KR960003644B1 - Preparation process of isomalto-oligosaccharide - Google Patents

Abstract

The preparation method for isomalto-oligosaccharide (I) consists of (1) culturing Aureobasidium pullulans L48 (KFCC 10245) in the medium containing maltose 5%, sucrose 0.5%, yeast extract 0.5%, calcium chloride 0.1% and magnesium sulfate 0.1% at pH 4.5-5.5 and 20-30 deg. C and centrifuging to obtain the alpha glucosidase-containing cell (II), and (2) converting maltose syrup with (II) at pH 3.5-5.5 and 35-55 deg. C to 50% conversion.

Description

Method for preparing isomaltooligosaccharide

In the present invention, the microorganism strain belonging to the genus Aureobasidium (Aureobasidium flulans L48 (Aureobasidium pullulans L48: KFCC 10245) by culturing aerobic in a culture medium to produce alpha glucosidase (α-glucosidase) The present invention relates to a method for producing isomaltooligosaccharide by acting on maltose syrup containing maltose or maltose, maltotriose, and glucose as a major constituent sugar by using the cells as biocatalysts.

Isomaloligosaccharide is a generic term for oligosaccharides composed only of glucose with α-1,6 glucoside bonds. The constituent sugars belonging to the isomaltooligosaccharides are isomaltose, panose, isomaltotriose, isomaltotetraose, isomaltosylmaltose and isopanose. isopanose). Isomaltooligosaccharides are also known as branched oligosaccbarides. Generally, maltose syrup with a purity of 75-85% of maltose syrup is treated with purified alphaglucosidase, and the content of isomaltoligosaccharide is 50% or more, and non-isomaltooligosaccharides such as glucose, maltose and maltotriose occupy the rest. A mixed sugar syrup obtained by mixing isomaltooligosaccharide and other sugars is obtained, which is commonly referred to as isomaltooligosaccharide. Unless otherwise specified, the above-mentioned mixed sugar syrup is referred to as isomaltooligosaccharide.

The sweetness of isomaltooligosaccharide is slightly lower than that of maltose, stable against acid and heat, and the progress of coloring under amino acid coexistence is less than that of isosugar and glucose and is similar to maltose. It is difficult to be degraded by various enzymes, and when degraded, the degradation rate is extremely low. Isomaltooligosaccharide is not fermented by yeast and remains in the product when used in liquor, bread, etc., and plays a role of improving quality. In addition, it is very hygroscopic and is non-crystalline so that it is good for wetting composition of various foods, improving the texture and taste, and maintaining quality. Isomaltooligosaccharide has low caries and anticaries and has selective growth activity of Bifidus, an enterobacteriaceae.

Conventional isomaltooligosaccharide production methods can be classified into three types according to the enzyme used. First, a method of producing low-purity isomaltooligosaccharide from glucose using sugar condensing enzymes such as glucoamylase (JP83-76063, JP86-181354, JP88-258556, JP86-124389, JP86-212296, JP86-219345, JP86 -219392, JP86-16599, JP89-265867), and secondly, by separating and purifying sugar potential enzymes such as alpha glucosidase from Aspergillus microorganism, and reacting with maltose syrup or starch hydrolysate. There is a method for preparing oligosaccharides (JP85-30695, JP91-175989). Third, hydrolyzed by appropriate enzymes or acids, using pullulan, dextran, etc., having many α-1,6 bonds as substrates. Thereafter, there is a method of separating and purifying only isomaltooligosaccharide.

While the method of using a sugar condensation enzyme has the advantage of relatively easy purification and low price of the sugar condensation enzyme, the conversion rate from glucose to isomaltooligosaccharide is low, so that the content of more than 50% isomaltooligosaccharide can be obtained. There is a disadvantage that the sugar refining process is necessary to raise. On the other hand, the method of using a sugar dislocation enzyme has the advantage that the conversion rate from starch hydrolyzate or maltose syrup to isomaltooligosaccharide is higher than other methods, whereas in the production of sugar dislocation enzymes, when liquid culture is performed, With a large amount of amylase from the cells, a small amount of glycolocation enzyme is released in the liquid phase, and in the case of solid culture, water extraction is performed for enzyme collection, and also with a large amount of amylase as in the liquid culture. A small amount of glycolocation enzyme is released, amylases interfere with the action of glycolocation enzymes, so in order to produce sugar potential enzymes, it is difficult to purify and purified from amylases. While the first two methods are actually used for industrial production, the method for producing isomaltooligosaccharides from flulan and dextran is rarely used industrially because flulan and dextran themselves are expensive.

Accordingly, it is an object of the present invention to improve the disadvantages of conventional isomaltooligosaccharide production methods to provide a new production method that is simpler and easier to produce.

The production method of the present invention is an improvement of the method for producing isomaltooligosaccharide from starch hydrolyzate or maltose syrup using sugar dislocation enzyme. Aureobasidium pullulans L48 (microbial Aureobasidium pullulans L48) used in this manufacturing method, unlike conventional microorganisms do not release the glycolocation enzyme out of the cell, but almost all accumulate in the cell, the cell itself is used as a sugar potential enzyme By directly acting on the substrate, more than 50% of isomaltooligosaccharide can be obtained. In other words, it is no need for a separate enzymatic separation and purification process from other substances including other amylases and proteins to obtain a sugar dislocation enzyme, which is a major disadvantage of the conventional method for preparing isomaltooligosaccharide using a sugar dislocation enzyme.

The present invention is described in detail as follows.

The composition of the production medium for the production of alpha glucosidase using Oreobashdium Flulans L48 is 50g / l malt, 5g / l sugar, 5g / l yeast extract, 2g / l potassium phosphate, 1g / l potassium chloride, sulfuric acid Magnesium is 1 g / l, pH is 4.5-7.0 and more preferably 4.5-5.5. The culture temperature is 20-30 ° C and more preferably 25 ° C. When shaking culture in the Erlenmeyer flask, the rotation speed of the incubator is good at around 200rpm and the incubation time is suitable for 3-5 days. The culture termination solution is centrifuged at 5,000 g for 20 minutes to collect the cells. The enzyme unit is defined as 1 unit of enzyme that generates 1 μmole of pannose per hour in 20% maltose aqueous solution when reacted at a temperature of 4.5 and 60 ° C. The obtained cells can be used for the production of branched oligosaccharide as it is, and if necessary, the cells can be crushed using chitase and the like, and then purified using only alpha glucosidase and used for the production of branched oligosaccharides.

The reaction conditions for producing branched oligosaccharides using the cells containing the produced alpha glucosidase enzyme are as follows. Branch oligosaccharides are produced by joining the cells to the substrate of 20% maltose syrup (prepared based on 0.02% acetic acid buffer solution) to about 2.0-5.0 units / ml. The reaction pH is preferably between 3.5-5.5 and more preferably 4.0-5.0. The reaction temperature is preferably 35-55 ° C, more preferably 45-50 ° C. While the reaction proceeds, stir well so that the reaction occurs evenly. If maltose syrup has been sufficiently transferred to isomaltoligosaccharide with a content of 50% or more, the enzymatic reaction is stopped by heat treatment at 100 ° C. for 10 minutes to prevent the reaction from proceeding further. Isomaltooligosaccharide content is determined by high performance liquid chromatography. On the other hand, it is also possible to use purified maltose aqueous solution instead of maltose syrup as a raw material for isomaltooligosaccharide.

Analysis of isomaltooligosaccharide by high-performance liquid chromatography is as follows. The developing solution is a mixed solution of acetonitrile and water at a mixing ratio of 60:40 using a MicroBondapak hydrocarbon column (manufactured by Waters). The temperature of the column is maintained at 25 ℃ and the type and concentration of the separated sugars are measured using a differential refractometer. If necessary, Ramnose may be used as an internal standard. The flow rate is 0.8 ml / min and the column pressure is about 130 kg / cm ² .

After completion of the reaction, the isomaltooligosaccharide mixture is centrifuged to remove the cells, desalting and decolorizing to increase the purity of the isomaltooligosaccharide, and then concentrated to obtain a syrup type colorless and odorless isomaltooligosaccharide.

The present invention is explained in detail in the following examples.

Example 1

Add 2.6 units of non-dried cells to 10 ml of 20% pure maltose aqueous solution at pH 4.5 and react for 20 hours in a 55 ℃ shake incubator. After completion of the reaction, the mixture was heated for 10 minutes in boiling water to dissipate enzymatic activity, filtered, and analyzed by high-performance liquid chromatography for the constituent sugar and content of isomaltooligosaccharide. The constituent sugar and content of the obtained isomaltooligosaccharide are as follows (Table 1).

TABLE 1

Example 2

The alphaglucosidase activity of the cells at pH 3, 4, 5, 6, 7, 8 was compared using 0.2N citrate-phosphate buffer and 0.2N phosphate buffer. At this time, the amount of cells treated was 50 units / ml, the reaction temperature was 40 ℃, the reaction time was 1 hour and the results are as follows (Table 2).

TABLE 2

enzyme activity according to pH

Example 3

In order to investigate the stability of alpha glucosidase of Oreovacidium flulans L48 with pH, 0.1 N citrate-phosphate buffer solution and 0.2 N phosphate buffer solution were used for pH 3, 4, 5, 6, 7, 8 and Cells were treated for 1 hour at 40 ° C. and then the residual activity of alphaglucosidase was compared. The amount of cells treated was 50 units / ml and the results are as follows (Table 3).

TABLE 3

Enzyme Stability According to pH

Example 4

In order to investigate the change in alphaglucosidase activity of Oreobacidis plulans L48 with temperature, 1, at 20, 30, 40, 50, 60, 70, 80 After the reaction, the enzyme activity was compared. The amount of cells treated was 50 units / ml, and the results are as follows (Table 4).

TABLE 4

Enzyme Activity According to Temperature

Example 5

In order to examine the stability of alpha glucosidase of Oreovacidium flulans L48 according to temperature, the residual enzyme activity of the non-dried cells was compared after treatment for 1 hour at 30, 40, 50, 60 and 70 ° C. The treated cell mass was 50 units / ml, the reaction pH was 5.0, and the reaction time was 1 hour. The results are as follows.

TABLE 5

Enzyme Stability with Temperature

Example 6

Isomaltooligosaccharides were prepared using industrial malt sugar from Hayashi, Japan. The composition of the industrial maltose of Hayashi Corporation used for production is as follows (Table 6).

TABLE 6

Hay malt's industrial maltose composition

After preparing maltose aqueous solution of pH 5.0 and 20% of solid content, add 5 units / ml of non-dry cell weight to 20 ml of this aqueous solution, and then react for 24 hours in a shaker at 40 ° C. to inactivate the cells. After that, the cells were removed by centrifugation to obtain isomaltooligosaccharide. The constituent sugar and the content of the obtained isomaltooligosaccharide are as follows (Table 7).

TABLE 7

Example 7

Isomaltooligosaccharide was prepared using maltose syrup from Korea's Japan-based industry. The maltose syrup composition of the Japan-Japan industry used in the experiment is as follows.

TABLE 8

Established malt sugar syrup

Using the above maltose syrup, 20 ml of maltose aqueous solution having a pH of 5.0 and a solid content of 20% was prepared, and the non-dried cell mass was added to 5 units / ml, and then reacted in a shaking reactor at 40 ° C. for 24 hours, followed by heat treatment. After inactivation and centrifugation, the cells were removed to obtain isomaltooligosaccharide. The constituent sugar and content of the obtained isomaltooligosaccharide are as follows.

TABLE 9

Claims (2)

Aureobasidium pullulans L48 (KFCC 10245), a microbial variant of the genus Aureobasidium, was cultured aerobically in a nutrient medium, followed by centrifugation of the cells containing alpha glucosidase. Method for producing isomaltooligosaccharide, characterized in that maltose or maltose, maltotriose and glucose are acted on maltose syrup containing maltose as the major constituent sugar at 3.5-5.5 and reaction temperature of 35-55 ° C. . The method of claim 1, wherein the medium contains maltose, sugar, yeast extract, potassium phosphate, potassium chloride, and magnesium sulfate.