JP2009148211A - Method for fermentatively producing d-arabitol and microorganism used for performance thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for fermentatively producing D-arabitol, by which the D-arabitol, a functional saccharide, can effectively efficiently be produced, at a low cost from glycerol massively by-produced in the oil and fat industry as a main raw material, and to provide a microorganism used for efficiently performing the method. <P>SOLUTION: Provided is the method for producing the D-arabitol, culturing at least one microorganism selected from Candida quercitrusa, Debaryomyces hansenii, Pichiastipitis, Metschnikowia zobelli, and Metschnikowia lunata in a liquid culture medium containing glycerol as a carbon source. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention efficiently fermentatively produces D-arabitol, which is a functional carbohydrate, by using glycerol, particularly inexpensive glycerol produced in large quantities as a by-product in the fat and oil industry, as a main raw material in a culture medium for microorganisms. The present invention relates to a method for producing D-arabitol and a microorganism used for efficiently carrying out the method.

  D-arabitol is a sugar alcohol conventionally produced by a chemical reduction method of D-arabinose. The starting material D-arabinose itself is an expensive sugar, and as a result, D-arabitol has also become very expensive. However, since D-arabitol is a compound with little demand mainly used as a chemical reagent, it has not been regarded as important in terms of high cost.

  However, recently, interest in the function of sugar alcohol has increased, and future demand is expected to increase. For example, xylitol as a trigger is lower in calories than sucrose and has a sweetness comparable to sucrose, so it is not only promising as a low-calorie sweetener in the future, but also has anti-cariogenic sweetness. It is also used as a fee. Furthermore, since xylitol does not increase blood glucose level, it is also used as an infusion for the treatment of diabetes.

  Similarly, D-arabitol itself has been found useful as a functional material having a moisturizing effect and as a sweetener having anti-cariogenic properties. Furthermore, it has been reported that D-arabitol can be used as a starting material for the production of the above-mentioned xylitol, which is already in great demand (Patent Documents 1, 2, Non-Patent Document 1), and future demand expansion as an extremely useful carbohydrate. Is expected.

However, as described above, D-arabitol is very expensive. Therefore, in response to the recent increase in demand, a method that does not use expensive D-arabinose as a starting material, for example, a fermentation method using an inexpensive raw material has been studied. It is being advanced. Glucose, as microorganisms relates very inexpensive sugar as raw materials D- arabitol production fructose, for example, Debaryomyces-Hanzenii (Debaryomyces hansenii) (Non-Patent Document 2), Metschnikowia (Metschnikowia) genus (Patent Document 3), Candy A yeast belonging to Candida parapsilosis (Patent Document 4) has been reported. Since these fermentation methods are highly economical and can produce only D-arabitol specifically from glucose or fructose, D-arabitol can be separated very easily after the reaction.

  On the other hand, the use of biomass energy has been promoted recently from the viewpoints of global warming prevention measures and the development of a sustainable society, and enormous amounts of sugars have been used as bioethanol production raw materials. Raw material prices are rising. Glucose is now not a cheap raw material. In contrast, glycerol is produced in large quantities as a by-product in fields such as the development of materials using vegetable oil as raw materials and the production of biodiesel as the same renewable natural resources. These surplus glycerol is expected to continue to increase in the future, and it is desired to develop a treatment method or an effective utilization method.

Recently, in conjunction with the above-mentioned background of the times, research to produce functional substances using glycerol as a fermentation raw material has been actively promoted. As a report example to be noted here, there are old report examples (Patent Document 5, Non-Patent Document 3) related to a production method for fermentative production of D-arabitol using glycerol as a raw material. Here, Candida polymorpha (Candida polymorpha), Saccharomyces rouxii (Saccharomyces rouxii), using a microorganism belonging to Torulopsis prongs (Torulopsis famata), glycerol in media as a main carbon source D- arabitol fermentation production I did, but the production was not enough. There are no examples of fermentative production of D-arabitol using glycerol as a raw material by microorganisms other than the above three examples.

European Patent Publication No. 403392 European Patent Publication No. 421882 JP 2001-8682 A JP 2002-191388 A Japanese Patent Publication No.47-20394 H. H. Onishi, T. Suzuki, “Applied Microbiology”, Vol. 18, p1031-1035 (1969). M. F. Noble, M.F. S. Dacosta (M. S. Dacosta), “Canadean Journal of Microbiology”, Vol. 31, p467-471 (1985). H. H. Onishi, T. Suzuki, “J. Ferment. Technol.”, 48, p563-566 (1970).

  In view of the above circumstances, in the present invention, glycerol, which is a less expensive raw material, particularly glycerol produced as a by-product in a large amount in the fat and oil industry, is used as a main raw material, so that D-arabitol, which is a functional carbohydrate, is inexpensive and efficient. It aims at providing the microorganisms used in order to carry out the method of fermentative production, and implementing it efficiently.

  As a result of intensive studies to solve the above problems, the present inventor used D-arabitol by culturing it in a liquid medium to which glycerol was added as a main raw material, using microorganisms screened from the environment and related microorganisms. It has been found that it can be produced at a higher yield and at a lower cost than conventional fermentation production methods, and has led to the present invention.

That is, the present invention is shown in the following [1] to [9].
[1] In a liquid medium containing glycerol as a carbon source, Candida quercitrusa , Debaryomyces hansenii , Pichia stipitis , Metschnikowia zobelli or Metschnikowia zobelli A method for producing D-arabitol, wherein at least one microorganism selected from Metschnikowia lunata ) is cultured.
[2] Candida-Kuerushitorusa (Candida quercitrusa) is a Candida, Kuerushitorusa (Candida quercitrusa) 17-2A (FERM P -21420), a method of D- arabitol production according to [1].
[3] The method for producing D-arabitol according to [1], wherein the microorganism is Candida quercitrusa 17-2A (FERM P-21420).
[4] The method for producing D-arabitol according to any one of [1] to [3], wherein glycerol is a by-product of decomposition or transesterification of vegetable oil.
[5] A microorganism Candida quercitrusa that can grow at 37 ° C.
[6] A microorganism Candida quercitrusa that can grow on a vitamin-deficient medium.
[7] A microorganism Candida quercitrusa having L-arabinitol utilization.
[8] A microorganism Candida quercitrusa that can be grown at 37 ° C., can be grown on a vitamin-deficient medium, and has L-arabinitol utilization.
[9] Microbial Candida quercitrusa 17-2A (FERM P-21420).

According to the present invention, high-value-added D-arabitol can be produced using glycerol, which is an inexpensive raw material, in particular, by-products such as the fats and oils industry, or inexpensive glycerol produced in large quantities as industrial waste. it can. Up to now, there have been reports of manufacturing technology using carbohydrates such as glucose, fructose, sucrose, etc. as raw materials, but by using glycerol, which is a cheaper raw material, to provide D-arabitol to the market at a lower price. Can do.
Microorganisms having the ability to produce D- arabitol glycerol as a raw material, so far, three cases of a microorganism belonging to Candida polymorpha (Candida polymorpha), Saccharomyces rouxii (Saccharomyces rouxii), Torulopsis prongs (Torulopsis famata) However, the microorganisms used in the present invention can produce D-arabitol in a yield exceeding these, and can also be produced from low-purity glycerol.
In particular, from the background of recent times, the price of the sugar is expected to rise, whereas the amount of surplus glycerol produced is expected to increase steadily. The effect of reducing the price of arabitol is extremely large.
At the same time, the present invention provides a method for treating and effectively using surplus glycerol in the industry, which has been limited in its use so far, and greatly contributes to the efficient circulation of natural resources. Is.

Hereinafter, the present invention will be described in more detail.
< Microorganisms that can be used to produce D-arabitol >
The microorganisms used to practice the present invention, Candida-Kuerushitorusa (Candida quercitrusa), Debaryomyces, Hanzenii (Debaryomyces hansenii), Pichia stipitis (Pichia stipitis), Metschnikowia-Zoberi (Metschnikowia zobelli), or Metschnikowia lunata And at least one microorganism selected from ( Metschnikowia lunata ). Moreover, the mutant obtained by these microorganisms based on the conventional method is also included.

Further, among the above-mentioned Candida quercitrusa , the present inventors newly discovered Candida quercitrusa 17-2A ( Candida quercitrusa ) from a plant (flower) sample collected in Naha City, Okinawa Prefecture. FERM P-21420) was found to have the ability to selectively produce D-arabitol from glycerol and found it could be used more effectively in the present invention. Therefore, in the method of the present invention, the Candida quercitrusa 17-2A (FERM P-21420) can be used in combination with the above-mentioned other microorganisms or alone. Hereinafter, the mycological properties of this new strain will be described.

<Mycological properties of 17-2A strain>
The 17-2A strain forms a colony having a diameter of about 2 to 3 mm by culturing on a YM agar medium at 25 ° C. for 4 days (shape: circular, raised state: cushion shape, peripheral edge: almost all edges, Surface shape: smooth or slightly wrinkled, color tone: white to cream, gloss and properties: chalky, buttery). Furthermore, according to microscopic morphological property observation with an optical microscope, it is confirmed that the vegetative cells of this strain after 4 days of culture are elliptical and proliferate by multipolar budding. Pseudohyphae formation is also observed. In addition, the formation of sexual reproductive organs is not observed on the culture medium after 1 month of culture.

In the 17-2A strain, the nucleotide sequence (rDNA sequence) of the 26S rDNA-D1 / D2 region of the ribosomal RNA gene was determined, and BLAST homology search (SF) using Apollon DB-FU (Ver.1.0) and the international nucleotide sequence database. Altschul et al., Nucleic Acids Research, 25, 3389-3402 (1997)) showed a 99.8% match with the rDNA sequence of Candida quercitrusa . The molecular phylogenetic tree of this strain is shown in FIG. Furthermore, the result of the physiological property test of this strain (Table 1) was combined, and this strain was named Candida quercitrusa 17-2A strain. This strain was deposited on October 30, 2007 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (IPOD) (1-1-3 Higashi 1-1-3, Tsukuba, Ibaraki) under the accession number FERM P-21420 Has been. Conventional Candida quercitrusa does not show L-arabinitol assimilation ability and does not show growth in vitamin-deficient medium and at 37 ° C. (Kurtzman, CP and Fell, JW 1998. The Yeasts, a taxonomic study) , 4th edition, Elsevier, Amsterdam, Netherlands). On the other hand, this strain has L-arabinitol assimilation ability and exhibits growth in a vitamin-deficient medium and growth at 37 ° C. Accordingly, the present strain has an increased ability to utilize L-arabinitol, thereby increasing the types of nutrient sources that can be used. By exhibiting growth in a vitamin-deficient medium, it can be cultured in an oligotrophic medium. Furthermore, by showing the growth at 37 ° C, in the fermentation production using microorganisms generally accompanied by heat generation, the cooling process is not required as in the case of using the conventional Candida quercitrusa, and the cost is reduced. Can be reduced. As described above, this strain has excellent advantages in performing fermentation production.

表中の「＋」は反応が陽性、「−」は反応が陰性、「Ｗ (weak)」は弱い陽性反応、「S (slow)」は試験開始後２週間から３週間にかけて徐々に陽性反応が認められたこと、「Ｌ (latent)」は試験開始後２週間以降に急速に陽性反応が認められたことを示す。

“+” In the table indicates a positive reaction, “−” indicates a negative reaction, “W (weak)” indicates a weak positive reaction, and “S (slow)” indicates a gradually positive reaction from 2 to 3 weeks after the start of the test. “L (latent)” indicates that a positive reaction was rapidly observed after 2 weeks from the start of the test.

< Method for producing D-arabitol >
The method for producing D-arabitol of the present invention is carried out by culturing the above microorganisms in a medium using glycerol as a carbon source. Details of the method are described below.
<Medium and culture conditions>
In the present invention, it is desirable to aerobically culture at least one or two or more of the above microorganisms in a liquid medium.
As the liquid medium, a solvent obtained by adding a nutrient such as a carbon source, a nitrogen source, inorganic salts, vitamins to a solvent such as water can be used.
In the method for producing D-arabitol of the present invention, glycerol is used as a main carbon source. As the glycerol, a commercially available high-purity glycerol reagent can be used. In the present invention, waste glycerol produced as a by-product of decomposition or transesterification of vegetable oil in the oil and fat industry can be used as it is. is there. Furthermore, it is better to remove microorganism production inhibitors (such as trace metals) contained in the waste glycerol by activated carbon treatment and use it in the culture solution. Specifically, activated carbon (granular form, manufactured by Wako Pure Chemical Industries, Ltd.) (for example, 200 to 500 g with respect to 1 L of waste glycerol) is added to the above-mentioned waste glycerol, and after stirring for 60 minutes or more at room temperature, the activated carbon is filtered. It is desirable to use the removed filtrate as activated glycerol-treated waste glycerol. The concentration condition of pure glycerol added to the liquid medium is 50 to 500 g / L, preferably 100 to 400 g / L, more preferably 150 to 350 g / L, and most preferably 200 to 300 g / L. It is preferable. Glycerol can be added, for example, at an initial stage of 100 to 400 g / L, and then added stepwise by 100 to 200 g / L at intervals of 2 to 4 days. In addition to glycerol, a carbon source can be obtained by adding about 10 to 50 g / L of a commonly used saccharide that can be assimilated by the microorganism used in the practice of the present invention, such as glucose, galactose, sucrose, or molasses. May be used together.

Nitrogen compounds that can be used by microorganisms such as organic compounds (yeast extract, peptone, malt extract, casamino acid, corn steep liquor, amino acids, urea, etc.), inorganic substances (nitrates such as sodium nitrate, ammonium salts, etc.) Any of these can be used alone or in combination, and yeast extract can be preferably used from the viewpoint of enhancing fermentation productivity.
Inorganic salts may be used alone or in combination as a mineral component such as phosphates, calcium salts (for example, calcium chloride), magnesium salts, manganese salts, iron content, zinc content and the like. Furthermore, various vitamins, antifoaming agents, and the like can be appropriately selected and added as necessary, and their use may be carried out in the usual manner.
In the method of the present invention, the concentration of the nitrogen source is in the range of 0.1 to 20 g / L, preferably 1 to 10 g / L, more preferably 5 to 9 g / L, and most preferably 6 to 8 g / L. It is preferable to increase the fermentation productivity. Moreover, it is a viewpoint which makes the density | concentration of the said calcium salt the range of 0.1-20 g / L, Preferably it is 0.2-10 g / L, More preferably, it is the range of 0.5-2 g / L which improves fermentation productivity. To preferred.
Hereinafter, the medium used for producing D-arabitol as described above is referred to as “D-arabitol production medium”.

<Example of D-arabitol production method>
For example, the cells of the microorganism grown on an agar medium containing 10 g / L of D-glucose, 3 g / L of yeast extract, 3 g / L of malt extract, and 5 g / L of peptone are directly added to the D-arabitol production medium. Inoculation is carried out by inoculating D-arabitol production medium with a seed culture solution obtained by inoculation or separately by preculture. The seed culture solution is prepared by, for example, transferring the cells on the agar medium to a liquid medium containing sterilized glycerol 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, and peptone 5 g / L. One platinum ear is inoculated and cultured aerobically at 25-30 ° C. for 20-30 hours. The culture temperature of the culture for producing D-arabitol (main culture) is 20 to 40 ° C, preferably 25 to 34 ° C. The pH of the D-arabitol production medium is usually adjusted to 3 to 10, preferably 5 to 8. Since the culturing period varies depending on the concentration of glycerol used, it is desirable to terminate the cultivation period at the most effective time when the amount of bacterial cell growth and the amount of D-arabitol produced in the range of 3 to 14 days settles to a steady state.

<Quantification, recovery and purification of D-arabitol>
The amount of D-arabitol produced in the culture solution can be measured using a known method such as high performance liquid chromatography (HPLC) or gas chromatography. Regarding isolation and purification of D-arabitol from the obtained culture broth, commonly used operations such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, and crystallization are appropriately combined as necessary. Done. For example, the cells are removed from the culture solution by centrifugation or the like, and then this solution is treated with activated carbon to remove colored substances, desalted with an ion exchange resin, and then concentrated from a concentrated syrup such as ethanol or acetone. Crystallization in the presence or absence of an organic solvent makes it possible to obtain pure D-arabitol crystals.
Hereinafter, the present invention will be described with reference to examples and comparative examples, but the scope of the present invention is not limited thereto. In addition, in the case of the concentration (g / L), all means the concentration of the substance with respect to 1 L of the culture solution. All cultures were performed under aerobic conditions unless otherwise specified.

Example 1
(D-arabitol production using waste glycerol)

  As a microorganism for producing D-arabitol from glycerol, Candida quercitrusa NBRC1022 strain was used.

  The above strain is cultured in an agar medium containing 10 g / L of D-glucose, 3 g / L of yeast extract, 3 g / L of malt extract, 5 g / L of peptone, and 20 g / L of agar. (Glycerol 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L, sterilized at 121 ° C. for 20 minutes) Inoculate a test tube containing 5 mL, and culture with shaking at 28 ° C. and 150 rpm for 24 hours. This was used as a seed culture solution.

  This seed culture solution 1 L was sterilized at a D-arabitol production medium (activated carbon-treated waste glycerol 250 g / L, yeast extract 6 g / L, potassium dihydrogen phosphate 1 g / L, pH 6.5, 121 ° C. for 20 minutes) ) Inoculated into a 300 mL Erlenmeyer flask containing 30 mL, and cultured with shaking at 28 ° C. and 250 rpm for 7 days. The activated glycerol-treated waste glycerol used here is a two-fold dilution of a waste glycerol solution by-produced in a transesterification reaction of vegetable oils and fats (85% by mass of waste glycerol glycerol manufactured by Lion Corporation). This is a filtrate obtained by adding 40% by mass (based on glycerol) (manufactured by Wako Pure Chemical Industries, Ltd.) and stirring for 1 hour at room temperature, and removing activated carbon by filtration. Hereinafter, unless otherwise specified, when described as “activated carbon-treated glycerol”, it means glycerol that has been subjected to the above treatment. There was no change in the concentration of glycerol before and after the activated carbon treatment.

The concentration of arabitol produced in the culture solution was measured by removing the bacterial cells from a partially collected culture solution by centrifugation, and then diluting the supernatant appropriately and subjecting it to high performance liquid chromatography (HPLC). . For HPLC analysis, Shodex Sugar SC1011 was used for the column, the column temperature was 80 ° C., pure water was used for the mobile phase, and the flow rate was 1.0 mL / min. As a detector, a differential refractive index (RI) detector was used. As an example, FIG. 1 shows an HPLC chart of the culture supernatant of Candida quercitrusa NBRC1022 strain. When this was seen, only the peak of D-arabitol was detected other than the medium component and glycerol as a carbon source, indicating that only D-arabitol was selectively produced. The amount of D-arabitol produced was calculated based on a calibration curve prepared using a D-arabitol standard product (trade name D-(+)-arabitol manufactured by Wako Pure Chemical Industries, Ltd.).
(Example 2 to Example 7)
Examples of microorganisms that produce D-arabitol from glycerol include Mechinicobia zoberi NBRC1680 strain (Example 2), Methicobia runata NBRC1605 strain (Example 3), Debaryomyces hansenii var. Fabryi NBRC0015 strain (Example) 4) Debaryomyces hansenii var. Hansenii NBRC0083 strain (Example 5), Pichia stipitis NBRC1687 strain (Example 6) or Candida quercitosa 17-2A (FERM P-21420) strain (implementation) Example 7) was used and was carried out as in Example 1.

(Examples 8 and 9)
(D-arabitol production using pure glycerol)
Using commercially available pure glycerol reagents (manufactured by Wako Pure Chemical Industries, Ltd.), D- of Candida quercitrusa NBRC1022 strain (Example 8) and Candida quercitrusa 17-2A (FERM P-21420) strain (Example 9) The arabitol productivity was examined. Culture and analysis were performed in the same manner as in Example 1.

(Comparative Examples 1 and 2)
(D-arabitol production from waste glycerol by existing D-arabitol-producing bacteria)
As comparative examples, Metschnikowia pulcherrima NBRC1678 strain (Comparative Example 1), Metschnikowia leucawi ( Metschnikowia ), which is known from a conventional report (Patent Document 3) to produce D-arabitol using glucose as a carbon source. reukaufii ) NBRC1679 strain (Comparative Example 2) was used to examine D-arabitol productivity from waste glycerol. Culture and analysis were performed in the same manner as in Example 1.
(Comparative Example 3 to Comparative Example 6)
(D-arabitol production from waste glycerol by other Candida yeasts)
As comparative examples, Candida fragi NBRC 10759 (Comparative Example 3), Candida natalensis NBRC 1981 (Comparative Example) considered to be closely related to Candida quercitrusa in FIG. 4) Further, using Candida vanderwaltii NBRC 10319 (Comparative Example 5) and Candida azyma NBRC 10406 (Comparative Example 6), the productivity of D-arabitol from waste glycerol was examined. Culture and analysis were performed in the same manner as in Example 1.

The analysis results of the production amounts of D-arabitol obtained in Examples 1 to 9 and Comparative Examples 1 to 6 are summarized in Table 2. In the examples, production of D-arabitol from waste glycerol was confirmed in the range of 6.6 to 41.7 g / L in all the strains used. In Candida quercitrusa NBRC1022 strain and Candida quercitrusa 17-2A (FERM P-21420) strain, it is possible to produce D-arabitol which is almost the same as when pure glycerol is used from waste glycerol by simple activated carbon treatment. Indicated.
In the comparative examples, two strains known to be able to produce D-arabitol from glucose, two strains considered to be closely related to Candida quercitrusa in molecular phylogeny, and two other strains belonging to the genus Candida. In all studies of the strain, D-arabitol could not be produced from waste glycerol.

(Example 10)
(Effect of glycerol concentration on D-arabitol production)
D-arabitol productivity was examined using Candida quercitrusa NBRC1022 strain and a D-arabitol production medium having a glycerol concentration of 150 to 350 g / L. A commercially available pure glycerol reagent (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the carbon source, and the others were carried out in the same manner as in Example 1.
The analysis results of the production amount of D-arabitol obtained in Example 10 are summarized in Table 3. Production of D-arabitol was confirmed in the range of 7.9 to 45.0 g / L at all the glycerol concentrations used, and the production amount of D-arabitol was maximized particularly when culturing was performed at a glycerol concentration of 250 g / L. It became.

(Example 11)
(Influence of nitrogen source on D-arabitol production)
Using Candida quercitrusa NBRC1022 strain, D-arabitol productivity was examined when various nitrogen sources were used in the D-arabitol production medium. The concentration of the test nitrogen source was 5 g / L, and yeast extract was added to 1 g / L to supplement nutrient sources such as trace elements. Others were the same as in Example 1.
The analysis results of the production amount of D-arabitol obtained in Example 11 are summarized in Table 4. The production of D-arabitol was confirmed in the range of 3.7 to 45.0 g / L at all the nitrogen source concentrations used, and the production amount of D-arabitol was maximized when the yeast extract was used.

Example 12
(Effect of pH in D-arabitol production)
Candida quercitrusa FERM P-21420 strain was used to examine D-arabitol productivity under various pH conditions in a D-arabitol production medium. The pH of the D-arabitol production medium was adjusted to 3-9, and the other procedures were performed in the same manner as in Example 1.
The analysis results of the production amount of D-arabitol obtained in Example 12 are summarized in Table 5. The production of D-arabitol was confirmed in the range of 12.6 to 35.6 g / L at all pH tested, and effectively produced D-arabitol particularly in the range of pH 5-9. .

(Example 13)
(Effect of yeast extract concentration on D-arabitol production)
Candida quercitrusa NBRC1022 strain was used to examine D-arabitol productivity at a yeast extract concentration of 2 to 8 g / L in the D-arabitol production medium. The same procedure as in Example 1 was performed except for the concentration of the yeast extract in the D-arabitol production medium.
The analysis results of the production amount of D-arabitol obtained in Example 13 are summarized in Table 6. Production of D-arabitol was confirmed in the range of 13.5 to 45.0 g / L at all the yeast extract concentrations used, particularly when the yeast extract concentration was used in the range of 6 to 8 g / L. Production increased.

(Example 14)
(Effect of calcium on D-arabitol production)
Using Candida quercitrusa NBRC1022 strain, the productivity of D-arabitol in the presence of calcium in the D-arabitol production medium was examined. Calcium chloride dihydrate was added to the D-arabitol production medium so as to be 0 to 2 g / L, and the other processes were performed in the same manner as in Example 1.
The analysis results of the production amount of D-arabitol obtained in Example 13 are summarized in Table 6. At all the calcium chloride concentrations used, the formation of D-arabitol was confirmed in the range of 45.0-58.2 g / L, especially when 0.5 g / L or more of calcium chloride dihydrate was added, D-arabitol was produced effectively.

(Example 15)
(D-arabitol production from waste glycerol using fermenter in Candida quercitrusa NBRC1022 strain and Candida quercitrusa 17-2A (FERM P-21420) strain)
Candida quercitrusa NBRC1022 strain and Candida quercitrusa 17-2A (FERM P-21420) strain were cultured under the same conditions as the seed culture of Example 1, and this was used as seed culture solution 1. 1 mL of this seed culture solution was added to 30 mL of seed culture medium 2 (D-glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L, sterilized at 121 ° C. for 20 minutes) Were inoculated into two 300 mL Erlenmeyer flasks, and cultured with shaking at 28 ° C. and 250 rpm for 1 day.

  The total amount of the two seed cultures was added to D-arabitol production medium (activated carbon-treated waste glycerol 250 g / L, yeast extract 6 g / L, potassium dihydrogen phosphate 1 g / L, pH 6.5, 121 ° C.) Sterilized for minutes) Inoculated into a 2000 mL fermentor containing 800 mL and cultured for 7 days at 28 ° C., 800 rpm, and aeration of 1 vvm. The culture solution was analyzed in the same manner as in Example 1. 3 and 4 show graphs in which the cell growth amount and the D-arabitol production amount were traced along the culture time for each strain.

The amount of D-arabitol produced on the 7th day of culture was 50.1 g / L for the NBRC1022 strain and 45.0 g / L for the 17-2A (FERM P-21420) strain.
(Example 16)
(Production of D-arabitol from waste glycerol under calcium addition conditions using a fermenter in Candida quercitrusa NBRC1022 strain)
The Candida quercitrusa NBRC1022 strain was cultured under the same conditions as in the seed cultures 1 and 2 of Example 14.
The total amount of these two seed cultures was added to D-arabitol production medium (activated carbon-treated waste glycerol 250 g / L, yeast extract 6 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride dihydrate 2 g / L L, pH 6.5, sterilized at 121 ° C. for 20 minutes) was inoculated into a 2000 mL fermentor containing 800 mL, and cultured for 7 days at 28 ° C., 800 rpm, aeration rate 1 vvm. The culture solution was analyzed in the same manner as in Example 1. FIG. 5 shows a graph in which the bacterial cell growth amount and D-arabitol production amount were traced along the culture time for each strain.
The amount of D-arabitol produced on the 7th day of culture was 65.3 g / L. This is more than twice the amount of D-arabitol produced from pure glycerol (30.4 g / L) using Candida polymorpha ATCC 20213 strain shown in the conventional report (Non-Patent Document 2). It was confirmed that D-arabitol was produced very efficiently. As described above, according to the present invention, it is possible to produce D-arabitol with high yield by using inexpensive waste glycerol as a raw material.

The molecular phylogenetic tree created based on the base sequence obtained from Candida quercitrusa 17-2A strain is shown. In Example 1, the analysis result by the high performance liquid chromatography (HPLC) of the culture supernatant of Candida quercitrusa NBRC1022 strain is shown. In Example 2, it is the graph which followed the cell growth amount and the D-arabitol production amount by the culture test of the Candida quercitrusa NBRC1022 strain | stump | stock conducted using the fermenter along the culture time. The graph which followed the cell growth amount and D-arabitol production amount according to the culture | cultivation time by the culture test of the Candida quercitrusa 17-2A (FERM P-21420) strain | stump | stock performed in Example 2 using the fermenter. It is. In Example 15, it is the graph which followed the cell growth amount and the D-arabitol production amount by the culture | cultivation test on the calcium addition condition of Candida quercitrusa NBRC1022 strain | stump | stock performed using the fermenter along culture | cultivation time. .

Claims (9)

In liquid medium glycerol as a carbon source, Candida-Kuerushitorusa (Candida quercitrusa), Debaryomyces, Hanzenii (Debaryomyces hansenii), Pichia stipitis (Pichia stipitis), Metschnikowia-Zoberi (Metschnikowia zobelli) or Metschnikowia lunata (Metschnikowia lunata) A method for producing D-arabitol, comprising culturing at least one microorganism selected from the group consisting of: Candida & Kuerushitorusa (Candida quercitrusa) is a Candida, Kuerushitorusa (Candida quercitrusa) 17-2A (FERM P -21420), a method of D- arabitol production according to claim 1. The method for producing D-arabitol according to claim 1, wherein the microorganism is Candida quercitrusa 17-2A (FERM P-21420).   The method for producing D-arabitol according to any one of claims 1 to 3, wherein glycerol is a by-product of decomposition or transesterification of vegetable oil. Candida quercitrusa , a microorganism that can grow at 37 ° C. The microorganism Candida quercitrusa , which can grow on vitamin-deficient media.   A microorganism Candida quercitrusa having L-arabinitol utilization. A microorganism Candida quercitrusa that can grow at 37 ° C., can be grown on a vitamin-deficient medium, and has L-arabinitol utilization. The microorganism Candida quercitrusa 17-2A (FERM P-21420).

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