JP4803434B2 - Highly efficient production method of mannosyl erythritol lipid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for highly efficiently producing a mannosylerythritol lipid (MEL) of a glycolipid of one kind of a biosurfactant by using microorganisms. <P>SOLUTION: Microorganisms having MFL-producing ability, Pseudozyma parantarctica, are cultured in an MEL-producing medium consisting essentially of oils and fats such as a vegetable oil and fat. At the time, the MEL can be efficiently produced by regulating the culture temperature at 33-37&deg;C. <P>COPYRIGHT: (C)2008,JPO&amp;INPIT

Description

  The present invention relates to a production method of mannosyl erythritol lipid capable of greatly improving the production efficiency (production amount, production rate, and yield) of mannosyl erythritol lipid which is a kind of biosurfactant.

  Glycolipids are substances in which 1 to several tens of monosaccharides are bound to lipids, are involved in information transmission between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. In addition, glycolipids are amphiphilic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. It is called a substance. Until the petrochemical industry prospered, surfactants (biosurfactants) derived from biological components such as lecithin and saponin were used. Synthetic surfactants have been developed in recent years due to the development of the petrochemical industry, and their production has dramatically increased, making it an indispensable substance for daily life. As a result, environmental pollution has spread and social problems have arisen. For this reason, it is desired to develop a highly biodegradable surfactant that is highly safe and can reduce the burden on the environment.

Conventionally, the surface active substances produced by microorganisms are classified into five types: surface active substances of glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer type. Among these, glycolipid-based surfactants are the most studied, and many types of surfactants from bacteria and yeast have been reported.
Examples of the bacteria, (see Non-Patent Document 3) rhamnolipid (Non-Patent Documents 1 and 2 refer) and Yusuchirajin acid by Pseudomonas spp, such as trehalose lipid (see Non-Patent Document 4) is known by the genus Rhodococcus. However, in all cases, the production amount is 15 g / L or less.
As said yeast, the sophorose lipid by Candida genus, the mannosyl erythritol lipid (refer patent document 1), etc. are known.
As for the sophorose lipid, it has been reported that it is possible to efficiently produce 180 g / L of sophorose lipid in 200 hours by the fed-batch culture method of glucose and oleic acid using Candida bombicola (non-patent document). Reference 5).

Regarding the mannosyl erythritol lipid (MEL), 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 6 and 7). Also, using Candida antarctica T-34 strain, it is possible to produce 38 g / L (production rate: 0.2 g / L / h, raw material yield: 48 mass%) from 8 mass% soybean oil in 8 days. (See Non-Patent Documents 8 and 9). Similarly, 110 g / L (production rate: 0.2 g / L / h, raw material yield) from 25% by mass of peanut oil after 24 days by Caddida antarctica T-34 strain using a total of 3 sequential feedings at 6-day intervals. It has been reported that production of MEL at a rate of 44 mass% is possible (see Non-Patent Document 10).

Candida sp. 50g / L (production rate: 0.25g / L / h, raw material yield: 50% by mass) can be produced in 200 hours by batch culture method from 10% by weight of vegetable oil using SY-16 strain In addition, 120 g / L (production rate: 0.6 g / L / h, raw material yield: 50 mass%) of MEL can be produced from 20 mass% of vegetable oil in 200 hours by fed-batch culture method. Has been reported (see Non-Patent Document 11).
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 in 24 hours by fed-batch culture method Has been reported to be possible (see Non-Patent Document 12).
Moreover, 17 g / L (production rate: 0.1 g / L) from 8% by weight soy sauce oil in 7 days using soy sauce oil (oil) produced as a by-product in the soy sauce brewing process using Candida antarctica T-34 strain / H, raw material yield: 21% by mass) has been proposed (see Patent Document 2).

JP 2002-45195 A JP 2002-101847 A S. Itoh, H.C. Honda, Ftonami and T. Suzuki: J. et al. Antibiotics, 23, 885 (1971). M.M. Yamaguchi, A .; Sato and R.M. Yukuyama: Chem. Ind. 17, 741 (1976). S. S. Bhattacharijee, R.A. H. Haskins and P.M. A. Golin: Carbohyd. Res. 13, 235 (1970). P. Rapp, H.M. Boch, V.D. Wary and F.M. Wagner: J.M. Gen. Microbiol. 115, 491 (1979). U. Rau, C.I. Manzke and F.M. Wagner: Biotechnol. Lett. , 18, 149 (1996). 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.M. 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.M. Yanagishita, T .; Nakane and T.K. Nakahara: Biotechnol. Lett. , 14, 305 (1992). Kim, Ito Univ., Toru Katsuragi, Yoshiki Tani: Abstracts of 1998 Annual Meeting of the Japanese Society for Biotechnology, p195. U. Rau, L. A. Naguyen, H.H. Roeper, H. Koch and S. Lang: Appl. Microbiol. Biotechnol. , (2005).

  On the other hand, in order to widely disseminate biosurfactants such as mannosyl erythritol lipid, which is said to have high biodegradability, low toxicity, environmental friendliness and novel physiological functions, in the food industry, pharmaceutical industry, chemical industry, etc. It is necessary to increase the production efficiency of mannosyl erythritol lipid and to reduce the production cost. 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 provides a method capable of efficiently producing mannosyl erythritol lipid (MEL) by using a microorganism having the ability to produce mannosyl erythritol lipid and optimizing the medium composition and culture conditions. For the purpose.

  As a result of intensive studies by the present inventors in order to solve the above problems, a microorganism having an ability to produce mannosyl erythritol lipid is cultured in a medium containing oils and fats such as vegetable oils as a main component, so that mannosyl erythritol lipids are obtained. In production, the inventors found that mannosyl erythritol lipid can be produced with high production efficiency by setting the culture temperature to 33 to 37 ° C., and the present invention has been completed.

That is, the present invention is as follows.
(1) When a mannosyl erythritol lipid is produced by culturing a microorganism capable of producing a mannosyl erythritol lipid in an oil-containing medium, the culture temperature is set to 33 to 37 ° C. Production method.

(2) The production method according to (1) above, wherein the microorganism having the ability to produce the mannosyl erythritol lipid is a microorganism belonging to the genus Pseudozyma .

(3) Pseudozyma (Pseudozyma) microorganism belonging to the genus characterized in that it is a microorganism belonging to Pseudozyma para Anta click Tikka (Pseudozyma parantarctica), method of production according to (2).

(4) The production method according to any one of (1) to (3) above, wherein the culture is performed in a medium having an initial oil and fat concentration of 2 to 30% by weight.

(5) Culturing is started in a medium having an initial oil and fat concentration of 2 to 30% by mass, and fats and / or other nutrient sources are supplied into the medium from 5 to 7 days after the start of the culture. The production method according to any one of (1) to (3) above.

(6) The production method according to any one of (1) to (5) above, wherein ammonium nitrate is used as a nitrogen source of the medium.

(7) The production method according to any one of (1) to (5) above, wherein the medium composition is as shown below.
Yeast extract: 0.1-2 g / L
Sodium nitrate: 0.1-1 g / L
Potassium dihydrogen phosphate: 0.1-2 g / L
Magnesium sulfate: 0.1-1 g / L
Vegetable oil and fat: 20-300 g / L

According to the present invention, when culturing a microorganism having the ability to produce mannosyl erythritol lipid in a fat-containing medium such as vegetable oil, the culture temperature is set to 33 to 37 ° C., particularly 35 ° C., so that mannosyl erythritol lipid is obtained. Can be produced very efficiently. In particular, when Pseudozyma parantarctica JCM 11752, which is a microorganism belonging to the genus Pseudozyma, is used, the production rate is increased about twice.

  The present invention makes it possible to reduce temperature control energy when producing mannosyl erythritol lipids. That is, during the production of mannosyl erythritol lipid, the fermentation temperature is generated, and thus the culture temperature must be controlled. In contrast, the culture temperature in the previous production method is 30 ° C. or lower, whereas in the present invention, the culture temperature is 35 ° C. It can be set as described above, and the cooling cost (energy) of 5 ° C. or more can be reduced.

  As production efficiency improves, simplification of culture means for obtaining high productivity can be expected. That is, in the previous production method, monitoring the culture state and adding nutrient sources during the course of several weeks were required in order to obtain mannosylerythritol lipids of 100 g / L or more. On the other hand, according to the present invention, mannosyl erythritol lipids of 100 g / L or more can be obtained only by shaking culture for 1 week, and special equipment, monitoring of the culture state, and intermediate addition of nutrient sources are unnecessary. Moreover, in order to obtain a higher production volume, if the culture state is monitored and nutrient sources are added during the course of several weeks, the production volume that naturally surpasses the previous production method can be achieved.

Furthermore, in this invention, since the consumption rate of fats and oils, such as vegetable oil, also improves, as a result, mixing in the oil component of these origin is reduced, and the effect can be expected also in the separation and purification of mannosyl erythritol lipid.
Therefore, the present invention greatly contributes to the development of biosurfactant production technology expected to be used for various uses such as medicine.

(Target product)
Mannosyl erythritol lipid (MEL), which is a target product 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 14 carbon atoms, preferably 3 to 12 carbon atoms. Represents a saturated fatty acid residue.
The mannosyl erythritol lipid (MEL) has a high surface activity and is used as various surfactants or fine chemical catalysts. When mannosyl erythritol lipid is allowed to act on human acute promyelocytic leukemia cell line HL60, it induces leukemia cell differentiation that differentiates the granule system, and mannosyl erythritol lipid acts on PC12 cells derived from rat adrenal medullary pheochromocytoma It has a physiological activity 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 (X. Zhao et. Al., Cancer Research, 59 , 482-486 (1999)) 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.

(Used microorganism)
The microorganism used in the present invention is not particularly limited as long as it has the ability to produce mannosyl erythritol lipids, and examples thereof include microorganisms belonging to the genus Pseudozyma. Among them, particularly preferable microorganisms include Pseudozyma paraantactica. Can be mentioned. The microorganism was not known as a mannosyl erythritol-producing microorganism, but was first confirmed by the present inventors to have productivity equivalent to that of known mannosyl erythritol lipid-producing bacteria such as pseudozyma and antactica. It is. Moreover, the microorganism belonging to Pseudozyma parantarctica has a particularly high productivity improvement effect when cultured at 33 to 37 ° C., for example, Pseudozyma parantarctica ( Pseudozyma parantarctica) JCM 11752 In the case of the strain, it is noteworthy in that it reaches about twice when the culture temperature is 35 ° C.

(Mannosyl erythritol lipid production)
In culturing the microorganisms used in the present invention, the medium contains fatty acid esters such as fatty acids and fatty acid triglycerides, or fats and oils such as vegetable oils. Conditions other than temperature conditions are not particularly limited and are appropriately selected. be able to. For example, a medium generally used for yeast can be used, and examples of such a medium include YPD medium (yeast extract 10 g, polypeptone 20 g, and glucose 100 g).
In order to increase the production amount of MEL, it is preferable to set the culture temperature to 33 to 37 ° C., and as a result of culturing by changing the culture temperature at the start of the culture, the present inventors have set the culture temperature to 35 It has been found that particularly good MEL production rates, yields and yields can be obtained when set to ° C.

Microorganisms use of the present invention, the preferred medium composition of especially when producing mannosylerythritol lipid using the Pseudozyma para Anta click Tikka (Pseudozyma parantarctica JCM 11752) strain is as follows.
The yeast extract is preferably 0.1 to 2 g / L, particularly preferably 1 g / L. Sodium nitrate is preferably 0.1 to 1 g / L, particularly preferably 0.5 g / L.
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.
As for vegetable oil and fat, 80 g / L or more is preferable and 180 g / L is especially preferable.

The method for producing the mannosyl erythritol lipid of the present invention is not particularly limited and may be appropriately selected according to the purpose. For example, the seed culture, the main culture, and the mannosyl erythritol lipid production culture may be scaled up in this order. desirable.
Examples of culture media and culture conditions in these cultures are as follows.
a) Seed culture: 20 g / L of glucose, 1 g / L of yeast extract, 1 g / L of sodium nitrate, 0.5 g / L of potassium dihydrogen phosphate, and 4 mL of a liquid medium having a composition of 0.5 g / L of magnesium sulfate A platinum loop is inoculated into a test tube and cultured with shaking at 30 ° C. for 1 day.

b) Main culture: a predetermined amount of fats and oils such as vegetable oil and fat, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L A Sakaguchi flask containing 100 mL of the liquid medium having the composition is inoculated and cultured at 33 to 37 ° C. for 2 days.
c) Mannosyl erythritol lipid production culture; predetermined amount of oils and fats such as vegetable oil and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L A jar fermenter containing 1.4 L of a liquid medium having the following composition is inoculated and cultured at 33-37 ° C. at a stirring speed of 800 rpm. In this culturing, it is desirable that the vegetable fats and oils flow down into the culture vessel from the middle of the culturing to maintain the fats and oils concentration in the medium at 40 to 200 g / L.

Hereinafter, production of mannosyl erythritol lipid in the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.
In the following Examples, the medium for main culture of b) above was used as a medium for production test of mannosyl erythritol lipid.

( Pseudozyma parantarctica JCM 11752 strain culture)
a) Pseudozyma parantarctica JCM 11752 stored in a storage medium (malt extract 3 g / L, yeast extract 3 g / L, peptone 5 g / L glucose 10 g / L, agar 30 g / L), glucose 20 g / L, yeast 1 platinum ear was inoculated into a test tube containing 1 mL of extract, 1 g / L of sodium nitrate, 0.5 g / L of potassium dihydrogen phosphate and 0.5 g / L of magnesium sulfate. Shake culture at 0 ° C, then
b) Composition of the obtained cell culture broth with a predetermined amount of vegetable oil and fat, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L Was inoculated into a Sakaguchi flask containing 20 mL of the liquid medium and cultured at 35 ° C. with shaking.
c) A liquid medium having a predetermined amount of soybean oil and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L in a liquid medium 1.4 L The jar fermenter was inoculated and cultured at 35 ° C. with a stirring speed of 800 rpm. Further, vegetable oil (soybean oil) was allowed to flow down into the culture container every week from the middle of the culture.

Tests shown in the following (1) to (7) were performed using the cell culture broth obtained by each of the above cultures a) to c).
(Test method, results)
(1) Confirmation of mannosylerythritol lipid (MEL) production ability of Pseudozyma parantarctica JCM 11752 strain
After culturing a) for 1 day, the culture solution after b) culturing for 7 days in a soybean oil-added medium was collected and used to reduce the productivity of Pseudozyma parantarctica JCM 11752 biosurfactant. Confirmed by layer chromatography. As a standard for mannosyl erythritol lipid, Pseudozyma antarctica JCM 10317 strain was cultured under the same conditions as above, and a purified preparation from which impurities such as raw oils and fats were removed was used. In the mannosyl erythritol lipid standard, MEL-A, MEL-B and MEL-C are respectively in the general formulas (R1, 2 = fatty acid residue having 1 to 14 carbon atoms, R3, 4 = acetyl group) and (R1 2 = fatty acid residue having 1 to 14 carbon atoms, R3 = hydrogen atom, R4 = acetyl group) and the same (R1,2 = fatty acid residue having 1 to 14 carbon atoms, R3 = acetyl group, R4 = hydrogen) Atom) is shown.
The results are shown in FIG. According to this, both strains produce mannosyl erythritol lipid (MEL) in a soybean oil-containing medium. In contrast to the standard of mannosyl erythritol lipid, it can be seen that the biosurfactant produced by Pseudozyma parantarctica JCM 11752 is mannosyl erythritol lipid.

(2) Mannosyl erythritol lipid (MEL) production in the same medium
Pseudozyma parantarctica JCM 11752 was used, and a) was cultured for 1 day, and then b) was cultured in a soybean oil-added medium for 7 days. The culture solution was collected and purified, and the produced MEL was detected by high performance liquid chromatography. As a comparative example, Pseudozyma antarctica JCM 10317 strain was cultured under the same conditions as described above and detected by high performance liquid chromatography in the same manner. The results are shown in FIG. In addition, FIG. 2 is the result of having detected the ethyl acetate soluble part in a culture solution, and having detected it by the high performance liquid chromatography, and corresponds with the thing of the known mannosyl erythritol lipid. According to FIG. 2, the Pseudozyma parantarctica JCM 11752 strain has a MEL production capacity similar to the Pseudozyma antarctica JCM 10317 strain.

(3) Structural analysis of mannosylerythritol lipid (MEL) produced by Pseudozyma parantarctica JCM 11752
Pseudozyma parantarctica JCM 11752 was used, and a) was cultured for 1 day, and then b) was cultured in a soybean oil-added medium for 7 days. The culture solution was collected, and MEL in the culture solution was extracted with ethyl acetate. After purification, the structure of the product was analyzed by ¹ H NMR (upper). In addition, as an example of a typical structural analysis result of MEL, an analysis result of MEL produced by Pseudozyma antarctica JCM 10317 strain is shown in the lower part. According to FIG. 3, it is clear that the substance produced by Pseudozyma parantarctica JCM 11752 has a typical MEL structure.

(4) Effect of nitrogen source on MEL production of Pseudozyma parantarctica JCM 11752
Using Pseudozyma parantarctica JCM 11752, a) was cultured for 1 day, b) was cultured in a soybean oil-added medium for 7 days, the culture was collected, and the amount of MEL produced was measured by high performance liquid chromatography. Detected with. At this time, the nitrogen sources in the culture solution were each prepared to have the composition described in the figure. The results are shown in FIG. According to this result, in the MEL production by Pseudozyma parantarctica JCM 11752, the most suitable nitrogen source is sodium nitrate.

(5) Effects of vegetable oils and fats on MEL production of Pseudozyma parantarctica JCM 11752
Using Pseudozyma parantarctica JCM 11752 strain, a) was cultured for 1 day, b) was cultured for 7 days, and then the culture broth was collected, and the amount of MEL produced was detected by high performance liquid chromatography. At this time, vegetable oils and fats in the culture solution were prepared to the compositions described in the figure. The results are shown in FIG. According to this result, Pseudozyma parantarctica JCM 11752 produced MEL from various vegetable oils, and soybean oil showed the best result.

(6) Effect of vegetable oil concentration on MEL production of Pseudozyma parantarctica JCM 11752
Using Pseudozyma parantarctica JCM 11752 strain, a) was cultured for 1 day, b) was cultured for 7 days, and then the culture broth was collected, and the amount of MEL produced was detected by high performance liquid chromatography. At this time, soybean oil in the amount shown in the figure was added to the culture solution. The results are shown in FIG. According to this result, the production amount of MEL by Pseudozyma parantarctica JCM 11752 strain is about 50 g / L.

(7) Effect of culture temperature on MEL production of Pseudozyma parantarctica JCM 11752
Using Pseudozyma parantarctica JCM 11752, a) was cultured for 1 day, b) was cultured in a soybean oil-added medium for 7 days, the culture was collected, and the amount of MEL produced was measured by high performance liquid chromatography. Detected with. At this time, the culture temperature was set as shown in FIG. The results are shown in FIG. According to this result, the optimum production temperature of Pseudozyma parantarctica JCM 11752 is 35 ° C, and the production of MEL is more than twice as high as that at 30 ° C.

(8) MEL production culture of Pseudozyma parantarctica JCM 11752 The above-mentioned a) was cultured for 1 day, then b) was cultured at 35 ° C. for 10 days, followed by c). C) was cultured at 35 ° C. using a medium containing 160 g / L soybean oil. From the middle of the culture, 160 g / L of vegetable oil was allowed to flow down into the culture container every week. FIG. 8 is a graph prepared by collecting a culture solution every week and quantifying the MEL production amount by high performance liquid chromatography. The arrow indicates the time when the carbon source is added. According to FIG. 8, the amount of MEL produced in 14 days reaches 190 g / L or more.

It is a thin layer chromatographic photograph about this culture | cultivation which shows that Pseudozyma parantarctica JCM11752 strain | stump | stock can produce mannosyl erythritol lipid in a soybean oil containing medium. It is a figure which shows the analysis result of the high performance liquid chromatography about this culture | cultivation which shows that Pseudozyma parantarctica JCM11752 strain | stump | stock can produce mannosyl erythritol lipid in a soybean oil containing medium. It is a figure which shows the result of the structural analysis by 1H NMR about this culture | cultivation which shows that Pseudozyma parantarctica JCM11752 strain | stump | stock can produce mannosyl erythritol lipid in a soybean oil containing medium. It is a graph which shows the influence of a nitrogen source with respect to the production of mannosyl erythritol lipid by Pseudozyma parantarctica JCM11752 strain. It is a graph which shows the influence of vegetable oil and fats on the production of mannosyl erythritol lipid by Pseudozyma parantarctica JCM 11752 strain. It is a graph which shows the influence of vegetable oil and fat concentration on the production of mannosyl erythritol lipid by Pseudozyma parantarctica JCM 11752 strain. It is a graph which shows the influence of culture | cultivation temperature with respect to the production of mannosyl erythritol lipid by Pseudozyma parantarctica JCM11752 strain | stump | stock. It is a graph which shows the result of the mannosyl erythritol lipid production culture of Pseudozyma parantarctica JCM11752 strain | stump | stock.

Claims (3)

Upon possess the ability to produce the mannosylerythritol lipid in fats and oils containing medium, to produce mannosylerythritol lipid by culturing a microorganism belonging to the Pseudozyma para Anta click Atlantica (Pseudozyma parantarctica), set the culture temperature to 33~37 ℃ A method for producing mannosyl erythritol lipids, characterized in that: Initial fats and oils concentration and performing culture in a medium which is 2 to 30% by weight, method of production according to claim 1. The culture starts with a medium having an initial oil and fat concentration of 2 to 30% by mass, and oils and / or other nutrients are supplied into the medium from 5 to 7 days after the start of the culture. The production method according to claim 1 or 2 .

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