Salt and sugar separation method

Abstract

A process for separating and recovering a first carbohydrate from a feed solution comprising two or more carbohydrates and a salt by using a chromatographic separation system comprising one or more strong acid cation exchange resins and recovering at least one fraction rich in the first carbohydrate.

Description

[0001] The present disclosure relates to a method for separating and purifying carbohydrates. More specifically, the present specification discloses a method for separating and purifying allulose from salts and other sugars.

[0002] The separation and purification of biomolecules such as carbohydrates remains one of the most challenging aspects of synthetic carbohydrate chemistry. The many variations in the physical and functional properties of carbohydrates complicate the development of methods for the effective analysis, separation, and purification of such carbohydrates. Some carbohydrates such as rare sugars and steviol glycosides are present in very small amounts in nature and are therefore typically prepared using, for example, fermentation or enzymatic conversion. The resulting carbohydrates need to be efficiently (in terms of time and cost) separated and recovered from other materials from their potential natural sources or from their fermentation or conversion media. For various commercial and therapeutic applications, the acceptable carbohydrate purity levels are very high (e.g., greater than 90%). A variety of chromatographic methods, including liquid chromatography separations, have attracted considerable interest due to their industrial and analytical applications.

[0003] Batch methods such as chromatography can be used to separate carbohydrates such as sugars from a feed solution by passing the feed solution through a fixed bed of an ion exchange column and then through an eluent such as deionized water. These carbohydrates are then separated from the feed solution via the eluent, which flows through a portion of the fixed resin together with the feed solution. This chromatographic separation can be carried out in a long column packed with a fixed ion exchange resin or by using a simulated moving bed (SMB). SMB can be used on an industrial scale in continuous processes for separating and / or purifying carbohydrates. Thus, compared to batch separation methods including crystallization and stepwise chromatographic separation, SMB technology offers significant economic advantages in manufacturing operations. SMB can also be used to separate chemical compounds that are difficult or impossible to separate by any other means. Considerable interest exists in using ion exchange technology to desalt carbohydrates such as sugars (i.e., separate the sugar and the salt to provide a salt-free fraction). While ion exchange technology can be used to separate salts from carbohydrates such as sugars, using ion exchange is not always effective or economically viable, especially when the salt load is too high. Therefore, in methods involving the preparation of carbohydrates such as sugars, there is a need for simple, efficient, and cost-effective separation and purification techniques to provide high-purity carbohydrates.

[0004] In one aspect, the technology disclosed in this specification relates to a method for separating and recovering a first carbohydrate from a feed solution containing two or more carbohydrates and salts, the method comprising: passing the feed solution through a chromatographic separation system comprising one or more strong acid cation exchange resins; and recovering a first fraction rich in the first carbohydrate and a second fraction containing one or more carbohydrates and salts. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Details of one or more embodiments are set forth in the accompanying drawings and the following description. Other features, aspects, and advantages of the present disclosure will become apparent from the description, the drawings, and the claims.

[0006] Figure 1 A process flow diagram depicting a method for separating and recovering one or more carbohydrate molecules in an exemplary embodiment of a rapid reaction method is shown.

[0007] Figure 2 A process flow diagram depicting an alternative method for separating and recovering one or more carbohydrate molecules in an exemplary embodiment of a rapid reaction method is shown.

[0008] Figure 3 Shows the chromatographic elution curves (Brix versus bed volume (BV)) corresponding to Example 2 when using DOWEX MONOSPHERE TM 99Ca / 310 resin, and these chromatographic elution curves show the elution curves corresponding to allulose, glucose, and fructose.

[0009] Figure 4 Shows the chromatographic elution curves corresponding to the individual components of the injection mixture of Example 2 when using DIAION TM UBK-555 99Ca / 220 resin, and these chromatographic elution curves show the elution curves corresponding to allulose, glucose, and fructose.

[0010] Figure 5A Shows the chromatographic elution curves (Brix versus bed volume (BV)) of carbohydrates corresponding to Example 3 when using fresh or feed solution-exposed ( "poisoned") DOWEX MONOSPHERE TM 99Ca / 310 resin. The curves of salts (measured as conductivity response) were also measured and plotted on the same graph ( Figure 5A ).

[0011] Figure 5B Shows the chromatographic elution curves (Brix versus bed volume (BV)) corresponding to Example 3, and these chromatographic elution curves only show fresh DOWEX MONOSPHERE TMTotal solids elution of 99Ca / 310 resin, used resin (“poisoned”), and used resin regenerated with 10% calcium chloride.

[0012] In at least one embodiment, the strong acid cation exchange resin comprises divalent cations, monovalent cations, cations balanced with the cations present in the feed solution, or combinations thereof. In at least one embodiment, the divalent cations are selected from the group consisting of: Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , and the monovalent cations are selected from the group consisting of: Na + , K + or combinations of cations with variable ratios. In at least one embodiment, the strong acid cation exchange resin is in the form of gel beads. In at least one embodiment, the strong acid cation exchange resin has a median bead diameter of 100 microns to 500 microns. In at least one embodiment, the strong acid cation exchange resin is a resin having a polystyrene matrix. In at least one embodiment, the strong acid cation exchange resin is crosslinked with divinylbenzene. In at least one embodiment, the strong acid cation exchange resin is functionalized with alkaline earth metal ions or combinations of two or more of them. In at least one embodiment, the eluent for the ion chromatography separation system is water.

[0013] In at least one embodiment, the feed solution is a cell lysis solution, which comprises an aqueous mixture of inorganic salts, biological fragments, metabolites of the cells, enzymes released by the cells, and carbohydrates. In at least one embodiment, one or more carbohydrate molecules comprise rare sugars, and the method includes recovering a fraction rich in rare sugars. In at least one embodiment, the rare sugar is allulose. In at least one embodiment, the rare sugar fraction in the feed solution has a purity of 10% to 80% based on dry solids. In at least one embodiment, based on dry solids, the purity of the recovered rare sugar fraction is at least 10% higher than the purity of the rare sugar fraction in the feed solution. In at least one embodiment, the purity of the recovered rare sugar fraction has a purity of 60% or higher based on dry solids. In at least one embodiment, the recovered rare sugar fraction has a purity of greater than 90% based on dry solids. In at least one embodiment, the method provides a rare sugar yield of at least 80%.

[0014] In at least one embodiment, the rare sugar component is separated from the components of the feed solution selected from the group consisting of sucrose, fructose, dextrose, oligomers of dextrose, fragments of RNA and / or DNA, endotoxins, amino acids, and inorganic salts.

[0015] In at least one embodiment, the chromatographic separation system includes a chromatographic method selected from the group consisting of simulated moving bed, sequential simulated moving bed, batch process, intermittent simulated moving bed, or combinations thereof. In at least one embodiment, the chromatographic separation system includes ion chromatography or sequential simulated moving bed.

[0016] In at least one embodiment, the chromatographic separation system includes a plurality of separation zones and collection zones. In at least one embodiment, the chromatographic separation system includes a regeneration zone for continuous or periodic regeneration of the resin, wherein a regeneration solution is applied to the bed. In at least one embodiment, the regeneration solution comprises NaOH, NaCl, KOH, KCl, Ca(OH) 2 or CaCl 2 .

[0017] Another aspect of many embodiments of the present invention relates to a method for separating and recovering a first carbohydrate from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a first chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with monovalent metal ions; recovering a first fraction rich in the two or more carbohydrates and a second fraction comprising salts and an admixture of carbohydrates not recovered in the first fraction; subjecting the first fraction to an ion exchange purification process to obtain a third fraction comprising two or more carbohydrates; passing the third fraction through a second chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with divalent metal ions; and recovering a fourth fraction rich in the first carbohydrate and a fifth fraction comprising one or more additional carbohydrates.

[0018] In at least one embodiment, the monovalent metal ion is selected from Na + , K + or combinations thereof. In at least one embodiment, the divalent metal ion is selected from the group consisting of Ca 2+ , Co 2+ , Mg 2+ , Mn 2+or a combination of two or more of them. In at least one embodiment, the first chromatographic separation system includes ion chromatography. In at least one embodiment, the second chromatographic separation system includes a sequential simulated moving bed method. In at least one embodiment, the first carbohydrate comprises a rare sugar, and the method includes recovering a fraction rich in rare sugar. In at least one embodiment, the rare sugar is allulose. In at least one embodiment, the method provides a rare sugar yield of at least 80%. In at least one embodiment, one or more additional carbohydrate molecules in the fifth fraction are selected from the group consisting of fructose, dextrose, and sugars having a degree of polymerization greater than 2.

[0019] Another aspect of many embodiments of the present invention relates to a method for separating and recovering allulose from a feed solution, the method comprising: passing the feed solution through a chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with Ca 2+ and recovering at least one fraction rich in allulose from the feed solution, wherein the fraction further comprises a reduced content of salts, bioderived fragments, and other sugars.

[0020] Yet another aspect of many embodiments of the present invention relates to a method for separating and recovering allulose from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a first chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with Na + recovering a first fraction rich in the two or more carbohydrates and a second fraction comprising salts; subjecting the first fraction to an ion exchange purification process to obtain a third fraction comprising two or more carbohydrates; passing the third fraction through a second chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with Ca 2+ and recovering a fourth fraction rich in allulose and a fifth fraction comprising one or more carbohydrates.

[0021] Another aspect of many embodiments of the present invention relates to a method for separating and recovering allulose from a feed solution, the method comprising: passing the feed solution through a simulated moving bed comprising one or more strongly acidic cation exchange resins functionalized with Ca 2+ and recovering at least one fraction rich in allulose and free of salts, bioderived fragments, and other sugars.

[0022] One aspect of many embodiments of the present invention relates to a chromatographic system for separating and recovering one or more carbohydrate molecules from a feed solution comprising one or more strong acid cation exchange resins, wherein the resin comprises a polystyrene matrix crosslinked with an aromatic crosslinker, and wherein the strong acid cation exchange resin is functionalized with Na + , K + , Co 2+ , Mn 2 + , Mg 2+ or Ca 2+ or a combination thereof.

[0023] Another aspect of many embodiments of the present invention relates to the use of a chromatographic system for improving the efficiency of separating and recovering carbohydrate molecules from an aqueous mixture of salts, sugars and carbohydrate molecules.

[0024] Various embodiments are described below. It should be noted that specific embodiments are not intended as an exhaustive description or as a limitation on the broader aspects discussed herein. An aspect described in connection with a particular embodiment is not necessarily limited to that embodiment and can be practiced by any other embodiment.

[0025] For purposes of clear and concise description, features may be described herein as part of the same or separate aspects or embodiments of the present technology. Those skilled in the art will understand that the scope of the present technology may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

[0026] The various embodiments of the present technology described herein relate to methods for separating, recovering and purifying various carbohydrate molecules. These methods are intended to simplify and combine multi-step methods that include separating various components, such as salts and sugars, from a carbohydrate-containing composition using ion separation, ion exchange, evaporation and simulated moving bed (SMB) techniques. As provided below, the present invention relates to a more effective and efficient method for simultaneously separating a desired carbohydrate from a complex mixture comprising salts, the desired carbohydrate and other undesired carbohydrates. The method is particularly effective because it permits the separation of high purity specific desired carbohydrates from raw materials containing contaminants such as inorganic salts, organic salts, coloring materials, disaccharides, oligosaccharides and other similar carbohydrates. In addition, it has been found that the methods according to the present technology have higher yields and lower production costs. The inventors have found that it is not necessary to use separate steps, such as ion separation, to separate salts from sugars and then use chromatographic methods to separate one sugar from other sugars. It has been found that purification by ion separation and chromatographic separation using resins can result in the separation and recovery of high purity carbohydrates even from feeds containing large amounts of inorganic salts and / or organic salts.

[0027] Aspects of the present technology relate to a method for separating and recovering one or more carbohydrate molecules from a carbohydrate-containing feed solution. The feed solution can include one or more carbohydrates and one or more salts. The method includes: passing the feed solution through an ion chromatography separation system comprising one or more strong acid cation exchange resins; and recovering at least one fraction enriched in one or more carbohydrate molecules. The method can further include: recovering a second fraction comprising salts. In addition to salts, the second fraction can further include one or more carbohydrate molecules. In various embodiments, one or more carbohydrate molecules in the second fraction are different or not identical to one or more carbohydrate molecules in the first fraction.

[0028] Suitable carbohydrates that can be separated and purified using the methods of the present technology can include: rare sugars; steviol glycosides; and saccharides, including for example but not limited to monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Non-limiting examples of suitable saccharides include for example but not limited to glucose (dextrose), fructose (levulose), galactose, sucrose, maltose, trehalose, cellobiose, chitobiose, lactose, maltodextrin, starch, and combinations thereof. Non-limiting examples of suitable steviol glycosides include for example but not limited to rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside D4, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, steviolbioside, rubusoside, other steviol glycosides found in the Stevia rebaudiana plant, and mixtures of any of the foregoing, as well as stevia extracts, glycosylated steviol glycosides, and steviol glycosides prepared by chemical, enzymatic synthesis, or fermentation by recombinant microorganisms. Non-limiting examples of oligosaccharides include for example but not limited to fructooligosaccharides, inulin, inulin oligosaccharides, maltooligosaccharides, and combinations of any of the foregoing.

[0029] In at least one embodiment, one or more carbohydrate molecules recovered in the first fraction include one or more rare sugars. Suitable rare sugars include, for example but not limited to, psicose (D - psicose), tagatose (D - tagatose), allose, apiose, melezitose, sorbose, and combinations of any of the foregoing. In at least one embodiment, the carbohydrate is psicose (also known as tagatose). Psicose differs from other sugars in that it is almost calorie - free, producing less than about 0.2% of the metabolic energy of an equal amount of sucrose and causing a negligible increase in blood glucose or insulin levels, which makes it attractive as a nutritive sweetener in various applications. Psicose is a rare sugar that is present in small amounts in jackfruit, figs, molasses, and isomerized sugars. It can also be prepared enzymatically from D - fructose using epimerase, since D - psicose is the C - 3 epimer of D - fructose. In at least one embodiment, one or more carbohydrates that are isolated and recovered are psicose, and the method includes recovering a psicose - rich fraction. Thus, the method results in the separation, purification, and recovery of psicose from both salts and sugars (e.g., fructose).

[0030] In at least one embodiment, one or more carbohydrate molecules recovered in the first fraction are rare sugars. In at least one embodiment, one or more carbohydrate molecules recovered in the first fraction are psicose. In at least one embodiment, one or more carbohydrate molecules recovered in the second fraction are different from one or more carbohydrate molecules recovered in the first fraction. In at least one embodiment, one or more carbohydrate molecules recovered in the second fraction include one or more of fructose, dextrose, and sugars having a degree of polymerization greater than 2 (DP2+).

[0031] A feed solution containing carbohydrates can be obtained from natural sources or can be produced by biosynthetic pathways such as fermentation or enzymatic methods for producing carbohydrates. For example, carbohydrates can be produced by cell-based manufacturing methods, and at the end of the process, the cell precipitate suspended in the culture medium can be lysed and collected to form the feed solution. Thus, in addition to one or more carbohydrates, the feed solution can also contain other components produced by the manufacturing process, such as carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, phosphonucleosides, diphosphonucleosides, triphosphonucleosides, organic salts and inorganic salts, borates, colored materials, organic acids and inorganic acids, bases, inorganic materials or organic materials, enzymes, solvents, buffer solutions, biological fragments or bio-derived fragments (such as nucleic acids and endotoxins), metabolites of the cells or enzymes released by the cells, toxins, water, etc. In at least one embodiment, the feed solution can be a cell lysis solution that contains an aqueous mixture of inorganic salts, biological fragments, cell metabolites, enzymes released by the cells, and carbohydrates.

[0032] The carbohydrate fraction in the feed solution can have a purity of greater than about 10%, greater than about 15%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50%, and less than about 65%, less than about 60%, less than about 55%, less than about 50%, or less than about 40% based on dry solids. In at least one embodiment, the carbohydrate fraction in the feed solution contains rare sugars. In at least one embodiment, the carbohydrate fraction in the feed solution has a purity of about 10% to about 80%, about 15% to about 70%, about 20% to about 60%, about 25% to about 50%, about 30% to about 55%, about 35% to about 45%, or about 30% to about 40%, or includes any arbitrary value among these values and / or any range between any two of these values based on dry solids. In at least one embodiment, the carbohydrate fraction in the feed solution contains a rare sugar fraction having a purity of 10% to 80% based on dry solids. In at least one embodiment, the rare sugar fraction in the feed solution has a purity of 15% to 55% based on dry solids. In at least one embodiment, the rare sugar fraction in the feed solution has a purity of 25% to 50% based on dry solids.

[0033] The methods disclosed herein increase the purity of the recovered carbohydrates such that, based on dry solids, the purity of the recovered carbohydrates (e.g., rare sugar fraction) is at least 10% higher than the purity of the carbohydrates in the feed solution. In at least one embodiment, based on dry solids, the purity of the recovered carbohydrates (e.g., rare sugar fraction) is at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% higher than the purity of the carbohydrates in the feed solution.

[0034] In Figure 1 , according to one embodiment of the present technology, a functional flowchart for separating and recovering allulose using a two-step ion separation and SSMB method is provided. The feed solution containing allulose described herein can first be subjected to membrane filtration and concentrated by evaporation. Then, ion separation is performed on the feed solution. In this step, the feed solution is desalted by passing it through a Na + or K + resin (such as AmberLite TM CR99 Na / 310 ion exchange resin available from DuPont). The resulting extract mainly contains all carbohydrates, while the raffinate mainly contains salts and optionally some carbohydrates (especially DP2+) as additives. Then, ion exchange is performed to remove the excess salts. After desalting, a sequential simulated moving bed (SSMB) separation using a column filled with one or more strong acid cation exchange resins in the Ca 2+ form, such as AmberLite TM CR99 Ca / 310 available from DuPont and DIAION TM UBK-555 (in Ca form) available from Mitsubishi Chemical, is used to separate the extract containing a mixture of allulose, carbohydrates, and optionally residual salts. This SSMB system can separate allulose from a mixture composed of two or more carbohydrates, recover an extract stream lacking other carbohydrates and rich in allulose (90% or higher), and recover a raffinate stream containing all other carbohydrates (such as dextrose, fructose, DP2+) and a large amount of residual salts (if any). The extract containing the separated allulose is concentrated by evaporation, and then subjected to activated carbon decolorization, an ion exchange purification step for refining, and a final evaporation step to obtain a liquid allulose product. The raffinate can be further processed to recover other carbohydrates. This embodiment advantageously provides separate product streams for salts, allulose, and other carbohydrates.

[0035] In Figure 2 , according to another embodiment of the present technology, a functional flowchart for separating and recovering allulose using a one-step ion separation method combining ion separation and SSMB steps is provided. The feed solution containing allulose described herein can first be subjected to membrane filtration and concentrated by evaporation. Then, the feed solution is passed through a Ca 2+ resin (such as DOWEX MONOSPHERE TM99Ca / 310) to effect ion separation. The resulting extract is rich in allulose (90% or more), while the raffinate contains a mixture of all other carbohydrates (such as dextrose, fructose, DP2+) and a large amount of salts. The extract containing the separated allulose is concentrated by evaporation, followed by decolorization with activated carbon, an ion exchange purification step for removing any residual cations and salts, and a final evaporation step to obtain a liquid allulose product. The resin can be regenerated with 10% CaCl 2 solution.

[0036] Aspects of the present technology relate to a method for separating a first carbohydrate from an aqueous feed comprising a first carbohydrate and at least one other distinct component selected from sugars and salts. The method includes passing the aqueous feed over one or more resins and collecting a portion of the aqueous phase containing the first carbohydrate that exits the resins, wherein the one or more resins include strong acid cation exchange resins.

[0037] The feed solution can contain two or more carbohydrates, including the first carbohydrate to be separated, and salts. In one embodiment, a single chromatographic separation step can be used to separate the first carbohydrate from the other distinct carbohydrates and salts. Thus, in one embodiment, a method for separating and recovering a first carbohydrate from a feed solution includes passing the feed solution through a chromatographic separation system that includes one or more strong acid cation exchange resins; and recovering a first fraction rich in the first carbohydrate and a second fraction containing one or more carbohydrates and salts.

[0038] In another embodiment, two chromatographic separation steps can be used to separate the first carbohydrate from the other carbohydrates and salts, one step for separating most of the carbohydrates from the salts and a second step for separating the first carbohydrate from the other distinct carbohydrates. Thus, in one embodiment, a method for separating and recovering a first carbohydrate from a feed solution containing two or more carbohydrates and salts includes passing the feed solution through a first chromatographic separation system that includes one or more strong acid cation exchange resins functionalized with monovalent metal ions; recovering a first fraction rich in the two or more carbohydrates and a second fraction containing salts and an admixture of the un-recovered carbohydrates from the first fraction carbohydrates; subjecting the first fraction to an ion exchange purification process to obtain a third fraction containing two or more carbohydrates; passing the third fraction through a second chromatographic separation system that includes one or more strong acid cation exchange resins functionalized with divalent metal ions; and recovering a fourth fraction rich in the first carbohydrate and a fifth fraction containing one or more carbohydrates.

[0039] A feed solution containing two or more carbohydrates (including a first carbohydrate to be separated) and salts can be passed through one or more ion chromatography separation systems that comprise one or more strong acid cation exchange resins. Alternatively, zwitterionic exchange resins can also be used. Suitable zwitterionic resins can have quaternary ammonium groups and carboxyl groups bonded to a crosslinked polystyrene backbone. Suitable resin structures can include macroporous or gel resins. In at least one embodiment, the strong acid cation exchange resin is in the form of gel beads. The strong acid cation exchange resin can have a polystyrene (PS) matrix and can have carbonyl groups or sulfonic acid groups, with hydrogen, potassium, and sodium as counterions. The strong acid cation exchange resin can be in the form of monovalent or divalent cations. In at least one embodiment, the strong acid cation exchange resin is functionalized with monovalent ions (e.g., alkali metal ions) or a combination of two or more of them. In at least one embodiment, the strong acid cation exchange resin is functionalized with divalent metal ions (e.g., alkali metal ions) or a combination of two or more of them. In certain embodiments, monovalent cations such as K + 、Na + ) can be present on the surface of the cation resin. In at least one embodiment, the cation composition is defined and balanced with the cations present in the feed solution. Thus, the strong acid cation exchange resin can be functionalized with Ca 2+ 、Co 2+ 、Na + 、K + 、Mg 2+ 、Mn n+ or a combination of cations with variable ratios. The strong acid cation exchange resin is suitable for crosslinking with aromatic crosslinking agents such as divinylbenzene (DVB), divinyltoluene, divinyldimethylbenzene, divinylnaphthalene, trivinylbenzene, divinyldiphenyl sulfone, alkylene diacrylate, and alkylene dimethacrylate). In at least one embodiment, the crosslinking agent is divinylbenzene. The DVB content can be varied to obtain the desired separation performance. The polystyrene-divinylbenzene (PS-DVB) stationary phase has a hydrophobic nature and high chemical and thermal stability. In at least one embodiment, the styrene-divinylbenzene resin is in the divalent form and is functionalized with Ca 2+ . In at least one embodiment, the calcium-functionalized resin can be used to first separate the desired carbohydrate from salts and different carbohydrates, and then an ion exchange purification step can be performed to obtain a fraction containing the purified first carbohydrate.

[0040] The strong acid cation exchange resin has a median bead diameter of from about 100 microns to about 500 microns (including but not limited to from about 150 microns to about 450 microns, from about 200 microns to about 400 microns, from about 250 microns to about 350 microns, or from about 300 microns to about 350 microns). In at least one embodiment, the strong acid cation exchange resin has a median bead diameter of from about 300 microns to about 350 microns. Non-limiting examples of representative resins include DOWEX MONOSPHERE TM 99Ca / 310, available from DuPont's AmberLite TM CR99 Na / 310 and AmberLite TM CR 99Ca / 310, and DIAION TM UBK-555 (Ca form), available from Mitsubishi Chemical. Non-limiting examples of suitable amphoteric resins include Macronet TM MN202 and DIAION TM AMP03, available from Mitsubishi Chemical.

[0041] The feed solution is introduced into the resin-packed bed, and one or more carbohydrate-rich product fractions can be recovered during the feed stage and / or one or more other stages. During the elution stage, an eluent is fed into the resin-packed bed. The feed solution and the eluent can be fed separately or simultaneously. The method can also include a recycle stage, in which substantially no feed solution or eluent is fed into the resin bed and no product is recovered. Suitable liquid eluents (such as water, glycerol, or a portion of the collected residual fraction) can be used to elute and collect one or more carbohydrate-rich fractions. In at least one embodiment, the eluent for the ion chromatography separation system is water.

[0042] Separation can be carried out using any suitable chromatographic method known in the art (including but not limited to simulated moving bed (SMB), sequential simulated moving bed (SSMB) method, batch method, intermittent simulated moving bed (ISMB) method, other proprietary chromatographic methods, or combinations thereof). In at least one embodiment, the separation is performed by the sequential simulated moving bed method. Examples of simulated moving bed separation systems that can be used for chromatographic separation are the systems described in WO2016073881 and / or the references cited therein, the entire teachings of which are incorporated herein by reference in their entirety. In a continuous SSMB system, all fluid flows are continuous and can include a feed solution and an eluent feed, a separation curve cycle, and product recovery. In a sequential SMB system, all steps are carried out in a predetermined order and can optionally be repeated. In at least one embodiment, the first chromatographic separation system is an ion chromatography system that comprises one or more strongly acidic cation exchange resins functionalized with alkali metal ions (e.g., Na + ). In at least one embodiment, the second chromatographic separation system is a sequential simulated moving bed that comprises one or more strongly acidic cation exchange resins functionalized with alkaline earth metal ions (e.g., Ca 2+ ).

[0043] The chromatographic separation system can include multiple zones, including a separation zone, a collection zone, a washing zone, an equilibration zone, and / or a regeneration zone. The chromatographic separation system can provide multiple separation zones (e.g., 3 or more separation zones) and options for collecting multiple products and by-products (e.g., 2 or more collection zones). For example, in one application, the product can be recovered from zone 1 of the chromatographic system by eluting with water, zone 2 can be used to separate the component of interest from other components, zone 3 can be dedicated to injecting the feed and simultaneously separating the component of interest from other components, and zone 4 can be used for resin regeneration or as a safety zone. Additional zones can be added to assist in resin regeneration, act as a safety zone, or be used to recover other fractions of interest (where 3 or more fractions need to be recovered). In at least one embodiment, the chromatographic separation system includes multiple separation zones and collection zones that allow the recovery of two or more fractions or streams.

[0044] A chromatographic separation system can provide one or more zones for continuous or periodic regeneration of chromatographic resin. The latter can be achieved by applying a regeneration solution to the packed bed. Depending on the chemical nature of the chromatographic medium, the solution can be a strongly basic solution, a strongly acidic solution, a chaotropic buffer, an organic solvent (e.g., ethanol), an aqueous buffer supplemented with an organic solvent, or an aqueous buffer with an ionic or non-ionic detergent. The regeneration solution enables the removal of bound fractions from the resin. The regeneration solution can be the feed solution itself, or it can be different from the feed solution and applied to the chromatographic medium separately from the feed solution. Suitable regeneration solutions can include, but are not limited to, appropriate concentrations of NaOH, Ca(OH) 2 , NaCl, KOH, KCl, and CaCl 2 or combinations thereof.

[0045] Aspects of the present technology relate to a method for improving the efficiency of separating and recovering carbohydrate molecules from an aqueous mixture of salts, sugars, and other carbohydrate molecules. In at least one embodiment, the method includes: improving the efficiency of separating and recovering allulose from an aqueous mixture of salts and other carbohydrate molecules.

[0046] In one aspect, a method for separating and recovering allulose from a feed solution is provided. The method includes: passing the feed solution through a chromatographic separation system that includes one or more strongly acidic cation exchange resins functionalized with Ca 2+ ; and recovering at least one fraction that is rich in allulose and free of salts, bio-derived fragments, and other sugars.

[0047] In another aspect, a method for separating and recovering allulose from a feed solution containing two or more carbohydrates and salts is provided. The method includes: passing the feed solution through a first chromatographic separation system that includes one or more strongly acidic cation exchange resins functionalized with Na + ; recovering a first fraction rich in the two or more carbohydrates and a second fraction containing salts and optionally residual carbohydrates not recovered in the first fraction; subjecting the first fraction to an ion exchange purification process to obtain a third fraction containing two or more carbohydrates; passing the third fraction through a second chromatographic separation system that includes one or more strongly acidic cation exchange resins functionalized with Ca 2+ ; and recovering a fourth fraction rich in allulose and a fifth fraction containing one or more carbohydrates.

[0048] At the end of the chromatographic separation process, at least one fraction rich in one or more carbohydrate molecules is recovered. The desired carbohydrate (such as, for example, a rare sugar) is separated from other carbohydrate components and non-carbohydrate components, which other carbohydrate components and non-carbohydrate components include but are not limited to sucrose, fructose, glucose, dextrose, oligomers of dextrose, fragments of RNA and / or DNA, endotoxins, amino acids, inorganic salts, and combinations thereof. In at least one embodiment, the recovered carbohydrate includes a rare sugar selected from psicose (D - psicose), tagatose (D - tagatose), allose, apiose, melezitose, sorbose, or combinations thereof. In at least one embodiment, one or more carbohydrate molecules include psicose, and the method includes recovering a fraction rich in psicose. In at least one embodiment, the recovered psicose is separated from one or more of glucose, fructose, dextrose, sugars having a degree of polymerization greater than 2 (DP2+), RNA and / or DNA fragments, endotoxins, amino acids, inorganic salts, and combinations thereof. In at least one embodiment, the recovered psicose is separated from salts, DP2+, dextrose, and fructose.

[0049] The recovered first carbohydrate fraction can be subjected to further processing steps including, but not limited to, ion exchange purification, evaporation, and decolorization. The first carbohydrate thus obtained can have a purity of greater than about 60% or higher based on dry solids. In at least one embodiment, the recovered first carbohydrate comprises a recovered rare sugar having a purity of about 60% or higher. This includes a purity of about 65% or higher, about 70% or higher, about 75% or higher, about 80% or higher, about 85% or higher, about 90% or higher, about 95% or higher, or about 98% or higher based on dry solids. In at least one embodiment, the recovered first carbohydrate has a purity of about 90% or higher, about 91% or higher, about 92% or higher, about 93% or higher, about 94% or higher, about 95% or higher, about 96% or higher, about 97% or higher, about 98% or higher, or about 99% or higher based on dry solids. In at least one embodiment, the first carbohydrate has a purity of about 90% to about 99%, about 90% to about 98%, about 90% to about 97%, about 90% to about 96%, or about 90% to about 95%, or includes any two of these values and / or any range between any two of these values based on dry solids. In at least one embodiment, the rare sugar has a purity of 90% to 99% based on dry solids. In at least one embodiment, based on dry solids, the purity of the first carbohydrate is at least 10% higher than the purity of the carbohydrate in the feed solution. In at least one embodiment, the first carbohydrate is allulose having a purity of 90% to 99% based on dry solids. The final carbohydrate solution has an allulose concentration of 70 degrees Brix or higher (e.g., 75 degrees Brix or higher, 80 degrees Brix or higher, or 85 degrees Brix or higher). In one or more embodiments, the final carbohydrate solution has an allulose concentration of 70 degrees Brix to 95 degrees Brix (e.g., 75 degrees Brix to 90 degrees Brix or 80 degrees Brix to 85 degrees Brix).

[0050] The recovered carbohydrate-rich fraction can be used as is or further processed to obtain one or more carbohydrates in solid or liquid form and having a desired purity. The recovered carbohydrates can optionally be further purified using known methods such as chromatographic purification, crystallization, ultrafiltration, and nanofiltration, etc., or combinations thereof.

[0051] The method can provide a carbohydrate yield of at least 80% (such as at least 85%, at least 90%, or at least 95%). In at least one embodiment, the method provides a yield of about 80% to about 99% (such as about 85% to about 95%, or about 95% to about 98%, or includes any two of these values and / or any range between any two of these values).

[0052] The method may optionally include one or more additional processing steps after separation and recovery. The one or more post-treatment steps may include, but are not limited to, carbon treatment, evaporation, and ion exchange purification. Thus, one or more of these post-treatment steps (including evaporation, carbon treatment, and ion exchange purification) may be further performed on the recovered carbohydrate-rich fraction. Ion exchange purification may employ the use of strongly acidic cation exchange resins or weakly basic anion exchange resins, such as PPC150SH, A510SMBPLUS, and A133S, AmberLite TM IRA96, _AmberLite TM FPC88MB, and AmberLite TM FPA66, etc.

[0053] Aspects of the present technology relate to a method for separating and recovering allulose from a feed solution. The method may include: contacting the feed solution with a suitable resin using ion chromatography or simulated moving bed, wherein the resin comprises one or more strongly acidic cation exchange resins functionalized with Ca 2+ , Co 2+ , Na + , K + , Mg 2+ , Mn n+ (e.g., Mn 2+ ) or a combination of cations with variable ratios. In at least one embodiment, the strongly acidic cation exchange resin is functionalized with alkaline earth metal ions or a combination of two or more of them. In at least one embodiment, the strongly acidic cation exchange resin is in the divalent cation form. The method may further include: recovering at least one fraction rich in allulose and free of salts, bio-derived fragments, and other sugars.

[0054] In another aspect, provided is a chromatographic system for separating and recovering one or more carbohydrate molecules from a feed solution. The chromatographic system comprises one or more strongly acidic cation exchange resins, wherein the resin comprises a polystyrene matrix crosslinked with an aromatic crosslinker, and wherein the strongly acidic cation exchange resin is in the form of Ca 2+ . The chromatographic system is used to improve the efficiency of separating and recovering carbohydrate molecules from an aqueous mixture of salts, sugars, and carbohydrate molecules.

[0055] The methods and apparatuses disclosed herein advantageously provide for the separation and purification of carbohydrates, such as allulose, not only from other carbohydrates, such as fructose, glucose, and oligomers (DP2+), but also from salts, such as inorganic salts, small charged biomolecules, and phosphates of carbohydrates. The method is even effective for separating complex mixtures of carbohydrates and large amounts of salts, which are 2-fold, 5-fold, or even 10-fold more than classical mixtures, where conventional methods, such as ion exchange and SSMB, prove ineffective. When large amounts of salts are present, the purification process typically requires more advanced desalination processes (e.g., capacitive deionization (CDI)). Figure 1 and Figure 2 The ion separation / SSMB one-step or two-step method depicted in Figure 1 and described herein for separating carbohydrates, such as allulose, from any other substances is superior to multi-step methods that require ion separation, evaporation, and chromatographic separation, as it reduces costs, time, and complexity, and increases yield and purity. The method also provides a sidestream containing valuable carbohydrates as well as salts and biomolecules. The sidestream can have added value by being recycled back into the process or used in other processes (e.g., fermentation).

[0056] The following terms are used throughout the disclosure and are defined as follows.

[0057] Strong acid cation exchange resins are known in the industry and are available from different suppliers. Without limiting the full scope of the term as understood in the art, strong acid cation exchange resins are generally products that contain a moiety that acts as a strong acid in operation. Although not intended to limit the full scope of the term, generally this moiety is a sulfonic acid group.

[0058] As used herein and in the appended claims, unless otherwise indicated herein or clearly contradicted by context, singular forms such as "a," "an," "the," and similar referents in the context of describing an element (especially in the context of the following claims) are to be understood to cover both the singular and the plural. Unless otherwise indicated herein, the recitation of a range of values herein is merely intended to be a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were recited herein individually. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. Unless otherwise stated, the use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the embodiments and does not pose a limitation on the scope of the claims. Any language in this specification should not be construed as indicating any non-claimed element as essential.

[0059] The embodiments described illustratively herein may be suitably practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. should be read expansively and not restrictively. Additionally, the terms and expressions used herein have been used as terms of description and not of limitation, and are not intended to exclude any equivalents or portions of the features shown and described, but it should be recognized that various modifications are possible within the scope of the technology claimed herein. Additionally, the phrase "consisting essentially of" will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel features of the technology claimed herein. The phrase "consisting of" excludes any element not specified. The recitation "including" means "including but not limited to." Thus, there may be other materials, additives, carriers or steps not mentioned. Unless otherwise stated, "a" or "an" means one or more.

[0060] Unless otherwise indicated, all numbers expressing quantities of properties, parameters, conditions, etc. used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. Any numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When used before a numerical designation (e.g., temperature, time, amount, and concentration, including ranges), the term "about" denotes an approximation that may vary by (+) or (-) 10%, 5%, or 1%.

[0061] Unless otherwise specified, all percentages specified herein are by weight.

[0062] As will be understood by those skilled in the art, for any and all purposes, particularly in the context of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be readily identified as being fully described and such that the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. Those skilled in the art will also understand all language such as "at most," "at least," "greater than," "less than," etc., including the recited numbers and refer to ranges that can then be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual member.

[0063] The technology described in this specification can be better understood with reference to the following aspects, which are illustrative and not intended to limit the full scope of the invention disclosed in this specification.

[0064] 1. A method for separating and recovering a first carbohydrate from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a chromatographic separation system comprising one or more strong acid cation exchange resins; and recovering a first fraction rich in the first carbohydrate and a second fraction comprising one or more carbohydrates and salts.

[0065] 2. The method according to claim 1, wherein the strong acid cation exchange resin comprises divalent cations, monovalent cations, and cations balanced with the cations present in the feed solution, or combinations thereof.

[0066] 3. The method according to claim 1 or claim 2, wherein the divalent cations are selected from the group consisting of: Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ or combinations of two or more of them; and the monovalent cations are selected from the group consisting of:

[0067] Na + , K + or combinations thereof.

[0068] 4. The method according to claim 3, wherein the strong acid cation exchange resin is in the form of gel beads.

[0069] 5. The method according to any one of the preceding claims, wherein the strong acid cation exchange resin has a median bead diameter of 100 microns to 500 microns.

[0070] 6. The method according to any one of the preceding claims, wherein the strong acid cation exchange resin is a resin having a polystyrene matrix.

[0071] 7. The method according to claim 6, wherein the strong acid cation exchange resin is crosslinked with divinylbenzene.

[0072] 8. The method according to any one of the preceding claims, wherein the feed solution is a cell lysate solution, the cell lysate solution comprising an aqueous mixture of inorganic salts, biological fragments, metabolites of cells, enzymes released by cells, and carbohydrates.

[0073] 9. The method according to any one of the preceding claims, wherein the first carbohydrate comprises rare sugars, and the method comprises recovering a fraction rich in rare sugars.

[0074] 10. The method according to claim 9, wherein the rare sugar is allulose.

[0075] 11. The method according to claim 10, wherein the rare sugar fraction in the feed solution has a purity of 10% to 80% based on dry solids.

[0076] 12. The method according to any one of claims 9 to 11, wherein the purity of the recovered rare sugar fraction is at least 10% higher than the purity of the rare sugar fraction in the feed solution, based on dry solids.

[0077] 13. The method according to any one of claims 9 to 12, wherein the recovered rare sugar fraction has a purity of 60% or higher based on dry solids.

[0078] 14. The method according to claim 13, wherein the recovered carbohydrate fraction has a purity of greater than 90% based on dry solids.

[0079] 15. The method according to any one of claims 9 to 14, wherein the method provides a rare sugar yield of at least 80%.

[0080] 16. The method according to any one of claims 9 to 15, wherein the rare sugar is separated from a feed solution comprising sucrose, fructose, dextran, oligomers of dextran, fragments of RNA and / or DNA, endotoxins, amino acids, and inorganic salts.

[0081] 17. The method according to claim 1, wherein the one or more carbohydrate molecules in the second fraction are selected from the group consisting of fructose, dextran, and sugars having a degree of polymerization greater than 2.

[0082] 18. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises a chromatographic method selected from the group consisting of ion chromatography, simulated moving bed, sequential simulated moving bed, batch method, intermittent simulated moving bed, or a combination thereof or two or more of them.

[0083] 19. The method according to claim 18, wherein the chromatographic separation system comprises ion chromatography or sequential simulated moving bed.

[0084] 20. The method according to claim 19, further comprising ion exchange purification.

[0085] 21. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises a plurality of separation zones and collection zones configured to allow the recovery of two or more fractions.

[0086] 22. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises a regeneration zone for continuous or periodic regeneration of the resin, wherein a regeneration solution is applied to the bed.

[0087] 23. The method according to claim 22, wherein the regeneration solution comprises NaOH, NaCl, KOH, KCl, Ca(OH) 2 or CaCl 2 .

[0088] 24. A method for separating and recovering a first carbohydrate from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a first chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with monovalent metal ions; recovering a first fraction rich in the two or more carbohydrates and a second fraction comprising salts; subjecting the first fraction to an ion exchange purification process to obtain a third fraction comprising two or more carbohydrates; passing the third fraction through a second chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with divalent metal ions; and recovering a fourth fraction rich in the first carbohydrate and a fifth fraction comprising one or more carbohydrates.

[0089] 25. The method according to claim 24, wherein the monovalent metal ion is selected from Na + , K + or a combination thereof.

[0090] 26. The method according to claim 24, wherein the divalent metal ion is selected from the group consisting of: Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ or a combination of two or more of them.

[0091] 27. The method according to any one of claims 24 to 26, wherein the first chromatographic separation system comprises ion chromatography.

[0092] 28. The method according to any one of claims 24 to 27, wherein the second chromatographic separation system comprises a sequential simulated moving bed method.

[0093] 29. The method according to any one of claims 24 to 28, wherein the first carbohydrate comprises rare sugars, and the method comprises recovering a fraction rich in rare sugars.

[0094] 30. The method according to claim 29, wherein the rare sugar is psicose.

[0095] 31. A method according to any one of claims 24 to 30, wherein the method provides a rare sugar yield of at least 80%.

[0096] 32. A method according to any one of claims 24 to 31, wherein the one or more carbohydrate molecules in the fifth fraction are selected from the group consisting of fructose, dextrose, and sugars having a degree of polymerization greater than 2.

[0097] 33. A method for separating and recovering allulose from a feed solution, the method comprising: passing the feed solution through a chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with Ca 2+ ; and recovering at least one fraction rich in allulose and having a reduced content of salts, bioderived fragments, and other sugars.

[0098] 34. A method for separating and recovering allulose from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a first chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with Na + ; recovering a first fraction rich in the two or more carbohydrates and a second fraction comprising salts; subjecting the first fraction to an ion exchange purification process to obtain a third fraction comprising two or more carbohydrates; passing the third fraction through a second chromatographic separation system comprising one or more strongly acidic cation exchange resins functionalized with Ca 2+ ; and recovering a fourth fraction rich in allulose and a fifth fraction comprising one or more carbohydrates.

[0099] 35. A method according to claim 33 or claim 34, wherein the feed solution is a cell lysate solution comprising an aqueous mixture of inorganic salts, bioderived fragments, metabolites of the cells, enzymes released by the cells, and carbohydrates.

[0100] 36. A method according to claim 34, wherein the one or more carbohydrate molecules in the fifth fraction are selected from the group consisting of fructose, dextrose, and sugars having a degree of polymerization greater than 2.

[0101] 37. A chromatographic system for separating and recovering one or more carbohydrate molecules from a feed solution comprising two or more carbohydrates and salts, the system comprising one or more strongly acidic cation exchange resins, wherein the resin comprises a polystyrene matrix crosslinked with an aromatic crosslinker, and wherein the strongly acidic cation exchange resin is functionalized with Na+ , K + , Co 2+ , Mn 2+ , Mg 2+ or Ca 2+ or functionalized by their combination.

[0102] 38. A method for separating and recovering a first carbohydrate from a feed solution containing two or more carbohydrates and salts, the method comprising: passing the feed solution through a chromatographic separation system comprising one or more strong acid cation exchange resins to separate a first fraction rich in the first carbohydrate and a second fraction containing a second carbohydrate and one or more salts from the feedstock; and recovering the first fraction by applying steps selected from the group consisting of evaporation, decolorization, purification, and mixtures thereof, wherein the first carbohydrate is a rare sugar.

[0103] 39. The method according to claim 38, wherein the strong acid cation exchange resin comprises divalent cations, monovalent cations, and cations balanced with the cations present in the feed solution, or combinations thereof.

[0104] 40. The method according to any one of the preceding claims, wherein the strong acid cation exchange resin is crosslinked with divinylbenzene.

[0105] 41. The method according to any one of the preceding claims, wherein the feed solution is a cell lysate solution, the cell lysate solution comprising an aqueous mixture of inorganic salts, biological fragments, metabolites of the cells, enzymes released by the cells, and carbohydrates.

[0106] 42. The method according to any one of the preceding claims, wherein the rare sugar is allulose.

[0107] 43. The method according to any one of the preceding claims, wherein the recovered rare sugar fraction has a purity of 60% or higher or greater than 90% based on dry solids.

[0108] 44. The method according to any one of the preceding claims, wherein the method provides a rare sugar yield of at least 80%.

[0109] 45. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises ion chromatography.

[0110] 46. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises a plurality of separation zones and collection zones configured to allow the recovery of two or more fractions.

[0111] 47. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises a regeneration zone for continuous or periodic regeneration of the resin, and the method further comprises the step of applying a regeneration solution to regenerate the resin.

[0112] 48. A method for separating and recovering a first carbohydrate from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a chromatographic separation system comprising means for separating a first fraction rich in the first carbohydrate from a second fraction comprising one or more carbohydrates and salts; and recovering the first fraction by applying steps selected from the group consisting of evaporation, decolorization, purification, and mixtures thereof, wherein the first carbohydrate is a rare sugar.

[0113] 49. The method according to claim 48, wherein the rare sugar is allulose.

[0114] 50. The method according to claim 48 or 49, wherein the recovered rare sugar fraction has a purity of 60% or higher or greater than 90% based on dry solids.

[0115] 51. The method according to any one of claims 48 or 50, wherein the method provides a rare sugar yield of at least 80%.

[0116] 52. The method according to any one of claims 48 to 51, wherein the means for separating a first fraction rich in the first carbohydrate from a second fraction comprising one or more carbohydrates and salts comprises a strong acid cation exchange resin.

[0117] 53. The method according to any one of claims 48 to 52, wherein the chromatographic separation system further comprises a regeneration zone for continuous or periodic regeneration of the resin, and the method further comprises the step of applying a regeneration solution to regenerate the resin.

[0118] 54. The method according to any one of claims 52 or 53, wherein the strong acid cation exchange resin is crosslinked with divinylbenzene.

[0119] 55. The method according to claims 48 to 54, wherein the feed solution is a cell lysate solution comprising an aqueous mixture of inorganic salts, biological fragments, metabolites of the cells, enzymes released by the cells, and carbohydrates.

[0120] 56. The method according to any one of the preceding claims, wherein the one or more carbohydrate molecules in the second fraction are selected from the group consisting of fructose, dextrose, and sugars having a degree of polymerization greater than 2.

[0121] 57. The method according to claim 40 or 54, wherein the regeneration solution comprises NaOH, NaCl, KOH, KCl, Ca(OH) 2 or CaCl 2 .

[0122] The various embodiments will be further illustrated by the following examples, which are in no way intended to limit the present disclosure thereto. Examples

[0123] Example 1 - Pulse Test

[0124] To test the feasibility of chromatographic separation of allulose from salts and other carbohydrates, a pulse test experiment was performed. Chromatographic pulse tests were carried out using the test conditions summarized in Table 2.

[0125] Table 2: Chromatographic Pulse Test Parameters

[0126]

[0127] Selected cationic resins and the amphoteric resin DOWEX MONOSPHERE were pulse tested with ~52% DS syrup TM 99Ca / 310, Macronet TM MN202, DIAION TM UBK-555 (Ca form) and DIAION TM AMP03. All resins showed signs of sugar-salt separation. The 99Na / 310 resin allowed sugar-salt separation, and the 99Ca / 310 resin allowed separation of allulose from salts, DP2+, dextrose, and fructose.

[0128] Heat the sample resin listed above to 45 °C and preheat the injected feed to 45 °C. Pump water through the column at the tested flow rate (~2.5 BV / hr). To start the test, inject 0.067 BV (10 mL) of the heated 52°Bx feed solution into the column. Immediately start flowing the eluent (DI water at 45 °C) after injection. After 0.3 BV (45 mL DI water) of elution, start collecting samples. Manually collect samples in 15 mL to 20 mL portions (accurately measured using an analytical balance). Analyze the total solid concentration of each fraction using a calibrated handheld Brixmeter and analyze the conductivity of each fraction using a conductivity meter (Traceable, model 89094-958). Analyze the concentration of carbohydrates (except DP2+) in selected fractions with solid content above zero using LC / MS. Based on the analysis results, plot a pulse test curve graph with the bed volume on the x-axis (calculated from the fraction number and the volume collected up to that point) and the response (Brix or conductivity) on the y-axis. Figure 3 Illustrates the pulse test results of DOWEX MONOSPHERE TM 99Ca / 310. The results show that the 99Ca / 310 resin can separate allulose from other sugars and salts; that is, the peaks of conductivity overlap with fructose, glucose, and DP2+ (not shown in the graph), while allulose elutes at a longer residence time. Figure 4 Shows that allulose can be separated from glucose and fructose using the UBK-555 99Ca / 220 μm resin.

[0129] Example 2 - Regeneration Test of 99Ca / 310 Resin

[0130] The feed solution contains approximately ~15 g / L of different salts, including salts of calcium, cobalt, magnesium, manganese, sodium, and potassium, etc., where the counterions are represented by chloride ions, phosphate ions, nitrate ions, etc. Charged organic molecules (such as organic phosphates, phospholipids, nucleic acid fragments) can be part of the fermentation broth. Therefore, it is naturally expected that the resin (e.g., 99Ca / 310) can partially reduce its original separation efficiency.

[0131] To test whether the resin will lose its separation efficiency when processing ~20 BV of the feed solution through the resin bed during operation. Repeat a pulse test similar to the one described in Example #1. After that, regenerate the resin with 4 BV of a 10% w / w CaCl 2 deionized aqueous solution. The test results are as shown in Figure 5A and Figure 5B shown.

[0132] Figure 5AThe curves in [graph] show the elution curves (total solids and conductivity) of the feed eluted from fresh (fr.), poisoned (pois. and pois.rep.), and regenerated (regen.) 99Ca / 310 resin. Figure 5B The graph in [graph] shows only the total solids elution curves of fresh, poisoned, and regenerated resins. As can be seen from the two graphs, fresh, poisoned, and regenerated resins show similarities, showing two distinct elution peaks. Based on Figure 3 the results presented, the first peak can be related to the elution of salts, DP2+, glucose, and fructose. The second peak can be related only to the elution of psicose.

Claims

1. A method for separating and recovering a first carbohydrate from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a chromatographic separation system comprising one or more strong acid cation exchange resins to separate a first fraction rich in the first carbohydrate and a second fraction comprising a second carbohydrate and one or more salts from the feedstock; and recovering the first fraction by applying steps selected from the group consisting of evaporation, decolorization, purification, and mixtures thereof wherein the first carbohydrate is a rare sugar.

2. The method according to claim 1, wherein the strong acid cation exchange resin comprises divalent cations, monovalent cations, and cations balanced with the cations present in the feed solution, or combinations thereof.

3. The method according to any one of the preceding claims, wherein the strong acid cation exchange resin is crosslinked with divinylbenzene.

4. The method according to any one of the preceding claims, wherein the feed solution is a cell lysate solution comprising an aqueous mixture of inorganic salts, biological fragments, metabolites of the cells, enzymes released by the cells, and carbohydrates.

5. The method according to any one of the preceding claims, wherein the rare sugar is allulose.

6. The method according to any one of the preceding claims, wherein the recovered rare sugar fraction has a purity of 60% or higher or greater than 90% based on dry solids.

7. The method according to any one of the preceding claims, wherein the method provides a rare sugar yield of at least 80%.

8. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises ion chromatography.

9. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises a plurality of separation zones and collection zones configured to allow the recovery of two or more fractions.

10. The method according to any one of the preceding claims, wherein the chromatographic separation system comprises a regeneration zone for continuous or periodic regeneration of the resin, and the method further comprises the step of applying a regeneration solution to regenerate the resin.

11. A method for separating and recovering a first carbohydrate from a feed solution comprising two or more carbohydrates and salts, the method comprising: passing the feed solution through a chromatographic separation system comprising means for separating a first fraction rich in the first carbohydrate from a second fraction comprising one or more carbohydrates and salts; and recovering the first fraction by applying steps selected from the group consisting of evaporation, decolorization, purification, and mixtures thereof wherein the first carbohydrate is a rare sugar.

12. The method according to claim 11, wherein the rare sugar is allulose.

13. The method according to claim 11 or 12, wherein the recovered rare sugar fraction has a purity of 60% or higher or greater than 90% based on dry solids.

14. The method according to any one of claims 11 or 13, wherein the method provides a rare sugar yield of at least 80%.

15. The method according to any one of claims 11 to 14, wherein the means for separating the first fraction rich in the first carbohydrate from the second fraction comprising one or more carbohydrates and salts comprises a strong acid cation exchange resin.

16. The method according to any one of claims 11 to 15, wherein the chromatographic separation system further comprises a regeneration zone for continuous or periodic regeneration of the resin, and the method further comprises the step of applying a regeneration solution to regenerate the resin.

17. The method according to any one of claims 15 or 16, wherein the strong acid cation exchange resin is crosslinked with divinylbenzene.

18. The method according to claims 11 to 17, wherein the feed solution is a cell lysate solution, the cell lysate solution comprising an aqueous mixture of inorganic salts, biological fragments, metabolites of the cells, enzymes released by the cells, and carbohydrates.

19. The method according to any one of the preceding claims, wherein the one or more carbohydrate molecules in the second fraction are selected from the group consisting of fructose, dextrose, and sugars having a degree of polymerization greater than 2.

20. The method according to claim 10 or 16, wherein the regeneration solution comprises NaOH, NaCl, KOH, KCl, Ca(OH) 2 or CaCl 2 .

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