JP2008187902A - Method for producing deacylated product of glycolipid type biosurfactant - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a biosurfactant, such as glycolipid, having high biodegradability and low toxicity and which is environmentally friendly and has improved water solubility in order to attempt wide spreadof the biosurfactant in the food industry, cosmetic industry, medicalindustry, chemical industry, and environmental field, or the like. <P>SOLUTION: The method for producing a deacylated product of a glycolipid type biosurfactant comprises regioselectively cleaving the acyl group of a glycolipid type biosurfactant by using a hydrolase. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing a deacylated form of a glycolipid biosurfactant. More specifically, the present invention relates to a method for producing a deacylated form of a glycolipid type biosurfactant produced by a microorganism.

  Biosurfactants such as glycolipids are said to have high biodegradability, low toxicity, environmental friendliness, and novel physiological functions. Therefore, it is expected to be widely spread in the food industry, the cosmetic industry, the pharmaceutical industry, the chemical industry, the environmental field, etc. For that purpose, many types of biosurfactants are required. However, the number of biosurfactants discovered to date is as small as 20 or so.

  Mannosyl erythritol lipid (hereinafter abbreviated as “MEL” where appropriate), which is a type of glycolipid, is a natural surfactant produced by yeast, and various physiological actions have been reported (Non-patent Document 1). Recently, a mannosyl mannitol lipid (hereinafter abbreviated as “MML” as appropriate) in which erythritol replaces mannitol has been found (Patent Document 1). Biosurfactants are useful as anti-inflammatory agents and antiallergic agents (Patent Document 2), hair nourishing / hair-growth agents (Patent Document 3), antibacterial action (Patent Document 4) and surface tension reducing action (Patent Document 2) Document 5) is known.

  There are four types of MELs, MEL-A, MEL-B, MEL-C, and MEL-D, depending on whether or not the 4,6 position of mannose is acetylated. That is, MEL-A is acetylated at both positions 4 and 6 of mannose. MEL-B is acetylated at the 6-position, and MEL-C is acetylated at the 4-position. MEL-D is one in which both the 4th and 6th positions are not acetylated.

  Since MEL-A, MEL-B and MEL-C have an acetyl group, their water solubility is low. Therefore, there is a drawback that it is difficult to use in applications that require water solubility.

  Therefore, for applications that require water solubility, it is desired to use a MEL with many hydroxyl groups such as MEL-D after deacylation.

  Currently, MEL can be produced in large quantities by culturing yeast that can produce MEL using an appropriate medium. For example, a method of culturing yeast (Candida antarctica T-34 strain) using a medium containing soybean oil as a carbon source has been found (Non-patent Document 2).

However, the MEL produced by this method is a MEL containing an acetyl group such as MEL-A, MEL-B or MEL-C. Only a very small amount of MEL-D can be produced by the above-described production method.
JP 2005-104837 A JP 2005-68015 A JP 2003-261424 A JP-A-57-145896 JP-A-61-205450 Journal of Bioscience and Bioengineering, 94,187 (2002) Biotechnology Letter, 23,1709 (2001)

  As described above, a method for mass production of MEL-A, MEL-B, and MEL-C has been found, but deacyl MEL having an increased number of hydroxyl groups such as MEL-D is mass produced. No method has been found.

  The present invention has been made in view of the above circumstances, and biosurfactants such as glycolipids having high degradability, low toxicity and environmental friendliness are widely spread in the food industry, cosmetic industry, pharmaceutical industry, chemical industry, environmental fields, etc. Therefore, an object of the present invention is to provide a method for producing a biosurfactant with improved water solubility. More specifically, it aims at providing the manufacturing method of MEL which hydrolyzed the acyl group of mannosyl erythritol lipid and improved water solubility.

  As a result of intensive studies to solve the above problems, the present inventors have found that a deacylated product can be produced with high efficiency and low cost by cleaving the acyl group of a glycolipid type biosurfactant. It came to complete.

  That is, the present invention provides the following inventions.

  (1) A method for producing a deacylated form of a glycolipid type biosurfactant, wherein the acyl group of the glycolipid type biosurfactant is cleaved regioselectively.

  (2) The method for producing a deacylated form of a glycolipid type biosurfactant according to (1), wherein the glycolipid type biosurfactant is mannosyl erythritol lipid (MEL) or mannosyl mannitol lipid (MML).

  (3) The deacylated form of the glycolipid type biosurfactant is represented by the formula (I)

(Wherein R ₁ and R ₂ each independently represents a hydrocarbon group or an oxygen atom-containing hydrocarbon group), and the glycolipid according to (2), For producing a deacylated form of a type biosurfactant

  (4) The glycolipid-type bio of any one of (1) to (3), wherein the ester bond is cleaved by reacting a glycolipid-type biosurfactant with a hydrolase. A method for producing a deacylated form of a surfactant.

  (5) Deacylate of glycolipid biosurfactant according to (4), wherein the hydrolase is at least one hydrolase selected from the group consisting of lipase, protease and esterase Manufacturing method.

  According to the present invention, a deacylated form of a glycolipid type biosurfactant can be produced with high efficiency and at low cost.

  In particular, the acetyl group added to the 4-position and / or 6-position of MEL can be converted to MEL-D, which is a deacetylated form of MEL that has been deacetylated to improve water solubility.

  Hereinafter, the present invention will be described in detail. In addition, all the nonpatent literatures and patent literatures described in this specification are used as reference in this specification.

  The method for producing a deacylated form of a glycolipid type biosurfactant of the present invention (hereinafter abbreviated as “the production method of the present invention”) can be any one that regioselectively cleaves the acyl group of a glycolipid type biosurfactant. Good.

  “Biosurfactant” is a general term for substances having surface-active ability and emulsifying ability produced by living organisms, and not only exhibits excellent surface activity and high biodegradability, but also has various physiological functions. Therefore, there is a possibility of developing different behaviors and functions from synthetic surfactants.

  Among biosurfactants, a biosurfactant comprising a saccharide and a fatty acid bonded to the saccharide via an ester bond is referred to as a “glycolipid type biosurfactant”. In the present specification, “fatty acid” includes acetic acid.

  In the present specification, “regioselectively cleave the acyl group of a glycolipid-type biosurfactant” means that the ester bond formed between the fatty acid and the sugar is selectively selected depending on the carbon position of the sugar. It means that the acyl group contained in the fatty acid is cleaved by hydrolysis.

  Therefore, the “deacylated form of glycolipid biosurfactant” produced by the production method of the present invention is a glycolipid biosurfactant having a structure in which an acyl group at a specific position of a sugar carbon is eliminated. Note that “cleaving the acyl group of a glycolipid type biosurfactant” and “deacylating a glycolipid type biosurfactant” are synonymous.

  The acyl group is a functional group represented by “RCO— (R represents a hydrocarbon group)”, and examples thereof include an acetyl group.

  Fatty acids can be classified into saturated fatty acids and unsaturated fatty acids.

  Examples of the saturated fatty acid include acetic acid, propionic acid, butyric acid, valeric acid, heptanoic acid, caproic acid, caprylic acid, pelargonic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, palmitic acid, stearic acid And arachidic acid.

  Examples of the unsaturated fatty acid include decenoic acid, dodecenoic acid, tetradecenoic acid, hexadecenoic acid, octadecenoic acid, icosenic acid, decadienoic acid, dodecadienoic acid, hexadecadienoic acid, octadecadienoic acid, linolenic acid, arachidonic acid and the like. Can be mentioned.

  As glycolipid type biosurfactant, mannosyl erythritol lipid (MEL), mannosyl mannitol lipid (MML), sophorolipid, rhamnolipid, trehalose lipid and the like are known.

  In the production method of the present invention, among the glycolipid type biosurfactants, a glycolipid type biosurfactant in which a part of the acyl group is an acetyl group (hereinafter abbreviated as “acetyl group-containing glycolipid type biosurfactant”) It is preferable to use it.

  As the acetyl group-containing glycolipid type biosurfactant, mannosyl erythritol lipid (hereinafter abbreviated as “MEL”) and mannosyl mannitol lipid (hereinafter abbreviated as “MML”) are known, but not limited thereto.

  As a specific structure of the deacylated form of the glycolipid type biosurfactant produced by the production method of the present invention, for example, the following formula (I)

(Wherein, R ₁ and R ₂ each independently represents a hydrocarbon group or an oxygen atom-containing hydrocarbon group). In said formula (I), the hydrocarbon group of R < ₁ >, R < ₂ > may have only a saturated bond and may have an unsaturated bond. When it has an unsaturated bond, it may have a plurality of double bonds. The carbon chain may be linear or branched. In the case of an oxygen atom-containing hydrocarbon group, the number and position of oxygen atoms contained are not limited.

In formula (I), R ₁ and R ₂ preferably have 6 to 20 carbon atoms. R ₁ and R ₂ form an ester bond with the hydroxyl groups at the 2nd and 3rd positions of mannose as an aliphatic acyl group (RCO-).

  Hereinafter, as an example of the production method of the present invention, a method for producing a deacylated product by regioselectively cleaving the acyl group of MEL will be described. However, the production method of the present invention is not limited to MEL, and an acetyl group-containing glycolipid biosurfactant such as MML can also be used. This is because the structure of MML is the same as that of MEL except that it has a sugar alcohol having 2 more carbon atoms than erythritol. Therefore, those skilled in the art readily understand that if deacylation easily proceeds in MEL, deacylation also proceeds easily in MML.

Formula (II) shows the structure of MEL. In formula (II), R ₁ and R ₂ represent a hydrocarbon group or an oxygen atom-containing hydrocarbon group. R ₃ and R ₄ represent an acetyl group or H. Furthermore, four types of MEL, MEL-A, MEL-B, MEL-C, and MEL-D, are known depending on the presence or absence of an acetyl group. MEL-A is a compound in which, in formula (II), R ₁ and R ₂ are a hydrocarbon group having 6 to 20 carbon atoms or an oxygen atom-containing hydrocarbon group, and R ₃ and R ₄ are acetyl groups. .

MEL-B is R ₃ and R ₄ in formula (II) are H and acetyl groups, respectively, and MEL-C is R ₃ and R ₄ in formula (II) are acetyl groups and H, respectively. In D, in formula (II), R ₃ and R ₄ are both H. In this specification, three types of MEL having an acetyl group, that is, MEL-A, MEL-B, and MEL-C are collectively referred to as acetyl group-containing MEL.

  As described above, the “acyl group” is a functional group containing an “acetyl group”. Therefore, regioselectively cleaving the acyl group of MEL specifically comprises the 2-position acyl group, 3-position acyl group, 4-position acetyl group, and 6-position acetyl group of mannose of MEL. It means that one or more functional groups selected from the group are selectively cleaved.

<Method for producing MEL>
MEL that can be used in the production method of the present invention is not particularly limited, but can be produced by arbitrarily selecting a fermentation method using a microorganism. Examples of the microorganism include, for example, Pseudozyma antarctica NBRC 10736, Candida antarctica, Candida sp. , P.M. Examples include, but are not limited to, tsukubaensis. It is a well-known fact that MEL can be easily obtained with any microorganism. The microorganism having the ability to produce MEL is not particularly limited and can be appropriately used depending on the purpose.

  Among the microorganisms exemplified above, MEL-A can be mainly selectively produced by using Pseudozyma antarctica NBRC 10736. By using tsukubaensis, MEL-B can be mainly selectively produced.

  The medium used for the fermentation culture of microorganisms capable of producing the MEL is a nitrogen source such as yeast extract or peptone, a carbon source such as glucose or fructose, and an inorganic nitrogen source, dipotassium hydrogen phosphate, magnesium sulfate heptahydrate. A medium containing an inorganic salt such as oil added with oils and a water-insoluble substrate can be used. In addition, fatty acids such as myristic acid and esters thereof may be used instead of the oils.

  As fats and oils, vegetable fats and oils are preferable. There is no restriction | limiting in particular as vegetable fats and oils, According to the objective, it can select suitably. Examples include soybean oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, palm oil, sunflower oil, corn oil, canola oil, coconut oil, and among these, acetyl group-containing MEL Soybean oil is particularly preferable in that the production efficiency (production amount, production rate, and yield) can be improved. Moreover, these vegetable fats and oils may be used individually by 1 type, or may use 2 or more types together.

  As the water-insoluble substrate, hydrocarbons such as liquid paraffin and tetradecane can be used. These water-insoluble substrates may be used alone or in combination of two or more.

  There is no restriction | limiting in particular as an inorganic nitrogen source, Although it can select suitably according to the objective, For example, ammonium nitrate, urea, sodium nitrate, ammonium chloride, ammonium sulfate, etc. are mentioned.

  The conditions for fermentation culture (culture temperature, culture time, pH of the medium, etc.) can be arbitrarily set as optimum conditions for the yeast to be used. For example, in the following Example 1, the present inventors used Pseudozyma antarctica NBRC 10936 for 7 to 14 days using 5 L-jar under the conditions of 30 ° C., 300 rpm (stirring rotation), and 0.5 L / min (Air). It is in culture.

  In Example 2 below, P.I. tsukubaensis is cultured for 8 days using 5 L-jar under the conditions of 26 ° C., 300 rpm (stirring rotation), 1/4 VVM, 0.5 L / min (Air). However, it is not limited to these.

  The culture solution after fermentation contains MEL. Therefore, the culture solution after fermentation can be used as it is for the production method of the present invention. Furthermore, it is possible to appropriately add operations such as filtration, centrifugation, extraction, purification, and sterilization to the culture solution after fermentation as necessary, and the obtained extract can be diluted, concentrated, and dried.

  There is no restriction | limiting in particular as a collection | recovery and refinement | purification method of MEL, According to the objective, it can select suitably. For example, it can be recovered by centrifuging the culture solution to recover the oil and extracting and concentrating with ethyl acetate.

  Examples of the extraction solvent include water, alcohols (for example, lower alcohols such as methanol, absolute ethanol, and ethanol, or polyhydric alcohols such as propylene glycol and 1,3-butylene glycol), ketones such as acetone, diethyl ether, and dioxane. In addition, esters such as acetonitrile and ethyl acetate, and organic solvents such as xylene, benzene and chloroform can be used alone or in combination of two or more kinds. Moreover, what combined each solvent extract can also be used.

  The extraction method is not particularly limited, but usually it may be in the range of the boiling point of the solvent from normal temperature to normal pressure. After extraction, it is filtered or ion exchange resin is used for adsorption, decolorization, and purification. A solution, paste, gel, or powder may be used. In this way, a crude product of MEL can be obtained. In many cases, it can be used in the state of the crude product, but if necessary, purification treatment such as deodorization and decolorization may be further added as long as the effect is not affected.

  As a means for purification treatment such as deodorization and decolorization, an activated carbon column or the like may be used, and a normal means generally applied depending on the extracted substance may be arbitrarily selected. In this way, a highly purified MEL product can be obtained. Moreover, you may refine | purify further using a silica gel column as needed.

  Hereinafter, as an example of the production method of the present invention, a method for producing MEL-D, which is a deacetylated form, by selectively cleaving the acetyl groups at the 4-position and 6-position of mannose of MEL will be described.

  However, the method for producing a deacylated form of MEL is not limited to this. The method for producing a deacylated form of MEL includes one or more functional groups selected from the group consisting of the 2-position acyl group, 3-position acyl group, 4-position acetyl group, and 6-position acetyl group of mannose of MEL It is sufficient that a deacylated form of MEL can be produced by regioselectively cleaving.

<Method for producing MEL-D>
In the production method of MEL-D, the above-described acetyl group-containing MEL (MEL-A, MEL-B, and MEL-C) can be suitably used. Among these acetyl group-containing MELs, one kind may be used alone, or two or more kinds may be used in combination.

  Moreover, as an aspect of the said acetyl group containing MEL, it is the culture solution after fermentation mentioned above, the precipitate produced | generated in a culture solution, a rough refined material, or a refined material.

  The manufacturing method of MEL-D should just include the process of selectively cut | disconnecting the acetyl group of 4-position and 6-position of the mannose of acetyl group containing MEL.

  Examples of the method for selectively cleaving the acetyl group include “a method for hydrolyzing the acetyl group of an acetyl group-containing MEL in an acidic or alkaline solution”, “a method using an enzyme”, and the like. . The “method using an enzyme” may be any method including a step of reacting an acetyl group-containing MEL with a hydrolase.

  As the hydrolase, lipase, protease, esterase and the like can be preferably used. Of these, lipase and esterase are preferable, and lipase is more preferable. By using these hydrolases, only the acetyl group can be efficiently cleaved without cleaving the fatty acid portion of the acetyl group-containing MEL. At least one selected from the above hydrolases may be used, or a plurality of hydrolases may be used.

  Thus, even those skilled in the art cannot predict that MEL-D can be produced efficiently by selectively cleaving only the acetyl group of the acetyl group-containing MEL by using the hydrolase.

  The method for reacting the acetyl group-containing MEL and the hydrolase is not particularly limited, and for example, it can be carried out by adding the hydrolase to a solution containing the acetyl group-containing MEL.

  As the solution, an aqueous solution, an organic solvent, or a mixed solution of an aqueous solution and an organic solvent can be used. As said aqueous solution, well-known aqueous solutions for those skilled in the art, such as 0.1M phosphate buffer (pH 7) and a tris buffer solution, can be used.

  The organic solvent is not limited as long as it can solubilize all or part of the acetyl group-containing MEL. The organic solvent may be a mixture of a plurality of organic solvents. For example, methanol, ethanol, propanol, butanol, acetone, propanone, butanone, pentan-2-one, 1,2-ethanediol, 2,3-butanediol, dioxane, acetonitrile, 2-methyl-butan-2-ol, Tertiary butanol, 2-methylpropanol, and 4-hydroxy-2-methylpentanone, tetrahydrofuran, hexane, DMF, DMSO, pyridine, methyl ethyl ketone, and the like can be given. Among these organic solvents, methanol, acetone, tetrahydrofuran, tertiary butanol, acetonitrile, or dioxane is preferable, and methanol or acetone is more preferable.

  In a solution containing acetyl group-containing MEL at a concentration of 1 mg / ml to 1000 mg / ml, more preferably 5 mg / ml to 500 mg / ml, 0.1 mg / ml to 100 mg / ml, more preferably 1 mg / ml to 10 mg / ml. An acetyl group can be efficiently cleaved by adding a hydrolase and reacting with stirring so as to have a concentration of ml, and a deacetylated acetyl group-containing MEL can be produced.

  The reaction temperature is preferably 10 to 100 ° C, more preferably 20 to 50 ° C, and further preferably 25 to 40 ° C. The reaction time is preferably 1 hour to 7 days.

  The MEL-D produced after the above reaction can be recovered, but the recovery method is not particularly limited and can be appropriately selected depending on the purpose. For example, it can be recovered by adding ethyl acetate to the solution after the reaction and extracting and concentrating.

  Examples of the extraction solvent include water, alcohols (for example, lower alcohols such as methanol, absolute ethanol, and ethanol, or polyhydric alcohols such as propylene glycol and 1,3-butylene glycol), ketones such as acetone, diethyl ether, and dioxane. In addition, esters such as acetonitrile and ethyl acetate, and organic solvents such as xylene, benzene and chloroform can be used alone or in combination of two or more kinds. Moreover, what combined each solvent extract can also be used.

  The extraction method is not particularly limited, but usually it may be in the range of the boiling point of the solvent from normal temperature to normal pressure. After extraction, it is filtered or ion exchange resin is used for adsorption, decolorization, and purification. A solution, paste, gel, or powder may be used. In this way, a crude product of MEL-D can be recovered. Furthermore, if necessary, purification treatment such as deodorization and decolorization may be added within a range that does not affect the efficacy.

  As a means for purification treatment such as deodorization and decolorization, an activated carbon column or the like may be used, and a normal means generally applied depending on the extracted substance may be arbitrarily selected. In this way, a highly purified product of MEL-D can be obtained. Moreover, you may refine | purify further using a silica gel column as needed.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention. The following examples further illustrate the present invention in detail, but the present invention is not limited thereto.

<Example 1-Method for deacetylating MEL-A using hydrolase>
[Production of MEL-A]
Inoculum culture was performed by inoculating Pseudozyma antarctica NBRC 10736 in a seed medium (20 ml / 500 ml Sakaguchi flask) for 1 loop. Cultured overnight at 30 ° C. The obtained culture broth was used as an inoculum. The composition of the seed medium was 4% Glucose, 0.3% NaNO ₃ , 0.02% MgSO ₄ · H ₂ O, 0.02% KH ₂ PO ₄ , and 0.1% yeast extract.

In culture, 75 ml of the above inoculum is inoculated into 1.5 L (5 L-jar) of the production medium, and 7 to 14 using 5 L-jar under the conditions of 30 ° C., 300 rpm (stirring rotation), 0.5 L / min (Air). Cultured for days. The composition of the production medium was 3% soybean oil, 0.02% MgSO ₄ · H ₂ O, 0.02% KH ₂ PO ₄ , and 0.1% yeast extract.

  The obtained culture solution (250 mL) was centrifuged (6500 rpm, 30 min). The supernatant was removed, and the precipitate (bacteria) was collected. 50 ml of ethyl acetate was added to the precipitate, and after sufficient stirring, the mixture was centrifuged (8500 rpm, 30 min), divided into a precipitate and a supernatant, and the supernatant was concentrated with an evaporator. Using silica gel, elution was performed with hexane: acetone = 5: 1 and hexane: acetone = 1: 2 to obtain a MEL-A fraction.

  In addition, it confirmed that it was MEL-A by NMR.

[Deacetylation of MEL-A]
1 g of MEL-A produced as described above was suspended in 50 ml of a mixed solution having the following composition 1-5. Thereafter, 1 g of lipase LPL-311 (Toyobo) was added, and the mixture was reacted by shaking at 30 ° C. for 12 hours.

1-methanol 2-0.1 M phosphate buffer (pH 7.0): methanol = 1: 3
3-0.1 M phosphate buffer (pH 7.0): methanol = 1: 1
4-0.1 M phosphate buffer (pH 7.0): methanol = 3: 1
5-0.1M phosphate buffer (pH 7.0)
After the reaction, the reaction mixture was extracted and concentrated with ethyl acetate, and the product was isolated with 20 g of silica gel with ethyl acetate: methanol 10: 1 to obtain 100 mg of a colorless oily product.

  FIG. 1 is a diagram showing TLC (developing solvent: ethyl acetate: methanol: water = 85: 10: 5, sulfuric acid coloration) of each mixed solution after the reaction. In addition, 6 has shown TLC of MEL-A used as a control | contrast. According to FIG. 1, one product spot having higher polarity than MEL-A was observed in 1 to 5.

A product having a higher polarity than MEL-A was roughly isolated, and its structure was confirmed by ¹³ C-NMR and ¹ H-NMR.

Table 1 is a table showing ¹³ C-NMR (solvent: DMSO-d6 chemical shift value (ppm) of each carbon atom between MEL-A and the product (MEL-D). E1 in Table 1 , E2, E3, E4, M1, M2, M3, M4, M5, and M6 represent carbon atoms of a mannosylerythritol skeleton of the following formula (III).

  Compared with MEL-A, in the product (MEL-D), the 4th and 6th carbons of the mannose residue of MEL are shifted in a high magnetic field, and the adjacent 3rd and 5th carbons are shifted in a low magnetic field. This confirmed that the acetyl group of MEL-A was deacetylated.

Table 2 is a table showing ¹ H-NMR (solvent: DMSO-d6, chemical shift value (ppm) of each hydrogen atom between MEL-A and the product (MEL-D). E1 in Table 2 , E2, E3, E4, M1, M2, M3, M4, M5 and M6 represent carbon atoms of the mannosylerythritol skeleton of the above formula (III).

^{From 1} H-NMR, in the product, the peak of the acetyl group disappeared, the hydroxyl group at the 6th position of mannose appeared, and the protons at the 4th and 6th positions were shifted in the high magnetic field in comparison with MEL-A. I understood that. As a result of the assignment as shown in Table 2, it was confirmed that the product was MEL-D.

Example 2 Method for Deacetylating MEL-B Using Enzyme
[Production of MEL-B]
0.2 ml of P.I. The tsukubaensis frozen stock was inoculated into a YM seed medium (20 ml / 500 ml Sakaguchi flask) and cultured overnight at 26 ° C. and 180 rpm. The obtained culture broth was used as an inoculum.

  For culturing, 20 ml of the above inoculum was inoculated into 2 L of YM medium (5 L-jar), and cultured for 8 days under the conditions of 26 ° C., 300 rpm (stirring rotation), 1/4 VVM, 0.5 L / min (Air).

  The obtained culture solution was dispensed into six 500 ml centrifuge tubes and centrifuged (7900 rpm, 60 min, 4 ° C.). The supernatant was removed and the cells (including MEL-B) were collected. 80 ml of ethyl acetate was added to each of the bacterial cell fractions × 6 and stirred up and down so that the bacterial cells were sufficiently suspended. After stirring, the mixture was centrifuged (7900 rpm, 60 min, 4 ° C.) to separate into a supernatant and a precipitate.

  The resulting supernatant was collected in an Erlenmeyer flask. Again, 80 ml of ethyl acetate was added to the precipitate and centrifuged in the same manner to separate the precipitate and the supernatant. The supernatant was collected in the Erlenmeyer flask.

  The obtained supernatant was placed in a separatory funnel, the supernatant and an equal amount of saturated saline were added, and the mixture was sufficiently stirred. The aqueous layer was discarded and an ethyl acetate layer was obtained. An appropriate amount of anhydrous sodium sulfate was added to the ethyl acetate layer and allowed to stand for 30 minutes. The MEL-B crude product was obtained by evaporation.

  The obtained MEL-B crude product (20 g) was subjected to a silica column (200 g, hexane swelling), hexane: acetone = 5: 1 (1500 ml), hexane: acetone = 5: 2 (700 ml), hexane: acetone = 200 ml each was collected at 1: 1 (1000 ml) to obtain a MEL-B fraction.

  In addition, it confirmed that it was MEL-B by NMR.

[Deacetylation of MEL-B]
Using 1 g of MEL-B produced as described above, the reaction was carried out in the same manner as in Example 1, followed by purification to obtain 95 mg of a colorless oily product.

  FIG. 2 is a diagram showing TLC (developing solvent ethyl acetate: methanol: water = 85: 10: 5, sulfuric acid coloration) of the mixed solution after the reaction. 1 in FIG. 2 indicates that the composition of the mixed solution is 0.1 M phosphate buffer (pH 7.0): methanol = 1: 1. 2 indicates the TLC of MEL-A as a control, and 3 indicates the TLC of MEL-B as a control.

  According to FIG. 2, as in Example 1, one spot of a product having a polarity higher than that of MEL-A was observed in 1, and it was considered that MEL-D was generated.

  According to the present invention, a deacylated form of a glycolipid biosurfactant can be produced efficiently. More specifically, MEL with improved water solubility can be produced by reacting MEL with a hydrolase and deacylating by hydrolysis.

  That is, according to the manufacturing method of the present invention, when applying MEL, MEL-D which cannot be manufactured by the conventional method can be provided. Therefore, it can be widely used in industries such as food industry, cosmetics industry, pharmaceutical industry, chemical industry, and environmental field.

FIG. 1 is a diagram showing a TLC pattern of the reaction solution shown in Example 1. FIG. 2 is a diagram showing a TLC pattern of the reaction solution shown in Example 2.

Claims (5)

  A method for producing a deacylated form of a glycolipid type biosurfactant, wherein the acyl group of the glycolipid type biosurfactant is cleaved regioselectively.   The method for producing a deacylated form of a glycolipid type biosurfactant according to claim 1, wherein the glycolipid type biosurfactant is mannosyl erythritol lipid (MEL) or mannosyl mannitol lipid (MML). The deacylated form of the glycolipid type biosurfactant has the formula (I)
The glycolipid according to claim 2, wherein R ₁ and R ₂ are each independently a glycolipid represented by a hydrocarbon group or an oxygen atom-containing hydrocarbon group. For producing a deacylated form of a type biosurfactant   4. The glycosyl biosurfactant deacylated product according to any one of claims 1 to 3, wherein an ester bond is cleaved by reacting a glycolipid biosurfactant with a hydrolase. Production method.   5. The method for producing a deacylated form of a glycolipid biosurfactant according to claim 4, wherein the hydrolase is at least one hydrolase selected from the group consisting of lipase, protease and esterase. .