KR950007223B1 - Preparation method of branched oligosaccharides - Google Patents

Abstract

The branched oligosaccharide is prepd. by i) innoculating aureobasidium L48 (KFCC 10245) into a medium and fermenting at 30 deg.C and 200 rpm for 2 days to obtain the starter, iii) innocluating the obtd. starter into a medium and re-fermenting at 30 deg.C and 20 rpm for 48 hrs., iii) centrifuging the re-fermented broth to obtain the cell and suspendiung in the distillated water, iv) extracting transglucosidase of the cell by adding kitalase into the cell-suspended water and reacting at 40 deg.Cm pH 5.9 for 2 hrs., and v) reacting the obtd. transglucosidase with 20% maltose soln. to obtain the final product.

Description

Method of producing branched oligosaccharides

In the present invention, transurecocci by aerobic culture of microorganism strains belonging to the genus Aureobasidium (Aure obasidium L48: KFCC 10245) in nutrient medium containing carbon atoms, nitrogen atoms and other microorganisms are required. The present invention relates to a method for producing branched oligosaccharides by generating a gluase (Transglucosidase) and then reacting the resulting transglucosidase with starch sugar.

Branched oligosaccharides are composed mainly of 2-4 sugars composed of alpha 1 → 6 bonds. Iso-maltose, panosoe, Iso-maltotriose, and isomaltovetrose. maltotetrose) is a constituent. Efficacy in preventing indigestion and tooth decay. It is widely used as a bifidus growth factor, low calorie sweetener, health food, animal feed and medicine.

As a conventional method for preparing such a transglucosidase, a method using Aspergillus niger (published JP 61-124389) and a method using Taralamys duponti (USP 4,6689,296) However, there was no industrial use using Aureobasidium pullulans, a kind of yeast.

Therefore, in the present invention, by producing a high titer of transglucosidase by cultivating a new type of bacterium Oriobacidium flulans, which is not an existing branched oligosaccharide producing strain, in a nutrient optimal medium containing a carbon source, a nitrogen source, amino acids, and minerals, A high yield of transglucosidase in cells was extracted using a special enzyme, kitalase, and then, it was added to a high concentration of maltose solution to industrially produce large amounts of branched oligosaccharides.

The culture temperature of the strain of the present invention is suitable in the range of 25-32 ℃, preferably in the range of 30-32 ℃ but the optimum fold of the pH amount is near the neutral and when the aeration for 1-3 days, transglucosidase in the cells Is generated.

Although a method of destroying the cells has been used as a method for recovering intracellular transglucosidase, the present inventors have used a special enzyme, namely, Kitaraso (Kitalaso Nippon Iku Chemical Co., Ltd.) without the disruption of the cells. It was found that the cidases can be extracted in high yield.

The separated enzyme filtrate can be purified by conventional enzyme purification methods such as intraocular filtration, sodium sulfate staining and ion chromatography. Transglucose sugars can also industrially produce branched oligosaccharides in high concentrations of malt solutions. After the end of the enzyme reaction, the enzyme is deactivated at a high temperature, and then the branched oligosaccharide is decolorized and purified to produce a product.

The content of branched oligosaccharides was analyzed using high performance liquid chromatography. The titer of the enzyme was determined by measuring the amount of generated panoose because the branched oligosaccharide component Panoose (Parnose) is produced by the enzyme transfer reaction, substrate concentration 20w / v%. One unit was defined as the amount of enzyme which produces 1 micromole of phanous per minute under the condition of the temperature of 40 degreeC and pH5.0.

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

Example 1

Spawn culture

After inoculating 100 ml of the strain of the present invention (KFCC 10245) into a 250 ml shake culture flask containing 50 ml of sterilized germinated medium (2% malt, 0.2% powdered yeast, pH5.0), at 30 ° C Incubated for 2 days while shaking culture (200rpm).

Present culture

1 ml of the culture solution obtained from the above seed culture was aseptically sterilized into a 250 ml flask containing 50 ml of the present culture medium having the following composition.

The culture medium composition is as follows.

Maltose 15%, powdered yeast 2%, sulfuric acid-magnesium sulfate 0.05%, potassium chloride 0.1%, potassium phosphate 0.1% and then incubated with shaking at 20 ℃ (20rpm) for 48 hours. The transglucosidase produced in the cells after completion of the culture was 10-15 units / ml.

Example 2

In order to extract the transglucosidase in the cells produced by the main culture of Example 1, the culture medium was centrifuged, and then the cells as solids were suspended in water to adjust the concentration to about 20%.

Powder chitase was added thereto at a concentration of 1.2 w / w% per g of cells, and then stirred at 100 rpm for 2 hours at pH 5.9 and 40 ° C., followed by centrifugation to recover the enzyme. The titer of the recovered enzyme was 14 units / ml.

Example 3

The transglucosidase obtained in Example 2 was added to a 20% maltose solution at a concentration of 1 unit per g of maltose, and then reacted at pH 5.0 at 40 ° C. for 24 hours. Then, decolorized and purified and heated at 100 ° C. for 10 minutes to recapture the enzyme and simultaneously sterilize, and cooled to prepare a branched oligosaccharide. The composition ratio (%) of the solid content of the prepared branched oligosaccharides is as follows.

Glucose 36%, maltose 12%, isomaltose 13.3%, maltotriose 1.47%, panos 29%, isomaltotriose 8.3%.

Claims (2)

The microorganism strain Oriobacidium L-48 (KFCC-10245) belonging to the genus Oriobasidium was cultured aerobicly in a nutrient medium to generate transglucosidase, and then the transglucosidase in cells was extracted to act on maltose. Method for producing a branched oligosaccharide, characterized in that. The method for producing a branched oligosaccharide according to claim 1, wherein the transglucosidase produced in the microorganism of the genus Orobasidium microorganism (KFCC-10245) is treated with chitase to extract and recover.