JP2011182660A - New microorganism and method for producing sugar type biosurfactant using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To isolate a microorganism having an ability for producing a sugar type biosurfactant from an edible plant and then use the microorganism to produce the sugar type biosurfactant suitable for uses requiring higher safety, in order to provide the sugar type biosurfactant more excellent in adaptability than conventional biosurfactants for uses requiring higher safety for living bodies, such as cosmetics, foods, and agricultural livestock. <P>SOLUTION: There is provided the microorganism belonging to Pseudozyma churashimaensis isolated from sugarcane. The microorganism is a new kind, and is suitable for producing mannosyl erythritol lipid (MEL)-A which is one of biosurfactants. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a microorganism isolated from sugarcane having the ability to produce a sugar-type biosurfactant, and its use.

  Glycolipids are substances in which 1 to 10 monosaccharides are bound to lipids, are involved in the transmission of information between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. On the other hand, glycolipids are amphipathic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. being called.

  On the other hand, some microorganisms are known to produce these surfactants efficiently, and this biological surfactant (biosurfactant) is highly safe and biodegradable with low environmental impact. Research is progressing as an excellent environmentally advanced surfactant. Currently, the surface active substances produced by microorganisms are classified into five types: sugar type, acyl peptide type, phospholipid type, fatty acid type, and polymer compound type. Among these, surfactants of the sugar type in particular are included. There are many types of substances that have been best studied and produced by bacteria and yeast.

  These biosurfactants are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. For this reason, the wide application of these biosurfactants in the food industry, cosmetic industry, pharmaceutical industry, chemical industry, environmental field, etc. has both the realization of a sustainable society and the provision of high-performance products. Meaningful.

  Examples of sugar-type biosurfactants include the following.

Rhamnolipid (hereinafter abbreviated as RL) was first discovered from the culture solution of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) as an antibiotic of Mycobacterium tuberculosis (see Non-Patent Document 1).
In addition, four kinds of homologues have been reported so far from bacteria of the genus Pseudomonas , and the initial production was about several g / L, but now it is possible to produce over 100 g / L. (See Non-Patent Document 2).

Trehalose lipid (hereinafter abbreviated as TL) has been discovered as a cell surface material such as Corynebacterium . Similar substances have also been reported from Mycobacterium , Nocardia and Rodococcus bacteria (see Non-Patent Document 3).
In general, production is low due to binding to the cell wall, but it has been reported that succinoyl trehalose lipid is produced at a rate of 32 g / L when cultivating Rhodococcus erythropolis under nitrogen limitation. (Refer nonpatent literature 4).

Sophorose lipids (also referred to as “sophorolipids”; hereinafter abbreviated as SL) A. It has been discovered by Gorin et al. In a culture solution of Starmerella ( Candida ) bombicola (see Non-Patent Document 5).
Later, other yeasts such as Candida bogoriensis , Candida magnoliae , Candida gropengisseri , and Candida apicola were also used in the culture. It has been reported that it is produced in a relatively large amount (see Non-Patent Document 6).
Furthermore, production of 300 g / L or more is now possible (see Non-Patent Document 7).

Cellobiose lipid (hereinafter abbreviated as CL) is a glycolipid having high antimicrobial activity, also called ustilagic acid or flocculosin.
In addition, CL is produced by Ustilago maydis over 15 g / L (see Non-Patent Document 8), Cryptococcus humicola (see Non-Patent Document 9), Pseudozyma flocculosa ( Pseudozyma flocculosa ) ( Pseudozyma flocculosa ) Non-patent document 10), Pseudozyma fusiformata (see non-patent document 11) and the like have been reported.

  Mannosyl erythritol lipid (MEL) has a high surface activity and is used as a surfactant or as a catalyst for fine chemicals. When mannosyl erythritol lipid is allowed to act on human acute promyelocytic leukemia cell line HL60, it has a leukemia cell differentiation inducing action that differentiates the granule system, and mannosyl erythritol lipid acts on PC12 cells derived from rat adrenal medullary pheochromocytoma And has a physiologically active action such as a neural cell line differentiation-inducing action that causes neurite outgrowth. Furthermore, for the first time as a glycolipid produced by microorganisms, it becomes possible to induce apoptosis of melanoma cells, and has an effect of suppressing cancer cell growth. In view of these physiological actions, mannosyl erythritol lipid is expected to be used as a medicine such as an anticancer agent. In addition, mannosyl erythritol lipid (MEL) is biodegradable and is considered to have high safety (see Non-Patent Document 12, etc.).

MEL is a phospholipid having mannose or mannose partially hydroxylated and erythritol as a sugar skeleton (hydrophilic group) and 1 to 3 fatty acids as hydrophilic groups. Formula 1 of MEL is shown in Chemical Formula 1.
In the general formula 1, R _{1 to} R ₄ may be the same as or different from each other, and represent a hydrogen atom, an acetyl group, or a saturated or unsaturated fatty acid residue having 3 or more carbon atoms.
In this formula, a structure in which R ₃ and R ₄ are acetyl groups is MEL-A, and a structure in which R ₄ is an acetyl group and R ₃ is hydrogen is MEL-B, a structure in which R ₃ is an acetyl group, and R ₄ A structure in which is hydrogen is defined as MEL-C, and a structure in which R ₃ and R ₄ are hydrogen is defined as MEL-D.

  Currently, MEL is being used industrially in a wide range of fields such as detergents and cosmetics. Use of MEL as a gene introduction agent (see Patent Document 1) and use as a liposome forming agent (see Patent Document 2), MEL skin care In addition, there are reports on utilization as hair care agents (see Patent Document 3), emulsifiers / solubilizers (see Patent Document 4), protein separation carriers (see Patent Document 5) and the like.

For mannosyl erythritol lipid (MEL), Candida sp. It is reported that 95 g / L (production rate: 0.475 g / L / h, raw material yield: 45%) of MEL can be produced from 211 g / L soybean oil in 200 hours using SY16 strain. (See Non-Patent Document 13). In addition, it has been reported that 47 g / L (production rate: 0.32 g / L / h) of MEL can be produced from soybean oil in 6 days using Candida antarctica T-34 strain (non-patent literature). 14). Using Pseudozyma aphidi s strain, 80% by mass of vegetable oils and fats by the fed-batch method for 13.9 g / L (production rate: 0.57 g / L / h, raw material yield: 92% by mass) in 24 hours It has been reported that production is possible (see Non-Patent Document 15).

JP 2005-281146 A JP 2006-174727 A WO2007 / 060956 JP 2007-181789 A JP 2006-310959 A

“Journal of the American Chemical Society” (J. Am. Chem. Soc.), 71, p4124-4126 (1949). “Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.)”, 51, p22-32 (1999). “Biosurfactant and Biotechnology” (USA), Marshall Decker, New York (1987). "Journal of Biotechnology (J. Biotechnol.)" (UK), Vol. 13, p257-266 (1990). “Canadian Journal of Chemistry”, Vol. 39, p. 848-855 (1961). “Journal of Biotechnology”, Vol. 33, p147-155 (1990). “Biotechnol. Lett.”, 20, p805-807 (1998). "Applied Microbiology and Biotechnology" (Appl. Microbiol. Biotechnol.), (Germany), Springer-Verlag, 51, p33-39 (1999). “Biochimica Biophysica Acta”, (Netherlands), Elsevier, 1558, p161-170 (2002). “Antimicrobial Vial Agents and Chemiotherapy, Vol. 49, p 1597-1599 (2005). "FEMS Yeast Res." (Netherlands), Elsevier, Vol. 5, p919-923 (2005). “Journal of Bioscience and Biotechnology, Vol. 94, p187-201 (2002). “Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.)”, 70, p391-396 (2006). “Biotechnology. Lett.”, Vol. 14, p305-310 (1992). “Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.)”, 68, p607-613 (2005).

  Biosurfactants are superior in environmental compatibility and biocompatibility compared to general-purpose surfactants, and various physiological activities are recognized, but they are used in a wide range of applications similar to existing surfactants. No. For example, there are restrictions on use for food applications. This is due to the fact that although the pathogenicity of the produced microorganism is not known in many cases, the source of isolation from the natural world is not clear, and thus knowledge about safety is poor. In particular, in order to develop a microbial product for food use, an isolation source of microorganisms to be used is regarded as important. For example, budding yeast, lactic acid bacteria, Bacillus subtilis (natto), and the like used for the production of alcoholic beverages and bread are generally well known as microorganisms that play an active role in the food field. These microorganisms are all microorganisms that can be easily isolated from food.

  Furthermore, in order to increase the production efficiency of biosurfactants, reduce production costs, and develop homologue production technologies with new functionality and deploy them to a wide range of applications, new biosurfactant producing bacteria The current situation is that search / acquisition is strongly desired.

  Therefore, if a biosurfactant-producing bacterium can be newly isolated from an edible plant and a biosurfactant production technology using the microorganism can be developed, the resulting biosurfactant has a great advantage in terms of safety. It is expected that the application of the entire product including the production microorganism will be greatly expanded.

  In view of the above circumstances, in the present invention, safety to living bodies such as cosmetics, foods, food additives, medicines, agricultural and livestock industry-related products (feeds, feed additives, fertilizers, agricultural chemicals, fish foods), detergents, etc. The purpose of the present invention is to provide a microorganism isolated from a safe environment and a method for fermenting a biosurfactant using the same, in order to provide a biosurfactant that is more adaptable than before.

As a result of searching for the biosurfactant-producing bacteria to solve the above-mentioned problems, the present inventor has developed a new yeast, Pseudozyma churashimaensis, from sugarcane, which is a raw material for sugar that is used daily. ) ( Ustilago esculenta ) was successfully isolated. The yeast can produce mannosyl erythritol lipid (MEL) which is a kind of biosurfactant, and can produce not only existing MEL but also triacetyl monoacyl mannosyl erythritol lipid which can hardly be produced by existing producing bacteria. As a result, the present invention has been completed.

That is, the present invention is as follows.
[1] A microorganism belonging to Pseudozyma churashimaensis, which has the ability to produce triacetyl monoacyl-type mannosyl erythritol lipids.
[2] The microorganism according to the above [1], which has mycological properties represented by the following (a) and (b).
(A) Morphological properties
(B) Physiological properties
[3] The microorganism according to [1] or [2] above, which is Pseudozyma Chulashima Ensis OK-96 (Accession No. FERM P-21896).
[4] Mannosylerythritol lipid, wherein the microorganism according to any one of [1] to [3] above is cultured, and mannosylerythritol lipid-A is collected from the obtained culture or isolated cells. Production method.
[5] Culturing the microorganism according to any one of [1] to [3] above in a medium containing saccharides as a carbon source, and collecting triacetyl monoacyl mannosyl erythritol lipid from the obtained culture A process for producing a triacetyl monoacyl mannosyl erythritol lipid, characterized in that

According to the present invention, it is possible to provide a microorganism that is resident in sugarcane, which is a widely eaten plant for a long time, more specifically, Pseudozyma churashimaensis .
By using the microorganism as a production microorganism, the safety of the obtained biosurfactant is ensured, and as a surfactant that can be directly used for food use, livestock feed, fish food, and agricultural use that have been limited in use so far. Expected to be used, it is expected to contribute significantly to the spread of biosurfactants.

  Furthermore, according to the manufacturing method which concerns on this invention, MEL-A can be selectively produced, without producing a by-product. That is, in the conventional method, it is possible to obtain MEL-B that does not contain MEL-B and MEL-C, which require a purification process, only by culturing microorganisms. In particular, since MEL can be produced even if a medium containing only a carbohydrate (glucose or sucrose) as a carbon source is used without using a hydrophobic substrate such as fat or oil, isolation of MEL becomes simple. Furthermore, the microorganism of the present invention has an ability to produce triacetyl type MEL. This triacetyl monoacyl type MEL belongs to MEL-A as defined above, and its surface activity itself is not much different from conventional MEL-A (diacetyl diacyl type). Since the number of fatty acids is one, the affinity for water molecules is improved as compared with the conventional MEL-A having two fatty acids. Further, there is a difference in the interaction between molecules due to the difference in molecular structure, and application as a nanomaterial different from the conventional MEL-A is expected.

  As described above, according to the present invention, only MEL-A can be produced. For example, when used as a surfactant, the purification step can be simplified, so that the MEL production cost can be reduced. It becomes. In particular, the triacetyl monoacyl type MEL described above is notable because it has an improved affinity for water molecules and can be expected to be used as a nanomaterial different from the conventional MEL-A.

In Example 1, it is a graph which shows the result of TLC which investigated the influence with respect to MEL production of the kind of carbon source added to a culture medium. In Example 1, it is a graph which shows the result of the HPLC fixed_quantity | assay which investigated the influence with respect to MEL production of the kind of carbon source added to a culture medium. In Example 2, it is the figure which analyzed the glycolipid component just after extraction from a culture solution, and the glycolipid component after refinement | purification by TLC analysis. In Example 2, it is a figure which shows the result of the 1H-NMR analysis of each glycolipid component (GL-A, GL-B, GL-C). In Example 2, it is a figure which shows the result of the MALDI-TOF / MS analysis of each glycolipid component (GL-A, GL-B, GL-C). In Example 3, it is the figure which spotted the surface tension value of each component (GL-A, GL-B, GL-C) of each density | concentration on the log-log graph. In Example 4, it is a figure which shows the result of the microscope observation which investigated the self-assembly formation ability of each component (GL-A, GL-B, GL-C).

Hereinafter, the present invention will be described in more detail.
<Microorganism of the present invention>
The microorganism according to the present invention is a new yeast belonging to the genus Pseudozyma having the ability to produce MEL. The yeast produces mannosyl erythritol lipid, and the mannosyl erythritol lipid is desirable because of its high physiological activity and excellent practical properties. In addition, the yeast has a characteristic property particularly in that it produces a triacetyl monoacyl MEL. Examples of such microorganisms include the OK-96 strain belonging to the genus Pseudozyma . This strain is a strain isolated from a plant (sugar cane) collected by the present inventors in Japan. This strain is cultured on a YM agar medium, a PDB agar medium and a 5% malt extract medium at 25 ° C. for 4 days. And forms a cream to light pink colony with full edge, conical, smooth, bright and moist. On the third day of culture in YM liquid medium and PD liquid medium at 25 ° C., the cells are elliptical to cylindrical and proliferate by extreme budding. In addition, formation of hyphae with septa and pseudohyphae is observed. In addition, no apparent sexual genital formation is observed even after one month of culture. These morphological properties are consistent with the general morphological characteristics of the genus Pseudozyma. In addition, the strain of Ok-96 does not exhibit sugar fermentability, and is also characterized by the utilization of nitrate as a nitrogen source. Therefore, this strain can be said to be a strain belonging to the genus Pseudozyma.

For the isolated OK-96 strain, the nucleotide sequence (rDNA sequence) of the 26S rDNA-D1 / D2 region of the ribosomal RNA gene was determined (SEQ ID NO: 1), the DNA database (DDBJ) was accessed, and the FASTA program ( The homology search using http://www.ddbj.nig.ac.jp/search/fasta-j.html) revealed the highest homology with the rDNA sequence of Pseudozyma aphidis CBS517.83T. (99.5%). However, the nucleotide sequence (SEQ ID NO: 2) of the ITS1-5, 8SrDNA-ITS2 region of the OK-96 strain does not show a homology of 99% or more on the database, and the 8SrDNA-ITS2 of Ustilago trichophora 1334 strain The best was 94.1% homology. 92.6% homology (Pseudozyma hubeiensis CBS10077) was the highest for various yeasts of the genus Judezaima, and only 90.1% homology with the Pseudozyma aphidis CBS517.83T sequence. . In addition, as a result of the physiological property test tail, OK-96 strain and Pseudozyma aphidis (Pseudozyma aphidis) CBS517.83T showed that this strain did not assimilate erythritol, assimilated ethanol, and showed growth in a vitamin-deficient medium. Clearly different in respect. From these points, this strain was found to be a new species of the genus Pseudozyma, a kind of basidiomycete, and was designated as Pseudozyma churashimaensis OK-96 strain.

The morphological observation results and physiological property test results of this strain are summarized in Tables 1 and 2 below.
This strain was deposited on January 26, 2010 at the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center (IPOD) (Higashi 1-1-3 Tsukuba, Ibaraki) under the accession number FERM P-21896. .

(A) Morphological properties

(B) Physiological properties

(Production biosurfactant)
The biosurfactant produced by the microorganism of the present invention is mannosylerythritol (MEL), specifically, MEL-A having 4-O-β-D-mannopyranosyl-erythritol as a sugar skeleton, as MEL-A Is a compound in which R ₃ and R ₄ are acetyl groups and R ₁ and R ₂ are saturated or unsaturated aliphatic acyl groups having 4 or more carbon atoms, and R _1, R ₃ And R ₄ are both acetyl groups, and R ₂ is an aliphatic acyl group having 3 or more carbon atoms.

In the present specification, the diacetyl diacyl type mannosyl erythritol lipid (MEL) means the former compound, and the triacetyl monoacyl type mannosyl erythritol lipid (MEL) means the latter compound.

When the microorganism of the present invention is cultured using a saccharide as a carbon source, it accumulates in the culture solution as a mixture of diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl monoacyl mannosyl erythritol lipid (MEL). The Since there is no great difference between the properties of the surfactants, they may be used as a surfactant in the form of a mixture. Also, the affinity of triacetyl monoacyl mannosyl erythritol lipid (MEL) for water molecules and / or molecules If attention is paid to the interaction, diacetyldiacyl-type mannosylerythritol lipid (MEL) and triacetylmonoacyl-type mannosylerythritol lipid (MEL) may be further separated.
On the other hand, when the microorganism of the present invention is used as a carbon source for culturing oils and fats, fatty acids or fatty acid esters as a carbon source, only diacetyldiacyl mannosylerythritol lipid (MEL) is obtained. The diacetyldiacyl-type mannosylerythritol lipid (MEL) accumulated depending on the carbon source of the medium has a slightly different carbon number of the aliphatic acyl group at the 2-position of mannosyl (in the above general formula, R ₁ ).

<Medium and culture conditions>
The culture form of the microorganism of the present invention is batch culture using a liquid medium or fed-batch culture in which a carbon source and / or an organic nitrogen source are continuously added to a culture system, and it is desirable to aeration and agitate. The initial pH of the medium is not particularly limited as long as it is in the acidic to neutral range, but it is preferably adjusted to 3.0-9.0, more preferably 6.0-8.0. The temperature range suitable for the culture is 20-30 ° C, more preferably 22-27 ° C.

In the present invention, a general semi-synthetic medium may be used as the medium, but when a mixture of diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl monoacyl mannosyl erythritol lipid (MEL) is accumulated in the culture solution. Uses sugars as a carbon source.
On the other hand, in order to accumulate only diacetyldiacyl mannosylerythritol lipid (MEL) in the culture solution, fats and oils are used as a carbon source.
As the nitrogen source, it is desirable to use a combination of an organic nitrogen source and an inorganic nitrogen source.
The saccharide is not particularly limited as long as it can assimilate the microorganism of the present invention. For example, monosaccharides such as glucose, galactose and fructose, and disaccharides such as sucrose and maltose are preferably used. Is glucose. The initial culture concentration is 10-200 g / L, preferably 50-150 g / L.

The fats and oils to be used are not particularly limited, and for example, vegetable oils, fatty acids or esters thereof may be used.
Examples of vegetable oils to be used include soybean oil, rapeseed oil, corn oil, cottonseed oil, sunflower oil, kapok oil, sesame oil, rice oil, peanut oil, safflower oil, olive oil, flaxseed oil, gill oil, castor oil, palm oil, palm Examples thereof include nuclear oil, coconut oil, and mixtures thereof.
As the fatty acid and fatty acid ester to be used, a fatty acid or fatty acid ester having a carbon chain of 10-24, preferably 16-18 carbon chains can be used. These fatty acids or fatty acid esters may contain 1-3 unsaturated bonds in the molecule. For example, saturated fatty acids such as decanoic acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, behenic acid, lignoceric acid, etc. Acids, Linderic acid, Tuzic acid, Myristoleic acid, Palmitoleic acid, Petroselinic acid, Oleic acid, Elaidic acid, Vaccenoic acid, Erucic acid, Sorbic acid, Linoleic acid, Linoleic acid, Gamma-linolenic acid, Linolenic acid, Arachidone Examples thereof include unsaturated fatty acids such as acids or esters thereof.
The concentration of the fats and oils added to the medium is in the range of 20-100 g / L, preferably 50-80 g / L. When adopting a fed-batch culture mode, it is added continuously or intermittently during the culture period so that the concentration in the medium falls within the above range.

As the organic nitrogen source to be used, one or a combination of two or more of yeast extract, malt extract, peptone, polypeptone, corn steep liquor, casamino acid, urea and the like can be used. Yeast extract is preferably used in a concentration range of 1-8 g / L.
As the inorganic nitrogen source, one or a combination of two or more of sodium nitrate, potassium nitrate, ammonium nitrate, ammonium sulfate, and ammonia can be used. Sodium nitrate is preferably used.

(Separation of mannosyl erythritol lipid (MEL))
MEL-A accumulated in the culture solution can be obtained by being separated from the culture solution by extracting the culture solution after culturing the present strain. Extraction may be performed according to a method commonly used in the art using an organic solvent such as ethyl acetate, chloroform, methanol, ethanol, hexane, propanol or the like.
When MEL-A extracted with an organic solvent is a mixture of diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl monoacyl mannosyl erythritol lipid (MEL), diacetyl diacyl mannosyl erythritol lipid (MEL) and triacetyl Monoacyl type mannosyl erythritol lipid (MEL) may be separated by silica gel column chromatography or the like.

<Determining the structure of MEL>
The structure of the glycolipid component obtained as described above is determined as follows. The structure of MEL obtained by culturing glucose or soybean oil as a carbon source using Pseudozyma churashimaensis OK-96 strain This will be explained below using the determination method as an example).
The isolated glycolipid component can be judged to be a glycolipid component by being colored blue-green with an anthrone sulfate reagent on a TLC plate. The glycolipid is subjected to ¹ H, ¹³ C-NMR analysis, and the obtained spectrum is compared with MEL data having a known structure to perform structural analysis.

<Quantitative analysis method of MEL>
The total glycolipid amount can be measured by using the anthrone sulfate method. The content and abundance ratio of MEL congeners can be measured by using thin phase chromatography (TLC) method and high performance liquid chromatography (HPLC).

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1
(Production of MEL using Pseudozyma churashimaensis ; Influence of carbon source used)
<Seed culture>
30 mL of YM medium (glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, malt extract 3 g / L, peptone 5 g / L) was stored frozen in a deep freezer (−80 ° C.) in a pseudo- Zima Churashima Ensis ( Pseudozyma churashimaensis ) OK-96 strain. L) was inoculated with 0.5 mL and cultured with shaking at 28 ° C. for 1 day.
<Main culture>
MEL production media were prepared as shown in Tables 3 and 4 (composition of MEL production medium) in a 300 mL Erlenmeyer flask with the carbon source contained in 30 mL, and a medium sterilized by autoclaving at 121 ° C. for 20 minutes was prepared. 1.5 mL of the above seed culture solution was inoculated, and shaking culture was performed at 25 ° C. for 5 days. The MEL preparation was extracted with ethyl acetate from the culture broth after completion of the culture. The MEL concentration in the extract was quantitatively analyzed using HPLC connected to a silica gel column (Inertosyl Sil-100A). The eluent was a chloroform / methanol mixed solvent and eluted with a gradient system set so that the mixing ratio was changed from 10: 0 to 0:10 at a flow rate of 1 mL / min. For detection, an evaporative light scattering detector (ELSD-LT, manufactured by Shimadzu Corporation) was used. A calibration curve was created using the purified MEL, and the MEL concentration was calculated from the peak area.

<Thin phase chromatography analysis>
As shown in FIG. 1, two spots showing glycolipid production were obtained in the medium supplemented with glucose. That is, GL-A with a mobility of 0.66 and GL-B with a mobility of 0.62 were obtained. On the other hand, one spot was obtained in the medium supplemented with soybean oil. That is, GL-C having a mobility of 0.66 was obtained. The TLC results were the same whether the type of sugar added as the carbon source or the type of vegetable oil was changed. Further, from the result of FIG. 1, the mobility of the conventional MEL-A used for the standard is 0.66, whereas the mobility of GL-B is 0.62, and the mobility of GL-B It can be seen that the mobility is clearly low (high polarity). That is, it can be seen that the affinity of GL-B for water molecules is larger than that of conventional MEL-A. The result of measuring the production amount of MEL is shown in FIG.

Example 2
Structural analysis of glycolipid produced by Pseudozyma churashimaensis OK-96 strain (Purification of glycolipid (MEL))
Glycolipids were extracted and collected from the culture broth cultured in the same manner as in Example 1 using ethyl acetate, and each component (GL-A, GL-B, GL-C) was purified using a silica gel column. The result of analyzing each purified glycolipid component by TLC analysis is shown in FIG.
(NMR analysis of glycolipid components)
About each component (GL-A, GL-B, GL-C) obtained above, the structure was identified by ¹ H-NMR analysis using deuterated methanol (CD ₃ OD) as a solvent. As a result, it was found that all glycolipids produced by Pseudozyma churashimaensis OK-96 strain were MEL-A having acetyl groups at positions 4 and 6 of mannose (in FIG. 4). Peaks d and e). That is, GL-A, GL-B, and GL-C showed almost the same chemical shift as known MEL. Furthermore, in GL-A and GL-B, a peak indicating the presence of an acetyl group at the 2-position of mannose was obtained, so that the fatty acid at the 2-position of mannose became extremely short, and acetyl It was found that the base structure was included as one of the main components (peak c in FIG. 4). Moreover, in GL-B, since the peak which shows the fatty acid chain (acyl group) of the 2nd position of mannose was not seen, most of MEL-A contained in GL-B is the 2nd position of mannose. Can be said to be a structure having an acetyl group (peak a in FIG. 4). Table 5 summarizes the chemical shifts assigned in ¹ H-NMR analysis for each glycolipid.

(MALDI-TOF / MS analysis of glycolipid components)
As a result of analyzing the molecular weight of each component (GL-A, GL-B, GL-C) obtained above by MALDI-TOF / MS, the molecular weight of the main component of GL-A is 704.8, 676.8. The molecular weight of the main component of GL-B was 648.1, and the molecular weight of the main component of GL-C was 704.1 and 676.1. FIG. 5 shows the molecular weight peak obtained by MALDI-TOF / MS. From these results, GL-A is 4-O-[(6 ′, 4′-di-O-acetyl-, 3′-O-alka (e) noyl-2′-O-caproyl) -β- D-mannopyranosyl] -D-erythritol (general formula 3), 4-O-[(6 ', 4'-di-O-acetyl-, 3'-O-alka (e) noyl-2'- O-butanoyl ) -β-D-mannopyranosyl] -D-erythritol (general formula 4), 4-O-[(6 ', 4', 2'-tri-O-acetyl-, 3'-di-O-alka (e ) noyl) -β-D-mannopyranosyl] -D-erythritol (general formula 5), a fraction of a MEL-A mixture, GL-B is 4-O-[(6 ′, 4 ′, 2 '-tri-O-acetyl-, 3'-di-O-alka (e) noyl) -β-D-mannopyranosyl] -D-erythritol (general formula 5) as a main component, MEL-A fraction, GL- C is 4-O-[(6 ′, 4′-di-O-acetyl-, 3′-O-alka (e) noyl-2′-O-caproyl) -β-D-mannopyranosyl] -D-erythritol (General formula 3), 4-O-[(6 ', 4'-di-O-acetyl-, 3'-O-alka (e) noyl-2'-O-butanoyl) -β-D-mannopyranosyl] It was found to be a mixture of MEL-A mainly composed of -D-erythritol (general formula 4). Formula 3 shows the general formula 3. Formula 4 shows the general formula 4. Formula 5 shows the general formula 5.

Example 3
(Measurement of MEL surface activity)
<Surface tension measurement>
In Example 2, the surface tension reducing ability of each purified component (GL-A, GL-B, GL-C) was examined. Aqueous solutions of various components at various concentrations were prepared, and the surface tension was measured using a Wilhelmy type surface tension meter (CBVP-A3, manufactured by Kyowa Interface Science).

About each component (GL-A, GL-B, GL-C), surface tension reduction ability was measured using said method. The surface tension for each component at each concentration showed the values shown in FIG. From the curve of FIG. 6, the critical micelle concentration (cmc) of GL-A was 4.86 × 10 ⁻⁶ M, and the surface tension value (γcmc value) at that time was 29.2 mN / m. The critical micelle concentration (cmc) of GL-B was 1.36 × 10 ⁻⁶ M, and the surface tension value (γcmc value) at that time was 29.3 mN / m. The critical micelle concentration (cmc) of GL-C was 2.08 × 10 ⁻⁶ M, and the surface tension value (γcmc value) at that time was 28.7 mN / m. Compared with existing synthetic surfactants, they showed sufficiently small cmc values and γcmc values. Therefore, it can be inferred that the MEL produced in the present invention can exhibit a sufficient function as a surfactant.

Example 4
(MEL self-assembly ability)
In Example 2, the ability of the purified components (GL-A, GL-B, GL-C) to form self-assemblies was examined. Each component was applied onto a glass plate and covered with a cover glass, water was then allowed to enter between the glass plate and the cover glass, and the boundary between each component and water was observed with an optical microscope and a polarizing microscope. The results are shown in FIG. As a comparison object, the result of the conventional MEL-A is also shown. According to FIG. 7, the liquid crystal formation pattern of the conventional MEL-A and the liquid crystal formation patterns of GL-A, GL-B, and GL-C are completely different, and the MEL-A produced in the present invention is the conventional one. It can be seen that the self-assembly forming ability is different from that of the type MEL-A.

Claims (5)

  A microorganism belonging to Pseudozyma churashimaensis, which has the ability to produce triacetyl monoacyl-type mannosyl erythritol lipids. The microorganism according to claim 1, which has mycological properties represented by the following (a) and (b).
(A) Morphological properties
(B) Physiological properties
  The microorganism according to claim 1 or 2, which is Pseudoma Churashima Ensis OK-96 (Accession No. FERM P-21896).   A method for producing mannosyl erythritol lipid, comprising culturing the microorganism according to any one of claims 1 to 3 and collecting mannosyl erythritol lipid-A from the obtained culture or isolated cells.   A microorganism according to any one of claims 1 to 3 is cultured in a medium containing saccharides as a carbon source, and triacetyl monoacyl mannosyl erythritol lipid is collected from the obtained culture. A method for producing an acetyl monoacyl mannosyl erythritol lipid.