KR0178085B1 - Mutant strains of the genus Tricosporonoides and preparation method of erythritol using the same - Google Patents

Abstract

1. TECHNICAL FIELD OF THE INVENTION

Preparation of microorganisms and organic substances

2. The technical problem to be solved by the invention

Increased yield of erythritol and development of microorganisms

3. Summary of Solution to Invention

A mutant strain tricosphoronoides producing erythritol in high yield and a method of culturing this strain to produce erythritol.

4. Important uses of the invention

food

Description

Mutant strains of the genus Tricosporonoides and preparation method of erythritol using the same

The present invention relates to a novel microorganism producing erythritol and a method for producing erythritol using the microorganism. More specifically, the present invention relates to artificial mutants of microorganisms belonging to the genus Tricosporonoides and to a method for producing erythritol in high yield by aerobic culturing this strain in nutrient medium.

The applicant named the mutant strain developed in the present invention as Tricosporonoides madida and deposited it with the accession number KCTC 8712P at the Biotechnology Research Institute.

Erythritol is a natural substance present in the body fluids of fermented foods, plants such as mushrooms, and mammals, and has a sweetness of 70 to 80% of sugar. Erythritol is a low-calorie sweetener whose calories are less than 1/10 (0.4 Kcal / g) of sugar. It is endothermic when dissolved and has a cool sweetness. In addition, the decayed Streptococcus mutans (S. mutants) can not decompose and use erythritol, so it does not produce acid or glucan, and the disadvantage of conventional sugar alcohols is that it causes diarrhea when ingested in large quantities. It is known that erythritol and erythritol have a physiological feature that causes diarrhea.

Conventional methods for producing erythritol are methods using microorganisms of the genus Aureobasidium (JP 62-24716), methods using Tolua microorganisms (EPO136,805), Torigonopsis variabilis (JP 49-118889) and Candida lipolytica (USP 3,756,917), but the above strains have a long fermentation period and low culture sugar concentration (cultivation time: 168 to 200 hours, Sugar concentration: 20 to 40%) The fermentation yield is low (40% yield level) has a disadvantage, and in addition to erythritol tends to by-produce other sugar alcohols such as glycelulol has a lot of problems when purifying erythritol from fermentation broth.

Therefore, an object of the present invention is to develop a high titer strain that can overcome the disadvantages of the conventional erythritol production method, and to improve the production yield of erythritol using it.

The present inventors incubated the wild strain Tricosporonoides sp. CFW001 in a medium containing 20% glucose or 20% sugar and 1% powder enzyme, and then centrifuged the culture solution to separate the supernatant and the cells. The solution was diluted with a buffer solution containing N-methyl-N'-nitro-N-nitrosoguanidine at a concentration of 1 mg / ml for 30 minutes, followed by agar medium containing 50% glucose and 0.5% powdered yeast. Among the colonies produced by the above, a mutant tricosphoronoides madida having excellent erythritol production ability (named Trichosporonoides sp. ST1 at the time of publication at the 29th Biochemical Symposium) was obtained.

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

1) Creamy colonies are formed in yeast malt agar medium and become black colonies over time. In the single cell form, acute mycelium may be formed depending on conditions. It reproduces by sprouting and forms segmental spores, sprouted spores, and conidia.

2) Physiological Properties

Growth temperature: up to about 40 ℃

Optimum growth temperature: 28-32 ℃

Growth pH: 2.5 to 8.0

Optimal pH: 4.0 to 7.0

Urease Generation: Yes

DBB (Diazonium B Blue) Dyeing: Positive

KNO ₃ Availability: Yes

Generate Hyphae: Yes

3) Fermentation of Sugar

4) assimilation of sugar organic acids

The strain of the present invention having the above microbiological properties was found to be a different strain from the microorganisms belonging to the genus Tricosporonoides known to date when classified according to The Genera of Fungi Sporulating in Pure Culture (J. A. Von Arx: 1974).

Accordingly, the present invention provides novel strains of the genus Tricosphoronoides.

In another aspect, since the mutant strain according to the present invention produces erythritol in high yield, the present invention provides an improved method for producing erythritol by culturing tricosphoronoides madida strains.

Carbon sources that can be used for culturing the high titer strain according to the present invention include glucose, sugar, fructose, and the like. Powdered yeast, heptone, and urea may be used as organic and inorganic nitrogen sources.

The culture temperature of the modified strain of the present invention is suitable in the range of 25 to 37 ℃, preferably in the range of 30 to 32 ℃. The optimum pH for culturing is around neutral, and erythritol is produced by culturing with aeration and agitation for 4 to 5 days. Erythritol is mainly produced in the culture medium, and a small amount of glycerol is produced. The lower the amount of glycerol, the better the crystallization yield during the erythritol purification process. The purification process can use a conventional purification method.

The following examples more particularly illustrate the method of the present invention. However, it should not be understood that these examples are intended to limit the present invention.

Example 1

Tricosphoronoides Madida strains were plated in agar preservation medium containing 40% glucose and 1% powder enzyme and incubated at 32 ° C for 3 days. One hundred micrograms of cells obtained from the agar preservation medium culture were taken and inoculated into a 250 ml Erlenmeyer flask containing 50 ml of culture medium containing 40% glucose, 1% yeast powder, and 0.1% urea. It was then incubated with shaking (200 rpm) for 120 hours at 32 ℃. After completion of the culture, the erythritol yield and the glycerol yield were 45% and 20%, respectively.

Example 2

50 ml of a medium containing 40% of sugar, 1% of powdered enzyme, and 0.1% of urea was placed in a 250 ml Erlenmeyer flask and inoculated aseptically with platinum of tricosporonoides madida cells obtained from agar preservation medium culture. It was then incubated with shaking (200 rpm) for 120 hours at 32 ℃. After completion of the culture, the yield of erythritol was 40%, and the glycerol yield was 4%.

Example 3

3L of medium containing 40% glucose, 1% powdered yeast, 0.1% urea was added to a fermenter of 5L capacity, and a 500 rpm silicone antifoaming agent was added, followed by sterilization at 121 ° C., 1.2 atm, and 15 minutes. 150 ml of Tricosporonoides Madida cell culture medium was inoculated and incubated for 4 days under conditions of aeration of 1 vvm, 32 ° C, and 600 rpm. The erythritol yield was 48% and the glycerol yield was 1.4% after the completion of the culture.

Example 4

20 L of medium containing 40% of glucose, 1% of powdered yeast, and 0.1% of urea was added to a 30 L fermenter, and 500 ppm of silicone oil-based antifoam was added and sterilized at 121 ° C., 1.2 atm for 15 minutes. 1 L of Tricosporonoides Madida cell culture medium was inoculated and incubated for 4 days under conditions of aeration 0.7vvm, 32 ° C, and 400 rpm. After completion of the culture, the yield of erythritol was 50% and the glycerol yield was 1.2%.

Example 5

800L of medium containing 40% of glucose, 1% of powdered yeast and 0.1% of urea was added to a 1.2KL fermenter, and 500ppm of silicone oil-based antifoam was added and sterilized at 121 ° C., 1.2 atm for 15 minutes. 60 L of Tricosporonoides Madida cell culture medium was inoculated and incubated for 4 days under conditions of aeration 0.5vvm, 32 ° C and 250ppm. After completion of the culture, the yield of erythritol was 50% and the glycerol yield was 1%.

Claims (4)

Mutant strain tricosphoronoides Madida producing erythritol in high yield KCTC 8712P. Method for producing erythritol, characterized in that the strain according to claim 1 in aerobic culture. The method of claim 2 wherein the carbon source used in the medium is glucose, fructose or sugar, or a mixture thereof, and the nitrogen source is powdered yeast, peptone or urea, or a mixture thereof. The method of claim 2 or 3, characterized in that the incubation with stirring for 4 to 5 days at a temperature of 25 to 37 ℃ and neutral pH.