper (Quantofix Peroxide 100 TM ) measured the concentration of hydrogen peroxide. Three PS cleavage reagents (ammonium hydroxide, ammonium bicarbonate, and sodium hydroxide) were first incubated with locust bean gum treated with hydrogen peroxide and Fe(II). The use of ammonium hydroxide at room temperature was shown to quickly eliminate the presence of hydrogen peroxide. However, in contrast, neither ammonium bicarbonate nor sodium hydroxide had an effect on the concentration of hydrogen peroxide ( Figure 3 ). This demonstrates that while existing Fenton-based methods use a strong Arrhenius base to quench the Fenton reaction, they do not quench or eliminate the residual hydrogen peroxide itself, or any residual free radicals generated prior to the introduction of the strong Arrhenius base.

[0375] According to a particular aspect of the invention, the following mechanism: This supports applicants' concept that when ammonia is generated (or otherwise introduced into the reaction), some residual hydrogen peroxide or its free radical will be quenched / eliminated.

[0376] To further test the proposed mechanism, the reactions with the three cleavage reagents were heated for one hour at increasing temperatures (up to 65°C) to drive the ammonium bicarbonate solution to produce more ammonia. In fact, the reaction pH increased with increasing temperature, indicating the presence of hydroxide ions, accompanied by ammonia gas, and thus the observed simultaneous quenching of hydrogen peroxide ( Figure 4 ). In addition, the reaction occurs rapidly at 40°C and ultimately results in hydrogen peroxide levels below the detectable limit at 65°C. Notably, there is no significant difference in the hydrogen peroxide concentration in the heated sodium hydroxide solution. This data demonstrates that hydrogen peroxide is rapidly quenched / eliminated when incubated, for example, with the presently disclosed nitrogen-based cleavage reagents, rather than with traditional strong Arrhenius bases (e.g., Na + OH - , K + OH - or Ca 2+ (OH - ) 2 ). Example 4 (Ammonium hydroxide produces unique oligosaccharide profiles from spent grain fractions)

[0377] The two spent grain fractions were ground into fine powders and subjected to the process described in Example 1, while using ammonium hydroxide as the peroxide quenching / PS cleavage reagent. The grain samples represented the "intact" spent fraction and the protein-depleted fraction produced and recovered from the bioethanol production process. The LC-MS (MS) spectra obtained from the depolymerization products of the two fractions were Figure 5A and Figure 5B ) showed that both fractions included abundant hexose oligomers in the range of 3 to 10 monomer units; however, larger structures were also abundant but were not examined under the present conditions. Figure 5A ) compared to the protein-depleted fraction ( Figure 5B ) contained a higher concentration of total carbohydrates and produced approximately 2 times the concentration of oligosaccharides. The non-carbohydrate components of a complex mixture can inhibit the effect of the reaction by competing for the oxidation potential and cleavage potential of the reaction (Stadtman and Berlett 1991). However, this result demonstrates the broad validity of the disclosed CDPG method (e.g., involving the use of non-Arrhenius bases and weak Arrhenius bases as peroxide quenching / PS cleavage reagents after a previous Fenton oxidation). Example 5 (In the disclosed COG reaction, the initiation of peroxide quenching can be before, during or after the initiation of polysaccharide (PS) cleavage)

[0378] Table 1 above lists exemplary PS cleaving agents and / or peroxide quenchers.

[0379] In terms of the preferred COG method, as disclosed and discussed herein above, the COG method overcomes substantial problems in the art by using a hydrogen peroxide quencher ("peroxide quencher" agent) to reduce or eliminate off-target side reactions after the PS cleavage step is initiated. + OH - , K + OH - or Ca 2+ (OH - ) 2 ) as a PS cleavage agent, which is said to "quench" the initial Fenton reaction (i.e., by flocculating the metal ion reactants), such strong Arrhenius base PS cleavage agents do not quench / eliminate residual peroxides or peroxide radicals themselves (as disclosed herein; e.g., see Example 3 above), and thus the prior art methods are prone to unwanted side reactions.

[0380] In preferred COG methods, preferably, the PS cleavage initiator also acts as a peroxide quencher to quench (e.g., substantially reduce or eliminate) residual hydrogen peroxide and / or its radicals themselves to minimize or eliminate off-target side reactions. In these method aspects, the initiation of peroxide quenching (and also quenching the Fenton reaction) is simultaneous with the initiation of PS cleavage. Although such COG reactions can be simply viewed as a two-step reaction aspect (including a Fenton oxidation aspect followed by a PS cleavage aspect), it should be understood that the quenching of the peroxide (and / or the quenching of the Fenton reaction) may be immediate or well-defined or may not be immediate or well-defined, and may also occur in at least part of the PS cleavage aspect; that is, despite the use of a peroxide quencher as disclosed herein, there may be at least some degree of overlap between the Fenton reaction aspect, the peroxide quenching aspect, and / or the PS cleavage aspect of such COG reactions. The degree of overlap may vary depending on the nature and amount of the peroxide quencher used.

[0381] In a specific COG method, the PS cleavage agent (cleavage initiator) may or may not be a peroxide quencher, and in either case may be used in combination with an additional compatible peroxide quencher, which may or may not itself be a cleavage agent. In these aspects, the additional compatible peroxide quencher may be introduced into the reaction before, during, or after the introduction of the PS cleavage agent.

[0382] In a specific aspect, the introduction of an additional compatible peroxide quencher in the reaction is simultaneous with the introduction of the PS cleavage agent. While such COG reaction aspects may be viewed simply as two-step reaction aspects (including a Fenton oxidation aspect followed by a PS cleavage aspect), it is understood that the quenching of the peroxide (and / or the quenching of the Fenton reaction) may or may not be immediate or well-defined, and may also occur in at least part of the PS cleavage aspect; that is, despite the use of a peroxide quencher as disclosed herein, there may be at least some degree of overlap between the Fenton reaction aspect, the peroxide quenching aspect, and / or the PS cleavage aspect of such COG reactions. The degree of overlap may vary depending on the nature and amount of the peroxide quencher used.

[0383] In a specific aspect, an additional compatible peroxide quencher is introduced into the reaction prior to the introduction of the PS cleavage agent. Although such COG reaction aspects may be simply viewed as two-step reaction aspects (including a Fenton oxidation aspect followed by a PS cleavage aspect), or three-step reaction aspects (including a Fenton oxidation aspect followed by a peroxide quenching aspect followed by a PS cleavage aspect), it is understood that the quenching of the peroxide (and / or the quenching of the Fenton reaction) may or may not be immediate or well-defined, and may also occur in at least part of the PS cleavage aspect; that is, despite the use of a peroxide quencher as disclosed herein, there may be at least some degree of overlap between the Fenton reaction aspects, the peroxide quenching aspects, and / or the PS cleavage aspects of such COG reactions. The degree of overlap may vary depending on the nature and amount of the peroxide quencher used.

[0384] In a specific aspect, an additional compatible peroxide quencher is introduced into the reaction after the PS cleavage agent is introduced. Although such COG reaction aspects can be simply viewed as two-step reaction aspects (including Fenton oxidation aspects, followed by PS cleavage aspects), or three-step reaction aspects (including Fenton oxidation aspects, followed by PS cleavage aspects, followed by peroxide quenching aspects), it should be understood that the quenching of the peroxide (and / or the quenching of the Fenton reaction) may or may not be immediate or well-defined, and may also occur in at least part of the PS cleavage aspects; that is, despite the use of peroxide quenchers as disclosed herein, there may be at least some degree of overlap between the Fenton reaction aspects, peroxide quenching aspects, and / or PS cleavage aspects of such COG reactions. The degree of overlap may vary depending on the nature and amount of the peroxide quencher used.

[0385] According to a preferred aspect of the present invention, in all of the above COG method aspects, a peroxide quencher is used to quench (eg, substantially reduce or eliminate) residual hydrogen peroxide and / or its free radicals themselves, thereby minimizing or eliminating off-target side reactions.

[0386] According to preferred aspects of the present invention, in all of the above-mentioned COG method aspects, the use of specific weak Arrhenius bases and / or non-Arrhenius bases (e.g., nitroperoxide quenching / PS cleavage reagents, etc.; for example, see Table 1) not only provides improved high-yield oligosaccharide production (relative to the strong Arrhenius bases used in the art), but also eliminates the need for expensive and time-consuming post-reaction concentration and desalting steps. Example 6 (COG provides enhanced biological activity compared to similar approaches)

[0387] In certain aspects, the oligosaccharides produced by COG can be used to promote the growth of bacteria and / or the microbial flora of humans and animals (intestinal tract, skin, respiratory tract, vagina, eye, oral cavity) in fermentation (biotechnology, ethanol production, food processing). Common methods for evaluating the ability of microorganisms to consume specific oligosaccharides and oligosaccharide groups require monitoring them by optical density throughout the growth period. However, these results may be wrong if the oligosaccharides are contaminated by endogenous or exogenous substances. Compounds such as salts, acids, metals and oxidants / reducing agents can inhibit bacterial growth in in vitro systems.

[0388] Production of Oligosaccharides: In this exemplary aspect, pullulan (550 mg) was dissolved in 20 ml of HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55°C and 85 RPM for 20 minutes. The pH of the solution was adjusted to 5.2. Hydrogen peroxide (5 ml) and iron (II) sulfate (2.75 mg in 50 μL of water) were added to the reaction mixture and mixed thoroughly. The reaction in the capped reaction vessel was allowed to proceed in a shaking incubator at 55°C and 65 RPM for two hours. The closed reaction was cooled to 12°C in a -20°C refrigerator. Ammonium hydroxide (1 ml, 28% v / v, to pH 10.2) or NaOH (600 μl, 10.45 M) was used to adjust the pH, and the sample was reacted in a shaking incubator at 45°C and 20 RPM for 1 hour, with the lid loosened to allow the release of oxygen, ammonia, and carbon dioxide gases. The sample was then frozen and lyophilized and then stored at -80°C. In a 50 mL Bio-Scale TM Mini Size exclusion chromatography was performed on a P-6 desalting column using 0.03 M ammonium bicarbonate buffer at a flow rate of 10 mL / min. For desalting purposes, a 50 mL elution window was collected after the void volume and the sample was lyophilized to complete dryness. The resulting material was analyzed for iron, hydrogen peroxide and sulfate concentrations, pH, oxidation / reduction potential (ORP) and electrical conductivity (EC).

[0389] Carbohydrate analysis: The oligosaccharide samples after cleavage were reconstituted in water and subjected to sugar alcohol reduction. The samples were reduced with 2M sodium borohydride at 65°C for 1 hour and then immediately subjected to C18 solid phase extraction. Before loading and collecting the samples, the solid phase column was washed with three volumes of acetonitrile and two volumes of water as a direct flushing solution (flow-through). The samples extracted by the C18 column were then subjected to non-porous graphitized carbon (NPGC) solid phase extraction. The NPGC column was pre-washed with two volumes of water, two volumes of 80% acetonitrile containing 0.01% (v / v) TFA aqueous solution, and another two volumes of water. The sample extracted by the C18 column was then loaded and washed with five volumes of water, and then eluted with 40% acetonitrile containing 0.05% (v / v) TFA. Finally, the samples after NPGC were completely dried by evaporative centrifugation and stored at -20°C until analysis.

[0390] Oligosaccharide analysis was performed on an Agilent 1290 Infinity II HPLC coupled to an Agilent 6530 Accurate-Mass Q-TOF MS. Chromatographic separation was performed on a Thermo Scientific Hypercarb PGC column with a binary gradient consisting of solvent A: 3% acetonitrile / water + 0.1% formic acid, and solvent B: 10% water / acetonitrile + 0.1% formic acid. The flow rate was 0.15 mL / min and the gradient was run for 60 min: 2-15% B, 0 to 20 min; 15-60% B, 20 to 45 min; 60-99% B, 45 to 45.10 min; 99-99% B, 45.10 to 51 min; 99-2% B, 51 to 51.10 min; 2-2% B, 51.10 to 60 min. The mass spectrometer was operated in positive ion mode with a reference mass of 922.0098 m / z. Gas temperature and flow rate were set to 150 ° C and 11 l / min, respectively. The voltages of the nozzle, fragmentor, and skimmer were set to 1500 volts, 75 volts, and 60 volts, respectively. Using tandem mass spectrometry, fragmentation was performed at a collision energy of 1.45 × (m / z)-3.5. Data were processed using Agilent MassHunter Workstation Quantitative Analysis 10.1 software. The main peaks corresponding to the mass of oligosaccharides in the chromatogram were integrated. The responses of oligosaccharides with a DP of 2 to 10 were added to represent the total oligosaccharide peak area.

[0391] In addition, the monosaccharide composition of the samples was analyzed as described by Amicucci et al. (Amicucci, Galermo, et al. 2019).

[0392] Bacterial Growth Methods: The ability of the generated oligosaccharide fractions to support bacterial growth was evaluated by culturing Bifidobacterium breve (model organism) in minimal medium supplemented with 3% (m / v) oligosaccharide fractions under anaerobic conditions at 37°C. The minimal medium used for these experiments was basal MRS (Ruiz-Moyano et al. 2013). Prior to inoculation, basal MRS was mixed with lactose and each oligosaccharide fraction, the pH was adjusted to 6.8, filter sterilized and placed in an anaerobic chamber for approximately 12 hours to remove oxygen. Positive controls (1% lactose only) and negative controls (without carbohydrates) were included, and each treatment was inoculated in triplicate with 2% of fresh Bifidobacterium culture and incubated under anaerobic conditions. Growth was determined based on absorbance measurements at 600 nm for 24 hours. The sterility of the medium was tested by culturing uninoculated medium.

[0393] Results: The oligosaccharide yields of oligosaccharides produced by ammonium hydroxide cleavage were higher than those produced by sodium hydroxide ( Figure 6 ), and as quantitative monosaccharide analysis ( Figure 7 ), also produced a purer final product. In addition, ammonium hydroxide showed a lower ORP, indicating less residual hydrogen peroxide, and showed similar electrical conductivity (EC), indicating lower ion content (Table 2). 4 Oligosaccharides generated with OH showed a stronger growth response than those generated with NaOH ( Figure 8 ). NH 4 OH oligosaccharide supported the growth of bifidobacteria to a maximum OD (620 nm) of 0.974, but the cell density using NaOH oligosaccharide only reached a maximum OD (620 nm) of 0.64. This suggests that bifidobacteria are sensitive to NH 4 The substrate preference of OH oligosaccharides was superior to that of NaOH oligosaccharides and suggests that this and other COG fractions will enrich other bacterial groups in a similarly superior manner. Table 2. Physical properties of the oligosaccharide library. Example 7 (Oligosaccharide spectrum depends on the choice of base)

[0394] In one exemplary aspect, pullulan (200 mg) was dissolved in 7 ml of HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55° C. and 85 RPM for 20 minutes. The pH of the solution was adjusted to 5.2. Hydrogen peroxide (1.75 ml) and iron (II) sulfate (1 mg in 25 μL of water) were added to the reaction mixture and mixed thoroughly. The reaction in the capped reaction vessel was allowed to proceed in a shaking incubator at 55° C. and 65 RPM for two hours. The closed reaction was cooled to 12° C. in a -20° C. refrigerator. Seven bases were tested: pyridine (100 ul), N,N-diisopropylethylamine (DIPEA), NaOH (35 μl, 10 M), CsOH (35 μl, 10 M), Ca(OH) 2 (45 μl, 10 M), KOH (35 μl, 10 M) and NH 4 OH (500 μl, 28% v / v). All bases were added to the reaction mixture to pH 10, except pyridine to pH 9. All seven conditions were reacted in a shaking incubator at 45°C and 20 RPM for 1 hour, with the lid loosened to allow the release of oxygen, ammonia, and carbon dioxide gases. Samples were frozen and lyophilized and then stored at -80°C before cleanup and subsequent mass spectrometry analysis.

[0395] Carbohydrate analysis: The oligosaccharide samples after cleavage were reconstituted in water and subjected to sugar alcohol reduction. The samples were reduced with 2M sodium borohydride at 65°C for 1 hour and then immediately subjected to C18 solid phase extraction. Before loading and collecting the samples, the solid phase column was washed with three volumes of acetonitrile and two volumes of water as direct rinses. The samples extracted by the C18 column were then subjected to non-porous graphitized carbon (NPGC) solid phase extraction. The NPGC column was pre-washed with two volumes of water, two volumes of 80% acetonitrile containing 0.01% (v / v) TFA aqueous solution, and another two volumes of water. The sample extracted by the C18 column was then loaded and washed with five volumes of water, and then eluted with 40% acetonitrile containing 0.05% (v / v) TFA. Finally, the samples after NPGC were completely dried by evaporative centrifugation and stored at -20°C until analysis.

[0396] (Amicucci, Galermo et al. 2019). Oligosaccharide analysis was performed on an Agilent 1290 Infinity II HPLC coupled to an Agilent 6530 Accurate-Mass Q-TOF MS. Chromatographic separation was performed on a Thermo Scientific Hypercarb PGC column with a binary gradient consisting of solvent A: 3% acetonitrile / water + 0.1% formic acid, and solvent B: 10% water / acetonitrile + 0.1% formic acid. The flow rate was 0.15 mL / min, and the gradient was run for 60 min: 2-15% B, 0 to 20 min; 15-60% B, 20 to 45 min; 60-99% B, 45 to 45.10 min; 99-99% B, 45.10 to 51 min; 99-2% B, 51 to 51.10 min; 2-2% B, 51.10 to 60 min. The mass spectrometer was operated in positive ion mode with a reference mass of 922.0098 m / z. The gas temperature and flow rate were set to 150°C and 11 l / min, respectively. The voltages of the nozzle, fragmentor, and skimmer were set to 1500 volts, 75 volts, and 60 volts, respectively. Using a tandem mass spectrometer, fragmentation was performed at a collision energy of 1.45×(m / z)-3.5. Data were processed using Agilent MassHunterWorkstation Quantitative Analysis 10.1 software. The main peaks corresponding to the mass of oligosaccharides in the chromatogram were integrated. The responses of oligosaccharides with a DP of 2 to 10 were added to represent the total oligosaccharide peak area.

[0397] Oligosaccharide analysis showed different oligosaccharide yields both at the aggregate level and at the specific structural level. All bases used in this experiment produced oligosaccharide products. Ammonium hydroxide produced the highest concentration of oligosaccharides, which was almost twice that of sodium hydroxide ( Fig. 9 ). In addition, the nitrogenous base (DIPEA) produced the second highest concentration of oligosaccharides, while pyridine produced oligosaccharides comparable to the Arrhenius base. This provides further evidence for the broad classification of "nitrogen-based cleavage agents" that are considered good cleavage agents. Example 8 (Iron(II) and the production of locust bean oligosaccharides)

[0398] In some aspects, the oxidation state of the metal can be varied to obtain similar or different results. In the described aspects, iron (II) is used to produce oligosaccharides from locust bean gum polysaccharide. Locust bean gum comprises a galactomannan polymer containing a β1,4 mannose backbone with terminal branches of α1,6 galactose.

[0399] Production of Oligosaccharides: Locust bean gum (550 mg) was dissolved in 20 ml of HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55°C and 85 RPM for 20 minutes. The pH of the solution was adjusted to 5.2. Hydrogen peroxide (5 ml) and iron (II) sulfate (2.75 mg in 50 μL water) were added to the reaction mixture and mixed thoroughly. The reaction in the capped reaction vessel was allowed to proceed in a shaking incubator at 55°C and 65 RPM for two hours. The closed reaction was cooled to 12°C in a -20°C refrigerator. Ammonium hydroxide (1 ml 28% v / v, to pH 10.2) was used to adjust the pH, and the sample was reacted in a shaking incubator at 45°C and 20 RPM for 1 hour, with the lid loosened to allow the release of oxygen, ammonia and carbon dioxide gases. The sample was then frozen and lyophilized and then stored at -80°C. The freeze-dried oligosaccharide mixture was rehydrated with the minimum amount of water required to allow a free-flowing solution. The solution was then loaded onto a column containing 15 mL of mixed bed ion exchange resin per gram (dry weight) of crude material, and the effluent was collected in a plastic freezer bag. Once the material was loaded onto the column, the column was then rinsed with 3 bed volumes of water. Finally, the effluent was sealed and frozen in a bag, then carefully pulverized and lyophilized.

[0400] Carbohydrate analysis: The oligosaccharide samples after cleavage were reconstituted in water and subjected to sugar alcohol reduction. The samples were reduced with 2M sodium borohydride at 65°C for 1 hour and then immediately subjected to C18 solid phase extraction. Before loading and collecting the samples, the solid phase column was washed with three volumes of acetonitrile and two volumes of water as direct rinses. The samples extracted by the C18 column were then subjected to non-porous graphitized carbon (NPGC) solid phase extraction. The NPGC column was pre-washed with two volumes of water, two volumes of 80% acetonitrile containing 0.01% (v / v) TFA aqueous solution, and another two volumes of water. The sample extracted by the C18 column was then loaded and washed with five volumes of water, and then eluted with 40% acetonitrile containing 0.05% (v / v) TFA. Finally, the samples after NPGC were completely dried by evaporative centrifugation and stored at -20°C until analysis.

[0401] (Amicucci, Galermo et al. 2019). Oligosaccharide analysis was performed on an Agilent 1290 Infinity II HPLC coupled to an Agilent 6530 Accurate-Mass Q-TOF MS. Chromatographic separation was performed on a Thermo Scientific Hypercarb PGC column with a binary gradient consisting of solvent A: 3% acetonitrile / water + 0.1% formic acid, and solvent B: 10% water / acetonitrile + 0.1% formic acid. The flow rate was 0.15 mL / min, and the gradient was run for 60 min: 2-15% B, 0 to 20 min; 15-60% B, 20 to 45 min; 60-99% B, 45 to 45.10 min; 99-99% B, 45.10 to 51 min; 99-2% B, 51 to 51.10 min; 2-2% B, 51.10 to 60 min. The mass spectrometer was operated in positive ion mode with a reference mass of 922.0098 m / z. The gas temperature and flow rate were set to 150°C and 11 l / min, respectively. The voltages of the nozzle, fragmentor, and skimmer were set to 1500 volts, 75 volts, and 60 volts, respectively. Using a tandem mass spectrometer, fragmentation was performed at a collision energy of 1.45×(m / z)-3.5. Data were processed using Agilent MassHunterWorkstation Quantitative Analysis 10.1 software. The main peaks corresponding to the mass of oligosaccharides in the chromatogram were integrated. The responses of oligosaccharides with a DP of 2 to 10 were added to represent the total oligosaccharide peak area.

[0402] Results: Oligosaccharides generated from Fe(II) oxidation and cleavage of locust bean gum were structurally similar to their parent locust bean polysaccharide but with a much shorter degree of polymerization. Monosaccharide analysis showed high purity (>90%) and similar monomer composition to the parent polymer, with a mannose:galactose ratio of 3.17:1 vs. 4.52:1 ( Fig.10 Oligosaccharide analysis revealed oligosaccharides ranging from 3 to 8 hexoses in length, which contained a large number of isomers ( Fig.11 ). Locust bean gum is known to contain galactomannan polysaccharides comprising a β1,4 mannose backbone from which a single α1,6 linked galactose branches. Example 9 (Araboxylan oligosaccharides derived from corn fiber.)

[0403] Corn fiber is a highly abundant waste stream from residual fermentation of corn for ethanol production. This material includes several abundant polysaccharides, including β-glucans, arabinoxylans, cellulose, and residual amylose and amylopectin. The arabinoxylan fraction provides an opportunity to produce arabinoxylan-oligosaccharides, which have been shown to modulate the gut microbiome (Neyrinck et al. 2012).

[0404] Corn fiber was purified by chloroform extraction, where 5 g of material was suspended in 100 mL of chloroform and mixed for about 2 hours. The resulting mixture was then crushed with 50 mL of 0°C water to produce a viscous material. The mixture was centrifuged at 6500 rpm for 30 minutes and the liquid layer was discarded. The bottom layer was then resuspended in 10 ml of water and crushed with absolute ethanol at 0°C. Two additional subsequent washes were performed with absolute ethanol at 0°C to produce a white polysaccharide precipitate. The material was dried by lyophilization to produce 4.8 g.

[0405] The material was subjected to COG reaction under the following conditions. 550 mg of the material was dissolved in 20 ml of HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55° C. and 85 RPM for 20 minutes. The pH of the solution was adjusted to 5.2. Hydrogen peroxide (5 ml) and copper (II) sulfate (2.75 mg in 50 μL water) or iron (II) sulfate (2.75 mg in 50 μL water) were added to the reaction mixture and mixed thoroughly. The reaction in the capped reaction vessel was carried out in a shaking incubator at 55° C. and 65 RPM for two hours. The closed reaction was cooled to 12° C. in a -20° C. refrigerator. The pH was adjusted to 8, 9 or 10 using ammonium hydroxide (1 ml 28% v / v, to pH 10.2), and the samples were reacted in a shaking incubator at 45° C. and 20 RPM for 45 minutes, 60 minutes or 90 minutes, and the lid was loosened to allow the release of oxygen, ammonia and carbon dioxide gases. The samples were then frozen and lyophilized.

[0406] The oligosaccharide mixture of freeze drying is rehydrated with the minimum amount of water that allows free flow solution.Then this solution is loaded onto the post containing 15mL mixed bed ion exchange resin of every gram (dry weight) crude material, and the effluent is collected in a plastic freezing bag.Once the material is loaded onto the post, the post is then rinsed with 3 bed volumes of water.Finally, the effluent is sealed and frozen in the bag, then carefully pulverized and freeze dried.

[0407] Carbohydrate analysis: The oligosaccharide samples after cleavage were reconstituted in water and subjected to sugar alcohol reduction. The samples were reduced with 2M sodium borohydride at 65°C for 1 hour and then immediately subjected to C18 solid phase extraction. Before loading and collecting the samples, the solid phase column was washed with three volumes of acetonitrile and two volumes of water as direct rinses. The samples extracted by the C18 column were then subjected to non-porous graphitized carbon (NPGC) solid phase extraction. The NPGC column was pre-washed with two volumes of water, two volumes of 80% acetonitrile containing 0.01% (v / v) TFA aqueous solution, and another two volumes of water. The sample extracted by the C18 column was then loaded and washed with five volumes of water, and then eluted with 40% acetonitrile containing 0.05% (v / v) TFA. Finally, the samples after NPGC were completely dried by evaporative centrifugation and stored at -20°C until analysis.

[0408] (Amicucci, Galermo et al. 2019). Oligosaccharide analysis was performed on an Agilent 1290 Infinity II HPLC coupled to an Agilent 6530 Accurate-Mass Q-TOF MS. Chromatographic separation was performed on a Thermo Scientific Hypercarb PGC column with a binary gradient consisting of solvent A: 3% acetonitrile / water + 0.1% formic acid, and solvent B: 10% water / acetonitrile + 0.1% formic acid. The flow rate was 0.15 mL / min, and the gradient was run for 60 min: 2-15% B, 0 to 20 min; 15-60% B, 20 to 45 min; 60-99% B, 45 to 45.10 min; 99-99% B, 45.10 to 51 min; 99-2% B, 51 to 51.10 min; 2-2% B, 51.10 to 60 min. The mass spectrometer was operated in positive ion mode with a reference mass of 922.0098 m / z. The gas temperature and flow rate were set to 150°C and 11 l / min, respectively. The voltages of the nozzle, fragmentor, and skimmer were set to 1500 volts, 75 volts, and 60 volts, respectively. Using a tandem mass spectrometer, fragmentation was performed at a collision energy of 1.45×(m / z)-3.5. Data were processed using Agilent MassHunterWorkstation Quantitative Analysis 10.1 software. The main peaks corresponding to the mass of oligosaccharides in the chromatogram were integrated. The responses of oligosaccharides with a DP of 2 to 10 were added to represent the total oligosaccharide peak area.

[0409] Results: Oligosaccharides produced from corn fiber by Cu(II) oxidation and cleavage at pH 10 for 60 min proved to be the most successful in producing oligosaccharides ( Fig.12). The oligosaccharide has a DP range of 3 to 4 and has a monosaccharide and bond profile representative of arabinoxylan. The oligosaccharide has a xylose:arabinose ratio of 1.36:1 and has bonds including terminal xylose, terminal arabinose, terminal galactose, 4-xylose, 3,4-xylose, and 4-glucose. For reference, Fig.15 Annotated chromatograms of the ligation analysis of corn fiber are provided in . Fig.12 Four unique corn fiber oligosaccharide profiles are shown. Condition 1 produced the most oligosaccharides, which was obtained by adding NH 4 The solution was heated at 45 °C for 1 h to reach pH 10. Condition 2 produced about 5 times less oligosaccharides than condition 1, which was due to the addition of NH 4 The solution was heated at 45 °C for 1.5 h to reach pH 9. Condition 3 produced slightly fewer oligosaccharides than condition 1, which was due to the addition of NH 4 The solution was heated at 45 °C for 0.75 h to reach pH 8. These results indicate that pH, time, and temperature are important factors in optimizing oligosaccharide yields. In addition, condition 4 produced only a few oligosaccharides, which was due to the addition of NH 4 The results are as follows: OH was used to reach pH 10 and the solution was heated at 45°C for 1 hour. This result suggests that some polysaccharide sources are more susceptible to depolymerization when copper rather than iron is used in the oxidation step. Example 10 (Material composition)

[0410] The ability of many polysaccharide-rich materials to be dissociated by COG was evaluated. Due to the previous lack of mechanism, each material produced many unexpected oligosaccharide products and was characterized at the library level (multiple oligosaccharides) and the single oligosaccharide level. Where possible, the library was described by their monosaccharide and glycosidic bond spectra, 2D-NMR (Table 5) and liquid chromatography / quadrupole time of flight mass spectrometry (LC / Q-TOF MS). In addition, single oligosaccharides were identified and characterized by their mass, retention time and fragmentation pattern.

[0411] Oligosaccharide production: Arabinogalactan II, lichenin, 1,4B-mannan, xylan, pullulan, arabinoxylan, β-glucan, galactan, galactomannan, glucomannan, xyloglucan and locust bean gum (550 mg) were dissolved in 20 ml of HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55°C and 85 RPM for 20 minutes. The pH of the solution was adjusted to 5.2. Hydrogen peroxide (5 ml) and iron (II) sulfate (2.75 mg in 50 μL water) were added to the reaction mixture and mixed thoroughly. The reaction in the capped reaction vessel was allowed to proceed in a shaking incubator at 55°C and 65 RPM for two hours. The closed reaction was cooled to 12°C in a -20°C refrigerator. Ammonium hydroxide (1ml, 28% v / v, to pH 10.2) is used to adjust pH, and the reaction is carried out in a shaking incubator at 45°C and 20RPM for 45 minutes, 60 minutes or 90 minutes, and the lid is loosened to allow the release of oxygen, ammonia and carbon dioxide gas. The sample is then frozen and lyophilized, and then stored at-80°C. The freeze-dried oligosaccharide mixture is rehydrated with the minimum amount of water required to allow free flow solution. The solution is then loaded onto a column containing 15mL mixed bed ion exchange resin per gram (dry weight) of crude material, and the effluent is collected in a plastic freezing bag. Once the material is loaded onto the column, the column is then rinsed with 3 bed volumes of water. Finally, the effluent is sealed and frozen in a bag, then carefully pulverized and lyophilized.

[0412] Curdlan and corn fiber (550mg) were dissolved in 20ml HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55°C and 85RPM for 20 minutes. The pH of the solution was adjusted to 5.2. Hydrogen peroxide (5ml) and copper (II) sulfate (2.75mg in 50μL water) were added to the reaction mixture and mixed thoroughly. The reaction in the capped reaction vessel was carried out in a shaking incubator at 55°C and 65RPM for two hours. The closed reaction was cooled to 12°C in a -20°C refrigerator. Ammonium hydroxide (1ml 28% v / v, to pH 10.2) was used to adjust the pH, and the sample was reacted in a shaking incubator at 45°C and 20RPM for 1 hour, and the lid was loosened to allow the release of oxygen, ammonia and carbon dioxide gas. The sample was then frozen and lyophilized and then stored at -80°C. The freeze-dried oligosaccharide mixture was rehydrated with the minimum amount of water required to allow a free-flowing solution. The solution was then loaded onto a column containing 15 mL of mixed bed ion exchange resin per gram (dry weight) of crude material, and the effluent was collected in a plastic freezer bag. Once the material was loaded onto the column, the column was then rinsed with 3 bed volumes of water. Finally, the effluent was sealed and frozen in a bag, then carefully pulverized and lyophilized.

[0413] Monosaccharide analysis was performed in the manner of Amicucci et al. (Amicucci, MJ, Galermo, AG, et al. (2019). International Journal of Mass Spectrometry 438: 22-28.), but this was adapted for use on an Agilent 6530Q-TOF mass spectrometer. Glycosidic bond analysis was performed in the manner of Galermo, AG, Nandita, E., et al. (2018). Analytical Chemistry 90 (21): 13073-13080 and in the extended retention time library provided in Galermo, AG, Nandita, E., et al. (2019). Analytical Chemistry 91 (20): 13022-13031, which was adapted for use on an Agilent 6530Q-TOF mass spectrometer. Oligosaccharide analysis was performed as described in Amicucci, MJ, Nandita, E., et al. (2020). Nature Communications 11(1): 1-12. Oligosaccharide peak volumes were generated using the “Search by Molecular Feature” function of Agilent MassHunter Qualitative Analysis B.10. For NMR analysis, oligosaccharides were dissolved in D 2 O, and its HSQC spectrum was analyzed on a 600 MHz Bruker NMR spectrometer.

[0414] Monosaccharide composition: The monosaccharide composition of the oligosaccharide pool produced by the COG reaction was analyzed as shown in Table 3. Seven monosaccharides were measured in the 14 samples subjected to the COG reaction. Table 3. Monosaccharide composition of claimed library compositions. Units represent relative abundance by mass. The symbol "--" represents that the monosaccharide is present in an amount less than 2% of the total weight of the polymer.

[0415] Glycosidic bond analysis: The monosaccharide composition of the oligosaccharide pool produced by the COG reaction was analyzed as shown in Table 4. 16 glycosidic bond positions were identified in the 14 samples subjected to the COG reaction. Table 4. Glycosidic bond composition of the claimed material library composition. The unit represents the relative abundance of the peak area. The symbol "--" represents that the amount of glycosidic bond present is less than 2% of the total weight of the polymer. 1H-13CHSQCNMR: performed on all samples except galactan. This analysis provides a fingerprint for each sample in order to compare the similarity between these samples and future oligosaccharide libraries. Table 5 lists the cross-peak coordinates of the anomeric region of the spectrum. Fig.13 shown. Table 5. 1H-13C HSQC NMR correlations for oligosaccharides produced by the COG process. The pairs listed correspond to those major peaks in the anomeric region. 1H-13CHSQCNMR: Oligosaccharides are present in two forms. Tables 6-19 show the "Find by Molecular Feature" data, which shows the mass, retention time, composition, and relative abundance of oligosaccharides. In addition, we have Fig.14 Annotated oligosaccharide chromatograms are provided in .

[0416] Amylopectin refers to a polysaccharide with an α-1,4 main chain and α-1,6 branches extending in linear α-1,4 branches that can be similar branches. The oligosaccharides we produced closely matched this composition. The glucose composition was 98.19% (Table 3), and the glycosidic bond composition was 17.6% terminal glucose, 70.23% 4-linked glucose, and 3.83% 4,6-linked glucose (Table 4). 29 oligosaccharides were observed in the library, ranging in length from 3 pentoses to 7 pentoses. The most abundant structures were represented by linear α-1,4 glucose polymers (3Hex, 4.11 min; 4Hex, 9.29 min; 5Hex, 12.31 min; 6Hex, 14.058; 7Hex, 15.254 min; 8Hex, 16.394; 9Hex, 18.013 min; 10Hex, 21.99 min; 11Hex, 22.911; 12Hex, 24.55 min). Other isomers were also found, which were represented by structures with at least one α-1,6 branch. A complete list of oligosaccharide peaks and abundances is shown in Table 6. The oligosaccharide pool can be further distinguished by its 1H-13C2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 6. Oligosaccharides produced from COG depolymerization of amylopectin. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose includes only glucose.

[0417] Arabinoxylans are polysaccharides with a β-1,4 xylose backbone with α-1,3 and α-1,2 arabinose branches in a ratio of 1 to 2. The oligosaccharides we generated closely matched this composition. The xylose composition was 60.28%, followed by 36.99% arabinose and 2.08% galactose (Table 3). The glycosidic bond composition was 30.55% terminal arabinose, 31.20% 4-linked xylose, 22.22% 3,4-linked xylose and 2.65% terminal xylose (Table 4). 22 oligosaccharides were observed in the library, ranging in length from 3 pentoses to 7 pentoses. The most abundant structures are represented by 3pent, 8.612 min and 14.346 min; 4pent, 20.455 min; 5pent, 20.812 min and 25.947 min; 6pent, 24.969 min; 7pent, 27.697 min; the complete list of oligosaccharide peaks and abundances is shown in Table 7. The oligosaccharide pool can be further distinguished by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 7. Oligosaccharides produced from COG depolymerization of arabinoxylan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Pentose refers to arabinose and xylose.

[0418] Xyloglucan refers to a polysaccharide with a β-1,4 glucose backbone with α-1,6 xylose branches. The branches can be further extended in a 1 to 2 ratio by adding β-2,1 galactose. The oligosaccharides we produced closely matched this composition. The glucose composition was 48.75%, followed by 36.99% xylose and 14.14% galactose (Table 3). The glycosidic bond composition was 28.23%, 20.49%, 5.63% and 4.23% 4-glucose, 4,6 glucose, 6 glucose, terminal glucose, and 20.62% terminal galactose, respectively (Table 4). In addition, further connections were considered to be 10.78% terminal xylan and 5.81% 2-xylan (Table 4). 42 oligosaccharides were observed in the library, ranging in length from 2Hex1Pent to 5Hex3Pent. The most abundant structures are represented by 2Hex1Pent, 6.596 min; 2Hex2Pent, 14.055 min; 3Hex1Pent, 12.735; 3Hex2Pent, 23.712 min and 22.6 min; 4Hex2Pent 24.966 min and 29.18 min; 4Hex3Pent 26.017. A complete list of oligosaccharide peaks and abundances is shown in Table 8. The oligosaccharide pool can be further distinguished by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13). Notable peaks include those shown in Table 5. Table 8. Oligosaccharides produced from COG depolymerization of xyloglucan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose refers to glucose and galactose. Pentose refers to xylose.

[0419] β-Glucan refers to a polysaccharide with a 4:1 ratio of β-1,4 and β-1,3 glucose backbone. The oligosaccharides we generated closely matched this composition. The glucose composition was 97.04% (Table 3). The glycosidic bond composition was 48.91%, 30.95% and 17.06% of 4-glucose, 3-glucose and terminal glucose, respectively (Table 4). Fifteen oligosaccharides were observed in the library, ranging in length from 3 to 6 hexoses. The most abundant structure was represented by 3Hex, 14.158 minutes; 4Hex 9.81 minutes and 11.27 minutes; 5Hex 7.33 minutes and 11.24 minutes; 6Hex, 34.032 minutes. A complete list of oligosaccharide peaks and abundances is shown in Table 9. The oligosaccharide library can be further distinguished by its 1H-13C 2D-NMR (HSQC) fingerprint spectrum ( Fig.13 ). Notable peaks include those shown in Table 5. Table 9. Oligosaccharides produced from COG depolymerization of β-glucan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose refers to glucose only.

[0420] Galactomannans are polysaccharides with a β-1,4 mannose backbone and 22% α-1,3 galactose branches. The oligosaccharides we generated closely matched this composition. The mannose composition was 78.14% and galactose was 18.91% (Table 3). The glycosidic bond composition was 47.34%, 20.76% and 6.52% 4-mannose, terminal mannose and 4,6-mannose, respectively, 17.85% terminal galactose and 2.34% 4-glucose (Table 4). 54 oligosaccharides were observed in the library, ranging in length from 3 hexoses to 7 hexoses. The most abundant structures are represented by 3Hex, 1.489 min; 4Hex 4.109 min and 5.122 min; 4Hex1HexA, 10.301 min; 4Hex1Pent, 9.614 min; 5Hex, 7.65 min; 6Hex, 11.077 min; 7Hex, 13.245 min. A complete list of oligosaccharide peaks and abundances is shown in Table 10. The oligosaccharide pool can be further distinguished by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 10. Oligosaccharides produced from COG depolymerization of galactomannan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose refers to galactose and mannose.

[0421] Arabinogalactan II refers to a polysaccharide with a β-1,3 galactose backbone, extensive branching including α-1,6 arabinose, β-1,6 galactose-β-1,6 galactose, β-1,6 galactose-α-1,4 arabinose, and β-1,4 galactose-β-1,6 galactose. The oligosaccharides we produced closely matched this composition. The galactose composition was 87.28%, and arabinose was 7.23% (Table 3). The glycosidic bond composition was 50.75%, 17.33%, 14.18%, and 11.83% of terminal galactose, 1,3 galactose, 1,3,6 galactose, and 6 galactose, respectively, and 3.28% of terminal arabinose (Table 4). 62 oligosaccharides were observed in the library, ranging in length from 3 hexoses to 6 hexoses. The most abundant structures are represented by 3Hex, 2.53 min and 5.552 min; 4Hex, 3.534 min and 8.843 min; 5Hex, 10.555 min and 11.7 min; and 6Hex, 12.269. A complete list of oligosaccharide peaks and abundances is given in Table 11. The oligosaccharide pool can be further distinguished by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 11. Oligosaccharides produced from COG depolymerization of arabinogalactan II. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Pentose refers to arabinose and hexose refers to galactose.

[0422] Curdlan refers to a polysaccharide with a β-1,3 glucose backbone. The oligosaccharides we generated closely matched this composition. The glucose composition was 99.04% (Table 3). The glycosidic bond composition was 74.84% 1,3 glucose and 8.82% terminal glucose (Table 4). Ten oligosaccharides were observed in the library, ranging in length from 2 hexoses to 6 hexoses. The most abundant structures were represented as 2Hex, 1.456 min, 3Hex, 1.456 min; 2Hex1Pent, 12.672 min; 4Hex, 24.35 min; 5Hex, 30.063 min; 6Hex, 36.833. A complete list of oligosaccharide peaks and abundances is shown in Table 12. The oligosaccharide library can be further distinguished by its 1H-13C 2D-NMR (HSQC) fingerprint spectrum ( Fig.13 ). Notable peaks include those shown in Table 5. Table 12. Oligosaccharides produced from COG depolymerization of curdlan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose refers to glucose.

[0423] Lichenin refers to a polysaccharide with a β-1,4 glucose backbone alternating with β-1,3 glucose at a 33% chance. The oligosaccharides we generated closely matched this composition. The glucose composition was 80.21%, galactose and mannose were both 8.64% (Table 3). The glycosidic bond composition was 67.02%, 8.95% and 6.82% 4-mannose, 4,6-mannose and terminal mannose, respectively, and 19.58% terminal galactose (Table 4). 42 oligosaccharides were observed in the library, ranging in length from 3 hexoses to 8 hexoses. The most abundant structures were represented as 3Hex1Pent, 7.59 min; 4Hex 17.74 min; 4Hex1Pent, 6.96 min; 5Hex 15.88 min; 5Hex1HexA, 12.747 min, 6Hex, 10.877 min; 7Hex, 13.039 min. A complete list of oligosaccharide peaks and abundances is shown in Table 13. The oligosaccharide pool can be further differentiated by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 13. Oligosaccharides produced from COG depolymerization of lichenan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, Deoxyhex refers to deoxyhexose. Hexose refers to glucose.

[0424] Mannans refer to polysaccharides with a β-1,4 mannose backbone. The oligosaccharides we generated closely matched this composition. The mannose composition was 83.8%, followed by galactose, glucose, and arabinose at 7.61%, 4.48%, and 2.99%, respectively (Table 3). The glycosidic bond composition was 58.31% and 34.6% 4-mannose and terminal mannose, respectively, and 3.63% terminal galactose (Table 4). 46 oligosaccharides were observed in the library, ranging in length from 1 hexose and 1 pentose to 5 hexoses and 2 pentoses. The most abundant structures are represented by 2Hex1Pent, 6.624 min and 9.655 min; 2Hex1Pent, 13.77 min; 3Hex1Pent 12.727 min; 3Hex2Pent 16.706 min and 23.412 min; 4Hex1pent, 19.731 min; 4Hex2pent, 24.422 min. A complete list of oligosaccharide peaks and abundances is shown in Table 14. The complete list of oligosaccharides can be further distinguished by their 1H-13C 2D-NMR (HSQC) fingerprints ( Fig.13 ). Notable peaks include those shown in Table 5. Table 14. Oligosaccharides produced from COG depolymerization of mannan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose refers to mannose.

[0425] Xylans refer to polysaccharides with a β-1,4 xylose backbone and 13% α-1,2 glucose-4-OMe. The oligosaccharides we generated closely matched this composition. The xylose composition was 85.48%, followed by glucose, mannose, and galactose at 5.36%, 4.9%, and 2.04%, respectively (Table 3). The glycosidic bond composition was 54.71% 1,4 xylose, 15.28% 1,4 mannose, 13.61% 1,4 glucose, 7.18% terminal xylose, and 5.19% terminal glucose (Table 4). 15 oligosaccharides were observed in the library, ranging in length from 2 pentoses to 6 hexoses and 1 pentose. The most abundant structures are represented by 3Pent, 8.429 min; 4Pent, 16.521 min; 4Pent1HexAoMe, 21.15 min; 5Pent, 23.199; 6Pent, 26.735; 6Hex1Pent, 18.422 min. A complete list of oligosaccharide peaks and abundances is shown in Table 15. The oligosaccharide pool can be further distinguished by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 15. Oligosaccharides produced from COG depolymerization of xylan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, Deoxyhex refers to deoxyhexose. Pentose refers to xylose. 1 HexAOMe refers to methylated glucuronic acid.

[0426] Galactans refer to polysaccharides with a β-1,4 galactan backbone. The oligosaccharides we generated closely matched this composition. The galactan composition was 80.06%, followed by arabinose, rhamnose, and galacturonic acid at 9.28%, 4.59%, and 3.04%, respectively (Table 3). The glycosidic bond composition was 61.68% and 33.73% of 4-galactose and terminal galactose, respectively, and 2.02% of terminal arabinose (Table 4). 17 oligosaccharides were observed in the library, ranging in length from 3 hexoses to 6 hexoses and one hexuronic acid. The most abundant structures are represented as 3Hex, 2.69 minutes; 2Hex1Pent, 3.038 minutes; 4Hex6.614 minutes; 3Hex1Pent, 7.292; 3Hex1hexA, 8.937 minutes; 5Hex 9.652 minutes; 4Hex1Pent, 10.112 minutes, 6Hex, 11.525 minutes, 4Hex1HexA, 11.857 minutes; 5Hex1HexA, 13.573 minutes. A complete list of oligosaccharide peaks and abundances is shown in Table 16. The oligosaccharide library can be further distinguished by its 1H-13C2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 16. Oligosaccharides produced from COG depolymerization of galactans. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, Deoxyhex refers to deoxyhexose. Hexose refers to galactose.

[0427] Glucomannan refers to a polysaccharide with a backbone of 60% β-1,4 mannose and 40% β-1,4 glucose. The oligosaccharides we generated closely matched this composition. The mannose composition was 60.45%, followed by 36.73% glucose (Table 3). The glycosidic bonds were 47.58% and 20.23% 4-mannose and terminal mannose, respectively, and 31.52% 4-glucose (Table 4). 87 oligosaccharides were observed in the library, ranging in length from 3 hexoses to 8 hexoses. The most abundant structures are represented as 3Hex, 6.695 minutes; 3Hex1Pent, 18.947 minutes; 4Hex 16.802 minutes and 17.38 minutes; 4Hex1Pent, 20.328 minutes; 5Hex 18.549 minutes and 25.896 minutes; 6Hex, 22.854 minutes; 7Hex, 24.537 minutes. A complete list of oligosaccharide peaks and abundances is shown in Table 17. Table 17. Oligosaccharides produced from COG depolymerization of glucomannan. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose refers to glucose and mannose.

[0428] Locust bean gum refers to a polysaccharide with a 73% β-1,4 mannose backbone, of which 23% is modified with β-1,4 galactose. The oligosaccharides we generated closely matched this composition. The mannose composition was 72.91%, followed by 22.98% galactose (Table 3). The glycosidic bond composition was 62.02%, 8.95% and 6.82% 4-mannose, 4,6-mannose and terminal mannose, respectively, and 19.58% terminal galactose (Table 4). 39 oligosaccharides were observed in the library, ranging in length from 3 hexoses to 7 hexoses. The most abundant structures were represented as 3Hex, 11.02 min; 4Hex 4.188 min; 4Hex1Pent, 9.688 min; 5Hex7.755 min; 6Hex, 11.153 min; 7Hex, 13.293 min. A complete list of oligosaccharide peaks and abundances is shown in Table 18. The oligosaccharide pool can be further differentiated by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13 ). Notable peaks include those shown in Table 5. Table 18. Oligosaccharides produced from COG depolymerization of locust bean gum. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Hexose refers to galactose and mannose.

[0429] Corn fiber refers to a polysaccharide or a mixture of polysaccharides extracted from spent distiller's grains or other corn streams. In some aspects, corn fiber refers to alkali-soluble substances extracted from distiller's grains or other corn streams. In some aspects, corn fiber refers to acid-soluble substances extracted from distiller's grains or other corn streams. In some aspects, corn fiber refers to insoluble substances from distiller's grains or other corn streams. Corn fiber oligosaccharides are composed of 3.07% glucose, 6.78% galactose, 35.76% arabinose, and 48.68% xylose (Table 3). The glycosidic bond composition includes 5.83% 4-glucose, 16.33% 4-xylose, 6.21% 3,4-xylose, 25.05% terminal xylose, and 27.79% terminal arabinose (Table 4). 29 oligosaccharides were observed in the library, ranging in length from 3 hexoses to 12 hexoses. The most abundant structures are represented by 3Hex, 4.11min; 4Hex 9.29min; 5Hex 12.31min; 6Hex, 14.058; 7Hex, 15.254min; 8Hex, 16.394; 9Hex, 18.013min; 10Hex, 21.99min; 11Hex, 22.911; 12Hex, 24.55min. A complete list of oligosaccharide peaks and abundances is shown in Table 19. The oligosaccharide library can be identified by its 1H-13C 2D-NMR (HSQC) fingerprint ( Fig.13 ) further distinguished that the fingerprint showed cross-sectional coordinates similar to those of arabinoxylan. The significant peaks included those shown in Table 5. Table 19. Oligosaccharides produced from COG depolymerization of corn fiber. Hex refers to hexose, Pent refers to pentose, HexA refers to hexuronic acid, and Deoxyhex refers to deoxyhexose. Pentose refers to xylose and arabinose. Hexose refers to glucose and galactose. Embodiment 11 (Comparison between COG products and FITDOG products)

[0430] Oligosaccharides produced by COG are expected to be different from those produced by a similar method called FITDOG in PCT Application No. PCT / US2018 / 038350 (published as WO / 2018 / 236917). Some homogeneity of oligosaccharides between libraries was found; however, substantial differences were also encountered. COG was applied to galactomannans, arabinoxylans, xyloglucans, glucomannans, lichens, mannans, galactans, beta-glucans, curdlans, and xylans. The results were analyzed by mass spectrometry and the peaks corresponding to the oligosaccharides were compared to those described in PCT Application No. PCT / US2020 / 035748.

[0431] COG oligosaccharide production: Galactomannan, arabinoxylan, xyloglucan, glucomannan, lichenin, mannan, galactan, β-glucan, curdlan and xylan (550 mg) were dissolved in 20 ml of HPLC grade water in a capped reaction vessel and placed in a shaking incubator at 55°C and 85 RPM for 20 minutes. The pH of the solution was adjusted to 5.2. Hydrogen peroxide (5 ml) and iron (II) sulfate (2.75 mg in 50 μL water) were added to the reaction mixture and mixed thoroughly, except that copper (II) sulfate was used for curdlan. The reaction in the capped reaction vessel was allowed to proceed in a shaking incubator at 55°C and 65 RPM for two hours. The closed reaction was cooled to 12°C in a -20°C refrigerator. Ammonium hydroxide (1ml, 28% v / v, to pH 10.2) is used to adjust pH, and the sample is reacted 1 hour in the shaking incubator of 45 ℃ and 20RPM, and the lid is loosened to allow the release of oxygen, ammonia and carbon dioxide gas. The freeze-dried oligosaccharide mixture is rehydrated with the minimum amount of water required to allow free flow solution. Then this solution is loaded onto the column containing 15mL mixed bed ion exchange resin of every gram (dry weight) crude material, and the effluent is collected in a plastic freezing bag. Once the material is loaded onto the column, the column is rinsed with 3 bed volumes of water. Finally, the effluent is sealed and frozen in the bag, then carefully pulverized and freeze-dried.

[0432] Data analysis of COG products: Oligosaccharide analysis was performed in the manner of Amicucci, MJ, Nandita, E., et al. (2020). Nature Communications 11(1): 1-12. Oligosaccharide peak volumes were generated using the “Search by Molecular Feature” function of Agilent Mass Hunter Qualitative Analysis B.10.

[0433] FITDOG oligosaccharide production: A solution containing 95% (v / v) sodium acetate buffer (adjusted to pH 5 with glacial acetic acid), 5% (v / v) hydrogen peroxide (30% w / w) and 65 nM of the metal complex to be studied was prepared. The mixture was vortexed and added to galactomannan, arabinoxylan, xyloglucan, glucomannan, lichenin, mannan, galactan, β-glucan, curdlan and xylan to make a final solution of 1 mg / ml. The reaction was incubated at 100°C for 60 minutes. After the reaction, half the reaction volume of cold 2M NaOH was added and vortexed, and then 0.6% of the initial reaction volume of glacial acetic acid was added for neutralization.

[0434] Oligosaccharides were separated using a non-porous graphitized carbon column (GCC-SPE). The column was washed with 80% acetonitrile and nano-pure water in 0.1% (v / v) trifluoroacetic acid (TFA). Oligosaccharides were loaded and washed with 5 column volumes of nano-pure water. Oligosaccharides were eluted with 40% acetonitrile containing 0.05% (v / v) TFA.

[0435] Data analysis of FITDOG products: Oligosaccharides of FITDOG were manually annotated from their parent masses by Agilent Mass Hunter qualitative analysis. For this example, data were obtained directly from PCT application number PCT / US2020 / 035748.

[0436] Several trends were noted when comparing the COG and FITDOG samples. The full data is presented in Table 20; unique oligosaccharides for each process are noted, while similar oligosaccharides can be inferred from the differences in Table 20 from Tables 7-10 and Tables 12-18. In addition, the masses of the compounds in Table 20 can be referenced to the compositional properties of Tables 7-10 and Tables 12-18. For galactomannan, COG produced many compounds composed of 3 to 5 hexoses and a single pentose; while the FITDOG process included two different 7Hex isomers. For arabinoxylan, COG produced several unique small DP3 and DP4 pentose oligosaccharides, while FITDOG produced several other isomers ranging from DP3 to DP11, with many high DP isomers not produced by COG. For xyloglucan, FITDOG tended to produce more large DP isomers, while COG produced shorter oligosaccharides. For glucomannans, COG produced many isomers containing hexose and a single pentose unit, while FITDOG produced none of these isomers. For galactans, COG produced many unique isomers containing hexose and a single pentose unit, while FITDOG produced some larger DP8 and DP9 oligosaccharides not found in COG. For β-glucans, FITDOG produced more DP6 and DP7 isomers. For lichenan, COG produced many unique isomers containing hexose and a single pentose unit, while FITDOG produced many unique DP3 to DP10 oligosaccharides not found in COG. For mannans, COG produced many unique isomers containing hexose and a single pentose unit, while FITDOG produced many unique DP4 to DP9 oligosaccharides not found in COG. For xylans, the FITDOG process produced more unique oligosaccharides with methylated glucuronic acid residues. For curdlan, COG produced unique oligosaccharides with unique isomers containing a hexose and a single pentose unit and a unique DP3 oligosaccharide.

[0437] Disclosed herein are synthetic oligosaccharides, including oligosaccharide libraries produced by a COG process, comprising at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or all of the oligosaccharides specific to the COG process mentioned in Table 20. Also disclosed herein are synthetic oligosaccharides produced by a COG process, including oligosaccharide libraries, but wherein the oligosaccharides specific to the FITDOG process mentioned in Table 20 are not present at detectable levels in the COG-produced oligosaccharides. Table 20: GalMan = galactomannan, ArabXyl = arabinoxylan, XylGlc = xyloglucan, GlcMan = glucomannan, Lich = lichenin, Man = mannan, Gal = galactan, β-Glc = β-glucan, Curd = curdlan, Xyl = xylan

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Claims

1. A synthetic oligosaccharide composition comprising oligosaccharides, wherein each oligosaccharide independently comprises a backbone having 3 to 30 monomers, wherein the backbone comprises 3 or more glucose monomers, and wherein the glucose monomers in the backbone are linked by a combination of β-1,4 glucose bonds and β-1,3 glucose bonds.

2. The synthetic oligosaccharide composition of claim 1, wherein the synthetic oligosaccharide composition is described by the mass and retention time identifiers in Table 9 or Table 13.

3. The synthetic oligosaccharide composition of claim 1 or claim 2, wherein the glucose monomers in the backbone chain comprise β-1,4 glucose and β-1,3 glucose alternating in a repeating manner.

4. The synthetic oligosaccharide composition of claim 1 or claim 2, wherein the sum of Compound 2, Compound 4, Compound 12 and Compound 14 constitutes at least 42% or 35% to 50% of the peak volume shown in Table 9.

5. The synthetic oligosaccharide composition of claim 1 or claim 2, wherein the sum of Compound 1, Compound 2, Compound 4, Compound 6, Compound 7 and Compound 12 constitutes at least 62% or 55% to 75% of the peak volume shown in Table 9.

6. A synthetic oligosaccharide composition as described in claim 1 or claim 2, wherein the sum of Compound 5, Compound 11, Compound 14, Compound 16, Compound 20, Compound 22, Compound 27, Compound 31, Compound 32 and Compound 33 constitutes at least 73% or 65% to 85% of the peak volume shown in Table 13.

7. A synthetic oligosaccharide composition as described in claim 1 or claim 2, wherein the sum of Compound 1, Compound 5, Compound 6, Compound 14, Compound 16, Compound 21, Compound 27, Compound 33, Compound 38 and Compound 40 constitutes at least 51% or 40% to 60% of the peak volume shown in Table 13.

8. The synthetic oligosaccharide composition of any one of claims 1-7, wherein the synthetic oligosaccharide comprises 1H-13C 2D-NMR (HSQC) peaks within 10% of those described as lichenin or beta glucan in Table 5.

9. The synthetic oligosaccharide composition of any one of claims 1-7, wherein the composition comprises 20% to 40%, 40% to 60%, 60% to 80%, or at least 80% terminal glucose, β-1,4 glucose linkages, and β-1,3 glucose linkages.

10. A synthetic oligosaccharide comprising a β-1,4 xylose backbone, wherein the total number of monomers in the synthetic oligosaccharide is 3 to 30; preferably, the synthetic oligosaccharide is produced from corn fiber.

11. A synthetic oligosaccharide comprising a β-1,4 glucose backbone, a β-1,4 galactose backbone, a β-1,3 galactose backbone, a β-1,3 glucose backbone, a repeating linear β-1,4 mannose backbone, or a backbone having β-1,4 mannose and β-1,4 glucose, wherein the total number of monomers in the synthetic oligosaccharide is 3 to 30.

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