JPH03180189A - Production of high-purity fructoligosaccharide - Google Patents

Abstract

PURPOSE:To efficiently obtain a high-purity fructoligosaccharide without requiring removal of unnecessary substances by immobilizing glucose isomerase and fructofuranosidase on the same carrier and then bringing the resultant carrier into contact with a sucrose solution. CONSTITUTION:Glucose isomerase 3 capable of producing fructose (F) from glucose (G) and fructofuranosidase 4 capable of producing a fructoligosaccharide by transfer reaction are immobilized on a carrier 2 and arranged in a bioreactor 1. A solution of sucrose (GF) is then fed into the aforementioned bioreactor 1 to convert the glucose molecules (G) into fructose molecules (F) with the glucose isomerase 3. The activated fructose molecules (F) just after the formation thereof are bound to other molecules of the glucose (G) or sucrose (GF) with the fructofuranosidase 4 to produce fructoligosaccharides (GFF), (GFFF), etc. Nonoligosaccharide ingredients (G) and (GF), as necessary, are subsequently separated and removed by using a loose reverse osmosis membrane 5.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing highly pure fructooligosaccharides by increasing the concentration of fructooligosaccharides in a sucrose solution.

(Prior Art) Fructooligosaccharide has recently attracted attention as a sugar that is low in calories, has an effect on the growth of intestinal bifidobacteria, and is less likely to cause dental caries.

Fructooligosaccharide is a polysaccharide in which two or more fructose molecules F are bonded to a glucose molecule G, and fructose transferase (β-fructofuranosidase) is attached to sucrose.
It is generated by applying

Known production methods include a batch method as shown in JP-A-56-154967, a column method as shown in JP-A-60-41497, and a forced membrane permeation method using a ceramic membrane on which enzymes are immobilized. However, all of them contain large amounts of monosaccharide and disaccharide non-oligosaccharide components.

Therefore, as shown in Fig. 4, a solution of sucrose molecules GF is permeated through a bioreactor 11 in which an enzyme is immobilized, and then this solution is supplied to a membrane module 12 to separate and remove non-oligosaccharide components G and GF. It is considered to do.

(Problems to be Solved by the Invention) In the conventional method described above, non-oligosaccharides such as glucose must be removed as unnecessary substances, leading to waste of materials. Moreover, it takes time and effort to remove unnecessary substances.

(Means for Solving the Problems) In order to solve the above problems, the present inventors focused on the fact that fructose immediately after being produced by the action of glucose isomerase on glucose has high activity and can serve as a substrate for producing fructooligosaccharides. Two types of enzymes, ie, glucose isomerase and fructofuranosidase, were immobilized on a carrier for enzyme immobilization, and these enzymes were brought into contact with a sucrose solution.

(Function) Since glucose isomerase and fructofuranosidase are immobilized at high density on the same carrier, highly active fructose immediately after being produced by glucose isomerase is immediately converted to other glucose molecules or sucrose molecules by fructofuranosidase. Combined with 3
It can be a fructooligosaccharide that is more than sugar.

(Example) Embodiments of the present invention will be described below based on the accompanying drawings.

FIG. 1 is a diagram showing the steps of the method of the present invention. In the method of the present invention, a solution of sucrose GF is passed through a bioreactor 1 to produce fructooligosaccharide G F F.

Let it be a solution of GFFF,...

Here, in the bioreactor 1, as shown in FIG. 2, glucose isomerase 3 and fructofuranosidase 4 are immobilized as enzymes at high density on a carrier 2 for enzyme immobilization. The carrier 2 is, for example, a cylindrical porous ceramic made of At*os, and the surface on which the enzyme is immobilized is covered with silica. As a method for immobilization, the OH groups on the surface of the silica are silanized to form NH, groups, and the NH, groups are cross-linked with the NH, groups possessed by the enzyme using glutaraldehyde for immobilization.

In addition to the above immobilization method, a solution of a zirconium crosslinking agent such as zirconyl ammonium carbonate is refluxed through the carrier 1 to physically bond the zirconium crosslinking agent to the pore surface of the immobilization carrier, and then immobilization is performed again. The enzyme solution may be refluxed through the carrier to chemically bond the enzyme to the zirconium-based crosslinking agent.

In addition, the reaction mechanism within the bioreactor 1 is
As shown in the figure, glucose isomerase 3 converts glucose molecule G into fructose molecule F, and since this fructose molecule F is activated immediately after generation, it can be converted into other glucose G or sucrose by contacting fructofuranosidase 4. It combines with GF molecules to form trisaccharide or more fructooligosaccharides GFF, GFFF, etc. Therefore, the non-oligosaccharide components G and GF in the fructooligosaccharide solution coming out of the bioreactor 1 are significantly reduced compared to the conventional method.

Therefore, a sufficiently high concentration of fructooligosaccharides can be obtained through the above steps, but if necessary, non-oligosaccharide components may be further separated and removed using the loose reverse osmosis membrane 5.

(Effect of the invention) Figure 3 shows residual amount of sucrose/total sugar and quesdose amount/total sugar when glucose isomerase is added to β-fructofuranosidase (example of the present invention) and when it is not added (comparative example). This is a graph showing the relationship between the two, that is, the influence of glucose isomerase on the sucrose transfer reaction by β-fructofuranosidase.

Here, the specific conditions of the examples and comparative examples of the present invention are as follows.

(Example) 0.1MM0PS-NaC1 buffer with pH 7.0 100
An enzyme solution was prepared by dissolving and diluting β-fructofuranosidase (Sigma, baker's yeast type, 1nvertase) and glucose isomerase (Nagase Sangyo ■) in m1 to a concentration of 1800 U/ml.

40g of A1□O, which is an immobilization carrier (manufactured by Showa Denko ■, WA-3000, average particle size 10μm), was added to the above enzyme solution.
After mixing and opening at 4° C. for about 20 hours using a silver foil, β-fructofuranosidase and glucose isomerase were physically adsorbed onto the surface of the carrier.

The concentration of the substrate sucrose was 50% (W/W).
], and 0.1 MMO P 5-NaCl buffer was used as the solvent.

HPLC was used to measure the sugar composition (column: Tosoh ■
manufactured by TSK-gel-Amide-80, flow rate: 1. O
ml/min, temperature 280°C, mobile phase: 60% acetonitrile, detection: R1) Thus obtained β-fructofuranosidase, glucose isomerase, A1
The □03 complex was packed into a packing column, washed with water, a substrate solution was fed, and the water in the column was replaced with a sucrose solution. The column was kept at a constant temperature of 65°C in a hot water bath, and the amount of liquid sent to the column was 0.1 to 3.0 [ml/min].
The reaction was carried out. The amount of Kesdose, which is an oligosaccharide, reaches its maximum when sucrose accounts for about 40% of the total sugar, and the maximum value of Kesdose was 5.2%. (△ in the figure) (Comparative example) 0.1MM0P 5-NaCl buffer with pH 7.0 10
β-Fructofuranosidase (Sigma,
180007ml of Baker's Yeast (1nvertase)
An enzyme solution was prepared by dissolving and diluting the enzyme.

Add 40 g of A1□03 (manufactured by Showa Denko, WA-3000, average particle size 10 μm) as an immobilization carrier to the above enzyme solution.
The mixture was mixed with a silver foil at 4° C. for about 20 hours, and β-fructofuranosidase was physically adsorbed onto the surface of the carrier.

The concentration of the substrate sucrose was 50% (W/W).
], and 0.1MMOPS-NaCl buffer was used as the solvent.

HPLC was used to measure the sugar composition (column: Tosoh ■
manufactured by TSK-gel-Amide-80, flow rate: 1. O
ml/min, temperature = 80°C, mobile phase: 60% acetonitrile, detection: RI) The thus obtained β-fructofuranosidase and AhOs complex was packed into a packing column, and after washing with water, the substrate solution was added. was pumped, and the water in the column was replaced with a sucrose solution. Place the column in a hot water bath for 65 minutes.
℃ kept constant, and the amount of liquid sent to the column was adjusted to 0.1-3゜[ml
/win] to perform the reaction. The amount of Kesdose, which is an oligosaccharide, reached its maximum when sucrose accounted for about 40% of the total sugar, and the maximum value of Kesdose was 3.8%. (○ in the figure) As described above, it can be seen that when oligosaccharides are produced using β-fructofuranosidase, the oligosaccharide production efficiency is improved by adding glucose isomerase.

In other words, according to the present invention, glucose molecules, which conventionally had to be removed as unnecessary substances, are combined with other glucose molecules while remaining active fructose that can serve as a substrate for oligosaccharides. It is extremely advantageous because the oligosaccharide concentration can be increased without removing substances, the process can be simplified, and only one stage of equipment is required.

[Brief explanation of drawings]

Figure 1 is a diagram showing the method of the present invention in the order of steps, and Figure 2 is a diagram showing the method of the present invention in the order of steps.
An enlarged view of the main part of the figure, Figure 3 is a graph showing the relationship between the amount of sucrose remaining and the amount of Kesdose with and without the addition of glucose isomerase, and Figure 4 is a diagram showing the conventional method in the order of steps. . In the drawing, 1 is a bioreactor, 2 is a carrier, 3 and 4 are glucose isomerase and fructofuranosidase as enzymes, G is a glucose molecule, and F is a graphose molecule. Patent applicant: Totokiki Co., Ltd.

Claims (1)

[Claims] In a method for producing high-purity fructooligosaccharides in which the concentration of fructooligosaccharides is increased by contacting an enzyme immobilized on a carrier with a sucrose solution, the carrier contains fructooligosaccharides through a transfer reaction with glucose isomerase that generates fructose from glucose. A method for producing high purity fructo-oligosaccharides, characterized by immobilizing fructofuranosidase that produces.