DK167366B1 - PROCEDURE FOR PREPARING ISOMALTULOSE (6-O-ALFA-D-GLUCOPYRANOSIDO-D-FRUCTOSE) BY CONCERNING SACCHAROSE WITH IMMOBILIZED BACTERY CELLS - 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

in DK 167366 B1

The present invention relates to a process for the preparation of isomaltulose (6-O-α-D-glucopyranosido-D-fructose) by enzymatic conversion of sucrose by microorganisms capable of producing isomaltulose from sucrose to treat a pure sucrose solution. with dead, immobilized bacterial cells or contact the solution with such cells, after which the isomaltulose formed is recovered by crystallization. The method according to the invention is characterized by the characterizing part of claim 1.

Danish Patent Application No. 4349/81 discloses a process for the preparation of 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 65-20 75 wt%, through a reactor filled with immobilized cells. The isomaltulose thus formed is recovered in crystalline form in a manner known per se.

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

30

In carrying out this known process, it has now 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 in the unreacted state in the reaction solution. Since these by-products also inhibit the crystallization of the isomaltulose formed and thereby further decrease the yield of crystals, it is of great economic interest to provide a process which completely or at least partially prevents the formation of undesirable and annoying by-products.

5

It has now surprisingly been found that it is possible to recover isomaltulose of high purity and in great yield from sucrose, if the sucrose solution is shared with a concentration of 40-75% by weight, preferably 10 45-60% by weight , in two parts, 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 80-90% of the sucrose is converted and then the mother liquor obtained after crystallization of the formed isomaltulose, mixed with the second part of the sucrose solution, passes through another reactor which is also filled with immobilized dead cells of microorganisms capable of forming isomaltulose from sucrose at 25-40 ° C, preferably 30-40 ° C, in such a way that the sucrose is almost completely transformed, thereby recovering in the manner known per se isomaltulose from d in a crystalline form, the crystals dissolve in the already reacted first portion of the sucrose solution, induce crystallization and separate the isomaltulose crystals thus formed.

The mean residence time in the reactor for the sucrose solution to be reacted is dependent on the specific activity of the immobilized cells at any given time. The specific activity is defined as the amount of sucrose reacted per day. g of immobilized cells (dry weight) per minute. For example, a reactor of 100 mm in diameter and 450 mm in bed height contains 950 g of immobilized cells 35 (dry weight) with a specific activity of 60 units / g.

This reactor must be flushed with 1900 ml of sucrose solution (50% by weight) per liter. hourly to react 90% of the% sucrose added. Since the activity decreases continuously during the operating period, the flow rate must be constantly re-regulated to obtain a constant composition reaction product.

5

The process according to the invention is illustrated in Example 3 below. Examples 1 and 2 are comparative examples where Example 1 corresponds to Example 1 of DK Patent Application No. 4349/81.

EXAMPLE 1 (Comparative Example) a) Rinse cells from a seed of Protaminobacter rubrum (CBS 574.77) with 10 ml of a standard nutrient substrate consisting of 8 kg of thick juice from a sugar factory (dry matter content = 65%), 2 kg of corn cast water, 0.1 kg (NH 4) 2 HPO 4 and 89.9 kg of distilled water, adjusted to pH 7.2 as needed. This suspension serves as an inoculum for a pre-culture shaker in 1 liter of 20 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 25 liter mini fermentor containing 20 flasks (4 L) and fermented at 29 ° C with 20 l of air per day. and a stirring speed of 350 rpm. The increasing number of germs is determined microscopically. After obtaining a □ germ count of 5 x 10 g / ml, the contents of the fermentor 30 are transferred to another container to which a cationic flocculant (such as "Primafloc C7" from Rohm & Haas, Philadelphia, USA) is added. ). The precipitated cells are allowed to settle, decanted, washed with 0.1 M phosphate buffer (pH 7.0) and the water removed 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 extracted mixture 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 whose temperature 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 "20 This solution is transferred to a cooling crystallizer and cooled to 20" C, after which the crystallized isomaltulose is separated from the mother liquor for a sieve basket centrifuge week.

Yield 100 kg of a solution containing 70 kg of sucrose after the reaction contains 43.8 g 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 corresponds 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 expected).

Example 16

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 (0.1 M, pH 7.0) and filled under water on a column whose temperature can be regulated. 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 Glucose 1.8 g / 100 g "

Sucrose 0

Isomaltulose 78.4 g / 100 g "1-O-e-D-glucopyranosido-D-fructose 12.6 g / 100 g"

Oligosaccharides 3.6 g / 100 g "20

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 25 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.

Yield: 100 kg of a solution containing 50 kg of sucrose in the container after the reaction 39.2 kg of isomaltulose (78.4% of the dry matter content). Of this, 25.0 kg of pure isomaltulose and 25.6 kg of isomaltulose with 98% purity are recovered during the first crystallization. This corresponds to a total yield of 34.4 kg of pure isomaltic acid 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 at two columns whose temperature is regulated to 30 ° C.

10

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

One of the columns (1) continuously flows through a 50% by weight sucrose solution. The flow rate is regulated so that, when flowing through the column, only approx. 90% of the sucrose. The reaction solution thus obtained has the following composition (HPLC): 20 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" 25 Oligosaccharides 3.1 g / 100 g

Parallel to the first column, the second column (2) is supplied with a substrate consisting of a mixture of sucrose and mother liquor from a prior isomaltulose crystallization. This substrate has the composition (HPLC): 35 7 DK 167366 B1 Solids content 50% by weight

Fructose 2.0 g / 100 g dry matter

Glucose 1.0 g / 100 g "

Sucrose 76.2 g / 100 g "5 Isomaltulose 10.8 g / 100 g" 1-O-a-D-glucopyranosido-D-fructose 8.2 g / 100 g

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" 20 Oligosaccharides 2.5 g / 100 g

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 at this stage 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 in a sieve basket centrifuge. The mother liquor produced at this stage is used for the substrate preparation of the second column.

35 in DK 167366 B1 8

Yield

By proceeding in the manner described in this example, 100 kg sucrose 80.0 kg pure 5 isomaltulose.

10 15 20 25 30 35

Claims (3)

A process for the preparation of isomaltulose (6-Oa-5D-glucopyranosido-D-fructose) by enzymatic conversion of sucrose by microorganisms capable of producing isomaltulose from sucrose, thereby treating a pure sucrose solution with dead, immobilized cells of such microorganisms or bring the solution into contact with such cells, after which the isomaltulose formed is recovered by crystallization, characterized by first conducting a portion of the sucrose solution at a concentration of 40-75% by weight, preferably 45-60 wt.%, through a reactor filled with the 15 immobilized dead cells at 25-40 ° C, preferably at 30-40 ° C, and conducting the reaction in such a way that 80-90% of the sucrose is converted so that then the mother liquor formed after crystallization of the formed isomaltulose, mixed with the remaining part 20 of the sucrose solution, passes through another reactor which is also filled with imm. unilated dead cells, at 25-40 ° C, preferably at 30-40 ° C, so that the sucrose is almost completely reacted and that the isomaltulose 25 obtained in the crystalline form obtained in the crystalline form is known per se way, the crystals dissolve in the already reacted first part of the solution, causing the solution to crystallize and separate the obtained isomaltulose crystals. 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) and Leuconostoc mesenteroides (NRRL B-512 F (ATCC 10830 a)), preferably using Protaminobacter rubrum (CBS 574.77).