JPH0343091A - Method for producing erythritol by fermentation using novel microorganisms - Google Patents

Abstract

PURPOSE:To obtain the title compound in high yield by using highly concentrated glucide without forming foams in culture by culturing a new artificial variant belong to the genus Aureobasidium. CONSTITUTION:Aureobasidium sp. SN-G42 strain (FERM P-8940), a new artificial variant, is aerobically cultured in a medium containing a fermentable saccharide (e.g. glucose) as a main carbon source, erythritol is formed and accumulated in a culture solution and collected. The culture is preferably carried out at 35-37 deg.C in the medium having pH4-7.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a method for producing erythritol by fermentation using a new microorganism, and more particularly, to a method for producing erythritol by fermentation using a new microorganism, and more particularly, to a method for producing erythritol by fermentation using a new microorganism. The present invention relates to a method for producing erythritol by fermentation using a novel microorganism, which is characterized by converting fermentable sugars into erythritol using the Sidium sp. 5N-G42 strain and its mutant strain.

[Prior Art and Problems to be Solved by the Invention] Conventionally, the genus Cancida and the genus Devariosces have been used.

A method for producing erythritol from sugars such as glucose and glycerin by a fermentation method using microorganisms belonging to the genus Atribonobsis, Torulopsis, Hansenula, Moniliella, Aureobasidium, etc. is already known.

For example, Japanese Patent Publication No. 47-41549 describes a method for converting fermentable sugars such as glycerin into erythritol using microorganisms belonging to the genus Cancida and Trigonobsis. However, in this method, the sugar concentration used as the main carbon source (
There was a limit to the substrate concentration (substrate concentration), and it was necessary to carry out fermentation at a relatively low concentration.

Further, JP-A-60-110295 and the like disclose improved production of erythritol using microorganisms belonging to the genus Moniliella. This method is superior in that it has a high conversion rate to erythritol and the microorganism has a high sugar tolerance, but this microorganism produces a lot of foam and the commonly used antifoaming agents are useless, so expensive xanthan gum etc. It is necessary to add a large amount of , which has the disadvantage of increasing manufacturing cost.

Furthermore, in JP-A No. 61-31091, the present inventors reported that the 5N-45 strain of Aureopacidium sp.
Disclosed is a method for producing erythritol from fermentable sugars such as glucose using a method of producing erythritol from fermentable sugars such as glucose. This method is
Although it is possible to produce erythritol using a relatively high concentration of carbohydrates, the yield of erythritol is not sufficient compared to the amount of bacterial cells produced in the culture solution, and the production conditions of pH. This is not necessarily a satisfactory method as the optimum range is narrow.

[Means for Solving the Problems] In order to improve the drawbacks of the conventional methods as described above, the present inventors conducted various studies on improving microorganisms in order to increase the sugar tolerance of microorganisms belonging to the genus Aureobasidium. As a result, Aureobasidium sp, which belongs to the genus Aureobasidium and is a Deuteromycete, was isolated from the soil in a starch factory in Okinawa Prefecture.

The 5N-124A strain (Choken Bacteria No. 8745) was used as the parent strain, and the mutant strains obtained from this strain, Aureobasidium sp, and the 5N-G42 strain, had high erythritol production ability and high viscosity resistance. The present invention was achieved based on the discovery that the foams are obtained and that bubbles are not generated during culture.

That is, the present invention is characterized in that it has the ability to produce erythritol, its bacterial bodies (cells) are hydrophilic, non-aggregating, and substantially do not produce bubbles when cultured aerobically in a liquid medium. Aureobasidium sp. 5N-G42 strain was used to inoculate a medium containing fermentable sugars as the main carbon source,
The present invention relates to a method for producing erythritol by fermentation, which comprises culturing aerobically to produce and accumulate erythritol in a culture solution, and collecting the erythritol.

Aureobasidium sp related to the present invention.

5N-G42 strain is a parent strain, Aureobasidium sp, 5N-124^ strain, after irradiation with ultraviolet light.
This is a novel mutant strain induced by treatment with a mutagenic agent such as N-methyl-N'-nitro-N''-nitrosoguanidine.

The mycological properties of this mutant strain are shown below.

1. Growth status on medium l) Microscopic findings Size of vegetative cells (*1) Shape of 4-7 x 4-15μ vegetative cells (*1) Shapes such as hyphal and yeast-like unicellular, oval, etc. show.

Method for propagating vegetative cells (*1) Multipolar budding mycelia of hyphae and yeast-like cells (*2) Forms true hyphae and produces many whole osteogenic conidia at the tips and sides.

(Note) *Culture on IYM agar medium at 27℃ for 5 days *2 Slide culture on potato glucose agar 2) Agar slant (*3) Good growth Favorable condition Surface is smooth, glossy, dark color tone
As days pass, the colony changes from white to a pale yellow-black color.

(Note) *3 YM agar medium 3) Liquid culture (*4) Surface growth Thick film formation Turbidity Transparent Sedimentation
Dog (Note) *4 YM liquid medium 2, ascospore formation Potato glucose agar medium No formation, cornmeal agar medium No formation, YM agar medium
Carrot extract agar medium without formation V6 agar medium without formation No formation 3, Physiological properties Oxygen requirement Aerobic growth temperature Up to about 40℃ Optimum growth temperature 35-37℃ Growth pH 2.5-10
．． 0 Optimal growth p)! 4.0-7.0K
NOj Assimilation (*5) Yes Li(N)1
4) 2504 kR convertibility (*5) Yes Decomposition of urea No Liquefaction of gelatin
None Formation of carotenoids None Fast Blue B color test None Formation of organic acids None Formation of starch-like substances
None Vitamin requirements (*5) Yes
Liglucose concentration (*6) 50% growth Fence 60% growth Low salt concentration (*7) 2% growth +10% growth (Note) *5 J using Wickerham's synthetic medium
, Determined by the method of Lodder et al. - Xylose Galactose Erythritol D - Arabinose - Arabinose (*5) D - Ribose Sucrose L - Rhamnose Maltose Ethanol Methanol Cellobiose Salicin L - Sorbose Ribitol Galactitol Glycerol Trehalose Lactose Melibiose D - Mannitol Raffinose Meleditose α-Methyl -D-glucoside inulin inositol soluble starch DL-lactic acid - succinic acid citrate D-glucitol + glucuronic acid arbutin + p-glucosamine (H(R) 2-ketogluconic acid 15-ketogluconic acid 10 Mycological properties as above This strain has been mutated from the parent strain Aureobasidium sp. 5N-124A, and has properties similar to the parent strain, so it is a new mutant strain belonging to the genus Aureobasidium. Judging that there is, Aureobasidium sp, 5N
The strain was named -642.

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as "Microorganism Accession Number: Tetsukoken Bacteria Collection No. 8940."

In this way, Aureobasidium sp according to the present invention
The 5N-G42 strain is close to the mycological property X (see Japanese Patent Application No. 61-210669) of the parent strain Aureobasidium sp. , has some properties such as non-degradability of urea and ability to assimilate carbon sources, and has higher resistance characteristics than the parent strain (see Test Example 1 below), and can be used aerobically in a liquid medium. The fact that it does not substantially produce bubbles when cultured, and the bacterial cells (
There are also differences in that the cells) are parent-like and non-aggregating (see Test Example 2 below), and in the morphology of colonies when cultured stationary on a plate agar medium (see Test Example 3 below). ing.

The method for producing erythritol according to the present invention will be described below, and the percentages used in the present invention are expressed in volume (W/V)% unless otherwise specified.

The culture in the present invention is usually preferably carried out by stirring culture under aerobic conditions using a liquid medium.

The main carbon source of the liquid medium is glucose.

Carbohydrates such as fructose and sucrose are used, but the quantitative proportion of these sugars in the medium is 10
It is added in an amount of 55% to 55%, preferably 20 to 50%. As nitrogen sources, nitrogen compounds usable by microorganisms, such as yeast extract, peptone, malt extract, casamino acids, corn steep liquor, etc., are used. Also,
Examples of inorganic salts added to the medium include ferrous sulfate, potassium chloride, sodium chloride, potassium dihydrogen phosphate,
Salts such as calcium hydroxide are used. Furthermore, various organic substances necessary for yeast growth as necessary.

Inorganic substances or commonly used antifoaming agents can be added.

Cultivation is carried out by directly inoculating microbial cells into a liquid medium having the above composition, or by separately inoculating a seed culture obtained by pre-cultivation. To prepare this seed culture solution, for example, one platinum loop of bacteria cultured on a slant by a conventional method is inoculated into a liquid medium of pH 4 to 6 containing 33.5% glucose and 4.5% corn steabric acid at 34 to 36°C. This is done by culturing at a temperature of 2 to 4 days.

The culture temperature is within the range where microorganisms can grow, that is, 30 to 38
The temperature is preferably 35 to 37°C.

Further, the pH of the medium is adjusted within the range of 4 to 9, preferably 4 to 7.

The culture period varies depending on the type of medium used and the concentration of carbohydrates, which are the main carbon sources, but is usually about 4 to 8 days.

It is desirable that the culture in the present invention be terminated when the nutrient source of the medium is fully utilized and the amount of erythritol produced in the culture solution reaches its maximum.

The amount of erythritol produced in the culture solution can be quickly measured using a well-known method such as gas chromatography or high performance liquid chromatography.

Thus, the erythritol accumulated in the culture medium is
It is isolated from the culture solution using conventional methods. That is, in such cases, well-known means commonly used in the field,
For example, operations such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, distillation, and crystallization may be used in appropriate combinations as necessary. - For example, bacterial cells are removed from the culture solution by filtration, centrifugation, etc., then this solution is treated with activated carbon to remove colored substances, and then deionized with an ion exchange resin, and then the solution is concentrated. Next, erythritol is crystallized and separated from this syrup. If glycerin is contained in the mother liquor after separating erythritol, this can be further purified by vacuum distillation to obtain glycerin.

[Example] Next, the present invention will be explained in detail using test examples and examples.

Below, Aureobasidium sp according to the present invention,
The characteristics of the 5N-G42 strain were compared with the parent strain Aureobacitium sp, 5N-124A strain.

Test Example 1 (glucose tolerance test) - Method Yeast extract (manufactured by Difco) 1.0% (indicates w/v%. The same applies hereinafter), glucose 22.0-45
．． A medium containing 0% was placed in a 500 mJ) Erlenmeyer flask, and steam sterilized at 120°C for 15 minutes.

After cooling, adjust the medium p)l to 5.5, and add 6 of the seed culture solution to this.
%, rotational culture was performed at 30° C. and 180 rpL11 for 7 days.

-Results The next page shows the erythritol yield per C vs. consumed glucose.

As is clear from the table, the parent strain, Aureobasidium sp.
A good conversion rate was shown between 6.5 and 38.3%, but when the glucose concentration reached 39.5% or more, a rapid decrease in the conversion rate was observed as the glucose concentration increased.

On the other hand, in Aureobasidium sp. 5N-042 strain, which is a mutant strain of the present invention, no decrease in the conversion rate to erythritol was observed even when the glucose concentration of the medium exceeded 39.5%, and the glucose concentration When the strain became 45.0%, the erythritol yield was approximately 2.3 times higher than that of the parent strain.

Test Example 2 (Foamability, Agglomeration and Hydrophilicity Test)/Method A medium containing 1.0% yeast extract and 33.5% glucose was placed in a test tube and steam sterilized at 120°C for 15 minutes. After cooling, adjust the pH of the medium to 5.5, and add seed culture solution 6 to this.
After inoculating %, shaking culture was performed at 30°C for 5 days. After the culture was completed, the cells were allowed to stand for 10 to 15 minutes, and the state of foam formation and aggregation of bacterial bodies (cells) were observed (foaming property, aggregation property).

Subsequently, the same amount of benzene as the culture solution was added and stirred strongly, and the mixture was allowed to stand for about 30 minutes to observe the state of migration of microbial bodies (cells) to the benzene layer (hydrophilicity).

・Results Parent strain Aureopasidium sp, 5N-124
Strain A showed significant foaming when cultured aerobically in a liquid medium, and the foam did not disappear for a long time even after stirring (shaking) was stopped. Furthermore, when the stirring was stopped, the bacterial bodies (cells) in the culture solution quickly aggregated to form a precipitate.

On the other hand, the Aureobasidium sp. 5N-G42 strain, which is a mutant strain of the present invention, shows no foaming at all even when cultured under the same conditions as above, and even when stirring (shaking) is stopped, the bacterial cells remain No aggregation occurred.

In addition, an appropriate amount of benzene was added to each culture solution of the parent strain and the mutant strain of the present invention cultured under the same conditions as above, and the benzene-water 2
When a layer system is used, the bacterial cells of the parent strain migrate to the benzene layer, and nothing remains in the water layer, but in the case of Iiju, the parent strain transfers to the benzene layer and nothing remains in the water layer.
There was no migration to the main 0-benzene layer in layer r11'.

Test Example 3 (morphological observation) - Method Yeast extract 1.0%, glucose, 33.5%, agar t
The strain was inoculated onto a plate agar medium consisting of .

・Results Parent strain Aureobasidium sp, 5N-124
Strain A produces colonies with wrinkled surfaces on flat agar plates, but the mutant strain of the present invention, Aureobasidium sp. 5N-642, produces colonies with smooth surfaces and is significantly different in appearance. are doing.

Example 1 (Creation of mutant strain) Aureobasidium sp. 5N-124A strain was precultured for 2 days in a medium containing 0.5% yeast extract and 22% glucose, diluted 100 times, and isolated from a distance of 30 cm.
40 5W UV lamps (Toshiba Germicidal Lamp rGLIJ)
Irradiate for minutes. After diluting this, agar medium (yeast extract 1
．． 0%, glucose 33.5%, and agar 1.5%), and those that grew were selected.

Next, the strains selected in the above treatment were again irradiated with ultraviolet light for 20 minutes, and those that grew in the same manner were selected.

Furthermore, the bacterial strains selected by the above treatment were treated with N-methyl-N'-nitrosoguanidine at a concentration of 1 mg/ml for 30 minutes, and those that grew in the same manner were selected and mutated. The strain Aureobasidium sp, 5N
-G42 strain (FERM P-8940) was obtained.

Example 2 Aureobasidium sp was grown on a slant medium consisting of 33.5% glucose, 1.0% yeast extract and 1.5% l'Js.
, 5N-042 strain (FERM P-8940) is applied and cultured stationary at 35° C. for 2 to 3 days.

Next, 1 liquid medium (p) containing 20% glucose and 1.6% cornstarch liquor (made by egg corn starch)
4.0) One platinum loop of the above cultured cells was inoculated into a 5,000-volume Erlenmeyer flask containing ISOmN, and cultured at 35°C for 3 days.
It was prepared by inoculating a 50Oej volume Erlenmeyer flask containing 50fllJ and culturing with shaking at 35°C for 3 days.

Main culture glucose 20.0% and corn steep liquor 1,
Put 15 ft of medium containing 6% into a 30 ft fermenter,
Antifoaming agent ("Silicone KS-66J Shin-Etsu Chemical ■") 30
After adding 0 ppm, steam sterilize at 120°C for 20 minutes. After cooling, adjust the pH of the medium to 4.0 using caustic soda.
After adjusting to this, Aureopacidium sp,
Add 6% seed culture solution of 5N-G42 strain (FERM P-8940), temperature 35°C, aeration volume 0.2Svvi+
Culture was carried out for 7 days at a % rotation speed of 23 rpm.

After the culture was completed, analysis of the culture solution revealed that glucose had been completely consumed, the yield of erythritol in the culture solution was 49.3%, and the yield of glycerin was 2.2%.

Next, take a part of this culture solution, remove the bacterial cells by centrifugation, and further decolorize with activated carbon and ion exchange resin C.
IRA-410: IR-120B-2: 1')
Desalination was carried out by walking. The eluate was concentrated to a sugar concentration of 50% or more and cooled slowly to obtain crystals, which were then dissolved in water and recrystallized in the same manner.

The resulting polyhedral-like white crystals have a mild sweet taste,
Its melting point was 121t. Furthermore, as a result of measurements such as liquid chromatography, gas chromatography optical rotation, and nuclear magnetic resonance spectrum, the above crystals were identified as erythritol.

Example 3 Glucose 20.0% and Corn Steep Liquor 1,
Put the medium 15JZ containing 6% into a 30j 2 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilization is performed at 120° C. for 20 minutes. After cooling, the pH of the medium is reduced using caustic soda.
Aureobasidium sp, 5N-642 strain (FERMP-8!1) prepared according to the method of Example 2 using a medium with the same composition as the above medium after adjusting
40) <73 im Kyoshunsei 60A l;fkaff1-
Temperature ff35℃, ventilation amount 0.50vvm, rotation speed 230
Culture was performed under rp- conditions for 7 days.

After completion of the culture, analysis of the culture solution revealed that glucose was completely consumed, and the yield of erythritol and glycerin in the culture solution was 49.2% and 1.2%, respectively.

Example 4 Glucose 20.0% and corn stew liquor-1,
Culture medium 151 containing 6% is placed in a 30-liter fermenter, 300 ppm of antifoaming agent is added, and then steam sterilized at 120° C. for 20 minutes. After cooling, the pH of the medium was adjusted to 4.0 using caustic soda, and Aureopacidium sp. -8940
) was added, and the temperature was 35°C and the aeration rate was 0.75.
Culture was carried out for 7 days under the conditions of vvm and rotation speed of 230 rpm.

After completion of the culture, analysis of the culture solution revealed that glucose was completely consumed, erythritol yield in the culture solution was C±46-5%, and glycerin Uvmt was 2.7%.

Example 5 Glucose 20.0% and corn steep liquor 1,
Medium 15j2 containing 6% was placed in a fermenter of 3 (IQ volume),
After adding 300 ppm of antifoaming agent, steam sterilize at 120 tons for 20 minutes. After cooling, the pH of the medium was adjusted to 4.0 using caustic soda, and Aureobasidium sp., 5N-G42 strain (FERMP -89
Add 6% of the seed culture solution from 40), maintain the temperature at 35°C, and the aeration rate at 1.
Culture was carried out for 7 days under the conditions of 00vvm and 230rpa+ rotation speed.

After completion of the culture, analysis of the culture solution revealed that glucose had been completely consumed, and the yield of erythritol in the culture solution was 41.8% and the yield of glycerin was 1.4%.

Example 6 Glucose 33.5% and Corn Stew Liquor 4,
Five cubes of medium containing 47% are placed in a seven cube fermenter, 300 ppm of antifoaming agent is added, and then steam sterilized at 120 tons for 20 minutes. After cooling, the pH of the medium was adjusted to 4.2 using caustic soda, and Aureobasidium sp., 5N-G42 strain (FERMP- 8940)
Add 6% seed culture solution, temperature 35℃, aeration volume 0.25V
Culture was carried out for 5 days under the conditions of 500 rpm and 500 rpm.

After completion of the culture, analysis of the culture solution revealed that glucose was completely consumed, and the yield of erythritol in the culture solution was 39.7% and the yield of glycerin was 7.9%.

Example 7 Glucose 39.5% and Corn Stew Liquor 4,
A medium containing 0% 5° C. is placed in a 71 L fermenter, 300 ppm of antifoaming agent is added, and the fermenter is steam sterilized at 120 tons for 20 minutes. After cooling, p)l of the medium using caustic soda
Aureobasidium sp, 5N-G42 strain (FERMP-8940
) was added, the temperature was 35°C, and the aeration rate was 0.25.
vv11. Culture was performed for 6 days at a rotation speed of 500 rpm.

After completion of the culture, analysis of the culture solution revealed that glucose was completely consumed, and the yield of erythritol and glycerin in the culture solution was 38.6% and 9.5%, respectively.

Example 8 Glucose 39.5% and Corn Stew Liquor-5,
A medium 52 containing 3% was placed in a seven-sided fermenter, and an antifoaming agent 3 was added to the fermenter.
After adding 00 ppm, steam sterilize at 120 t for 20 minutes. After cooling, adjust the pH of the medium to 4 using caustic soda.
．． 2, add 6% of the seed culture of Aureobasidium sp. °C, ventilation amount 0.25VV
Culture was performed for 5 days under the conditions of O1 and rotation speed of 500 rpm.

After the culture was completed, analysis of the culture solution revealed that glucose had been completely consumed, and the yield of erythritol and glycerin after 7 nights of culture was 37.5% and 7.2%, respectively.

Example 9 Glucose 39.5% and Corn Stew Liquor 6,
After putting 5 il of medium containing 6% into a heptagonal fermenter and adding 300 ppm of antifoaming agent, 120'+? : Steam sterilize for 2% minutes. After cooling, the pH of the medium was adjusted to 4.2 using caustic soda, and Aureobasidium sp., 5N-G42 strain (FERMP- 89
Add 6% of the seed culture solution from 40), and maintain the temperature at 35°C and the aeration rate at 0.
Culture was carried out for 4 days under the conditions of 25vv+n and 500 rpm.

After completion of the culture, analysis of the culture solution revealed that glucose was completely consumed, and the yield of erythritol in the culture solution was 37.1% and the yield of glycerin was 4.8%.

Example 10 Glucose 45.0% and Cohn-Steebly force-6,
Medium 1 containing 0%! [was placed in a 30 fl fermenter, 300 ppm of antifoaming agent was added, and then steam sterilized at 120'C for 20 minutes. After cooling, the pH of the medium was adjusted to 4°0 using caustic soda, and Aureobasidium sp., 5N-G42 strain (FERMP- 89
Add 6% of the seed culture solution from 40), and maintain the temperature at 35°C and the aeration rate at 0.
Culture was carried out for 8 days under conditions of 25 vvm and 230 rpm.

After completion of the culture, analysis of the culture solution revealed that glucose was completely consumed, and the yield of erythritol in the culture solution was 37.3% and the yield of glycerin was 4.5%.

Example 11 Sucrose 33.5% and Corn Stew Liquor-4
, 5% of the culture medium was placed in a 7J2 fermenter, 300 ppm of antifoaming agent was added thereto, and then steam sterilized at 120° C. for 20 minutes. After cooling, adjust the pH of the medium using caustic soda.
4.1, add 6% of the seed culture of Aureopacidium sp. 5N-G42 strain prepared according to the method of Example 2 using a medium with the same composition as the above medium,
Temperature 35℃, ventilation amount 0.25vv*, rotation speed 500r
Cultured for 6 days under pm conditions.

After the culture was completed, analysis of the culture solution revealed that sucrose was completely consumed, and the yield of erythritol in the culture solution was 41.9% and the yield of glycerin was 7.4%.

Example 12 Sucrose 39.5% and Corn Steep Liquor 5
, 3% of medium 5j2 is placed in a 7j2 volume fermenter, 300 ppm of antifoaming agent is added, and then steam sterilized at 120° C. for 20 minutes. After cooling, the culture medium was diluted with 9H using caustic soda.
Aureobasidium sp. 5N-G42 strain (FEBMP-8940) prepared according to the method of Example 2 using a medium with the same composition as the above medium.
) was added, the temperature was 35°C, and the aeration rate was 0.25.
Culture was performed for 6 days under the conditions of vvm and rotation speed of 500 rpm.

After completion of the culture, analysis of the culture solution revealed that sucrose was completely consumed, and the yield of erythritol in the culture solution was 42.4% and the yield of glycerin was 5.8%.

Example 13 Sucrose 4560% and Corn Steep Liquor 6
, 0% was placed in a 7°C fermenter, 300 ppm of an antifoaming agent was added, and then steam sterilized at 120°C for 20 minutes. After cooling, the pH of the medium was adjusted to 4.1 using caustic soda, and Aureopacidium sp., 5N-G42 strain (FERMP- 8940)
Add 6% seed culture solution, temperature 35℃, aeration volume 0.25V
Culture was carried out for 6 days under the conditions of vm and rotation speed of 50 Orpm+.

After the culture was completed, analysis of the culture solution revealed that sucrose was completely consumed, and the yield of erythritol in the culture solution was 41.7% and the yield of glycerin was 3.8%.

[Effects of the invention] The novel microorganism of the present invention, Aureobasidium sp.
, 5N-642 strain has excellent sugar tolerance and heat resistance, high erythritol production ability, and generates very little foam during fermentation culture, so it is very convenient for industrial use.

Claims (1)

[Claims] (1) Aureobasidium sp. A method of producing erythritol by fermentation, which is characterized in that the SN-G42 strain is inoculated into a medium containing fermentable sugars as the main carbon source, and cultured aerobically to produce and accumulate erythritol in the culture solution, which is then collected. Production method.