JP2001069999A - Production of erythritol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for industrially producing erythritol, reduced in culture medium-preparing operation and using an inexpensive nitrogen source having slight purification load. SOLUTION: This method for producing erythritol comprises inoculating a culture medium containing a carbon source with a microorganism having erythritol-producing ability and culturing the microorganism while gradually adding ammonia gas as a main nitrogen source of the culture medium from a feed line of air to the culture medium until ammonia gas concentration expressed in terms of nitrogen concentration become 0.2-20 g/L while maintaining pH of the culture medium to 3-7.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing erythritol by culturing a microorganism capable of producing erythritol, and more particularly to reducing the burden of the purification step by using ammonia gas as the main nitrogen source of the medium. A method for producing erythritol to reduce.

[0002]

2. Description of the Related Art The following method is known as a method for producing erythritol by culturing microorganisms. Glycerol as a carbon source, a casein hydrolyzate in a medium containing a nitrogen source, Trigonopsis genus (Trigonopsis) or Candida microorganisms process for producing erythritol by cultivating (JP 47-41549 JP) of (Candida). Hydrocarbons as a carbon source, yeast extract, ammonium chloride, in medium containing urea as a nitrogen source, Candida (Candida), Torulopsis (Torulopsis), Hansenula (Hansenul
a ), Pichia or Debaromyces ( Debar
method of producing cultured erythritol a microorganism belonging to the Yomyces) (JP-B 51-21072 Patent Publication).

[0003] A medium containing glucose or the like as a carbon source and corn steep liquor, yeast extract, ammonium sulfate, urea or the like as a nitrogen source, is a medium of the genus Moniliella (JP-A-60-110295, JP-A-10-21588).
7), Aureobasidium (JP-B-63-9831) or Yarrowia ( Yarrow )
ia ) A method for producing erythritol by culturing a microorganism belonging to (JP-A-10-21587).

Using sugars such as glucose as a carbon source, yeast extract, peptone, malt extract, corn steep liquor,
A medium containing ammonia water, ammonium salts, urea, nitrates and the like as a nitrogen source, and is a medium containing Tricosporonoidesoedocephalis (Japanese Unexamined Patent Publication No.
252765), Trichosporonoides megachiliensis
(JP-A-10-96) or Tricosporonoides madida (JP-A- 10-96 )
A method for producing erythritol by culturing a microorganism belonging to U.S. Pat. Also, in a method for producing erythritol using yeast having an erythritol-producing ability, a method using ammonia as a pH adjuster (Japanese Patent Application Laid-Open No. 49-118889) is known.

[0005]

As the nitrogen source of the erythritol producing medium, various nitrogen compounds as described above are known. However, yeast extract, casein hydrolyzate, and peptone are very expensive. Corn steep liquor is inexpensive but contains a large amount of salt, so that the load of the purification step after the completion of the culture is large and economically disadvantageous. Ammonium sulphate requires a large number of pH adjusting agents because the sulfate groups remain in the medium after the microorganisms grow and assimilate the nitrogen source, resulting in a decrease in the pH during cultivation. The purification load increases. Urea has a small burden on the purification process, but when cultivated under strains with high urease activity or culture conditions that increase urease activity, urea is degraded and the pH of the culture solution rises, adversely affecting the culture. Exert. Urea and ammonium sulfate require a dissolution operation at the time of preparation of the culture medium, and when performed on a large scale, the dissolution operation requires time and a large dissolution tank is required. Other ammonium chloride, aqueous ammonia, and the like are listed as nitrogen sources, but no specific usage is reported.

[0006] An object of the present invention is to provide a method for industrially and efficiently producing erythritol using a nitrogen source which reduces the work required for preparing a medium, stably performs cultivation, is inexpensive, and has a low purification load. Is to do.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have found that erythritol can be efficiently produced by using ammonia as a main nitrogen source. The present invention has been completed. That is, the gist of the present invention is to provide a method for producing erythritol by culturing a microorganism having erythritol-producing ability, inoculating the microorganism with a carbon source-containing medium, and then adding ammonia as a main nitrogen source of the medium, A method for producing erythritol, which comprises culturing.

According to a preferred embodiment of the present invention, Trichosporonoides megachiliensis SN-G42 is added to a medium containing a carbon source.
(FERM BP-1430), and then ammonia gas was used as the main nitrogen source of the medium from the air supply line to the medium to convert the medium into a nitrogen concentration so as to maintain the pH of the medium at 3 to 7. And then culturing while gradually adding the erythritol to the range of 0.2 to 20 g / L.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The microorganism used in the present invention is not particularly limited as long as it is a microorganism capable of producing erythritol, and examples thereof include the following microorganisms. Aureobasidium described in JP-B-63-9831
Microorganisms belonging to Japanese Patent Publication No. 4-635, Japanese Unexamined Patent Publication No.
154589, JP-A-9-252765,
JP-A-10-96, JP-A-10-94398, JP-A-10-215887 or JP-A-11-94
A microorganism belonging to the genus Tricosporonoides described in JP-A-32754 . JP-A-60-1
A microorganism belonging to the genus Moniliella described in JP 10295, JP-A-9-154589 or JP-A-10-21587.

[0010] Yarrowia genus described in JP-A-9-154590 discloses or Hei 10-215887 (Yarrowia), Ustilago spp (Ustillago), Firobadjiumu genus (Filobasidium) or Trigonopsis (Trigon
Opsis) microorganism belonging to the genus; Trigonopsis genus are described in JP-B 47-41549 Patent Publication (Trigonopsis) or microorganism belonging to the genus Candida (Candida). Tokiko Sho 51
No. 21072, Candid genus ( Candid
a), Torulopsis (Torulopsis), Hansenula (H
ansenula ), a microorganism belonging to the genus Pichia or the genus Debaryomyces .

Biotechnology Letters (Biot)
echnol. lett. , 7 (4), 119-234.
(1985)) Han Xenia Supora genus described (Hans
eniaspor ), Saccharomyces ( Saccharomyces ), Kloeckera ( Kloeckera ), Kluveromyces ( Kluyv )
eromyces ) or a microorganism belonging to the genus Torulaspora . Microorganisms belonging to the genus Aspergillus (SA Barer, J. Chem. So
c. , 2538-2586 (1958)).

Experimental Mycology (Ga
by. E. FIG. , Experimental Mycolo
gy, 4, 160-170 (1980)), a microorganism belonging to the genus Eupenicillium , the genus Monascus , the genus Penicillium , or the genus Verticillium . The genus Zygopichia (currently the genus Yamada
zyma ) (Suzuki, Fermentation and Industry, 35 (6), (1977). Rhodot ( Rhodot )
orula ) (J. Gen, Microbi)
ol. , 18, 269 (1958)).

The preferred microorganism used in the present invention is a microorganism belonging to the genus Trichosporonoides, in particular, Trichosporonoides megatiliensis strain SN-G42 (FERM BP-1430, formerly known as Aureobaideum SN). -G42 strain).

As the carbon source of the medium for culturing the microorganism of the present invention, sugars such as glucose, fructose, sucrose and the like, starch saccharified solution containing these sugars, sweet potato molasses,
Carbohydrates such as sugar beet molasses can be mentioned, but glucose is preferably used. The sugar concentration in the medium is 10 to 60% (W /
V), preferably in the range of 30 to 50% (W / V). Ammonia is used as the main nitrogen source in the medium for culturing the microorganism of the present invention. The amount added is 0.2 to 20 g when the ammonia concentration in the medium is converted to the nitrogen concentration.
/ L, preferably 0.4 to 10 g / L.

The microorganism used in the present invention cannot grow or grows weakly when the culture medium is alkaline. Since ammonia is strongly alkaline, the medium to which ammonia is added also becomes alkaline. Therefore, as a method of adding ammonia to the medium, the microorganism is inoculated in advance into the medium containing the medium components excluding the nitrogen source, and then the ammonia is gradually added so as to maintain the pH of the medium at 3 to 7. If the amount of ammonia is large,
If ammonia is added and the microorganism is cultured to allow the microorganism to consume the ammonia, the required amount of ammonia can be added while maintaining the pH of the medium at 3 to 7.

The form of ammonia used in the present invention may be any form of liquefied ammonium, ammonia gas or aqueous ammonia, or may be used in combination. However, in consideration of the workability at the time of addition, the most preferable method is to purchase in the form of liquefied ammonium, convert it to a gaseous state after decompression, and supply it as ammonia gas to the culture medium in the fermenter. Further, instead of directly adding ammonia gas to the fermenter, a method of introducing ammonia gas into sterilized water to prepare ammonia water and adding the ammonia water to the culture medium of the fermenter may be used.

In the case of ammonia gas, the most preferable method for supplying ammonia to the culture medium is a method for supplying ammonia from a supply line of air to the fermenter. Specifically, when air is supplied before the filter for removing bacteria, the risk of bacterial contamination can be reduced. In the case of aqueous ammonia, the normal p
A method of supplying from the upper part of the fermentation tank as in the case of the H preparation agent may be used.
The addition of ammonia to the culture medium can be easily automated, and the operations such as measurement, dissolution, and sterilization required when using another nitrogen source can be omitted, and the operation can be reduced.

As a component of the medium for culturing the microorganism of the present invention, phosphoric acid, magnesium, potassium, calcium, iron and the like are used as inorganic salts in addition to the carbon source and the nitrogen source. In addition, components that assist the growth of microorganisms such as vitamins and nucleotides can be added as necessary. Furthermore, when culturing under aeration and stirring conditions, it is preferable to add an antifoaming agent, a pH adjuster, and a buffer. As the defoaming agent, an inorganic or organic defoaming agent is used, for example, polyvinyl alcohol, sorbitan fatty acid ester, a silicon-based compound, and as the pH adjusting agent, sodium hydroxide, potassium hydroxide, sulfuric acid, Examples include hydrochloric acid and nitric acid, and examples of the buffer include sodium lactate and various phosphates.

The process for producing erythritol according to the present invention comprises the steps of producing erythritol in a conventional batch fermentation by a known method similar to the usual batch fermentation except that ammonia is used as a nitrogen source and a special addition device is installed. Can be manufactured. The same applies to fed-batch fermentation and continuous fermentation. Therefore, culturing conditions such as pH, culturing temperature, and aeration conditions during culturing are different depending on the microorganism used, but it is not necessary to change the conditions during each batch culturing. In addition, the cultivation was performed by appropriately measuring the erythritol concentration in the culture solution and the concentration of the raw material substrate by a known method such as high performance liquid chromatography or gas chromatography, and the erythritol concentration reached the highest point or the raw material substrate was consumed. It may be stopped at the time.

Since all of the ammonia is consumed by the microorganisms during the culture, no ammonia is detected in the culture solution after the culture. Accordingly, unlike the method for producing erythritol using corn steep liquor or ammonium sulfate as a main nitrogen source in which a large amount of salt remains, the burden on the purification step after culture can be reduced. For example, one of the indexes for determining the burden of the decolorization / desalting step during purification includes the degree of coloration of the culture solution.When ammonia was used as the nitrogen source, corn steep liquor was used as the nitrogen source. It can be seen that the degree of coloring is lower than in the case, and the load on the desalting step is reduced. The degree of coloring of the culture solution can be easily measured with a spectrophotometer.

One of the indexes for judging the burden of the desalting step at the time of purification is electric conductivity. When ammonia is used as the nitrogen source, corn steep liquor or ammonium sulfate is used as the nitrogen source. It can be seen that the electrical conductivity is lower than in the case where the load was reduced, and the load on the desalting step is reduced. The electric conductivity of the culture solution can be easily measured by an electric conductivity meter. The method of collecting erythritol produced in the culture solution,
What is necessary is just to carry out according to a well-known method (Japanese Patent Publication No. 34748/1995). For example, after removing the microbial cells from the culture solution by centrifugation or filtration, the mother liquor can be desalted and decolorized with an ion exchange resin and activated carbon to crystallize erythritol, which can be obtained after drying.

[0022]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 A 50 mL medium containing 30% glucose (W / V) and 1% yeast extract was placed in a 500 mL Erlenmeyer flask, cotton-plugged, sterilized at 120 ° C. for 20 minutes, and Trichosporonoides mega serrata was obtained from the slant medium. Chileensis SN-G42
(FERM BP-1430). This was cultured with shaking at a temperature of 35 ° C. for 3 days. Next, glucose 30%
(W / V), 1.5 L of a medium containing 3.7% of corn steep liquor was placed in a 3 L fermenter, sterilized at 120 ° C. for 20 minutes, and inoculated with a culture obtained by culturing in a 500 mL Erlenmeyer flask first. The mixture was cultured at 35 ° C. for 2 days, and the obtained culture was used as a seed culture.

Main culture medium 15 L containing 40% glucose
To the medium, while gradually adding ammonia gas to the medium so as to maintain the pH of the medium at 6, while passing aeration volume of 0.5 vvm and stirring speed of 330 rpm.
The culture was performed under the conditions of m, pressure 0.5 kg / cm ² . The amount of ammonia gas added was 2 g / L in terms of nitrogen concentration with respect to the medium. The culture was terminated when glucose was consumed (92 hours after the start of the culture). As a result of measuring the erythritol concentration in the culture solution by high performance liquid chromatography, it was 202 g / L. The degree of coloration (absorbance at 420 nm) of the culture supernatant after heat-sterilization of the bacteria was measured by a spectrophotometer and found to be 0.59, and the electrical conductivity was 1.
It was 3 mS / cm.

Example 2 Seed culture and main culture were performed in the same manner as in Example 1. However, ammonia water was used as the main nitrogen source in the main culture, and ammonia was gradually added from the upper part of the fermenter until the amount of added ammonia became 2 g / L in terms of nitrogen concentration with respect to the medium while maintaining the pH of the medium at 6. . 9 after start of culture
As a result of stopping the culture for 4 hours, the erythritol concentration in the culture solution was 195 g / L. The degree of coloration (absorbance at 420 nm) of the culture supernatant was measured with a spectrophotometer. As a result, it was 0.53, and the electric conductivity was 1.3 mS / cm.

Comparative Example 1 Seed culture and main culture were performed in the same manner as in Example 1. However, the main nitrogen source of the main culture was corn steep liquor 8%. As a result, the erythritol concentration at 94 hours was 172 g / L, the degree of coloring (absorbance at 420 nm) of the culture supernatant after heat sterilization of the bacteria was 2.8, and the electric conductivity was 6 mS / cm. Was.

Comparative Example 2 Seed culture and main culture were carried out in the same manner as in Example 1. However, ammonium sulfate was used as the main nitrogen source in the main culture, and the amount added to the medium was 2.2 g / L in terms of the nitrogen concentration in the medium. As a result, the erythritol concentration at the 94th hour was 167 g / L, the degree of coloring (absorbance at 420 nm) of the culture supernatant after heating and sterilizing the bacteria was 0.8, and the electric conductivity was 5.6 mS / cm. there were.

[0027]

According to the method of the present invention, erythritol can be efficiently produced by using a low-cost, low-purification nitrogen source capable of stably culturing the medium, reducing the work required for the preparation of the medium.

Claims (7)

[Claims] 1. A method for producing erythritol by culturing a microorganism having an erythritol-producing ability, wherein the microorganism is inoculated into a medium containing a carbon source, and then ammonia is added as a main nitrogen source of the medium, followed by culturing. A method for producing erythritol, characterized in that: 2. The erythritol according to claim 1, wherein the amount of ammonia added to the culture medium is in the range of 0.2 to 20 g / L in terms of nitrogen concentration with respect to the culture medium. Manufacturing method. 3. The method for producing erythritol according to claim 1, wherein the microorganism is cultured while ammonia is gradually added to the medium so as to maintain the pH of the medium at 3 to 7. 4. The method according to claim 1, wherein the ammonia is added in the form of liquefied ammonium, ammonium gas, or aqueous ammonia, or is added in combination. A method for producing erythritol according to one of the above. 5. The method for producing erythritol according to any one of claims 1 to 3, wherein ammonia gas is added to the culture medium from a supply line of air to the culture medium. 6. The method for producing erythritol according to claim 1, wherein the microorganism is a microorganism belonging to the genus Trichosporonoides. 7. The method for producing erythritol according to any one of claims 1 to 6, wherein the microorganism is Trichosporonoides megatiliensis SN-G42.