Production of Bioactive Oligosaccharides

Abstract

Methods of producing oligosaccharides are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 522,604, filed on June 20, 2017, which is incorporated herein by reference for all purposes. Background Art

[0003] Short chains of sugar molecules 3-20 monosaccharides long are called oligosaccharides and are of particular interest due to their biological activity. This includes their ability to act as prebiotics, modulate the immune system, and hinder the adhesion of pathogens in the intestine [Hooper, LV; Midtvedt, T.; Gordon, JI, Annual review of nutrition 2002, 22 (1), 283-307]. The best examples of these oligosaccharides are those produced in human milk [LoCascio, RG et al., Journal of agricultural and food chemistry 2007, 55 (22), 8914-8919; Marcobal, A. et al., Journal of agricultural and food chemistry 2010, 58 (9), 5334; Wang, M. et al., Journal of pediatric gastroenterology and nutrition 2015, 60 (6), 825]. These compounds are known to have a high degree of specificity for enriching specific bacteria in the intestines of newborns.

[0004] There have been many attempts to collect oligosaccharides from plant and animal products. Examples include galacto-oligosaccharides (GOS) [Lamsal, BP, Journal of the Science of Food and Agriculture 2012, 92 (10), 2020-2028] and fructo-oligosaccharides (FOS) [Yoshikawa, J. et al., Biotechnology letters 2008, 30 (3), 535-539]. These products are currently used for nutritional and pharmaceutical purposes.

[0005] With the exception of human milk oligosaccharides, short uncoupled oligosaccharide chains are not easily found in nature. In addition, these compounds are very expensive to synthesize and difficult to isolate because oligosaccharides are not abundant in most natural products. Polysaccharides (i.e. sugar molecules up to 100,000 monosaccharides in length) occur naturally in very high abundance in plants, bacteria and yeast, but they are too large to be biologically active.

[0006] Fenton chemistry is based on the generation of hydroxyl and peroxide radicals using an iron catalyst [Neyens, E.; Baeyens, J., Journal of Hazardous materials 2003, 98(1), 33-50; Walling, C., Accounts of chemical research 1975, 8(4), 125-131], and these free radical products can in turn oxidize many different substrates [Kuo, W., Water Research 1992, 26(7), 881-886; Lin, SH; Lo, CC, Waterresearch 1997, 31(8), 2050-2056; Watts, RJ et al., Hazardous Waste and Hazardous Materials 1990, 7(4), 335-345; Zazo, J. et al., Environmental science & technology 2005, 39(23), 9295-9302]. It is noteworthy that Fenton oxidation has been used as an analytical tool to determine the interaction between nucleic acid complexes and proteins by selectively cleaving their respective polymers [Meares, CF et al., Methods in enzymology 2003, 371, 82-106; Rana, TM; Meares, CF, Journal of the American Chemical Society 1990, 112 (6), 2457-2458]. Carbohydrate oxidation and oxidative degradation with or without iron catalysts have received extensive attention, especially in the degradation of functionalized starch and cellulose and wood polysaccharides [Emery, JA et al., Wood Science and Technology 1974, 8 (2), 123-137; Xu, G.; Goodell, B., Journal of Biotechnology 2001, 87 (1), 43-57; Haskins, JF; Hogsed, MJ, The Journal of Organic Chemistry 1950, 15 (6), 1264-1274; VS et al., Polymer Degradation and Stability 2007, 92(8), 1476-1481; Parovuori, P. et al., 1995, 47(1), 19-23]. Many researchers have studied the mechanism and results of Fenton-type oxidation of monosaccharides, disaccharides and oligosaccharides. Fenton oxidation of carbohydrates often has inherent specificity. It has been shown that disaccharides are more susceptible to oxidation than monosaccharides and sugar alcohols [Morelli, R. et al., Journal of Agricultural and Food Chemistry 2003, 51(25), 7418-7425], and (1→6) linkages are more unstable than (1→4) linkages [Uchida, K.; Kawakishi, S., Carbohydrate Research 1988, 173(1), 89-99].

[0007] Recently, the Fenton system has been used for the depolymerization of chondroitin sulfate, heparin, and other glycosaminoglycans [Achour, O. et al., Carbohydrate Polymers 2013, 97(2), 684-689; Li, J.-h. et al., Marine Drugs 2016, 14(9), 170; Petit, AC et al., Carbohydrate Polymers 2006, 64(4), 597-602; Wu, M. et al., Carbohydrate Polymers 2010, 80(4), 1116-1124]. All of these documents use acid-containing substrates in the form of iduronic acid or glucuronic acid residues. SUMMARY OF THE INVENTION

[0009] A method for forming oligosaccharides from polysaccharides is provided. In some embodiments, the method comprises treating the polysaccharide with hydrogen peroxide and Fe 3+ , Fe 2+ , Cu 2+ or other metal reactions described herein, followed by cleavage of the glycosidic bonds in the polysaccharide with a base, thereby producing oligosaccharides from the polysaccharide. This reaction is referred to herein as "FITDOG".

[0010] We have developed a method to produce bioactive oligosaccharides by digesting polysaccharides from plants, bacteria, and yeast. 3+ ,Fe 2+ ) or other transition metals (including but not limited to Cu 1+ ,Co 2+ In some embodiments, the oligosaccharides produced are in the range of DP 3-20 (DP refers to degree of polymerization). In some embodiments, the method will produce oligosaccharides for analysis and bioactive foods, such as prebiotics, anticancer, pathogen blocking or foods with other functions.

[0011] The method can be used to convert polysaccharides (e.g., from plants, bacteria or yeast) into biologically active oligosaccharides. 3+ Reacts with hydrogen peroxide. Other metal ions, including but not limited to Fe 2+ and Cu 2+ The same results can also be produced with different effectiveness. In some embodiments, the reaction is allowed to proceed for 30 minutes (or, for example, 10 minutes to 4 hours, for example, 15 minutes to 2 hours, or 10 minutes to 1 hour). The reaction is subsequently quenched with a base (e.g., aqueous sodium hydroxide, calcium hydroxide, potassium hydroxide, etc.).

[0012] In some embodiments, the oligosaccharide properties obtained can be determined. In some embodiments, high performance liquid chromatography-mass spectrometry (LC-MS) analysis of the product mixture shows a number of oligosaccharide structures, ranging in size from a degree of polymerization of 3 to 20 (or, for example, 3-200), depending on the source of the polysaccharide. The oligosaccharide structure and composition depend on the source of the polysaccharide.

[0013] In some embodiments, the production of plant-derived oligosaccharides consisting of a degree of polymerization (DP) of 3-20 (or, for example, 3-200) is provided. Polysaccharides may include, for example, those obtained from known food sources, such as rice, bananas, squash, wheat flour, and polysaccharides that are byproducts of food production. In some embodiments, polysaccharides may come from waste food products and sources that are not commonly recognized as food. In some embodiments, polysaccharide sources are processed foods and plant products.

[0014] In some embodiments, the method provides for producing oligosaccharides (eg, oligosaccharides having a degree of polymerization of DP 3-20 (or, eg, 3-200)) from bacterial cell wall polysaccharides.

[0015] In some embodiments, the method provides for producing oligosaccharides (eg, oligosaccharides having a degree of polymerization of DP 3-20 (or, eg, 3-200)) from yeast cell wall polysaccharides.

[0016] In some embodiments, the method provides for producing oligosaccharides (eg, oligosaccharides having a degree of polymerization of DP 3-20 (or, eg, 3-200)) from algal polysaccharides.

[0017] In some embodiments, the oligosaccharide is a biologically active oligosaccharide. In some embodiments, the biologically active oligosaccharide is consumed by bacteria that are beneficial to the human intestinal tract. In some embodiments, the biologically active oligosaccharide can regulate the immune system. Oligosaccharides can cause the immune system to respond insufficiently or excessively to known or unknown stimuli. In some embodiments, the effect of the biologically active oligosaccharide is to block pathogens.

[0018] In some embodiments, oligosaccharides are selective carbon substrates for stimulating soil microbial growth. In some embodiments, oligosaccharides are added to the soil after soil fumigation or disinfection. If not controlled, accessible organic carbon will drive soil ecology to develop in the direction of pathogens. By providing specific oligosaccharides that selectively stimulate the growth of beneficial soil microbial communities, the soil pathogen community in the soil can be reduced. In some embodiments, a combination of one or more oligosaccharides prepared as described herein can be added to soil containing one or more microorganisms (e.g., beneficial soil microorganisms).

[0019] In some embodiments, one or more oligosaccharides prepared as described herein can be used to produce prebiotics for food supplements. In some embodiments, the oligosaccharides can be used for appetite control and / or energy (caloric) intake in overweight and obese children.

[0020] In some embodiments, a method for producing soluble fiber from an insoluble fiber comprising a polysaccharide using the reaction conditions described herein is provided. By only allowing the reaction to run to a certain extent, a composition (e.g., a gel or ointment) with desirable properties can be produced. The soluble fiber product can be used for many purposes, including but not limited to pharmaceutical products and devices, food products (i.e., thickeners, nutrition improvements, flavor regulators), soil conditioners (for enriching specific beneficial soil microbial composition components) and fiber production (i.e., novel fabrics, ropes, biodegradable packaging, etc.). In some embodiments, for example, the insoluble fiber is cotton.

[0021] In some embodiments, a method for producing oligosaccharides from polysaccharides is provided. In some embodiments, the method includes reacting a polysaccharide in a reaction mixture with hydrogen peroxide and a transition metal or an alkaline earth metal; then quenching the reaction with a base and / or cleaving the glycosidic bonds in the polysaccharide with a base, thereby producing an oligosaccharide mixture from a polysaccharide. In some embodiments, the reaction mixture comprises a transition metal. In some embodiments, the transition metal is selected from iron (e.g., Fe3+, Fe2+), copper (e.g., Cu2+), manganese, cobalt, or molybdenum. In some embodiments, the reaction mixture comprises an alkaline earth metal. In some embodiments, the alkaline earth metal is selected from calcium or magnesium.

[0022] In some embodiments, the transition metal or alkaline earth metal concentration in the reaction mixture is at least 0.65nM. In some embodiments, the transition metal or alkaline earth metal concentration in the reaction mixture is 0.65nM to 500nM. In some embodiments, the concentration of hydrogen peroxide in the reaction mixture is at least 0.02M. In some embodiments, the concentration of hydrogen peroxide in the reaction mixture is 0.02M-1M. In some embodiments, the base is sodium hydroxide, potassium hydroxide or calcium hydroxide. In some embodiments, the base concentration is at least 0.1M. In some embodiments, the base concentration is 0.1M-5.0M. In some embodiments, the polysaccharide comprises one or more of amylose, amylopectin, β-glucan, pullulan, xyloglucan, arabinogalactan I and arabinogalactan II, polyrhamnogalacturonic acid I, polyrhamnogalacturonic acid II, galactan, arabinan, arabinoxylan, xylan (e.g., beechwood xylan), glycogen, mannan, glucomannan, curdlan, or inulin. In some embodiments, the polysaccharide is from a plant or animal source. In some embodiments, the polysaccharide is from a bacterial or algae source. In some embodiments, the polysaccharide is in the form of a plant material (optionally freeze-dried). In some embodiments, the plant material is a banana, chickpea, or millet plant material. In some embodiments, the method further comprises purifying one or more oligosaccharides from the oligosaccharide mixture.

[0023] In some embodiments, before reacting, the method comprises contacting the polysaccharide with one or more polysaccharide degrading enzymes. In some embodiments, the one or more polysaccharide degrading enzymes comprise amylase, isoamylase, cellulase, maltase, polyglucosidase or a combination thereof.

[0024] Also provided are compositions comprising a mixture of oligosaccharides produced by the methods described above or elsewhere herein or purified one or more oligosaccharides produced by the methods described above or elsewhere herein.

[0025] Also provided are the oligosaccharides in Table 1 or mixtures of two or more oligosaccharides in Table 1.

[0026] Also provided is a method for stimulating microorganisms in vivo or in vitro. In some embodiments, the method comprises contacting a microorganism (e.g., bacteria, fungi, yeast) and a composition comprising an oligosaccharide mixture (e.g., Table 1) or oligosaccharides in the table produced by the method described above or elsewhere herein under conditions that selectively stimulate microbial growth. In some embodiments, the microorganism is a prebiotic microorganism. In some embodiments, the microorganism is in the intestinal tract of an animal and the composition is applied to the animal. In some embodiments, prebiotic microorganisms are applied to animals independently of or together with the composition. In some embodiments, the prebiotic microorganism is Bifidobacterium pseudocatenulatum. In some embodiments, the microorganism is a soil microorganism, an oral microorganism (e.g., a bacterium) or a skin microorganism (e.g., a bacterium).

[0027] definition

[0028] The "degree of polymerization" or "DP" of an oligosaccharide refers to the total number of sugar monomer units that are part of a particular oligosaccharide. For example, tetragalacto-oligosaccharide has a DP of 4, with 3 galactose moieties and one glucose moiety.

[0029] The term "Bifidobacterium" and its synonyms refer to a genus of anaerobic bacteria 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 has health benefits for its host. See, for example, Guarner F and Malagelada JR. Lancet (2003) 361, 512-519 for further description of Bifidobacteria in normal intestinal flora.

[0030] "Prebiotics" or "prebiotic nutrients" are generally non-digestible food ingredients that, when ingested, can have 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 "prebiotics" refers to such non-digestible food ingredients in a non-naturally occurring state (e.g., purified, chemically or enzymatically synthesized, as opposed to, for example, in whole human milk).

[0031] "Prebiotics" refer to live microorganisms that confer a health benefit on the host when administered in adequate amounts.

[0032] As described herein, when used to modify a numerical value or an amount defined in a range, the term "about" or "approximately" refers to the value and relatively reasonable deviations known to those skilled in the art, such as ±20%, ±10% or ±5%, are within the meaning of the stated value.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 .Exemplary MALDI-MS of xyloglucan oligosaccharides produced by FITDOG.

[0035] Figure 2 Optimize the iron (III) sulfate concentration to obtain optimal oligosaccharide abundance.

[0036] Figure 3 Optimize buffer pH to obtain optimal oligosaccharide abundance.

[0037] Figure 4 .Optimize NaOH and hydrogen peroxide concentrations.

[0038] Figure 5 .Optimize time and temperature conditions.

[0039] Figure 6 . HPLC-MS spectra of oligosaccharides from green split pea produced using CuCl2, FeSO4 and Fe3(SO4)2.

[0040] Figure 7 . Relevant monosaccharide composition of foods subjected to FITDOG treatment.

[0041] Figure 8 Annotated base peak chromatograms of banana peel, chickpea and millet subjected to FITDOG treatment. Annotations correspond to the compounds named in Table 1.

[0042] Fig. 9 Growth of Bifidobacterium pseudocatenulatus MP80 on oligosaccharides produced by Fenton oxidation of butternut squash.

[0043] Fig.10 The xylan-annotated base peak chromatogram is shown.

[0044] Fig.11 The annotated base peak chromatogram of arabinoxylan is shown.

[0045] Fig.12 The annotated base peak chromatogram of lichenan is shown.

[0046] Fig.13 The annotated base peak chromatogram of galactomannan is shown.

[0047] Fig.14 The base peak chromatogram of the amylopectin annotation is shown.

[0048] Fig.15 The base peak chromatogram of amylose annotated is shown.

[0049] Fig.16 The base peak chromatogram annotated with polyrhamnogalacturonic acid I is shown.

[0050] Fig.17 The annotated base peak chromatogram of xyloglucan is shown.

[0051] Fig.18 The base peak chromatogram of the thermocoagulation annotation is shown.

[0052] Fig.19 The annotated base peak chromatogram of galactan is shown.

[0053] Fig. 20 The annotated base peak chromatogram of mannan is shown.

[0054] Fig.21 Annotated base peak chromatogram of glucomannan is shown.

[0055] Fig. 22 Annotated base peak chromatogram of larch arabinogalactan is shown.

[0056] Fig.23 The polygalacturonic acid annotated base peak chromatogram is shown.

[0057] Fig.24 The annotated base peak chromatogram for inulin is shown.

[0058] Fig.25 Annotated base peak chromatograms of amylose are shown, where different metals were used in the FITDOG method.

[0059] Fig.26 Annotated base peak chromatograms of amylose are shown, where different metals were used in the FITDOG method.

[0060] Fig. 27 The base peak chromatogram of amylose annotated is shown.

[0061] Fig.28 Annotated base peak chromatograms of yeast cell wall polysaccharides are shown.

[0062] Fig.29 Annotated base peak chromatogram of butternut squash subjected to upscaled FITDOG is shown, showing the degree of polymerization of 1-200+ hexasaccharides. The data were analyzed on a nanochip-HPLC / Q-TOF mass spectrometer. Hexasaccharides with a degree of polymerization of 4-7 showed the greatest abundance. DETAILED DESCRIPTION

[0063] Fenton's Initiation Toward DefinedOligosaccharide Groups (FITDOG) is a method for controlled degradation of polysaccharides into oligosaccharides. In some embodiments, crude polysaccharides are first subjected to an initiated oxidation treatment with hydrogen peroxide and a transition metal or alkaline earth metal (e.g., iron (III) sulfate) catalyst to make the glycosidic bonds more unstable. Alkali-induced cleavage is then performed with NaOH or other bases to obtain various oligosaccharides. Neutralization is immediately performed to reduce peeling reactions. This method can produce a large amount of bioactive oligosaccharides from various carbohydrate sources.

[0064] If desired, the polysaccharide can be optionally treated with one or more polysaccharide degrading enzymes to reduce the average size or complexity of the polysaccharide, and then the resulting polysaccharide can be treated with an oxidative treatment and a metal catalyst. Non-limiting examples of polysaccharidases include, for example, amylases, isoamylases, maltase, glucanase, or a combination thereof.

[0065] The initial oxidation treatment may include hydrogen peroxide and a transition metal or alkaline earth metal. Metals of different oxidation states, sizes, groups of the periodic table of elements, and coordination numbers have been tested to understand the application of the FITDOC process. Each different metal shows activity in the FITDOG reaction. Although these metals are available for any polysaccharide, different metals can be used to produce oligosaccharides with a preferred degree of polymerization. The oxidation treatment is followed by alkali treatment. The method can produce oligosaccharides from polysaccharides having different degrees of branching and different monosaccharide compositions, including natural and modified polysaccharides. The method is effective for polysaccharides from any source. Exemplary polysaccharide substrates include, but are not limited to, one or more of the following: amylose, amylopectin, β-glucan, amylopectin, xyloglucan, arabinogalactan I and arabinogalactan II, polyrhamnogalacturonic acid I, polyrhamnogalacturonic acid II, galactan, arabinan, arabinoxylan, xylan (e.g., beechwood xylan), glycogen, mannan, glucomannan, thermocoagulated polysaccharide, or inulin.

[0066] In some embodiments, the resulting oligosaccharide mixture produced by the method may have an average degree of polymerization of 2-200, such as between 2-100 or 3-20 or 5-50.

[0067] The resulting oligosaccharide mixture produced by the method can have a variety of uses. In some embodiments, the oligosaccharide mixture can be used as a prebiotic to selectively stimulate the growth of one or more probiotics. In some embodiments, the oligosaccharide composition can be applied as a prebiotic preparation (i.e., without bacteria) or as a prebiotic preparation (i.e., containing the desired bacteria such as bifidobacteria described herein). In general, any food or beverage that can be ingested by humans or animals can be used to prepare a preparation containing a composition of prebiotics and prebiotic oligosaccharides. Exemplary foods include those of semi-liquid consistency, which allow for the easy and uniform dispersion of the prebiotics and prebiotic compositions described herein. However, other consistencies (e.g., powders, liquids, etc.) can also be used without restriction. Therefore, these foods include, but are not limited to, dairy products such as cheese, cottage cheese, yogurt, and ice cream. Processed fruits and vegetables, including those for infants and young children, such as applesauce or dehydrated beans and carrots are also suitable for use with the oligosaccharides of the present invention. Infant rice cereals such as rice or oat-based rice cereals and adult cereals such as Musilix are also suitable for use with oligosaccharides. In addition to foods intended for human consumption, animal feed may also be supplemented with compositions containing prebiotics and prebiotic oligosaccharides.

[0068] Alternatively, compositions containing probiotics and prebiotic oligosaccharides can be used to supplement beverages. Examples of these beverages include, but are not limited to, infant formulas, older infant formulas, toddler beverages, milk, fermented milk, fruit juice, fruit-based beverages and sports drinks, etc. Many infant and toddler formulations are known in the art and are commercially available, including, for example, Nutrish A / B produced by Carnation GoodStart (Nestle Nutrition subsidiary, Glendale, California) and Mayfield Dairy (Athens, Tennessee). Other examples of infant or infant formulations include those disclosed in U.S. Patent No. 5,902,617. Other beneficial formulations of the composition include supplements of animal milk such as milk.

[0069] Alternatively, the composition containing prebiotics and prebiotic oligosaccharides can be formulated into pills or tablets, or encapsulated in capsules, such as gelatin capsules. Tablet forms can optionally include, for example, one or more lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants and pharmaceutically compatible carriers. Cache or candy forms can include flavored compositions containing carriers known in the art in addition to the active ingredients, such as sucrose, and soft lozenges containing the composition in an inert matrix, such as gelatin and glycerin or sucrose and gum arabic emulsions, gels, etc. The preparation containing prebiotics or prebiotic oligosaccharides can also include conventional food supplement fillers and bulking agents such as rice flour.

[0070] In some embodiments, the composition containing prebiotics or prebiotic oligosaccharides also contains non-human proteins, non-human liquids, non-human carbohydrates, or other non-human components. For example, in some embodiments, the composition contains cow (or other non-human) milk protein, soy protein, rice protein, β-lactoglobulin, whey, soybean oil or starch.

[0071] The dosage of the composition containing prebiotics and prebiotic oligosaccharides can vary according to individual needs and will take into account factors such as age (infants versus adults), weight, reasons for loss of beneficial intestinal bacteria (e.g., antibiotic treatment, chemotherapy, disease or age). According to the present specification, the amount administered to an individual should be sufficient to establish a colony of beneficial bacteria in the intestine over time. The size of the dose may also be determined by the presence, nature and extent of any adverse side effects associated with the administration of a composition containing prebiotics or prebiotic oligosaccharides. In some embodiments, the dosage range is effective as a food supplement for the reconstruction of beneficial bacteria in the intestine. In some embodiments, 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 embodiments, the dosage range of the oligosaccharide composition of the present invention is about 100 micrograms / L to about 15 grams / L of oligosaccharides. In some embodiments, the dosage range of the oligosaccharide composition of the present invention is about 1 gram / L to about 10 grams / L of oligosaccharides. Exemplary bifidobacterium doses include, but are not limited to, about 10 per dose. 4 to 10 12 Another advantageous dosage is about 10 6 to 10 10 CFU.

[0072] The formulation containing prebiotics or prebiotic oligosaccharides can be administered to any individual in need. In some embodiments, the individual is an infant or a toddler. For example, in some embodiments, 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 embodiments, the individual is between 3-18 years old. In some embodiments, the individual is an adult (e.g., 18 years old or older). In some embodiments, the individual is over 50, 55, 60, 65, 70, or 75 years old. In some embodiments, the individual has an immunodeficiency (e.g., an individual suffering from AIDS or undergoing chemotherapy).

[0073] Exemplary bifidobacteria that can be used in the prebiotic 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 depends in part on the target consumer.

[0074] It should be understood that for some applications, it is advantageous to include other bifidus factors in the preparations described herein. These additional ingredients may include, but are not limited to, oligofructose, such as Raftilose (Rhone-Poulenc, Cranbury, New Jersey), inulin (Imperial Holly, Sugar Land, Texas) and Nutraflora (Golden Technologies, Westminister, Colorado), as well as lactose, oligoxylose, soy oligosaccharides, lactulose / lactitol and oligogalactose, etc. In some applications, other beneficial bacteria such as Lactobacillus, Rumminococcus, Akkermansia, Bacteroides, Faecalibacterium may be included in the preparation.

[0075] Oligosaccharides as described herein can be used to stimulate any type of microorganism. Examples of microorganisms that can be stimulated by oligosaccharides include soil microorganisms (e.g., mycorrhizal fungi and bacteria and 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, Nitrosocus, Nitrosolobus, and Nitrosovibrio.

[0076] In some embodiments, the oligosaccharide composition is administered to a person or animal in need. For example, in some embodiments, the oligosaccharide composition is administered to a person or animal having at least one of the following symptoms: inflammatory bowel disease syndrome, constipation, diarrhea, colitis, Crohn's disease, colon cancer, functional bowel disorder (FBD), irritable bowel syndrome (IBS), excess sulfate-reducing bacteria, inflammatory bowel disease (IBD) and ulcerative colitis. Irritable bowel syndrome (IBS) is characterized by abdominal pain and discomfort, flatulence, and changes in intestinal function, constipation and / or diarrhea. There are three types of IBS: constipation-type IBS (C-IBS), alternating IBS (A-IBS) and diarrhea-type IBS (D-IBS). The oligosaccharide composition is useful, for example, for inhibiting or prolonging the recurrence period of ulcer patients. Oligosaccharide compositions can be administered to treat or prevent any form of functional bowel disorder, in particular irritable bowel syndrome (IBS), such as constipation-type IBS (C-IBS), alternating IBS (A-IBS) and diarrhea-type IBS (D-IBS); functional constipation and functional diarrhea. FBD is a general term for some chronic or semi-chronic gastrointestinal diseases that combine intestinal pain, intestinal dysfunction and social disturbance.

[0077] In other embodiments, the oligosaccharide compositions are administered to a subject in need of immune system stimulation and / or are used to promote resistance to bacterial or yeast (eg, Candidiasis) infections or diseases induced by sulfate-reducing bacteria.

[0078] Example

[0079] Example 1

[0080] The ability to build oligosaccharides from polysaccharides by depolymerization is an attractive solution. Currently, there are two ways to depolymerize polysaccharides, acid hydrolysis and enzymatic treatment with glycosyl hydrolases. Both technologies have their own disadvantages. Acid hydrolysis means that a large amount of monosaccharides and very small amounts of oligosaccharides are produced. On the other hand, enzymatic treatment can produce a large amount of oligosaccharides, but each glycosyl hydrolase can only cut at very specific sites depending on the monosaccharide arrangement of the attachment position, stereochemistry and surrounding environment. This means that even if an enzyme capable of depolymerizing a specific polysaccharide is known and available, it cannot degrade the structurally variable polysaccharides present in natural products on a large scale.

[0081] Although depolymerization of cellulose and acid-containing polysaccharides has been reported, there is still a need for a method that can effectively depolymerize neutral polysaccharides using the Fenton system. In addition, the preparation of large amounts of oligosaccharides from crude natural products in high yield using the Fenton system has not been reported. The purpose of the following examples is to show the analytical and preparative scale methods and applications of Fenton-type initiated oligosaccharide group dissociation (FITDOG) to produce oligosaccharides in high yield from polysaccharide isolates and natural products.

[0082] method

[0083] Material

[0084] Food was obtained from a local supermarket. Sodium acetate, hydrogen peroxide (30% w / w), sodium hydroxide, iron (III) sulfate pentahydrate, and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO).

[0085] Small-scale Fenton-induced oligosaccharide group dissociation (FITDOG)

[0086] 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 iron (III) sulfate was prepared. The mixture was vortexed and added to the dry polysaccharide standard to achieve a final solution of 1 mg / ml. The reaction was incubated at 100° C. for 20 minutes. After the reaction, half the reaction volume of cold 2M NaOH was added, vortexed, and then 0.6% of the initial reaction volume of concentrated acetic acid was added for neutralization.

[0087] Purification of oligosaccharides produced by small-scale FITDOG

[0088] Oligosaccharides were separated using a non-porous graphitized carbon cartridge (GCC-SPE). The cartridge was washed with 80% acetonitrile dissolved in 0.1% (v / v) trifluoroacetic acid (TFA) and nanopure water. Oligosaccharides were loaded and washed with 5 column volumes of nanopure water. Oligosaccharides were eluted with 40% acetonitrile containing 0.05% (v / v) TFA.

[0089] Large-scale Fenton-induced oligosaccharide group dissociation (FITDOG)

[0090] The freeze-dried food was completely dry and ground into a fine powder using a KRUPS F203 grinder (Millville, NJ). 950 ml of a solution of 40 mM sodium acetate buffer adjusted to pH 5 with glacial acetic acid was boiled in a 2-liter round-bottom flask equipped with a cold water column condenser. 1.0 g of the ground freeze-dried food, 32 mg of iron (III) sulfate, and 50 ml of 30% (w / w) aqueous hydrogen peroxide solution were added to the flask. The solution was allowed to react for 20 minutes with a stirring rod. To induce cleavage, 500 ml of ice-cold 2M NaOH was added, stirred for 30 seconds, and then 61.5 ml of ice-cold acetic acid was added to neutralize the reaction.

[0091] Purification of oligosaccharides produced by FITDOG on a large scale

[0092] Filtration through a 0.45 μm polyvinylidene fluoride (PVDF) filter membrane removed flocculated iron. For CombiFlash Rf200 (Teledyne Isco) flash liquid chromatography, a self-packaged 50 g porous graphitized carbon (PGC) flash liquid chromatography (Flash-LC) column was used. The column was loaded at 7 ml / min. A two-phase solvent system was used, which consisted of the following: A: nanopure water, and B: acetonitrile (HPLC grade). The sample was washed with 100% solvent A at 10 ml / min for 20 minutes, then washed with a solvent B gradient (0% to 80%) for 140 minutes, and then maintained isocratically at 80% solvent B for 20 minutes. The eluent was directed to waste, except that the eluent at 40-100 minutes was collected in a glass bottle. The collected eluent was dried on a rotary evaporator for 1 hour to remove acetonitrile. The remaining liquid was evaporated on a freeze dryer.

[0093] Analysis by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)

[0094] The samples were reconstituted in nanopure water. 1 μl of the sample was spread directly on a stainless steel MALDI plate. 0.3 μl of 0.01 M NaCl and 0.7 μl of 25 mg / ml 2,5-dihydroxybenzoic acid were added and mixed with a pipette tip. The sample was then vacuum dried. The sample was run on a Bruker UltraFlextreme MALDI-TOF / TOF instrument. The instrument was operated in positive mode, using 95% of the maximum laser power.

[0095] Chip-HPLC-nano / quadrupole time-of-flight mass spectrometry (Chip-HPLC-nano / Q-TOFMS) analysis

[0096] The samples were reconstituted in nanopure water and then analyzed by chip-HPLC-nano / Q-TOF MS. The system includes two pumps: a capillary pump for sample loading and a nanopump for analytical separation. In this system, an Agilent 1200 series HPLC and an Agilent 6520Q-TOF mass spectrometer were coupled via a chip cube interface. The chip contained a 40nl enrichment column and a 75μm×43mm analytical column, both of which were packed with PGC. Samples were loaded with 3% (v / v) acetonitrile / water + 0.1% formic acid at a flow rate of 4μl / min. Chromatographic separation was performed with the following two-phase gradient: solvent A: (3% (v / v) acetonitrile / water + 0.1 formic acid) and solvent B: (90% acetonitrile / water + 0.1% formic acid) at a flow rate of 0.4μl / min. The gradient was run for 60 min: 1% B to 5% B for 2 min, then 5% to 30% for 33 min, then 30% to 99% for 5 min, then maintained at 99% for 10 min, then 99% to 1% for 1 min, then maintained at 1% for 9 min before the next run.

[0097] Data were collected in positive mode and calibrated with internal calibration ions in the range of m / z 118.086 to 2721.895. Drying gas was set at 325°C with a flow rate of 5 l / min. Fragment, separator and Octapole 1RF voltages were set at 175, 60 and 750 volts, respectively. Fragmentation was performed at 0.63 spectra / second. Collision energy was based on compound mass and expressed as (collision energy)=1.8*(m / z)-2.4.

[0098] Monosaccharide composition

[0099] Dried butternut squash and FITDOG oligosaccharides were tested for monosaccharide composition as follows. Briefly, 10 mg of sample was reconstituted with 138 μl of nanopure water. Approximately 100 mg of 1.4 mm stainless steel beads were added, the sample was melted by vigorous impact in a Next Advance Storm 24 bullet mixer (Averill Park, NY), and boiled at 100°C for 1 hour. Next, 68 μl of concentrated TFA was added to form a 4M solution and incubated at 100°C for 2 hours. To quench the reaction, 800 μl of ice-cold nanopure water was added to dilute the solution 1000 times. 50 μl of the diluted solution was vacuum dried. The dried sample was then derivatized with 3-methyl-1-phenyl-2-pyrazol-5-one and dried. The sample was then reconstituted in nanopure water, extracted twice with chloroform, and the aqueous layer was used for analysis. The sample was injected into an Agilent 1290 Infinity II UHPLC equipped with an Agilent 6495QqQ MS. Separation was performed on an Agilent Zorbax EclipsePlus C18 column (2.1 mm x 150 mm id, 1.8 μm particle size). The solvent system consisted of: A: 25 mM ammonium acetate, adjusted to pH 8.2 using 5% acetonitrile / water solution of NH4OH. All other instrument parameters were followed.

[0100] Bacterial uptake

[0101] Several bacteria, mostly bifidobacterium strains, were screened for their ability to metabolize the FITDOG oligosaccharide pool. To achieve this, pure cultures were collected in the late logarithmic phase and used to inoculate a basic MRS containing the following different carbon sources: 2% glucose (positive control), 2% and 5% w / v butternut squash oligosaccharides (BSL) and no carbon source (negative control). The cultures were incubated under anaerobic conditions at 37°C for 96 hours and their growth was monitored every 30 minutes using a Biotek plate reader EON 120928C (Biotek Instruments, Winooski, VT, USA). After shaking for 30 seconds, the absorbance was measured at 600 nM to monitor growth.

[0102] result

[0103] Optimization of FITDOG reaction conditions

[0104] The Fenton reaction was optimized under various conditions using commercial xylan (polysaccharide) standards. The concentrations of iron (III) sulfate and hydrogen peroxide, reaction time, temperature, reaction pH, and quenching NaOH concentration were optimized in sequence using the analytical FITDOG method. Oligosaccharide products were monitored using MALDI-TOFMS. Relative oligosaccharide concentrations were directly obtained using MS abundance. MALDI TOF-MS spectra ( Figure 1 ) to obtain the relative abundance of each product.

[0105] 1. Optimization of Fe2(SO4)3 concentration

[0106] The concentration was varied from 0.0065 nM to 65 nM to optimize iron (III) sulfate. No oligosaccharide abundance was observed below 0.65 nM. Figure 2 The overall OS abundance was shown to increase with concentration. The signal reached a maximum at about 10 nM Fe2(SO4)3 concentration and increased slightly at 65 nM. Therefore, the latter concentration was used for the preparation.

[0107] 2. Optimization of buffer pH

[0108] The pH conditions were optimized by adding appropriate amounts of acid and base (acetic acid and NaOH (aqueous solution)) corresponding to pH 2-12 in increments of 1 pH unit using sodium acetate buffer. A pH less than 5 did not produce observable amounts. The maximum abundance of oligosaccharides was obtained at pH 5, while the abundance obtained above 6 was very small ( Figure 3 ). Therefore, we choose pH 5 as the optimal value.

[0109] 3. Hydrogen peroxide and NaOH concentrations.

[0110] An important feature of this method is the use of alkaline NaOH solution to terminate the reaction. This step is critical for producing abundant oligosaccharide products. To optimize the conditions, a full factorial design was used by varying the NaOH concentration, H2O2 concentration, and oligosaccharide abundance. The concentrations of NaOH were 0, 0.1, 1.0, 2.0, and 3.0 M. The concentrations of H2O2 were 0.02, 0.12, 0.24, and 0.48 M hydrogen peroxide. When no NaOH was added (0.0 M, Figure 4 ), there was little or no detectable oligosaccharide in the reaction mixture. Similarly, no detectable oligosaccharide was observed when the H2O2 concentration was below 0.02M. When no H2O2 was added to the solution, there was no detectable oligosaccharide signal. Based on this study, we found that 2.0M NaOH and 0.12M H2O2 worked well.

[0111] 4. Reaction time and temperature

[0112] Time and temperature were also analyzed using a full factorial design. Time points of 0, 20, 40, 60, and 120 minutes were tested. Temperatures of 25, 50, 75, and 100°C were used. Minimal oligosaccharide signal was observed below 75°C. Maximum abundance was observed at 100°C with a reaction time of 20 minutes. ( Figure 5 ).

[0113] 5. Effects of other transition metals

[0114] It has been shown that Fenton-type reactions can be performed with compounds containing Fe(III), Fe(II), and Cu(II). Iron(III) sulfate, iron(II) sulfate, and copper(II) chloride were reacted with green peas under previously optimized conditions. All three transition metals produced oligosaccharides ( Figure 6 ). Copper(II) chloride produced a high abundance of oligosaccharides 3-7 hexoses in length, with Hex4 being the most abundant oligosaccharide. Iron(II) sulfate also produced oligosaccharides 3-7 hexoses in length, but the abundance was 10-fold lower than that of the copper(II) chloride sample. Iron(III) sulfate produced oligosaccharides 3-10 hexoses in length with an abundance comparable to or higher than that of copper(II) chloride. This sample also produced several hexose isomers, including three Hex3 isomers and two Hex4 isomers. We found from this data that different transition metals and oxidation states can produce oligosaccharides from polysaccharides. We also found that different transition metals produced different oligosaccharide distributions, suggesting that each metal has different specificities.

[0115] Application of Fenton reaction in food polysaccharides

[0116] Millet, chickpea and banana peels were treated with FITDOG to show the types of oligosaccharides produced from different starting polysaccharides. Figure 7 Shown is the monosaccharide composition determined using the method described previously. Glucose was the most abundant component in all foods, however, for banana peel, xylose and fructose were more abundant. For chickpeas, arabinose and galactose were more abundant. Millet contained mainly glucose (95.17%) ( Figure 7 ). This indicates that the hexose oligosaccharides released from FITDOG are composed of glucose.

[0117] The results of the three polysaccharides were summarized by LC-MS chromatography ( Figure 8 ). The peaks and their oligosaccharide compositions are listed in Table 1. Each chromatogram obtained different peaks due to its respective monosaccharide composition.

[0118] Banana peel (above) mainly obtained short oligomers, including three Hex3 isomers (hexose triose A, B and S). Hex or hexose can be glucose or fructose, but is more likely to be glucose. The most abundant oligosaccharide corresponds to Hex5 (J). Hex4 isomers (F and H) were also observed. Chickpea was found to have the Hex2HexA1 category (D), with HexA corresponding to hexuronic acid. Other abundant compounds Hex4 (C, E, G), Hex5 (I), Hex6 (K), and Hex7 (N and P) were observed. Millet (below) obtained Hex4 (C, E and G), with low abundance Hex3. Hex5 was present, corresponding to I with the greatest abundance, while K corresponded to Hex6. A mixed combination was observed in L, which consisted of Hex5PentA1, where PentA represents penturonic acid.

[0119] Prebiotic Activity of Butternut Squash Oligosaccharides

[0120] Several oligosaccharides from various food polysaccharides FITDOG were tested for their prebiotic activity. Oligosaccharides from butternut squash were used as an example. The oligosaccharide profiles were similar to those obtained above. In this example, Bifidobacterium pseudocatenulatum MP80 grew on oligosaccharides produced by Fenton oxidation of butternut squash. In contrast to the other strains tested, Bifidobacterium pseudocatenulatum MP80 was able to grow on both 2% and 5% solutions of butternut squash oligosaccharides. Growth eventually reached a maximum OD of 0.962 in the 5% solution. Growth on butternut squash oligosaccharides was significantly higher than the negative control (maximum OD 0.378), but lower than the positive control ( Fig. 9 ). Since other strains tested did not grow on butternut squash oligosaccharides, this result suggests that these oligosaccharides selectively enable the growth of certain bacteria.

[0121] Table 1: Most abundant library compounds found in FITDOG treatment of several foods. "X" represents the food where the compound was found.

[0122]

[0123] Example 2

[0124] The results below show the use of the FITDOG process on complex mixtures of polysaccharides and pure polysaccharides. The complex mixture of polysaccharides included butternut squash (note the production of 3-200 oligosaccharides ( Fig.29 )). This method can be used for yeast cell wall ( Fig.28 The method can also be used to purify polysaccharides including: xylan, rye arabinoxylan, lichenan, galactomannan, gelatin starch, amylose, polyrhamnogalacturonic acid I, xyloglucan, thermogelatin, galactan, mannan, glucomannan, larch arabinoglucan, polygalacturonic acid, inulin ( Figure 10-24 and 27).

[0125] Different metals are used in the FITDOG process. They include Fe, Ni, Ca, Cu and Mg. They produce oligosaccharides with different DP distributions ( Figure 25-26 ).

[0126] Material:

[0127] Xylan, rye arabinoxylan, lichenan, galactomannan, amylose, polyrhamnogalacturonic acid I, xyloglucan, thermogellan, galactan, mannan, glucomannan, larch arabinogalactan, polygalacturonic acid and inulin were purchased from Megazyme (UK). Gum starch was purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0128] Sodium acetate, hydrogen peroxide (30% w / w), sodium hydroxide, iron (III) sulfate pentahydrate, iron (II) sulfate heptahydrate, copper (II) sulfate heptahydrate, cuprous (I) chloride, manganese (II) sulfate, cobalt (II) sulfate heptahydrate, magnesium sulfate, calcium chloride, nickel (II) chloride, and glacial acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0129] method:

[0130] Oligosaccharide production

[0131] 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 the dry polysaccharide standard to achieve 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, vortexed, and then 0.6% of the initial reaction volume of glacial acetic acid was added for neutralization.

[0132] Oligosaccharide purification

[0133] Oligosaccharides were separated using nonporous graphitized carbon core (GCC-SPE). The cartridge was washed with 80% acetonitrile dissolved in 0.1% (v / v) trifluoroacetic acid (TFA) and nanopure water. Oligosaccharides were loaded and washed with 5 column volumes of nanopure water. Oligosaccharides were eluted with 40% acetonitrile containing 0.05% (v / v) TFA.

[0134] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and those skilled in the art should understand various modifications or changes made therefrom, and they are included in the subject matter and interests of this application and the scope of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. A method for producing oligosaccharides from polysaccharides, the method comprising: reacting the polysaccharide with a metal catalyst and hydrogen peroxide in the pH range of 5-6, wherein the metal catalyst is selected from iron, copper, manganese, cobalt, calcium or magnesium to form a reaction mixture, wherein the hydrogen peroxide concentration is at least 0.02M and wherein the concentration of the metal catalyst is at least 0.65nM; then Adding a base to the reaction mixture to quench the reaction and / or cleave the glycosidic bonds in the polysaccharide, thereby producing an oligosaccharide mixture from the polysaccharide, wherein the base is selected from sodium hydroxide, potassium hydroxide or calcium hydroxide, wherein the base is added to at least 0.1M; The step of reacting the polysaccharide with hydrogen peroxide and the metal catalyst is carried out for 20 minutes or longer, and the reaction is carried out at a temperature of at least 50°C.

2. The method of claim 1, wherein the metal catalyst is selected from iron, copper, manganese, or cobalt.

3. The method of claim 1, wherein the metal catalyst is selected from calcium or magnesium.

4. The method of claim 1 or 2, wherein the concentration of the metal catalyst in the reaction mixture is 0.65 nM to 500 nM.

5. The method of claim 1, wherein the concentration of hydrogen peroxide in the reaction mixture is 0.02 M to 1 M.

6. The method of claim 1, wherein the concentration of the base is 0.1M-5.0M.

7. The method of claim 1, wherein the polysaccharide comprises one or more of the following: amylose, amylopectin, β-glucan, amylopectin, xyloglucan, arabinogalactan I and arabinogalactan II, polyrhamnogalacturonic acid I, polyrhamnogalacturonic acid II, galactan, arabinan, xylan, glycogen, mannan, thermogelatin or inulin.

8. The method of claim 1, wherein the polysaccharide comprises arabinoxylan.

9. The method of claim 1, wherein the polysaccharide comprises glucomannan.

10. The method of claim 1, wherein the polysaccharide is from a plant or animal source.

11. The method of claim 1, wherein the polysaccharide is from a bacterial, fungal or algal origin.

12. The method of claim 1, wherein the polysaccharide is in the form of plant material.

13. A method as claimed in claim 12, wherein the plant material is banana, chickpea or millet plant material.

14. The method of claim 1, further comprising purifying one or more oligosaccharides from the oligosaccharide mixture.

15. The method of claim 1, wherein prior to reacting, the method comprises contacting the polysaccharide with one or more polysaccharide degrading enzymes.

16. The method of claim 15, wherein the one or more polysaccharide degrading enzymes comprises amylase, isoamylase, cellulase, maltase, glucanase, or a combination thereof.

17. The method of claim 1, wherein the step of reacting the polysaccharide with hydrogen peroxide and the metal catalyst is reacted for 20 minutes to 120 minutes.

18. The method of claim 1, wherein the reaction mixture in the step of reacting the polysaccharide with hydrogen peroxide and the metal catalyst is carried out at a pH of 5.

19. The method of claim 1, wherein the concentration of the metal catalyst is between 0.65 nM and 65 nM.

20. The method of claim 1, wherein the oligosaccharide mixture comprises oligosaccharides having an average degree of polymerization of 3 to 20.

21. The method of claim 2, wherein the metal catalyst is Fe3+ or Fe2+.

22. The method of claim 2, wherein the metal catalyst is Cu2+.

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