JPH11137285A - Production of ribitol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To industrially and advantageously obtain ribitol from a cheap and easily obtainable raw material by culturing a microorganism belonging to the genus Trichosporonoides in a culture medium containing a fermentable saccharide as a main source of carbon and taking the product from the resultant culture. SOLUTION: This method is conducted by aerobically culturing a microorganism (e.g. Trichosporonoides oedocephalis ATCC 16958 or the like) belonging to the genus Trichosporonoides in a culture medium containing a fermentable saccharide as a main source of carbon in a saccharide concentration of 10 to 45 wt.% at pH of 3 to 5.5 at 24 to 38 deg.C, feeding the obtained culture into a column packed with a strong acid cation exchange resin of any one of aluminum, manganese and iron types, and taking ribitol by a chromatography fractionation adapted to separate a ribitol fraction, thus obtaining the objective ribitol industrially and advantageously by using an easily obtainable and cheap raw material instead of an expensive ribose.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to a method for producing ribitol,
More particularly, the present invention relates to a method for industrially advantageously producing ribitol by a fermentation method using a microorganism.

[0003]

2. Description of the Related Art

[0004] Ribitol is a Western species of Adonis
vernalis L. (Ranunculaceae) and Mishimasaiko Bupl
eurum falcatum is a kind of pentasaccharide alcohol contained in the root of L. (Apiaceae).

[0005] This ribitol can be obtained by catalytic hydrogen reduction of ribose in the presence of various solid catalysts. Alternatively, ribitol can be obtained by inoculating Candida polymorpha or Torulopsis hamada to ribose and fermenting it. No. 45-2071), a method of culturing a strain belonging to Corynebacterium in a medium containing ribose as a main carbon source (Japanese Patent Publication No. 49-12718).
Etc. can be manufactured.

[0006] However, ribose used as a raw material is produced mainly by chemical decomposition of purine nucleotides, but is expensive and is not suitable as a practical industrial raw material for producing ribitol.

[0007] In addition, there has been reported a method of producing ribitol by irradiating algae for photosynthesis with irradiation of light (Japanese Patent Publication No. 44-16350). However, ribitol is accumulated in a culture solution. By the time, a long time was required, the amount of accumulation was small, and therefore, it was not always a satisfactory method.

An object of the present invention is to find a method for industrially producing ribitol using readily available and inexpensive raw materials.

[0009]

[Means for Solving the Problems]

The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, the present inventors have found that microorganisms belonging to the genus Trichosporonoides can be cultured in a medium containing fermentable sugars such as glucose, fructose or sucrose as a main carbon source. It has been found that culturing produces ribitol, and furthermore, it has been found that ribitol is produced at a high yield under specific culturing conditions, thereby completing the present invention.

That is, means for solving the problems of the present invention are as follows.

First, a microorganism belonging to the genus Trichosporonoides is cultured in a medium containing fermentable carbohydrate as a main carbon source.
A method for producing ribitol, comprising collecting ribitol from the obtained culture. Secondly, the method for producing ribitol according to the first aspect, wherein the microorganism belonging to the genus Trichosporonoides is Trichosporonoides oedespharis. Third, the cultures have carbohydrate concentrations of 10-45% by weight, p
The method for producing ribitol according to the first or second aspect, wherein the method is performed under aerobic conditions at a temperature of H3 to 5.5 and a temperature of 24 to 38 ° C. Fourth, harvesting ribitol from the culture
The first to third chromatographic fractions, in which the culture is supplied to a column filled with a cation exchange resin to separate a ribitol fraction,
The method for producing ribitol according to any one of the above. Fifth,
The method for producing ribitol according to the above item 4, wherein the cation exchange resin is a strongly acidic cation exchange resin in any of an aluminum form, a manganese form and an iron form.

The microorganism used in the present invention is not particularly limited as long as it belongs to the genus Trichosporonoides and has the ability to produce ribitol from fermentable sugars such as glucose, fructose or sucrose. .

Examples of microorganisms belonging to the genus Trichosporonoides include, for example, Trichosporonoides oedocephalis and Trichosporonoides megachiriensis (Trichosporonoides me).
gachiliensis), and in particular, Trichosporonoides oeedespharis is most suitable for producing ribitol.

The strains of these microorganisms are C
entral Bureau voor Schimmelcultures (CBS) and Mary, USA
land is readily available from the American Type Culture Collection (ATCC); specifically, for example, Trichosporonoides oedespharis is available from the ATCC in the United States as "A
TCC 16958 ”and Trichosporonoides megachiriensis from the Dutch CBS“ CB
S 191.92 ".

As the carbon source of the present invention, carbohydrates such as glucose, fructose and sucrose can be employed. When glucose is used as the carbon source, the cultivation time for producing ribitol is short, and the conversion to ribitol is further reduced. Is also preferred.
These carbon sources are usually used alone, but may be used in combination of two or more.

The concentration of sugar as a carbon source is 10 to 45% by weight, preferably 30 to 40% by weight.

When the concentration of the sugar as a carbon source is lower than 10% by weight, the amount of handling in the purification step after fermentation increases, a large treatment facility is required, and more energy is required for concentrating the purified liquid. Required and industrially disadvantageous.

The concentration of sugar as a carbon source is 45% by weight.
If it is higher, it takes much time until all the sugar is consumed, which is not preferable.

The cultivation in the present invention is carried out under aerobic conditions using a liquid medium, and is particularly preferably carried out with aeration and agitation.

The cultivation time varies depending on the type of medium and the concentration of sugar as a carbon source, but is usually about 5 to 15 days, for example, until all of the sugar as a carbon source is consumed or the production amount of ribitol. The culture is stopped at the time when is maximum.

The amounts of sugar and ribitol as a carbon source in the culture solution can be easily analyzed by high performance liquid chromatography.

The cultivation of the present invention is carried out by adjusting the pH to the range of 3 to 5.5.

The cultivation of the present invention is carried out within a temperature range in which microorganisms can grow, that is, at 24 to 38 ° C.

As the nitrogen source, there can be used nitrogen compounds such as yeast extract, corn steep liquor and urea which can be used by microorganisms.

In addition, various organic substances, inorganic substances, antifoaming agents and the like can be added to the culture solution, if necessary.

The ribitol produced in the culture solution is collected as a ribitol solution by removing the cells from the culture solution by centrifugation, filtration, etc., and further purifying using activated carbon treatment or an ion exchange resin. Can be.

The resulting ribitol solution is concentrated as it is and then crystallized, or passed through a packed column filled with a cation exchange resin, and subjected to chromatographic fractionation utilizing the difference in the adsorptivity of each component to the resin. By increasing the content of ribitol and then crystallization, ribitol crystals can be obtained.

As the cation exchange resin used in the chromatographic fractionation of the present invention, most commercially available resins can be used.
Among them, a strongly acidic cation exchange resin in which a sulfonic acid group is bonded to a crosslinked polymer of styrene-divinylbenzene is commercially available, and charged with ions such as aluminum, magnesium, barium, manganese, iron, and potassium by a conventional method. In particular, aluminum, manganese, or iron-type strongly acidic cation exchange resin charged with aluminum ion, manganese ion, or iron ion to the strongly acidic cation exchange resin can be used to separate ribitol from the culture medium. Excellent for separating from the components of

The chromatographic fractionation of the present invention can be any of a batch type or a simulated moving bed type, a single column type or a multiple column type.

[0031]

【Example】

Hereinafter, the method of the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to the following examples. In the following examples,% represents% by weight unless otherwise specified.

[0033]

Embodiment 1

(Preculture) 100 ml of a medium containing 200 g / l of anhydrous crystalline glucose and 15 g / l of corn steep liquor was placed in a 500 ml Sakaguchi flask.
Trichosporonoides Oedes Separis (ATCC
16958) was inoculated and shake-cultured at 35 ° C. for 2 days to obtain a pre-culture solution.

(Main culture) 3 kg of anhydrous crystalline glucose, 225 g of corn steep liquor, 600 ml of preculture liquid (six preculture liquids), and 4.5 g of antifoaming agent (Nippon Oil & Fats Co., Ltd., Deform CA-123) 15 liters of the culture medium into a 30 liter incubator, pH 4.5, temperature 35
Culturing was performed for 8 days at a temperature of 800 ° C., a rotation speed of 800 rpm, and an aeration rate of 1.0 vvm. At this time, all the glucose in the culture solution had been consumed. The culture solution was analyzed using liquid chromatography, and it was found that 5 ml of ribitol per 1 ml of the culture solution was used.
7.4 mg (28.7% yield), erythritol
It contained 6 mg (19.3% yield) and 4.6 mg glycerin (2.3% yield). Next, the cells were separated from the culture using a centrifugal separator, stirred with 50 g of activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd., Shirasagi) at a temperature of 50 ° C. for 1 hour, and the activated carbon was filtered off. 300 ml of cation exchange resin (IR-120B, manufactured by Organo Co., Ltd.) and 50 anion exchange resin (IRA-410, manufactured by Organo Co., Ltd.)
After passing through a column filled with 0 ml, the solution was concentrated to a concentration of 60% to obtain 2510 g of a ribitol-containing solution.

(Chromatographic Fractionation Apparatus) As shown in the schematic diagram of FIG. 1, the chromatographic fractionation apparatus used in this example is a 1-liter jacketed glass tower (with an inner diameter of 3.6 c).
m, 105cm in length) A to F are connected in series,
Is connected to a raw material charging pump G via a preheater K and a valve H, and to a water charging pump I via a preheater K and a valve J. In addition, the chromatographic fractionation apparatus attaches effluent receiving tanks L and M to the lower part of the column F via valves N and O,
The valve P is attached to the tip side of the valve O. Each of the towers A to F was filled with 1000 ml of an aluminum form of a strongly acidic cation exchange resin (CR-1310, manufactured by Organo Corporation) per one tower.

(Chromatographic fractionation) While maintaining the columns A to F at 60 ° C., the valves H and P were opened, and the valves J, N and O
Is closed, and the above-mentioned 60% concentration of ribitol-containing liquid 3
00 ml was flowed at a rate of 50 ml per minute. Next, valve H
Is closed, valve J is opened, and water is pumped 50 m / h from pump I.
Feed at a rate of 1. The concentration at the outlet of valve P is 0.2%
At this time, the valve P was closed and the valve N was opened. 2
Two minutes later, valve N was closed and valve O was opened. Valve O
When the concentration at the outlet of became 0.2% or less, the valve O was closed and the valve P was opened. This operation was repeated. Table 1 shows the concentrations, weights, and sugar compositions of the effluents obtained in the tanks L and M.

[0038]

[Table 1]

(Crystallization) The effluent of the tank L is made to have a concentration of 77%.
The solution was transferred to a crystallizer having a capacity of 500 ml, and cooled from room temperature to 70 ° C. over 10 hours. On the way 60
At 0 ° C., a small amount of crystalline ribitol powder was added as a seed. The generated crystals were separated using a centrifuge and washed with a small amount of water. The obtained crystals weighed 144 g after drying. This crystal had a melting point of 102.8 ° C., a purity of 99.9% by liquid chromatography analysis, and was confirmed to be ribitol by analysis of liquid chromatography, gas chromatography, infrared absorption spectrum and NMR spectrum. did.

[0040]

Embodiment 2

(Main culture) 1.5 kg of anhydrous crystalline glucose,
Corn steep liquor 112g, Example 1 (pre-culture)
600 ml of a pre-culture liquid and 4.5 g of an antifoaming agent (manufactured by NOF CORPORATION, Deform CA-1)
15 liters of the medium containing 23) was placed in a 30 liter incubator, and the pH was 4.5, the temperature was 35 ° C., and the rotation speed was 800 rpm.
Culture was carried out for 8 days at a flow rate of 0.8 vvm. At this time, all the glucose in the culture solution had been consumed. The culture solution was analyzed using liquid chromatography. As a result, 29.5 mg of ribitol (yield 29.5%) and 17.4 mg of erythritol (1% yield) per ml of the culture solution were analyzed.
7.4%) and 1.7 mg of glycerin (1.7% yield). Next, cells were separated from the culture solution using a centrifugal separator, and then 20 g of activated carbon (Takeda Pharmaceutical Co., Ltd.)
After stirring for 1 hour at a temperature of 50 ° C. with Shirasagi), the activated carbon is filtered off, and the filtrate is a cation exchange resin (Organo Co., Ltd.)
(IRA-120B) and 300 ml of an anion exchange resin (IRA-410, manufactured by Organo Corporation), and then concentrated to a concentration of 60% to obtain 1205 g of a liquid containing ribitol.

(Chromatographic fractionation) The following chromatographic fractionation was carried out using the same chromatographic fractionation apparatus as in Example 1 except that the strongly acidic cation exchange resin was in the manganese form.
While keeping the towers A to F at 60 ° C., the valves H and P were opened and the valves J, N and O were closed,
300 ml of a liquid containing 10% ribitol was flowed at a rate of 50 ml per minute. Next, the valve H was closed, the valve J was opened, and water was supplied from the pump I at a rate of 50 ml / h. Valve P
When the concentration at the outlet of was 0.2%, the valve P was closed and the valve N was opened. After 20 minutes, valve N was closed and valve O was opened. When the concentration at the outlet of the valve O became 0.2% or less, the valve O was closed and the valve P was opened. This operation was repeated. Table 2 shows the concentrations, weights, and sugar compositions of the effluents obtained in the tanks L and M.

[0043]

[Table 2]

(Crystallization) The effluent of the tank L is made to have a concentration of 77%.
The solution was transferred to a crystallizer having a capacity of 500 ml, and cooled from room temperature to 70 ° C. over 10 hours. On the way 60
At 0 ° C., a small amount of crystalline ribitol powder was added as a seed. The generated crystals were separated using a centrifuge and washed with a small amount of water. The obtained crystals weigh 82 g after drying, and the purity by liquid chromatography analysis is 100%.
Met.

[0045]

Embodiment 3

Cultivation was carried out in the same manner as in Example 1 except that the raw material for producing ribitol was crystalline fructose. The obtained culture solution contained 45.0 m of ribitol per ml.
g (yield 22.5%), erythritol 45.2 mg
(Yield 22.6%), glycerin 0.8 mg (yield 0. 2%).
4%). Next, the cells were separated from the culture using a centrifugal separator, and stirred with 50 g of activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd., Shirasagi) at a temperature of 50 ° C. for 1 hour.
The activated carbon was filtered off, and the filtrate was 300 ml of cation exchange resin (IR-120B, manufactured by Organo Co., Ltd.) and 500 m of anion exchange resin (IRA-410, manufactured by Organo Co., Ltd.)
After passing through a column filled with 1, the mixture was concentrated to a concentration of 60% to obtain 2268 g of a ribitol-containing liquid.

(Chromatographic fractionation) The following chromatographic fractionation was carried out using the same chromatographic fractionation apparatus as in Example 1 except that the strongly acidic cation exchange resin was in the iron form. AF
While maintaining the temperature of each column at 60 ° C., the valves H and P were opened, and the valves J, N and O were closed, and 300 ml of the above-mentioned 60% concentration of ribitol-containing liquid was flowed at a rate of 50 ml per minute. Next, the valve H was closed, the valve J was opened, and water was supplied from the pump I at a rate of 50 ml / h. When the concentration at the outlet of the valve P became 0.2%, the valve P was closed and the valve N was opened. After 22 minutes, valve N was closed and valve O was opened. When the concentration at the outlet of the valve O became 0.2% or less, the valve O was closed and the valve P was opened. This operation was repeated. Table 3 shows the concentrations, weights, and sugar compositions of the effluents obtained in the tanks L and M.

[0048]

[Table 3]

(Crystallization) The effluent of the tank L is made to have a concentration of 77%.
The solution was transferred to a crystallizer having a capacity of 500 ml, and cooled from room temperature to 70 ° C. over 10 hours. On the way 60
At 0 ° C., a small amount of crystalline ribitol powder was added as a seed. The generated crystals were separated using a centrifuge and washed with a small amount of water. The obtained crystals weigh 104 g after drying and have a purity of 99.9 according to liquid chromatography analysis.
%Met.

[0050]

Embodiment 4

Cultivation was carried out in the same manner as in Example 1 except that the raw material for producing ribitol was sugar. Obtained culture solution 1
49.0 mg of ribitol per ml (yield 24.
5%), 45.2 mg of erythritol (yield 22.6)
%) And 1.4 mg of glycerin (yield 0.7%).

[0052]

Embodiment 5

As a microorganism belonging to the genus Trichosporonoides, Trichosporonoides megachiliensis (CBS1)
91.92), except that the main culture was performed for 11 days.
Culture was performed in the same manner as in Example 1. 22.6 mg of ribitol (yield 1
1.3%) and 48.6 mg of erythritol (yield 24.
3%) and 4.8 mg of glycerin (2.4% yield).

[0054]

【The invention's effect】

By practicing the present invention, ribitol can be industrially produced from easily available and inexpensive raw materials.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a chromatographic fractionation apparatus.

[Explanation of symbols]

 A to F tower G Raw material charging pump H valve I Water charging pump J valve K Preheater L Outflow liquid receiving tank M Outflow liquid receiving tank N valve O valve P valve

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshiaki Tateno 1333-55, Atsuhara, Fuji City, Shizuoka Prefecture (72) Inventor Naoki Okamoto 186-2 Onemoto, Matsudo City, Chiba Prefecture

Claims (5)

[Claims] 1. A method for producing ribitol, comprising culturing a microorganism belonging to the genus Trichosporonoides in a medium containing fermentable carbohydrate as a main carbon source, and collecting ribitol from the obtained culture. 2. The method for producing ribitol according to claim 1, wherein the microorganism belonging to the genus Trichosporonoides is Trichosporonoides oeedespharis. 3. The method according to claim 1, wherein the culturing is carried out at a carbohydrate concentration of 10 to 45% by weight,
The method for producing ribitol according to claim 1, wherein the method is performed under aerobic conditions at a temperature of H3 to 5.5 and a temperature of 24 to 38 ° C. 4. The method according to claim 1, wherein the collection of ribitol from the culture is a chromatography fraction in which the culture is supplied to a column packed with a cation exchange resin to separate a ribitol fraction. The method for producing ribitol according to the above. 5. The cation exchange resin is in the form of aluminum,
The method for producing ribitol according to claim 4, which is a strongly acidic cation exchange resin in either a manganese form or an iron form.