Optimized Method For Producing A Composition Containing Isomaltulose

Abstract

The present invention relates to a method for producing a composition containing isomaltulose from a substrate containing sucrose comprising the steps of: a) contacting the substrate containing sucrose with a particulate carrier-immobilized sucrose isomerase biomass and b) obtaining a composition containing isomaltulose, characterized in that the median particle size d(0.5) of the carrier-immobilized sucrose isomerase biomass is from 370 to 550 m. The carrier can be an alginate or a polyvinyl alcohol carrier.

Claims

1. A method for producing a composition containing isomaltulose from a substrate containing sucrose, comprising the steps of: a) contacting the substrate containing sucrose with a particulate carrier-immobilized sucrose isomerase biomass, wherein the carrier is an alginate or polyvinyl alcohol carrier and b) obtaining a composition containing isomaltulose, characterized in that the median particle size d(0.5) of the carrier-immobilized sucrose isomerase biomass is from 370 to 500 m.

2. The method according to claim 1, wherein the particulate carrier-immobilized sucrose isomerase biomass has d(0.5) from 420 to 470 m.

3. The method according to claim 1, wherein the particulate carrier-immobilized sucrose isomerase biomass is spherical.

4. The method according to claim 1, wherein the weight ratio of sucrose isomerase biomass to carrier is 10 to 6 parts sucrose isomerase biomass to 6 to 2 parts of the carrier (in each case dry weight).

5. The method according to claim 1, wherein the composition containing isomaltulose contains trehalulose.

6. The method according to claim 1, wherein the sucrose isomerase biomass is carrier-immobilized by bonding, crosslinking or entrapment immobilization.

7. The method according to claim 1, wherein the sucrose isomerase biomass is a sucrose isomerase, a microorganism cell with sucrose isomerase activity or a cell-extract with sucrose isomerase activity.

8. The method according to claim 1, wherein the sucrose isomerase biomass originates from microorganisms of the genera Escherichia, Salmonella, Serratia, Erwinia, Enterobacter, Klebsiella, Raoultella, Pectobacterium, Pseudomonas, Azotobacter, Pantoea, Leucanea, Protaminobacter, Leuconostoc, Agrobacterium or Bacillus sp.

9. The method according to claim 8, wherein the microorganism is selected from the group consisting of: Protaminobacter rubrum, Klebsiella strain LX3 or strain NK33-98-8, Klebsiella pneumoniae strain 342; Enterobacter sp. strain SZ62 or strain FMB1, Erwinia tasmaniensis strain Et1/99; Pectobacterium atrosepticum strain SCRI 1043; Pectobacterium carovotum subspecies brasiliensis strain PBR 1692, Azotobacter vinelandii, Leucanea leucocephalia, Erwinia rhapontici, Raoultella planticola, Pseudomonas mesoacidophila, Leuconostoc mesenteroides, Pantoea dispersa, Serratia plymuthica, Serratia marcescens or Agrobacterium radiobacter.

10. Method according to claim 1, wherein the method steps a) and b) are carried out in a fixed bed or stirred tank.

11. The method according to claim 1, wherein the obtained composition containing isomaltulose is catalytically hydrogenated.

12. The method of claim 1 further comprising a catalytic hydrogenation of the resultant composition containing isomaltulose to obtain a composition containing sugar alcohol.

13. The method of claim 13, wherein the composition containing sugar alcohol is isomalt or an isomalt variant.

Description

DESCRIPTION OF THE DRAWINGS

[0089] The figures show:

[0090] FIG. 1 graphically illustrates the size distribution of a first inventive particle preparation (coarse grain),

[0091] FIG. 2 shows the size distribution of a second inventive particle preparation (normal grain), and

[0092] FIG. 3 shows the size distribution of a third inventive particle preparation (fine grain).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

[0093] Determination of particle size distribution of immobilized biocatalysts by means of laser diffraction using a particle size analyzer (MASTERSIZER 2000, Malvern Instruments Ltd.) dry measurement.

Execution

[0094] With the help of laser diffraction measurement, the particle size distribution of the biocatalyst (immobilisate) according to the invention is determined. To describe the size distribution, the parameters d.sub.0,1, d.sub.0,5, d.sub.0,9 and the uniformity are shown.

Short Description

[0095] The measurement principle is based on the scattered light/laser diffraction spectroscopy ISO 13320. Scattered particles are brought in low concentration into a laser beam. The introduction of the particles is effected by sucking the dry biocatalyst sample into the measuring cell (dry measurement). Depending on the diameter of the particles, there is a diffraction of the laser light that is collected by detectors as the scattered radiation. The result of the measurement is initially in the form of light intensities measured at the detectors and must be converted into a particle size distribution. This is achieved using the customary evaluation software for particles >1 m given here by an approximation of Joseph von Fraunhofer (1814).

Equipment/Means

[0096] Test sieve with a diameter of 200 mm in accordance with DIN ISO 3310-1 of mesh size 1.25 mm [0097] Collecting tray and cover for the test sieve [0098] Malvern MASTERSIZER 2000 with dry dispersion unit

[0099] Scirocco 2000 (A), including associated control and analysis software, Malvern Instruments Ltd.

Sample Preparation

[0100] All weighing operations are performed at 0.01 g (reading accuracy). [0101] Determine tare weights (tare) of the sieve and of the collecting tray [0102] Assemble collecting tray and sieve [0103] Determine weight of the complete biocatalyst sample (usually in a 250-mL vial) of each of the biocatalyst batch [0104] Transfer sample quantitatively onto the sieve, cover it and sieve by hand [0105] After sieving, weigh both the sieve with the residue and the collecting base with the passed volume (gross) [0106] Evaluation/calculation: the percentage of coarse fraction of the biocatalyst sample is determined from the sieve residue [0107] The sieve residue is discarded and the passed volume in the collecting tray is returned to the original sample bottle

[0108] Measurement with the Malvern MASTERSIZER 2000

Dosing/Feeding of the Sample

[0109] The dosage of the samples is carried out via a vibrating channel with adjustable gap width. A coarse sieve with several balls is used as sieve strainer.

[0110] The dosage (gap width) needs to be adjusted depending on the product so that the measured concentration (green area) is achieved.

Dispersing Medium

[0111] Air with a certain pressure is used as a dispersing medium.

[0112] The measurements are performed with the MASTERSIZER 2000, dispersing unit Scirocco 2000 (A).

Example 2

A. Preparation of the Biocatalyst

[0113] Cells from an inoculation of the strain Protaminobacter rubrum (CBS 574.77) are washed off with 10 ml sterile nutrient substrate consisting of 8 kg thick juice from a sugar plant (dry substance content=65%), 2 kg of corn steeping liquor, 0.1 kg (NH.sub.4).sub.2HPO.sub.4 and 89.9 kg distilled water, adjusted to pH 7.2 if necessary. This suspension is used as inoculum for the shaking machine pre-culture in 1 liter flasks containing 200 ml of nutrient solution of the above composition.

[0114] After a 30-hour incubation at 29 C., 18 liters of nutrient solution of the above composition are inoculated with 10 flasks each (total content 2 liter) in a 30-liter small fermenter and fermented at 29 C. with 20 liters of air per minute and a stirrer speed of 350 rpm.

[0115] After reaching germinal counts of more than 510.sup.9 germs/ml, the fermentation is stopped; the cells are harvested from the fermenter solution by centrifugation and suspended in a 2% sodium alginate solution.

[0116] By using a rotation immobilization, a jet cutting process or pulsating drip method, in particular using electrostatic methods, blow-off or vibration methods, in particular by using a lateral blow-off pressure, rotating disks or rotating nozzles, the suspension is dripped in a 2% calcium chloride solution and the inventively preferred particle diameters and particle size distributions, in particular with a particle diameter dp of 250-500 m (based on dry particles) are provided. Multi-nozzle systems using a belt dryer are also used.

[0117] Table 1 shows the characterizing parameters achieved for three exemplary obtained biocatalyst preparations, namely for a first preparation, referred to as coarse grain, a second preparation, referred to as normal grain, and a third preparation, referred to as fine grain.

TABLE-US-00001 TABLE 1 Vol. mean value d(0, 1) d(0, 5) d(0, 9) D[4, 3] D[3, 2] Spec. surface (m) (m) (m) (m) (m) (m.sup.2/g) Uniformity Coarse grain 392.690 539.575 749.682 559.012 524.745 0.0114 0.207 Normal grain 335.826 457.546 626.839 470.798 444.496 0.0135 0.198 Fine grain 280.522 381.883 523.390 393.435 371.278 0.0162 0.202

[0118] The resulting immobilization balls are washed with water. This biocatalyst can be stored at +4 C. for several weeks.

B. Production of the Composition Containing Isomaltulose

[0119] The immobilized cells obtained as described in A are filled into a temperature-controlled column reactor, heated up to 25 to 30 C. and continuously passed through by a sucrose solution with 35 to 45% dry matter content. The flow rate is set such that at least 97% of the sucrose used is rearranged.

TABLE-US-00002 TABLE 2 Turnover rate Weight dm Used Feed stream sucrose/w. dm rel. activity biocatalyst [mL/h] immobilisates/h % Mean value 120 2.3 95 coarse grain Mean value 126 2.4 100 normal grain Mean value 149 2.8 118 fine grain Production 4500 l/h 2.25 94 (800 kg dm)

[0120] Table 2 shows the catalytic activity of immobilisates of different particle diameters according to Table 1 in a 100 ml fixed bed.

[0121] A HPLC analysis of the composition containing isomaltulose emerging from the column reactor resulted in the following composition:

TABLE-US-00003 TABLE 3 residual Fructose Glucose Sucrose Isomaltulose Trehalulose Isomaltose DP-3 Isomelizitose saccharides 2.7 2.0 0.9 85.1 8.0 0.8 0.3 0.3 <0.1 2.7 2.0 0.9 85.2 8.0 0.7 0.2 0.2 <0.1 2.7 2.0 0.9 85.3 7.8 0.8 0.2 0.3 <0.1

[0122] Product range according to Table 2 (coarse grain, normal grain, fine grain) (indications in g/100 g dry matter).

D. Hydrogenation of the Composition Containing Isomaltulose

[0123] The respective preparations, freed from the residual sucrose, of the composition containing isomaltulose were continuously hydrogenated on Raney nickel at 80 C. with hydrogen gas under pressure of about 10 MPa. After nickel separation and purification by ion exchange, the preparations of the composition containing isomaltulose hydrogenated under neutral conditions had about the following composition:

TABLE-US-00004 TABLE 4 Mannitol 1.5% of dry matter Sorbitol 4.0% of dm 1,6-GPS 44.4% of dm 1,1-GPS 3.8% of dm 1,1-GPM 45.3% of dm hydrogenated and non- 1.0% of dm hydrogenated oligomers

Example 3

[0124] To prepare this biocatalyst, cells from an inoculation of the strain Pseudomonas mesoacidophila MX-45 (FERM 1 1808) were washed off with 10 ml sterile nutrient substrate consisting of 8 kg thick juice from a sugar plant (dry substance content=65%), 2 kg of corn steeping liquor, 0.1 kg (NH.sub.4).sub.2HPO.sub.4 and 89.9 kg distilled water adjusted to pH 7.2. This suspension is used as inoculum for the shaking machine pre-culture in a 1-liter flask containing 200 ml of nutrient solution.

[0125] After a 30-hour incubation at 29 C., 18 liters of nutrient solution of the above composition were inoculated with 10 flasks each (total content 2 liter) in a 30-liter small fermenter and fermented at 29 C. with 20 liters of air per minute and a stirrer speed of 350 rpm.

[0126] After reaching germinal counts of more than 510.sup.9 germs/ml, the fermentation was stopped; the cells were harvested from the fermenter solution by centrifugation and suspended in a 2% sodium alginate solution.

[0127] By using rotation immobilization, a jet cutting process or pulsating drip method, in particular using electrostatic methods, blow-off or vibration methods, in particular by using a lateral blow-off pressure, rotating disks or rotating nozzle, the suspension is dripped into a 2% calcium chloride solution and the inventively preferred particle diameters and particle size distributions, in particular with a particle diameter dp according to table 1 (based on dry particles) are provided. Multi-nozzle systems using a belt dryer are also used.

[0128] The resulting immobilization balls were washed with water. This biocatalyst can be stored at +4 C. for several weeks.

[0129] In order to produce a composition containing isomaltulose, the immobilized cells of Pseudomonas mesoacidophila MX-45 (FERM 11808) obtained in such a way were filled into a temperature-controlled column reactor, heated up to 25 to 30 C. and continuously passed through by a sucrose solution with 35 to 45% dry matter content. The flow rate is set such that at least 97% of the sucrose used was rearranged.

[0130] A HPLC analysis of the composition containing isomaltulose emerging from the column reactor resulted in the following composition:

TABLE-US-00005 TABLE 5 Fructose 0.2% of dm Glucose 0.2% of dm Sucrose 1.0% of dm Isomaltulose 12.5% of dm Isomaltose 0.2% of dm Trehalulose 85.7% of dm Oligomers (DP > 3) 0.2% of dm

[0131] The composition containing isomaltulose produced in this way was freed from the residual sucrose and continuously hydrogenated on Raney nickel at about 80 C. with hydrogen gas under pressure of 8 to 12 MPa.

[0132] After nickel separation and purification by ion exchange, the composition containing isomaltulose hydrogenated under neutral conditions had the following composition:

TABLE-US-00006 TABLE 6 Mannitol 0.4% of dm Sorbitol 1.0% of dm 1.1-GPM 57.7% of dm 1.1-GPS 34.4% of dm 1.6-GPS 6.4% of dm hydrogenated and non- 0.2% of dm hydrogenated oligomers

[0133] In order to remove the hydrogenated and non-hydrogenated oligomers as well as sorbitol from the product by chromatographic separation, the chromatographic separation, after the hydrogenation with a chromatographic separation column, was performed with a strongly acidic cation exchange resin loaded with sodium or potassium ions.