KR890001127B1 - Producing method for oligo-fructose - Google Patents

Abstract

A method for preparing oligo-fructose is presented. Thus, Aureobasidium L-48 (KFCC-10245) was cultured in a medium contg. 0.75% KH2PO4, 1.5% NaNO3, 20% sucrose, 0.05% MgSO4.7H2O and 2.5% dried yeast at 32≰C for 96hr (180 rpm) to give 480-500 unit/ml of fructotransferase. Five unit of fructotransferase per 1g of sucrose was reacted with 70% sucrose soln. at pH 5.5 and 55≰C for 22hrs to give oligo-fructose.

Description

Method for preparing fructo-oligo sugar

In the present invention, a microorganism mutant strain (Aure -obasidium L48: KFCC-10245) belonging to the genus Aureobasidium is cultured aerobically in a nutrient medium containing a carbon source, a nitrogen source, and other microorganisms required, and puructosyl trans The present invention relates to a method for producing furucto-oligosaccharides by producing a fructosyltransferase and then reacting the produced fructosyltransferase with sugar. Fructooligosaccharides are composed mainly of Kestose, which has one fructose in sugar, and Nystose, which has two fructose in sugar. It is effective in preventing indigestion and tooth decay, and is a Bifidus growth factor. It is widely used as a low-calorie sweetener, health food, animal feed and medicine.

Conventional methods for producing such fructosyltransferases include Oribasidium pullulan (Aureobasidium). pullulans) (US Pat. No. 4,309,505), Aspergillus niger (Japanese Patent No. 58,162,292) and Penicillium (Penicillium) and Fusarium (Fusarium) (Japanese Patent No. 6,819,839) and the like, but these methods had a disadvantage in that it is difficult to use industrially due to low enzyme titer (200 to 440 units / ml). Therefore, the present invention is a modified strain Oriobashdium L48 (KFCC-10245) of the present invention artificially mutated to solve these problems in the conventional method comprising a carbon source, nitrogen source, amino acids, minerals and inorganic salts, and triggers By incubating in the optimal medium for the preparation of fuructosyltransferase to produce a high titer, using a special enzyme, kinase (Kitalase) to extract the high yield of fructosyltransferase in the cells, and then added to a high concentration of sugar solution The production of large amounts of fructooligosaccharides was possible.

The present invention will be described in more detail as follows. Parent Oriobadium pullulan (ATCC-9348) is germinated medium (sugar 5%, powder yeast 0.4%, potassium phosphate 0.3%, salt 0.1%, ammonium sulfate 0.06%, magnesium sulfate 0.02%, iron sulfate 0.01% After incubation for 24 to 48 hours, the supernatant was separated by centrifugation, suspended in sterilized 0.1 M magnesium sulfate solution, and then irradiated with ultraviolet rays (0.1-1% survival) for 20 minutes to induce mutations. Then, spread on agar preservation medium (2% sugar, 0.4% powdered yeast, 0.3% potassium monophosphate, 0.1% salt, 0.06% ammonium sulfate, 0.02% magnesium sulfate, 0.01% iron sulfate, 1.5% agar), After culturing at 32 ° C. for 24 to 48 hours, the isolated colonies were flask cultured to prepare a mutant strain of the present invention (KFCC-10245) having a high capacity for producing fructosyltransferase.

The mutant strains of the present invention have the following strain characteristics.

1) It is an incomplete fungus whose morphology changes from yeast to mycelium by asexual reproduction depending on environmental conditions. In other words, initially, colonies aggregate to grow into round pellets and grow into a grayish white yeast form, and then, as time passes, the yeast form tends to grow in a mycelium form.

2) If the environmental conditions are good at the time of growth in the mycelium form, the mycelium can continue the growth of the yeast form, in which the enzyme is vigorously produced.

3) When environmental conditions deteriorate, that is, nutrient depletion of the medium occurs, viable thick-filmed spores form in the hyphae and gradually start to produce pigments on the surface of the colony. After a week or so, black spots appear on the surface of the colony, gradually spreading through the cells and darkening the front of the colony.

Carbon sources that can be used in the culturing of high titer mutants of the present invention include glucose, lactose, fructose, sugar, mannitol and glycerol, and organic and inorganic nitrogen sources include powdered yeast, peptone, sodium nitrate, ammonium nitrate and amino acids. Can be. As the mineral and inorganic salts, magnesium sulfate, zinc sulfate, first potassium phosphate, dibasic potassium phosphate, and the like can be used.

The culture temperature of the modified strain of the present invention is suitable in the range of 25 ~ 32 ℃, preferably in the range of 30 ~ 32 ℃. The optimum pH for culturing is around neutral, and when cultured with aeration and agitation for 3 to 4 days, fuructosyltransferase is produced in and between cells. Extracellular enzymes can also be used for the production of fructooligosaccharides, but they also contain enzymes other than the fructosyltransferases, such as invertase, isomerase, and the like, thus producing fructose due to sugar breakdown and glucose isomerization. Will cause.

Therefore, it is preferable to use only the intracellular enzymes separately since the extracellular enzymes result in a decrease in the degree of production of fructooligosaccharides from sugar. As a method of recovering the fuructosyltransferase in the cell, a method of crushing the cell has been used. However, the inventors of the present invention use a special enzyme, ie, a kitase (Kitalase, Japan's Gumi Eye Chemical Co., Ltd.). It has been found that the fructosyltransferase can be extracted in high yield. The separated enzyme filtrate may be purified by conventional enzyme purification methods such as ultrafiltration, sodium sulfate salting, and ion chromatography.

Fructosyltransferase produced by culturing the mutant strains of the present invention causes the following transfer reaction.

Unlike the hydrolysis reaction, the enzymatic reaction proceeds well in a high concentration of sugar solution, so that the enzyme of the present invention can be industrially prepared from fructooligosaccharide from a high concentration of sugar solution. After the end of the enzyme reaction, the enzyme is re-reduced at a high temperature, and then, after discoloring and purifying the fulucto oligosaccharide to prepare the product, the content of the fructooligosaccharide in the reaction termination solution is preferably 50% or more. The content of fructooligosaccharide was analyzed using high performance liquid chromatography.

The titer of the enzyme was determined by measuring the amount of glucose released by the enzyme transfer reaction, and the enzyme yielded 1 μmol of glucose per minute under a substrate concentration of 60 w / v%, a temperature of 55 ° C., and a pH of 5.5. The amount was defined as 1 unit.

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

Example 1

Germination

After inoculating 1 ml of the strain (KFCC-10245) of the present invention into a 500 ml shake culture flask containing 100 ml of sterilized germination medium as described above, the mixture was shaken at 32 ° C. (180 rpm) for 2 days. Incubated.

Spawn culture

In a 500 ml shake culture flask containing 100 ml of sterilized germination medium, 5 ml of the culture solution obtained from germination culture was inoculated, and cultured for 1 day under the same conditions as in germination culture, followed by use as a seed.

Present culture

1 ml of the culture solution obtained from the above seed culture was aseptically grown in a 500 ml flask containing 100 ml of the culture medium of the following composition.

Main culture medium composition

0.75% potassium monophosphate, 1.5% sodium nitrate, 20% sugar, 0.05% magnesium sulfate, 2.5% powdered yeast.

Then incubated with shaking rotation (180 rpm) for 96 hours at 32 ℃. After completion of the culture, the fructosyltransferases produced in and outside the cells were 480-500 units / ml.

Example 2

Fermentation It carried out similarly to Example 1 except adding 0-5% of amino acid valine to the composition of this medium. The amount of fuctosyltransferase produced following the addition of valine is shown in Table 1.

Example 3

Fermentation It carried out like Example 1 except adding 0-1.0% of D-mannitol to the composition of this medium. The amount of fuctosyltransferase produced by the addition of D-mannitol is shown in Table 2.

Example 4

Fermentation It was carried out as in Example 1, except that 0 'to 10 mM of 3', 5'-cyclic adenosine monophosphoate (3 ', 5-CAM P) was added to the composition of the medium. The amount of fuctosyltransferase produced following addition of 3 'and 5'-cyclic adenosine monophosphate is shown in Table 3.

Example 5

In order to extract the fructosyltransferase in the cells produced by the main culture of Example 1, the culture medium was centrifuged, and 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 45 ° C., followed by centrifugation to recover the enzyme. The titer of recovered enzyme was 250 units / ml.

Example 6

The fructosyltransferase obtained in Example 5 was added to a 70% sugar solution at a concentration of 5 units per g of sugar, and then reacted at pH 5.5 and 55 ° C. for 22 hours. Then, decolorized and purified, heated at 100 ° C for 10 minutes to deactivate the enzyme and at the same time sterilization, and then cooled to prepare a fructooligosaccharide. The composition ratio (%) of the solid content of the produced fructooligosaccharide is as follows.

Fructose 0.43%, Glucose 19.8%, Sugar 16.6%, Kestose 45.8%, Nittos 17.2%

Example 7

Partially purified extracellular fuructosyltransferase obtained from this culture was added to a 70% sugar solution at a concentration of 5 units per gram of sugar, followed by reaction at pH 5.5 and 55 ° C. for 22 hours. Then, decolorized and purified and heated at 100 ° C. for 10 minutes to recapture the enzyme and at the same time sterilize, and then cooled to prepare a fructooligosaccharide. The composition ratio (%) of the solid content of the produced fructooligosaccharide is as follows.

Fructose 1.2%, Glucose 23.8%, Sugar 22.2%, Kestose 39.6%, Nittos 13.2%

Claims (5)

The microorganism mutant Oriobassidium L-48 (KFCC-10 245) belonging to the genus Oribasidium is cultured aerobicly in a nutrient medium to produce fuructosyltransferase, and then the fuructosyltransferase in cells is extracted to act on sugar. Method for producing a fructooligosaccharide characterized in that. The method for producing furoktooligosaccharide according to claim 1, wherein the nutrient medium is cultured by adding 2 to 3% of valine. The method for producing furoktooligosaccharide according to claim 1, wherein the nutrient medium is added with 0.5 to 0.8% of mannitol and cultured. The method of claim 1, wherein cyclic adenosine monophosphate is added to the nutrient medium as an inducer for 6 to 8 mM culture. The method for producing furoktooligosaccharide according to claim 1, wherein the fuluctosyltransferase produced in the microorganism strain of the genus Orobasidium microorganism strain (KFCC-10245) is extracted and recovered by treatment with chitase.