JP6420536B2 - Production method of sophorolipid - Google Patents

Description

  The present invention relates to a method for producing sophorolipid.

Amphiphiles produced by microorganisms such as yeast are called biosurfactants (biosurfactants), and are materials that have both environmental compatibility and functionality in the fields of food, cosmetics, life science, environment and energy. Application has been studied.
Among them, sophorolipid is one of the typical biosurfactants currently used in industry.

It is known that sophorolipid is fermented and produced at a relatively high production amount using an oily substance such as a hydrocarbon or fats and oils as a substrate. In particular, it is known that when saccharides and fats and oils are combined as a carbon source substrate, sophorolipid is efficiently produced by fermentation.
As fats and oils used for sophorolipid fermentative production, animal and vegetable oils, fatty acids, fatty acid esters, and the like are used. For example, Patent Document 1 discloses oleic acid in addition to Candida yeast in addition to sugar and vegetable oil. A method for fermenting sophorolipids in a high yield and a high yield by culturing in a medium mixed with free fatty acids such as is disclosed.

JP 2002-45195 A

Like the prior art, the carbon source substrate of sophorolipid generally uses liquid fats and oils in view of the productivity of sophorolipid, and solid fats and the like having a melting point higher than the culture temperature are It was considered difficult to use suitably because of the low utilization of microorganisms. So far, there has been no report that sophorolipid was produced with high efficiency by using solid oils and fats at the culture temperature.
Therefore, the present invention relates to providing a method capable of efficiently producing sophorolipid from a substrate containing solid oils and fats.

  As a result of intensive studies to solve the above problems, the present inventors have combined solid oils and fats with specific surfactants, thereby improving the utilization of solid oils and fats. It has been found that it can be converted to sophorolipid very efficiently.

That is, the present invention includes the following components (A) and (B):
(A) Solid fats and oils (B) (b1) HLB 1-7 nonionic surfactant and / or (b2) HLB 12-18 nonionic surfactant The present invention provides a method for producing sophorolipid, which comprises a step of obtaining sophorolipid by fermentation using a microorganism having the ability to produce forolipid.

  According to the present invention, a sophorolipid can be efficiently produced using a substrate containing solid oils and fats. Therefore, it can be expected to use a cheaper or new carbon source in the production of sophorolipid.

The method for producing the sophorolipid of the present invention comprises the following components (A) and (B):
(A) Solid fats and oils (B) (b1) HLB 1-7 nonionic surfactant and / or (b2) HLB 12-18 nonionic surfactant The method includes a step of obtaining sophorolipid by fermentation using a microorganism having the ability to produce phorolipid.

Sophorolipid is a glycolipid composed of soforose and hydroxy fatty acid. Soforus is a disaccharide in which glucose is β-1,2 linked, and hydroxy fatty acid is a fatty acid having a hydroxy group at the ω-position or ω-1 position. The fatty acid portion of the hydroxy fatty acid is not particularly limited, but a saturated or unsaturated fatty acid having 10 to 30 carbon atoms is preferable. Soforus also includes those in which the hydroxy group is partially acetylated.
Sophorolipids are broadly divided into acid types in which the carboxyl group of hydroxy fatty acid is liberated and lactone types in combination with Sophorose in the molecule, and are generally known to be obtained as a mixture of acid and lactone types in fermentation production. ing.

The microorganism having the ability to produce the sophorolipid used in the present invention may be any microorganism that has the ability to produce the sophorolipid from the substrate and to produce it outside the cell. For example, it belongs to the genus Candida. Examples include microorganisms.
Examples of the microorganisms belonging to the genus Candida, Candida bombicola, Candida bogoriensis, Candida magnoliae, Candida gropengiesseri, Candida apicola , and the like. Among these, Candida bombicola is preferable from the viewpoint of productivity of sophorolipid .

The culture medium used by this invention contains a component (A) solid fats and oils.
Here, the solid fats and oils are fats that are solid or semi-solid at the culture temperature. Examples of such oils and fats include animal and vegetable oils, fatty acids, and fatty acid esters, and these can be used alone or in combination of two or more.
Animal and vegetable fats and oils include palm oil, palm olein, palm stearin, palm oil, palm kernel oil, cocoa butter, monkey fat, shea fat and other vegetable fats, lard, beef tallow, butter fat and other animal fats and oils Of the transesterified oil, hydrogenated oil or fractionated oil. Moreover, what was solidified by hydrogenation of liquid oils, such as soybean oil, cottonseed oil, and fish oil, is also included.

As the fatty acid, a fatty acid having 10 to 30 carbon atoms is preferable. For example, capric acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, henicosanoic acid, behen Examples thereof include saturated fatty acids such as acid, tricosanoic acid and lignoceric acid, and unsaturated fatty acids such as palmitoleic acid, petrothelic acid, elaidic acid, vaccenic acid, erucic acid, sorbic acid, linoelaidic acid and punicic acid.
Examples of fatty acid esters include esters of the above fatty acids with monohydric to trihydric alcohols, such as octadecyl laurate, tetradecyl myristate, dodecyl palmitate, methyl stearate, ethyl stearate, propyl stearate, Examples include octyl stearate, methyl arachidate, and ethyl arachidate.
Among solid oils and fats, fatty acid esters are preferable and saturated fatty acid esters are more preferable from the viewpoint of obtaining a sophorolipid having a single fatty acid composition. The fatty acid ester is preferably an alkyl ester of a fatty acid having 12 to 24 carbon atoms, preferably 16 to 22 carbon atoms, more preferably 16 to 18 carbon atoms, more preferably methyl palmitate, methyl stearate or ethyl stearate. As melting | fusing point of the alkyl ester of a fatty acid, 25-55 degreeC is preferable, More preferably, it is 28-45 degreeC, More preferably, it is 32-40 degreeC.

  The initial content of (A) solid fats and oils in the medium is not particularly limited, but is 1% (w / v) or more, and further 5% (w / v) from the viewpoint of efficient sophorolipid production. v) or more, preferably 20% (w / v) or less, more preferably 15% (w / v) or less. Moreover, 1 to 20% (w / v) is preferable, and 5 to 20% (w / v) is more preferable.

Moreover, the culture medium used by this invention contains the nonionic surfactant whose component (B) (b1) HLB is 1-7, and / or (b2) the nonionic surfactant whose HLB is 12-18.
Here, HLB (Hydrophilic-Lypophilic Balance) indicates the molecular weight of the hydrophilic group in the total molecular weight of the surfactant. The HLB of the nonionic surfactant can be obtained by the Griffin equation. In addition, HLB in the case of being composed of two or more kinds of nonionic surfactants is obtained by averaging the HLB values of the respective nonionic surfactants based on the blending mass ratio as shown in the following formula. .
Mixed HLB = Σ (HLBx × Wx) / ΣWx
(In the formula, HLBx represents the HLB value of the nonionic surfactant X, and Wx represents the mass (g) of the nonionic surfactant X having the value of HLBx.)

Examples of the nonionic surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyethylene glycol fatty acid ester, sucrose fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl Examples include ether, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene glycerin fatty acid ester, and polyoxyethylene hydrogenated castor oil. These can be used alone or in combination of two or more.
When a nonionic surfactant having an ethylene oxide chain is used, the number of moles of ethylene oxide added is preferably 4 to 250, more preferably 20 to 160, and still more preferably 20 to 80. Moreover, carbon number of the fatty acid part and alkyl part of a nonionic surfactant becomes like this. Preferably it is 8-24, More preferably, it is 12-22.

  Component (b1) Nonionic surfactants having an HLB of 1 to 7 have an HLB of 1.2 or more, more preferably 1.5 or more, and further 1.8 or more from the viewpoint of efficient sophorolipid production. It is preferably 6 or less, further 4.3 or less, and further 4 or less. The HLB is preferably 1.2 or more and 7 or less, more preferably the HLB is 1.2 or more and 6 or less, and still more preferably the HLB is 1.5 or more and 4 or less. Moreover, (b1) As a kind of nonionic surfactant whose HLB is 1-7, sorbitan fatty acid ester is preferable. Of the sorbitan fatty acid esters, sorbitan trifatty acid esters are preferable, and sorbitan trioleate is more preferable.

  Further, the nonionic surfactant having a component (b2) HLB of 12 to 18 is preferably one having an HLB of 14 or more and 18 or less from the viewpoint of efficient production of sophorolipid. More preferably, HLB is 14 or more and 17 or less. As a kind of nonionic surfactant, polyoxyethylene sorbitan fatty acid ester is preferable. Among these, polyoxyethylene sorbitan monolaurate is preferable.

The initial content of component (B) in the medium is 0.001% (w / v) or more, more preferably 0.02% (w / v) or more, from the viewpoint of efficient sophorolipid production. It is preferably 0.04% (w / v) or more, more preferably 0.05% (w / v) or more, and from the viewpoint of the amount of foam of the medium, 0.3% (w / v) or less, and further 0 .2% (w / v) or less, further 0.15% (w / v) or less, further 0.12% (w / v) or less, and further preferably 0.1% (w / v) or less. . Moreover, it is preferable that it is 0.001-0.3% (w / v), More preferably, it is 0.02-0.20% (w / v), More preferably, it is 0.04-0.15% ( w / v), more preferably 0.04 to 0.12% (w / v).
“Content of component (B)” is the content of one of components (b1) and (b2), and includes both components (b1) and (b2). It is the total amount.

In addition to the components (A) and (B), a carbon source, a nitrogen source, inorganic salts, and other necessary nutrient sources that can be assimilated by microorganisms can be appropriately used for the medium used in the present invention. Moreover, a conventionally well-known culture medium can be used, for example, commercially available solid culture media or liquid culture media, such as a YM culture medium, can be used.
Examples of the carbon source include sugars (glucose, arabinose, xylose, mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, mannitol, inosit, glycerin, soluble starch, waste molasses, invert sugar, etc.), fats and oils ( Liquid oils such as soybean oil, rapeseed oil, fish oil, fatty acids such as oleic acid, linoleic acid, γ-linolenic acid, linolenic acid, arachidonic acid, etc.), organic acids that can be assimilated such as acetic acid, alcohols such as ethanol And the like. Especially, it is preferable to contain saccharide | sugar and also glucose from the point of proliferation.
The content of the initial carbon source in the medium is preferably 1 to 15% (w / v) from the viewpoint of growth.
Examples of the nitrogen source include ammonia, inorganic / organic ammonium salts, urea, corn gluten meal, soybean flour, yeast extract, meat extract, fish extract, polypeptone, peptone, various amino acids, and soy bean meal.

In the present invention, in the step of obtaining sophorolipid by fermentation using a microorganism having the ability to produce sophorolipid, the sophorolipid may be fermented and produced while culturing under conditions where the microorganism grows. The sophorolipid may be fermented and produced by culturing in a state of resting cells, that is, in a state where the growth of the microorganism is stopped. From the viewpoint of the time required for growth, it is preferable to fermentatively produce sophorolipid in the state of resting cells.
When the resting cells are used, the concentration of the microorganism having the ability to produce sophorolipid is preferably 10 to 200 as the absorbance at 660 nm (OD660). Moreover, as time to perform resting cell reaction, 50 to 200 hours are preferable.

The culture conditions may be any conditions as long as a microorganism having the ability to produce sophorolipid is fermentatively produced sophorolipid.
The culture method is preferably under aerobic conditions, and general methods such as aeration stirring culture and shaking culture can be applied.
The culture temperature is not particularly limited as long as the microorganism to be used is grown, but is usually preferably 20 to 33 ° C, more preferably 25 to 30 ° C, and further preferably 28 to 30 ° C. At this time, the initial pH (30 ° C.) of the culture solution is preferably 2 to 7, and more preferably 3 to 6.

  Examples of the buffer that adjusts the pH of the culture solution include organic acids such as carbonic acid, acetic acid, citric acid, fumaric acid, malic acid, lactic acid, gluconic acid, and tartaric acid, or salts thereof, inorganic substances such as phosphoric acid, hydrochloric acid, and sulfuric acid. Examples thereof include acids or salts thereof, hydroxides such as sodium hydroxide, ammonia or aqueous ammonia, and these can be used alone or in combination of two or more.

Since sophorolipid accumulates in the medium by such culture, the sophorolipid can be collected from the medium by an appropriate separation / purification means after completion of the culture.
For example, sophorolipid can be obtained by solvent extraction using ethyl acetate or the like and then using fractional precipitation, liquid-liquid distribution, column chromatograph, high performance liquid chromatograph or the like alone or in combination.

According to the present invention, sophorolipid can be produced from a substrate at a conversion rate of 15 to 50%.
The sophorolipid conversion rate is the molar amount of sophorolipid production contained in 1 L of the culture broth in terms of fatty acid equivalent of the fat and oil substrate (initially charged fat and oil substrate) charged in the medium for sophorolipid fermentation production. Divided value. Details of the method for calculating the sophorolipid conversion rate are described in the Examples.

  The sophorolipid obtained by the present invention is expected to be used as a surfactant, a cosmetic base, a raw material for various intermediates, and the like.

  In relation to the above-described embodiment, the present invention further discloses the following manufacturing method.

<1> The following components (A) and (B):
(A) Solid fats and oils (B) (b1) HLB 1-7 nonionic surfactant and / or (b2) HLB 12-18 nonionic surfactant A method for producing sophorolipid, comprising a step of obtaining sophorolipid by fermentation using a microorganism having an ability to produce forolipid.

<2> (A) The method for producing a sophorolipid according to <1>, wherein the solid fat is preferably one or more selected from solid animal and vegetable fats, fatty acids and fatty acid esters.
<3> Solid animal and vegetable oils and fats are preferably palm oil, palm olein, palm stearin, palm oil, palm kernel oil, cacao butter, monkey fat, shea fat, lard, beef tallow, butter fat, or esters thereof Exchange oil, hydrogenated oil or fractionated oil, solid fatty acids are preferably capric acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecylic acid, arachidic acid, Henicosanoic acid, behenic acid, tricosanoic acid, lignoceric acid, palmitoleic acid, petrothelic acid, elaidic acid, vaccenic acid, erucic acid, sorbic acid, linoelaidic acid, or punicic acid, solid fatty acid ester, preferably lauric acid Octadecyl acid, tetradecyl myristate, dodecyl palmitate, Methyl stearic acid, ethyl stearate, propyl stearate, octyl stearate, Seo Roman lipid producing method according to methyl arachidate, or arachidic acid ethyl <2>.
<4> (A) The solid oil or fat is preferably an alkyl ester of a fatty acid having 12 to 24 carbon atoms, more preferably 16 to 22 carbon atoms, still more preferably 16 to 18 carbon atoms, and further preferably palmitic acid. The method for producing sophorolipid according to <1>, which is methyl acid, methyl stearate, or ethyl stearate.
<5> (A) The melting point of the solid oil or fat is preferably 25 to 55 ° C, more preferably 28 to 45 ° C, still more preferably 32 to 40 ° C, and any one of <1> to <4> A method for producing the sophorolipid described in 1.
<6> The content of (A) solid oil or fat in the medium is preferably 1% (w / v) or more, more preferably 5% (w / v) or more, and preferably 20%. (W / v) or less, preferably 1 to 20% (w / v), more preferably 5 to 20% (w / v), <1> to <5> Of manufacturing sophorolipid.
<7> (B) (b1) A nonionic surfactant having an HLB of 1 to 7, preferably an HLB of 1.2 or more, more preferably an HLB of 1.5 or more, more preferably an HLB of 1.8 or more. Preferably, HLB is 6 or less, more preferably 4.3 or less, more preferably HLB is 4 or less, and preferably HLB is 1.2-7, more preferably HLB is 1.2. -6, More preferably, the manufacturing method of the sophorolipid as described in any one of <1>-<6> which is a nonionic surfactant with HLB of 1.5-4.
<8> (B) (b1) The nonionic surfactant having an HLB of 1 to 7 is preferably sorbitan fatty acid ester, more preferably sorbitan trifatty acid ester, and further preferably sorbitan trioleate <1> to <7 The manufacturing method of the sophorolipid of any one of>.
<9> (B) (b2) A nonionic surfactant having an HLB of 12 to 18, preferably an HLB of 14 or more, preferably an HLB of 17 or less, and preferably an HLB of 14 to 18, More preferably, the method for producing a sophorolipid according to any one of <1> to <8>, which is a nonionic surfactant having an HLB of 14 to 17.
<10> (B) (b2) The nonionic surfactant having an HLB of 12 to 18 is preferably polyoxyethylene sorbitan fatty acid ester, more preferably polyoxyethylene sorbitan monolaurate <1> to <9> A method for producing a sophorolipid according to any one of the above.
<11> The content of the component (B) in the medium is preferably 0.001% (w / v) or more, more preferably 0.02% (w / v) or more, and further preferably 0.04% ( w / v) or more, more preferably 0.05% (w / v) or more, preferably 0.3% (w / v) or less, more preferably 0.2% (w / v) or less. More preferably, it is 0.15% (w / v) or less, more preferably 0.12% (w / v) or less, more preferably 0.1% (w / v) or less, and preferably 0 0.001 to 0.3% (w / v), more preferably 0.02 to 0.2% (w / v), still more preferably 0.04 to 0.15% (w / v), still more preferably The method for producing a sophorolipid according to any one of <1> to <10>, which is 0.04 to 0.12% (w / v).
<12> The method for producing a sophorolipid according to any one of <1> to <11>, wherein the medium preferably contains sugar, more preferably glucose.
<13> The method for producing a sophorolipid according to any one of <1> to <12>, wherein the reaction using a microorganism having the ability to produce a sophorolipid is preferably a resting cell reaction.
<14> The microorganism having the ability to produce sophorolipid is preferably a microorganism belonging to the genus Candida , more preferably Candida bombicola , Candida bogoriensis , Candida majoriand , Candida group , Candida group , Candida group , Candida group The method for producing a sophorolipid according to any one of <1> to <13>, which is preferably Candida bombicola .

  In the following examples and comparative examples, “%” means “% (w / v)”.

[Analysis of Sophorolipid]
10 mL of the culture solution was sampled, washed twice with 10 mL of hexane, and then extracted twice with 10 mL of ethyl acetate for recovery. Thereafter, the solvent was distilled off, and the ethyl acetate fraction was collected. The dry weight of the collected ethyl acetate fraction was calculated as the sophorolipid production amount.

[Calculation of Sophorolipid conversion rate]
The sophorolipid conversion rate was calculated by the following equation.
Conversion rate (%) = (Mole amount of sophorolipid contained in 1 L of culture solution) / (Mole amount in terms of fatty acid of initially charged fat and oil substrate) × 100

[Culture method]
1. Plate culture
Candida bombicola NBRC10243 strain was used.
One platinum ear inoculum was inoculated in a petri dish of an agar medium containing 1% glucose, 1% yeast extract, 1% tryptone and 1.5% agar, and cultured at 30 ° C. for 2 days.

2. Preculture 100 mL of a culture solution containing 1% glucose, 1% yeast extract, and 1% tryptone was placed in a Sakaguchi flask and sterilized at 121 ° C. for 20 minutes at a high temperature. After cooling, 1 platinum loop and inoculation were performed from the plate, and stirring culture was performed for 2 days under the conditions of a temperature of 30 ° C. and a stirring rotation speed of 120 r / min.

3. Main culture A culture solution containing 10% glucose, 2% yeast extract, and 1.2% urea was adjusted to pH 5.0 and made up to 1.2 L. After sterilizing the culture solution in a 2 L jar fermenter, inoculate 24 mL of the preculture solution, and for 48 hours under conditions of a temperature of 30 ° C., a stirring speed of 600 r / min, and an aeration rate (aeration rate) of 0.6 L / min Stirring culture was performed.

4). Recovery of the bacteria The culture solution obtained in the main culture was centrifuged at 6,000 r / min for 20 minutes, and the operation of recovering the bacterial cells was repeated twice. A resting cell reaction was performed using the collected cells.

Example 1
Test tube with 10% ethyl stearate (melting point 36 ° C.), 10% glucose, sorbitan trioleate (Leodol® SP-O30V, HLB = 1.8) 0.1%, 20 mM phosphate buffer pH 4.0 10 mL was placed and sterilized at 121 ° C. for 20 minutes at high temperature. The resting cells recovered after cooling were put into the medium so that the OD660nm was 30, and cultured for 144 hours under the conditions of a temperature of 30 ° C. and a stirring speed of 240 r / min.
After culturing, the sophorolipid conversion rate was determined to be 45.9%.

Example 2
Incubation was performed in the same manner as in Example 1 except that sorbitan monooleate (Leodol SP-O10V, HLB = 4.3) was used instead of sorbitan trioleate.
After culturing, the sophorolipid conversion rate was determined to be 37.2%.

Example 3
Culture was performed in the same manner as in Example 1 except that sorbitan monopalmitate (Emazole (registered trademark) P-10V, HLB = 6.7) was used instead of sorbitan trioleate.
After culturing, the sophorolipid conversion rate was determined to be 17.6%.

Example 4
Incubation was carried out in the same manner as in Example 1 except that polyoxyethylene sorbitan monolaurate (Leodol TW-L120, HLB = 16.7) was used instead of sorbitan trioleate.
After culturing, the sophorolipid conversion rate was determined to be 32.7%.

Example 5
Culturing was performed in the same manner as in Example 1 except that the amount of sorbitan trioleate was 0.05%.
After culturing, the sophorolipid conversion rate was determined to be 46.2%.

Example 6
Culturing was carried out in the same manner as in Example 1 except that the amount of sorbitan trioleate was 0.03%.
After cultivation, the sophorolipid conversion rate was determined to be 43.8%.

Example 7
Culturing was performed in the same manner as in Example 1 except that the amount of sorbitan trioleate was 0.01%.
After culturing, the conversion rate of sophorolipid was determined to be 41.8%.

Comparative Example 1
Culturing was performed in the same manner as in Example 1 except that culturing was performed without adding sorbitan trioleate.
After cultivation, the sophorolipid conversion rate was determined to be 9.8%.

Comparative Example 2
Culture was performed in the same manner as in Example 1 except that sorbitan monolaurate (Emazole L-10V, HLB = 8.6) was used instead of sorbitan trioleate.
After culturing, the sophorolipid conversion rate was determined to be 4.6%.

Comparative Example 3
Culture was performed in the same manner as in Example 1 except that polyoxyethylene sorbitan trioleate (Leodol TW-O320V, HLB = 11.0) was used instead of sorbitan trioleate.
After culturing, the sophorolipid conversion rate was determined to be 8.5%.
Table 1 shows the conditions and results of each example and comparative example.

  As shown in Table 1, it was confirmed that a sophorolipid can be obtained at a high conversion rate by combining solid oils and fats with a specific surfactant. Moreover, it was confirmed that the addition of the surfactant is particularly effective in a specific range of concentrations.

Reference example 1
10 mL of 10% butyl stearate (melting point 27.5 ° C.), 10% glucose, and 20 mM phosphate buffer pH 4.0 was placed in a test tube and sterilized at 121 ° C. for 20 minutes at a high temperature. 3. After cooling, the above [Culture method] The resting cells collected by collecting the cells were put into the medium so that the OD660nm was 30, and cultured for 144 hours under the conditions of a temperature of 30 ° C. and a stirring speed of 240 r / min.
After culturing, the conversion rate of sophorolipid was determined to be 35.9%.

Reference example 2
Culturing was carried out in the same manner as in Reference Example 1 except that 0.1% sorbitan trioleate (Leodol SP-O30V, HLB = 1.8) was added to the medium.
After culturing, the sophorolipid conversion rate was determined to be 31.8%.
Thus, when general liquid fats and oils were used as a substrate, the effect of adding a nonionic surfactant on the sophorolipid conversion rate was not recognized.

Claims (6)

The following components (A) and (b1):
(A) One or two or more solid oils and fats selected from fatty acids and fatty acid esters (b1) In a medium containing a nonionic surfactant whose HLB is a sorbitan fatty acid ester of 1 to 7, A method for producing sophorolipid, comprising a step of obtaining sophorolipid by fermentation using a microorganism belonging to the genus Candida having an ability to produce forolipid. The following components (A) and (b2):
(A) Solid fats and oils that are one or more selected from fatty acids and fatty acid esters (b2) In a medium containing a nonionic surfactant that is a polyoxyethylene sorbitan fatty acid ester having an HLB of 12 to 18 And a method for producing sophorolipid, comprising a step of obtaining sophorolipid by fermentation using a microorganism belonging to the genus Candida having the ability to produce sophorolipid, wherein the first content of component (A) in the medium is included The production method, wherein the amount is 5% (w / v) or more.   The method for producing a sophorolipid according to claim 1 or 2, wherein the content of the component (b1) or (b2) in the medium is 0.001 to 0.3% (w / v).   The method for producing a sophorolipid according to any one of claims 1 to 3, wherein the component (A) is an alkyl ester of a fatty acid having 12 to 24 carbon atoms.   The melting point of a component (A) is 25-55 degreeC, The manufacturing method of the sophorolipid of any one of Claims 1-4.   The method for producing sophorolipid according to any one of claims 1 to 5, wherein the reaction using a microorganism belonging to the genus Candida having the ability to produce sophorolipid is a resting cell reaction.