A PROCESS FOR PRODUCING ALTERNAN-OLIGOSACCHARIDE

Abstract

A process (51) for producing alternan-oligosaccharide (8), comprising contacting in a reactor (11) sucrose (9) with a catalytically effective amount of alternansucrase enzyme (13) and acceptor molecules (12), wherein the alternansucrase enzyme (13) and acceptor molecules (12) are present in the reactor (11) in an aqueous liquid (4) and the sucrose (9) is continuously or half-continuously fed to the reactor (11), and wherein the sucrose (9) and the acceptor molecules (12) are converted to alternan-oligosaccharide (8), and fructose (6) is formed as a by-product, continuously or half-continuously removing at least a part of the fructose (6) from the reactor (11) by membrane filtration (17).

Claims

1. A process for producing alternan-oligosaccharide, comprising contacting in a reactor sucrose with a catalytically effective amount of alternansucrase enzyme and acceptor molecules, wherein the alternansucrase enzyme and acceptor molecules are present in the reactor in an aqueous liquid and the sucrose is continuously or half-continuously fed to the reactor, and wherein the sucrose and the acceptor molecules are converted to alternan-oligosaccharide, and fructose is formed as a by-product, continuously or half-continuously removing at least a part of the fructose from the reactor by membrane filtration.

2. The process of claim 1, wherein the membrane filtration is a diafiltration.

3. The process of claim 1, wherein removing of at least a part of the fructose comprises continuously or half-continuously circulating the content of the reactor through a device comprising a membrane and contacting the content of the reactor with the membrane, wherein at least a portion of the fructose and a portion of the aqueous liquid pass the membrane, and wherein a remainder is returned to the reactor.

4. The process of claim 1, further comprising continuously or half-continuously feeding further aqueous liquid to the reactor.

5. The process of claim 3, further comprising continuously or half-continuously feeding further aqueous liquid to a reactor system comprising the reactor and the device comprising a membrane.

6. The process of claim 1, further comprising removing at least a part of an alternan-polysaccharide, which is formed as a further by-product, and at least a part of the alternansucrase enzyme, preferably by a further membrane filtration, wherein in the further membrane filtration the alternan-oligosaccharide is comprised in a permeate.

7. The process of claim 1, further comprising concentrating the alternan-oligosaccharide.

8. The process of claim 1, wherein an average degree of polymerization DPw or DPn of the alternan-oligosaccharide is regulated by the amount of the sucrose which is fed to the reactor.

9. The process of claim 1, wherein the feeding of the sucrose is stopped when a desired average degree of polymerization DPw or DPn of the alternan-oligosaccharide is reached.

10. The process of claim 1, wherein the weight average degree of polymerization DPw of the alternan-oligosaccharide is in a range of 5-30, as determined with GPC-RI.

11. The process of claim 1, wherein the weight average degree of polymerization DPw of the alternan-oligosaccharide is in a range of 10-20, as determined with GPC-RI.

12. The process of claim 1, wherein the acceptor molecules are maltose molecules.

13. The process of claim 1 wherein the sucrose is continuously fed and a rate of feed of sucrose, in molar amount of sucrose per time, is equal or substantially equal to a rate of continuous removal of fructose, in a molar amount of fructose per time, or wherein the ratio of the rate of feed of sucrose to the rate of removal of fructose is in the range of 1.2:1 to 1:1.

14. The process of claim 1, wherein a molar ratio of sucrose:acceptor molecules, which is the total amount of fed sucrose in relation to the total amount of acceptor molecules used in the process, is 10:1-30:1.

15. The process of claim 1, wherein further alternansucrase enzyme is fed to the reactor, prcfcrably continuously or half continuously.

16. The process of claim 1, wherein a ratio of alternansucrase enzyme:sucrose, which is the total amount of fed sucrose, is 1000-10000 units (enzyme activity):1000 g of sucrose.

17. The process of claim 2, wherein further alternansucrase enzyme is fed to the reactor.

18. The process of claim 3, wherein further alternansucrase enzyme is fed to the reactor.

19. The process of claim 4, wherein further alternansucrase enzyme is fed to the reactor.

20. The process of claim 5, wherein further alternansucrase enzyme is fed to the reactor.

Description

BRIEF DESCRIPTION OF FIGURES

[0113] FIG. 1a shows a scheme of the process according to the prior art;

[0114] FIG. 1b shows a scheme of the process of the invention;

[0115] FIG. 2a shows a process design of the prior art process;

[0116] FIG. 2b shows a process design of the process of the invention;

[0117] FIG. 3 is a chart showing the increase of molecular weight over the process time when constant feed of sucrose is applied.

EXAMPLES

Methods

Determination of DP with HPAEC-PAD

[0118] Values for DP were measured by HPAEC-PAD after reducing and hydrolyzing glucose-based saccharides. Two milliliters of a solution containing 6 g/mL of digestible carbohydrate composition were treated with 0.2 mL of a NaBH4 solution (40 mg/mL) in 0.5 M ammonia at 40° C. for 30 min. Reduced samples were subsequently hydrolyzed with 0.5 mL 2 M Trifluoroacetic acid heated at 121° C. for 1 h to release monomers. The released monomers were quantified by injecting sample solutions on a Thermo Scientific™ Dionex™ ICS-6000 ion chromatograph system equipped with a CarboPac™ MA1 and fed with eluents (water and NaOH 1000 mM) at 0.4 mL/min. DP values are calculated with the following formula:

[00001]$\mathrm{DP}=\frac{\left(\frac{\mathrm{sugar}\mathrm{alcohols}}{182}\right)+\left(\frac{\mathrm{glucose}\mathrm{content}}{180}\right)}{\left(\frac{\mathrm{sugar}\mathrm{alcohols}}{182}\right)}$

Determination of DP with GPC-RI

[0119] Values for DP were alternatively measured by GPC-RI. Samples were diluted with water and subjected to gel permeation chromatography in water (0.5 mL/min) using 2×Tosoh TSK GEL G2500 PWXL columns coupled in series. Oligosaccharides are separated during permeation by their relative molecular weight and quantified using refractometric detection. Molecular weight quantification was based on the external standard approach using standards ranging from 342 to 5000 Da (PSS-Polymer Standards). The Chromeleon Extension Pack V2.0 was used according to Dionex instructions for calibration and calculation.

Working Examples

Example 1

[0120] The prior art process is known for example from WO 0047727 A2 and WO 2009095278 A2, and comprises following four steps P1-P4 of FIG. 1a:

[0121] In step P1 bioconversion 2 of sucrose and maltose is done in in batch reactor 1 for a duration of about 20 h at T=37° C. The sucrose : maltose ratio is chosen to 7:1 (w/w or mol/mol). Step P1 is done in the batch reactor 1 for bioconversion 2 which is shown in FIG. 2a.

[0122] P2 is a step of ultrafiltration 3 for removal of alternan-polymer (alternan polysaccharide) and alternansucrase enzyme (AlSu). This step is done in the ultrafiltration device 3′ shown in FIG. 2a.

[0123] In P3, fructose is removed by nanofiltration 5. This step is done in the nanofiltration device 5′ shown in FIG. 2a. Here, water 4′ is added and a mixture of water 4″ and fructose 6 is removed.

[0124] In the final step P4 the product from P3 is concentrated by evaporation 7. This is done in the evaporation device 7′ in FIG. 2a and maltose-alternan oligosaccharide (MAOS) 8 is obtained.

[0125] The process of the invention, in a specific embodiment, comprises three steps S1-S3, shown in FIG. 1b:

[0126] In comparison to the prior art process of FIG. 1a, the bioconversion 2 in step S1 comprises continuous feed 10 of sucrose 9 and continuous removal of fructose 6. Half-continuous feed and removal is possible. The mass ratio of sucrose 9 (total amount added) to maltose 12 is 19:1 (19 kg sucrose per 1 kg maltose) and the duration is about 72 h. Step P3 of the prior art can be omitted because fructose 6 is removed in step S1 already. Removing fructose 6 makes possible using a higher ratio of sucrose 9 to maltose 12 and reaching a higher degree of polymerization of the maltose-alternan-oligosaccharide 8. Step S1, a bioconversion, is done in the reactor 11 in FIG. 2b, wherein maltose 12 and alternansucrase enzyme (AlSu) 13 are present in water 4. A stated above, bioconversion is done with continuous feed 10 of sucrose 9, continuous removal of fructose 6, for a duration about 72 h (variable), T=37° C., and a sucrose:maltose ratio of 19:1 (w/w or mol/mol). Sucrose 9 is added as feed (dissolved in water) 10 and the reactor 11 content is stirred. In the bioconversion in the reactor 11, alternan oligosaccharide comprising acceptor molecule maltose, also called maltose alternan oligosaccharide 8 (MAOS), is formed as main product and alternan polymer 14, fructose 6 and leucrose 15 are formed as by-products. Content from the reactor 11 is continuously circulated through the membrane cell (diafiltration cell) 16, where water 4″, fructose 6 and leucrose 15 are removed in membrane filtration 17, which in this example is a nanofiltration, done as a constant volume diafiltration. Leucrose 15 content is reduced from about 30% to less than 10%, in comparison with the prior art. Removed water 4″ is replaced by the water 4′ feed stream.

[0127] The reactor 11 and the membrane cell 16 form a combined bioconversion and nanofiltration device, also called reactor system.

[0128] Process step S2 of this embodiment corresponds to step P2 of the prior art. Here, alternan polysaccharide (alternan polymer) 14, as by product, and alternansucrase enzyme (AlSu) 13 are removed by ultrafiltration 3 in device 3′. The step is beneficial in case that more alternan polymer 14 as desired has been formed, or in order to steer desired DPw of the alternan species remaining.

[0129] Process step S3 of this embodiment corresponds to step P4 of the prior art. Here the product is concentrated by evaporation 7 in device 7′.

[0130] The composition of the reaction solution used during the production is shown in Table 1. The 2.1 L solution gives a total of about 5.6 L with the water in the system (dead volume).

[0131] The process is run without depletion for the first hour to minimize potential loss of maltose across the membrane. Subsequently, fructose is constantly depleted via a nanofiltration membrane Filmtec NF270-2540 (DOW). Upon completion of the chain extension, the nanofiltration module was replaced with a TRISEP 2540-UE50-QXF ultrafiltration module (Microdyn Nadir). This separated the maltose-alternan oligosaccharide (MAOS) fraction from the longer aging chains and the enzyme. The membranes used during the process and process parameters used are summarized in Table 2. The filtrate was finally concentrated to a dry matter content of >72%.

TABLE-US-00001 TABLE 1 Composition of the reaction solution Component Amount feed rate maltose 451 g Batch sucrose 8500 g ~100 g/h sodium acetate 57 g Batch alternansucrase 1900 U Batch water ad 2.1 L Batch

TABLE-US-00002 TABLE 2 membranes and parameters process step filter pressure temperature nanofiltration Filmtec NF270-2540 5-30 bar, 30-40° C., (DOW) actually actually used 15 bar used 37° C. ultrafiltration TRISEP 2540-UE50-QXF 2-15 bar, 30-60° C., (Microdyn Nadir) actually actually used 10 bar used 40° C.

[0132] FIG. 3 shows the increase in chain length over the course of the process. The values were recorded by GPC-RI measurements. The relationship for calculation of DPw from the Mw values of FIG. 3 is as follows: DPw=Mw/(162 Da). So it can be seen that at the end of the process, a DPw of about 15.4 is reached (2500/162).

[0133] In order to reach an average chain length DPw of about 15, 19 kg of sucrose (in total) were used per 1 kg of maltose.

Comparative Example

[0134] Alternan-oligosaccharide was made according to a process as shown in FIG. 1a. A ratio of 21:1 (kg Sucrose: kg Maltose) was used and a DPw of 9.9 was obtained. For the measurement of DPw, GPC-RI was used, but not exactly according to the method-protocol as mentioned above in the method section. Nevertheless, in comparison with the results of FIG. 3 it is shown that by the present invention alternan with higher DPw can be obtained.

[0135] DPw values for two samples obtained by the method of the invention that were analyzed with HPAEC-PAD method are summarized in the table 3 below:

TABLE-US-00003 TABLE 3 Tested Product Avg DP (DPw) Comparative Sample DCC-1  6.9 ± 0.02 Sample DCC-2 12.4 ± 0.24 Sample DCC-3 17.3 ± 0.53 P2

[0136] Samples DCC-2 and DCC-3 were produced by a process of the invention.

[0137] Glycosidic linkage profiles for glucose-based oligosaccharides were measured with partially-methylated alditol acetates by GC-MS. Briefly, samples were dissolved in anhydrous DMSO, deprotonated by an addition n-Butyl Lithium (Sigma 230707) and methylated with Methyl iodide (Sigma 289566). The methylated samples were subsequently hydrolyzed with 2 N TFA (60 min at 121° C.). The hydrolyzed samples were evaporated under a nitrogen air draft, re-dissolved in 1 M ammonium hydroxide and aldehyde groups were reduced with a DMSO solution containing sodium borodeuteride (20 mg/ml). Glacial acetic acid was added drop wise to stop reaction and acetylation was done by addition of 1-methylimidazole and acetic anhydride. Partially methylated alditol acetates in acetone were quantified by GC-MS (7890A-5975C MSD, Agilent

[0138] Technologies, Inc., Santa Clara, Calif., USA) using a Supelco 24111-U SP-2380 capillary column (injector volume, 0.5 μl; injector temperature, 250° C.; detector temperature, 250° C.; carrier gas, helium: 30 mL/min; split ratio, 40:1; temperature program, 100° C. for 3 min, 4° C./min to 270° C. for 20 min. Electron impact spectra were acquired at 69.9 eV over 50-550 Da mass range.

TABLE-US-00004 Glycosydic-linkage DCC-1 DCC-2 DCC-3 Terminal-Glc 36 31.5 29.3 1,3-D-Glc 13 15.2 16.8 1,6-D-Glc 39 44.1 44.3 1,4-D-Glc 11 6.8 5.0 1,3,6-D-Glc 1.4 2.4 4.7

[0139] Higher values of 1,6-glycosidic linkages are explained by the leucrose content in the digestible carbohydrate compositions that contributes to the amounts of 1,5,6-Tri-O-acetyl-1-deuterio-2,3,4-tri-O-methyl-D-glucitol observed. In addition, leucrose, as well as monomeric glucose, contributes to the amounts of terminal-Glc in the digestible carbohydrate compositions.

Example 2

[0140] In this example, parameters were varied as shown in the following table and alternansucrase was given to the reactor in 4 equal portions, the first portion being present before sucrose was fed to the reactor.

TABLE-US-00005 1. experiment- 2. experiment - Parameter 72 h process 24 h process Sucrose amount 15.5 kg 16.5 kg Feeding rate 250 g/h 775 g/h Enzyme activity 56 kU 79.2 kU Temperature 37° C. 43° C. Time 72 h 27 h

[0141] Reaction time of the process of the invention (not including here further process steps like removing alternan-polysaccharide and alternansucrase enzyme by a further membrane filtration, or concentrating a retentate which is obtained in the further membrane filtration) could be reduced by increasing the sucrose amount, increasing the sucrose feeding rate, increasing the enzyme activity and increasing the temperature.

LIST OF REFERENCE SYMBOLS

[0142] P1 Bioconversion in batch

[0143] P2 ultrafiltration

[0144] P3 nanofiltration

[0145] 4 concentration

[0146] S1 Bioconversion

[0147] S2 ultrafiltration

[0148] S3 concentration

[0149] 1 batch reactor

[0150] 2 bioconversion

[0151] 3 membrane filtration, in the example: ultrafiltration

[0152] 3′ membrane filtration device, in the example: ultrafiltration device

[0153] 4 aqueous liquid, in the example: water

[0154] 4′ aqueous liquid, in the example: water

[0155] 4″ aqueous liquid, in the example: water

[0156] 5 nanofiltration

[0157] 5′ nanofiltration device

[0158] 6 fructose

[0159] 7 evaporation

[0160] 7′ evaporation device

[0161] 8 alternan-oligosaccharide, in the example: maltose alternan oligosaccharide (MAOS)

[0162] 9 sucrose

[0163] 10 feed

[0164] 11 reactor

[0165] 12 acceptor molecules, in the example: maltose

[0166] 13 alternansucrase (AISu)

[0167] 14 alternan polymer (alternan polysaccharide)

[0168] 15 leucrose

[0169] 16 device comprising a membrane, in the example: membrane cell

[0170] 17 membrane filtration; in the example: bioconversion and nanofiltration (constant volume diafiltration)