JP2009148212A - Method for fermentatively producing mannitol and microorganism used for performance thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for fermentatively producing mannitol, 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 mannitol, culturing at least one microorganism selected from Candida azyma, Candida magnoliae, Sporidiobolus pararoseus, Candidasp. 7-12G (FERM P-21419), Hansenula anomala, and Torulopsis bombicola in a liquid culture medium containing glycerol as a carbon source. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention uses glycerol, particularly inexpensive glycerol produced in large quantities as a by-product in the fats and oils industry, as a main raw material in a culture medium for microorganisms, thereby efficiently producing mannitol, which is a functional carbohydrate, by fermentation. The present invention relates to a production method and microorganisms that can be used for the production.

  Mannitol is naturally contained in brown algae, mushrooms, fungi and the like as a hexose sugar alcohol. Mannitol has 30-40% sweetness of sugar and is not only used as an alternative sweetener in food production where the use of sugar is restricted, but also cold drinking, low hygroscopicity, fluidity, etc. Because of its excellent properties, it is widely used for confectionery additives, pharmaceutical fillers, surfactants, waterproofing agents and the like. In addition, it is used as an intermediate substance for therapeutic agents for hypotension, and also used as a coating agent for reducing bitterness in the manufacturing process of various pharmaceutical preparations.

  Mannitol is present in various fruits and vegetables, but in this case, since it exists only in a very small amount, extraction from fruits and vegetables is not economical from an industrial point of view. Therefore, the commercial production method of mannitol is produced by separating fructose produced by hydrolysis from sugar or the like and adding hydrogen to this fructose in the presence of a catalyst at high temperature and high pressure. However, since sorbitol is produced as a by-product, not only a separate purification process is required, but also the conversion yield is very low and the manufacturing cost is high. There are also dangers associated with high temperature and pressure reactions and waste disposal problems.

In order to solve the above-mentioned problems, many studies on methods for fermentative production of mannitol by microorganisms are in progress. Glucose, sugars fructose as microorganisms associated with mannitol production of a raw material, for example, Torulopsis (Torulopsis) genus (Patent Document 1, Non-Patent Document 1), Candida magnoliae (Candida magnoliae) (Patent Document 2) Yeasts belonging to have been reported. Since these fermentation methods are highly economical and can produce only mannitol specifically from glucose or fructose, the separation and purification step of mannitol after the reaction can be made very easy.

  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 material development using vegetable oil as a raw material that can be regenerated and biodiesel production. These surplus glycerols are expected to continue to increase, and the development of treatment or effective utilization methods is desired.

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 3, Non-Patent Document 2) related to a production method for fermentative production of mannitol using glycerol as a raw material. Here, Torulopsis Man nits chopsticks Enns (Torulopsis mannitofaciens), Torulopsis Basachirisu (Torulopsis versatilis), using a microorganism belonging to Torulopsis anomala (Torulopsis anomala), the fermentative production of mannitol glycerol in medium mainly carbon source I did, but the production was not enough. There is no example of fermentative production of mannitol using glycerol by a microorganism other than the above three examples.

JP-A-61-35789 JP 2002-335985 A Japanese Patent Publication No. 47-20393 H. H. Onishi, T. Suzuki, “Applied Microbiol.”, Vol. 16, p. 1847-1952 (1968). H. H. Onishi, T. Suzuki, “J. Ferment. Technol.”, 48, p563-566 (1970).

  In view of the above circumstances, in the present invention, by using glycerol, which is a by-product in large quantities in the fat and oil industry, as a main raw material, a method for efficiently and efficiently producing mannitol, which is a functional sugar, at low cost, and efficiently The purpose is to provide a novel microorganism used for implementation.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor used microbials screened from the environment and their related bacteria, etc., and cultured them in a liquid medium to which glycerol was added as a main raw material. It has been found that it can be produced at a higher yield and at a lower cost than the fermentation production method, and has led to the present invention.

That is, the present invention is shown in the following [1] to [4].
[1] a in a liquid medium as a carbon source glycerol, Candida-Ajima (Candida azyma), Candida magnoliae (Candida magnoliae), Sporidiobolus Pararoseusu (Sporidiobolus pararoseus), the genus Candida yeast (Candida sp.) 7- 12G (FERM P-21419), Hansenula anomala (Hansenula anomala) or culturing at least one microorganism selected from Torulopsis Bonbikora (Torulopsis bombicola), manufacturing method of mannitol.
[2] The method for producing mannitol according to [1], wherein the microorganism is Candida sp. 7-12G (FERM P-21419).
[3] The method for producing mannitol according to [1] or [2], wherein glycerol is a by-product of decomposition or transesterification of vegetable oil.
[4] Microbial Candida sp. 7-12G (FERM P-21419).

According to the present invention, mannitol having high added value can be produced using glycerol, which is an inexpensive raw material, in particular, by-products such as the fat and oil industry, or inexpensive glycerol produced in large quantities as industrial waste. So far, there have been reports of manufacturing technology using sugars such as glucose, fructose, sucrose, etc. as raw materials, but mannitol can be provided to the market at a lower price by using glycerol, which is a cheaper raw material. .
Microorganism having an ability to produce mannitol glycerol as a raw material, so far Torulopsis Man nits chopsticks Enns (Torulopsis mannitofaciens), Torulopsis Basachirisu (Torulopsis versatilis), reports a 3 example Torulopsis anomala (Torulopsis anomala) Yes. On the other hand, the microorganism used in the present invention can produce mannitol in a yield exceeding these, and can use low-purity glycerol as a raw material produced as a by-product in the fat and oil industry.
In particular, from the background of the current era, the price of the sugar is expected to rise, whereas the amount of surplus glycerol produced is expected to continue to increase. The effect on price decline is extremely large.
At the same time, the present invention provides a method for the treatment and effective use of 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 mannitol >
Examples of the microorganism used for carrying out the present invention include Candida azima, Candida magnolia, Sporidioborus pararoseus, Candida sp. 7-12G (FERM P-21419), Hansenula anomala Or at least 1 sort (s) of microorganisms selected from tortropsis bombibicola is mentioned, The microorganisms which belong to Candida azima are especially preferable. Moreover, the mutant obtained by these microorganisms based on the conventional method is also included.

The present inventors belong to the genus Candida, and were newly discovered from a plant (flower) sample collected in Naha City, Okinawa Prefecture, the above Candida sp. 7-12G (FERM P-21419) In addition, the inventors have found that the present invention has the ability to selectively produce mannitol from glycerol, and found that the present invention can be used more effectively. Hereinafter, the mycological properties of this new strain will be described.

<Mycological properties of 7-12G strain>
The 7-12G 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, color: white to cream, gloss and properties: bright, wet. Furthermore, according to microscopic morphological observation with an optical microscope, it is confirmed that the vegetative cells of this strain after 4 days of culture are from elliptical to wide elliptical and proliferate by multipolar budding. In addition, the formation of sexual reproductive organs, hyphae, and pseudohyphae is not observed on the culture medium after one month of culture.

In the 7-12G 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)). As a result, there was a difference of 5 bases from the rDNA sequence of the Candida azyma reference strain, and the homology rate was 99.1%. Indicated. The molecular phylogenetic tree of this strain is shown in FIG. Generally, if the number of bases different from the reference strain is 0 to 3 bases, it is highly likely that they are the same species or sister species, and if the difference is 1% or more, it is likely that they are different species. In the case of this strain, although it forms a phylogenetic group with Candida azima, the distance from the Candida azima reference strain is recognized in the phylogenetic tree, so a closely related candy slightly different from Candida azima It is highly likely that it is a new species of the genus Da. Furthermore, as a result of the physiological property test of this strain (Table 1), it showed physiological and biochemical characteristics almost similar to Candida azima, whereas Candida azima showed 0.1% cycloheximide resistance ( Kurtzman, CP and Fell, JW 1998. The Yeasts, a taxonomic study, 4th edition, Elsevier, Amsterdam, Netherlands). This strain differs from Candida azima in that it did not grow in the presence of 0.1% cycloheximide. showed that. Combining the above results, this strain was named Candida sp. 7-12G strain closely related to Candida azima. 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-21419 Has been.

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.

< Mannitol production method >
The method for producing mannitol according to 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 mannitol according to 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. Further, when the viscosity of waste glycerol is high, the same operation may be performed after diluting with water 1.5 times to 5 times. 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 300 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. You may use together.

Nitrogen sources include nitrogen compounds that can be used by microorganisms, such as organic substances (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, magnesium salts (for example, calcium chloride), 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 3 to 7 g / L. It is preferable from the viewpoint. Further, the concentration of the calcium salt is set to a range of 0.1 to 20 g / L, preferably 0.5 to 10 g / L, more preferably 1 to 6 g / L, and most preferably 2 to 6 g / L. From the viewpoint of improving fermentation productivity.
Hereinafter, the medium used for the production of mannitol as described above is referred to as “mannitol production medium”.

<Specific examples of mannitol production>
For example, cells 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 can be inoculated directly into the mannitol production medium or separately. The seed culture solution obtained by the preculture is inoculated into a mannitol production medium. 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 (main culture) for producing mannitol is 20 to 40 ° C, preferably 25 to 34 ° C. The pH of the mannitol 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 microbial growth and the amount of mannitol produced in the range of 3 to 14 days settles to a steady state.

<Quantification, recovery, and purification of mannitol>
The amount of mannitol produced in the culture can be measured using a known method such as high performance liquid chromatography (HPLC) or gas chromatography. Isolation and purification of mannitol from the obtained culture broth is performed by appropriately combining commonly used operations such as filtration, centrifugation, ion exchange or adsorption chromatography, solvent extraction, and crystallization as necessary. . 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 mannitol crystals.
Hereinafter, the present invention will be described in detail 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. In addition, when not specifically described below, the culture was performed under aerobic conditions.

Example 1
(Mannitol fermentation production from waste glycerol)
Candida azima NBRC10406 strain was used as a microorganism for producing mannitol from glycerol.

  The above-mentioned strain is cultured on 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. Ears were inoculated into a test tube containing 5 mL of seed culture medium (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) The culture was carried out with shaking at 150 ° C. for 24 hours, and this was used as a seed culture solution.

  1 mL of this seed culture solution is mannitol production medium (250 g / L of waste glycerol treated with activated carbon, 6 g / L of yeast extract, 1 g / L of potassium dihydrogen phosphate, pH 6.5, sterilized at 121 ° C. for 20 minutes) A 300 mL Erlenmeyer flask containing 30 mL was inoculated 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 (produced by Lion Co., Ltd .: waste glycerol: glycerol content 85% by mass) that is by-produced in the transesterification of vegetable oils. 40% by mass of activated carbon (manufactured by Wako Pure Chemical Industries, granules) (vs. waste glycerol) was added and stirred at room temperature for 1 hour, and the activated carbon was removed by filtration. Hereinafter, unless otherwise specified, when describing as “activated carbon-treated glycerol”, it means the 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 mannitol produced in the culture solution was measured by removing the cells from the 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 a culture supernatant of Candida azima NBRC10406 strain. From this, only the peak of mannitol was detected in addition to the medium component and glycerol as the carbon source, indicating that only mannitol was selectively produced. The production amount of mannitol was calculated based on a calibration curve prepared using a mannitol standard product (trade name D-(-)-mannitol, manufactured by Wako Pure Chemical Industries, Ltd.).
(Examples 2 to 6)
As microorganisms for producing mannitol from glycerol, Sporidioboras pararoseus NBRC0376 strain (Example 2), Hansenula anomala NBRC10213 strain (Example 3), Candida magnolia NBRC0705 strain (Example 4), Tolropsis bonbicola NBRC10243 strain (implementation) Example 5) was carried out in the same manner as in Example 1 except that Candida sp. 7-12G (FERM P-21419) (Example 6) was used.

(Examples 7 and 8)
(Mannitol production of Candida azima NBRC10406 strain and Candida sp. 7-12G (FERM P-21419) using pure glycerol)
Using a commercially available pure glycerol reagent (manufactured by Wako Pure Chemical Industries, Ltd.), Candida azima NBRC10406 strain (Example 7) and Candida sp. 7-12 (FERM P-21419) (Example) The mannitol productivity in 8) was examined. Culture and analysis were performed in the same manner as in Example 1.

The analysis results of the production amount of mannitol obtained in the examples are summarized in Table 1. In all the strains used, production of mannitol from waste glycerol was confirmed in a range of 3.8 to 31.8 g / L by simple activated carbon treatment. Candida azima NBRC10406 showed the same amount of mannitol production as that of pure glycerol in waste glycerol. As described above, according to the present invention, glycerol can be selectively fermented and produced using glycerol with very high efficiency.

Example 9
(Effect of glycerol concentration on mannitol production)
Using Candida azima NBRC10406 strain, mannitol productivity was examined when the glycerol concentration in the mannitol production medium was 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 other procedures were carried out in the same manner as in Example 1.
The analysis results of the production amount of mannitol obtained in Example 9 are summarized in Table 3. At all the glycerol concentrations used, the production of mannitol was confirmed in the range of 18.3 to 35.5 g / L, and the mannitol production was maximized when culturing was performed at a glycerol concentration of 250 g / L.

(Example 10)
(Influence of nitrogen source on mannitol production)
Using Candida azima NBRC10406 strain, mannitol productivity was examined in a mannitol production medium using various nitrogen sources. 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. Other than that was carried out in the same manner as in Example 1.
The analysis results of the production amount of mannitol obtained in Example 10 are summarized in Table 4. Production of mannitol was confirmed in the range of 5.3 to 35.5 g / L at all nitrogen source concentrations used, and the production amount of mannitol was maximized particularly when yeast extract was used.

(Example 11)
(Effect of yeast extract concentration on mannitol production)
Candida azima NBRC10406 strain was used, and mannitol productivity was examined when yeast extract was used at a concentration of 2 to 8 g / L in mannitol production medium. The same procedure as in Example 1 was performed except for the concentration of the yeast extract in the main culture medium.
The analysis results of the production amount of mannitol obtained in Example 11 are summarized in Table 5. Production of mannitol was confirmed in the range of 30.1 to 38.2 g / L in all the yeast extract concentrations used, and most produced mannitol when the yeast extract concentration was used in the range of 4 to 8 g / L. .

Example 12
(Effect of pH on mannitol production)
Using Candida azima NBRC10406 strain, mannitol productivity under each pH condition was examined. The pH of the mannitol production medium was adjusted to 3 to 9, and the rest was performed in the same manner as in Example 1.
The analysis results of the production amount of mannitol obtained in Example 12 are summarized in Table 6. Production of mannitol was confirmed in the range of 26.1 to 37.7 g / L at all pHs subjected to the culture test, and mannitol was effectively produced particularly in the range of pH 5 to 7.

(Example 13)
(Mannitol production from waste glycerol using fermenter)
Candida azima NBRC10406 strain was 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.

Sterilize the whole amount of the two seed cultures at mannitol 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 2000 mL fermentor containing 800 mL and cultured at 28 ° C., 800 rpm, aeration rate 1 vvm for 7 days. The culture solution was analyzed in the same manner as in Example 1. FIG. 3 shows a graph in which the cell growth amount and the mannitol production amount are traced along the culture time.
The amount of mannitol produced on the 7th day of culture was 36.2 g / L.

(Example 14)
(Mannitol production from waste glycerol under calcium addition conditions using a fermentor in Candida azima NBRC10406)
Candida azima NBRC10406 strain was cultured under the same conditions as seed cultures 1 and 2 of Example 13.
The total amount of the seed culture 2 liquid was mixed with mannitol production medium (waste glycerol treated with activated carbon 250 g / L, yeast extract 6 g / L, potassium dihydrogen phosphate 1 g / L, calcium chloride dihydrate 2 g / L, Sterilized at pH 6.5, 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, and aeration rate of 1 vvm. The culture solution was analyzed in the same manner as in Example 1. FIG. 4 shows a graph in which the cell growth amount and the mannitol production amount were traced along the culture time for each strain.
The amount of mannitol produced on the seventh day of culture was 50.8 g / L.

(Example 15)
(Effect of calcium on mannitol production)
Using Candida azima NBRC10406 strain, mannitol productivity in the presence of calcium in mannitol production medium was examined. Calcium chloride dihydrate was added to the mannitol production medium so as to be 0 to 8 g / L, and the others were carried out in the same manner as in Example 1.
The analysis results of the production amount of mannitol obtained in Example 15 are summarized in Table 7. At all calcium chloride concentrations used, the formation of mannitol was confirmed in the range of 35.5-64.1 g / L, especially when 4 g / L of calcium chloride dihydrate was added, most effectively mannitol. Was produced (64.1 g / L). This is more than twice the amount of mannitol produced from pure glycerol (24.0 g / L) using toluropsis Versatilis CBS1752 as shown in the previous report (Non-patent Document 2), which is extremely efficient. Was confirmed to be produced. As described above, according to the present invention, mannitol can be produced with high yield by using inexpensive waste glycerol as a raw material.

Genus Candida yeast (Candida sp.) Shows a molecular phylogenetic tree was prepared based on the nucleotide sequence obtained from 7-12G strain. In Example 1, the analysis result by the high performance liquid chromatography (HPLC) of the culture solution supernatant of Candida azima NBRC10406 strain is shown. In Example 9, it is the graph which followed the cell growth amount and the mannitol production amount according to the culture | cultivation time by the culture test of the Candida azima NBRC10406 strain | stump | stock performed using the fermenter. In Example 14, it is the graph which followed the microbial cell growth amount and the mannitol production amount according to the culture | cultivation time by the culture | cultivation test of the Candida azima NBRC10406 strain | stump | stock carried out using the fermenter in the calcium addition conditions.

Claims (4)

In liquid medium glycerol as a carbon source, Candida-Ajima (Candida azyma), Candida magnoliae (Candida magnoliae), Sporidiobolus Pararoseusu (Sporidiobolus pararoseus), the genus Candida yeast (Candida sp.) 7-12G (FERM P-21419), at least one microorganism selected from Hansenula anomala ( Hansenula anomala ) or Torulopsis bombicola ( Torulopsis bombicola ), a method for producing mannitol. The method for producing mannitol according to claim 1, wherein the microorganism is Candida sp. 7-12G (FERM P-21419).   The method for producing mannitol according to claim 1 or 2, wherein glycerol is a by-product of decomposition or transesterification of vegetable oil. Microbial Candida sp. 7-12G (FERM P-21419).

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