JPH09154590A - Production of erythritol - Google Patents

Abstract

PROBLEM TO BE SOLVED: To profitably obtain a compound from an inexpensive raw material by culturing a microorganism belonging to the genus Yarrowia, Ustilage, Filobasidium or Trigonopsis and capable of producing the erythritol from a fermentable saccharide. SOLUTION: This method for producing the erythritol comprises culturing a yeast (e.g. Yarrowia lipolytica ATCC 8661 strain) belonging to the genus Yarrowia, the genus Ustilago, the genus Filobasidium, the genus Trigonopsis, etc., and having an ability to produce the erythritol in a culture medium containing a fermentable saccharide such as glucose, fructose or glycerol as a main carbon source in a concentration of 20-50% at a culture temperature of 25-35 deg.C and subsequently collecting the product from the culture product. Thus, the objective erythritol is efficiently from the inexpensive raw material such as the fermentable saccharide in a high yield.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a method for producing erythritol, and more particularly to a method for industrially advantageously producing erythritol by fermentation using yeast.

2. Description of the Related Art As a method for producing erythritol, a method of culturing yeasts of the genera Trigonobosis and Candida in a medium containing glycerol as a carbon source (Japanese Examined Patent Publication No. 47).
No. 41549), a yeast of the genus Candida, the genus Tolulopsis, and the genus Hansenula are cultured in a medium containing a hydrocarbon or the like as a carbon source to produce (Japanese Patent Publication No. 51-21072).
Is known. However, these methods have not yet been industrialized because raw materials used as carbon sources are not suitable for actual industrial production. In addition, Moniella Tomentosa Bar Polinis ( Mo
nielliatomentosa var. po
llinis ) is cultivated in a medium containing a sugar such as glucose as a carbon source to prepare (JP-B-60-11029).
No. 5, etc.) are also known. This method is excellent in that it uses glucose, which is an inexpensive and safe raw material, and has high productivity, but foaming during culture is remarkable, and it is useless with commonly used antifoaming agents. However, it is necessary to add a large amount of expensive xadadan gum or the like, which is not always an advantageous method in industrial production.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing erythritol from a raw material that is inexpensive and can be easily supplied at a high yield and at a low cost.

Means for Solving the Problems The present inventors have conducted extensive research on a method for producing erythritol from an inexpensive raw material, and as a result, many yeasts belonging to the genus Yerobia, the genus Ustilago, the genus Filovacidium and the genus Trigonopsis,
The inventors have found that erythritol is produced in large quantities from fermentable sugars such as glucose and fructose, and completed the present invention. That is, the present invention, the genus Yerobia, genus Ustilago, genus Filovacidium or genus Trigonopsis, a microorganism having the ability to produce erythritol from a fermentable sugar, is cultured in a medium having a fermentable sugar as a main carbon source, and cultured. A method for producing erythritol, which comprises collecting erythritol from a product. According to a preferred embodiment of the present invention, the above-mentioned production method, wherein the yeast belonging to the genus Hierobia is Hierbia lipolytica; the yeast belonging to the genus Ustilago is Ustilago cruz-galli, Ustilago Sinodontis, Ustilago maides or Ustilago. The above-mentioned production method which is Lavenfostiana; the yeast belonging to the genus Filovacidium is Filobacidium capsulignus, the above-mentioned production method; the yeast which belongs to the genus Trigonopsis is the production method described above, which is Trigonopsis variabilis; , Glucose, fructose or glycerol; the above-mentioned production method, wherein the concentration of fermentable sugar in the medium is in the range of 20 to 50%;
There is provided the above-mentioned production method, wherein the culture temperature is within the range of 25 ° C to 35 ° C.

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below. The yeast used in the present invention may be any yeast as long as it belongs to the genus Yerobia, the genus Ustilago, the genus Filovasidium or the genus Trigonopsis and has the ability to produce erythritol from fermentable sugars. Specifically, yeasts belonging to the genus Yerobia include, for example, Yarrowia lipolytica ( Yarrowowi)
a lipolytica) and the like; Ustilago The yeast belonging to the genus example, Ustilago Cruz - gully (Us
tilago crus-galli ), ustilago
Sinodonchis ( Ustilago cynodonti
s ), Ustilago maides ( Ustilago ma)
ydis ) and Ustilago lavenfostiana ( Us
tilago rabenhorstiana ) and the like; yeasts belonging to the genus Filovasidium include, for example, Filobasidium capsuligienus ( Filobasidi).
um capsuligenum ) and the like; yeasts belonging to the genus Trigonopsis include, for example, Trigonopsis variabili.
Lis ) and the like are preferred. The above yeast was irradiated with UV, treated with N-methyl-N-nitrosoguanidine (NTG), and ethyl methanesulfonate (EMS).
It may be any strain such as a mutant strain by treatment, nitrite treatment, acridine treatment or the like, or a gene recombinant strain induced by a genetic technique such as cell fusion or gene recombination. As the yeast used in the present invention, yeast belonging to the genus Yerobia is particularly preferable. All of the above yeasts are known strains deposited in the depository institution and can be easily obtained from the depositary institution. In other words, Yelovia Ripolitica is an American type culture
Collection (American Type Cult
ure Collection (ATCC) to ATCC
Deposited as 8661, Ustilago Cruz-
Galli, Ustilago Sinodontis, Ustilago Maydes, Ustilago Ravenfostiana, Firobacillium capsulighenum and Trigonopsis variabilis are the Institute of Fermentation Research Institute
Fermentation, Osaka; IFO)
, IFO31947, IFO9758, I
Deposited as FO6907, IFO8995, IFO1119 and IFO0671. Fermentable sugars such as glucose, fructose and glycerol are used as the main carbon source of the medium used for culturing these yeasts. These fermentable sugars can be used alone or in combination. The concentration to be used is not particularly limited, but it is advantageous to make it as high as possible within a range that does not inhibit the production of erythritol, and a preferable concentration is 20 to 50.
It is within the range of% (W / V). As the nitrogen source, various organic and inorganic nitrogen compounds such as ammonia salt, urea, peptone, yeast extract and corn stapler are used. As the inorganic salt, various phosphates, sulfates, and metal salts such as magnesium, potassium, manganese, iron, and zinc are used. In addition, factors that promote yeast growth, such as vitamins, nucleotides and amino acids, are added as necessary. It is also desirable to add an appropriate amount of a commercially available antifoaming agent in order to suppress foaming during culturing. PH 3 at the beginning of fermentation
To 7, preferably pH 3 to 4.5. The temperature during the culture is 25 ° C to 35 ° C, preferably 27 ° C to 32 ° C.
Adjust to ℃. At the time of culturing, the cells may be directly inoculated into the medium from the slant culture, but it is preferable to inoculate the culture solution obtained by culturing in a liquid medium for 1 to 4 days.
Further, the culture is performed under aerobic conditions such as aeration and stirring, shaking. The culture time is preferably until the main carbon source is consumed, and usually 3 to 8 days. The amount of erythritol produced in the culture broth can be measured by a method such as gas chromatography or high performance liquid chromatography. Erythritol thus accumulated in the culture solution is separated and purified from the culture according to a conventional method. Specifically, erythritol can be separated and purified by removing solid matter by centrifugation, filtration and the like, decolorizing and desalting with activated carbon and an ion exchange resin, and crystallizing from the solution.

EXAMPLES The present invention will be described more specifically below with reference to Examples.
The present invention will be described below unless the gist thereof is exceeded.
The method of listing is not limited. Example 1 Culture containing 20% glucose (W / V) and 1% yeast extract
Place in a 500 ml Erlenmeyer flask with 50 ml ground
And sterilized at 120 ° C for 20 minutes.
Inoculated with lipolytica ATCC 8661 strain, 7 days at 27 ℃
The cells were cultured with shaking. Increase the erythritol concentration in the culture
As a result of measurement by high performance liquid chromatography, the concentration is
It was 92 g / L. This is a glucose yield of 46%
there were. The culture solution is centrifuged and the solid matter is filtered off.
Decolorized by. This clarified liquid is a strongly acidic cation exchange resin.
(Diaion SKIB H type) and weakly basic anion
Uses on-exchange resin (Diaion WA30 OH type)
And desalted. This solution was concentrated under reduced pressure at 50 ° C,
Concentrate to 60% thritol and leave it in a cool dark place.
More crystals were obtained. This crystal has a melting point of 121 ° C and has a sweet taste.
I was The molecular formula is C _FourH_TenO_FourAnd a liquid chromatograph
Fee retention time and infrared absorption spectrum are
In agreement, it was identified as erythritol. Example 2 Medium 50 containing 1.0% yeast extract and 20% glucose
Add 500 ml Erlenmeyer flask to 2 at 120 ℃
Sterilize for 0 minutes, and then add this to Yerobia lipolytica ATCC
The 8661 strain was inoculated and shake-cultured at 27 ° C. for 4 days.
This culture was inoculated into the above medium at a rate of 10%
Incubate in a medium containing glucose at various concentrations as described below.
Inoculated into the ground and cultured. That is, yeast extract 1.0%
And 50 ml of medium containing 20-60% glucose
Place in a 500 ml Erlenmeyer flask and inoculate as described above.
And cultured for 7 days with shaking. After the culture is complete,
High-performance liquid chromatographic measurement of lysitol concentration
did. As a result, erythritol at each glucose concentration
The production amount of the alcohol was as follows.Glucose concentration (%) Erythritol production (g / L) 20 72.6 30 98.6 40 73.6 50 57.2 60 3.7 Example 3 Fructose 20% (W / V) and yeast extract 1
50 ml of the medium containing 50% of the medium, and 500 ml of a triangular plug with a cotton plug.
Place in a rasco and sterilize at 120 ° C for 20 minutes.
27 inoculated with Erovia lipolytica ATCC 8661 strain
The culture was carried out with shaking at 7 ° C for 7 days. Erythritol in culture
As a result of measuring the concentration by high performance liquid chromatography,
Was 53.4 g / L. Example 4 Glycerol 20% (W / V), yeast extract 1%
Place 50 ml of medium in a 500 ml Erlenmeyer flask with a cotton stopper.
And sterilized at 120 ° C for 20 minutes.
Inoculated with lipolytica ATCC 8661 strain at 27 ° C for 7 days
Shake culture was performed. High-speed erythritol concentration in culture solution
As a result of measurement by liquid chromatography, the concentration is 4
It was 3.2 g / L. Example 5 50 ml of medium containing 30% glucose and 1% yeast extract
Put each into a 500 ml Erlenmeyer flask with 6 cotton stoppers,
Sterilized at 120 ℃ for 20 minutes
Ritchica ATCC 8661 strain was inoculated and shaken at 27 ° C for 4 days.
It was cultured. Contains 30% glucose and 1% yeast extract
Add 17 L of culture medium to a 30 L culture tank to remove 500 ppm
Add the foaming agent CA330 (made by NOF CORPORATION), and
The preculture was inoculated into a sterilized product at 0 ° C for 20 minutes, and 27
Culturing was carried out at 4 ° C for 4 days. Aseptically use 1.7 L of this culture solution.
40% glucose (W / V), yeast extract 2%, salt
Medium containing 0.1% thiamine acid and adjusted to pH 4.0
Add 17 L to a 30 L culture tank and add 500 ppm defoamer
Sterilized by adding CA330 and sterilizing at 120 ° C for 20 minutes
The cells were inoculated and cultured at 27 ° C. 96 hours after the start of culture
Erythritol at the time of complete consumption of Lucose
Was 205.5 g / L. Also during the culture
No foaming was observed. Example 6 Culture containing 20% glucose (W / V) and 1% yeast extract
Place in a 500 ml Erlenmeyer flask with 50 ml ground
Sterilized at 120 ° C for 20 minutes.
Sinodonchis IFO 9758 strain was inoculated and kept at 27 ° C for 7 days
The cells were cultured with shaking. Increase the erythritol concentration in the culture
As a result of measurement by high performance liquid chromatography, the concentration is
It was 103 g / L. This gives a yield of 51.
5%. The fermentation broth is centrifuged and the solid matter is filtered off,
Decolorized with activated carbon. This clarified liquid is subjected to strong acid cation exchange.
Replacement resin (Diaion SKIB H type) and weak base
Anion Exchange Resin (Diaion WA30 OH
Desalting was carried out using Concentrate this solution under reduced pressure at 50 ° C.
Then, concentrate it to 60% erythritol and leave it in a cool and dark place.
By doing so, crystals were obtained. This crystal has a melting point of 121 ° C and is sweet
Had a taste. The molecular formula is C_FourH_TenO_FourAnd liquid black
The retention time and infrared absorption spectrum of
Consistent with the quasi substance, it was identified as erythritol. Example 7 Culture containing 20% glucose (W / V) and 1% yeast extract
Place 50 ml of ground into a 500 ml Erlenmeyer flask with a cotton stopper.
Sterilized at 120 ° C for 20 minutes.
Mades IFO 6907 strain, Ustilago Lavenphosti
Ana IFO 8995 strain, Ustilago Cruz-Gali IF
O31947 strain, Phylobacillus capsuligenum I
FO1119 strain, Trigonopsis variabilis IFO
Each of the 0671 strains was inoculated and shaken at 27 ° C for 7 days.
Nourished. After culturing, adjust the concentration of erythritol in the culture solution.
It was measured by high performance liquid chromatography. As a result, each
The amount of erythritol produced by the strain was as follows.Strain Erythritol production (g / L) IFO6907 28.0 g / L IFO8895 33.3 g / L IFO31947 3.7 g / L IFO1119 21.9 g / L IFO0631 15.1 g / L Example 8 Yeast extract 1.0%, medium 20 containing glucose 50% 50
Add 500 ml Erlenmeyer flask to 2 at 120 ℃
Sterilize for 0 minutes, and then add this to Yerobia lipolytica ATCC
The 8661 strain was inoculated and shake-cultured at 27 ° C. for 4 days.
This culture was inoculated into the above medium at a rate of 10%
Incubate in a medium containing glucose at various concentrations as described below.
Inoculated into the ground and cultured. That is, yeast extract 1.0%
And 50 ml of medium containing 10-60% glucose
Place in a 500 ml Erlenmeyer flask and inoculate as described above.
Then, the cells were shake-cultured for 7 days. After the culture is complete,
High-performance liquid chromatographic measurement of lysitol concentration
did. As a result, erythritol at each glucose concentration
The production amount of the alcohol was as follows.Glucose concentration (%) Erythritol production (g / L) 10 0 20 60.6 30 77.9 40 92.7 50 37.2 60 2.3 Example 9 Medium 50 containing 1.0% yeast extract and 20% glucose
Add 500 ml Erlenmeyer flask to 2 at 120 ℃
Sterilize for 0 minutes, and then add this to Yerobia lipolytica ATCC
The 8661 strain was inoculated and shake-cultured at 27 ° C. for 4 days.
Yeast extract 1.0%, glucose 40%, 500ppm
1L culture of 500ml medium containing antifoam agent CA330
Precultured in a tank and sterilized at 120 ° C for 20 minutes
And the temperature was changed between 20 and 37 ° C,
Cultured for 6 hours. Erythritol in the culture medium after culturing
Concentration was measured by high performance liquid chromatography. That
As a result, the amount of erythritol produced at each temperature was
It was a cage.Temperature (℃) Erythritol production (g / L) 20 30.0 25 61.4 30 75.4 32 88.8 35 47.4 37 0

According to the method of the present invention, erythritol can be efficiently produced in high yield from inexpensive fermentable sugars such as glucose.

Claims (9)

[Claims] 1. A yeast having the ability to produce erythritol from fermentable sugars belonging to the genus Yerobia, the genus Ustilago, the genus Filovasidium or the genus Trigonopsis, is cultivated in a medium containing fermentable sugar as a main carbon source, and a culture product. A method for producing erythritol, which comprises collecting more erythritol. 2. The method according to claim 1, wherein the yeast belongs to the genus Yerobia. 3. The method according to claim 1 or 2, wherein the yeast belonging to the genus Yerobia is Yerobia lipolytica. 4. The method according to claim 1, wherein the yeast belonging to the genus Ustilago is Ustilago cruz-galli, Ustilago Sinodontis, Ustilago Maydes or Ustilago Lavenfostiana. 5. The production method according to claim 1, wherein the yeast belonging to the genus Firobacillium is Firobacillus capsulighenus. 6. The method according to claim 1, wherein the yeast belonging to the genus Trigonopsis is Trigonopsis variabilis. 7. The method according to claim 1, wherein the fermentable sugar is glucose, fructose or glycerol. 8. The concentration of fermentable sugar in the medium is 20 to 50.
The manufacturing method according to claim 1 or 2, which is in the range of%. 9. The production method according to claim 1, wherein the culture temperature is in the range of 25 ° C. to 35 ° C.