JP2014047311A - Method for separating plant sterol and triterpene alcohol and method for producing fatty acid ester thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for separating a plant sterol and a triterpene alcohol from a mixture of a plant sterol and a triterpene alcohol, and to provide a method for producing a fatty acid ester of the plant sterol and a fatty acid ester of the triterpene alcohol separately from the mixture of the plant sterol and the triterpene alcohol.SOLUTION: By using a column chromatography, it is possible to separate a plant sterol and a triterpene alcohol from a mixture of a plant sterol and a triterpene alcohol. By esterifying the plant sterol and the triterpene alcohol which are separated, a fatty acid ester of the plant sterol and a fatty acid ester of the triterpene alcohol are produced separately.

Description

  The present invention relates to a method for separating plant sterol and triterpene alcohol and a method for producing these fatty acid esters.

  Sterol is a general term for compounds having 27 to 30 carbon atoms having a steroid skeleton, having a hydroxyl group at the C-3 position and a hydrocarbon side chain at the C-17 position. Among these, those contained in plants are called plant sterols, and are widely distributed in plants. As main ones, β-sitosterol, campesterol, stigmasterol and the like are known.

  Triterpene alcohol is a kind of sterol having 30 carbon atoms that is abundant in rice, and has a structure and properties similar to plant sterols. Triterpene alcohol is distributed in rice bran, olive seeds, corn seeds, aloe, and the like, and main compounds include cycloartenol, 24-methylenecycloartanol, cycloartanol, cyclobranol and the like. These are also known as partial structures of γ-oryzanol, which is a typical functional substance of rice bran.

  As a main physiological function of plant sterols, cholesterol lowering action is known. In addition, physiological functions such as improvement of dysuria due to prostatic hypertrophy, cancer cell proliferation inhibitory effect, and inflammation inhibitory effect have been reported. On the other hand, although the physiological function of triterpene alcohol is similar to the physiological function of plant sterols, it has been reported that triterpene alcohol has a physiological function inherent in triterpene alcohol that reduces triglycerides and inhibits lipase. It has been reported to synergistically inhibit lipid absorption.

  As a method for producing plant sterols, a method is generally used in which by-products such as soap stock and deodorized scum generated in the process of refining vegetable oil are used as raw materials, extracted with a solvent such as acetone or hexane, and purified by crystallization. Triterpene alcohol can also be obtained by almost the same method, but separation of plant sterol and triterpene alcohol is difficult. If plant sterols and triterpene alcohols can be separated from raw materials such as soap stock and deodorized scum, it is considered that each physiological function can be effectively used separately.

  There is no industrially established method for producing triterpene alcohol. For example, methods obtained by hydrolyzing γ-oryzanol have been reported (Patent Documents 1 to 4). Although these methods easily provide a mixture of plant sterols and triterpene alcohols at high concentrations, separation of plant sterols and triterpene alcohols is still difficult.

  Since sterols are inherently difficult to dissolve in water and oil and are easily oxidized, stability and versatility can be improved by esterification with fatty acids and derivatization. It is also known that esterification facilitates separation and purification by crystallization. Therefore, plant sterol fatty acid esters and triterpene alcohol fatty acid esters are used as important raw materials for cosmetics, pharmaceuticals, and foods, and are particularly excellent in skin moisturizing action, anti-aging action, and anti-inflammatory action. Expectation is increasing (Patent Document 5).

  Plant sterol fatty acid esters are synthesized from plant sterols by organic synthesis. However, enzymatic synthesis is currently being studied from the viewpoint of the progress of side reactions, coloring, odor, catalyst removal, and the like. As plant sterol fatty acid ester synthase, there are reports using lipase (Patent Document 6) and cholesterol esterase (Patent Documents 7 and 8). As the origin of lipase, Alcaligenes genus, Chromobacterium genus, Pseudomonas genus, Humicola genus and the like have been reported (Patent Document 6). As the origin of cholesterol esterase, a sterol ester production technique using cholesterol esterase derived from Pseudomonas genus, Candida genus, Streptomyces genus and several other microorganisms is disclosed (Patent Documents 7 and 8). Although these enzyme synthesis methods are specific and efficient esterification reactions of plant sterols and free fatty acids, no examples of applying them to triterpene alcohols have been reported.

JP 2012-41304 A Japanese Patent Publication No.55-2440 JP 2006-273762 A JP 05-331101 A JP 2006-176422 A JP 2001-040388 A JP 2009-55819 A Japanese Patent Publication No. 05-33712

  The present invention provides a method for separating plant sterol and triterpene alcohol from a mixture of plant sterol and triterpene alcohol, and a method for separately producing plant sterol fatty acid ester and triterpene alcohol fatty acid ester from a mixture of plant sterol and triterpene alcohol. This is the issue.

The present invention includes the following inventions in order to solve the above problems.
[1] A method for separating plant sterol and triterpene alcohol from a mixture of plant sterol and triterpene alcohol, the method comprising using column chromatography.
[2] The separation method according to the above [1], wherein the mixture of plant sterol and triterpene alcohol is derived from soap stock or deodorized scum generated in the process of refining vegetable oil.
[3] The separation method according to [1] or [2], wherein silica gel is used as a column chromatography carrier.
[4] The separation method according to [3], wherein a mixed liquid of hexane: ethyl acetate = 70: 30 to 95: 5 is used for a mobile phase of column chromatography.
[5] A method for producing a plant sterol fatty acid ester and / or a triterpene alcohol fatty acid ester, wherein the plant sterol and the triterpene alcohol are separated using the separation method according to any one of [1] to [4]. A production method characterized in that at least one of plant sterol and triterpene alcohol is subsequently subjected to fatty acid esterification.
[6] The production method according to [5], wherein the fatty acid esterification is performed by an esterification reaction using an enzyme as a catalyst.
[7] The method according to [6], wherein the reaction is allowed to proceed while reducing water generated by the esterification reaction.
[8] The production method of [6] or [7], wherein the enzyme is lipase.
[9] The production method according to [8] above, wherein the lipase has a property of non-specifically esterifying a mixture of triterpene alcohol and plant sterol.
[10] The method according to [9], wherein the lipase is derived from a Candida genus microorganism.
[11] A plant sterol fatty acid characterized in that a plant sterol fatty acid ester is produced from a mixture of plant sterol, triterpene alcohol and fatty acid using an enzyme that specifically esterifies plant sterol, and the plant sterol fatty acid ester is separated. Ester production method.
[12] The production method according to [11], wherein the enzyme is a lipase derived from at least one microorganism selected from the group consisting of Pseudomonas genus, Penicillium genus and Candida genus.
[13] A plant sterol fatty acid ester is produced from a mixture of plant sterol, triterpene alcohol and fatty acid using an enzyme that specifically esterifies plant sterol, and the plant sterol fatty acid ester is separated to obtain unreacted triterpene alcohol. A method for separating triterpene alcohol.

  According to the present invention, plant sterol and triterpene alcohol can be separated from a mixture of plant sterol and triterpene alcohol, and plant sterol fatty acid ester and triterpene alcohol fatty acid ester can be produced separately. If plant sterols and triterpene alcohols obtained separately or their fatty acid esters are used, each physiological function can be effectively utilized separately.

It is a figure which shows RI chromatogram which isolate | separated PS and TA from the mixture of plant sterol and triterpene alcohol using silica gel chromatography. It is a figure which shows the result of having examined the addition amount of various lipases and TA ester production amount in the esterification reaction of TA.

[Method for separating plant sterol and triterpene alcohol]
The method for separating plant sterols and triterpene alcohols of the present invention is a method for separating plant sterols and triterpene alcohols from a mixture of plant sterols and triterpene alcohols using column chromatography.
The mixture of plant sterol and triterpene alcohol is not particularly limited as long as it contains both plant sterol and triterpene alcohol. It may consist of only two components of plant sterol and triterpene alcohol, or may contain components other than these.

  The mixture of plant sterol and triterpene alcohol is preferably derived from soap stock or deodorized scum generated in the process of refining vegetable oil. Soap stock is also referred to as alkaline foots or alkaline oil cake. Deodorized scum is also referred to as deodorized distillate. Both are obtained as by-products in the process of refining vegetable oils, and are known to be rich in plant sterols and triterpene alcohols. Soap stock or deodorized scum may be subjected to column chromatography as it is, but it is preferable to use a mixture of plant sterol and triterpene alcohol that has been subjected to a purification operation as column material for column chromatography. The purification operation is not particularly limited, and a known purification method can be used. Examples of the mixture of plant sterol and triterpene alcohol that have undergone purification operations include a crude γ-oryzanol hydrolyzate obtained from soapstock by crystallization treatment and the like.

  The type of column chromatography is not particularly limited, and examples thereof include open column chromatography, flash column chromatography, HPLC (High performance liquid chromatography), and the like. The carrier used for column chromatography is not particularly limited, and examples thereof include silica gel, ODS gel, hydrophobic resin, ion exchange resin and the like. Silica gel is preferred. The diameter and length of the column and the amount of the carrier can be appropriately optimized according to the kind of the mixture of plant sterol and triterpene alcohol to be provided and the amount of impurities.

A mobile phase is not specifically limited, It can optimize suitably according to the kind of the support | carrier to be used and the mixture of the plant sterol and triterpene alcohol to provide. When the carrier is silica gel, a mobile phase generally used for silica gel chromatography can be used. For example, hexane / ethyl acetate mixed solution, chloroform / methanol mixed solution, toluene and the like can be mentioned. Preferably, it is a hexane / ethyl acetate mixture, and the mixing ratio is preferably hexane: ethyl acetate = 70: 30 to 95: 5, more preferably hexane: ethyl acetate = 80: 20 to 95: 5, still more preferably. Hexane: ethyl acetate = 85: 15-95: 5. When the carrier is an ODS gel, a mixture of alcohols such as methanol, ethanol and propanol, a mixture of alcohol and water, and the like can be used as the mobile phase.
Other column chromatography conditions are not particularly limited, and appropriate conditions can be set by appropriately selecting and combining known column chromatography conditions.

The detector is not particularly limited as long as it can detect plant sterols and triterpene alcohols. Specific examples include a differential refractive index (RI) detector, an ultraviolet / visible light (UV / VIS) detector, and an evaporative light scattering detector (ELSD). Among them, it is preferable to use a differential refractive index (RI) detector.
Plant sterols and triterpene alcohols can be separated and obtained by fractionating the fractions at the time of peak detection.

[Production method of fatty acid ester]
The present invention provides a method for producing a plant sterol fatty acid ester and / or a triterpene alcohol fatty acid ester (hereinafter referred to as “the production method of the present invention”).
The production method of the present invention is a method in which the plant sterol and triterpene alcohol separated by the method for separating a plant sterol and triterpene alcohol of the present invention are separately fatty acid esterified. The production method of the present invention includes a method in which only one of the separated plant sterols and triterpene alcohols is fatty acid esterified. The method of esterification of a plant sterol or triterpene alcohol with a fatty acid is not particularly limited. For example, a known synthesis method such as a chemical synthesis method using a chemical catalyst such as acid or alkali, an enzyme synthesis method using an enzyme as a catalyst, etc. Can be used. Preferred is an enzyme synthesis method using an enzyme as a catalyst.

  The fatty acid may be either a saturated fatty acid or an unsaturated fatty acid, for example, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid Behenic acid, docosapentaenoic acid, docosahexaenoic acid and the like. Either a linear fatty acid or a branched fatty acid may be used, but a linear fatty acid is preferred. The fatty acid may be derived from an animal or a plant, but is preferably derived from a plant. The ratio of the plant sterol or triterpene alcohol and the fatty acid is not particularly limited, but the molar ratio is preferably a plant sterol or triterpene alcohol: fatty acid = 1: 0.5-10.

  The enzyme is not particularly limited as long as it is an enzyme that can catalyze an esterification reaction between a plant sterol or triterpene alcohol and a fatty acid. As such an enzyme, for example, lipase, cholesterol esterase and the like are known and can be suitably used in the production method of the present invention. Examples of the origin of these enzymes include, but are not limited to, microorganisms such as Pseudomonas genus, Penicillium genus, Candida genus, Streptomyces genus, Alcaligenes genus, Chromobacteria genus, and Humicola genus. Among them, as the enzyme used in the production method of the present invention, it is preferable to use a lipase derived from a microorganism belonging to the genus Candida in terms of excellent esterification efficiency of triterpene alcohol. Among the genus Candida, a lipase derived from Candida rugosa is more preferable, and a lipase derived from Candida rugosa (Type VII, product number L1754-100G) manufactured by SIGMA or a lipase having an activity equivalent thereto is more preferable. The lipase derived from Candida rugosa manufactured by SIGMA is Type VII, and it has been reported to contain three types of isozymes (RCC CHANG, et al., Multiple forms and functions of Candida rugosa biotype, Appl.Biochem., Vol. 19, 93-97 (1994)).

  Conditions such as the amount of enzyme added, reaction temperature, reaction time, etc. can be appropriately set according to the enzyme used. For example, when a lipase derived from Candida rugosa is used, it is usually an enzyme of 10 to 5000 units per gram of plant sterol or triterpene alcohol (where the unit is the amount of enzyme that produces 1 μmol of free fatty acid per minute in the hydrolysis of olive oil). And may be reacted at 20 ° C. to 50 ° C. for 30 minutes to 48 hours.

  In the esterification reaction using an enzyme as a catalyst, it is preferable to proceed the reaction while reducing the water generated by the esterification reaction. Thereby, it is possible to prevent the ester synthesis and the ester decomposition in the reaction system from reaching equilibrium and reducing the esterification efficiency. The method of proceeding the reaction while reducing the water generated by the esterification reaction is not particularly limited. The method of allowing the reaction liquid to stand still in the middle of the reaction, removing the separated water and continuing the reaction, or centrifuging in the middle of the reaction. Examples include a method of separating and removing the separated water and continuing the reaction, a method of adding a dehydrating agent, a method of allowing the reaction to proceed while dehydrating under reduced pressure conditions, and a method of combining two or more of these. Preferably, the reaction proceeds while dehydrating under reduced pressure. As decompression conditions, for example, 0.1 kPa to 10 kPa is preferable. The reaction may proceed while reducing the water content from the start of the esterification reaction, or the reaction may proceed while reducing the water content during the esterification reaction. In the case of performing from the middle of the esterification reaction, it is preferable to start after the esterification reaction reaches equilibrium.

  The method for separating the plant sterol fatty acid ester or triterpene alcohol fatty acid ester from the reaction solution is not particularly limited. Examples include column chromatography, preparative thin layer chromatography, molecular distillation, short path distillation, solvent fractionation, and liquid-liquid extraction. Column chromatography is preferred.

[Selective plant sterol fatty acid ester production method and triterpene alcohol separation method]
The method for producing a selective plant sterol fatty acid ester according to the present invention comprises generating a plant sterol fatty acid ester from a mixture of plant sterol, triterpene alcohol and fatty acid using an enzyme that specifically esterifies plant sterol, It is characterized by separating. A mixture of plant sterol, triterpene alcohol and fatty acid can be prepared by adding a fatty acid to a mixture of plant sterol and triterpene alcohol. As the mixture of plant sterol and triterpene alcohol, a mixture of plant sterol and triterpene alcohol used in the above-mentioned “method for separating plant sterol and triterpene alcohol” can be preferably used. The ratio of the mixture of plant sterol and triterpene alcohol and the fatty acid is not particularly limited, but the molar ratio is preferably a mixture of plant sterol and triterpene alcohol: fatty acid = 1: 1-5.

When an enzyme that specifically esterifies plant sterol is allowed to act on a mixture of plant sterol, triterpene alcohol, and fatty acid, the following formula (PS selectivity) = (PS ester amount) / [(PS ester amount) + (TA ester amount)]
The PS selectivity (plant sterol selectivity) determined in (1) is preferably 0.80 or more, more preferably 0.90 or more, still more preferably 0.95 or more, and 0.98 or more. More preferably it is.
As such an enzyme, a lipase derived from a microorganism belonging to the genus Pseudomonas, a lipase derived from a microorganism belonging to the genus Penicillium, or a lipase derived from a microorganism belonging to the genus Candida can be preferably used. As a microorganism belonging to the genus Pseudomonas, Pseudomonas cepacia or Pseudomonas stutzeri is preferable, and as a microorganism belonging to the genus Penicillium, Penicillium camembertii is preferable, and Candida rugosa is preferable as a microorganism belonging to the genus Candida. Among the lipases derived from Candida rugosa, lipase OF manufactured by Meisho Sangyo Co., Ltd. or a lipase having an activity equivalent to this is more preferable. In addition, it has been reported that lipase OF manufactured by Meikatsu Sangyo Co., Ltd. contains only one type of isozyme (RC CANG, et al., Multiple forms and functions of Candida rugosa lipase, Biotechnol. Appl. Biochem. , Vol. 19, 93-97 (1994)).

  Conditions such as the amount of enzyme added, reaction temperature, reaction time, etc. can be appropriately set according to the enzyme used. Usually, 10 to 3000 units of enzyme per gram of a mixture of plant sterol and triterpene alcohol (where the unit is the amount of enzyme that produces 1 μmol of free fatty acid per minute in the hydrolysis of olive oil) is added, and 10 ° C. to 40 ° C. For 30 minutes to 48 hours.

  Unreacted triterpene alcohol can be obtained by separating the produced plant sterol fatty acid ester from the reaction solution. That is, the present invention provides a method for separating triterpene alcohol from a mixture of plant sterols, plant sterols, triterpene alcohols and fatty acids. Examples of the method for separating the produced plant sterol fatty acid ester from the reaction solution include column chromatography, preparative thin layer chromatography, molecular distillation, short path distillation, solvent fractionation, liquid-liquid extraction, and the like. Preferably, column chromatography is used. In the reaction solution from which the plant sterol fatty acid ester has been separated, unreacted triterpene alcohol and fatty acid remain. A triterpene alcohol fatty acid ester can be synthesized using this reaction solution. As the enzyme used at this time, an enzyme having an excellent esterification efficiency of triterpene alcohol is preferably used. As such an enzyme, a lipase derived from a microorganism belonging to the genus Candida is preferable, and among them, a lipase derived from Candida rugosa is more preferable. A lipase derived from Candida rugosa (Type VII, product number L1754- 100G) manufactured by SIGMA or a lipase having an activity equivalent to this is more preferable.

  It is also possible to separate the triterpene alcohol from the reaction solution from which the plant sterol fatty acid ester has been separated. As a method for separating triterpene alcohol from the reaction solution from which the plant sterol fatty acid ester has been separated, a method using column chromatography can be used as in the method for separating plant sterol and triterpene alcohol. The separated triterpene alcohol may be used as it is, or may be reacted with a fatty acid and used as a triterpene alcohol fatty acid ester.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these.

[Example 1: Separation of plant sterol and triterpene alcohol]
From a mixture of plant sterol (hereinafter referred to as “PS”) and triterpene alcohol (hereinafter referred to as “TA”), separation of PS and TA was attempted using silica gel chromatography.
8.0 g of PS / TA mixture obtained by hydrolysis of γ-oryzanol was dissolved in 40 mL of acetone and adsorbed on Wakogel C-200 (trade name, Wako Pure Chemical Industries). Acetone was removed with a rotary evaporator, suspended in hexane / ethyl acetate = 9/1 (v / v), and packed in a glass column (15 × 150 mm). Connect the column to ULTRA PACK Glass Column (silica gel, 37 × 300 mm, Yamazen), pass hexane / ethyl acetate = 9/1 (v / v) at 14 mL / min, and detect the peak using RI detector The time fraction was dispensed into an Erlenmeyer flask.

  The obtained RI chromatogram is shown in FIG. As is clear from FIG. 1, PS and TA could be separated. After TLC analysis of each fraction, fractions containing only the same substance were combined, and the solvent was removed using a rotary evaporator to obtain PS and TA. The yield of PS was 3.0 g and the purity was 66%, and the yield of TA was 4.5 g and the purity was 97%. In addition, silica gel aluminum sheet for TLC (Merck, Tokyo) was used for TLC analysis, developing solvent was hexane / ethyl acetate = 7/3 (v / v), and sprayed with 50% ethanol sulfate solution on a hot plate. Colored on heating.

[Example 2: Esterification reaction with lipase and separation of ester from reaction solution]
The PS or TA obtained in Example 1 and oleic acid were mixed at a molar ratio of 1: 2.5, respectively, and the same weight of enzyme (lipase) solution (Candida rugosa, SIGMA (Type VII, product no. L-1754-100G); 5000 U / mL) was added and stirred at 30 ° C. After the reaction reaches equilibrium (reaction rate 75%), the reaction was performed under reduced pressure at 2.1 kPa using a diaphragm type vacuum pump DIVAC 0.6L (Tokyo Rika Kikai), and the reaction was performed under normal pressure without reducing pressure. When the reaction was performed under normal pressure, the reaction rate did not change, but the reaction rate under the reduced pressure was improved to 89%. From this result, in order to improve the efficiency of the esterification reaction by lipase, it was found that it is preferable to perform the reaction while dehydrating under reduced pressure.

Unreacted oleic acid was removed from the reaction solution obtained by the reaction at normal pressure using a 1 mol / L NaOH aqueous solution. Next, the sample was loaded on a silica gel column ULTRA Pack Glass Column (300 mm × 37 mm id), and eluted with hexane / ethyl acetate = 9/1 (v / v) as the mobile phase to obtain unreacted PS. And TA were removed. The obtained ester fraction was further separated and purified by an HPLC preparative column (COSMOSIL Cholester; 250 × 10 mm id, Nacalai Tesque). Methanol: isopropyl alcohol = 1: 1 (v / v) was used as the mobile phase, and the flow rate was 5 mL / min. The fraction was collected every 2.5 min from a retention time of 70 to 120 min, and each fraction was analyzed by TLC. The fractions containing only the same substance were combined. TLC analysis was performed in the same manner as in Example 1.
When reacted under normal pressure, 6.1 g of PS ester and 3.0 g of TA ester were obtained from 27 g of the reaction solution.

[Example 3: Examination of enzyme suitable for esterification reaction of TA]
TA and oleic acid are mixed at a molar ratio of 1: 2.5, and the amount of lipase added is set to 5 to 7 steps within a range of 50 to 2000 U. As in Example 2, the reaction reaches equilibrium. Then, the reaction was performed while reducing the pressure at 2.1 kPa. As lipases, lipase (Candida rugosa, SIGMA, Type VII, product number L1754-100G), lipase TL (Pseudomonas stutzeri, famous sugar industry), lipase SL (Pseudomonas cepacia, famous sugar industry), and lipase A “Amano” 6 ( Aspergillus niger and Amano Enzyme) were used. The amount of TA ester produced in the reaction solution was measured by the same method as in Example 2.

  The results are shown in FIG. When lipase (Candida rugosa, SIGMA, Type VII, product number L1754-100G) was used, the amount of TA ester produced increased with the amount of enzyme. On the other hand, when the other three kinds of lipases are used, it becomes clear that the fluidity of the reaction solution decreases and the reaction efficiency decreases as the amount of enzyme increases, and as a result, the esterification reaction proceeds sufficiently. I could not. From this result, it became clear that Candida rugosa-derived lipase is suitable as the lipase used for esterification of TA.

[Example 4: Examination of enzyme suitable for selective esterification reaction of PS]
After adding 0.75 g of PS / TA mixture and 1.25 g of oleic acid (molar ratio 1: 5) to a 10 mL vial, 2 mL of each of the following enzyme aqueous solutions was added. The mixture was reacted at 30 ° C. for 24 hours, and the reaction solution was analyzed by HPLC. Enzymes include lipase SL (Pseudomonas cepacia), lipase TL (Pseudomonas stutzeri), lipase G “Amano” 50 (Penicillium camembertii, Amanoenzyme), lipase (Candida rugosa, SIGMA, Type VII, Five types, product number L-1754-100G) and lipase OF (Candida rugosa, Meito Sangyo) were used. Enzyme amounts are lipase SL and lipase (Candida rugosa, SIGMA, Type VII, product number L-1754-100G) 20 U, 50 U, 100 U, 200 U, 500 U, 1000 U and 2000 U, lipase TL 50 U, 100 U, Six stages of 200 U, 500 U, 1000 U and 2000 U, and lipase G “Amano” 50 and lipase OF were set to five stages of 100 U, 200 U, 500 U, 1000 U and 2000 U.

Table 1 shows the PS selectivity of each enzyme used. PS selectivity was determined by the following formula.
(PS selectivity) = (PS ester amount) / [(PS ester amount) + (TA ester amount)]
As is clear from Table 1, lipase G “Amano” 50 and lipase OF had a PS selectivity of 1 at any enzyme amount of 100 U to 2000 U. Lipase SL had a PS selectivity close to 1 up to an enzyme amount of 2000 U, and showed a high PS selectivity. Lipase TL also showed high PS selectivity, although somewhat inferior to lipase SL. On the other hand, lipase (Candida rugosa, Type VII, product number, L-1754-100G) decreased in PS selectivity as the amount of enzyme increased. From this result, for the selective synthesis of PS ester, a lipase from Penicillium camembertii (lipase G “Amano” 50), a lipase from Pseudomonas cepacia (lipase SL) and a lipase from Pseudomonas stutzeri (lipase TL g and lipase TL) Lipase (lipase OF) was shown to be suitable. On the other hand, in the case of lipase derived from Candida rugosa, it was found that some manufacturers and components are not suitable for synthesis of PS selectivity.

  Therefore, if an enzyme with high PS selectivity is used, it is possible to selectively synthesize PS ester from a PS / TA mixture, separate PS ester by crystallization, etc., and obtain unreacted TA by distillation or the like. It becomes. Furthermore, the obtained TA can be esterified with a lipase to produce a TA ester in which no PS ester is mixed.

  The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims, and technical means disclosed in different embodiments are appropriately combined. The obtained embodiment is also included in the technical scope of the present invention. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

Claims (13)

  A method for separating plant sterol and triterpene alcohol from a mixture of plant sterol and triterpene alcohol, the method comprising using column chromatography.   2. The separation method according to claim 1, wherein the mixture of the plant sterol and the triterpene alcohol is derived from soap stock or deodorized scum generated in the process of refining the vegetable oil.   3. The separation method according to claim 1, wherein silica gel is used as a carrier for column chromatography.   The separation method according to claim 3, wherein a mixed liquid of hexane: ethyl acetate = 70: 30 to 95: 5 is used for a mobile phase of column chromatography. A process for producing plant sterol fatty acid esters and / or triterpene alcohol fatty acid esters,
A method for producing a fatty acid esterified product comprising separating at least one of plant sterol and triterpene alcohol after separating the plant sterol and triterpene alcohol using the separation method according to claim 1.   6. The process according to claim 5, wherein the fatty acid esterification is performed by an esterification reaction using an enzyme as a catalyst.   The production method according to claim 6, wherein the reaction is allowed to proceed while reducing water generated by the esterification reaction.   The method according to claim 6 or 7, wherein the enzyme is lipase.   The method according to claim 8, wherein the lipase has a property of non-specifically esterifying a mixture of triterpene alcohol and plant sterol.   The method according to claim 9, wherein the lipase is derived from a microorganism of the genus Candida.   Production of a plant sterol fatty acid ester characterized by producing a plant sterol fatty acid ester from a mixture of plant sterol, triterpene alcohol and fatty acid using an enzyme that specifically esterifies the plant sterol, and separating the plant sterol fatty acid ester Method.   The method according to claim 11, wherein the enzyme is a lipase derived from at least one microorganism selected from the group consisting of the genera Pseudomonas, Penicillium, and Candida.   To produce plant sterol fatty acid esters from a mixture of plant sterols, triterpene alcohols and fatty acids using enzymes that specifically esterify plant sterols, isolate plant sterol fatty acid esters, and obtain unreacted triterpene alcohols A method for separating triterpene alcohol.