DK172544B1 - Process for Preparation of 1-O-alpha-D-glucopyranosido-D-fructose by enzymatic conversion of sucrose - Google Patents

Abstract

1. In a process for preparing isomaltulose (6-0-alpha-D-glucopyranosido-D-fructose) wherein sucrose solution is treated with or brought into contact with dead immobilized cells of a microorganism and the formed isomaltulose is obtained by crystallizing characterized in passing one part of the sucrose solution having a concentration of 40% by weight to 75% by weight, preferably 45% by weight to 60% by weight via a reactor, which is filled with immobilized dead cells at 25 degrees C to 40 degrees, preferably 30 degrees C to 40 degrees C so that 80% to 90% of the sucrose is converted, in passing the first mother liquor obtained after having crystallized the formed isomaltulose with a further part of the sacrose solution via a second reactor, which is also filled with immobilized, dead cells at 25 degrees C to 40 degrees C, preferrably 30 degrees C to 40 degrees C so that the sucrose is almost completely converted, and by obtaining the isomaltulose resulting from the second part of the solution in crystalline form by methods known per se, in dissolving and crystallizing the crystals in the converted first part of the solution and in separating the resultant isomaltulose crystals.

Description

DK PR 172544 B1 i

The present invention relates to a particular process for the preparation of 1-O-α-glucopyranosido-D-fructose by enzymatic conversion of sucrose by microorganisms capable of producing isomaltulose (6-O-α-D-glu-copyranosido-D-fructose ) from sucrose. The process according to the invention is peculiar to the characterizing part of claim 1.

Danish Patent Application No. 4349/81 discloses a method for producing isomaltulose by enzymatic conversion of sucrose, thereby contacting pure sucrose solutions with dead, immobilized cells of microorganisms capable of producing isomaltulose from sucrose. In this process, at a temperature of 40-65 ° C, sucrose solutions in the concentration range are 45-75 wt%, preferably 65-75 wt%, through a reactor loaded with immobilized cells. The isomaltulose thus formed is recovered in crystalline form in a manner known per se.

20

The microorganisms used in this known method can e.g. are Protaminobacter rubrum (CBS 574.77), Serratla plymuthica (ATCC 15928), Serratia marescescens (NCIB 8285) or Leuconostoc mesenteroides (NRRL 25 B-512 F (ATCC 10830a)), but are preferably Protaminobacter rubrum (CBS 574.77). .

In carrying out this known process, it has been found that a large proportion (up to 1/3) of the added sucrose is frequently not converted to the desired compound (isomaltulose) but to by-products and / or remains 1 unreacted state in the reaction solution. Since these by-products also inhibit the crystallization of the formed isomaltulose, thereby further lowering the yield of crystals, it has been of great economic interest to provide a method which completely or at least partially prevents the formation of undesirable and annoying by-products. .

This succeeded with the stock application of the present application (Danish Patent Application No. 1229/83), according to which it is possible to recover isomaltulose with high purity and in great yield from sucrose if the sucrose solution is shared with a concentration of 40-75. wt%, preferably 45-60 wt%, in two parts and passing the first part through a reactor loaded with immobilized dead cells of microorganisms capable of producing isomaltulose from sucrose at 25-40 * C, preferably 30-40 * C in such a way that 80-90% of the sucrose is converted. Then, the second portion of the sucrose solution is mixed with the mother liquor from a prior isomaltulose crystallization, and this solution is passed through another reactor filled with immobilized dead cells of microorganisms capable of forming isomaltulose. from sucrose, at 25-40 ° C, preferably 30-40 20 ° C, in such a way that the sucrose is almost completely converted. Finally, in the known manner, the obtained isomaltulose is obtained from this solution in crystalline form, dissolves the crystals in the first obtained isomaltulose solution and produces a crystallization, separates the thus formed isomaltulose crystals and uses the mother liquor to mix with another sucrose solution.

One of the aspects of the process of Danish Patent Application No. 1229/83 is that, after prior concentration, an aqueous solution containing 1-O-α-D-glu-copyranosido-D-fructose can be recovered from the mother liquor from iso-maltulose recovery. From this aqueous solution one can then obtain, in a manner known per se, 1-O-α-D-glu-35 copyranosido-D-fructose in dry form. The prior concentration is effected by crystallization of an additional 3 part of the isomaltulose from methanol and removal of the yeast digestible sugars by treatment with free or immobilized yeast and subsequent chromatographic separation with ion exchangers or other suitable separation materials.

5

This particular process for preparing 1- O -α-glucopyranosido-D-fructose is the subject of the present application.

The process of the invention is illustrated in Example 3 below. Examples 1 and 2 are Comparative Examples, wherein Example 1 corresponds to Example 1 of DK Patent Application No. 4349/81.

EXAMPLE 1 (Comparative Example) (a) Cells are rinsed from a seed of Protami-nobacter rubrum (CBS 574.77) with 10 ml of a sterile nutrient substrate consisting of 8 kg of thick juice from a sugar-20 plant (dry matter content - 65% ), 2 kg of maize cast water, 0.1 kg (NH «) j HPO« and 89.9 kg of distilled water, adjusted to pH 7.2 as required. This suspension serves as an inoculum for a pre-culture shaker machine in 1 liter flasks containing 200 ml of nutrient solution of the composition described above.

After 30 hours of cultivation at 39 ° C, 16 liters of nutrient solution of the above composition is seeded in a 30 liter ml fermentor containing 20 flasks (4 liters) and 30 fermented at 29 ° C with 20 liters of air per liter. and a stirring speed of 350 rpm. The increasing number of germs is determined microscopically. After obtaining a seed count of 5 x 109 g / ml, the contents of the fermentor are transferred to another container to which a cation-active flocculant is added (such as "Primafloc C7" from Rohm & Haas, Philadelphia, USA). . The flocculated cells are allowed to settle, decant, wash with 0.1 M phosphate buffer (pH 7.0) and remove the water by centrifugation. The pulp is then extruded into rods which are air dried and ground.

B) The sieve fraction 0.3-0.8 mm of the above-recovered preparation is stirred in a 0.1% glutaraldehyde solution for 10 minutes, washed with phosphate buffer (0.1 M, pH 7.0) and filled under water on a column , the temperature of which can be controlled. The column is then heated to 50 ° C and continuously flowed with a 70% by weight sucrose solution. The flow rate is adjusted so that no sucrose can be detected at the end of the column. The isomaltulose solution thus obtained has the following average composition (HPLC = high pressure liquid chromatography):

Fructose 7.4 g / 100 g dry matter

Glucose 0.3 g / 100 g "Sucrose 0.1 g / 100 g"

Isomaltulose 62.6 g / 100 g "1-O-α-D-glucopyranosido-D-fructose 16.6 g / 100 g"

Oligosaccharides 13.0 g / 100 g "25 This solution is transferred to a cooling crystallizer and cooled to 20 ° C, after which the crystallized isomaltulose is separated from the mother liquor in a sieve basket centrifuge.

Yield 100 kg of a solution containing 70 kg of sucrose after the reaction contains 43.8 kg of isomaltulose (62.6% of the dry matter content). Of this, at the first crystallization 15.9 kg of pure isomaltulose is obtained and at the second crystallization 13.4 kg of isomaltulose with 98% purity. This is equivalent to a total yield of 29 kg of pure isomaltulose from 70 kg of sucrose or 41.4% of the raw material. (In this example and in the following examples, anhydrous isomaltulose is considered).

EXAMPLE 2

The sieve fraction 0.3-0.8 mm of an analog of Example 1a is stirred for 10 minutes in a 0.1% glutraldehyde solution, washed with phosphate buffer (0.1 M, pH

7.0) and filled under water on a column whose temperature can be controlled. The column is then heated to 30 ° C and continuously flowed with a 50% by weight sucrose solution. The flow rate is adjusted so that no sucrose can be detected at the outlet of the column.

The isomaltulose solution thus obtained has the following average composition (HPLC):

Fructose 3.6 g / 100 g dry matter 20 Glucose 1.8 g / 100 g "

Sucrose O.

isomaltulose 78.4 g / 100 g "1-O-a-D-glucopyranosido-D-fructose 12.6 g / 100 g"

Oligosaccharides 3.6 g / 100 g "25

This solution is evaporated to 78-82% solids content, transferred to a cooling crystallizer and cooled to 20 ° C, after which the crystallized isomaltulose is separated from the mother liquor in a sieve basket centrifuge. The mother liquor 30 is again evaporated to 78-82% solids content and subjected to another cooling crystallization step to 20 ° C, whereupon the crystallized second isomaltulose amount is separated from the mother liquor in a sieve basket centrifuge.

35 6 DK PR 172544 B1

Yield: 100 kg of a solution containing 50 kg of sucrose after the reaction contains 39.2 kg of isomaltulose (78.4% of the dry matter content). Of this, 25.0 kg of pure isomaltulose is obtained in the first crystallization and in the second crystallization 9.6 kg of isomaltulose with 98% purity. This corresponds to a total yield of 34.4 kg of pure isomaltulose from 50 kg of sucrose or 68.8% of the raw material.

EXAMPLE 3

The sieve fraction 0.3-0.8 mm of an analog of Example 1a is stirred for 10 minutes in a 0.1% glutaraldehyde solution, washed with phosphate buffer (0.1M, pH

7.0) and filled under water on two columns whose temperature is adjusted to 30 ° C.

The process is further illustrated by the diagram shown in FIG. First

One of the columns (1) is continuously flowed by a 50% by weight sucrose solution. The flow rate is regulated so that, when flowing through the column 25, only approx. 90% of the sucrose. The resulting reaction solution has the following composition (HPLC):

Fructose 2.9 g / 100 g dry matter

Glucose 1.6 g / 100 g "Sucrose 10.8 g / 100 g"

Isomaltulose 75.2 g / 100 g "1-O-α-D-glucopyranosido-D-fructose 6.4 g / 100 g"

Oligosaccharides 3.1 g / 100 g "35 Parallel to the first column, a second substrate (2) is added to a substrate consisting of a mixture of sucrose and mother liquor from a prior isomaltulose crystallization. This substrate has the composition (HPLC): Solids content 50% by weight 5 Fructose 2.0 g / 100 g dry matter

Glucose 1.0 g / 100 g "

Sucrose 76.2 g / 100 g "

Isomaltulose 10.8 g / 100 g "1-O-α-D-glucopyranosido-D-fructose 8.2 g / 100 g" 10 Oligosaccharides 1.8 g / 100 g "

The flow rate through this column is regulated in such a way that the solution at the exit from the column contains no more than 1 g sucrose per day. 100 g of dry matter. The reaction solution thus obtained has the following composition (HPLC):

Fructose 5.4 g / 100 g dry matter

Glucose 2.2 g / 100 g "Sucrose 1.0 g / 100 g"

Isomaltulose 72.3 g / 100 g "1-O-α-D-glucopyranosido-D-fructose 16.6 g / 100 g"

Oligosaccharides 2.5 g / 100 g "25 This solution is evaporated to 78-82% solids content and subjected to cooling crystallization at 20 ° C, after which the crystallized isomaltulose is separated from this second mother liquor in a sieve basket centrifuge.

The isomaltulose obtained in this step is dissolved in the reaction solution obtained from the first column which is evaporated to 78-82% solids content. The solution is cooled in a cooling crystallizer to 20 ° C, and the crystallized, pure Isomaltulose is separated from the mother liquor 35 in a sieve basket centrifuge. The mother liquor produced on this step is used for the substrate manufacture of the second column.

The second mother liquor, also known as molasses, 5 contains approx. 30-40% of the dry matter in the form of 1-O-α-D-glucopyranosido-D-fructose and according to the invention can be used as raw material in the extraction of this sugar. The extraction of this sugar can e.g. is carried out by chromatographic separation on an ion exchanger or on another suitable separation material after a preliminary concentration by crystallizing another portion of the isomaltulose from methanol and removing sugars fermentable with yeast by treatment with free or immobilized yeast.

15

Yield

By proceeding in the manner described in this example, 100 kg of sucrose 80.0 kg of pure isomaltulose.

Claims (2)

A process for the preparation of 1-O-α-glucopyranosi-5-do-fructose by enzymatic conversion of sucrose by microorganisms capable of producing isomaltulose from sucrose, characterized by sharing a sucrose solution with a concentration of 40-75% by weight, preferably 45-60% by weight in two, and passing the first 10 part through a reactor filled with immobilized dead cells of such microorganisms at 25-40 ° C, preferably 30-40 ° C, on such a by mixing 80-90% of the sucrose and mixing another portion of the sucrose solution with the mother liquor from a prior isomaltulose crystallization and passing this solution through another reactor filled with immobilized dead cells of such microorganisms at 25 -40 ° C, preferably 30-40 ° C, in such a way that the sucrose is converted almost completely, and in a manner known per se to recover the isomaltulose thus formed in crystalline form, which after entrainment of the second mother liquor by crystallization of a further portion of the isomaltulose from methanol and removal of the yeast digestible sugars by treatment with free or immobilized yeast first recover the 1-Oa-glucopyranosido-D-fructose as aqueous solution by chromatography. separation with ion exchangers or other suitable separation materials and then by dry methods known per se. 30 Method according to claim 1, characterized in that the microorganisms used are selected from Protaminobacter rubrum (CBS 574.77), Serratia plymuthica (ATCC 15928), Serratia marcescens (NCIB 8285) or Leuconostoc mesenteroides (NRRL B-512 F ( ATCC 10830 (a)), preferably Protaminobacter rubrum (CBS 574.77).