JPH04635B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a method for producing erythritol by fermentation using a novel microorganism, and more particularly, to a method for producing erythritol by fermentation using a novel microorganism, and more specifically, Aureobasidium sp.SN, a novel artificial mutant strain belonging to the genus Aureobasidium. - A method for producing erythritol by fermentation using a novel microorganism, characterized by converting fermentable saccharides into erythritol using strain G42 and its mutant strain. [Prior art and problems to be solved by the invention] Conventionally, the genus Candida, the genus Debaryomyces,
A method for producing erthritol from sugars such as glucose and glycerin by a fermentation method using microorganisms belonging to the genus Trigonopsis, 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 Candida and the genus Trigonopsis. However, due to the low sugar tolerance of microorganisms in this method, there is a limit to the sugar concentration (substrate concentration) used as the main carbon source, and it is necessary to carry out fermentation at a relatively low concentration. Further, JP-A-60-110295 and the like disclose an improved method for producing 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 production cost. Furthermore, in Japanese Patent Application Laid-Open No. 61-31091, the present inventors reported that the Aureobasidium sp.
A method for producing erythritol from fermentable saccharides such as glucose using the method (No. 7594) is disclosed. Although this method makes it possible to produce erythritol using carbohydrates at a relatively high concentration, the yield of erythritol is insufficient compared to the amount of bacterial cells produced in the culture solution, and the production conditions PH that is
This method cannot necessarily be said to be satisfactory, as the optimal range of is narrow. [Means for Solving the Problems] In order to improve the drawbacks of the conventional methods as described above, the present inventors have made various improvements to microorganisms in order to increase the sugar tolerance of microorganisms belonging to the genus Aureobasidium. As a result of our investigation, we found that Aureobasidium sp.SN, which belongs to the genus Aureobasidium and is a Deuteromycete, was isolated from the soil in a starch factory in Okinawa Prefecture.
-124A strain (Feikoken Bacteria No. 8745) was used as the parent strain,
It was discovered that the mutant Aureobasidium sp. SN-G42 strain obtained from this has a high ability to produce erythritol, has acquired high sugar tolerance, and does not produce bubbles during culture. , this invention has been achieved. That is, the present invention has the ability to produce erythritol,
Aureobasidium whose bacterial bodies (cells) are hydrophilic and non-aggregating, and which substantially do not produce bubbles when cultured aerobically in a liquid medium.
Fermentation characterized by inoculating a medium containing fermentable sugars as a main carbon source using sp.SN-G42 strain, culturing aerobically to produce and accumulate erythritol in the culture solution, and collecting this The present invention relates to a method for producing erythritol. Aureobasidium sp.SN- related to the present invention
G42 strain is the parent strain Aureobasidium
A new mutant strain was induced by irradiating the sp.SN-124A strain with ultraviolet rays and subsequently treating it 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 (1) Microscopic findings Size of vegetative cells (*1) 4-7 x 4-
15μ Shape of vegetative cells (*1) Shows shapes such as fungal and yeast-like unicellular, oval, etc. Method for propagating vegetative cells (*1) Multipolar budding of hyphae and yeast-like cells Mycelium (*2) Forms true hyphae and produces many holoblastoid conidia at the tips and sides. (Note) *1 Cultured on YM agar medium at 27℃ for 5 days *2 Slide culture on potato glucose agar (2) Agar slope (*3) Good growth Morphology Smooth surface No gloss Color tone From white to pale yellow as days pass Changes to black colonies. (Note) *3 YM agar medium (3) Liquid culture (*4) Surface growth Thick film formation Turbidity Transparent Sediment Large (Note) *4 YM liquid medium 2 Ascospore formation Potato glucose agar medium No formation Cornmeal Agar medium No formation YM agar medium No formation Carrot extract agar medium No formation V ₈ Agar medium 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 Optimum growth PH 4.0-7.0 KNO ₃ assimilation (*5) Yes (NH ₄ ) ₂ SO ₄ assimilation (*5) Yes Urea decomposition None Gelatin liquefaction None Carotenoid production None First Blue B Color test None Production of organic acids None Production of starch-like substances None Requirement for vitamins (*5) Yes Glucose concentration (*6) 50% growth 60% growth Salt concentration (*7) 2% growth + 10% growth - ( Note) *5 J.I. using Ickerham's synthetic medium.
Determined by the method of Lodder et al. *6 Agar medium *7 Liquid medium 4 Sugar fermentability (*5) Glucose + lactose - galactose - melibiose - sucrose + ruffinose - maltose + D-arabinose - xylose - trehalose - inulin - 5 sugars, Assimilated glucose such as organic acids + D-xylose - galactose - erythritol + D-arabinose - L-arabinose - D-ribose + sucrose + L-rhamnose - maltose + ethanol + methanol ± cellobiose + salicin - L-sorbose + ribitol - galactitol - glycerol + trehalose - lactose - melibiose - D-mannitol - ruffinose + meleditose + α-methyl-D-glucoside - inulin + inositol - soluble starch - DL-lactic acid - succinic acid - citric acid - D-curcitol + glucuronide Acid - Arbutin + D-glucosamine (HCl) - 2-ketogluconic acid + 5-ketogluconic acid + This strain, which has the above mycological properties, was mutated from the parent strain Aureobasidium sp. SN-124A. SN-G42 strain was determined to be a new mutant strain belonging to the genus Aureobasidium and was named Aureobasidium sp. SN-G42 strain. . This bacterial strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as ``Microorganism Accession Number: Microbiological Research Institute No. 8940''. As described above, the Aureobasidium sp. SN-G42 strain according to the present invention has the mycological properties (see Japanese Patent Application No. 61-210669) of the parent strain Aureobasidium sp. SN-124A strain. However, it has slightly different properties such as weak nitrate assimilation, non-degradability of urea, and carbon source assimilation, and higher sugar tolerance than the parent strain (see below). (See Test Example 1), the fact that bubbles are not substantially generated when cultured aerobically in a liquid medium, and the bacterial bodies (cells) are hydrophilic and non-aggregating (see Test Example 2 below) There is also a difference in the morphology of colonies when statically cultured on a plate agar medium (see Test Example 3 below). 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, and the quantitative proportion of these sugars in the medium is 10 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. Further, as inorganic salts added to the medium, for example, salts such as ferrous sulfate, potassium chloride, sodium chloride, potassium dihydrogen phosphate, and 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, culture the bacteria by slant culture using a conventional method, add 33.5% glucose and 4.5% corn steep liquor.
Inoculate one platinum loopful into a liquid medium with a pH of 4 to 6 containing 34
This is done by culturing for 2-4 days at a temperature of ~36°C. The culture temperature is within the range where microorganisms can grow, i.e. 30°C.
It is carried out at -38°C, preferably in the range of 35-37°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. Note that 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. Erythritol thus accumulated in the culture solution is separated from the culture solution according to a conventional method. That is, in such cases, well-known means commonly used in the art, such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction,
Operations such as distillation and crystallization may be used in combination as appropriate. For example, after removing bacterial cells from the culture solution by filtration or centrifugation, this solution is then treated with activated carbon to remove colored substances, and further deionized with an ion exchange resin.
Concentrate the liquid to make syrup. 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 with reference to Test Examples and Examples. Below, Aureobasidium related to the present invention
The characteristics of sp. SN-G42 strain were compared with the parent strain Aureobasidium sp. SN-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~
A medium containing 45.0% was placed in each 500 ml Erlenmeyer flask, and steam sterilized at 120°C for 15 minutes. After cooling, the pH of the medium was adjusted to 5.5, and 6% of the seed culture solution was inoculated into the medium, followed by rotational culture at 30° C. and 180 rpm for 7 days. ●Results The next page shows the erythritol yield per glucose consumed.

[Table] As is clear from the table, the parent strain Aureobasidium sp. SN-124A has a good conversion rate of 36.5 to 38.3% to erythritol when the glucose concentration of the medium is 33.5% or less. However, when the glucose concentration exceeds 39.5%,
A rapid decrease in conversion rate was observed with increasing glucose concentration. On the other hand, in the Aureobasidium sp. SN-G42 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 in the medium exceeded 39.5%, and the glucose concentration decreased. At 45.0%, the erythritol yield was approximately 2.3 times higher than that of the parent strain. Test Example 2 (Foamability, flocculation 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, the pH of the medium was adjusted to 5.5, and 6% of the seed culture solution was inoculated thereto, followed by shaking culture at 30°C for 5 days. After the culture was completed, the mixture was 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 strongly stirred, and the mixture was allowed to stand for about 30 minutes to observe the state of migration of bacterial bodies (cells) to the benzene layer (hydrophilicity). ●Results Parent strain Aureobasidium sp.SN−
Strain 124A showed significant foaming when cultured aerobically in liquid medium, and the foam did not disappear for a long time even after agitation (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, Aureobasidium sp. SN-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 strain does not grow. No body aggregation occurred. Furthermore, when an appropriate amount of benzene is added to each culture solution of the parent strain and the mutant strain of the present invention cultured under the same conditions as above to form a benzene-water two-layer system, the bacterial cells of the parent strain migrate to the benzene layer, and the Although nothing remained in the layer, all the bacterial cells of the mutant strain of the present invention remained in the aqueous layer and did not migrate to the benzene layer at all. Test Example 3 (Morphological Observation) ●Method After inoculating bacterial cells using a platinum wire onto a plate agar medium consisting of 1.0% yeast extract, 33.5% glucose, and 1.5% agar, the cells were left to stand at 30°C for 5 days. , the colony morphology was observed. ●Results Parent strain Aureobasidium sp.SN−
The 124A strain produces colonies with wrinkled surfaces on flat agar plates, but the Aureobasidium sp. SN-G42 strain, which is the mutant strain of the present invention, produces colonies with smooth surfaces and is noticeably They are different. Example 1 (Creation of mutant strain) Aureobasidium sp. SN-124A strain was precultured for 2 days in a medium containing 0.5% yeast extract and 22% glucose, diluted 100 times, and cultured from a distance of 30 cm.
40 15W ultraviolet lamps (Toshiba germicidal lamp "GL15")
Irradiate for minutes. After diluting this, it was spread on an agar medium (1.0% yeast extract, 33.5% glucose, 1.5% agar), and those that grew were selected. Next, the strains selected in the above treatment were irradiated with ultraviolet light again for 20 minutes, and those that grew in the same manner were selected. Furthermore, the bacterial strains selected in the above treatment were treated with N-methyl-N'-nitro-N''-nitrosoguanidine at a concentration of 1 mg/ml for 30 minutes, and those that grew in the same manner were selected to identify mutant strains. A certain Aureobasidium sp. SN-G42 strain (FERM P-8940) was obtained. Example 2 Preparation of seed culture solution Glucose 33.5%, yeast extract 1.0%, agar 1.5%
Aureobasidium sp. on a slant medium consisting of %.
The cells of the SN-G42 strain (FERM P-8940) are applied and cultured at 35° C. for 2 to 3 days. Next, one platinum loop of the above cultured cells was inoculated into a 500 ml Erlenmeyer flask containing 150 ml of a liquid medium (PH4.0) containing 20% glucose and 1.6% corn steep liquor (manufactured by Oji Cornstarch Co., Ltd.). , cultured at 35°C for 3 days, and then inoculated 9 ml of this culture into a 500 ml Erlenmeyer flask containing 150 ml of the same liquid medium.
It was prepared by culturing with shaking at 35°C for 3 days. Main culture Glucose 20.0% and corn steep liquor
Add medium 15 containing 1.6% to a 30 volume fermenter,
Antifoaming agent (“Silicone KS-66” manufactured by Shin-Etsu Chemical Co., Ltd.)
After adding 300ppm, steam sterilize at 120℃ for 20 minutes. After cooling, adjust the pH of the medium using caustic soda.
After adjusting the temperature to 4.0, 6% of the seed culture of Aureobasidium sp. Culture was performed for 1 day. After the culture was completed, analysis of the culture solution revealed that glucose had been completely consumed, and the yield of erythritol in the culture solution was 49.3% and the yield of glycerin was 2.2%.
It was %. Next, take a part of this culture solution, remove the bacterial cells by centrifugation, decolorize with activated carbon, and use ion exchange resin (IRA-410:IR-120B=2:1).
Desalination was carried out by Crystals are obtained by concentrating the eluate to a sugar concentration of 50% or more and cooling slowly, and further,
After dissolving this crystal in water, recrystallization was performed in the same manner. The polyhedral-like white crystals obtained had a refreshing sweet taste and a melting point of 121°C. Furthermore, liquid chromatography, gas chromatography,
As a result of measurements such as optical rotation and nuclear magnetic resonance spectrum, the above crystal was identified as erythritol. Example 3 Glucose 20.0% and Corn Steep Liquor
Add medium 15 containing 1.6% to a 30 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.0, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 7 days at a temperature of 35°C, an aeration rate of 0.50 vvm, and a rotation speed of 230 rpm. After the culture was completed, analysis of the culture solution revealed that glucose had been completely consumed, and the yield of erythritol in the culture solution was 49.2% and the yield of glycerin was 1.2%.
It was %. Example 4 Glucose 20.0% and Corn Steep Liquor
Add medium 15 containing 1.6% to a 30 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.0, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 7 days at a temperature of 35° C., an aeration rate of 0.75 vvm, and a rotation speed of 230 rpm. After the culture was completed, analysis of the culture solution revealed that glucose had been completely consumed, and the yield of erythritol in the culture solution was 46.5% and the yield of glycerin was 2.7%.
It was %. Example 5 Glucose 20.0% and Corn Steep Liquor
Add medium 15 containing 1.6% to a 30 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.0, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 7 days at a temperature of 35°C, an aeration rate of 1.00 vvm, and a rotation speed of 230 rpm. After the culture was completed, 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%.
It was %. Example 6 Gurucose 33.5% and corn steep liquor
Put medium 5 containing 4.47% into a 7 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.2, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 5 days at a temperature of 35° C., an aeration rate of 0.25 vvm, and a 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 in the culture solution was 39.7% and the yield of glycerin was 7.9%.
It was %. Example 7 Glucose 39.5% and Corn Steep Liquor
Put medium 5 containing 4.0% into a 7 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.2, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 6 days at a temperature of 35° C., an aeration rate of 0.25 vvm, and a 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 in the culture solution was 38.6% and the yield of glycerin was 9.5%.
It was %. Example 8 Glucose 39.5% and Corn Steep Liquor
Put medium 5 containing 5.3% into a 7 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.2, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 5 days at a temperature of 35° C., an aeration rate of 0.25 vvm, and a rotation speed of 500 rpm. After the culture was completed, analysis of the culture solution revealed that glucose was completely consumed, and the yield of erythritol in the culture solution was 37.5% and the yield of glycerin was 7.2%.
It was %. Example 9 Glucose 39.5% and Corn Steep Liquor
Put medium 5 containing 6.6% into a 7 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.2, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 4 days at a temperature of 35° C., an aeration rate of 0.25 vvm, and a 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 in the culture solution was 37.1% and the yield of glycerin was 4.8%.
It was %. Example 10 Glucose 45.0% and Corn Steep Liquor
Put 15 medium containing 6.0% into a 30 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.0, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 8 days at a temperature of 35°C, an aeration rate of 0.25 vvm, and a rotation speed of 230 rpm. After the culture was completed, 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%.
It was %. Example 11 Medium 5 containing 33.5% sucrose and 4.5% corn steep liquor was placed in a 7 volume fermenter.
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
Seed culture solution 6 of Aureobasidium sp. SN-G42 strain prepared according to the method of Example 2 using a medium with the same composition as the above medium after adjusting the pH to 4.1
%, temperature 35℃, ventilation amount 0.25vvm, rotation speed
Culture was performed for 6 days at 500 rpm. 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 41.9%.
It was 7.4%. Example 12 Medium 5 containing 39.5% sucrose and 5.3% corn steep liquor was placed in a 7 volume fermenter.
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.1, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 6 days at a temperature of 35° C., an aeration rate of 0.25 vvm, and a rotation speed of 500 rpm. 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 42.4%, and the yield of glycerin was 42.4%.
It was 5.8%. Example 13 Medium 5 containing 45.0% sucrose and 6.0% corn steep liquor was placed in a 7 volume fermenter,
After adding 300 ppm of antifoaming agent, steam sterilize at 120°C for 20 minutes. After cooling, remove the medium using caustic soda.
After adjusting the pH to 4.1, Aureobasidium sp. SN-G42 strain (FERM P
-8940) was added and cultured for 6 days at a temperature of 35° C., an aeration rate of 0.25 vvm, and a rotation speed of 500 rpm. 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 41.7%.
It was 3.8%. [Effect of the invention] Aureobasidium sp. SN-G42 strain, which is a novel microorganism of the present invention, has excellent sugar tolerance and heat resistance.
It has a high erythritol production capacity and generates very little foam during fermentation and culture, making it very convenient for industrial use.

Claims (1)

[Claims] 1 Aureobasidium sp.SN-G42 strain,
Inoculate a medium containing fermentable sugars as the main carbon source,
A method for producing erythritol by fermentation, which comprises culturing aerobically to produce and accumulate erythritol in a culture solution, and collecting the erythritol.