JP2696092B2 - Method for producing erythritol using a novel microorganism - Google Patents

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to Aureobasidium sp. Having a high erythritol-producing ability. S
The present invention relates to a method for producing erythritol by fermentation, comprising converting a fermentable saccharide to erythritol using an N-124A strain (FERM P-8745). 2. Description of the Related Art Conventionally, microorganisms having erythritol-producing ability include:
For example, those belonging to the genus Candida, the genus Debaryomyces, the genus Trigonopsis, the genus Torulopsis, the genus Hansenula, the genus Aureobasidium and the like are known. For example, Japanese Patent Publication No. 47-41549 describes a method for converting fermentable sugars such as glycerin to erythritol using a microorganism belonging to the genus Candida or Trigonopsis. However, according to this method, the sugar concentration (substrate concentration) as a main carbon source is limited due to the problem of glucose tolerance of microorganisms, and it is necessary to perform fermentation at a relatively low concentration, and the conversion rate to erythritol is also low. Since it is not always sufficient, it has not yet been used industrially. The present inventors have previously proposed a novel microorganism SN-45 strain of the genus Aureobasidium (FERM P-759).
A method for producing erythritol from fermentable sugars such as glucose using 4) (JP-A-61-31091) was invented. This method made it possible to produce erythritol from a relatively high concentration of carbohydrate, and was of some value. However, in the above method, the yield of erythritol is not sufficient compared to the amount of bacterial cells,
Optimum pH and temperature ranges during production are narrow, and if they deviate slightly from these ranges, the yield of erythritol is remarkably reduced, which is not always industrially satisfactory. Means for Solving the Problems The inventors of the present invention have long searched for the usefulness of a microorganism as a biocatalyst for its ability to convert into a polyol.
A novel microorganism SN-12 belonging to the genus Aureobasidium, an imperfect fungus isolated from soil in a starch factory in Okinawa Prefecture
The present inventors have found that the 4A strain has a higher and higher erythritol-producing ability than the SN-45 strain, and have reached the present invention. That is, the present invention relates to Aureobasidium s
p. SN-124A strain (FERMP-8745) was inoculated into a medium containing fermentable saccharides such as glucose as a main carbon source, aerobically cultured, and allowed to produce and accumulate erythritol in the culture solution. The present invention relates to a method for producing erythritol by a characteristic fermentation. Hereinafter, the present invention will be described in detail. The microorganism Aureobasidium sp. S
The N-124A strain has the following mycological properties. 1. Growth condition on culture medium 1) Microscopic findings Size of vegetative cells (* 1) 4-7 × 4-15 μ Shape of vegetative cells (* 1) Hyphal and yeast-like solitary cells, oval, etc. Vegetative cell multiplication method (* 1) Multipolar budding mycelium of mycelium and yeast-like cells (* 2) Intrinsic hypha is formed, and a large number of all-budding conidia are formed on the tip and side. (Note) * 1 Cultured on YM agar medium at 27 ° C for 5 days * 2 Slide culture on potato glucose agar 2) Agar slant (* 3) Growth Good gloss No color tone White to black colonies with the passage of days * 3 YM agar medium 3) Liquid culture (* 4) Surface growth Thick film formation turbidity Transparent sedimentation Large (*) * 4 YM liquid medium [0008] Without V ₈ agar medium formed without carrot extract agar medium formed without YM agar medium formed without corn meal agar formed without forming formation potato glucose agar medium of ascospores [0009] 3. Physiological properties Oxygen demand Aerobic growth temperature Optimum growth temperature up to about 40 ° C 35-37 ° C Growth pH 2.5-9.5 Optimum growth pH 4-7 KNO ₃ assimilation (* 5) Yes (NH ₄ ) ) ₂ SO ₄ assimilation (* 5) Yes Decomposition of urea Yes Liquefaction of gelatin No Formation of carotenoids No formation of organic acids Yes Albumin No Formation of starch-like substances None Requirement of vitamins (* 5) Yes (Note) * 5 J. Lodder using Wickerham's synthetic medium
Judgment by these methods * 6 Agar medium * 7 Liquid medium 4. Fermentability of sugar (* 5) Glucose ++ Lactose-Galactose-Melibiose-Sucrose ++ Raffinose-Maltose + Cellobiose-Trehalose-Inulin- Glucose assimilating sugar, organic acid, etc. ++ D-xylose ± galactose-erythritol + D-arabinose-L-arabinose-D-ribose + sucrose + L-rhamnose-maltose + ethanol-cellobiose + salicin-L-sorbose- Ribitol-galactitol-glycerin + trehalose-lactose-melibiose-D-mannitol + raffinose-meletitose-α-methyl-D-glucoside-inulin ± inositol-soluble starch-DL-lactic acid-succinic acid ± citric acid + THE GENERA OF FUNGI SPORULATING I
As a result of studying with reference to N PURE CULTURE (JAVON ARX; 1974), this strain formed white to black colonies with the passage of days in the late stage of culture on YM agar medium and formed hyphae on vegetative cells. And produced many Blast spores and did not form chlamydospores. Therefore, it was determined to be a novel microorganism belonging to Aureobasidium sp., And Aureobasidium sp. SN-1
The strain was named 24A strain. That is, the microorganism of the present invention is purely isolated from soil in a starch factory in Okinawa according to a conventional method, belongs to the genus Aureobasidium, and has vegetative cells of 4 to 7 × 4 to 1
Proliferate by multipolar budding in the form of 5μ single cell, oval, etc.
Ascospores are not seen, true mycelia are seen, growth on YM agar medium is fast, and white to black colonies are formed in 3 days. In liquid medium, a film is formed, and fermentable sugars are mainly contained in erythritol and small Glycerin. This strain has been deposited with the National Institute of Bioscience and Biotechnology at the National Institute of Advanced Industrial Science and Technology as "FERM P-8745". Hereinafter, a method for producing erythritol according to the present invention will be described. The culture in the present invention is desirably usually performed by stirring culture under aerobic conditions using a liquid medium. Carbohydrates such as glucose, fructose, sucrose, and maltose are used as the main carbon source of the liquid medium, and the quantitative ratio of these sugars in the medium is 10 to 40% (w / w). It is particularly preferable to add and use in the range of 20 to 30% (w / w). As the nitrogen source, nitrogen compounds usable by microorganisms such as yeast extract, peptone, malt extract, casamino acid, corn steep liquor and the like are used. As the inorganic salts to be added to the medium, for example, salts such as ferrous sulfate, potassium chloride, sodium chloride, potassium dihydrogen phosphate, and calcium hydroxide are used.
Furthermore, if necessary, various organic substances necessary for the growth of yeast,
An inorganic substance or a commonly used antifoaming agent can be added. The cultivation is carried out by directly inoculating the microbial cells into a liquid medium having the above composition, or by inoculating a seed culture obtained by pre-culture. The seed culture is prepared by, for example, inoculating one loopful of bacteria cultured on a slope in a liquid medium of pH 5 to 6 containing 20% (w / w) of glucose and 2.0% of corn steep liquor at 34 to 36 ° C. It is performed by culturing at a temperature for 2 to 4 days. The cultivation temperature is within the range in which microorganisms can grow, that is, 30 to 38 ° C., and particularly preferably 35 to 38 ° C.
The range is 37 ° C. The pH of the medium is adjusted in the range of 4 to 9, preferably 5 to 7. The culture period varies depending on the type of medium used and the concentration of carbohydrate as a main carbon source, but is usually about 4 to 10 days. In the culturing in the present invention, it is desirable to terminate the culturing when the nutrient source of the medium is used to the maximum and the amount of erythritol produced in the culture solution reaches the maximum. [0017] The amount of erythritol produced in the culture solution can be quickly measured by a known method such as gas chromatography or high performance liquid chromatography. Thus, the erythritol accumulated in the culture solution is separated from the culture solution according to a conventional method. That is, in such a case, well-known means commonly used in the art, for example, filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, distillation, crystallization and the like are used in combination as needed as appropriate. . For example, the culture solution is filtered to remove bacterial cells by centrifugation, and then the solution is treated with activated carbon to remove coloring substances, and further deionized with an ion exchange resin. Syrup. Next, erythritol is crystallized and separated from the syrup. If glycerin is contained in the mother liquor after separation of erythritol,
This can be further purified by distillation under reduced pressure to obtain glycerin. Next, the present invention will be described in more detail with reference to Examples. Example 1 5 kg of a medium containing 20% of glucose (w / w), 2.0% of corn steep liquor previously adjusted to pH 6.0, 0.1% of sorbitan fatty acid ester and 0.03% of a silicon-based antifoaming agent were prepared. Into a liter fermenter, 120 ° C,
Sterilized for 20 minutes. After cooling, the medium pH was aseptically adjusted to 5.5.
Was adjusted. Aureobasidium sp. Cultured for 3 days in the same medium as above was added thereto. 200 ml of a seed culture of the SN-124A strain (FERM P-8745) was added, and the mixture was heated at 35 ° C., 400 rpm, and aerated at 1 liter / mi.
n for 7 days. As a result, 467 g of erythritol and trace amounts of glycerin were accumulated in this culture solution. The cells are removed from this culture by centrifugation, decolorized with activated carbon and ion-exchange resin (IRA-4).
10: IR-120B = 2: 1). Then, the mixture was concentrated and stored at 5 ° C. to obtain crystals. The crystals were dissolved and recrystallized by the same method. The obtained polyhedral white crystals had a refreshing sweet taste and the melting point was 121 ° C. This white crystal was identified as erythritol from the measurement results of NMR, optical rotation, liquid chromatography, gas chromatography and the like. EXAMPLE 2 50 g of a medium containing 30% (w / w) of glucose and 3.2% of corn steep liquor (pH 6.0 adjusted solution) was added to 50 g of a medium.
After placing in a 0 ml Erlenmeyer flask, sterilizing and cooling, the pH of the medium was adjusted to 6.0. The same seed culture solution 2 as in Example 1
ml, and shaking culture was performed at 35 ° C. and 180 rpm for 10 days. As a result, 5.9 g of erythritol and 1.4 g of glycerin were accumulated in this culture solution. Example 3 50 g of a medium containing 20% (w / w) of sucrose and 0.5% of yeast extract was placed in a 500 ml Erlenmeyer flask, and 1
Sterilized at 20 ° C. for 15 minutes. After cooling, the medium pH was adjusted to 6.0, to which Aureobasidium sp. SN-124
A strain (FERM P-8745) was inoculated at 35 ° C.
Shaking culture was performed at 180 rpm for 7 days. As a result, 4.2 g of erythritol is accumulated in the culture solution,
No glycerin accumulation was observed. Example 4 Yeast extract 0.5%, glucose 10-30% (w /
50 g of the medium containing w) was placed in a 500 ml Erlenmeyer flask, and sterilized at 120 ° C. for 15 minutes. After cooling, medium p
H was adjusted to 5.5, and a seed culture (Aureobasidium sp. SN-124A strain (FERM P-87) was added thereto.
45) was cultured in a medium consisting of 20% glucose and 0.5% yeast extract at 35 ° C for 3 days) 2 ml
, And shaking culture was performed at 35 ° C. and 180 rpm for 4 to 11 days. The results were as shown in Table 1. [Table 1] Example 5 Eight 500 ml Erlenmeyer flasks were charged with 50 g of a medium containing 20% (w / w) of glucose and 0.5% of yeast extract, and sterilized. After cooling, the pH of the medium was adjusted with 1N hydrochloric acid or 1N sodium hydroxide so as to be between 2.9 and 10.1. Next, a seed bacterium, Aureobasidium sp. SN-124A strain (FERM P-8745) was inoculated at 35 ° C., 18
Shaking culture was performed at 0 rpm for 7 days. As a result, erythritol was obtained in a yield as shown in Table 2. [Table 2] According to the present invention, erythritol can be produced very efficiently by a fermentation method using a strain belonging to the genus Aureobasidium.

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Claims (1)

(57) [Claims] Aureobasidium sp. SN-124A strain (FERMP-8745) was inoculated into a medium containing fermentable saccharides such as glucose as a main carbon source, aerobically cultured, and allowed to produce and accumulate erythritol in the culture solution. A method for producing erythritol by fermentation.