JP5361037B2 - Method for producing mannosyl erythritol lipid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for more inexpensively producing mannosylerythritol lipid (MEL), which finds a new microorganism capable of producing mannosylerythritol lipid and optimizing the medium composition and the culture conditions of mannosylerythritol lipid so as to shorten a period required for the production. <P>SOLUTION: In the method for producing mannosylerythritol lipid by culturing a microorganism capable of producing mannosylerythritol lipid in a culture solution containing a nutrient and a carbon source, the microorganism capable of producing mannosylerythritol lipid is Pseudozyma tsukubaensis. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a method for producing a mannosyl erythritol lipid capable of greatly reducing the time required for production of mannosyl erythritol lipid (hereinafter sometimes simply referred to as “MEL”).

  Mannosyl erythritol lipid (see Patent Document 1) is a substance in which a fatty acid and a sugar (mannose and erythritol) are combined. Since it is an amphiphilic substance that has both lipophilicity derived from the properties of lipids and hydrophilicity derived from the properties of sugars, it has surface activity and is a type of glycolipid biosurfactant that is produced by living organisms. . MEL has surface activity and has been reported to have antimicrobial activity, cell differentiation-inducing activity (all of which are reviewed in non-patent document 1), anti-allergic activity (see patent document 2), and the like. In addition, mannosyl erythritol lipid is biodegradable and is considered to have high safety, and its application to the food industry, pharmaceutical industry, chemical industry, environment-related fields, etc. has been widely studied. From such a background, development of an inexpensive MEL manufacturing method is desired.

For the production method of MEL, see Candida sp. It is possible to produce 35 g / L (production rate: 0.3 g / L / h, raw material yield: 70 mass%) of MEL from 5 mass% soybean oil using B-7 strain in 5 days. Have been reported (see Non-Patent Documents 2 and 3). Moreover, 38 g / L (production rate: 0.2 g / L / h, raw material yield: 48 mass%) in 8 days from 8 mass% soybean oil using Candida antarctica ( Candida antarctica ) T-34 strain | stump | stock. ) Has been reported to be possible (see Non-Patent Documents 4 and 5). Similarly, 110 g / L from 25 mass% peanut oil after 24 days of continuous addition of fats and oils at 6 day intervals using Candida antarctica T-34 strain (production rate: 0. 0). 2 g / L / h, raw material yield: 44% by mass) has been reported to be possible (see Non-Patent Document 6).

Candida sp. Production of MEL of 95 g / L (production rate: 0.48 g / L / h, raw material yield: 45% by mass) from 18% by mass soybean oil after 200 hours by fed-batch culture method using SY-16 strain It has been reported that this is possible (see Non-Patent Document 7). In addition, 17 g / L of 8% by mass of soy sauce oil is produced in 7 days using candida antarctica T-34 as a raw material of soy sauce oil produced as a by-product in the soy sauce brewing process. It has been proposed that MEL can be produced at a rate of 0.1 g / L / h and a raw material yield of 21% by mass (see Patent Document 3).

Production of MEL of 165 g / L (production rate: 0.58 g / L / h, raw material yield: 92% by mass) in 14 days by a fed-fed method of fats and oils using Pseudozyma aphidis DSM14930 It has been reported that this is possible (see Non-Patent Document 8).
In addition, the inventors of the present invention have also found that Kurtzmanomyces sp. It is possible to produce MEL of 153 g / L (production rate: 0.64 g / L / h, raw material yield: 85 mass%) in 10 days by batch culture method from 18 mass% of vegetable oil and fat using I-11 strain And the production of MEL of 307 g / L (production rate: 0.53 g / L / h, raw material yield: 77% by mass) from 40% by mass of fats and oils in 24 days by the fed-batch culture method of fats and oils. It is reported that this is possible (see Patent Document 4).

Furthermore, recently, production of 190 g / L (production rate: 0.28 g / L / h) of MEL in 28 days by fed-batch culture of vegetable oil and erythritol using Pseudozyma rugulosa NBRC 10877 (see Patent Document 5) Production of 2.7 g / L MEL from glucose using Pseudozyma antarctica KM-34 strain (see Patent Document 6) Production of 3 g / L MEL from glycerol using Pseudozyma antarctica JCM 10317 strain (see Patent Document 7) ), Pseudozyma parantarctica JCM 11752 strain for 14 days by feeding, the vegetable oil with 190 g / L (production speed: MEL production (see Patent Document 8 of 0.56g / L / h)), Pseudozyma g It has been reported that 13 g / L of MEL-C can be produced from soybean oil in 7 days using ramicocola CBS 10092 (see Patent Document 9).

Japanese Patent Publication No. 60-24797 JP 2005-68015 A JP 2002-101847 A JP 2004-254595 A JP 2007-185142 A JP 2007-209332 A JP 2007-209333 A JP 2007-252279 A JP 2008-079600 A D. Kitamoto, H .; Isoda and T.W. Nakahara: J. et al. Biosci. Bioeng. 94, 187 (2002). T.A. Nakahara, H .; Kawasaki, T .; Sugisawa, Y .; Takamori and T.K. Tabuchi: J. et al. Ferment. Technol. 61, 19 (1983). H. Kawasaki, T .; Nakahara, M .; Oogaki and T.K. Tabuchi: J. et al. Ferment. Technol. 61, 143 (1983). D. Kitamoto, S .; Akiba, C.I. Hioki and T.W. Tabuchi: Agric. Biol. Chem. , 54, 31 (1990). D. Kitamoto, K. et al. Haneishi, T .; Nakahara and T.A. Tabuchi: Agric. Biol. Chem. , 54, 37 (1990). D. Kitamoto, K. et al. Fijishiro, H .; Yanagishita, T .; Nakane and T.K. Nakahara: Biotechnol. Lett. , 14, 305 (1992). H. S. Kim, J. et al. W. Jeon, B.J. H. Kim, C.I. Y. Ahn, H .; M.M. Oh and B. D. Yoon: Appl Microbiol Biotechnol. , 70, 391 (2006). U. Rau, L .; A. Nguyen, H .; Roeper, H .; Koch and S.K. Lang: Appl Microbiol Biotechnol. 68, 607 (2005).

  In order to widely disseminate mannosyl erythritol lipid, which is said to have high biodegradability, low toxicity, environmental friendliness, and new physiological functions in the food industry, pharmaceutical industry, chemical industry, etc., the production efficiency of mannosyl erythritol lipid It is necessary to increase production and reduce production costs. However, as described above, the production rate of mannosyl erythritol lipid is low, and it takes a period of 10 days or more to produce a sufficient amount thereof. Therefore, development of a production method capable of shortening the production period of further mannosyl erythritol lipid is strongly desired.

  An object of the present invention is to meet such demands, solve the above-described problems, and achieve the following objects. That is, the present invention finds a new microorganism having an ability to produce mannosyl erythritol lipid, optimizes the medium composition and culture conditions, thereby shortening the time required for production and reducing mannosyl erythritol lipid (MEL) more inexpensively. It is an object of the present invention to provide a method capable of producing a).

As a result of intensive studies by the present inventors in order to solve the above problems, the following knowledge has been obtained. In other words, biosurfactants such as mannosyl erythritol lipid (MEL) are substances that are produced in order for microorganisms that exist widely in nature to survive, and biosurfactant-producing microorganisms that have been discovered so far are separated from nature. Based on the fact that various samples were collected from various parts of Japan and microorganisms producing mannosylerythritol lipid (MEL) were isolated, a new yeast, Pseudozyma tsukubaensis TM-181 strain ( NITE P-530) was obtained. The strain has excellent characteristics as a mannosyl erythritol lipid producing bacterium, and it has been found that mannosyl erythritol lipid (MEL) can be efficiently produced by examining the medium composition, culture conditions, and culture method.

In general, in fermentation production using microorganisms, it is known that the cell growth and the yield of the target product are greatly affected by the medium composition and culture conditions. Therefore, yeast extract, corn steep liquor, potassium dihydrogen phosphate, magnesium sulfate, and inorganic nitrogen source in the medium for MEL production using the above-mentioned Pseudozyma tsukubaensis TM-181 strain (NITE P-530). In addition to changing the concentration, the influence of pH, dissolved oxygen concentration and temperature on the production of MEL by changing the culture conditions was examined to determine the optimal medium composition and culture conditions for MEL production.

Moreover, in the conventional batch culture, it culture | cultivated by inoculating a preculture liquid to a culture tank. Usually, the preculture solution is inoculated to about 10% by volume with respect to the medium. However, since the cell concentration in the preculture solution is not high, the cell concentration at the start of the culture is not so high. For this reason, it takes about one day before MEL production starts. Although this period is called the induction period, it is considered that the culture time required for MEL production can be further reduced by shortening the induction period. In order to shorten the induction period, it is effective to increase the cell concentration at the start of culture. One of the methods for increasing the initial bacterial cell concentration is repeated batch culture. This method has the advantage that, in addition to increasing the initial bacterial cell concentration, there is no need for pre-culture and no washing of the culture tank. Have.
In repeated batch culture, culture is started in the same manner as in the batch culture described above, and after culturing for an arbitrary period of time to produce MEL, most of the culture solution containing the bacterial cells is discharged out of the culture tank, and part of it is discharged. A new medium (including fats and oils) left in the culture tank and sterilized in advance is aseptically added in a predetermined amount, and the next batch culture is started. Thereafter, the culture period required for MEL production can be shortened by repeating such operations.

In addition, four types of mannosyl erythritol lipids have been reported depending on the number and position of bonds of hydrogen atoms and acetyl groups to R ^{1 to} R ⁴ in the structural formula (1) described later. Since the acetyl group is a lipophilic group and decreases the solubility in water, mannosyl erythritol to which no acetyl group is bonded is desired. However, its existence was known until now, but the production amount was very small. Here, the present inventors further store the culture solution after batch culture or repeated batch culture at an optimal temperature, thereby acetylating R ^{1 to} R ⁴ in the structural formula (1) described later. It has been found that MELs having no group bonded thereto (MELs represented by structural formula (2) described later) can be produced.

Therefore, the pH of the mannosyl erythritol lipid (MEL) production medium is controlled using the Pseudozyma tsukubaensis TM-181 strain (NITE P-530), and the optimum medium composition and culture conditions are set. In addition, by performing batch culture and repeated batch culture, the culture period required for MEL production can be shortened, and mannosyl erythritol lipid in which R ^{1 to} R ⁴ are not bonded to R ^{1 to} R ⁴ in the structural formula (1) described later (structure described later) The inventors have found that mannosyl erythritol lipid represented by the formula (2) can be produced, and have made the present invention.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A method for producing mannosyl erythritol lipid by culturing a microorganism having an ability to produce mannosyl erythritol lipid in a culture solution containing a nutrient and a carbon source, wherein the microorganism having the ability to produce mannosyl erythritol lipid comprises a pseudo It is a manufacturing method of mannosyl erythritol lipid characterized by being Zima tsukubaensis ( Pseudozyma tsukubaensis ).
<2> The method for producing a mannosyl erythritol lipid according to <1>, wherein the microorganism having the ability to produce mannosyl erythritol lipid is Pseudozyma tsukubaensis TM-181 strain (NITE P-530). .
<3> The method for producing mannosyl erythritol lipid according to any one of <1> to <2>, wherein the culture is performed while controlling the pH of the culture solution in a range of 5.0 to 5.8.
<4> The method for producing mannosylerythritol lipid according to <3>, wherein the pH of the culture solution is controlled using ammonia.
<5> The method for producing mannosyl erythritol lipid according to any one of <1> to <4>, wherein the culture is performed while controlling the dissolved oxygen concentration of the culture solution in a range of 1 to 5 ppm.
<6> The method for producing mannosyl erythritol lipid according to any one of <1> to <5>, wherein the culture is performed by adding 0.1 to 4 g / L of corn steep liquor in the culture solution.
<7> The method for producing mannosyl erythritol lipid according to any one of <1> to <6>, wherein a linear aliphatic hydrocarbon having 6 to 24 carbon atoms is used as a carbon source.
<8> The method for producing mannosylerythritol lipid according to any one of <1> to <6>, wherein a fatty acid having 6 to 24 carbon atoms is used as a carbon source.
<9> The method for producing mannosyl erythritol lipid according to any one of <1> to <6>, wherein a triglyceride containing a linear aliphatic hydrocarbon group having 6 to 24 carbon atoms is used as a carbon source.
<10> The method for producing a mannosyl erythritol lipid according to any one of <1> to <6>, wherein a carbohydrate is used as a carbon source.
<11> The method for producing mannosyl erythritol lipid according to any one of <1> to <6>, wherein the waste oil is used as a carbon source.
<12> The method for producing mannosyl erythritol lipid according to any one of <1> to <11>, wherein the cultivation is performed in a carbon source concentration range of 10 to 32% by mass.
<13> The mannosyl according to any one of <1> to <12>, wherein the medium composition and culture conditions for producing mannosylerythritol lipid in a microorganism having the ability to produce mannosylerythritol lipid are as follows: This is a method for producing erythritol lipid.
Yeast extract: 0.1-2 g / L
Corn steep liquor: 0.1-4g / L
Ammonium nitrate: 0.1-1 g / L
Potassium dihydrogen phosphate: 0.1-2 g / L
Magnesium sulfate: 0.1-1 g / L
Carbon source: 100-320 g / L
Culture solution pH: 5.0 to 5.8
Dissolved oxygen concentration: 1-5ppm
Culture temperature: 26-32 ° C
<14> Cultivation is started in a carbon source concentration range of 10 to 30% by mass, and most of the culture solution containing the cells and mannosyl erythritol lipid is removed from the culture vessel at the end of the culture for 2 to 6 days after the start of the culture The mannosyl erythritol according to any one of <1> to <13>, wherein the mannosyl erythritol is discharged and left in a culture tank, a new medium is added, the next batch culture is started, and the operation is repeated. It is a manufacturing method of a lipid.
<15> Further, from the above <1> to <14>, the method further comprises a step of maintaining the culture solution after completion of the culture at 20 to 70 ° C. to obtain a mannosylerythritol lipid having a structure represented by the following structural formula (2) It is a manufacturing method of the mannosyl erythritol lipid in any one.
However, in the structural formula (2), R ^{1 to} R ⁴ may be the same as or different from each other, and represent a hydrogen atom or a saturated or unsaturated aliphatic acyl group having 1 to 20 carbon atoms. Represents (except acetyl group).
<16> The method for producing a mannosyl erythritol lipid according to any one of <1> to <15>, wherein the production amount of mannosyl erythritol lipid is 7% by mass or more (70 g / L or more).

  According to the present invention, it is possible to solve the conventional problems and achieve the above-mentioned object, by using a new microorganism having the ability to produce mannosylerythritol lipid, and by optimizing the medium composition and culture conditions, It is possible to provide a method capable of shortening the time required for production and producing mannosyl erythritol lipid (MEL) at a lower cost.

(Manufacturing method of mannosyl erythritol lipid)
The method for producing a mannosyl erythritol lipid according to the present invention is a method for producing a mannosyl erythritol lipid by culturing a microorganism having an ability to produce mannosyl erythritol lipid in a culture solution containing a nutrient and a carbon source. The microorganism having the ability to produce is characterized by being Pseudozyma tsukubaensis .

<Microorganisms capable of producing mannosyl erythritol lipid>
Pseudozyma tsukubaensis is used as the microorganism having the ability to produce the mannosyl erythritol lipid. There is no particular limitation on the strain to be used as the P. matsukubaensis, and examples thereof include TM-181 strain (NITE P-530), NBRC1940, ATCC24555, CBS6389 and the like. Pseudozyma tsukubaensis ) TM-181 strain (NITE P-530) is particularly preferred because of its high production rate of MEL.

The Pseudozyma tsukubaensis TM-181 strain is a novel yeast isolated by the present inventors, and is an independent administrative institution, National Institute for Product Evaluation and Technology, Japan Patent Microbiology Depositary Center. Deposited on Monday (NITE P-530). In addition, it has not been known at all that Pseudozyma tsukubaensis TM-181 strain has an excellent characteristic as a mannosyl erythritol lipid producing bacterium. This is a new finding.

Hereinafter, the Pseudozyma tsukubaensis TM-181 strain will be described.

(A) Growth state in each medium i. Colony observation In the culture for 7 days at 25 ° C. on YM agar medium, the formed colonies showed the following shapes.
Peripheral shape is almost full edge, raised state is cushion-like, surface state is smooth, gloss and properties are weak gloss and wet, color tone is pink to yellowish cream ii. Microscopic observation In 7 days of culture on a YM agar medium at 25 ° C., vegetative cells are oblong and proliferate by extreme budding. Formation of sexual reproductive organs is not observed 3 weeks after the start of culture.
(B) Physiological property test 1) Range in which growth is possible pH 2-7, temperature 10-37 ° C
2) Sugar fermentation test Glucose was not fermented.
3) Carbon source utilization test
4) Nitrogen source utilization test
5) No requirement for vitamins.
6) No generation of starch-like substances.
(C) 26SrDNA-D1 / D2 nucleotide sequence analysis The operations from extraction to cycle sequencing were performed based on each protocol.
1) DNA extraction: DNAeasy Plant Mini Kit (QIAGEN, Hilden, Germany)
2) PCR: puReTaq Ready-To-Go PCR beads (Amersham Biosciences, NJ, USA)
3) Cycle sequence: GigDye Terminator v3.1 Kit (Applied Biosystems, CA, USA)
4) Primers used: NL1, NL2, NL3 and NL4 (O'Donnell, 1993)
5) Sequence: ABI PRISM 3100 Genetic Analyzer System (Applied Biosystems, CA, USA)
6) Sequencing: Chromas Pro 1.4 (Technyllium Pty Ltd., Thewantin, AUS)
7) Homology search and simple molecular phylogenetic analysis:
Software Apollon 2.0
Database Apollon DB-FU 1.0
International base sequence database (GenBank / DDBJ / EMBL)
From the results of the above 26S rDNA-D1 / D2 nucleotide sequence analysis, simple morphological observation, and physiological / biochemical property tests, it was estimated to belong to Pseudozyma tsukubaensis .

<Culture>
The culture of the Pseudozyma tsukubaensis TM-181 strain is not particularly limited and can be appropriately selected according to the purpose. For example, a YPD medium (for example, a YPD medium (for example, A composition of 10 g yeast extract, 20 g polypeptone, and 20 g glucose) can be used. The optimum growth pH is 5.2, and the pH range in which growth is possible is 2-7. The optimum growth temperature is 30 ° C, and the temperature range in which growth is possible is 10 to 36 ° C. In order to increase the productivity of MEL, it is not sufficient to simply grow the Pseudozyma tsukubaensis TM-181 strain, and it is necessary to optimize the medium composition and culture conditions. Will be described later.

The carbon source for culturing the Pseudozyma tsukubaensis TM-181 strain is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include triglycerides, aliphatic hydrocarbons, and fatty acids. , Carbohydrates and the like.

  There is no restriction | limiting in particular as said triglyceride, A vegetable fat and oil, animal fats, etc. are mentioned, According to the objective, it can select suitably. Examples of the vegetable oil include soybean oil, linseed oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, balm oil, and among these, linseed oil, soybean oil, and rapeseed oil are preferable. Examples of animal fats include beef tallow, pork tallow, and fish tallow. You may use these individually by 1 type or in mixture of 2 or more types as appropriate. As said triglyceride, the triglyceride containing a C6-C24 linear aliphatic hydrocarbon group is preferable. In addition, as vegetable oils and fats, animal oils and fats, waste food oils after producing tempura and the like can be used.

There is no restriction | limiting in particular as said aliphatic hydrocarbon, According to the objective, it can select suitably. The hydrocarbon preferably has 6 to 24 carbon atoms, and may further contain 1 to 3 unsaturated bonds in each molecule. For example, saturated hydrocarbons such as dodecane, hexadecane, and octadecane, or unsaturated hydrocarbons such as 1-pentadecene (C ₁₅ H ₃₀ ) and 1-heptadecene (C ₁₇ H ₃₄ ), and mixtures thereof, more preferably It is possible to use 14 to 20 saturated or unsaturated hydrocarbons and mixtures thereof, more preferably mixtures containing octadecane as a main component. Further, the aliphatic hydrocarbon is preferably a linear aliphatic hydrocarbon.

  There is no restriction | limiting in particular as said fatty acid, According to the objective, it can select suitably. The fatty acid preferably has 6 to 24 carbon atoms, and may further contain 1 to 3 unsaturated bonds in each molecule. For example, saturated fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, or unsaturated fatty acids such as myristoleic acid, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and mixtures thereof, more preferably 14 to 20 saturated or unsaturated fatty acids and mixtures thereof, more preferably, linoleic acid and linoleic acid as a main component can be used.

  There is no restriction | limiting in particular as said carbohydrate, According to the objective, it can select suitably. For example, monosaccharides such as glucose, mannose, glycerol, mannitol and derivatives thereof, oligosaccharides such as sucrose, maltose, lactose, and mixtures thereof, preferably monosaccharides, more preferably glucose.

  Among these, vegetable oils are preferable as the carbon source, and linseed oil, soybean oil, and rapeseed oil are particularly preferable. As for content of the said carbon source, 10-32 mass% is preferable in a culture medium, and 14-30 mass% is especially preferable.

The nutrient for culturing the Pseudozyma tsukubaensis TM-181 strain is not particularly limited and may be appropriately selected according to the purpose. Examples thereof include yeast extract, malt extract, meat extract, Polypeptone, corn steep liquor, ammonium nitrate, urea, sodium nitrate, ammonium chloride, ammonium sulfate, potassium dihydrogen phosphate, potassium hydrogen phosphate, magnesium sulfate, etc., yeast extract, corn steep liquor, ammonium nitrate, dihydrogen phosphate Potassium, magnesium sulfate and the like are preferable.

The inorganic nitrogen source for culturing the Pseudozyma tsukubaensis TM-181 strain is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include ammonium nitrate, urea, sodium nitrate, Examples thereof include ammonium chloride and ammonium sulfate. Among these, ammonium nitrate and urea are preferable.

Suitable medium composition and culture conditions for producing mannosyl erythritol lipid using the Pseudozyma tsukubaensis TM-181 strain are as follows. The yeast extract is preferably 0.1 to 2 g / L, particularly preferably 1 g / L. The corn steep liquor is preferably 0.1 to 4 g / L, particularly preferably 2 g / L. As for ammonium nitrate, 0.1-1 g / L is preferable and 0.5 g / L is especially preferable. The potassium dihydrogen phosphate is preferably 0.1 to 2 g / L, particularly preferably 0.4 g / L. Magnesium sulfate is preferably 0.1 to 1 g / L, particularly preferably 0.2 g / L. 100-320 g / L is preferable and, as for carbon sources, such as vegetable oil, 140-300 g / L is especially preferable. The dissolved oxygen concentration is preferably 1 to 5 ppm, particularly preferably 3 to 5 ppm. The culture temperature is preferably 26 to 32 ° C, particularly preferably 30 ° C. The pH is preferably from 5.0 to 5.8, particularly preferably 5.2. In addition, it is preferable to adjust pH of a culture solution using ammonia which can become a nutrient of a microbial cell.

The production method of mannosyl erythritol lipid (MEL) of the present invention is not particularly limited and can be appropriately selected according to the purpose. For example, glucose 20 g / L, yeast extract 1 g / L, ammonium nitrate 1 g / L, phosphorus 1 Pseudozyma tsukubaensis TM-181 strain (NITE P-530) is added to a test tube containing 4 mL of a liquid medium having a composition of potassium dihydrogenate 0.5 g / L and magnesium sulfate 0.5 g / L. Inoculate platinum ears and incubate at 30 ° C for 1 day with shaking. This is inoculated into a Sakaguchi flask containing 100 mL of medium having the same composition, and cultured with shaking at 30 ° C. for 2 days. Furthermore, this is a composition of a predetermined amount of fat and oil and yeast extract 1 g / L, corn steep liquor 2 g / L, ammonium nitrate 0.5 g / L, potassium dihydrogen phosphate 0.4 g / L, and magnesium sulfate 0.2 g / L. Inoculate a jar fermenter containing 1.4 L of liquid medium and start main culture at 30 ° C. with an aeration rate of 1.5 L / min and an agitation rate of 800 rpm. The pH can be measured with a pH meter. The dissolved oxygen concentration can be measured with a dissolved oxygen concentration meter.

-Batch culture-
In batch culture, carbon sources such as fats and oils are not supplied during the culture, and the culture solution is aseptically collected 2 to 3 times a day, and each component in the culture solution is measured over time. For MEL, triglyceride, diglyceride, hydrocarbon, and fatty acid, add ethyl acetate to the collected culture and shake vigorously, collect the supernatant ethyl acetate layer, and add this ethyl acetate solution to The rod of Scan (manufactured by Yatron) is charged, and each component is quantitatively analyzed by a predetermined method.

  Here, in the batch culture, the pH of the culture solution is controlled in the range of 5.0 to 5.8, the culture solution contains an inorganic nitrogen source, and the inorganic nitrogen source is either ammonium nitrate or urea. It is preferable that In this case, it is preferable to perform the culture in the range of the initial carbon source concentration of 10 to 32% by mass, and it is more preferable to perform the culture in the range of 10 to 30% by mass.

-Repeated batch culture-
In the batch culture, culturing is performed by inoculating a preculture medium into a culture tank. Usually, preculture is inoculated with about 10% by volume with respect to the medium. However, since the cell concentration in the preculture is not high, the cell concentration at the start of the culture is not so high. For this reason, it takes about one day before MEL production starts. Although this period is called the induction period, it is considered that MEL can be produced in a shorter time by shortening the induction period. In order to shorten the induction period, it is effective to increase the cell concentration at the start of culture. One of the methods for increasing the initial bacterial cell concentration is repeated batch culture, and this method has the advantages that the initial bacterial cell concentration can be increased and that pre-culture is unnecessary and the culture tank is not required to be washed. doing.

  In repeated batch culture, culture is started in the same manner as in the batch culture described above, and after culturing for an arbitrary period of time to produce MEL, most of the culture solution containing the cells is discharged out of the culture tank, and a part of A new medium (including fats and oils) that has been sterilized in advance while remaining in the culture tank is aseptically added in a predetermined amount, and the next batch culture is started. Thereafter, such an operation is repeated.

  Here, in the repeated batch culture, the pH of the culture solution is controlled in the range of 5.0 to 5.8, the culture solution contains an inorganic nitrogen source, and the inorganic nitrogen source is any one of ammonium nitrate and urea. It is preferable that In this case, it is preferable to perform the culture in the range of the initial carbon source concentration of 10 to 36% by mass, and it is more preferable to perform the culture in the range of 14 to 30% by mass. The culture time is preferably 2 to 6 days. Moreover, it is preferable that the next batch culture is started by setting the amount of the culture solution remaining in the culture tank to 6 to 35% by volume of the entire culture solution.

<Mannosyl erythritol lipid>
Mannosyl erythritol lipid obtained by the method for producing mannosyl erythritol lipid of the present invention is a compound represented by the following structural formula (1).

In the structural formula (1), R ^{1 to} R ⁴ may be the same or different from each other, and may be a hydrogen atom, an acetyl group, or a saturated or unsaturated group having 1 to 20 carbon atoms, preferably 3 to 20 carbon atoms. Represents a saturated aliphatic acyl group.

-R ¹ to R ⁴ production of mannosylerythritol lipid which an acetyl group not bound to the -
As for mannosyl erythritol lipid, four types of substances have been reported in the structural formula (1) depending on the number and position of bonds of hydrogen atoms and acetyl groups to R ^{1 to} R ⁴ . Here, since the acetyl group is a lipophilic group and decreases the solubility in water, a mannosyl erythritol lipid having no acetyl group is desired.
This mannosyl erythritol lipid without an acetyl group is represented by the following structural formula (2). (In the structural formula (1) and the structural formula (2), the types of R ^{1 to} R ⁴ are different.)
In the structural formula (2), R ^{1 to} R ⁴ may be the same as or different from each other, and each represents a hydrogen atom or a saturated or unsaturated aliphatic acyl group having 1 to 20 carbon atoms ( However, acetyl group is excluded).

However, although MEL having no acetyl group represented by the structural formula (2) has been known to date, its production amount was very small.
Therefore, the present inventors further store the MEL-containing culture solution after performing batch culture or repeated batch culture as described above at an arbitrary temperature, whereby R ¹ to R ¹ in the structural formula (1) are stored. It was found that MEL in which an acetyl group is not bonded to R ⁴ (MEL represented by the structural formula (2)) can be produced. The method is as follows.

  The culture solution containing MEL after culturing is placed in a thermostatic bath at a temperature of 10 to 70 ° C. and kept warm. The lipid component is extracted with an equal volume of ethyl acetate at regular intervals, charged onto a TLC plate, and developed with chloroform: methanol: water = 65: 15: 2 (volume ratio). After completion of the development, the presence of MEL is confirmed with an orcinol sulfate reagent. Here, the storage temperature of the MEL-containing culture solution is preferably 20 to 70 ° C, particularly preferably 30 to 50 ° C. The storage time is preferably 2 to 30 days, and particularly preferably 3 to 20 days.

  The lipid component is extracted with an equal volume of ethyl acetate into the MEL-containing culture solution stored at a constant temperature, and the ethyl acetate is distilled off using an evaporator to recover the lipid component. This lipid component is dissolved in an equal amount of chloroform, and this is subjected to silica gel chromatography, and eluted with chloroform, chloroform: ethyl acetate solution (4: 1), acetone and methanol in this order. Each solution is charged on a TLC plate and developed with chloroform: methanol: water = 65: 15: 2 (volume ratio). After completion of the development, the presence of MEL is confirmed with an orcinol sulfate reagent. The eluate containing the MEL component that is thought to be free of acetyl groups was collected, and the solvent was distilled off to obtain the MEL component that was thought to be free of acetyl groups. The obtained MEL component is again dissolved in 1 mL of chloroform, subjected to iatrobead column chromatography, eluted with chloroform: methanol = 95: 5 (volume ratio), and a glycolipid component showing a single band is isolated by TLC. To do.

Identification of the binding position of the acyl group on the mannose residue can be performed by analysis of periodate oxidized glycolipid by NMR. In the 1H-NMR spectrum, the protons of sugars H-2 to H-6 are generally detected in the vicinity of 3.5 ppm, but when the hydrogen of the hydroxyl group is ester-bonded to the acyl group, the signal of the α-proton is low. It is known to shift to a magnetic field. In this MEL, two proton signals shifted by a low magnetic field, which are thought to be due to the binding of fatty acids, were observed, so that this MEL can be identified as having two molecules of acyl groups bound. Furthermore, although a signal indicating the presence of two molecules of —COCH ₂ — was observed, but no signal indicating the presence of an acetyl group (—COCH ₃ ) was observed, the acyl group bonded to mannose was It can be identified as two fatty acids.

  According to the method for producing the mannosyl erythritol lipid of the present invention as described above, the mannosyl erythritol lipid is preferably 7% by mass or more (70 g / L or more), more preferably 20% by mass or more (200 g / L or more). ), More preferably 20 to 40% by mass (200 to 400 g / L) at a high concentration in a short period of time.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to the following Example at all.

(Example 1: Selection of mannosylerythritol lipid-producing microorganism strain)
In Example 1, a microbial strain capable of efficiently producing mannosyl erythritol lipid in a short time was selected.

For 8 microorganisms with high glycolipid production (TM-179, TM-180, TM-181, TM-182, TM-183, TM-184, TM-185, TM-186) went. In addition, each said strain was isolate | separated from nature by the present inventors according to the method as described in Unexamined-Japanese-Patent No. 2005-104837, paragraph number [0023], [0049] etc. A liquid medium having a composition of glucose 20 g / L, yeast extract 1 g / L, ammonium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L (hereinafter abbreviated as preculture medium) ) One platinum loop of the strain was inoculated into a test tube containing 4 mL and cultured with shaking at 30 ° C. for 1 day. This was inoculated into a Sakaguchi flask containing 100 mL of the medium having the same composition, and cultured at 30 ° C. for 2 days with shaking. Furthermore, the liquid medium 1.4L of the composition of 18% by weight soybean oil and yeast extract 1 g / L, ammonium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L The jar fermenter was inoculated and main culture was started at 30 ° C. with an aeration rate of 1.5 L / min and a stirring speed of 800 rpm. The pH was maintained at 5.4 by supplying 14% by mass of ammonia using a pH controller. The culture solution is aseptically collected 2 to 3 times a day. Ethyl acetate is added to the culture solution aseptically shaken and allowed to stand, and the supernatant ethyl acetate layer is recovered. A rod (manufactured by Yatron) was charged and each component in the culture solution was measured over time. The amount of MEL produced in each strain and the culture time required to reach this concentration were determined as the reference strain Kurtzmanomyces sp. It shows in Table 3 with the result of I-11 strain (FERM P-18126).

  Depending on the strain, there was a difference in MEL production and the incubation time required to reach this concentration. TM-179, TM-181, TM-182, and TM-183 all produced 130 g / L or more of MEL, but TM-181 had the shortest period required for culture.

The TM-181 strain is available from Kurtzmanomyces sp. MEL production was 7 g / L lower than I-11 strain (FERM P-18126), but the time required for production was 144 hours, shortening the time required for production for 96 hours (4 days) did it.

This TM-181 strain is estimated to belong to Pseudozyma tsukubaensis from the results of 26S rDNA-D1 / D2 nucleotide sequence analysis, simple morphological observation, physiological and biochemical property tests, etc. As described above, the present inventors made a deposit (NITE P-530) on March 17, 2008 in the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation, a domestic depositary organization.
From the results in this Example 1, for the following examples, Pseudozyma tsukubaensis TM-181 strain (NITE P-530) was used as the mannosylerythritol lipid-producing microorganism strain.

(Example 2: Determination of optimum concentrations of medium components)
In Example 2, the optimum concentration of medium components for increasing the production efficiency of mannosyl erythritol lipids in Pseudozyma tsukubaensis TM-181 strain (NITE P-530) was determined.

In order to determine the optimal concentration of each medium component of yeast extract, corn steep liquor, ammonium nitrate, potassium dihydrogen phosphate, and magnesium sulfate as medium components, the concentration of each component is changed in 6 to 8 stages and batched as follows: Culture was performed. One platinum loop of the strain was inoculated into a test tube containing 4 mL of the preculture medium, and cultured with shaking at 30 ° C. for 1 day. This was inoculated into a Sakaguchi flask containing 100 mL of the medium having the same composition, and cultured with shaking at 30 ° C. for 2 days. Furthermore, based on the composition of 18% by weight soybean oil and yeast extract 1 g / L, ammonium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L, yeast extract ( 0-4 g / L), corn steep liquor (0-6 g / L), ammonium nitrate (0-4 g / L), potassium dihydrogen phosphate (0-4 g / L), and magnesium sulfate (0-4 g / L) Was inoculated into a jar fermenter containing 1.4 L of a liquid medium in which the concentration of 6 to 8 was changed, and main culture was started at 30 ° C. with an aeration rate of 1.5 L / min and an agitation rate of 800 rpm. In addition, pH was hold | maintained at 5.4 by supplying 14 mass% ammonia using a pH controller. The culture solution is aseptically collected 2 to 3 times a day. Ethyl acetate is added to the culture solution aseptically shaken and allowed to stand, and the supernatant ethyl acetate layer is recovered. A rod (manufactured by Yatron) was charged and each component in the culture solution was measured over time.
As a result, yeast extract 1 g / L, corn steep liquor 2 g / L, ammonium nitrate 0.5 g / L, potassium dihydrogen phosphate 0.4 g / L, and magnesium sulfate 0.2 g / L were the optimum concentrations.

(Example 3: Determination of optimum conditions of pH, dissolved oxygen concentration, culture temperature)
In Example 3, the optimum conditions of pH, dissolved oxygen concentration, and culture temperature were determined to increase the production efficiency of mannosylerythritol lipid in Pseudozyma tsukubaensis TM-181 strain (NITE P-530).

  In order to determine the optimum conditions of pH, dissolved oxygen concentration, and culture temperature, batch culture was performed by changing these three conditions to 3 to 7 stages. The pH of the culture solution was measured with a pH meter and controlled to a predetermined pH by dropping ammonia. The dissolved oxygen concentration was measured with a dissolved oxygen concentration meter and controlled to a predetermined dissolved oxygen concentration by automatically controlling the rotation speed. As a result, pH 5.2, dissolved oxygen concentration 3 to 5 ppm, and culture temperature 30 ° C. were the optimum conditions. Among these, the details of the method for determining the optimum conditions of pH and dissolved oxygen concentration are shown below.

-Basic culture conditions-
One platinum loop of Pseudozyma tsukubaensis TM-181 strain (NITE P-530) was inoculated into a test tube containing 4 mL of the preculture medium, and cultured with shaking at 30 ° C. for 1 day. This was inoculated into a Sakaguchi flask containing 100 mL of the medium having the same composition, and cultured with shaking at 30 ° C. for 2 days. Furthermore, this is made up of a predetermined amount of vegetable oil (linseed oil or soybean oil) and yeast extract 1 g / L, corn steep liquor 2 g / L, ammonium nitrate 0.5 g / L, potassium dihydrogen phosphate 0.4 g / L, and sulfuric acid. Inoculate a jar fermentor containing 1.4 L (batch culture) and 4.6 L (repeated batch culture) of a liquid medium (hereinafter abbreviated as main culture medium) having a composition of magnesium 0.2 g / L, and 30 The main culture was started at a temperature of 1.5 L / min (batch culture) and 5 L / min (repetitive batch culture) at 800 ° C. and a stirring speed of 800 rpm.

-Batch culture-
In batch culture, vegetable oil (linseed oil or soybean oil) is not supplied during the cultivation, and the culture solution is aseptically collected once or twice a day. It was measured. MEL, triglyceride, diglyceride, and fatty acid were added to ethyl acetate in the collected culture and shaken vigorously, and then allowed to stand to recover the supernatant ethyl acetate layer. This ethyl acetate solution was charged into an Iatroscan (Yatron) rod and each component was quantitatively analyzed by a predetermined method.

-PH selection of culture solution-
Next, in order to investigate the influence of the pH of the culture solution on MEL production, the initial fat concentration is set to 18% by mass and maintained at a constant value between pH 5.0 and 5.8 during the culture period using a pH controller. And cultured. In addition, as the alkaline solution used for adjusting the pH, ammonia (14% by mass ammonia solution) that can serve as nutrients for the cells was used.
Table 4 shows the amount of MEL produced at each pH and the incubation time required to reach this concentration. It was confirmed that culturing by controlling the pH to 5.0 to 5.8, particularly pH 5.2 is effective in increasing the production efficiency of MEL.

-Selection of dissolved oxygen concentration (DO) of culture solution-
Next, in order to examine the influence of the dissolved oxygen concentration of the culture solution on the MEL production, the initial fat and oil concentration was 20% by mass and the DO was controlled to 1, 3, and 5 ppm, and the culture was performed. At this time, the dissolved oxygen concentration was measured with a dissolved oxygen concentration meter and controlled to a predetermined dissolved oxygen concentration by automatically controlling the rotation speed. Table 5 shows the amount of MEL produced at each dissolved oxygen concentration and the incubation time required to reach this concentration. It was confirmed that culturing with DO controlled to 3 to 5 is effective in increasing the production efficiency of MEL.

(Example 4: Determination of optimum fat concentration)
Under the basic culture conditions set as in Example 3 above, the initial fat concentration was changed as shown in Table 6 to examine the influence of the initial fat concentration on MEL production. Considering efficient production of MEL, further higher concentration is desired. For this purpose, it is necessary to increase the initial fat concentration. Therefore, culturing was carried out while changing the oil and fat concentration from 10 to 36% by mass. Table 6 shows the amount of MEL produced in each culture and the culture time required to reach this concentration.

With the increase in the initial oil and fat concentration, the MEL production amount increased, and 220 g / L of MEL was produced in 126 hours at 30% by mass. At 34% by mass or more, the production amount dropped rapidly. Currently, MEL production is measured by Kurtzmanomyces sp. The highest is 307 g / L by fed-batch culture using I-11. In batch culture with Pseudozyma tsukubaensis TM-181 strain, 221 g / L is the highest value, but by using fed-batch culture method, it is possible to produce MEL at a higher concentration in a shorter time. Conceivable.

For reference, Kurtzmanomyces sp. The I-11 strain produced MEL of 153 g / L in 240 hours when the initial oil and fat concentration was 20% by mass, and the MEL production amount did not increase even when the oil and fat concentration was further increased. Pseudozyma tsukubaensis TM-181 strain is a strain of Kurtzmanomyces sp. The MEL production was 69 g / L higher than that of the I-11 strain, and production was possible in 114 hours.

(Example 5: repeated batch culture)
One platinum loop of the strain was inoculated into a test tube containing 4 mL of the preculture medium, and cultured with shaking at 30 ° C. for 1 day. This was inoculated into a Sakaguchi flask containing 100 mL of the medium having the same composition, and cultured with shaking at 30 ° C. for 2 days. In addition, 4 jars (total 0.4 L) were inoculated into 4.6 liters of 18% by weight flaxseed oil and main culture medium, and the aeration rate was 5 L / min at 30 ° C. and stirring at 800 rpm. The main culture was started at a speed. The pH was maintained at 5.2 by supplying 14% by mass of ammonia using a pH controller, and the first batch culture was performed for 96 hours. Vegetable oil (linseed oil) was rapidly decomposed into fatty acids and glycerol by lipase secreted by the cells. The generated intermediate metabolite, fatty acid, was taken up into the cell and metabolized to produce bacterial cells and MEL.
In the second batch culture, the rotation of the stirring blade was stopped, and 4.3 L of the culture solution was drawn from the jar famenter through the sampling tube, and 0.7 L was left in the jar famenter. Immediately, 4.3 L of a main culture medium containing 18% by mass of linseed oil, which had been sterilized in advance, was aseptically poured into the jar fermenter from the inoculation port, and culture was started at a stirring speed of 800 rpm. The pH was maintained at 5.2 by supplying 14% ammonia by volume using a pH controller.
In the third and subsequent batch cultures, the above operation was repeated, and the culture time was gradually shortened to repeat the batch culture for a total of 10 times. At this time, as shown in Table 7, the amount of the remaining culture solution in each batch culture was changed in the range of 0.3 to 1.75 L, and the same amount of a sterilized fresh medium as the extracted culture solution was added. Also, add ethyl acetate to the culture solution collected aseptically 2 to 3 times a day, shake it vigorously and let it stand, collect the supernatant ethyl acetate layer. A Toroscan (Yatron) rod was charged and each component in the culture was measured over time.
As shown in Table 7, all of the 10 cultures completely decomposed the fats and oils of the raw material, and the produced fatty acids were almost consumed, and about 130 g / L of MEL could be stably produced. There wasn't. Moreover, as a result of observing the culture solution with a microscope as needed, no bacteria such as bacteria were observed, and there was no contamination with bacteria. From these facts, it was considered that culture for a longer period was possible.

  In the first batch culture, it took 18 hours to start MEL (induction time). From the second time on, the induction time was shortened. The induction time was shortened in this way because the first bacterial cells in the culture tank were low in concentration since the first culture was inoculated with the culture solution from the shake flask. This is because the cell concentration at the start of a new batch culture is high. The MEL and cell concentration at the start of a new batch culture are affected by the residual liquid rate. Moreover, after starting MEL production, the MEL concentration increased almost linearly in any culture. Thereafter, the level of increase in MEL concentration gradually decreased before reaching the maximum production. The MEL production rate was calculated by the method of least squares from the slope of the portion where the MEL increased almost linearly with the aging of the culture. When the initial MEL concentration was high (= the amount of the remaining culture solution was large), the time required to reach the maximum production amount was shortened, and from the second time, the amount reached 130 g / L in 72 hours (3 days). Especially, in the 10th culture (residual liquid ratio: 0.35), it reached 130 g / L in 60 hours (2.5 days). This is considered to be because the bacterial cell concentration in the culture tank was increased by increasing the residual liquid ratio. Moreover, since lipase and MEL are present in a considerable amount from the beginning of the culture, it is considered that the decomposition of fats and oils was promoted, and the uptake of fatty acids into cells was promoted.

(Example 6: Mannosylerythritol lipid in which an acetyl group is not bonded to R ¹ to R ⁴ in Structural Formula (1) (= Mannosyl erythritol lipid represented by Structural Formula (2)))
One platinum loop of the strain was inoculated into a test tube containing 4 mL of the preculture medium, and cultured with shaking at 30 ° C. for 1 day. This was inoculated into a Sakaguchi flask containing 100 mL of the medium having the same composition, and cultured with shaking at 30 ° C. for 2 days. Furthermore, this was inoculated into a jar fermenter containing 18% by mass of flaxseed oil and main culture medium 1.4L, and the main culture was started at 30 ° C with an aeration rate of 1.5 L / min and an agitation rate of 800 rpm. did. During the culture period, a MEL-containing culture solution was prepared by culturing for 4 days while maintaining the pH at 5.2 using a pH controller. The MEL-containing culture solution was kept in a thermostat while being measured with a thermometer so that the MEL-containing culture solution was 20 to 70 ° C. Samples were taken at regular intervals, lipid components were extracted with an equal volume of ethyl acetate, charged on a TLC plate, and developed with chloroform: methanol: water = 65: 15: 2 (volume ratio). After the development, the presence of MEL was confirmed with an orcinol sulfate reagent. The results are shown in FIG.

  FIG. 1 shows a culture solution stored at a predetermined temperature for 7 days. Lane number 1 was stored before storage, 2 was 30 ° C., 3 was 20 ° C., 4 was 40 ° C., 5 was 50 ° C., 6 was 60 ° C., and 7 was 70 ° C. In the figure, the larger the spot, the greater the amount of MEL present. Before the start of storage, there was only MEL with one acetyl group bound, but after storage for 7 days, MEL with one acetyl group bound (the upper spot in FIG. 1) decreased and there was no acetyl group. The MEL spot (lower spot in FIG. 1) estimated to be larger. At 20 ° C in lane 3, the spot (upper spot) is larger for the MEL with one acetyl group bonded, but at 30-50 ° C in lanes 2, 4, and 5, the MEL spot with one acetyl group bonded There were almost no MEL spots, and almost all were MEL spots (bottom spots) presumed to have no acetyl group. Both spots were smaller at 60 and 70 ° C in lanes 6 and 7. From these results, it can be seen that the storage temperature of the MEL-containing culture solution is preferably 20 to 70 ° C, particularly preferably 30 to 50 ° C.

<Isolation of glycolipid>
The lipid component was extracted with an equal volume of ethyl acetate in an MEL-containing culture solution stored at a constant temperature, and the ethyl acetate was distilled off using an evaporator to recover the lipid component. This lipid component was dissolved in an equal amount of chloroform and subjected to silica gel chromatography, and eluted with chloroform, chloroform: ethyl acetate solution (4: 1), acetone and methanol in this order. Each solution was charged on a TLC plate and developed with chloroform: methanol: water = 65: 15: 2 (volume ratio). After the development, the presence of MEL was confirmed with an orcinol sulfate reagent. The eluate containing the MEL component that is thought to be free of acetyl groups was collected, and the solvent was distilled off to obtain the MEL component that was thought to be free of acetyl groups. The obtained MEL component is again dissolved in 1 mL of chloroform, subjected to iatrobead column chromatography, eluted with chloroform: methanol = 95: 5 (volume ratio), and a glycolipid component showing a single band is isolated by TLC. did.

<Determination of glycolipid structure>
The binding position of the acyl group was analyzed by NMR of periodate oxidized glycolipid. As a control, mannosylerythritol lipid in which two acetyl groups and a fatty acid are bonded to mannose (MEL: 4,6-di-O-acetyl-2,3-di-O-alkanoyl-β-D-mannopyranosyl 1-4) meso-erythritol).
Mannose was confirmed from the signal on the lower magnetic field side than 4.0 ppm. In addition to these signals, signals indicating the properties of acyl groups are observed in the high magnetic field region, and can be identified as —CH ₂ —proton signals of —COCH ₂ groups, respectively. You can see that it exists. On the other hand, since no signal indicating the presence of the methyl proton of the acetyl group (—COCH ₃ ) was observed, it was identified that the acetyl group was not bound to mannose. The results are shown in FIG. This compound is conventionally called MEL-D.

According to the present invention, by using Pseudozyma tsukubaensis TM-181 strain (NITE P-530) as a microorganism capable of producing mannosyl erythritol lipids, by optimizing the medium composition and culture conditions, The time required for the production of mannosyl erythritol lipid, a kind of biosurfactant, was greatly improved. Furthermore, the present invention enables mass production of mannosylerythritol lipids to which no acetyl group is bonded. The mannosyl erythritol lipid obtained by the present invention can be suitably used, for example, in the food industry, pharmaceutical industry, chemical industry, environment-related fields, and the like.

FIG. 1 is a diagram showing the results of confirming the type of MEL present by storing the MEL-containing culture solution at a predetermined temperature for 7 days and then developing it by thin layer chromatography (TLC). (Lane No. 1 is MEL-containing culture solution before starting storage, 2 is 30 ° C., 3 is 20 ° C., 4 is 40 ° C., 5 is 50 ° C., 6 is 60 ° C., 7 is MEL after storage at 70 ° C. (Indicates the culture medium contained.) FIG. 2 is a 1H-NMR spectrum of mannosylerythritol lipid according to Example 6. <1H-NMR chemical shift δ (assigned hydrogen, D ₂ O)> mannosyl erythritol part: 3.30-4.00 (Man-4-H, 5-H, 6-H ₂ , Ery-1-H ₂ , 2-H, 3-H, 4-H), 4.77 (Man-1-H), 4.93 (Man-3-H), 5.43 (Man-2-H); acyl moiety: 0 _{.86,0.95 (CH 3), 1.05-1.40,1.90-2.10 [} - (CH 2) n -], 1.60-1.70 (-CO-CH 2 - _{CH 2 -), 2.27,2.36 (-CO-} CH 2 -), 5.23 (-CH = CH-)

Claims (15)

A method for producing a mannosyl erythritol lipid, comprising culturing a Pseudozyma tsukubaensis TM-181 strain (NITE P-530) in a culture solution containing a nutrient and a carbon source. The method for producing mannosyl erythritol lipid according to claim 1, wherein the culture is performed while controlling the pH of the culture solution in a range of 5.0 to 5.8. The method for producing mannosyl erythritol lipid according to claim 2, wherein the pH of the culture solution is controlled using ammonia. The manufacturing method of the mannosyl erythritol lipid in any one of Claim 1 to 3 which culture | cultivates by controlling the dissolved oxygen concentration of a culture solution in the range of 1-5 ppm. The manufacturing method of the mannosyl erythritol lipid in any one of Claim 1 to 4 which culture | cultivates by making 0.1-4 g / L of corn steep liquor in a culture solution. The method for producing mannosyl erythritol lipid according to any one of claims 1 to 5, wherein a linear aliphatic hydrocarbon having 6 to 24 carbon atoms is used as the carbon source. The manufacturing method of the mannosyl erythritol lipid in any one of Claim 1 to 5 which uses a C6-C24 fatty acid as a carbon source. The manufacturing method of the mannosyl erythritol lipid in any one of Claim 1 to 5 using the triglyceride containing a C6-C24 linear aliphatic hydrocarbon group as a carbon source. The method for producing mannosyl erythritol lipid according to any one of claims 1 to 5, wherein a carbohydrate is used as a carbon source. The manufacturing method of the mannosyl erythritol lipid in any one of Claim 1 to 5 which uses food waste oil as a carbon source. The manufacturing method of the mannosyl erythritol lipid in any one of Claim 1 to 10 which culture | cultivates in the range whose carbon source concentration is 10-32 mass%. 12. The medium composition and culture conditions for producing mannosyl erythritol lipid in Pseudozyma tsukubaensis TM-181 strain (NITE P-530) are as follows. Manufacturing method of mannosyl erythritol lipid.
Yeast extract: 0.1-2 g / L
Corn steep liquor: 0.1-4g / L
Ammonium nitrate: 0.1-1 g / L
Potassium dihydrogen phosphate: 0.1-2 g / L
Magnesium sulfate: 0.1-1 g / L
Carbon source: 100-320 g / L
Culture solution pH: 5.0 to 5.8
Dissolved oxygen concentration: 1-5ppm
Culture temperature: 26-32 ° C Culturing is started at a carbon source concentration in the range of 10 to 36% by mass, and most of the culture solution containing the bacterial cells and mannosyl erythritol lipid is discharged out of the culture tank at the end of the culture for 2 to 6 days after the start of the culture. The method for producing mannosyl erythritol lipid according to any one of claims 1 to 12, wherein a part of the culture tank is left to add a new medium, the next batch culture is started, and the operation is repeated. The mannosyl according to any one of claims 1 to 13, further comprising a step of maintaining the culture solution after completion of the culture at 20 to 70 ° C to obtain a mannosyl erythritol lipid having a structure represented by the following structural formula (2). Method for producing erythritol lipid.
However, in the structural formula (2), R ¹ ~ R ⁴ May be the same as or different from each other, and each represents a hydrogen atom or a saturated or unsaturated aliphatic acyl group having 1 to 20 carbon atoms (excluding an acetyl group). The production method of mannosyl erythritol lipid according to any one of claims 1 to 14, wherein the production amount of mannosyl erythritol lipid is 7% by mass or more (70 g / L or more).