JP2016000017A - Method of producing sophorolipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method that makes it possible to produce sophorolipid from a substrate efficiently with a high conversion speed.SOLUTION: A method of producing sophorolipid comprises the step of obtaining sophorolipid by fermentation using microorganisms having the ability to produce sophorolipid in a culture medium comprising following components (A) and (B): (A) liquid oily material; and (B) solid oily material where the molar amount of the component (B) to the total molar amount of the oily material is 20-80 mol%.

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 a sugar such as glucose 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 fats and oils, fatty acids, fatty acid esters, and the like are used. For example, in Patent Document 1, Candida yeast is added to sugars and vegetable oils in addition to oleic acid. 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

It is known that the structure of the side chain fatty acid part, which is a non-saccharide part of sophorolipid, can be changed by appropriately selecting a carbon source substrate during cultivation, specifically an oily substance. However, depending on the type of the oily substance, the conversion rate (fermentation rate) from the oily substance as a substrate to sophorolipid may be slow, and it was considered that improvement of productivity suitable for industrial production is a major issue. Conventionally, in view of the productivity of sophorolipid, it has been common to use liquid oils and fats as a carbon source substrate of sophorolipid (for example, Patent Document 1), which has a melting point higher than the culture temperature. It is said that oily substances are difficult to use suitably due to low microbial assimilation ability. So far, sophorolipids have been produced with high efficiency using solid oily substances at culture temperatures. There are no reports.
Therefore, the present invention relates to providing a method capable of producing sophorolipid from a substrate with high conversion rate and efficiency.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have surprisingly been able to obtain a liquid by combining a liquid oily substance and a solid oily substance at a specific ratio at a culture temperature as a substrate. It was found that the conversion rate to sophorolipid was faster than that using only the oily substance in the form, and sophorolipid could be efficiently produced from the substrate.

That is, the present invention includes the following components (A) and (B):
(A) Liquid oily substance (B) A solid oily substance, and a sophorolipid in a medium in which the molar amount of component (B) is 20 to 80 mol% with respect to the total molar amount of the oily substance The present invention provides a method for producing sophorolipid, which comprises a step of obtaining sophorolipid by fermentation using a microorganism having an ability to produce.

  According to the present invention, a sophorolipid can be efficiently produced. In addition, since solid oily substances can be used, the use of new carbon sources according to the purpose can be expected in the production of sophorolipid.

The method for producing the sophorolipid of the present invention comprises the following components (A) and (B):
(A) Liquid oily substance (B) A solid oily substance, and a sophorolipid in a medium in which the molar amount of component (B) is 20 to 80 mol% with respect to the total molar amount of the oily substance The method includes a step of obtaining sophorolipid by fermentation using a microorganism having an ability to produce.
In addition, fermentation in this specification means using the function of microorganisms for substance production (in this specification, production of sophorolipid).
The oily substance in the present specification means an aliphatic compound having a solubility in 100 g of water at 20 ° C. of 2 g or less. The oily substance is either liquid or solid at the culture temperature.

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 part of the hydroxy fatty acid is not particularly limited, but a saturated or unsaturated fatty acid having 8 to 22 carbon atoms is preferable, and a saturated or unsaturated fatty acid having 12 to 22 carbon atoms is more 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) liquid oily substance.
Here, the liquid oily substance is an oily substance that is liquid at the culture temperature. As melting | fusing point of such an oily substance, 25 degrees C or less is preferable, More preferably, it is 5 to 25 degreeC, More preferably, it is 10 to 25 degreeC. In the present specification, the melting point is an ascending melting point in the case of fats and oils and fatty acids, and is a value measured according to the measuring method of the rising melting point in the “standard oil analysis test method” of the Japan Oil Chemical Society method.
Examples of the liquid oily substances include hydrocarbons, animal and vegetable oils, fatty acids, and fatty acid esters, and these can be used alone or in combination of two or more.
Examples of the hydrocarbon include aliphatic hydrocarbons and isoprenoid hydrocarbons, such as n-hexadecene, n-hexadecane, and squalene.
Examples of animal and vegetable oils and fats include vegetable oils such as soybean oil, rapeseed oil, rice bran oil, safflower oil, corn oil, sunflower oil, cottonseed oil, olive oil, sesame oil, perilla oil and linseed oil, or transesterification thereof. Oil, fractionated oil, waste oil and the like can be mentioned.

As the fatty acid, a free fatty acid having 8 to 22 carbon atoms is preferable. For example, saturated fatty acid such as caprylic acid and pelargonic acid; palmitoleic acid, linoleic acid, oleic acid, cis-vacenoic acid, α-linolenic acid, gondoic acid and the like Examples include unsaturated fatty acids.
Examples of fatty acid esters include esters of fatty acids with monohydric to trihydric alcohols, such as methyl caprylate, propyl caprylate, methyl caprate, ethyl caprate, butyl caprate, methyl laurate, and laurin. Ethyl acetate, butyl laurate, dodecyl laurate, methyl myristate, ethyl myristate, butyl myristate, ethyl palmitate, propyl palmitate, isopropyl palmitate, butyl palmitate, amyl palmitate, octyl palmitate, butyl stearate Etc.
As carbon number of a fatty acid part, More preferably, it is 12-22, More preferably, it is 16-22, More preferably, it is 16-18.

  Examples of the higher alcohol include lauryl alcohol, 2-pentadecanol, oleyl alcohol, linoleyl alcohol, and the like.

  Among liquid oily substances, hydrocarbons, fatty acids or esters thereof are preferred, and alkanes or saturated fatty acid esters are preferred, and n-hexadecene and n-hexadecane are preferred from the viewpoint of easily obtaining a sophorolipid having a single fatty acid composition. , Butyl laurate, dodecyl laurate, butyl myristate, ethyl palmitate, propyl palmitate, isopropyl palmitate, butyl palmitate, amyl palmitate, octyl palmitate, decyl palmitate or butyl stearate, more preferably n- Hexadecane, propyl palmitate, isopropyl palmitate or butyl palmitate are preferred.

Moreover, the culture medium used by this invention contains a component (B) solid oily substance.
Here, the solid oily substance is a solid oily substance at the culture temperature. The solid form includes a semi-solid form that is not liquid. As melting | fusing point of such an oily substance, 30 degreeC or more is preferable, More preferably, it is 40 to 80 degreeC, More preferably, it is 50 to 70 degreeC.
Examples of the solid oily substance include animal and vegetable oils, fatty acids, fatty acid esters, and higher alcohols, and these can be used alone or in combination of two or more.
As animal and vegetable oils and fats, for example, vegetable oils and fats such as palm oil, palm olein, palm stearin, palm oil, palm kernel oil, cocoa butter, monkey fat and shea fat, animal fats and oils such as lard, beef tallow and butter fat , Those transesterified oils, hydrogenated oils or fractionated oils. 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 free 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, Examples thereof include saturated fatty acids such as behenic acid, tricosanoic acid and lignoceric acid, and unsaturated fatty acids such as petroceric acid, elaidic acid, vaccenic acid, erucic acid, sorbic acid, linoelaidic acid and punicic acid.
Examples of the fatty acid ester include esters of the fatty acid and a monohydric to trihydric alcohol, such as octadecyl laurate, tetradecyl myristate, methyl palmitate, decyl palmitate, dodecyl palmitate, hexadecyl palmitate, Examples include methyl stearate, ethyl stearate, propyl stearate, octyl stearate, methyl arachidate, and ethyl arachidate.
As carbon number of a fatty acid part, More preferably, it is 12-24, More preferably, it is 16-22, More preferably, it is 16-18.

  Examples of the higher alcohol include myristyl alcohol, cetyl alcohol, stearyl alcohol, 2-hexadecanol, and 2-hexyldecanol.

  Among solid oily substances, fatty acids or esters thereof, higher alcohols are preferred, saturated fatty acids or esters thereof, higher alcohols are more preferred, and the number of carbon atoms in the fatty acid portion is easier to obtain a sophorolipid having a single fatty acid composition. 16-22 saturated fatty acids or esters thereof are preferred, and palmitic acid is more preferred.

In the culture medium used in the present invention, the molar amount of component (B) is 20 to 80 mol% with respect to the total molar amount of the oily substance in the culture medium. By setting it within such a range, the conversion rate from the substrate to the sophorolipid can be increased. In addition, foaming during culture can be suppressed.
Here, the total molar amount of the oily substance in the medium is the sum of the molar amount of the oily substance that is liquid at the culture temperature and the molar amount of the solid oily substance at the culture temperature. The molar amount of component (B) relative to the amount is given by the following formula (1):
{[Molar amount of component (B)] / [Total molar amount of oily substance]} × 100 (%) (1)
It is represented by
The molar amount of the component (B) with respect to the total molar amount of the oily substance in the medium is preferably 25 mol% or more, more preferably 30 mol% or more, and further 40 mol% from the viewpoint of efficient sophorolipid production. From the viewpoint of suppressing foaming, it is preferably 75 mol% or less, more preferably 70 mol% or less, and further preferably 60 mol% or less. Moreover, 25-75 mol% is preferable, 30-70 mol% is more preferable, Furthermore, 40-60 mol% is preferable.

  The initial content of the oily substance in the medium is not particularly limited, but is 1% (w / v) or more, and more preferably 5% (w / v) or more from the viewpoint of efficient sophorolipid production. Is 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.

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 as a fermentation medium of a sophorolipid, For example, commercially available solid culture media, such as a YM culture medium, or a liquid culture medium can be used.
Examples of the carbon source include sugars (glucose, arabinose, xylose, mannose, fructose, galactose, sucrose, maltose, lactose, sorbitol, mannitol, inositol, glycerin, soluble starch, waste molasses, invert sugar, etc.), acetic acid, etc. Examples include assimilated organic acids and lower alcohols such as ethanol. Especially, it is preferable to contain saccharides and also glucose from the point of proliferation and the production of sophorolipid.
The initial saccharide content 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 and reacting in a state of resting cells, that is, in a state where growth and proliferation of the microorganism are stopped. From the viewpoint of the time required for growth, it is preferable to 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, 50-200 hours are preferable as time to culture | cultivate and react in the state of a resting microbial cell.

The culture conditions may be any conditions as long as the sophorolipid is fermented and produced by a microorganism having the ability to produce the 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 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%, more preferably 18 to 45%.
The sophorolipid conversion rate is the value obtained by dividing the molar amount of sophorolipid production contained in the culture by the total amount of the oily substance substrate, that is, the initial charged oily substance charged in the medium for the fermentation production of sophorolipid. It is. 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 the following examples and comparative examples, “%” means “% (w / v)” except for mol%.

[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. To 0.01 g of the collected ethyl acetate fraction, 1 mL of methanol and 0.03 mL of concentrated hydrochloric acid were added using dodecane as an internal standard, and methanol exchange was performed in a 100 ° C. hot bath for 2 hours. After cooling, 1 mL of saturated saline and 1.5 mL of hexane were added and mixed, and the hexane phase was recovered after standing. The solvent of the obtained hexane phase was distilled off, 0.2 mL of silylating agent TMSI-H was added and silylation was performed at 70 ° C. for 20 minutes, and then 1.5 mL of water and 1.5 mL of hexane were added and mixed. After standing, the hexane phase was recovered and subjected to GC analysis.

[Calculation of Sophorolipid conversion rate]
The sophorolipid conversion rate was calculated by the following equation.
Conversion rate (%) = (Mole amount of sophorolipid contained in culture solution) / (Total amount of initially charged oily substance) × 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 by heating at 121 ° C. for 20 minutes. 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 the 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). Bacterial recovery The culture solution obtained in the main culture was centrifuged at 6,000 r / min for 20 minutes to recover the bacterial cells. After suspending in physiological saline, centrifugation was again performed at 6,000 r / min for 20 minutes to recover the cells. A resting cell reaction was performed using the collected cells.

Example 1
A medium (pH 4.0) containing 0.21 g of isopropyl palmitate (melting point: 12 ° C.) and 0.72 g of palmitic acid (melting point: 63 ° C.) as an oily substance and comprising the oily substance, glucose 10%, and 20 mM Na phosphate buffer. 10 mL was put into a test tube and sterilized at 121 ° C. for 20 minutes at a high temperature. The resting cells recovered after cooling were put into the medium so that the OD660nm was 30, and cultured for 76 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 19.1%.

Example 2
Culturing was carried out in the same manner as in Example 1 except that 0.42 g of isopropyl palmitate and 0.44 g of palmitic acid were replaced.
After culturing, the sophorolipid conversion rate was determined to be 22.9%.

Example 3
Culturing was performed in the same manner as in Example 1 except that 0.63 g of isopropyl palmitate and 0.36 g of palmitic acid were used.
After culturing, the sophorolipid conversion rate was determined to be 21.6%.

Example 4
Culture was performed in the same manner as in Example 1 except that 0.84 g of isopropyl palmitate and 0.18 g of palmitic acid were substituted.
After culturing, the conversion rate of sophorolipid was determined to be 20.4%.

Example 5
Culture was performed in the same manner as in Example 1 except that 0.16 g of isopropyl palmitate was replaced with 0.16 g of n-hexadecane (melting point: 18 ° C.) and 0.72 g of palmitic acid was used.
After culturing, the conversion rate of sophorolipid was determined to be 17.0%.

Example 6
Culture was performed in the same manner as in Example 5 except that 0.32 g of n-hexadecane and 0.54 g of palmitic acid were used.
After culturing, the conversion rate of sophorolipid was determined to be 19.7%.

Example 7
Culturing was carried out in the same manner as in Example 5 except that 0.48 g of n-hexadecane and 0.36 g of palmitic acid were replaced.
After culturing, the conversion rate of sophorolipid was determined to be 20.5%.

Example 8
Culture was performed in the same manner as in Example 5 except that 0.64 g of n-hexadecane and 0.18 g of palmitic acid were used.
After culturing, the conversion rate of sophorolipid was determined to be 21.9%.

Comparative Example 1
Culture was carried out in the same manner as in Example 1 except that the experiment was conducted with 0.90 g of palmitic acid.
After culturing, the conversion rate of sophorolipid was determined to be 14.5%.

Comparative Example 2
Culture was performed in the same manner as in Example 1 except that the experiment was performed with 1.05 g of isopropyl palmitate.
After culturing, the sophorolipid conversion rate was determined to be 17.3%.

Comparative Example 3
Culture was performed in the same manner as in Example 1 except that the experiment was performed with 0.80 g of n-hexadecane.
After cultivation, the sophorolipid conversion rate was determined to be 16.6%.

  Tables 1 and 2 show the conditions and results of the examples and comparative examples.

  As shown in Table 1 and Table 2, it is confirmed that a sophorolipid can be obtained at a high conversion rate by combining a liquid oily substance and a solid oily substance at a specific ratio at a culture temperature as a substrate. It was done. On the other hand, the conversion rate from substrate to sophorolipid is slow with only liquid oily substances or solid oily substances alone, and the amount of bubbles increases during cultivation with only solid oily substances, making it difficult to culture. It was.

Claims (7)

The following components (A) and (B):
(A) Liquid oily substance (B) A solid oily substance, and a sophorolipid in a medium in which the molar amount of component (B) is 20 to 80 mol% with respect to the total molar amount of the oily substance A method for producing sophorolipid, comprising a step of obtaining sophorolipid by fermentation using a microorganism having an ability to produce.   The method for producing a sophorolipid according to claim 1, wherein the component (A) is a liquid hydrocarbon, fatty acid or fatty acid ester.   The method for producing a sophorolipid according to claim 1 or 2, wherein the component (B) is a solid higher alcohol, fatty acid or fatty acid ester.   The melting point of a component (A) is 5 to 25 degreeC, The manufacturing method of the sophorolipid of any one of Claims 1-3.   The melting point of a component (B) is 40 to 80 degreeC, The manufacturing method of the sophorolipid of any one of Claims 1-4.   The method for producing a sophorolipid according to any one of claims 1 to 5, wherein the content of the oily substance in the medium is 1 to 20% (w / v).   The method for producing sophorolipid according to any one of claims 1 to 6, wherein the microorganism having the ability to produce sophorolipid is a microorganism belonging to the genus Candida.