JPH0731492A - Production of purified oligosaccharide - Google Patents

Abstract

PURPOSE:To provide a method for profitably producing a purified oligosaccharide in a high oligosaccharide concentration by separating off the by-produced glucose on the basis of a new mechanism quite different from conventional mechanisms. CONSTITUTION:This method for producing a fructo oligosaccharide by treating a sucrose aqueous solution with a transferase comprises allowing a boric acid compound to coexist in the reaction solution from the early time of the transition reaction for the formation of the complex of the by-produced glucose with the boric acid compound, and subsequently bringing the aqueous solution containing the complex into contact with an anion exchange resin for removal of the complex.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a purified oligosaccharide having a high oligosaccharide concentration by removing glucose by-produced when producing an oligosaccharide such as fructooligosaccharide.

[0002]

2. Description of the Related Art An aqueous solution of a sugar composition containing fructooligosaccharide as a main component (usually referred to as syrup) by reacting sucrose with fructosyltransferase.
Is obtained, but a considerable amount of glucose is produced as a by-product. Therefore, depending on the application, it is necessary to remove the glucose produced as a by-product to increase the oligosaccharide concentration.

[0003] In order to purify fructooligosaccharides from a reaction solution obtained by reacting sucrose with fructosyltransferase, an activated carbon chromatographic method or an ion exchange chromatographic method is generally adopted. Therefore, it is hard to say that it is an industrial method. Therefore, some proposals have been made regarding a method for purifying fructooligosaccharides.

[0004] For example, in JP-A-59-95895, when a fructooligosaccharide is isolated and purified from a sugar composition containing a monosaccharide, sucrose (that is, sucrose) and fructooligosaccharide, the sugar composition A method for purifying fructooligosaccharides in which sucrose is decomposed into monosaccharides by the action of α-glucosidase is shown. In the examples, after decomposing sucrose in the sugar composition into monosaccharides,
The activated carbon chromatographic method described above is applied.

In Japanese Patent Laid-Open No. 59-179100, a fixed bed filled with an alkali metal type or alkaline earth metal type strongly acidic cation exchange resin is mixed with oligosaccharides of trisaccharide or more and sugars of disaccharide or less. A method of separating a trisaccharide or higher oligosaccharide and a disaccharide or lower saccharide by a circulation method in which a stock solution is passed through in a downward flow or an upward flow is shown.

In JP-A-60-149596, a fructooligosaccharide solution having a nystose content of 60% or more is concentrated to a solid content concentration of 75 to 90%, and seed crystals containing crystalline nystose are added and dispersed. , A method for producing a fructooligosaccharide crystal powder, in which the crystal is solidified and aged, and then powdered. In this case, in order to obtain a fructooligosaccharide solution having a high content of nystose, a method is used in which the reaction solution is concentrated and then fractionated using a column such as activated carbon or an ion exchange resin.

In JP-A-62-14792, glucose is produced as a by-product when a sugar mixture containing fructooligosaccharide as a main component is obtained by a fructose transfer reaction in which an enzyme having a fructose transfer activity acts on sucrose. A method for producing a fructo-oligosaccharide-rich product, which is adapted to carry out an operation, is shown. The operation of reducing glucose by-produced here is specifically the following operation. (a) An operation of utilizing glucose by a microorganism, an operation of utilizing glucose by a microorganism in the coexistence of an agent that inhibits the action of the microorganism to decompose sucrose and fructooligosaccharide, or a method of utilizing glucose and sucrose by a microorganism. The operation to turn into. Here, the microorganisms are yeast and bacteria. (b) An operation of converting glucose by an enzyme (eg glucose oxidase).

[0008]

[Problems to be Solved by the Invention] Among the methods for purifying fructooligosaccharides from a reaction solution containing fructooligosaccharides as a main component, which is obtained by reacting sucrose with fructosyltransferase, it has been generally adopted conventionally. Since the activated carbon chromatographic method uses a liquid containing an organic solvent such as alcohol as a developing liquid, it is difficult to remove the organic solvent after that, the operation is complicated, and the oligosaccharide concentration in the separated eluate is low. However, there are problems that the column head is large with respect to the substrate (several%) (100 to 500 times), and the apparatus becomes large with respect to the throughput.

In JP-A-59-95895, sucrose is decomposed into monosaccharides by allowing α-glucosidase to act on the sugar composition. Since the activated carbon chromatographic method described above is adopted, there are the same problems as described above.

In the ion exchange chromatographic method, the concentration of the separated oligosaccharides is low, for example, 10 to 15%, and the concentration becomes lower when the yield is increased, so that the subsequent concentration is difficult. There is a problem. Such a problem is also caused by the improved method of JP-A-59-179100, in which the stock solution is passed through a fixed bed packed with an alkali metal type or alkaline earth metal type strongly acidic cation exchange resin by a circulation system. Essentially unsolvable.
In JP-A-60-149596, a method of concentrating the reaction solution as a pretreatment step and then fractionating using a column such as an ion exchange resin is adopted. The image treatment is unavoidable in that the yield of oligosaccharide is lowered.

Among the operations of reducing glucose by-product disclosed in Japanese Patent Laid-Open No. 62-14792, the method of assimilating glucose by microorganisms (yeast or bacteria) is a method of assimilating glucose in a short time (within about 24 hours). When trying to assimilate, many bacterial cells (0.5 to 1 times as much as the substrate) are required in the early stage, it is difficult to secure the bacterial cells, and there are many by-products due to the assimilation of the microorganisms, and the load of post-treatment is high. There are disadvantages such as being large.

Among the operations for reducing the by-product glucose disclosed in JP-A-62-14792, the method of converting glucose by an enzyme such as glucose oxidase has a low concentration of isolated oligosaccharides (10%) and is concentrated. Is difficult, the production of glucose oxidase is difficult, the cost is high, and aeration is required during the reaction.

A gel filtration chromatography method is also conceivable, but this method has a low concentration of separated oligosaccharides (10%).
Degree), concentration is difficult, and recovery rate is low (5
0-70%), there is a disadvantage that the recovery rate becomes worse as the purity is increased.

Under such circumstances, the present invention separates and removes glucose, which is a by-product, in the production of oligosaccharides based on a mechanism completely different from the conventional method, thereby producing purified oligosaccharides having a high concentration of oligosaccharides. It is intended to provide an industrially advantageous method for producing.

[0015]

Means for Solving the Problems The method for producing a purified oligosaccharide according to the present invention comprises the steps of reacting an enzyme with an aqueous saccharide to produce an oligosaccharide by an enzymatic reaction, and at any stage of the production process. The present invention is characterized in that a complex of by-product glucose and boric acid is formed by coexisting boric acid or boric acid consisting of a salt thereof with, and then the complex is separated and removed.

The present invention will be described in detail below.

Examples of oligosaccharides include fructooligosaccharides and galactooligosaccharides. Since fructooligosaccharides are particularly important, fructooligosaccharides will be mainly described below.

Fructooligosaccharides are produced by reacting sucrose in an aqueous solution with fructosyltransferase. This reaction is represented by the following equation.
For simplification of description, glucose units are represented by G, fructose units are represented by F, and GFF is represented by GF ₂ and GFFF is represented by GF ₃ as necessary.

In an actual reaction, in addition to GF _2, a large amount of GF ₃ is produced and a small amount of GF ₄ is also produced. The amount of GF ₅ or more produced is very small. Fructooligosaccharide is GF
₂ to about GF ₄ or GF ₅ .

From the above reaction formula, it is found that a considerable amount of glucose is inevitably produced as a by-product. However, since glucose does not have an intestinal regulating action, a bifidobacteria-proliferating action, and a lactic acid-bacterium-proliferating action, a method of reducing the ratio of glucose in the sugar composition as much as possible is advantageous. Although the reaction solution also contains a considerable amount of unreacted sucrose, the presence thereof often does not cause any particular problem for ordinary applications of fructooligosaccharides.

In the present invention, when an enzyme is reacted with a saccharide in an aqueous solution to produce an oligosaccharide by an enzymatic reaction, boric acid or a boric acid consisting of a salt thereof is present in the system at any stage of the production process. By doing so, a complex of by-product glucose and boric acid is formed.

Any step in the production process means that fructosyl transferase (which is a fructosyltransferase produced in the cells of a bacterium of the genus Aureobasidium or Aspergillus) is allowed to act on sucrose to produce fructooligosaccharide. Taking the case of production as an example, it refers to a suitable stage before the reaction such as charging of raw materials, an appropriate stage during the transfer reaction, and a stage for purification after the reaction.

By the way, by-product glucose is α
Type, but soon becomes equilibrium with β type. Since boric acid forms a complex only with α-type glucose,
Although boric acids can be allowed to coexist in the system at any stage of the production process, by allowing the boric acids to coexist in the system from the beginning of the transfer reaction, the α-type byproduct glucose immediately after production is immediately converted to boric acids. It is more advantageous to convert to the complex of

As boric acid, boric acid or borax is used. Since boric acids do not cause enzyme inhibition, they may be allowed to coexist in the system before or during the transfer reaction as well as after the transfer reaction.

The above-mentioned complex formation reaction is represented by the following formula. The structure of the complex is considered to be represented by the following chemical formula 1.

[0026]

[Chemical 1]

The amount of boric acid added is preferably set to a calculated amount according to the above equation (2) so as to be commensurate with the amount of by-product glucose, but a slight increase or decrease is allowed. Since the complex formation reaction proceeds under acidity, it is desirable to adjust the pH of the system to about 4-6. Since the formed complex is an acidic substance, the pH of the system decreases as the complex formation reaction proceeds. Therefore, care should be taken so that the pH of the system is maintained at about 4 to 6 by appropriately adding a pH adjuster.

After completion of the complex formation reaction, the complex is separated and removed from the system. Although various methods are adopted as the method for separating the complex, the present inventors have found that the above complex is adsorbed on the anion exchange resin almost quantitatively. Therefore, an aqueous solution containing the oligosaccharide and the above complex is anionic. The method of contact with the exchange resin is recommended.

Since the complex is almost completely removed by the treatment with the anion exchange resin, the pH of the treatment liquid is adjusted appropriately, and the product is further concentrated or powdered to obtain a product. Since the complex adsorbed on the anion exchange resin can be desorbed from the anion exchange resin by passing an aqueous alkali solution, the anion exchange resin can be repeatedly used. The eliminated complex can be decomposed into glucose and boric acid, if necessary.

[0030]

In the present invention, the byproduct glucose is removed from the aqueous solution of the sugar composition mainly comprising oligosaccharides by utilizing the complex forming ability of boric acids with α-type glucose. Since the complex formation reaction is an ionic reaction, the reaction rate is high and the reaction rate is high.

Boric acids can be added at any stage of the oligosaccharide production process. When oligosaccharides are produced by an enzymatic reaction, even if boric acids are added before or during the rearrangement reaction, the rearrangement reaction does not occur. It is not inhibited at all, and the transfer rate is substantially the same as that in the case where no boric acid is added.

The complex thus formed can be easily and surely removed by contact with an anion exchange resin, so that a purified oligosaccharide having a high oligosaccharide concentration can be produced.

[0033]

EXAMPLES Next, the present invention will be described in detail with reference to examples.

Example 1 A fructooligosaccharide syrup was prepared and glucose contained in the syrup was removed as follows.

<Purification of Enzyme> Fructosyltransferase-containing bacterium 2 of Aureobasidium pullulans
The cell membrane was disrupted (kg) and fructosyl transferase was released in 30 liters of water. At this time, the titer of fructosyltransferase was adjusted to 100 units / ml to obtain a crude fructosyltransferase aqueous solution. One unit is a titer that produces 1 micromol of 1-kestose per minute in a sucrose aqueous solution at a temperature of 50 ° C. and a concentration of 50% by weight.

Next, the above crude fructosyl transferase aqueous solution was subjected to ultrafiltration with a molecular weight cut off of 20,000.
Concentration and purification to 1.5 liters and a titer of 2000 units / ml gave a purified fructosyl transferase aqueous solution.

<Production of Fructooligosaccharide> Reference Example First, as a reference example, an experiment was carried out in the case where boric acid was not added. That is, sucrose (granulated sugar) 10
0 g was dissolved in 100 ml of water, 0.5 ml of the purified fructosyltransferase aqueous solution having a titer of 2000 units / ml obtained above was added, and the reaction was carried out at 55 ° C. for 24 hours with stirring.
After completion of the reaction, it was sterilized at 100 ° C. The results of analysis of the solid content in the reaction product, fructooligosaccharified granulated sugar syrup, are shown in Table 1 below. For nystose, GF ₃
In addition, GF ₄ and GF ₅ , which are produced in small amounts, are included. What is indicated by ^* is fructooligosaccharide, and the total amount is 61.7% by weight.

[0038]

[Table 1] Raw Granulated Sugar Syrup Glucose 0.1 wt% 24.5 wt% Sucrose 99.9 wt% 13.8 wt% 1- Kestose GF ₂ ^* 0.0 wt% 39.8 wt% Nistose GF ₃ ^* 0.0 wt% 21.9 wt%

Example Next, as an example, an experiment was carried out when boric acids were added. That is, sucrose (granulated sugar) 100
Dissolve g in 100 ml of water, add 4.2 g of boric acid, adjust the pH to 5.0 by adding a small amount of sodium hydroxide aqueous solution, and add 0.5 ml of the purified fructosyl transferase aqueous solution having a titer of 2000 units / ml obtained above. In addition, 55
Stirring reaction was performed at 24 ° C. for 24 hours. Since the pH gradually decreases during the progress of the reaction, an aqueous sodium hydroxide solution was added occasionally to adjust the pH to about 5. The amount of boric acid added was 4.2 g because the amount of glucose in the solids contained in the syrup at the end of the reaction in the above Comparative Example was
Since it is 24.5% by weight, 24.5 × 61.8 / (180 × 2) = 4.2% is set so that glucose (molecular weight 180) is 2 moles per 1 mole boric acid (molecular weight 61.8). .

<Purification of fructooligosaccharide> After completion of the reaction in the above example, the reaction solution was sterilized at 100 ° C., and the reaction solution was then subjected to anion exchange resin (“Amberlite IRA-410-OH type” sold by Organo Corporation). Filled with 250 ml of water and passed through the jacket with warm water at 40 ℃ to keep the inside diameter 6
The column having a length of 0 mm and a length of 350 mm was passed through an upflow at a space velocity of 0.3 for 1 cycle of 12 hours, and the passed solution was circulated through the column while adjusting the pH to 9.8 to 10.0 with an aqueous sodium hydroxide solution. "Amber Light IRA-4
The filling amount of 10 "was set to 250 ml because it was confirmed by preliminary experiments that the amount of glucose that can be exchanged and adsorbed per ml of the anion exchange resin was 0.10 to 0.11 g.

After the above operation, the product was concentrated to a solid content concentration of 70% by weight. The sugar composition in the obtained product was as follows. In addition to GF ₃ , nystose contains a small amount of GF ₄ and GF ₅ . Glucose 2.7 wt% Sucrose 16.0 wt% 1-Kestose GF ₂ 51.3 wt% Nystose GF ₃ 30.0 wt%

The total ratio of GF ₂ and GF ₃ is 81.3% by weight. The reason why the glucose removal rate does not reach 100% is that the amount of boric acid added was slightly less than the theoretical value due to the difference in the field between the comparative example and the example. If it is 100%, the glucose removal rate will be almost 100%.

1 (a) and 1 (b) are high performance liquid chromatograph charts for the sugar composition at the end of the transfer reaction and the sugar composition after the purification treatment by the anion exchange resin packed column in the above-mentioned Examples.

Example 2 The transfer reaction was carried out in the presence of boric acid in the same manner as in Example 1, and the reaction solution after completion of the transfer reaction was purified by "Amberlite IRA-410-OH type" in accordance with Example 1. went. The amount of sucrose charged was 400 g, the amount of boric acid added during the transfer reaction was 17.2 g with a glucose production rate of 25%, the amount of enzyme used was 10 u / sucrose, the pH was 5.0, and the temperature was 50 ° C. , Anion exchange resin filling amount 1000 ml, space velocity 0.8, temperature 40 ° C, 1 cycle 20 hours, adopting treatment liquid circulation system, solid content concentration 6
666 g of a 0 wt% reaction solution was supplied as an upflow.
Table 2 below shows changes in the sugar composition before the purification treatment and the sugar composition during the purification treatment together with the pH of the system.

[0045]

[Table 2] Treatment time Sugar composition (wt%) GF ₂ + GF ₃ pH (hr) G GF GF ₂ GF ₃ Total (wt%) 0 23.0 14.2 40.9 21.9 62.8 5.0 3 7.4 14.4 50.4 27.8 78.2 10.5 4 6.3 14.9 50.5 28.3 78.8 5 4.4 14.8 51.2 29.6 80.8 20 1.7 15.8 52.8 29.7 82.5 10.8

A small amount of glucose was also detected in the product after 20 hours of treatment, which is considered to be caused by the decomposition of sucrose after the completion of the transfer reaction.

EXAMPLE 3 Using Aureobasidium pullulans or Aspergillus japonicus as the fructosyltransferase, the transfer reaction was carried out in the presence or absence of boric acid in the same manner as in Example 1 to give 1-kestose GF ₂ . Nystose
The generation rate of GF ₃ (however, containing a small amount of GF ₄ and GF ₅ ) was measured over time. The transfer reaction conditions were 100 g of sucrose charge, 4.3 g of boric acid added during transfer reaction with glucose production of 25%, enzyme usage of 10 u / sucrose, pH of 5.0 and temperature of 50 ° C. . The results are shown in Figure 2.

From FIG. 2, it can be seen that the addition of boric acid does not show any enzyme damage and the transfer rate is virtually the same depending on whether boric acid is added or not.

[0049]

INDUSTRIAL APPLICABILITY In the present invention, the complex forming ability of boric acids with α-type glucose is utilized. Since the complex formation reaction is an ionic reaction, the reaction rate is high and the reaction rate is high. High selectivity for removal over glucose means high recovery. Further, the concentration of the separated oligosaccharides can be increased (for example, 50% or more), so that the concentration is easy.

Boric acids can be added from the beginning of the process for producing oligosaccharides. When oligosaccharides are produced by an enzymatic reaction, the presence of boric acids does not inhibit the rearrangement reaction, and the rate of rearrangement is that no boric acid is added. It is no different from the case of.

The produced complex can be easily and surely removed by contact with the anion exchange resin.

In addition, the method of the present invention has an advantage that it can be applied not only to fructooligosaccharides but also to other oligosaccharides that produce glucose by-product, such as galactooligosaccharides. It is also industrially advantageous that boric acids are inexpensive.

[Brief description of drawings]

FIG. 1 is a graph showing a sugar composition at the end of a transfer reaction and a sugar composition after a purification treatment with an anion exchange resin packed column.

FIG. 2 is a graph showing the generation rates of 1-kestose GF ₂ and nystose GF ₃ over time.

Claims (4)

[Claims] 1. When an oligosaccharide is produced by an enzyme reaction by reacting an enzyme with a saccharide in an aqueous solution to produce an oligosaccharide, boric acid or a boric acid consisting of a salt thereof is allowed to coexist in the system at any stage of the production process. A method for producing a purified oligosaccharide, which comprises forming a complex of by-product glucose and boric acid, and then separating and removing the complex. 2. The by-product glucose is immediately converted to a complex with boric acid by allowing boric acid or a boric acid consisting of a salt thereof to coexist in the system from the beginning of the enzymatic reaction. Manufacturing method. 3. The complex in the aqueous solution is separated and removed by contacting an aqueous solution containing a complex of by-produced glucose and boric acid with an anion exchange resin.
Or the manufacturing method according to 2. 4. The method according to claim 1, wherein the oligosaccharide is fructooligosaccharide.