Process for making allulose without ion exchange prior to chromatography separation

Abstract

The technology disclosed in this specification pertains to methods of obtaining a rare from a source solution comprising a source sugar. The method uses a single ionic moiety that can be removed from a mixed sugar solution comprising the source sugar and rare sugar without use of ion exchange before chromatographic separation.

Description

PROCESS FOR MAKING ALLULOSE WITHOUT ION EXCHANGE PRIOR TO CHROMATOGRAPHY SEPARATION

[0001] Disclosed herein is a process for making allulose. Advantageously, the disclosed processes allows for simplification of allulose manufacturing processes by eliminating steps and equipment necessary for reducing conductivity prior to chromatographic enrichment of allulose .

[0002] Rare sugars are naturally occurring epimers of more common sugar molecules like glucose and fructose. Rare sugars are of interest commercially. They are naturally occurring, can provide sweetness like glucose and fructose. But are not digested by humans in the same way; so rare sugars generally provide fewer calories pure unit of use.

[0003] Rare sugars can be obtained from a solution of source sugars using bioconversion methods followed by various costly and complex separation steps to obtain a purified rare sugar solution. For example, it is commonly necessary to separate the rare sugar from unconverted sources sugars or other occurring unwanted sugars arising from the bioconversion process. As another example, bioconversion systems commonly require use of various co-factors or pH control chemicals or chemical stabilizing agents to support the enzymes used, which also must be removed to purify the rare sugar solution. But the unwanted sugars and other chemical are removed using different technologies. The rare sugars are separated from the source sugars using a chromatography process. Co-factors, pH control chemicals and stabilizing chemicals, which are commonly ions, however, are removed using ion exchange systems, which are placed before chromatography systems to protect chromatography resins from becoming “poisoned” by the chemicals. An illustrative system is provided in WO2024086623.

[0004] In one aspect the technology disclosed in this specification is a simplified method for making rare sugars that can eliminate the need an ion exchange system prior to chromatographic separation. The method converts a source sugar to a rare sugar using an enzyme without the use of a co-factor or other supporting ion or molecule. Enzymes, preferably epimerase enzymes, capable of converting a source sugar to a rare sugar without the use of cofactors.

[0005] The technology disclosed in this specification can be best understood with reference to the following figures, which are provided illustrative purposes and are not intended to limit the full scope of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0006] Figure 1 is a graph showing changes over time of fructose and allulose content in an aqueous solution comprising an epimerase that does not require use of cofactors or other supporting ions.

[0007] Figure 2 is a graph showing the in content of various sugars and the conductivity of a aqueous solutions comprising allulose, other sugars, and calcium ions as volumes of the solution are passed through a calcium type chromatographic separation resin.

[0008] In any embodiment, this specification describes a method of making a rare sugar comprising: a) adjusting, using an ionic agent, the pH of a source solution comprising a source sugar b) contacting the source sugar with an enzyme to convert at least a part of the source sugar to a rare sugar c) separating the rare sugar from the source sugar in a chromatographic purification system comprising a resin of the same ionic type as the ionic agent and d) recovering the rare sugar as a syrup wherein the method does not use an ion exchange system prior to the chromatographic purification system; and wherein the method does not comprise adding additional ionic moieties other than the ionic agent.[00091 For any method described in the specification, any suitable ionic agent may be used provided that the agent is of the same ionic type as the resin used in the chromatographic purification system. A preferred agent comprises calcium ions, or Ca2+ions. A preferred ionic agent is calcium oxide (CaO) or calcium hydroxide (Ca(OH)2). Accordingly, in preferred embodiments the resin used in the chromatographic system is a calcium type resin. Additionally, in any embodiment this specification describes a method further comprising maintaining the pH of the source solution during the conversion by adding further amounts of the ionic agent as necessary to maintain the pH.

[0010] Within this specification rare sugars cover all rare sugars including but not limited to D-psicose (also called allulose), D-allose and D-altrose, D-tagatose, D-talose, D-sorbose, D- gulose, and D-idose. Preferred rare sugars obtainable by the method include but are not limited to allulose, allose, and tagatose. In at least some embodiments the rare sugar obtainable from the methods described in this specification is allulose (D-allulose). Accordingly, source sugars useful in the method include fructose or glucose. Depending on the method source sugars may be provided as fructose or glucose or may be provided as an oligosaccharide or polysaccharide. In such cases enzymes in addition to an epimerase may be needed to digest the polysaccharide or oligosaccharide to make glucose or fructose available or to convert fructose to glucose orglucose to fructose. In all such cases the enzymes are chosen such that the method does not comprise adding additional ionic moieties other than the ionic agent.

[0011] In at least some preferred embodiments, the source sugar is contacted with only one type of enzyme, preferably wherein the enzyme is an epimerase. In such embodiments, the source sugar is preferably fructose or glucose and more preferably is fructose.

[0012] In any embodiment described in this specification the pH of a source solution is adjusted to a favorable pH for converting the source sugar to the rare sugar. In any embodiment described in this specification the enzyme chosen converts a source sugar to a rare sugar most efficiently at a pH near neutral. In any embodiment, this specification describes a method wherein the source solution is adjusted to and maintained at a pH from 4 to 8 or from 5.5 to 7.5 or from 6 to 7.

[0013] In any embodiment of the methods described in this specification the source solution has a dry solids content from 40% to 80% (wt.%) or to 75% or to 70% or to 65% or to 60%, or from 45% to 80% (wt.%) or to 75%, or to 70%, or to 65%, or to 60%, or 55% to 80%, or to 75%, or to 70%, or to 65%, or 60%.

[0014] In any embodiment described in this specification a source solution is adjusted to a temperature favorable for converting the source sugar to the rare sugar. In any embodiment described in the method further comprising heating the source solution to a temperature from 30°, or from 35° or from 40° or from 45° to 70 °C, or from 30°, or from 35° or from 40° or from 45° to 65 °C. In any embodiment described in this specification the enzyme used to convert the source sugar to the rare sugar optimally converts the rare sugar at a temperature from 30°, or from 35° or from 40° or from 45° to 70° C, or from 30°, or from 35° or from 40° or from 45° to 65° C.

[0015] In any embodiment, this specification describes a method wherein an enzyme, preferably an epimerase enzyme is contacted with the sugar source in an amount from 0.01% to 0.5% on dry solid basis of substrate.

[0016] In any embodiment of the methods described in this specification, the chromatography step allows for recovery of a rare sugar syrup having high rare sugar content relative to all other solids in the syrup. In any embodiment of the methods described in this specification, the rare sugar syrup has rare sugar solids content greater than 90% (wt.%) or greater than 95% or greater than 97%, or greater than 98%. Additionally, in any embodimentthe methods described in this specification result in a syrup having high solids content. In any embodiment of the methods described in this specification the rare sugar syrup has dry solids content from 70% to 80% or from 70% to 75%.

[0017] Following chromatographic separation the rare sugar syrup can be subjected to any one of several conventional steps. For example, the syrup can be contacted with activated charcoal which can remove color or flavor. As another example, the syrups can be concentrated via evaporation using heat or vacuum in one or more steps, such as before and after contacting the syrup with carbon. As another example the rare sugar syrup can be contacted with an ion exchange system after chromatographic separation to remove any remaining ions from the ionic that were not separated during chromatographic separation.

[0018] This specification uses the term source sugar broadly. In some preferred embodiments a source sugar is fructose or glucose, most preferably fructose. Within this specification source sugar also includes disaccharides (maltose, isomaltose, sucrose), oligosaccharides and polysaccharides.

[0019] Use of “about” to modify a number is meant to include the number recited plus or minus 10%. Where legally permissible recitation of a value in a claim means about the value. Use of about in a claim or in the specification is not intended to limit the full scope of covered equivalents.

[0020] Recitation of the indefinite article “a” or the definite article “the” is meant to mean one or more unless the context clearly dictates otherwise.

[0021] While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the methods, and of the present technology. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed regarding any or all the other aspects and embodiments.

[0022] The present technology is also not to be limited in terms of the aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumeratedherein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to methods, conjugates, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. It is also to be understood that the terminology used herein is for the purpose of describing aspects only and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof. No language in the specification should be construed as indicating any non-claimed element as essential.

[0023] The embodiments illustratively described herein may suitably be 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. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. 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 characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0024] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the technology. This includes the generic description of the technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether the excised material is specifically recited herein.

[0025] As will be understood by one skilled in the art, for any and all purposes, particularly in terms 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 easilyrecognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member, and each separate value is incorporated into the specification as if it were individually recited herein.[0026| The technology disclose in this specification can be better understood with reference to the following aspects, which are for illustrative purposes and are not intended to limit the full scope of the technology described.

[0027] 1. A method of making a rare sugar comprising : a) adjusting, using an ionic agent, the pH of a source solution comprising a source sugar b) contacting the source sugar with an enzyme or series of enzymes to convert at least a part of the source sugar to a rare sugar c) separating the rare sugar from the source sugar in a chromatographic purification system comprising a resin of the same ionic type as the ionic agent and d) recovering the rare sugar as a syrup wherein the method does not use an ion exchange system prior to the chromatographic purification system; and wherein the method does not comprise adding additional ionic moieties other than the ionic agent.

[0028] 2. The method of claim 1 wherein the rare sugar is selected from the group consisting of allulose, allose, and tagatose.10029] 3. The method of claim 1 or 2 wherein the source sugar is fructose or glucose, wherein the source sugar is preferably fructose

[0030] 4. The method of any one of claims 1 to 3wherein the ionic agent comprises a calcium ion

[0031] 5. The method of any one of claims 1 to 4 wherein the ionic agent is calcium hydroxide and / or calcium oxide.

[0032] 6. Method of any one of claims 1 to 5 wherein the source solution is maintained at a pH from 4 to 8 or from 5.5 to 7.5 or from 6 to 7.

[0033] 7. The method of any one of claims 1 to 6 wherein the source solution has a dry solid content from 40% to 80% (wt.%) or to 75% or to 70% or to 65% or to 60%, or from45% to 80% (wt.%) or to 75%, or to 70%, or to 65%, or to 60%, or 55% to 80%, or to 75%, or to 70%, or to 65%, or 60%.

[0034] 8. The method of any one of claims 1 to 7 further comprising heating the source solution to a temperature from 30°, or from 35° or from 40° or from 45° to 70° C, or from 30°, or from 35° or from 40° or from 45° to 65° C.

[0035] 9. The method of any one of claims 1 to 8 wherein the epimerase is contacted with the sugar source in an amount from 0.01% to 0.25% on dry solids basis.

[0036] 10. The method of any one of claims 1 to 9 wherein rare sugar syrup comprises greater than 90% (wt.%) or greater than 96% or greater than 97%, or greater than 98% rare sugar solids.

[0037] 11. The method of claims 1 to 10 wherein the rare sugar syrup has dry solids content from 70% to 80% or from 70% to 75%.

[0038] 12. The method of any one of claims 1 to 11 further comprising controlling the pH of the source solution during the conversion by adding further amounts of the ionic agent.

[0039] 13. The method of any one of claims 1 to 12 wherein the resin is a cation ion type resin, preferably a calcium type resin.[0040| 14. The method of any one of claims 1 to 13 wherein the source sugar is contacted with only one type of enzyme, preferably wherein the enzyme is an epimerase.

[0001] The technology disclosed in this specification can be better understood with reference to the following Examples, which are for illustrative purposes and are not intended to limit the full scope for the technology described.EXAMPLE 1 - CONVERSION RATE OF FRUCTOSE TO ALLULOSE USING COFACTOR FREE EPIMERASE.

[0042] A co-factor free epimerase (available from IFF) was used to convert fructose to allulose. Starting material (source solution) was concentrated (55% dry solids weight) an approximately 80% high fructose (source sugar) com syrup. Source solution was adjusted to a target temperature of 55 °C and target pH of 6.5 using CaO. To source solution was added epimerase (about 0.06% ds / ds). The mixture was allowed to react for 48 hours (“mixed sugar solution”). Amount and timing of fructose to allulose conversion is graphed in Figure 1, whichshows the percent increase in allulose and corresponding percent decrease in fructose in the mixed solution (y-axis) as the reaction progresses (x-axis). Measured values for allulose graphed in Figure 1 are reported in Table 1.Table 1Percent Conversion Allulose to Fructose

[0043] Mixed sugar solution was purified using activated carbon filtration, and chromatographic separation using calcium enriched cationic resin. Separation obtained a syrup having greater than 98.5% allulose purity (dry basis). Separation profile of unwanted sugars and calcium salts is graphed in Figure 2, which shows the pH of the solution, total solids concentration (in Brix) (left hand vertical-axis), and conductivity (pS / cm, right hand verticalaxis) versus bed volumes (horizontal-axis). As shown, conductivity, glucose, and fructose substantially elute before allulose elution increases around 0.80 bed volumes. This demonstrates the efficacy of chromatography in separating out ions and sugars without the need of ion exchange filtration prior to the chromatography separation.

Claims

CLAIMSWhat is claimed is:

1. A method of making a rare sugar comprising : a) adjusting, using an ionic agent, the pH of a source solution comprising a source sugar b) contacting the source sugar with an enzyme or series of enzymes to convert at least a part of the source sugar to a rare sugar c) separating the rare sugar from the source sugar in a chromatographic purification system comprising a resin of the same ionic type as the ionic agent and d) recovering the rare sugar as a syrup wherein the method does not use an ion exchange system prior to the chromatographic purification system; and wherein the method does not comprise adding additional ionic moieties other than the ionic agent.

2. The method of claim 1 wherein the rare sugar is selected from the group consisting of allulose, allose, and tagatose.

3. The method of claim 1 or 2 wherein the source sugar is fructose or glucose, wherein the source sugar is preferably fructose.

4. The method of any one of claims 1 to 3wherein the ionic agent comprises a calcium ion.

5. The method of any one of claims 1 to 4 wherein the ionic agent is calcium hydroxide and / or calcium oxide.

6. Method of any one of claims 1 to 5 wherein the source solution is maintained at a pH from 4 to 8 or from 5.5 to 7.5 or from 6 to 7.

7. The method of any one of claims 1 to 6 wherein the source solution has a dry solid content from 40% to 80% (wt.%) or to 75% or to 70% or to 65% or to 60%, or from 45% to 80% (wt.%) or to 75%, or to 70%, or to 65%, or to 60%, or 55% to 80%, or to 75%, or to 70%, or to 65%, or 60%.

8. The method of any one of claims 1 to 7 further comprising heating the source solution to a temperature from 30°, or from 35° or from 40° or from 45° to 70° C, or from 30°, or from 35° or from 40° or from 45° to 65° C.

9. The method of any one of claims 1 to 8 wherein the epimerase is contacted with the sugar source in an amount from 0.01% to 0.25% on dry solids basis.

10. The method of any one of claims 1 to 9 wherein rare sugar syrup comprises greater than 90% (wt.%) or greater than 96% or greater than 97%, or greater than 98% rare sugar solids.

11. The method of claims 1 to 10 wherein the rare sugar syrup has dry solids content from 70% to 80% or from 70% to 75%.

12. The method of any one of claims 1 to 11 further comprising controlling the pH of the source solution during the conversion by adding further amounts of the ionic agent.

13. The method of any one of claims 1 to 12 wherein the resin is a cation ion type resin, preferably a calcium type resin.

14. The method of any one of claims 1 to 13 wherein the source sugar is contacted with only one type of enzyme, preferably wherein the enzyme is an epimerase.

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