JP5565796B2 - Novel microorganism and method for producing sugar-type biosurfactant using the same - Google Patents

Description

  The present invention provides a newly discovered microorganism having a high ability to produce a sugar-type biosurfactant in order to efficiently produce a sugar-type biosurfactant (a microorganism-derived amphiphilic lipid) that can be used as a skin care agent or the like. More specifically, the present invention relates to a production method that provides a microorganism that produces a large amount of mannosyl erythritol lipid, which is a kind of sugar-type biosurfactant, and enables highly efficient production of mannosyl erythritol lipid.

  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, the production amount is low because it is bound to the cell wall, but it has been reported that succinoyl trehalose lipid is produced at a rate of 32 g / L when culturing Rhodococcus erythropolis under nitrogen restriction. (Refer nonpatent literature 4).

Sophorose lipids (also referred to as “sophorose lipids”; 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 physiological activity such as neural cell 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.
The general formula 1, R _{1 to} R _{4, which} may be the same or different from each other, are a hydrogen atom, an acetyl group, or a saturated or unsaturated fatty acid residue having 1 to 14 carbon atoms, preferably 3 to 12 carbon atoms. Represents a group.
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 is hydrogen. A structure in which MEL-C, 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). Production of 13.9 g / L (production rate: 0.57 g / L / h, raw material yield: 92% by mass) of MEL from 80% by mass of vegetable oil using Pseudozyma aphidis strain by fed-batch culture for 24 hours Is reported to be possible (see Non-Patent Document 15).

By the way, among MEL-producing bacteria, Pseudozyma tsukubaensis is known to produce only MEL-B in which the configuration of erythritol is reversed and the sugar skeleton is an isomer. That is, this isomer MEL-B uses 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton, and 4-O-β-D-mannopyranosyl-erythritol produced by the above-mentioned Candida antarctica strain T-34 and the like. The sugar skeleton is different from MEL-B having a sugar skeleton. In addition, it is known that there is a difference between the physicochemical properties of the two, and the isomer MEL-B has improved hydration, high vesicle formation ability, and a promising biomaterial as a skin care agent and the like. Expected (see Non-Patent Document 16).

  However, there is no report on mass production of isomer MEL-B, and mass production by obtaining mass-producing bacteria of isomer MEL-B and examining production conditions is required.

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). "Carbohydrate Research (Carbohydr. Res.)," 343, p555-560 (2008).

Biosurfactants such as mannosyl erythritol lipids are superior in environmental compatibility and biocompatibility compared to general-purpose surfactants, and various physiological activities are recognized, but they are used in the same wide range as existing surfactants. For example, there are restrictions on the use in food applications and the like. 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.
On the other hand, mannosylerythritol lipid-B having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton (hereinafter sometimes referred to as isomer MEL-B) is 4-O-β-D-mannopyranosyl- Compared with MEL-B having erythritol as a sugar skeleton, hydration is improved, vesicle-forming ability is high, and further wide application development is expected, and higher productivity is required.

  Therefore, the object of the present invention is to provide a new microorganism which is collected from a safe isolation source and excellent in the productivity of isomer MEL-B, and mass-produces isomer MEL-B using the microorganism. Through this, the application of isomer MEL-B is expanded.

  In order to solve the above-mentioned problems, the present inventor has eagerly searched for an isomer MEL-B-producing bacterium. ) Was isolated, and it was found that the microorganism was excellent in the productivity of the isomer MEL-B, which led to the present invention.

That is, the microorganism of the present invention is shown below.
[1] Pseudozyma nsp. Which has the ability to produce mannosylerythritol lipid-B having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton and has the ability to utilize ribitol and mannitol. 1D9 (Accession number FERM P-21901) or Pseudozyma tsukubaensis 1E5 (Accession number FERM P-21902) .
[2] The pseudozyma nsp. Described in the above [1] having the mycological properties represented by the following (a) and (b): 1D9 (Accession number FERM P-21901) .
(A) Morphological properties
(B) Physiological properties
[3] Pseudozyma tsukubaensis 1E5 (Accession No. FERM P-21902) according to the above [1] having the mycological properties shown in the following (a) and (b ) .
(A) Morphological properties
(B) Physiological properties
[4] The microorganism according to any one of [1] to [3] above is cultured in a medium, and mannosylerythritol lipid having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton is obtained from the obtained culture. A method for producing mannosyl erythritol lipid-B having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton, wherein B is collected.

According to the present invention, a microorganism using perilla leaves as an isolation source, more specifically, pseudozyma nsp. (Pseudozyma nsp.) 1D9 strain and Pseudozyma tsukubaensis 1E5 strain can be provided. If the microorganism is used as a production microorganism, the safety of the obtained isomer MEL-B can be ensured and the production efficiency of the isomer MEL-B can be further improved, and the production cost can be reduced. Product costs can also be reduced. Therefore, the use as a surfactant that can be directly used for food use, pharmaceuticals, livestock feed, fish feed, agricultural use, where the use of isomer MEL-B has been limited so far in terms of safety and supply, It is expected to contribute significantly to the spread of biosurfactants.
As described above, by using the present invention, isomer MEL-B can be produced with high efficiency, MEL production cost can be reduced, and lower-priced MEL can be provided to the market. Become.

In Example 1, it is a figure which shows the result of TLC which detected the isomer MEL-B which 1D9 stock | strain and 1E5 stock | strain produced. In Example 1, it is a figure which shows the result of having investigated the production amount of isomer MEL-B which 1D9 stock | strain and 1E5 stock | strain produced. In Example 3, it is a figure which shows the result of having investigated the conditions with respect to production of isomer MEL-B by 1E5 stock | strain. In Example 4, it is a figure which shows the production result of isomer MEL-B by 1E5 stock | strain using a jar fermenter.

Hereinafter, the present invention will be described in more detail.
<Microorganism of the present invention>
The microorganism of the present invention is a microorganism suitable for production of isomer MEL-B, and examples of applicable microorganisms include pseudozyma nsp. (Pseudozyma nsp.) 1D9 strain and Pseudozyma tsukubaensis 1E5 strain. All of these strains are strains isolated from leaves of plants (Perilla) 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 5 days, and forms a full pink, flat, smooth, dull, wet, and pale pink colony. On YM plate medium at 25 ° C., on day 3 of culture, the cells are oval and grow by extreme budding. 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, and these strains can be said to belong to the genus Pseudozyma.

For the 1D9 and 1E5 strains, the base sequence (rDNA sequence) of the 26S rDNA-D1 / D2 region of the ribosomal RNA gene was determined, the DNA database (DDBJ) was accessed, and the FASTA program (http: //www.ddbj.nig The homology search using .ac.jp / search / fasta-j.html) was consistent with the rDNA sequence of Pseudozyma tsukubaensis (100%). Moreover, the base sequence of the ITS1-5.8 SrDNA-ITS2 region of the 1E5 strain showed high homology with the rDNA sequence of Pseudozyma tsukubaensis (over 99%). Combined with the results of these homology searches and the results of the physiological property test of the 1E5 strain, this strain is considered to be a strain belonging to Pseudozyma tsukubaensis .

However, the 1E5 strain is compared to the conventionally known Pseudozyma tsukubaensis isomer MEL-B producing strain Pseudozyma tsukubaensis ( Pseudozyma tsukubaensis ) BRC1940 (= C isomer). If the production yield is extremely high and the physiological properties are also closely examined, Pseudozyma tsukubaensis CBS6389 does not show L-sorbose utilization and cannot grow in the presence of 50% glucose (osmotic stress). The strains show L-sorbose assimilation and are different in that they can grow in the presence of 50% glucose (osmotic stress).

On the other hand, the nucleotide sequence (SEQ ID NO: 1) of the ITS1-5.8 SrDNA-ITS2 region of 1D9 strain showed the highest homology with the rDNA sequence of Pseudozyma tsukubaensis , but it was 98.1%. It showed no more than 99% homology. In addition, as a result of the physiological property test of the 1D9 strain, the growth temperature of Pseudozyma tsukubaensis CBS6389 is 30 ° C or less, whereas the heat resistance of the 1D9 strain is very high and can grow at 37 ° C. Is clearly different.

The 1E5 and 1D9 strains are common in that they have assimilation with ribitol and mannitol, whereas Pseudozyma tsukubaensis CBS6389, which is an isomer MEL-B producing bacterium, has assimilation with ribitol and mannitol. I don't have it.
From these points, the 1E5 strain is considered to be a new strain belonging to Pseudozyma tsukubaensis , and the 1D9 strain may be a new species of the genus Pseudozyma, a kind of basidiomycete.

  The results of the morphological observation and the physiological property test are as shown in Tables 1 and 2 (1D9 strain) and Tables 3 and 4 (1E5 strain). As of February 3, 2010, 1D9 and 1E5 shares will be registered with the National Institute of Advanced Industrial Science and Technology Patent Microbiology Depositary Center (IPOD) (1-1-3 Higashi 1-1-3, Tsukuba City, Ibaraki Prefecture) with the accession number FERM P-21901 ( 1D9 strain) and FERM P-21902 (1E5 strain).

(Production biosurfactant)
The biosurfactant produced by the microorganism of the present invention is MEL-B having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton. As MEL-B, R ₃ is hydrogen in the following general formula 2. , R ₄ is an acetyl group, and R ₁ and R ₂ are saturated or unsaturated fatty acids having 1 to 16 carbon atoms, preferably 4 to 14 carbon atoms.

<Medium and culture conditions>
The culture form 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 particular, the above-mentioned pseudozyma nsp. 1D9 strain of the present invention has a wide growth temperature range, and grows even at 30-37 ° C., which exceeds the upper limit temperature of growth of the conventional Pseudozyma tsukubaensis isomer MEL-B. MEL-B can be produced, temperature control during the production of isomer MEL-B is easy, and this also contributes to the improvement of production efficiency.

In the present invention, a general semi-synthetic medium may be used as the medium, and it is desirable to use sugars such as glucose, sucrose, and molasses as a main raw material, or fats and oils 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 the yeast can assimilate. For example, monosaccharides such as glucose, galactose and fructose, and disaccharides such as sucrose and maltose are used, and glucose is preferred. 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. 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, Petrothelic 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 10-100 g / L, preferably 20-60 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))
The isomer MEL-B accumulated in the culture broth by culturing the isomer MEL-B producing bacterium of the present invention can be separated from the culture broth by extracting the culture broth after culturing this 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.
Mannosyl erythritol lipid extracted with an organic solvent is only the isomer MEL-B, and no special means is required to isolate the isomer MEL-B. When fatty acid components derived from raw materials are mixed, means such as silica gel column chromatography is used for further purification.

<Determining the structure of MEL>
The structure of the glycolipid component obtained as described above is determined as follows. The structure determination method of MEL obtained by culturing Pseudozyma tsukubaensis 1E5 strain using soybean oil as a carbon source will be described below 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-NMR analysis, and the obtained spectrum is compared with the data of the isomer MEL-B having a known structure (see Non-Patent Document 16) 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 sugar-type biosurfactant by 1D9 and 1E5 strains)
<Seed culture>
30 mL of YM medium (glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L) of 1D9 and 1E5 strains stored frozen in a deep freezer (−80 ° C.) 0.5 mL was seeded and shake-cultured at 28 ° C. for 1 day.
<Main culture>
A 30 mL MEL production medium was prepared in a 300 mL Erlenmeyer flask as shown in Table 5 (MEL production medium composition), and a medium sterilized by autoclaving at 121 ° C. for 20 minutes was prepared. 1.5 mL of the above seed culture solution was seeded, 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 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, 1D9 strain and 1E5 strain produced a large amount of glycolipids having the same mobility as MEL-B used for the standard. That is, the mobility of glycolipid of 1D9 strain was 0.51, the mobility of glycolipid of 1E5 strain was 0.51, and the mobility of MEL-B used for the standard was also 0.51. In addition, Pseudozyma tsukubaensis NBRC1940 strain was used as a control strain. Furthermore, these structures were found to be isomer MEL-B from the results of structural analysis described in Example 2 below. The results of measuring the production amounts of isomer MEL-B when each carbon source is used are shown in FIG.

Example 2
Structural analysis of glycolipid produced by 1E5 strain (Glycolipid (MEL) purification)
From the culture medium cultured in the same manner as in Example 1, the glycolipid was extracted and collected using ethyl acetate, and the glycolipid was purified using a silica gel column. The following structural analysis was performed using the purified glycolipid component.
(NMR analysis of glycolipid components)
The structure of the glycolipid 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 the 1E5 strain were MEL-B having an acetyl group at the 6-position of mannose, and an isomer MEL-B having a coordination characteristic of erythritol. ^The chemical shifts assigned in ¹ H-NMR analysis are shown in Table 6. In addition, isomer MEL-B by Pseudozyma tsukubaensis NBRC1940 strain was described as a comparison object.

(MALDI-TOF / MS analysis of glycolipid components)
As a result of analyzing the molecular weight of the isomer MEL-B obtained above by MALDI-TOF / MS, the molecular weight of the isomer MEL-B was 634.

Example 3
(Examination of production conditions of isomer MEL-B by 1E5 strain: medium components)
The optimum vegetable oil (A), vegetable oil concentration (B), organic nutrient source (C), and yeast extract concentration (D) in the production of isomer MEL-B by the 1E5 strain were examined. The results are shown in FIG. The 1E5 strain was cultured in the same manner as in Example 1. All of the vegetable oils used were good carbon sources. The optimum vegetable oil (olive oil) concentration was 120 g / L. The optimal organic nutrient source was yeast extract, and the optimal concentration was 5.0 g / L.

Example 4
(Examination of production conditions of isomer MEL-B by 1E5 strain: jar fermenter culture)
A medium for MEL production of 3000 mL was prepared in a 5000 mL jar fermenter as shown in Table 5 (MEL production medium composition), and a medium sterilized by autoclaving at 121 ° C. for 20 minutes was prepared. The culture solution after culturing the 1E5 strain in the same manner as in Example 1 was transferred to this jar fermenter under aseptic conditions. Then, it culture | cultivated continuously on the conditions of 25 degreeC and 700 rpm. 55 hours after the start of culturing when the first olive oil was consumed, 100 g / L of olive oil was additionally added (flowing down), and the culturing was continued until 168 hours. FIG. 4 shows the isomer MEL-B, the amount of residual oil and fat, and the dry cell weight at this time. As a result, 1E5 strain produced and accumulated 70 g / L or more of isomer MEL-B in the culture solution in 168 hours.

Claims (4)

Pseudozyma nsp. Which has the ability to produce mannosylerythritol lipid-B having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton and has the ability to utilize ribitol and mannitol. 1D9 (Accession number FERM P-21901) or Pseudozyma tsukubaensis 1E5 (Accession number FERM P-21902) . The pseudozyma nsp. Of claim 1 having the mycological properties shown in the following (a) and (b). 1D9 (Accession number FERM P-21901) .
(A) Morphological properties
(B) Physiological properties
The pseudozyma tsukubaensis 1E5 (accession number FERM P-21902) of Claim 1 which has the mycological property shown by the following (a) and (b ) .
(A) Morphological properties
(B) Physiological properties
Culturing the microorganism according to any one of claims 1 to 3 in a medium, and collecting mannosylerythritol lipid-B having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton from the obtained culture. A process for producing mannosylerythritol lipid-B having 1-O-β-D-mannopyranosyl-erythritol as a sugar skeleton.