JPH0759585A - Production of pullulan oligosaccharide - Google Patents

Abstract

PURPOSE:To obtain a pullulan oligosaccharide, especially pullulan oligosacchhride having high polymerization degree by decomposing pullulan with an enzyme under specific condition. CONSTITUTION:Pullulan is brought into contact with a hydrolase capable of breaking the bond between glucose units constituting pullulan in a mixture of an organic solvent and water. A pullulan oligosaccharide having high polymerization degree unattainable in aqueous system can be produced by shifting the equilibrium of the reaction using an organic solvent system having high concentration.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing pullulan oligosaccharides, and more particularly to a method for producing pullulan oligosaccharides having a high degree of polymerization.

[0002]

2. Description of the Related Art In recent years, it has become known that carbohydrates are associated with physiological functions, and research is actively conducted.
Also, oligosaccharides having various actions such as low caries, those that act to normalize the intestinal microflora, those that have a bacteriostatic action, and those that have an inclusion action such as cyclodextrin. Became known and contributed to the food industry.

Some of such oligosaccharides can be directly obtained as a fermentation product, but many of them are usually produced by decomposing a polysaccharide. Conventionally, the decomposition of polysaccharides has been carried out by enzymatic decomposition or acid hydrolysis in a water system.

By the way, pullulan, which is a type of polysaccharide, is Aureobas, which is a type of imperfect bacterium called black yeast.
It is a water-soluble extracellular neutral polysaccharide obtained by culturing idium pullulans in a medium containing sucrose or a partial hydrolyzate of starch as a carbon source. Its molecular structure is a linear glucan in which maltotriose, which is a trimer of glucose, is repeatedly bound by α-1,6 glycoside bonds, and α-1,
Six bonds are cleaved.

Pullulan is an amorphous, non-crystalline, tasteless and odorless white powder, and is treated as a food in Japan without restriction of use or labeling, like starch, and is widely used in edible films and easily degradable plastics. Has been done. In addition, it has been reported that pullulan is indigestible by containing an α-1,6 glycoside bond and serves as an in-vivo growth promoter for bifidobacteria (JP-A-2-289520). It is expected that the degradation product, pullulan oligosaccharide, will have similar properties.

[0006]

As mentioned above, various oligosaccharides and their production methods have come to be known.
Little is reported about pullulan oligosaccharides.

The present invention has been made from the above viewpoint, and is intended to produce pullulan oligosaccharides, particularly pullulan oligosaccharides having a high degree of polymerization. Pullulan oligosaccharides are enzymatically decomposed using pullulan as a raw material. It is an object to provide a method for obtaining sugar.

[0008]

The inventors of the present invention have conducted various studies to solve the above problems, and as a result, enzymatically decompose pullulan in the presence of an organic solvent to produce pullulan oligosaccharides having a high degree of polymerization. The inventors have found that the above is the case and have reached the present invention.

That is, the present invention is a method for producing pullulan oligosaccharides by decomposing pullulan with a hydrolase which cleaves bonds between glucoses constituting pullulan, wherein pullulan and the hydrolase are combined. To
It is a method for producing pullulan oligosaccharide, which is characterized in that it is allowed to coexist in a mixed liquid of an organic solvent and water.

In the present specification, the high-polymerization degree pullulan oligosaccharide mainly refers to glucose of about 6 to 30 mer. Hereinafter, the present invention will be described in detail.

As described above, the method for producing a high degree of polymerization pullulan oligosaccharide of the present invention comprises enzymatically degrading pullulan as a raw material in a mixed system of an organic solvent and water. Here, pullulan as a raw material is, for example, Aureobasidium pu
It can be obtained by culturing llulans in a medium containing sucrose or a partial hydrolyzed starch as a carbon source. Moreover, you may use what is marketed.

The hydrolase used for enzymatic decomposition is an enzyme that cleaves the bond between glucoses constituting pullulan, and examples thereof include planase, isoamylase, amyloglycosidase, and the like, and particularly α-1 such as pullulanase. An enzyme that cleaves a 6,6 glycosidic bond is preferred.

This enzyme reaction may be carried out in the liquid phase, but it may also be carried out using a so-called immobilized enzyme in which the enzyme is bound to an insoluble carrier. In the present invention, the immobilization method is not particularly limited.

Examples of the organic solvent used in the enzymatic reaction include alcohols such as methanol, ethanol, propanol, isopropanol and butanol, ketones such as acetone, and hydrocarbons such as cyclohexane and dodecane. Especially, hydrophilic organic solvents are preferably used.

To obtain pullulan oligosaccharide in the present invention, an enzymatic reaction is carried out in a mixed system of the above organic solvent and water.
The water may be a buffer solution. The concentration of organic solvent is 5
-60 (v / v)% is preferable. Further, the concentration of pullulan as a raw material substrate is preferably 5 to 30 (w / v)% with respect to the reaction solution from the viewpoint of yield.

The organic solvent used and its concentration are appropriately set depending on the type of enzyme and the degree of polymerization to be obtained. The enzymatic reaction is preferably carried out under optimum conditions for the enzyme used, but the reaction may be carried out under mild conditions in order to increase the degree of polymerization of the oligosaccharide obtained. The optimum conditions are, for example, 20 to 50 ° C. when pullulanase is used as the enzyme,
pH 4.5-8.0 is mentioned.

The degree of polymerization of the pullulan oligosaccharide to be obtained is adjusted by changing the reaction conditions, particularly the reaction time, the amount of enzyme for pullulan, and the like. It is advisable to analyze the reaction product and determine the conditions in advance by HPLC (high performance liquid chromatography) or the like.

In order to obtain the desired degree of polymerization from the reaction products, it is separated by gel filtration or HPLC. Further, as described above, the pullulan oligosaccharide produced by using the enzyme that cleaves the α-1,6 glycoside bond is obtained as a glucose 3 × n monomer (n is an integer). When digested with glycosidase or the like, pullulan oligosaccharides having different compositions are obtained.

The pullulan oligosaccharide has an ethanol concentration of 6
Since most of it becomes insoluble at 5% (V / V) or more, it can be accumulated as a precipitate by centrifugation or the like. Further, it can be purified by gel filtration or the like if necessary after being dissolved in an appropriate concentration.

[0020]

Action Pullulanase is an endo-type hydrolase that cleaves the α-1,6 glucoside bond of pullulan, and is decomposed into maltotriose when the decomposition reaction of pullulan proceeds. However, when the concentration of water in the reaction system decreases, the equilibrium of the reaction changes to the reverse transfer or synthesis. That is, it is presumed that by carrying out such an enzymatic reaction in a high-concentration organic solvent system, the equilibrium of the reaction is shifted, and an oligosaccharide having a high degree of polymerization, which cannot be obtained in an aqueous system, can be obtained.

In the present invention, since the reaction is carried out in an organic solvent system, there is no fear of contamination by various bacteria, so that sterilization operation and equipment are unnecessary.

[0022]

EXAMPLES Examples of the present invention will be described below. As the pullulan, a research reagent (manufactured by Wako Pure Chemical Industries, Ltd.) was used in Examples 1 to 6, and a product for food industry (Hayashibara Corporation) was used in Example 7. Pullulanase is a pullulanase “Amano” (manufactured by Amano Pharmaceutical Co., Ltd .; 905 U / m
1) was used. In addition, 1U is a pullulan substrate,
This is the amount of the enzyme that causes an increase in the reducing power corresponding to 1 micromol of glucose per minute when pullulanase is allowed to act on this at pH 6.0 and 40 ° C.

The concentrations of the following organic solvents are V / V%, and all of them are mixed solutions with 50 mM phosphate buffer (pH 6.0). The reaction product is analyzed at 90 ° C after the reaction.
After inactivating the enzyme by heating at 10 ° C. for 10 minutes, the reaction was performed by HPLC, and the ratio of the oligosaccharide of each polymerization degree to the total amount of the obtained reaction product (excluding unreacted substrate) was calculated.

[0024] In HPLC, LC-6AD is used as a pump, differential refractometer RID-6A is used as a detector, and CTO-6A (above,
Shimadzu Corporation column, TSKg manufactured by Tosoh Corporation
The analysis was carried out using an el AMIDE80 at an oven temperature of 70 ° C.

In the following, G3 and G6 represent glucose trimers and hexamers.

[0026]

Example 1 Pullulan 1 g, pullulanase diluent (91
U / ml) 1 ml, ethanol 3 ml, phosphate buffer 6
ml was mixed, and the reaction was carried out in a constant temperature bath at 50 ° C. with gentle stirring using a magnetic stirrer. As Comparative Example 1, a phosphate buffer solution was added instead of ethanol, and the same reaction was performed.

A certain amount of the reaction solution was sampled over time, and the reaction products were analyzed for glucose trimer and hexamer and higher pullulan oligosaccharides. The percentage (%) of the weight of pullulan oligosaccharide with respect to the weight of added pullulan was taken as the conversion rate to pullulan oligosaccharide. The results are shown in Table 1.
In addition, the conversion rate for all products is shown in FIG.

[0028]

[Table 1] In addition, regarding the example, the product (3
FIG. 2 shows the conversion rate from the monomer to the monomer. The horizontal axis in the figure represents the degree of polymerization of the product with glucose as 1.

From these results, most of the reaction products in the water system are trimers (maltotriose), but in the ethanol-containing system, the amount of pullulan oligosaccharides of hexamers or more increases with the passage of reaction time. I understand.

[0030]

Example 2 12 U of amyloglucosidase (manufactured by Sigma) was added to the reaction solution obtained in Example 1 after 140 hours of reaction.
/ Ml or 10 U / m of α-amylase
The reaction mixture was added so that the concentration became 1 and reacted at room temperature for 1 hour, followed by HPLC.
The product was analyzed by. The results of amyloglucosidase treatment are shown in FIG. As is clear from FIG. 3, after the amyloglucosidase treatment, the glucose 3 × n monomer (n is an integer) was completely digested, and new pullulan oligosaccharide was produced. Further, pullulan oligosaccharide is not degraded by α-amylase and is expected to be indigestible.
Furthermore, the amyloglucosidase-treated pullulan oligosaccharide is considered to be a more resistant oligosaccharide.

[0031]

Example 3 The optimum enzyme concentration in a 35% ethanol system was examined. 1 ml of a reaction solution containing 10% pullulan and appropriately diluted pullulanase was placed in a thermostat at 20 ° C.
Let stand for 4 hours. The result of analysis of the reaction product is shown in FIG.

From these results, it is understood that under the above conditions, pullulan oligosaccharides having a glucose hexamer or higher with an enzyme concentration peaking at 10 U / ml are obtained, and above this concentration, decomposition into trimers proceeds.

[0033]

Example 4 Next, the influence of the ethanol concentration on the yield of pullulan oligosaccharide was examined. A reaction solution containing 10% pullulan, pullulanase at a final concentration of 9.1 U / ml, and 0 to 50% ethanol was placed in a thermostat bath at 20 ° C. and allowed to stand to carry out the reaction. The results of analyzing the reaction products after 22 hours and 43 hours are shown in FIGS. 5 (a) and 5 (b), respectively.

From these results, under the above-mentioned conditions, the ethanol concentration at which pullulan oligosaccharides having a glucose hexamer or higher were obtained at the highest conversion rate was 30% in 22 hours of reaction.
It was 40% in 43 hours of reaction, and the conversion rates at that time reached about 60% and about 80%, respectively.

Further, FIG. 6 shows the composition of pullulan oligosaccharide when the reaction was performed for 43 hours in the presence of 40% ethanol. It is clear that under these conditions, a hexamer or higher pullulan oligosaccharide can be obtained in a very high yield.

[0036]

Example 5 A method for recovering pullulan oligosaccharide obtained by the present invention was examined. 99.5% ethanol was added to the reaction liquid containing 30% ethanol of Example 4 for 22 hours while gently stirring. As the amount of addition increased, the transparent reaction solution gradually became cloudy and a precipitate was formed.

After adding ethanol so that the ethanol concentration of the reaction solution became 65% and 77% in this way, the mixture was gently stirred at room temperature (20 ° C.) for 3 hours, and the reaction solution was filtered to form a precipitate. Obtained. This precipitate was completely dried using a freeze dryer or the like to obtain a white powder.

The powder obtained above was redissolved in a certain amount of water, the composition of pullulan oligosaccharide of each polymerization degree was examined by HPLC, and the composition was examined immediately after the reaction to compare with the composition examined immediately after the reaction. The recovery rate was calculated. The results are shown in Fig. 7. From this result, when the final concentration of ethanol is 65%,
Pullulan oligosaccharides having a degree of polymerization of 21 (maltotriose heptamer) or more can be recovered as a precipitate in an amount of 90% or more.
When the final concentration of ethanol was 77% or more, 90% or more of pullulan oligosaccharide having a degree of polymerization of 12 or more could be recovered as a precipitate.

[0039]

Example 6 Next, pullulan oligosaccharides were produced by changing the type of organic solvent. Pullulan 10%, final concentration 9.
The reaction was carried out by allowing a reaction solution containing 1 U / ml of pullulanase and various organic solvents having a final concentration of 30% to stand in a constant temperature layer at 20 ° C. The composition of pullulan oligosaccharide after the reaction for 18 hours was examined.

The results are shown in FIG. From these results, it can be seen that the more polar (hydrophilic) an organic solvent is, the higher the conversion rate of glucose hexamer or more into pullulan oligosaccharide is.

[0041]

Example 7 An example of mass production of pullulan oligosaccharides using pullulan for the food industry will be described.

As the pullulan, pullulan for the food industry (“Pullulan PF20” manufactured by Hayashibara Co., Ltd.) was used. This pullulan was 10%, ethanol 30%, final concentration 9.1.
50 ml of a reaction solution containing U / ml pullulanase
Place in a 0 ml Erlenmeyer flask and stir at 20 ° C for 2
Reaction was carried out for 0 hours. The composition of pullulan oligosaccharide in the reaction solution at this time is shown in FIG.

After completion of the reaction, 99% ethanol was added to a final concentration of 60%, and the mixture was gently stirred at room temperature for 3 hours. Filter this with filter paper (TOYO Filter paper No.6),
A precipitate was obtained. This precipitate was dried in a freeze dryer for 24 hours to obtain 21.5 g of a white powder. The final yield of pullulan oligosaccharide was 71.1% based on the raw material.

[0044]

INDUSTRIAL APPLICABILITY According to the present invention, pullulan oligosaccharides can be efficiently produced by enzymatically decomposing a polysaccharide in a mixed system of a hydrophilic organic solvent and water.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between reaction time and pullulan oligosaccharide yield.

FIG. 2 is a diagram showing the composition of pullulan oligosaccharides having various degrees of polymerization in a reaction solution.

FIG. 3 is a view showing the composition of pullulan oligosaccharides having various degrees of polymerization in a reaction solution.

FIG. 4 is a graph showing the influence of enzyme concentration on the composition of pullulan oligosaccharide.

FIG. 5 is a graph showing the influence of ethanol concentration on the composition of pullulan oligosaccharides.

FIG. 6 is a view showing the composition of pullulan oligosaccharides having various degrees of polymerization in a reaction solution.

FIG. 7 is a view showing a recovery rate of pullulan oligosaccharides of each polymerization degree.

FIG. 8 is a diagram showing the relationship between the type of organic solvent and the yield of pullulan oligosaccharide.

FIG. 9 is a view showing the composition of pullulan oligosaccharides having various degrees of polymerization in a reaction solution.

Claims (5)

[Claims] 1. A method for producing pullulan oligosaccharides by degrading pullulan using a hydrolase that cleaves a bond between glucoses constituting pullulan, wherein pullulan and the hydrolase are A method for producing pullulan oligosaccharide, which comprises allowing the solvent to coexist in a mixed solution of water. 2. The method for producing pullulan oligosaccharide according to claim 1, wherein the hydrolase is an enzyme that cleaves an α-1,6 glycoside bond. 3. The method for producing pullulan oligosaccharide according to claim 1, wherein the organic solvent is a hydrophilic organic solvent. 4. The concentration of the organic solvent is 5 to 60 (V /
V)% is the manufacturing method of the pullulan oligosaccharide according to any one of claims 1 to 3. 5. A method for producing pullulan oligosaccharide, which comprises further digesting the pullulan oligosaccharide obtained by the method according to any one of claims 1 to 4 with amyloglycosidase.