JP2000135096A - Surfactant, its production and use thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a surfactant useful for separating a saturated organic component from an organic composition containing an unsaturated organic component and the saturated organic component by producing a specific aromatic compound having a surface active action by using a microorganism. SOLUTION: A suspension of soil collected from Sagara town in Shizuoka Prefecture is inoculated into an agar medium to form a colony. The colony is sprayed with petroleum and a bacterial strain for forming a halo around the colony is selected to give Bacillus BSP-1 strain (FERM P-16,956) capable of producing a surfactant. Then the BSP-1 strain is inoculated into a medium, subjected to shaking culture at 37 deg.C for 16 hours, a supernatant liquid is separated and concentrated. The concentrate is extracted with chloroform and the extract is purified by chromatography to give the objective surfactant of formula I ((n) is 3-30; (m) is 2-20; two substituent groups bonded to the benzene ring are in ortho, meta or para positional relation) or formula II ((n), (m) and position of substituent groups are the same in formula I).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an organic composition comprising:
The present invention relates to a surfactant for separating a specific component, a production method thereof, and a separation method using the surfactant.

Specifically, the present invention relates to a surfactant for separating a saturated organic component from an organic composition having an unsaturated organic component and a saturated organic component, a method for producing the same, and a surfactant. It relates to the separation method used. More specifically, the present invention relates to a surfactant for separating saturated and unsaturated fats and oils in a composition containing a plurality of fats and oils, such as naturally occurring vegetable oils, oil palm oil and palm oil, The present invention relates to a production method and a separation method using the surfactant.

[0003]

2. Description of the Related Art Generally, fats and oils applied to the food field are:
The raw material is a natural fat or oil composition such as palm oil. Here, the natural fat and oil composition is a mixture of various fats and oils and contains a large amount of saturated fats and oils having a high melting point. Therefore, there is a problem that natural fats and oils have a high melting point in the mixed fats and oils in a consuming area where the temperature is low, even in a homogeneous solution in a place of origin where the temperature is high.

Conventionally, for example, so-called natural separation operation has been performed for palm oil. In this method, at a cooling temperature (typically −10 ° C. or lower) set according to the melting point of each component of the fat and oil composition, a high melting point saturated fat and oil is precipitated over a long period of several weeks, The product is separated from the liquid oil by filtration.

However, this method does not sufficiently separate high-melting-point saturated fats and oils. For this reason, there still remains a problem that the high melting point saturated fats and oils easily precipitate in the oil layer.
This method also has the disadvantage that the amount of energy required is huge and costly.

Therefore, in order to solve the problem of the precipitation of the high melting point saturated fat and oil component, a new method for selectively separating the high melting point saturated fat and oil component in advance is desired.

As another method of solving this problem, a method of adding a low melting point substance is known. This is a method in which another fat or oil component (sesame oil, falling flower oil, etc.) having a melting point lower by about 5 to 10 ° C. than the desired melting point in the final fat or oil product is mixed with the raw fat or oil composition by about 30 to 40% by volume ratio. . But,
The decrease in melting point (cloud point) in this method is 2-3 ° C., which is still not sufficient. Furthermore, such a method has a disadvantage that the original characteristics of fats and oils are lost. Therefore, development of a separation method free from such disadvantages is desired.

In the above description, palm oil has been described, but the situation is the same for other natural fats and oils.

[0009]

SUMMARY OF THE INVENTION In the first aspect,
An object of the present invention is to provide a surfactant for easily separating a saturated organic component from an organic composition having an unsaturated organic component and a saturated organic component.

[0010] In a second aspect, the present invention aims to provide a microorganism producing such a surfactant.

[0011] In a third aspect, the present invention aims to provide a method for producing such a surfactant.

In the fourth and fifth aspects, an object of the present invention is to provide a method for separating a saturated organic component from an organic composition having an unsaturated organic component and a saturated organic component by a simple operation. And For example, it is an object of the present invention to provide a method for separating a saturated oil or fat from an organic composition which is a mixture of an unsaturated oil or fat and a saturated oil or fat to lower the cloud point of the organic composition.

[0013]

In a first aspect, the surfactant of the present invention is used for separating a saturated organic component from an organic composition containing an unsaturated organic component and a saturated organic component. A surfactant represented by the following formula I or II: Formula I: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n OH Formula II: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n H where n is 3 to 30, m is 2 to 20, and the positional relationship between the two substituents bonded to the benzene ring is
Ortho, meta, or para.

[0014] In the second aspect, the microorganism of the present invention comprises:
This is the microorganism BSP-1 strain (Accession No. FERMP-16956).

[0015] In a third aspect, the production method of the present invention uses a microorganism to obtain a compound of the formula I: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n OH or the formula II: H (CH ₂ ) a surfactant represented by _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n H (where n is 3 to 30,
m is 2-20 and the positional relationship of the two substituents attached to the benzene ring is ortho, meta or para).

[0016] In one embodiment, the microorganism is
BSP-1 strain.

[0017] In a fourth aspect, the method of the present invention comprises:
Organic composition with saturated organic components and saturated organic components
A method for separating the saturated organic component from the product,
(A) Formula I: H (CH_Two)_mC₆H_Four(OCH_TwoCH_Two)_nOH or formula I
I: H (CH_Two)_mC₆H_Four(OCH_TwoCH_Two) _nSurfactant represented by H
(Where n is 3-30, m is 2-20,
And the positional relationship between the two substituents attached to the benzene ring
Is ortho, meta, or para).
And (B) adding the saturated organic component to the
And a layer mainly containing the unsaturated organic component
And the step of separating into

In one embodiment, steps (A) and (B) are performed under non-hydrous conditions.

In one embodiment, the method further comprises (D) contacting the cyclic lipopeptide with the organic composition after the step (A) and before the step (B).

In a fifth aspect, the method of the present invention is a method of separating a saturated organic component from an organic composition in which an unsaturated organic component and a saturated organic component are present,
(A ′) Cyclic lipopeptide or formula I: H (CH ₂ ) _m C ₆ H ₄ (OCH
₂ CH ₂ ) _n OH or a surfactant represented by the formula II: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n H (where n is 3 to 30,
Is 2 to 20, and the positional relationship of the two substituents bonded to the benzene ring is ortho, meta, or para) with the organic composition. (C) the unsaturated organic component And a step of cooling the crystallization temperature of the saturated organic component to not higher than the crystallization temperature of the higher component,
And (B) a step of separating into a layer mainly containing the saturated organic component and a layer mainly containing the unsaturated organic component,
Is included.

In one embodiment, the step (B) further comprises the step (B ′) of performing a centrifugal separation at a temperature not higher than room temperature and not lower than a temperature at which the entire organic composition is solidified, thereby obtaining the saturated organic component. And a layer mainly containing the unsaturated organic component.

In one embodiment, the method further includes, after the step (B) or (B ′), (E) a step of squeezing the liquid in the layer mainly containing the saturated organic component.

[0023] In one embodiment, the cyclic lipopeptide is arthrofactin.

[0024] In one embodiment, the organic composition is a mixture of a saturated fat and an unsaturated fat.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS << First Aspect >> The first aspect of the present invention will be described below.

The first aspect of the present invention relates to a surfactant.

The surfactant of the present invention has the formula I: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n OH or the formula II: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂₎ a surfactant represented by _n H.

In the present specification, the surfactant of the formula I is also referred to as "B-BS". B-BS may be produced biologically or may be chemically synthesized. Biological production has the advantage of low production costs.

The biological production method of B-BS will be described later in detail.

As a chemical synthesis method of the compound of the formula I, for example, there is a method in which ethylene oxide is addition-polymerized to the active hydrogen of phenol of an alkylphenol in the presence of an alkali catalyst.

The compounds of formula II may be produced biologically or may be synthesized chemically. As a chemical synthesis method of the compound of the formula II, for example, there is a method of reducing the terminal hydroxyl group of the compound of the formula I by a known method.

N in the above formula I or II must be 3 or more, preferably 5 or more;
More preferably, it is 7 or more. Further, it is necessary to be 30 or less, preferably 20 or less, and more preferably 15 or less. It should be noted that n in Formula I and Formula I
N in I is independently selected.

M in the above formula I or II needs to be 2 or more, preferably 4 or more, more preferably 5 or more. Further, it is necessary to be 20 or less, preferably 15 or less, more preferably 10
It is as follows. Note that m in Formula I and m in Formula II are:
Independently selected.

The combination of m and n is, specifically,
For example, those in which m is 4 to 15 and n is 5 to 20 are preferable, and those in which m is 5 to 10 and n is 7 to 15 are more preferable.

The positional relationship between the two substituents bonded to the benzene ring in the above formula I or II may be any of ortho, meta and para. It is preferable because of good interpenetration.

The molecular weight of the compound of the formula I or II is preferably at least 100, more preferably at least 200, even more preferably at least 300. Further, it is preferably 2000 or less, more preferably 1500 or less, and further preferably 1200 or less.

The HLB of the compound of formula I or II is preferably at least 0.3, more preferably at least 0.5, even more preferably at least 1.0. The HLB is also preferably less than or equal to 4.0, more preferably 2.0
Or less, and more preferably 1.5 or less.

The compounds of formula I or formula II CMC (critical micelle concentration), preferably is a 10 ^{-1 0} M or more, more preferably not 10 ^-8 M or more, more preferably,
10 ^-6 M or more. The CMC is also preferably 1 M or less, more preferably 10 ^-2 M or less, even more preferably 10 ^-4 M or less.

Here, HLB means Hydrohile / Lipoph
Abbreviation of ile Balance, (molecular weight of hydrophilic portion) /
(Molecular weight of hydrophobic part). CMC is measured as the inflection point of a graph of surface tension versus the common logarithm of the surfactant concentration of the invention.

The function of the surfactant of the present invention will be described below.

The surfactant of the present invention has a specific affinity in a mixture of a saturated organic substance and an unsaturated organic substance, which has a preferential affinity with a saturated organic substance component. In particular,
It has a property of easily entering between a saturated organic substance and an unsaturated organic substance and promoting aggregation of the saturated organic substance. For this reason, the saturated organic component is in a state surrounded by the surfactant. At this time, the saturated organic component undergoes a change in an apparent specific gravity increase due to being surrounded by the surfactant. Due to the increase in apparent specific gravity, saturated organic components and
There is a difference in apparent specific gravity between the unsaturated organic components.
Thereafter, if the mixture is left standing or centrifuged, the saturated organic component surrounded by the surfactant sinks due to the apparent specific gravity difference.
By this action, the saturated organic component and the unsaturated organic component can be easily and efficiently separated. Details of the separation method will be described later.

<< Second Aspect >> Hereinafter, the second aspect of the present invention will be described.

The second aspect of the present invention relates to a microorganism producing B-BS.

The organism producing B-BS includes, for example, a novel microorganism BSP-1 strain isolated by the present inventors. The BSP-1 strain was found to belong to a new species belonging to the genus Bacillus based on its mycological properties and comparison of the nucleotide sequence of its 16S rRNA with other sequences (see Example 1A). The BSP-1 strain has been deposited with the National Institute of Bioscience and Human Technology on August 25, 1998, and its accession number is FERM P-16956.

Further, as a screening method for a microorganism having the same B-BS producing ability as the above-mentioned deposited strain, the following method is possible. Microorganisms in a sample obtained from the natural environment (eg, soil) are inoculated on a suitable agar medium. The agar medium is incubated to allow colonies to form. The formed colonies are sprayed with oil. Isolate the microorganisms with halos around the colonies.

<< Third Aspect >> The third aspect of the present invention will be described below.

The third aspect of the present invention relates to a method for producing B-BS biologically.

The production of B-BS is carried out by culturing an organism producing B-BS, for example, the BSP-1 strain, and recovering the B-BS from the culture. Culture conditions are selected to be appropriate for the growth of B-BS producing organisms and B-BS production. For example, in the case of the BSP-1 strain, LB medium (10 g of tryptone, 5 g of yeast extract,
After culturing with shaking in 5 g of NaCl (pH 7.0) for 40 hours, the culture solution is centrifuged, and B-BS is purified from the obtained supernatant. Purification is performed using any of the purification methods available in the art (eg, salting out method, solvent precipitation method, ultrafiltration, isoelectric point precipitation, electrophoresis, ion exchange chromatography, gel filtration, affinity chromatography, etc.) alone or in combination. It is implemented by doing.

<< Fourth Aspect >> Hereinafter, the fourth aspect of the present invention will be described.

The fourth aspect of the present invention relates to a method for separating a saturated organic component from an organic composition containing an unsaturated organic component and a saturated organic component using B-BS.

1. Materials Used 1) Organic Composition The fourth aspect of the present invention is applicable to any organic composition containing an unsaturated organic component and a saturated organic component. In particular, it is suitably used for natural oils and fats such as animal oils, vegetable oils, fish oils and mineral oils, and compositions mainly containing purified and modified products thereof. Specifically, it is preferably applicable to a composition in which 60% or more of the composition is a component derived from natural fats and oils, and more preferably applicable to a composition in which 80% or more of the composition is a component derived from natural fats and oils. is there.

As the organic composition, typically, a mixed fat or oil which is a mixture of triglycerides can be used in the fourth aspect of the present invention. In particular, various types of fats and oils such as natural products, for example, animal products, fish products, and mineral products are suitable.

2) Unsaturated Organic Component The unsaturated organic component refers to an organic compound having a carbon-carbon ethylenically unsaturated bond in the molecule. Typically, unsaturated fats and oils are mentioned.

The term "unsaturated fat" refers to a glycerin ester (triglyceride) having at least one unsaturated fatty acid residue. As unsaturated fatty acids, those having 6 to 24 carbon atoms are common, and specifically, palmitoleic acid, oleic acid, linoleic acid, linolenic acid, gadrenic acid (eicosenoic acid), erucic acid (docosenoic acid), and the like. There is.

3) Saturated Organic Substance Component The saturated organic substance component is an organic substance having no carbon-carbon ethylenically unsaturated bond in the molecule. Typically, saturated fats and oils are mentioned. Here, the saturated fat means a glycerin ester of only a saturated fatty acid. Here, as the saturated fatty acid, one having 6 to 24 carbon atoms is generally used. , Behenic acid and lignoceric acid.

4) Surfactant (Compound of Formula I or Formula II) The surfactant used in the fourth aspect of the present invention includes:
Formula _{I: H (CH 2) m} C 6 H 4 (OCH 2 CH 2) n OH or the formula II: H (CH ₂₎
a surfactant represented by _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n H (where n
Is 3-30, m is 2-20, and the positional relationship of the two substituents attached to the benzene ring is ortho, meta, or para). As compounds of formula I or formula II used in this aspect of the invention, the surfactants mentioned above with respect to the first and second aspects of the invention may be used.

5) Cyclic lipopeptide Cyclic lipopeptide refers to a compound containing a lipid component and a cyclic peptide component, and usually comprises a lipid component and a cyclic peptide component. Cyclic lipopeptides are both naturally occurring and chemically synthesized, and both can be used in the separation methods of the present invention.

As natural cyclic lipopeptides, arthrofactin and its homologues, surfactin and its homologues, iturin and selawettin are known. The structures of these cyclic lipopeptides are shown below.

[0059]

Embedded image

Here, the homologue of arthrofactin refers to a natural compound having a different carbon number in the lipid component in arthrofactin. Specifically, those having 8 to 12 carbon atoms are known. A homologue of surfactin refers to a natural compound in which the lipid component in surfactin has a different number of carbon atoms.

For the production method of the cyclic lipopeptide,
For example, JP-A-6-184194 discloses a production method using bacteria of the genus Arthrobacter.

[0062] Arthrobacter sp. No. Thirty-eight strains are strains that have been certified by the Japan Institute of Microbiology No. 13198.

The cyclic lipopeptide may also be a chemically synthesized product. Chemically synthesized products can be synthesized, for example, by sequentially linking amino acids to functional groups of lipid components and then closing the ring. For sequential binding of amino acids, solid phase synthesis or liquid phase synthesis of peptides is applied. It can be done by doing.

The cyclic lipopeptide may be a substance classified as a biosurfactant. Biosurfactant generally refers to a substance produced by an organism inside or outside a cell or a substance derived from a substance produced inside or outside a cell by a living organism, and refers to a surfactant having a hydrophilic portion and a hydrophobic portion. Are classified into higher animal and plant surfactants and microbial surfactants. Microbial surfactants are classified into glycolipids, amino acids (acylheptides), phospholipids, fatty acids, and macromolecules based on the structure of the hydrophilic portion. Biosurfactants that can be used as cyclic lipopeptides include glycolipid surfactants such as trehalose lipid, rhamnoslipid, and sophorose lipid, and other amino acid-based compounds having an oligopeptide as a hydrophilic partial structure. Surface active substances. Arthrofactin and its homologues described above as cyclic lipopeptides, surfactin and its homologues, iturin, and serawetin are surfactants produced by microorganisms as amino acid-based biosurfactants, and are preferred examples of cyclic lipopeptides. is there.

2. Each Step of Separation Method <Step (A): Contacting Step> In the step (A), a compound of the formula I or II is added to the organic composition.

First, an organic composition is prepared. When the melting point of the organic composition is high, it is preferable to heat the organic composition to a temperature higher than the melting point.

The compound of the formula I or II is usually produced in the form of an aqueous solution, and the aqueous solution of the compound of the formula I or II in the form of an aqueous solution can be added to the organic composition as it is. If necessary, the density may be adjusted and used. When adjusting the concentration, 1 mg / l to 5 mg
g / l, preferably from 10 mg / l to 500 mg /
More preferably, the concentration is 1. Furthermore, when a pure compound of the formula I or II is produced, the pure compound of the formula I or II may be added as it is.

In this contacting step, if necessary,
Water may be added. However, in a preferred embodiment, the organic composition can be contacted with the compound of formula I or II without the addition of water, ie under non-hydrated conditions. When performed under non-hydrous conditions, better separation efficiency is easily obtained.

The added amount of the compound of the formula I or II is preferably
Preferably, unsaturated organic components and saturated organic components in the organic composition
Approximately 1 × 10 per gram of total amount of ingredients^-6g to about 1 ×
10 ^-2g, more preferably the unsaturated content in the organic composition.
Per gram total amount of organic and saturated organic components
About 1 × 10^-Fiveg to about 1 × 10^-3g, even more preferred
Or unsaturated organic components and organic components in organic compositions
Approximately 5 × 10 per gram^-Fiveg to about 3 × 10
^-Fourg.

By adding the compound of the formula I or II and stirring appropriately, an organic composition in an emulsified state is formed. The stirring time is preferably about 1 minute to 1 hour,
More preferably, it is for about 3 to 20 minutes.

The addition is also possible at room temperature. When the melting point of the organic composition is high, it is preferable to perform the heating while heating to a temperature near or above the melting point.

Mixing Container Any known mixing container can be used in the present invention. The mixing vessel desirably has a temperature control function in the vessel, a stirring function, and a lower outlet. The temperature control function in the container refers to, for example, a function of flowing hot or cold water through a pipe installed around the container. If there is a temperature control function in the container, temperature control during operation is easy. If there is a lower outlet, it is easy to take out only the lower layer after the liquid is separated.

Before the stirring operation, it is preferable to remove the oxygen component in the material by reducing the pressure. It is preferable that nitrogen gas is blown into the liquid layer during the stirring from the bottom of the container to suppress deterioration of the organic composition due to oxidative deterioration.

The stirring is preferably performed at a low speed. Specifically, about 30 rpm to 300 rpm is preferable, and about 50 rpm to 200 rpm
Is more preferred. The structure of the stirring blade is preferably a simple propeller structure. If the stirring speed is too rapid or the stirring causes excessive stress in the liquid, the organic composition is likely to deteriorate.

The stirring after mixing is preferably performed for about 1 minute to 1 hour, more preferably for about 3 minutes to 30 minutes.
During the stirring, it is preferable to keep the temperature higher than the melting point of each component in the container. Specifically, it is preferable to keep the temperature at, for example, 40 to 60 ° C.

<Step (B): Separation Step> In the separation step of step (B), the mixture is separated into a layer mainly containing a saturated organic component and a layer mainly containing an unsaturated organic component. Here, “mainly containing a saturated organic component” means that the ratio of the saturated organic component to the unsaturated organic component is higher than that of the organic composition before the separation step. “Mainly containing unsaturated organic components” means that the ratio of unsaturated organic components to saturated organic components is increased as compared to the organic composition before the separation step.

The separation method includes, for example, stationary separation and centrifugation, both of which can be used.

Static separation For example, after stirring is stopped, the liquid is separated by standing. The standing time is preferably about 10 minutes to 1 minute.
Days, more preferably about 30 minutes to 15 hours, even more preferably about 1 to 10 hours. Usually, the upper liquid layer after the standing separation is an oil layer, which contains more abundant unsaturated components.

Centrifugation A known centrifuge can be used for centrifugation. The rotation speed is preferably at least 1,000 rpm, more preferably at least 3,000 rpm, and even more preferably at least 10,000 rpm. The rotation speed is also preferably not more than 100,000 rpm, more preferably 50,000 rp
m, and more preferably 30,000 rpm or less. The acceleration is preferably 100 G or more, more preferably 300 G or more, and even more preferably 500 G or more. The acceleration is preferably 50,000 G or less, more preferably 30,000 G or less, and still more preferably 10,000 G
0G or less. If the rotation speed or acceleration is low,
It is difficult to obtain a sufficient separation effect. If the rotation speed or the acceleration is too high, facility problems are likely to occur.

The time for performing centrifugation is preferably 10 seconds or more, more preferably 1 minute or more, and further preferably 5 minutes or more. The time for performing the centrifugation is preferably 1 hour or less, and more preferably 30 hours.
Minutes or less, and more preferably 20 minutes or less. If the time for centrifugation is short, it is difficult to obtain a sufficient separation effect. Conversely, if the length is long, the efficiency of the entire process is likely to decrease.

When the centrifugation is completed, the uppermost liquid layer of the centrifuge tube usually becomes an oil layer, and contains more abundant unsaturated components. Then, a crystal layer is formed thereunder, and this crystal layer contains abundant saturated fatty acids. Further, when centrifugation is performed in the presence of water, an aqueous layer is formed below the crystal layer.

After separation, the separated oil layer is subjected to a known method,
For example, a liquid rich in a desired unsaturated organic component can be taken out by decantation or the like. Further, a liquid rich in a desired saturated organic component can be taken out of the separated aqueous layer by a known method, for example, discharging from a lower outlet of a container.

If necessary, by repeating the above operations (A) and (B) with respect to either the oil layer or the aqueous layer obtained here under different temperature conditions, components having different melting points can be obtained. It can be further separated.

Here, the degree of separation of the saturated organic component can be adjusted by appropriately adjusting the conditions such as the contact time, the temperature at the time of contact, the concentration of the surfactant solution, the amount of the surfactant, and the separation time. That is, by adjusting the above conditions, the iodine value and the melting point of the organic composition can be adjusted to desired values.

<Step (D): Further Contacting Step> In the separation method of the present invention, the effect can be sufficiently obtained only by the above steps (A) and (B). By providing it after A), the separation efficiency can be further improved.

The cyclic lipopeptide used in the step (D) is
The hydrophilic portion is a cyclic structure composed of a plurality of chemical partial structures such as amino acids, monosaccharides and the like, which is very different from a so-called ordinary surfactant. Due to this characteristic, the cyclic lipopeptide exhibits a specific and strong affinity for various organic substances (for example, saturated fats and oils) to which the compound of the formula I or II is adsorbed. Therefore, step (A)
In step (D), if the cyclic lipopeptide is added after the compound is adsorbed by the compound of the formula I or II in the step (D), the cyclic lipopeptide further acts on the saturated organic component to make the saturated organic component incompatible with the saturated organic component. The action of separating from the saturated organic component is promoted.

However, since the cyclic lipopeptide may not show strong affinity to fats and oils to which the compound of the formula I or II is not added, if the step (D) is performed before the step (A), It is difficult to obtain a sufficient separation effect.

In the step (D), the above-mentioned cyclic lipopeptide is brought into contact with an organic composition. As a method for contacting, any known method can be used. Specifically, a method in which both are mixed and stirred in a container is preferable.

The cyclic lipopeptide is an aqueous solution of any concentration dissolved in pure water, if necessary. For example, pure powder of arthrofactin is dissolved in a buffer to obtain an aqueous solution. As the buffer, for example, a solution in which a salt containing sodium phosphate as a main component is dissolved at 10 ⁻⁶ M can be used.

The amount of the cyclic lipopeptide to be added is preferably about 1 × 10 ⁻⁶ g to about 1 × 10 ⁻² g per gram of the total amount of the unsaturated organic component and the saturated organic component in the organic composition.
And more preferably about 1 × 1 per gram of the total amount of the unsaturated organic component and the saturated organic component in the organic composition.
0 ^-5 g to about 1 × 10 ⁻³ g, and more preferably about 5 × 10 ⁻⁵ g to about 3 × 10 3 per gram of the total amount of the unsaturated organic component and the saturated organic component in the organic composition. ^-4 g.

<< Fifth Aspect >> The fifth aspect of the present invention will be described below.

The fifth aspect of the present invention relates to a method for separating a saturated organic component from an organic composition containing an unsaturated organic component and a saturated organic component by utilizing a cooling step.

<Step (A ′): Contacting Step> In this aspect, first, a cyclic lipopeptide or a compound of the formula I or II is brought into contact with an organic composition containing an unsaturated organic component and a saturated organic component. .

Here, an organic composition, a cyclic lipopeptide,
As the compound of the formula I or II, those described above can be used. When the compound of the formula I or II is brought into contact with the organic composition, a method similar to the above step (A) is possible. When the cyclic lipopeptide is brought into contact with the organic composition, the same method as in the above step (D) can be used.

The cyclic lipopeptide and the compound of the formula I or II may be used alone or in combination. When used in combination, the two may be mixed and used at once, or the contacting step may be provided in two stages.

In one preferred embodiment, first, formula I
Alternatively, the step of contacting the compound of formula II is performed, followed by the step of contacting the cyclic lipopeptide. In this embodiment, better separation efficiency is obtained.

<Step (C): Cooling Step> In the fifth aspect of the present invention, of the unsaturated organic component and the saturated organic component, the component having the higher crystallization temperature (hereinafter referred to as “highly crystalline”) (Hereinafter referred to as “crystallization temperature component”). In the case of normal mixed fats and oils,
The high crystallization temperature component is a saturated fat component. The crystallization temperature of the high crystallization temperature component is a temperature at which the high crystallization temperature component crystallizes, and is easily measured by an analyzer such as a differential scanning calorimeter (DSC).

In this step, crystallization of the high crystallization temperature component proceeds by cooling. During crystallization, separation from low crystallization temperature components proceeds. Thereby, the separation efficiency is improved.

Here, the cooling temperature is equal to or lower than the crystallization temperature of the high crystallization temperature component, but may be lowered to the crystallization temperature of the low crystallization temperature component.

The specific cooling temperature is preferably -10 ° C. or lower, more preferably -15 ° C. or lower, and more preferably -15 ° C. or lower in the case of a normal edible oil / fat composition such as palm oil. -20 ° C or less. The cooling temperature is also preferably −50 ° C. or higher, and −40 ° C.
More preferably, it is the above.

The cooling rate is preferably 5 ° C. or more per hour, more preferably 10 ° C. or more per hour.
More preferably, the temperature is 20 ° C. or more per hour. The cooling rate is preferably 200 ° C. or less per hour,
The temperature is more preferably 150 ° C. or less per hour, and even more preferably 100 ° C. or less per hour.

The time for maintaining the cooling state is preferably 1
Minutes or more, more preferably 5 minutes or more, and still more preferably 10 minutes or more. The time for maintaining the cooling state is also preferably 2 hours or less, more preferably 1 hour or less, and further preferably 30 minutes or less.

It is preferable that stirring is continued even during cooling.

<Step (B ′): Low-Temperature Centrifugation Step> In one embodiment of the present invention, centrifugation is performed at a temperature of not higher than room temperature and not lower than the temperature at which the entire organic composition solidifies, so that the saturated organic components can be removed. It is separated into a layer mainly containing and a layer mainly containing unsaturated organic components. By performing centrifugation under such temperature conditions, the separation efficiency is further increased.

Specifically, the temperature at which centrifugation is performed is
Preferably, it is about 15 ° C. or less, more preferably about 10 ° C. or less, even more preferably about 5 ° C. or less. If the temperature is too high, it is difficult to obtain sufficient separation efficiency.
Further, the temperature is preferably about -15 ° C or more, more preferably about -10 ° C or more, and still more preferably about -5 ° C or more.

<Step (E): Squeezing Step> In one embodiment of the present invention, after the step (B '), the liquid in the layer mainly containing the saturated organic component is further squeezed.

Here, as a method of pressing the crystal layer,
For example, there is a filtration method and the like. Specifically, it is preferable to put on a filter paper and perform suction filtration or pressure filtration.

[0108]

Next, non-limiting examples of the present invention will be described.

Example 1 First, a method for producing a surfactant of the present invention will be described.

Example 1A Isolation and Properties of B-BS Producing Bacteria A surfactant-producing microbial strain was newly isolated from soil in Sagara-cho, Shizuoka Prefecture. The screening was performed using the surfactant-producing ability as an index as follows.
The soil suspension was inoculated on an LB agar medium to form a colony. Next, petroleum was sprayed on the formed colonies, and strains that formed halos around the colonies were selected.

One strain was selected and its bacteriological properties were examined.

(A) Morphological properties Size: 0.3-0.5 × 2-3 μm Shape: Bacillus Polymorphism: 10 or more may be continuous (b) Cultural properties Growth medium: LB medium (10 g of tryptone per liter) , Yeast extract 5g, and NaCl 5g (pH 7.0)) (c) Physiological properties Gram stainability: positive Catalase: positive Starch hydrolysis: negative Growth range: as described in Table 1

[0113]

[Table 1]

(D) Other properties Protease (substrate: gelatin, casein): positive (e) Chemical properties 16S rRNA homology with closely related species: as described in Table 2.

[0115]

[Table 2]

The gene encoding 16S rRNA was obtained by PCR using a primer DNA designed from a highly conserved sequence region. The nucleotide sequence was determined by the dideoxy method. The determined 16S rRNA gene sequence is shown in SEQ ID NO: 1 in the sequence listing. The determined nucleotide sequence is
The BLASTN program was used to compare to known 16S rRNA gene sequences registered in the GenBank database. As a result, the isolated strain belongs to the genus Bacillus,
It was suggested to be closely related to us licheniformis and Bacillus subtilis.

However, the results, such as being able to grow at 55 ° C. and not producing amylase, suggest that this strain is classified as a new species. This strain is called BSP
-1 stock. The BSP-1 strain was deposited on August 25, 1998 with the Institute of Biotechnology and Industrial Technology,
Its accession number is FERM P-16956.

Example 1B: Production of B-BS B-BS was produced as follows. BSP-1 strain was inoculated into LB medium and incubated at 37 ° C.
For 16 hours. Then, the culture solution was 8,000 × g
For 20 minutes to obtain a supernatant. This supernatant was subjected to ultrafiltration using a filter having a molecular fraction of 10,000 and concentrated 10-fold. The concentrated solution was extracted with chloroform, and the chloroform phase was subjected to silica column chromatography (chloroform: methanol: distilled water = 180: 19: 1) to roughly purify. This crude product was further subjected to reverse phase HPLC (5C18-AR,
Nacalai Tesque). The conditions of the reverse phase HPLC are as follows. 0 min: 20% acetonitrile, 80% distilled water; 0-20 min: linear gradient; 20 min-: 100% acetonitrile.

Example 2 Hereinafter, an example of the method for separating organic components according to the present invention will be described.

Here, the B-BS prepared in Example 1 was prepared, dissolved in water, and used at a concentration of 100 mg / l. (Hereinafter, this solution is referred to as B-BS solution.) Cyclic lipopeptide is arthrofactin (molecular weight: 135
The powder of 4) was prepared, dissolved in water, and used at the concentrations described below. (Hereinafter, this solution is referred to as an Arthrofactin solution.) (Example 2A-1) 4.0 ml of linoleic acid (carbon number 16, double bond number 0) and palmitic acid (carbon number 18, double bond number 2) 1.6 g was mixed well at 65 ° C. to obtain a sample mixture.
The unsaturated fatty acid in the mixture was 68.0%.

To the mixed fat / oil was added 200 μl of the B-BS solution.

40.0 ml of hard water (pH about 8 to 9) containing CaCl ₂
Was added.

At a temperature above about 40 ° C., mixing was carried out for 10 minutes.

While the entire system was emulsified, 300 μl of an arthrofactin solution (15 mg / l) was added.

The whole system was quenched to about 0 ° C. while mixing gently, and ice grains were added to form seed crystals.

After centrifugation at 15,000 rpm for 15 minutes at about 4 ° C., the composition of the supernatant was analyzed by capillary gas chromatography. As a result, it was 86.1% of unsaturated fatty acids.

(Example 2A-2) The same procedure as in Example 2A-1 was carried out except that the addition of the arthrofactin solution was not carried out. As a result, the unsaturated fatty acid in the supernatant was 87.5%.

The conditions and results of Examples 2A-1 and 2A-2 are shown in FIG.

Example 2B-1 2.0 ml of linoleic acid and 0.5 g of palmitic acid were sufficiently mixed at 65 ° C. to prepare a sample mixture. The unsaturated fatty acid in the mixture was 77.3%.
The saturated fatty acid was 22.7%.

100 μl of the B-BS solution was added to the mixed oil and fat.

6.0 ml of hard water (pH about 8 to 9) containing CaCl ₂ was added.

At a temperature above about 40 ° C., mixing was carried out for 10 minutes.

The whole system was quenched to about 0 ° C. while mixing gently, and ice particles were added to form seed crystals.

The mixture was centrifuged at about 4 ° C. and 15,000 rpm for 15 minutes. As a result, the mixture was separated from the top into three layers: a supernatant layer, a crystal layer, and a water tank. The composition of the supernatant layer and the crystal layer was analyzed by capillary gas chromatography. As a result, the unsaturated fatty acid in the supernatant layer was 88.8%, and the saturated fatty acid in the crystal layer was 38.7%.

(Example 2B-2) The same operation as in Example 2B-1 was performed except that hard water was not added. As a result, the unsaturated fatty acid in the supernatant was 84.7%, and the saturated fatty acid in the crystal layer was 47.5%.

The conditions and results of Examples 2B-1 and 2B-2 are shown in FIG.

(Example 2C-1) 2.0 ml of linoleic acid and 0.5 g of palmitic acid were sufficiently mixed at 65 ° C. to prepare a sample mixture.

100 μl of the B-BS solution was added to the mixed oil and fat.

At a temperature above about 40 ° C., mixing was carried out for 10 minutes.

The whole system was quenched to about 0 ° C. with gentle mixing, and ice grains were added to form seed crystals.

After centrifugation at room temperature (25-30 ° C.) at 4,500 rpm for 15 minutes, the composition of the supernatant layer and the crystal layer was analyzed by capillary gas chromatography. As a result, the unsaturated fatty acid in the supernatant layer was 84.7%, and the saturated fatty acid in the crystal layer was 47.5%.

(Example 2C-2) The linoleic acid used was converted to 6.
0 ml, used palmitic acid to 1.5 g, and used B-BS
The solution was made 300 μl, and centrifugation was performed at 15,000 rp at about 4 ° C.
The procedure was performed in the same manner as in Example 2C-1 except that the procedure was performed at m for 15 minutes.
As a result, the unsaturated fatty acid in the supernatant was 96.1%, and the saturated fatty acid in the crystal part was 61.9%.

The conditions and results of Examples 2C-1 and 2C-2 are shown in FIG.

Example 2D-1 6.0 ml of linoleic acid and 1.5 g of palmitic acid were sufficiently mixed at 65 ° C. to prepare a sample mixture.

0.3 ml of the B-BS solution was added to the sample mixture.

At a temperature above about 40 ° C., mixing was carried out for 10 minutes.

While the whole system was emulsified, 0.5 ml of an arthrofactin solution (15 mg / l) was added.

The whole system was quenched to about −30 ° C. while mixing gently, and ice grains were added to form seed crystals.

After centrifugation at 15,000 rpm for 15 minutes at about 4 ° C., the composition of the supernatant layer and the crystal layer was analyzed by capillary gas chromatography. As a result, the unsaturated fatty acid in the supernatant layer was 97%, and the saturated fatty acid in the crystal layer was 62%.

Further, filtration was performed to obtain a crystal pressing layer. The saturated fatty acid in the crystal pressing layer was 97%.

(Example 2D-2) The same procedure as in Example 2D-1 was carried out except that the B-BS solution was not added. As a result, the amount of the supernatant of the unsaturated fatty acid was small, and the saturated fatty acid in the crystal part was 23%.

(Example 2D-3) The same operation as in Example 2D-1 was performed except that the cooling temperature was changed to 0 ° C. instead of -30 ° C. As a result, the unsaturated fatty acid in the supernatant was 98%, the saturated fatty acid in the crystal part was 62%, and the saturated fatty acid in the crystal pressed layer was 99%.

(Example 2D-4) The same operation as in Example 2D-1 was carried out except that the B-BS solution was not added and the cooling temperature was changed to 0 ° C. instead of -30 ° C. As a result, the supernatant volume of the unsaturated fatty acid was small, and the saturated fatty acid in the crystal part was 23%.
%Met.

The conditions and results of Examples 2D-1 to 2D-4 are shown in FIG.

[0155]

According to the method of the present invention, a saturated organic component can be separated from an organic composition having an unsaturated organic component and a saturated organic component by a simple operation. For example, it is easy to separate the saturated fat from the organic composition which is a mixture of the unsaturated fat and the saturated fat, and to lower the melting point of the organic composition. Further, according to the present invention, the melting point and the iodine value of the fat or oil composition can be adjusted to desired values.

[0156]

[Sequence List] SEQUENCE LISTING <110> Imanaka, Tadayuki <120> Surfactant, method for producing the same, and method for using the same <130> J1-99432220 <150> JP P1998-244115 <151> 1998-08- 28 <160> 1 <170> PatentIn Ver. 2.0 <210> 1 <211> 1553 <212> DNA <213> BSP-1 <400> 1 tttatcggag agtttgatcc tggctcagga cgaacgctgg cggcgtgcct aatacatgca 60 agtcgagcgg acagatggga gcttgcgcggctagcggctagcggctagcggcggctagcggcgcagcggcgcagcggcggcagcggggcgggagc actgggataa ctccgggaaa ccggggctaa taccggatgg 180 ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt acagatggac 240 ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcgtagccga 300 cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 360 gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 420 aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt cgaatagggc 480 ggtaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 540 atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 600 ttaagtctga tgtgaaagcc cccg gctcaa ccggggaggg tcattggaaa ctggggaact 660 tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 720 ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt 780 ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 840 ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt 900 acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 960 tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct 1020 agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc 1080 gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1140 cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1200 gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa 1260 gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag 1320 tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1380 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1440 aagtcggtga ggtaaccttt taggagccag ccgcc gaagg tgggacagat gattggggtg 1500 aagtcgtaac aaggtagccg tatcggaagg tgcggctgga tcacctcctt tct 1553

[Brief description of the drawings]

FIG. 1 is a table and a graph showing conditions and results of Examples 2A-1 and 2A-2.

FIG. 2 is a table and a graph showing conditions and results of Examples 2B-1 and 2B-2.

FIG. 3 is a table and a graph showing conditions and results of Examples 2C-1 and 2C-2.

FIG. 4 is a table and a graph showing conditions and results of Examples 2D-1 to 2D-4.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:07) (C12N 1/20 C12R 1:07) (C12P 7/00 C12R 1:07)

Claims (12)

[Claims] 1. A surfactant used for separating a saturated organic component from an organic composition containing an unsaturated organic component and a saturated organic component, wherein the surfactant is represented by the following formula I or II. : formula _{I: H (CH 2) m} C 6 H 4 (OCH 2 CH 2) n OH formula _{II: H (CH 2) m} C 6 H 4 (OCH 2 CH 2) n H , where, n is 3 3030, m is 22020, and the positional relationship between the two substituents bonded to the benzene ring is
A surfactant that is ortho, meta, or para. 2. The microorganism BSP-1 strain (Accession No. FERM P-16)
No. 956). 3. Using a microorganism, formula I: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n OH or formula II: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂₎ ) a surfactant represented by _n H (where n is 3 to 30,
m is from 2 to 20 and the positional relationship of the two substituents attached to the benzene ring is ortho, meta or para). 4. The method according to claim 3, wherein the microorganism is a BSP-1 strain. 5. A method for separating a saturated organic component from an organic composition in which an unsaturated organic component and a saturated organic component are present, comprising: (A) a compound of the formula I: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n OH or a surfactant represented by the formula II: H (CH ₂ ) _m C ₆ H ₄ (OCH ₂ CH ₂ ) _n H, wherein n is 3 to 30,
m is 2-20, and the positional relationship of the two substituents bonded to the benzene ring is ortho, meta, or para) with the organic composition; and (B) the saturated organic compound A layer mainly containing the component and a layer mainly containing the unsaturated organic component. 6. The method according to claim 5, wherein said steps (A) and (B) are performed under non-hydrous conditions. 7. The method according to claim 5, further comprising: (D) contacting the cyclic lipopeptide with the organic composition after the step (A) and before the step (B). the method of. 8. A method for separating a saturated organic component from an organic composition containing an unsaturated organic component and a saturated organic component, comprising: (A ′) a cyclic lipopeptide or a compound of the formula I:
_{H (CH 2) m C 6} H 4 (OCH 2 CH 2) n OH or the formula _{II: H (CH 2) m} C 6 H 4
A surfactant represented by (OCH ₂ CH ₂ ) _n H (where n is 3
-30, m is 2-20, and the positional relationship of the two substituents attached to the benzene ring is ortho, meta, or para) with the organic composition;
(C) a step of cooling the unsaturated organic component and the saturated organic component to a crystallization temperature of a component having a higher crystallization temperature or lower, and (B) a layer mainly containing the saturated organic component. Separating into a layer mainly containing the unsaturated organic component. 9. The method according to claim 1, wherein the step (B) comprises: (B ′) performing centrifugal separation at a temperature not higher than room temperature and not lower than a temperature at which the entire organic composition solidifies, thereby forming The method according to claim 8, wherein the method comprises a step of separating into a layer mainly containing the unsaturated organic component. 10. The method according to claim 5, further comprising, after the step (B) or (B ′), (E) a step of squeezing a liquid in a layer mainly containing the saturated organic component.
10. The method according to any one of items 9 to 9. 11. The method according to claim 7, wherein the cyclic lipopeptide is arthrofactin. 12. The method according to claim 5, wherein the organic composition is a mixture of a saturated fat and an unsaturated fat.