WO2013018859A1 - Method for producing transesterified oil - Google Patents

Method for producing transesterified oil Download PDF

Info

Publication number
WO2013018859A1
WO2013018859A1 PCT/JP2012/069676 JP2012069676W WO2013018859A1 WO 2013018859 A1 WO2013018859 A1 WO 2013018859A1 JP 2012069676 W JP2012069676 W JP 2012069676W WO 2013018859 A1 WO2013018859 A1 WO 2013018859A1
Authority
WO
WIPO (PCT)
Prior art keywords
lipase
fatty acid
acid alkyl
alkyl ester
oil
Prior art date
Application number
PCT/JP2012/069676
Other languages
French (fr)
Japanese (ja)
Inventor
洋介 中村
裕子 外山
良枝 山内
裕子 箱田
秀隆 上原
Original Assignee
日清オイリオグループ株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 日清オイリオグループ株式会社 filed Critical 日清オイリオグループ株式会社
Publication of WO2013018859A1 publication Critical patent/WO2013018859A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/10—Ester interchange
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis

Definitions

  • the present invention relates to a method for efficiently producing a transesterified oil by performing a transesterification reaction in a state where the enzyme activity of lipase is enhanced.
  • Oils and fats produced by transesterification of fatty acid alkyl esters and vegetable oils by the action of lipases are used in various applications.
  • a method has been proposed in which hard butter such as cocoa butter substitute fat is produced using a 1,3-selective lipase on a specific fat and oil and utilizing a transesterification reaction (Patent Documents 1 to 5).
  • Patent Documents 1 to 5 describe the use of lysops lipase, Aspergillus lipase, mucor lipase, pancreatic lipase, and rice nuclease as 1,3-selective lipase. Since the lipase used here is expensive, it is required to efficiently use the lipase by transesterifying the lipase with as high activity as possible.
  • An object of the present invention is to provide a method for producing a transesterified oil that efficiently uses a lipase by transesterifying a lipase with as high an activity as possible.
  • the present invention differs in lipase activity in transesterification reaction due to the difference in the mixing order of lipase, fatty acid alkyl ester and vegetable oil. It was made based on the knowledge that this occurs. That is, the present invention mixes a fatty acid alkyl ester, which is an ester of a fatty acid having 16 to 22 carbon atoms and an alcohol having 1 to 3 carbon atoms, and a lipase, and maintains the mixed state for 10 minutes or more at 20 to 80 ° C.
  • a method for producing a transesterified oil is provided, wherein the fatty acid alkyl ester and vegetable oil / or vegetable oil are mixed with the mixture and subjected to a transesterification reaction.
  • the activity of the lipase to be used can be improved by a simple operation, and the transesterification reaction can be performed in a state where the activity of the lipase is improved. Further, by using an oil and fat rich in triglyceride having an oleoyl group at the 2-position as a vegetable oil and using an alkyl stearate, an oil and fat rich in SOS can be obtained efficiently.
  • the fatty acid having 16 to 22 carbon atoms constituting the starting fatty acid alkyl ester used for transesterification in the present invention is preferably a saturated fatty acid, particularly stearic acid, palmitic acid or behenic acid.
  • the alcohol having 1 to 3 carbon atoms constituting the fatty acid alkyl ester as a raw material used for transesterification in the present invention methanol, ethanol and isopropyl alcohol are preferable, and ethanol is preferable.
  • fatty acid alkyl ester methyl palmitate, methyl stearate, methyl behenate, ethyl palmitate, ethyl stearate and ethyl behenate are preferable, and ethyl stearate is particularly preferable.
  • an oil rich in triglyceride having an oleoyl group and / or a linoleic oil group at the 2-position is preferable.
  • shea fat low melting point portion for example, iodine value 70 to 80
  • high oleic sunflower oil for example, high oleic safflower oil, high oleic lorinolen rapeseed oil
  • examples thereof include palm oil, palm fraction oil, and mixed oils thereof.
  • Examples of fats and oils having a linoleic oil group at the 2nd position include hylinol safflower oil, soybean oil, and grape seed oil.
  • the shea fat low melting point portion high oleic sunflower oil, high oleic lorinolen rapeseed oil, palm oil, and palm fraction oil are preferred.
  • highlinol safflower oil is preferable.
  • the use ratio (mass ratio) of the fatty acid alkyl ester to the vegetable oil is preferably 1/10 or more, particularly preferably 1/1 to 5/1.
  • 1,3-dipalmitoyl-2 is used by using stearic acid and / or a lower alcohol ester thereof as at least one selected from linear saturated fatty acids having 16 to 22 carbon atoms and lower alcohol esters thereof.
  • POP 1,3-dipalmitoyl-2-linoleoylglycerol
  • SOS 1,3-distearoyl-2-oleoylglycerol
  • SLS 1,3-distearoyl-2- Linoleoylglycerol
  • POS 1,3-dibehenyl-2-oleoylglycerol
  • BOB 1,3-dibehenyl-2-oleoylglycerol
  • BLB 1,3-dibehenyl-2-linoleoyl glycerin
  • the “fat rich in SOS and / or SLS” means 1,3-distearoyl-2-oleoylglycerin and / or 1,3-distearoyl-2 in all triglyceride species constituting the fat.
  • -It means that 10% by mass or more of linoleoylglycerin is contained, and preferably that the largest amount of triglyceride species is SOS and / or SLS.
  • the upper limit is preferably 90% by mass.
  • lipases can be used. Of these, Rhizopus delemar and Rhizopus oryzae are preferred, and 1,3-specific lipase is preferred. Examples of these lipases include products from DSM Japan, Inc .: Picantase R8000, and products from Amano Enzyme, Inc .: Lipase F-AP15. The most suitable lipase is derived from Rhizopus oryzae, Amano Enzyme. A product of the company: Lipase DF “Amano” 15-K (also referred to as lipase D). This is a powder lipase. The lipase DF “Amano” 15-K has conventionally been derived from Rhizopus delmar.
  • the lipase used in the present invention may be obtained by drying a lipase-containing aqueous solution containing a lipase medium component or the like.
  • a powder lipase having a spherical shape and a water content of 10% by mass or less.
  • 90% by mass or more of the lipase powder preferably has a particle size of 1 to 100 ⁇ m.
  • those prepared by spray-drying a lipase-containing aqueous solution whose pH is adjusted to 7-8 are preferred.
  • granulated powder lipase also referred to as powder lipase obtained by granulating the above lipase using soybean powder and making it into powder can be used.
  • soybean powder it is preferable to use a soybean powder having a fat content of 5% by mass or more.
  • the fat content is preferably 10% by mass or more, more preferably 15% by mass or more, and on the other hand, it is 25% by mass or less. preferable.
  • Particularly preferred is soybean powder having a fat content of 18 to 23% by mass.
  • examples of the fat include fatty acid triglycerides and analogs thereof.
  • the fat content of soybean can be easily measured by a method such as Soxhlet extraction.
  • full-fat soybean powder can be used as such soybean powder.
  • soy milk can also be used as a raw material of soybean powder.
  • the soybean powder can be produced by pulverizing soybean by a conventional method, and the particle size is preferably about 0.1 to 600 ⁇ m.
  • the particle size can be measured by a method similar to the method for measuring the particle size of powder lipase.
  • the amount of soybean powder used relative to the lipase is preferably 0.1 to 200 times, more preferably 0.1 to 20 times, and more preferably 0.1 to 10 times the amount on a mass basis. Most preferred.
  • the powder lipase used in the present invention preferably has a water content of 10% by mass or less, particularly preferably 1 to 8% by mass.
  • the particle size of the powder lipase used in the present invention can be arbitrary, but 90% by mass or more of the powder lipase preferably has a particle size of 1 to 100 ⁇ m.
  • the average particle size is preferably 10 to 80 ⁇ m.
  • the shape of the powder lipase is preferably spherical.
  • the particle size of the powder lipase can be measured, for example, using a particle size distribution measuring device (LA-500) manufactured by Horiba, Ltd.
  • the powder lipase used in the present invention is produced by drying an aqueous solution in which lipase and soybean powder are dissolved and dispersed by any one drying method selected from spray drying, freeze drying, and solvent precipitation / drying. be able to.
  • the aqueous solution in which lipase and soybean powder are dissolved / dispersed is prepared by dissolving / dispersing lipase powder and soybean powder in water, mixing the lipase powder in an aqueous solution in which soybean powder is dissolved / dispersed, or explained later. It can be obtained by mixing soybean powder with the lipase-containing aqueous solution.
  • the particles of lipase and / or soybean powder are aggregated to form a granulated product containing lipase and soybean powder.
  • This granulated product may contain a medium component of lipase.
  • the powder lipase thus prepared can be used as it is for transesterification.
  • the amount of water in the aqueous solution in which lipase and soybean powder are dissolved and dispersed adjusts the mass of water with respect to the total mass of lipase and soybean powder.
  • the mass of water with respect to the total mass of lipase and soybean powder is preferably 0.5 to 1,000 times, more preferably 1.0 to 500 times, and 3.0 to 100 times is most preferable.
  • the mass of water relative to the total mass of lipase and soybean powder is preferably 2.0 to 1,000 times from the characteristics of the apparatus, and preferably 2.0 to 500 times.
  • the ratio is more preferably double, and most preferably 3.0 to 100.
  • the lipase-containing aqueous solution is used as a raw material and the lipase content in the lipase-containing aqueous solution is unknown, the lipase-containing aqueous solution is pulverized by freeze drying or other reduced-pressure drying to determine the lipase content, and the lipase mass is calculated. can do.
  • the lipase-containing aqueous solution includes a lipase culture solution from which bacterial cells have been removed, a purified culture solution, a lipase obtained by dissolving and dispersing the lipase in water again, and a commercially available powder lipase dissolved and dispersed in water again. And commercially available liquid lipase. Further, those obtained by removing low molecular components such as salts in order to further increase the lipase activity are more preferable, and those obtained by removing low molecular components such as sugars in order to further improve the powder properties.
  • the lipase culture solution examples include aqueous solutions containing soy flour, peptone, corn staple liquor, K 2 HPO 4 , (NH 4 ) 2 SO 4 , MgSO 4 .7H 2 O and the like. These concentrations include soybean powder 0.1-20% by weight, preferably 1.0-10% by weight, peptone 0.1-30% by weight, preferably 0.5-10% by weight, corn staple liquor 0.1 to 30% by mass, preferably 0.5 to 10% by mass, K 2 HPO 4 0.01 to 20% by mass, preferably 0.1 to 5% by mass.
  • (NH 4 ) 2 SO 4 is 0.01 to 20% by mass, preferably 0.05 to 5% by mass
  • MgSO 4 ⁇ 7H 2 O is 0.01 to 20% by mass, preferably 0.05 to 5%. % By mass.
  • the culture conditions are a culture temperature of 10 to 40 ° C., preferably 20 to 35 ° C., an aeration rate of 0.1 to 2.0 VVM, preferably 0.1 to 1.5 VVM, and a stirring speed of 100 to 800 rpm, preferably
  • the pH is controlled to 200 to 400 rpm and the pH is 3.0 to 10.0, preferably 4.0 to 9.5.
  • Separation of the cells is preferably performed by centrifugation, membrane filtration or the like.
  • removal of low molecular components such as salts and sugars can be performed by UF membrane treatment.
  • UF membrane treatment Specifically, by performing a UF membrane treatment, concentrating an aqueous solution containing lipase to a volume of 1 ⁇ 2 volume, and then adding the same amount of phosphate buffer as the concentrated solution 1 to 5 times, A lipase-containing aqueous solution from which low molecular components have been removed can be obtained.
  • Centrifugation is preferably performed at 200 to 20,000 ⁇ g, and membrane filtration is preferably performed at a pressure of 3.0 kg / m 2 or less with an MF membrane, a filter press or the like.
  • the cells In the case of intracellular enzymes, it is preferable to crush the cells with a homogenizer, Waring blender, ultrasonic crushing, French press, ball mill or the like, and remove cell residues by centrifugation, membrane filtration or the like.
  • the stirring rotation speed of the homogenizer is 500 to 30,000 rpm, preferably 1,000 to 15,000 rpm
  • the rotation speed of the Waring blender is 500 to 10,000 rpm, preferably 1,000 to 5,000 rpm.
  • the stirring time is 0.5 to 10 minutes, preferably 1 to 5 minutes.
  • the ultrasonic crushing is performed under the condition of 1 to 50 kHz, preferably 10 to 20 kHz.
  • the ball mill preferably uses glass spheres having a diameter of about 0.1 to 0.5 mm.
  • the lipase-containing aqueous solution may be concentrated.
  • concentration method is not particularly limited, but evaporator, flash evaporator, UF membrane concentration, MF membrane concentration, salting out with inorganic salts, precipitation method with solvent, adsorption method with ion-exchange cellulose, water absorption with water-absorbing gel Law.
  • UF membrane concentration and an evaporator are used.
  • the module for concentrating the UF membrane is preferably a flat membrane or hollow fiber membrane having a molecular weight cut-off of 3,000 to 100,000, preferably 6,000 to 50,000, and the material is preferably polyacrylonitrile or polysulfone.
  • Spray drying is preferably performed using a spray dryer such as a nozzle countercurrent type, a disk countercurrent type, a nozzle cocurrent type, and a disk cocurrent type.
  • the disk co-current type is preferred, the atomizer speed is 4,000 to 20,000 rpm, the heating is preferably controlled at an inlet temperature of 100 to 200 ° C., and an outlet temperature of 40 to 100 ° C., and spray drying is preferred.
  • the temperature of the aqueous solution containing lipase and soybean powder is preferably adjusted to 20 to 40 ° C.
  • Freeze-drying is preferably performed by, for example, a lab-size small-scale freeze-dryer or a shelf-type freeze-dryer. Furthermore, it can also be prepared by drying under reduced pressure.
  • solvent precipitation / drying an aqueous solution in which lipase and soybean powder are dissolved / dispersed is gradually added to the solvent used to form a precipitate, and the resulting precipitate is centrifuged using a centrifuge. After collecting the precipitate, it is dried under reduced pressure.
  • the series of operations is preferably performed under a low temperature condition of room temperature or lower.
  • the solvent used in the solvent precipitation include water-soluble solvents or hydrophilic solvents such as ethanol, acetone, methanol, isopropyl alcohol, and hexane, and a mixed solvent thereof can also be used.
  • ethanol or acetone is preferably used in order to further enhance the activity of the powder lipase.
  • the amount of the solvent to be used is not particularly limited, but it is preferable to use a solvent having a volume of 1 to 100 times the volume of the aqueous solution in which lipase and soybean powder are dissolved and dispersed, and a solvent having a volume of 2 to 10 times. It is more preferable to use.
  • the precipitate can be obtained by standing and then filtered, but can also be obtained by mild centrifugation at about 1,000 to 3,000 ⁇ g. The obtained precipitate can be dried, for example, by drying under reduced pressure.
  • the production process of the powder lipase can further include a step of adding a filter aid.
  • filter aids that can be used include silica gel, celite, cellulose, starch, dextrin, activated carbon, activated clay, kaolin, bentonite, talc, and sand. Of these, silica gel, celite, and cellulose are preferable.
  • the particle size of the filter aid may be arbitrary, but is preferably 1 to 100 ⁇ m, particularly preferably 5 to 50 ⁇ m.
  • the filter aid that can be used before, during, or during the transesterification reaction is preferably added in an amount of 1 to 500% by mass, preferably 10 to 200% by mass, based on the total mass of lipase and soybean powder. Further preferred. This is because if the amount is within this range, the burden during filtration is reduced, and filtration pretreatment such as large-scale filtration equipment and advanced centrifugation is not required.
  • a filter aid can be contained in the powder lipase.
  • a filter aid may be added either before or after drying.
  • drying is performed by a method of drying after solvent precipitation, it is preferable to add a filter aid to the powdered lipase obtained by drying.
  • the amount of the filter aid to be contained in the obtained powder lipase can be 1 to 500% by mass based on the total mass of the lipase and soybean powder, and more preferably 10 to 200% by mass.
  • a fatty acid alkyl ester that is an ester of a fatty acid having 16 to 22 carbon atoms and an alcohol having 1 to 3 carbon atoms is mixed with the lipase so that the entire lipase is covered with the fatty acid alkyl ester. It is preferable that the fatty acid alkyl ester in the oil-soluble component in the mixed state when the fatty acid alkyl ester and the lipase are mixed is highly purified. Especially when the lipase is repeatedly used in the reaction, a reaction product or the like is mixed and the fatty acid alkyl ester is mixed. The ester concentration decreases.
  • components other than fatty acid alkyl ester are 15 mass% or less. More preferably, it is 0 to 10% by mass, and still more preferably 1 to 10% by mass.
  • oil-soluble components other than fatty acid alkyl esters include free fatty acids and alcohols, further plant oil as a raw material for transesterification, and glycerides generated by transesterification.
  • the lipase and the fatty acid are used at a temperature of 20 to 80 ° C., more preferably 30 to 70 ° C., and even more preferably 40 to 60 ° C. so that the fatty acid alkyl ester can be used in a liquid state.
  • the contact time is preferably 10 minutes or longer, more preferably 30 minutes or longer. It is more preferable to stir at the time of contact.
  • the fatty acid alkyl ester and the lipase are contacted in one reaction vessel, there is no problem if the total amount of the fatty acid alkyl ester is contacted with the lipase, but the fatty acid alkyl ester is 5 to 50% by mass of the total amount of the fatty acid alkyl ester used.
  • lipase are preferably mixed, and more preferably 5 to 20% by mass of the total amount of fatty acid alkyl ester used is mixed.
  • the fatty acid alkyl ester and lipase thus mixed are brought into contact at 30 to 70 ° C. for 30 minutes or longer while maintaining the mixed state.
  • the fatty acid alkyl ester has an appropriate viscosity and can be sufficiently contacted.
  • fever can be prevented by making it contact at 70 degrees C or less.
  • More preferable contact conditions are 40 to 60 ° C. and 30 minutes or more.
  • the contact time of the fatty acid alkyl ester and the lipase is the time from the start of mixing the fatty acid alkyl ester and the lipase until the vegetable oil of the reaction substrate is added.
  • the mixture of fatty acid alkyl ester and lipase is added to the remaining fatty acid alkyl ester and vegetable oil, the remaining fatty acid alkyl ester and vegetable oil are mixed in advance at a temperature of 30 to 100 ° C. until uniform.
  • a mixture of fatty acid alkyl ester and lipase is added while maintaining this temperature.
  • the above mixing can be performed using a commonly used stirring means such as a stirring blade.
  • the transesterification reaction carried out in the present invention 0.01 to 10 parts by mass (preferably 0.01 to 2 parts by mass, more preferably 0.1 to 1 part by mass) of powder lipase per 100 parts by mass of the fatty acid alkyl ester and the vegetable oil raw material. 0.5 parts by mass) at a temperature of 30 to 100 ° C. (preferably 35 to 80 ° C., more preferably 40 to 60 ° C.) for 0.1 to 50 hours (preferably 0.5 to 30 hours, more preferably Is preferably 1 to 20 hours).
  • the reaction is preferably carried out batchwise.
  • the raw materials for the transesterification reaction fatty acid alkyl ester and vegetable oil
  • those having a water content of 100 to 2000 ppm are preferably used as the raw materials for the transesterification reaction.
  • transesterification reaction it is preferable to remove the unreacted fatty acid alkyl and the produced fatty acid alkyl ester.
  • a removal method it is preferable to use a distillation method.
  • processing such as purification and fractionation of ordinary fats and oils such as solvent removal, fatty acid removal, fatty acid lower alcohol ester removal, decolorization, and deodorization may be performed.
  • Fractionation may be before or after purification.
  • the separation may or may not use a solvent.
  • the solvent used in the solvent fractionation acetone, hexane, ethanol, hydrous ethanol and the like can be mentioned, and acetone and hexane are preferable.
  • transesterified oil thus obtained can be widely used as fats and oils for chocolate, fats and oils for margarine, and fats and oils for shortening.
  • the substrate and enzyme reaction used in the present invention are shown below.
  • the substrate ethyl stearate (manufactured by Inoue Fragrance Co., Ltd .: purity 99% by mass) and high oleic sunflower oil (manufactured by Nisshin Oillio Group Co., Ltd.) were mixed at a ratio (mass ratio) of 6: 4.
  • a substrate whose water content was adjusted to 300 ppm was used as a substrate.
  • Lipase enzyme composition Amano Enzyme Co., Ltd.
  • Lipase DF “Amano” 15-K also called Lipase D
  • deodorized whole fat soybean powder (trade name: Alpha Plus HS-600, JP (Seiyo Rio Group Co., Ltd.)
  • a powder lipase was obtained. What added cellulose powder as a filter aid to this powder lipase was used as a lipase enzyme composition.
  • POS indicates that the fatty acid residue constituting the triglyceride is a palmitic acid residue, an oleic acid residue, or a stearic acid residue.
  • SOS indicates that the fatty acid residue constituting the triglyceride is , Stearic acid residue, oleic acid residue, stearic acid residue.
  • XOX indicates that the fatty acid residue at the 2nd position constituting the triglyceride is the oleic acid residue, and the fatty acid residues at the 1st and 3rd positions are saturated fatty acid residues.
  • Example 1 The lipase enzyme composition is placed in 4 grams of ethyl stearate so that the lipase powder formulation concentration is 6.25 wt% (temperature 50 ° C.), stirred using a stirrer, and the entire lipase powder formulation is covered with ethyl stearate. (Oil-soluble components other than ethyl stearate were less than 1%). After contact for 1 hour in this state, this was put into 46 g of a substrate at 50 ° C. in which ethyl stearate and high oleic sunflower oil were uniformly mixed at a ratio (mass ratio) of 13:10 (of the lipase powder formulation).
  • the final concentration was 0.5 wt%, and the reaction was performed for 16 hours while stirring with a stirrer while maintaining the substrate temperature at 50 ° C. (finally the ethyl stearate of the substrate and high oleic sunflower oil were 6: 4 (mass ratio)) Adjusted to be). After the reaction, the lipase powder formulation was removed. The above operation was further repeated 4 times (in the state where the entire lipase powder formulation was covered with ethyl stearate, the oil-soluble component other than ethyl stearate was 6 to 10%).
  • the lipase enzyme composition was stirred in 4 grams of substrate so that the lipase powder formulation concentration was 6.25 wt%. After 1 hour contact in this state, this was added to 46 grams of substrate (final concentration of lipase powder formulation 0.5 wt%) and allowed to react for 16 hours with stirring with a stirrer while maintaining the substrate temperature at 50 ° C. After the reaction, the lipase powder formulation was removed. The above operation was further repeated 4 times.
  • the value obtained by subtracting the amount of XOX after 0 hours from the amount of XOX after 1 hour of the first reaction as 100 is taken as 100, and the value obtained by subtracting the amount of XOX after 0 hours from the amount of XOX after 1 hour of the subsequent reaction as a relative value. expressed.
  • the lipase enzyme composition was placed in 4 grams of high oleic sunflower oil so that the concentration of the lipase powder preparation was 6.25 wt%, and stirred using a stirrer. After 1 hour of contact in that state, this was added to 46 grams of substrate in which ethyl stearate and high oleic sunflower oil were mixed in a ratio (mass ratio) of 15: 8 (the final concentration of the lipase powder formulation was 0. 1). 5 wt%), and the reaction was continued for 16 hours while stirring with a stirrer while maintaining the substrate temperature at 50 ° C. (finally adjusted to 6: 4 (mass ratio) of the substrate ethyl stearate and high oleic sunflower oil) ).
  • Table 1 As is apparent from Table 1, the present invention, which was pretreated with ethyl stearate for 1 hour, had the highest stability. In Comparative Example 1 where the purity of ethyl stearate is low, no effect was obtained.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Fats And Perfumes (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Provided is a method for producing transesterified oil, the method comprising mixing lipase and a fatty acid alkyl ester, which is an ester of a C16-22 fatty acid and a C1-3 alcohol, effecting contact at 20°C to 80°C for 10 minutes or longer while maintaining a mixed state, and subsequently mixing a plant oil or the remaining fatty acid alkyl ester and a plant oil into the mixture and carrying out a transesterification reaction. According to this method for producing a transesterified oil, a transesterification reaction can be carried out with lipase in the highest possible state of activity, and the lipase can be used in an efficient manner.

Description

エステル交換油の製造方法Method for producing transesterified oil
　本発明は、リパーゼの酵素活性を高めた状態でエステル交換反応を行うことによって、効率的にエステル交換油を製造する方法に関するものである。 The present invention relates to a method for efficiently producing a transesterified oil by performing a transesterification reaction in a state where the enzyme activity of lipase is enhanced.
　脂肪酸アルキルエステルと植物油をリパーゼの作用によりエステル交換して製造される油脂は種々の用途に用いられている。例えば、カカオ脂代用脂などのハードバターを、特定の油脂に１，３選択性リパーゼを作用させ、エステル交換反応を利用して製造する方法が提案されている（特許文献１～５）。これらの文献には、１，３選択性リパーゼとして、リゾプス系リパーゼ、アスペルギルス系リパーゼ、ムコール系リパーゼ、パンクレアチックリパーゼ、米ヌカリパーゼを用いることが記載されている。
　ここで用いるリパーゼは価格が高いので、できるだけリパーゼを高い活性の状態でエステル交換反応させて、リパーゼを効率的に使用することが求められている。 Oils and fats produced by transesterification of fatty acid alkyl esters and vegetable oils by the action of lipases are used in various applications. For example, a method has been proposed in which hard butter such as cocoa butter substitute fat is produced using a 1,3-selective lipase on a specific fat and oil and utilizing a transesterification reaction (Patent Documents 1 to 5). These documents describe the use of lysops lipase, Aspergillus lipase, mucor lipase, pancreatic lipase, and rice nuclease as 1,3-selective lipase.
Since the lipase used here is expensive, it is required to efficiently use the lipase by transesterifying the lipase with as high activity as possible.
特開昭５５－０７１７９７JP 55-071797 特公平０３－０６９５１６JP 03-069516 特公平０６－００９４６５JP 06-009465 ＷＯ９６／１０６４３WO96 / 10643 ＷＯ０３／０００８３２WO03 / 000832
　本発明は、できるだけリパーゼを高い活性の状態でエステル交換反応させて、リパーゼを効率的に使用するエステル交換油の製造方法を提供することを目的とする。 An object of the present invention is to provide a method for producing a transesterified oil that efficiently uses a lipase by transesterifying a lipase with as high an activity as possible.
　本発明は、脂肪酸アルキルエステルと植物油をリパーゼの作用を利用して、エステル交換油を製造するに当たり、リパーゼと、脂肪酸アルキルエステル及び植物油との混合順序の違いにより、エステル交換反応におけるリパーゼ活性に差違が生じるとの知見に基づいてなされたのである。
　すなわち、本発明は、炭素数１６～２２の脂肪酸と炭素数１～３のアルコールとのエステルである脂肪酸アルキルエステルとリパーゼを混合し、混合状態を維持したまま、２０～８０℃で１０分以上接触させ、次いで、この混合物に、前記脂肪酸アルキルエステルと植物油／又は植物油を混合してエステル交換反応を行うことを特徴とするエステル交換油の製造方法を提供する。 In producing transesterified oil using fatty acid alkyl ester and vegetable oil using the action of lipase, the present invention differs in lipase activity in transesterification reaction due to the difference in the mixing order of lipase, fatty acid alkyl ester and vegetable oil. It was made based on the knowledge that this occurs.
That is, the present invention mixes a fatty acid alkyl ester, which is an ester of a fatty acid having 16 to 22 carbon atoms and an alcohol having 1 to 3 carbon atoms, and a lipase, and maintains the mixed state for 10 minutes or more at 20 to 80 ° C. A method for producing a transesterified oil is provided, wherein the fatty acid alkyl ester and vegetable oil / or vegetable oil are mixed with the mixture and subjected to a transesterification reaction.
　本発明によれば、簡単な操作で、使用するリパーゼの活性を向上させることができ、リパーゼの活性が向上した状態でエステル交換反応を行うことができる。
　又、植物油として２位にオレオイル基を有するトリグリセリドに富む油脂を用い、かつステアリン酸アルキルエステルを用いることにより、効率よくＳＯＳに富む油脂を得ることができる。 According to the present invention, the activity of the lipase to be used can be improved by a simple operation, and the transesterification reaction can be performed in a state where the activity of the lipase is improved.
Further, by using an oil and fat rich in triglyceride having an oleoyl group at the 2-position as a vegetable oil and using an alkyl stearate, an oil and fat rich in SOS can be obtained efficiently.
　本発明でエステル交換に用いる原料の脂肪酸アルキルエステルを構成する炭素数１６～２２の脂肪酸としては、飽和脂肪酸が好ましく、特に、ステアリン酸、パルミチン酸、ベヘン酸が好ましい。
　又、本発明でエステル交換に用いる原料の脂肪酸アルキルエステルを構成する炭素数１～３のアルコールとしては、メタノール、エタノール、イソプロピルアルコールが好ましく、このなかでもエタノールが好ましい。
　本発明では、上記脂肪酸アルキルエステルとしては、パルミチン酸メチル、ステアリン酸メチル、ベヘン酸メチル、パルミチン酸エチル、ステアリン酸エチル及びベヘン酸エチルが好ましく、特にステアリン酸エチルが好ましい。 The fatty acid having 16 to 22 carbon atoms constituting the starting fatty acid alkyl ester used for transesterification in the present invention is preferably a saturated fatty acid, particularly stearic acid, palmitic acid or behenic acid.
In addition, as the alcohol having 1 to 3 carbon atoms constituting the fatty acid alkyl ester as a raw material used for transesterification in the present invention, methanol, ethanol and isopropyl alcohol are preferable, and ethanol is preferable.
In the present invention, as the fatty acid alkyl ester, methyl palmitate, methyl stearate, methyl behenate, ethyl palmitate, ethyl stearate and ethyl behenate are preferable, and ethyl stearate is particularly preferable.
　一方、本発明でエステル交換に用いる原料の植物油脂としては、２位にオレオイル基及び／又はリノレオイル基を有するトリグリセリドに富む油脂が好ましい。具体的に、２位にオレオイル基を有するトリグリセリドに富む油脂として、シア脂低融点部分（例えば、ヨウ素価７０～８０）、ハイオレイックヒマワリ油、ハイオレイック紅花油、ハイオレイックローリノレン菜種油、パーム油、パーム分画油、これらの混合油などがあげられる。また、２位にリノレオイル基を有する油脂としてハイリノールサフラワー油、大豆油、グレープシードオイルなどがあげられる。
　これらのうち、上記シア脂低融点部分、ハイオレイックヒマワリ油、ハイオレイックローリノレン菜種油、パーム油、パーム分画油が好ましい。特に、ＳＯＳ及び／又はＰＯＳに富んだハードバターを製造するには、シア脂低融点部分やハイオレイックヒマワリ油を用いるのが好ましい。また、ＰＬＰに富んだハードバターやＳＬＳに富んだハードバターを製造するには、ハイリノールサフラワー油が好ましい。
　脂肪酸アルキルエステル対植物油の使用比率（質量比）は、１／１０以上であるのが好ましく、特に、１／１～５／１であるのが好ましい。 On the other hand, as the raw material vegetable oil used for transesterification in the present invention, an oil rich in triglyceride having an oleoyl group and / or a linoleic oil group at the 2-position is preferable. Specifically, as fats and oils rich in triglycerides having an oleoyl group at the 2-position, shea fat low melting point portion (for example, iodine value 70 to 80), high oleic sunflower oil, high oleic safflower oil, high oleic lorinolen rapeseed oil, Examples thereof include palm oil, palm fraction oil, and mixed oils thereof. Examples of fats and oils having a linoleic oil group at the 2nd position include hylinol safflower oil, soybean oil, and grape seed oil.
Of these, the shea fat low melting point portion, high oleic sunflower oil, high oleic lorinolen rapeseed oil, palm oil, and palm fraction oil are preferred. In particular, in order to produce hard butter rich in SOS and / or POS, it is preferable to use a shea fat low melting point portion or high oleic sunflower oil. Moreover, in order to produce hard butter rich in PLP and hard butter rich in SLS, highlinol safflower oil is preferable.
The use ratio (mass ratio) of the fatty acid alkyl ester to the vegetable oil is preferably 1/10 or more, particularly preferably 1/1 to 5/1.
　本発明では、炭素数１６～２２の直鎖飽和脂肪酸及びその低級アルコールエステルから選ばれる１種以上のものとして、ステアリン酸及び／又はその低級アルコールエステルを用いて、１，３－ジパルミトイル－２－オレオイルグリセリン（ＰＯＰ）、１，３－ジパルミトイル－２－リノレオイルグリセリン（ＰＬＰ）、１，３－ジステアロイル－２－オレオイルグリセリン（ＳＯＳ）、１，３－ジステアロイル－２－リノレオイルグリセリン（ＳＬＳ）、１－ステアロイル－２－オレオイル－３－パルミトイルグリセリン又は１－パルミトイル－２－オレオイル－３－ステアロイルグリセリン（ＰＯＳ）、１，３－ジベヘニル－２－オレオイルグリセリン（ＢＯＢ）、１，３－ジベヘニル－２－リノレオイルグリセリン（ＢＬＢ）などから選ばれる１種以上を製造することができる。この中で、１，３－ジステアロイル－２－オレオイルグリセリン（ＳＯＳ）及び／又は１，３－ジステアロイル－２－リノレオイルグリセリン（ＳＬＳ）に富んだ油脂を製造するのが好ましい。
　ここで、「ＳＯＳ及び／又はＳＬＳに富んだ油脂」とは、油脂を構成する全トリグリセド種中で、１，３－ジステアロイル－２－オレオイルグリセリン及び／又は１，３－ジステアロイル－２－リノレオイルグリセリンを１０質量％以上含むことをいい、好ましくは一番多い量のトリグリセド種がＳＯＳ及び／又はＳＬＳであることをいう。ここで上限は、９０質量％であるのが好ましい。 In the present invention, 1,3-dipalmitoyl-2 is used by using stearic acid and / or a lower alcohol ester thereof as at least one selected from linear saturated fatty acids having 16 to 22 carbon atoms and lower alcohol esters thereof. -Oleoylglycerol (POP), 1,3-dipalmitoyl-2-linoleoylglycerol (PLP), 1,3-distearoyl-2-oleoylglycerol (SOS), 1,3-distearoyl-2- Linoleoylglycerol (SLS), 1-stearoyl-2-oleoyl-3-palmitoylglycerol or 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS), 1,3-dibehenyl-2-oleoylglycerol (BOB), 1,3-dibehenyl-2-linoleoyl glycerin (BLB), etc. It is possible to produce one or more kinds barrel. Of these, it is preferable to produce fats and oils rich in 1,3-distearoyl-2-oleoylglycerol (SOS) and / or 1,3-distearoyl-2-linoleoylglycerol (SLS).
Here, the “fat rich in SOS and / or SLS” means 1,3-distearoyl-2-oleoylglycerin and / or 1,3-distearoyl-2 in all triglyceride species constituting the fat. -It means that 10% by mass or more of linoleoylglycerin is contained, and preferably that the largest amount of triglyceride species is SOS and / or SLS. Here, the upper limit is preferably 90% by mass.
　本発明では、種々のリパーゼを用いることができる。このうち、リゾプス属のリゾプス　デレマー（Ｒｈｉｚｏｐｕｓ　ｄｅｌｅｍａｒ）及びリゾプス　オリザエ（Ｒｈｉｚｏｐｕｓ　ｏｒｙｚａｅ）を用いるのが好ましく、又、１，３－特異性リパーゼであるのが好ましい。
　これらのリパーゼとしては、ディー・エス・エム　ジャパン株式会社の商品：ピカンターゼＲ８０００や、天野エンザイム株式会社の商品：リパーゼＦ－ＡＰ１５等が挙げられるが、最も適したリパーゼとしては　Ｒｈｉｚｏｐｕｓ　ｏｒｙｚａｅ由来、天野エンザイム株式会社の商品：リパーゼＤＦ“Ａｍａｎｏ”１５－Ｋ（リパーゼＤともいう）が挙げられる。このものは粉末リパーゼである。なお、このリパーゼＤＦ“Ａｍａｎｏ”１５－Ｋについては、従来Ｒｈｉｚｏｐｕｓ　ｄｅｌｅｍａｒ由来の表記であった。
　本発明で使用するリパーゼとしては、リパーゼの培地成分等を含有したリパーゼ含有水溶液を乾燥して得られたものでもよい。本発明では、粉末リパーゼとしては、球状で、水分含量が１０質量％以下であるものを用いるのが好ましい。特に、リパーゼ粉末の９０質量％以上が粒径１～１００μｍであるのが好ましい。又、ｐＨが７～８に調整されてなるリパーゼ含有水溶液を噴霧乾燥して製造されるものが好ましい。
　本発明では、大豆粉末を用いて上記リパーゼを造粒し、粉末化した造粒粉末リパーゼ（粉末リパーゼともいう）を用いることができる。 In the present invention, various lipases can be used. Of these, Rhizopus delemar and Rhizopus oryzae are preferred, and 1,3-specific lipase is preferred.
Examples of these lipases include products from DSM Japan, Inc .: Picantase R8000, and products from Amano Enzyme, Inc .: Lipase F-AP15. The most suitable lipase is derived from Rhizopus oryzae, Amano Enzyme. A product of the company: Lipase DF “Amano” 15-K (also referred to as lipase D). This is a powder lipase. The lipase DF “Amano” 15-K has conventionally been derived from Rhizopus delmar.
The lipase used in the present invention may be obtained by drying a lipase-containing aqueous solution containing a lipase medium component or the like. In the present invention, it is preferable to use a powder lipase having a spherical shape and a water content of 10% by mass or less. In particular, 90% by mass or more of the lipase powder preferably has a particle size of 1 to 100 μm. Further, those prepared by spray-drying a lipase-containing aqueous solution whose pH is adjusted to 7-8 are preferred.
In the present invention, granulated powder lipase (also referred to as powder lipase) obtained by granulating the above lipase using soybean powder and making it into powder can be used.
　ここで、大豆粉末としては、脂肪含有量が５質量％以上である大豆粉末を用いるのが好ましい。脂肪含有量が５質量％以上である大豆粉末としては、脂肪含有量が１０質量％以上であるのが好ましく、さらに１５質量％以上であるのが好ましく、一方、２５質量％以下であるのが好ましい。特に脂肪含有量が１８～２３質量％である大豆粉末が好ましい。
　ここで、脂肪としては脂肪酸トリグリセリド及びその類縁体があげられる。大豆の脂肪含量は、ソックスレー抽出法などの方法により容易に測定することができる。
　本発明では、このような大豆粉末として、全脂大豆粉を用いることができる。また大豆粉末の原料として豆乳を用いることもできる。大豆粉末は、大豆を常法により粉砕して製造することができ、その粒径は０．１～６００μｍ程度であるのが好ましい。粒径は、粉末リパーゼの粒径の測定方法と同様の方法により測定することができる。
　リパーゼに対する大豆粉末の使用量は、質量基準で０．１～２００倍の量であるのが好ましく、０．１～２０倍の量であるのがより好ましく、０．１～１０倍の量が最も好ましい。 Here, as the soybean powder, it is preferable to use a soybean powder having a fat content of 5% by mass or more. As the soybean powder having a fat content of 5% by mass or more, the fat content is preferably 10% by mass or more, more preferably 15% by mass or more, and on the other hand, it is 25% by mass or less. preferable. Particularly preferred is soybean powder having a fat content of 18 to 23% by mass.
Here, examples of the fat include fatty acid triglycerides and analogs thereof. The fat content of soybean can be easily measured by a method such as Soxhlet extraction.
In the present invention, full-fat soybean powder can be used as such soybean powder. Moreover, soy milk can also be used as a raw material of soybean powder. The soybean powder can be produced by pulverizing soybean by a conventional method, and the particle size is preferably about 0.1 to 600 μm. The particle size can be measured by a method similar to the method for measuring the particle size of powder lipase.
The amount of soybean powder used relative to the lipase is preferably 0.1 to 200 times, more preferably 0.1 to 20 times, and more preferably 0.1 to 10 times the amount on a mass basis. Most preferred.
　本発明で用いる粉末リパーゼは、水分含量が１０質量％以下であるのが好ましく、特に、１～８質量％であるのが好ましい。
　本発明で用いる粉末リパーゼの粒径は任意とすることができるが、粉末リパーゼの９０質量％以上が粒径１～１００μｍであるのが好ましい。平均粒径は１０～８０μｍが好ましい。又、粉末リパーゼの形状は球状であるのが好ましい。
　粉末リパーゼの粒径は、例えば、株式会社堀場製作所社の粒度分布測定装置（ＬＡ－５００）を用いて測定することができる。
　本発明で用いる粉末リパーゼは、リパーゼ及び大豆粉末を溶解・分散させた水溶液を、スプレードライ、フリーズドライ、及び溶剤沈澱・乾燥の中から選ばれるいずれか１種の乾燥方法で乾燥し、製造することができる。
　ここで、リパーゼ及び大豆粉末を溶解・分散させた水溶液は、リパーゼ粉末と大豆粉末を水に溶解・分散させたり、大豆粉末が溶解・分散した水溶液にリパーゼ粉末を混合したり、又は、後に説明するリパーゼ含有水溶液に大豆粉末を混合することにより得ることができる。 The powder lipase used in the present invention preferably has a water content of 10% by mass or less, particularly preferably 1 to 8% by mass.
The particle size of the powder lipase used in the present invention can be arbitrary, but 90% by mass or more of the powder lipase preferably has a particle size of 1 to 100 μm. The average particle size is preferably 10 to 80 μm. The shape of the powder lipase is preferably spherical.
The particle size of the powder lipase can be measured, for example, using a particle size distribution measuring device (LA-500) manufactured by Horiba, Ltd.
The powder lipase used in the present invention is produced by drying an aqueous solution in which lipase and soybean powder are dissolved and dispersed by any one drying method selected from spray drying, freeze drying, and solvent precipitation / drying. be able to.
Here, the aqueous solution in which lipase and soybean powder are dissolved / dispersed is prepared by dissolving / dispersing lipase powder and soybean powder in water, mixing the lipase powder in an aqueous solution in which soybean powder is dissolved / dispersed, or explained later. It can be obtained by mixing soybean powder with the lipase-containing aqueous solution.
　リパーゼ及び大豆粉末を溶解・分散させた水溶液を乾燥させる過程では、リパーゼ及び／又は大豆粉末の粒子が凝集して、リパーゼ及び大豆粉末を含有する造粒物が形成される。この造粒物は、リパーゼの培地成分を含んでいてもよい。
　このようにして調製した粉末リパーゼは、そのままエステル交換に使用することができる。
　リパーゼ及び大豆粉末を溶解・分散させた水溶液中の水の量は、リパーゼ及び大豆粉末との合計質量に対して水の質量を調整する。具体的には、リパーゼ及び大豆粉末との合計質量に対する水の質量が、０．５～１，０００倍であるのが好ましく、１．０～５００倍であるのがより好ましく、３．０～１００倍が最も好ましい。
　特に、スプレードライにより粉末リパーゼを製造する場合は、装置の特性からリパーゼ及び大豆粉末との合計質量に対する水の質量が、２．０～１，０００倍であるのが好ましく、２．０～５００倍であるのがより好ましく、３．０～１００倍が最も好ましい。
　なお、リパーゼ含有水溶液を原料として使用する場合で、リパーゼ含有水溶液中のリパーゼ含量が不明な時は、フリーズドライ、その他の減圧乾燥によりリパーゼ含有水溶液を粉末化してリパーゼ含量を求め、リパーゼ質量を算出することができる。 In the process of drying the aqueous solution in which lipase and soybean powder are dissolved and dispersed, the particles of lipase and / or soybean powder are aggregated to form a granulated product containing lipase and soybean powder. This granulated product may contain a medium component of lipase.
The powder lipase thus prepared can be used as it is for transesterification.
The amount of water in the aqueous solution in which lipase and soybean powder are dissolved and dispersed adjusts the mass of water with respect to the total mass of lipase and soybean powder. Specifically, the mass of water with respect to the total mass of lipase and soybean powder is preferably 0.5 to 1,000 times, more preferably 1.0 to 500 times, and 3.0 to 100 times is most preferable.
In particular, when powder lipase is produced by spray drying, the mass of water relative to the total mass of lipase and soybean powder is preferably 2.0 to 1,000 times from the characteristics of the apparatus, and preferably 2.0 to 500 times. The ratio is more preferably double, and most preferably 3.0 to 100.
If the lipase-containing aqueous solution is used as a raw material and the lipase content in the lipase-containing aqueous solution is unknown, the lipase-containing aqueous solution is pulverized by freeze drying or other reduced-pressure drying to determine the lipase content, and the lipase mass is calculated. can do.
　ここで、リパーゼ含有水溶液としては、菌体を除去したリパーゼ培養液、精製培養液、これらから得たリパーゼを再度水に溶解・分散させたもの、市販の粉末リパーゼを再度水に溶解・分散させたもの、市販の液状リパーゼ等が挙げられる。さらに、リパーゼ活性をより高めるために塩類等の低分子成分を除去したものがより好ましく、また、粉末性状をより高めるために糖等の低分子成分を除去したものがより好ましい。
　リパーゼ培養液としては、例えば、大豆粉、ペプトン、コーン・ステープ・リカー、Ｋ2ＨＰＯ4、（ＮＨ4）2ＳＯ4、ＭｇＳＯ4・７Ｈ2Ｏ等含有する水溶液があげられる。これらの濃度としては、大豆粉０．１～２０質量％、好ましくは１．０～１０質量％、ペプトン０．１～３０質量％、好ましくは０．５～１０質量％、コーン・ステープ・リカー０．１～３０質量％、好ましくは０．５～１０質量％、Ｋ2ＨＰＯ4　０．０１～２０質量％、好ましくは０．１～５質量％である。又、（ＮＨ4）2ＳＯ4は０．０１～２０質量％、好ましくは０．０５～５質量％、ＭｇＳＯ4・７Ｈ2Ｏは０．０１～２０質量％、好ましくは０．０５～５質量％である。培養条件は、培養温度は１０～４０℃、好ましくは２０～３５℃、通気量は０．１～２．０ＶＶＭ、好ましくは０．１～１．５ＶＶＭ、攪拌回転数は１００～８００ｒｐｍ、好ましくは２００～４００ｒｐｍ、ｐＨは３．０～１０．０、好ましくは４．０～９．５に制御するのがよい。 Here, the lipase-containing aqueous solution includes a lipase culture solution from which bacterial cells have been removed, a purified culture solution, a lipase obtained by dissolving and dispersing the lipase in water again, and a commercially available powder lipase dissolved and dispersed in water again. And commercially available liquid lipase. Further, those obtained by removing low molecular components such as salts in order to further increase the lipase activity are more preferable, and those obtained by removing low molecular components such as sugars in order to further improve the powder properties.
Examples of the lipase culture solution include aqueous solutions containing soy flour, peptone, corn staple liquor, K 2 HPO 4 , (NH 4 ) 2 SO 4 , MgSO 4 .7H 2 O and the like. These concentrations include soybean powder 0.1-20% by weight, preferably 1.0-10% by weight, peptone 0.1-30% by weight, preferably 0.5-10% by weight, corn staple liquor 0.1 to 30% by mass, preferably 0.5 to 10% by mass, K 2 HPO 4 0.01 to 20% by mass, preferably 0.1 to 5% by mass. Further, (NH 4 ) 2 SO 4 is 0.01 to 20% by mass, preferably 0.05 to 5% by mass, and MgSO 4 · 7H 2 O is 0.01 to 20% by mass, preferably 0.05 to 5%. % By mass. The culture conditions are a culture temperature of 10 to 40 ° C., preferably 20 to 35 ° C., an aeration rate of 0.1 to 2.0 VVM, preferably 0.1 to 1.5 VVM, and a stirring speed of 100 to 800 rpm, preferably The pH is controlled to 200 to 400 rpm and the pH is 3.0 to 10.0, preferably 4.0 to 9.5.
　菌体の分離は、遠心分離、膜濾過などで行うのが好ましい。また、塩類や糖等の低分子成分の除去は、ＵＦ膜処理により行うことができる。具体的には、ＵＦ膜処理を行い、リパーゼを含有する水溶液を１／２量の体積に濃縮後、濃縮液と同量のリン酸バッファーを添加するという操作を１～５回繰り返すことにより、低分子成分を除去したリパーゼ含有水溶液を得ることができる。
　遠心分離は２００～２０，０００×ｇ、膜濾過はＭＦ膜、フィルタープレスなどで圧力を３．０ｋｇ／ｍ2以下にコントロールするのが好ましい。菌体内酵素の場合は、ホモジナイザー、ワーリングブレンダー、超音波破砕、フレンチプレス、ボールミル等で細胞破砕し、遠心分離、膜濾過などで細胞残さを除去することが好ましい。ホモジナイザーの攪拌回転数は５００～３０，０００ｒｐｍ、好ましくは１，０００～１５，０００ｒｐｍ、ワーリングブレンダーの回転数は５００～１０，０００ｒｐｍ、好ましくは１，０００～５，０００ｒｐｍである。攪拌時間は０．５～１０分、好ましくは１～５分がよい。超音波破砕は１～５０ｋＨｚ、好ましくは１０～２０ｋＨｚの条件で行うのが良い。ボールミルは直径０．１～０．５ｍｍ程度のガラス製小球を用いるのがよい。
　乾燥工程前の途中の工程において、リパーゼ含有水溶液を濃縮してもよい。濃縮方法は、特に限定されるものではないが、エバポレーター、フラッシュエバポレーター、ＵＦ膜濃縮、ＭＦ膜濃縮、無機塩類による塩析、溶剤による沈殿法、イオン交換セルロース等による吸着法、吸水性ゲルによる吸水法等があげられる。好ましくはＵＦ膜濃縮、エバポレーターがよい。ＵＦ膜濃縮用モジュールとしては、分画分子量３，０００～１００，０００、好ましくは６，０００～５０，０００の平膜または中空糸膜、材質はポリアクリルニトリル系、ポリスルフォン系などが好ましい。 Separation of the cells is preferably performed by centrifugation, membrane filtration or the like. Moreover, removal of low molecular components such as salts and sugars can be performed by UF membrane treatment. Specifically, by performing a UF membrane treatment, concentrating an aqueous solution containing lipase to a volume of ½ volume, and then adding the same amount of phosphate buffer as the concentrated solution 1 to 5 times, A lipase-containing aqueous solution from which low molecular components have been removed can be obtained.
Centrifugation is preferably performed at 200 to 20,000 × g, and membrane filtration is preferably performed at a pressure of 3.0 kg / m 2 or less with an MF membrane, a filter press or the like. In the case of intracellular enzymes, it is preferable to crush the cells with a homogenizer, Waring blender, ultrasonic crushing, French press, ball mill or the like, and remove cell residues by centrifugation, membrane filtration or the like. The stirring rotation speed of the homogenizer is 500 to 30,000 rpm, preferably 1,000 to 15,000 rpm, and the rotation speed of the Waring blender is 500 to 10,000 rpm, preferably 1,000 to 5,000 rpm. The stirring time is 0.5 to 10 minutes, preferably 1 to 5 minutes. The ultrasonic crushing is performed under the condition of 1 to 50 kHz, preferably 10 to 20 kHz. The ball mill preferably uses glass spheres having a diameter of about 0.1 to 0.5 mm.
In the middle step before the drying step, the lipase-containing aqueous solution may be concentrated. The concentration method is not particularly limited, but evaporator, flash evaporator, UF membrane concentration, MF membrane concentration, salting out with inorganic salts, precipitation method with solvent, adsorption method with ion-exchange cellulose, water absorption with water-absorbing gel Law. Preferably, UF membrane concentration and an evaporator are used. The module for concentrating the UF membrane is preferably a flat membrane or hollow fiber membrane having a molecular weight cut-off of 3,000 to 100,000, preferably 6,000 to 50,000, and the material is preferably polyacrylonitrile or polysulfone.
　次に、リパーゼ及び大豆粉末を溶解・分散させた水溶液を乾燥する方法であるスプレードライ、フリーズドライ、又は溶剤沈澱・乾燥について説明する。
　スプレードライは、例えば、ノズル向流式、デイスク向流式、ノズル並流式、デイスク並流式等の噴霧乾燥機を用いて行うのがよい。好ましくはデイスク並流式が良く、アトマイザー回転数は４，０００～２０，０００ｒｐｍ、加熱は入口温度１００～２００℃、出口温度４０～１００℃で制御してスプレードライするのが好ましい。特に、リパーゼと大豆粉末を含有する水溶液の温度を２０～４０℃に調整し、次いで７０℃～１３０℃の乾燥雰囲気内に噴霧するのが好ましい。又、乾燥前に水溶液のｐＨを７．５～８．５に調整しておくのが好ましい。
　フリーズドライ（凍結乾燥）は、例えば、ラボサイズの少量用凍結乾燥機、棚段式凍結乾燥により行うのが好ましい。さらに、減圧乾燥により調製することもできる。 Next, spray drying, freeze drying, or solvent precipitation / drying, which is a method of drying an aqueous solution in which lipase and soybean powder are dissolved and dispersed, will be described.
Spray drying is preferably performed using a spray dryer such as a nozzle countercurrent type, a disk countercurrent type, a nozzle cocurrent type, and a disk cocurrent type. The disk co-current type is preferred, the atomizer speed is 4,000 to 20,000 rpm, the heating is preferably controlled at an inlet temperature of 100 to 200 ° C., and an outlet temperature of 40 to 100 ° C., and spray drying is preferred. In particular, the temperature of the aqueous solution containing lipase and soybean powder is preferably adjusted to 20 to 40 ° C. and then sprayed in a dry atmosphere of 70 to 130 ° C. Further, it is preferable to adjust the pH of the aqueous solution to 7.5 to 8.5 before drying.
Freeze-drying (freeze-drying) is preferably performed by, for example, a lab-size small-scale freeze-dryer or a shelf-type freeze-dryer. Furthermore, it can also be prepared by drying under reduced pressure.
　溶剤沈殿・乾燥は、リパーゼ及び大豆粉末を溶解・分散させた水溶液を、使用する溶剤に徐々に添加して沈殿物を生成させ、得られた沈殿物を遠心分離機を用いて遠心分離を行って沈殿を回収した後、減圧乾燥する。一連の操作は、粉末リパーゼの変性・劣化を防止するために、室温以下の低温条件下で行うのが好ましい。
　溶剤沈澱で用いる溶剤として、例えば、エタノール、アセトン、メタノール、イソプロピルアルコール、及びヘキサン等の水溶性溶剤又は親水性溶剤が挙げられ、これらの混合溶剤も使用することができる。その中でも粉末リパーゼの活性をより高めるために、エタノール又はアセトンを用いることが好ましい。
　使用する溶剤の量は特に限定されないが、リパーゼ及び大豆粉末を溶解・分散させた水溶液の体積に対し、１～１００倍の体積の溶剤を使用するのが好ましく、２～１０倍の体積の溶剤を使用するのがより好ましい。
　また、溶剤沈澱した後、沈殿物は静置後に濾過により得ることができるが、１，０００～３，０００×ｇ程度の軽度な遠心分離により得ることもできる。得られた沈殿物の乾燥は、例えば、減圧乾燥により行うことができる。 In solvent precipitation / drying, an aqueous solution in which lipase and soybean powder are dissolved / dispersed is gradually added to the solvent used to form a precipitate, and the resulting precipitate is centrifuged using a centrifuge. After collecting the precipitate, it is dried under reduced pressure. In order to prevent denaturation / deterioration of the powder lipase, the series of operations is preferably performed under a low temperature condition of room temperature or lower.
Examples of the solvent used in the solvent precipitation include water-soluble solvents or hydrophilic solvents such as ethanol, acetone, methanol, isopropyl alcohol, and hexane, and a mixed solvent thereof can also be used. Of these, ethanol or acetone is preferably used in order to further enhance the activity of the powder lipase.
The amount of the solvent to be used is not particularly limited, but it is preferable to use a solvent having a volume of 1 to 100 times the volume of the aqueous solution in which lipase and soybean powder are dissolved and dispersed, and a solvent having a volume of 2 to 10 times. It is more preferable to use.
Further, after the solvent precipitation, the precipitate can be obtained by standing and then filtered, but can also be obtained by mild centrifugation at about 1,000 to 3,000 × g. The obtained precipitate can be dried, for example, by drying under reduced pressure.
　粉末リパーゼの製造工程には、更に、濾過助剤を添加する工程を含むことができる。
　使用できる濾過助剤としては、シリカゲル、セライト、セルロース、でんぷん、デキストリン、活性炭、活性白土、カオリン、ベントナイト、タルク、砂等があげられる。このうち、シリカゲル、セライト、セルロースが好ましい。濾過助剤の粒径は任意でよいが、１～１００μｍが好ましく、５～５０μｍが特に好ましい。
　エステル交換反応前後又は反応中に使用できる濾過助剤は、リパーゼ及び大豆粉末の合計質量に対し１～５００質量％の量を添加することが好ましく、１０～２００質量％の量を添加することがさらに好ましい。この範囲の量を使用すると、濾過時の負担がより小さくなり、大規模な濾過設備や高度な遠心分離等の濾過前処理を必要としないからである。 The production process of the powder lipase can further include a step of adding a filter aid.
Examples of filter aids that can be used include silica gel, celite, cellulose, starch, dextrin, activated carbon, activated clay, kaolin, bentonite, talc, and sand. Of these, silica gel, celite, and cellulose are preferable. The particle size of the filter aid may be arbitrary, but is preferably 1 to 100 μm, particularly preferably 5 to 50 μm.
The filter aid that can be used before, during, or during the transesterification reaction is preferably added in an amount of 1 to 500% by mass, preferably 10 to 200% by mass, based on the total mass of lipase and soybean powder. Further preferred. This is because if the amount is within this range, the burden during filtration is reduced, and filtration pretreatment such as large-scale filtration equipment and advanced centrifugation is not required.
　また、粉末リパーゼ中に濾過助剤を含有させることもできる。スプレードライ又はフリーズドライにより乾燥を行って粉末リパーゼを得る場合には、乾燥の前又は後のどちらで濾過助剤を添加してもよい。
　溶剤沈殿後に乾燥させる方法によって乾燥を行う場合には、乾燥して得られた粉末リパーゼへ濾過助剤を添加するのが好ましい。
　得られた粉末リパーゼに含有させる濾過助剤の量は、リパーゼと大豆粉末の合計質量を基準として１～５００質量％とすることができ、１０～２００質量％であることがさらに好ましい。 Moreover, a filter aid can be contained in the powder lipase. When the powder lipase is obtained by drying by spray drying or freeze drying, a filter aid may be added either before or after drying.
When drying is performed by a method of drying after solvent precipitation, it is preferable to add a filter aid to the powdered lipase obtained by drying.
The amount of the filter aid to be contained in the obtained powder lipase can be 1 to 500% by mass based on the total mass of the lipase and soybean powder, and more preferably 10 to 200% by mass.
　本発明では、リパーゼが特定の脂肪酸アルキルエステルと接触することが重要である。そのため、炭素数１６～２２の脂肪酸と炭素数１～３のアルコールとのエステルである脂肪酸アルキルエステルとリパーゼを混合して、リパーゼ全体が前記脂肪酸アルキルエステルで覆われるようにする。脂肪酸アルキルエステルとリパーゼを混合した時の混合状態における油溶性成分中の脂肪酸アルキルエステルが高純度になることが好ましく、特に反応にリパーゼを繰り返し用いる際は、反応生成物等が混入し、脂肪酸アルキルエステルの濃度が低下する。従って、脂肪酸アルキルエステル以外の成分が１５質量％以下であることが好ましい。より好ましくは、０～１０質量％であり、さらに好ましくは１～１０質量％である。脂肪酸アルキルエステル以外の油溶性成分としては、遊離の脂肪酸やアルコール、さらにエステル交換反応原料の植物油、エステル交換反応で生成されたグリセリド等が挙げられる。
　脂肪酸アルキルエステルとリパーゼを混合する際、脂肪酸アルキルエステルが液体状態で使用できるように、温度２０～８０℃で、より好ましくは、３０～７０℃、さらに好ましくは、４０～６０℃でリパーゼと脂肪酸アルキルエステルとを接触させることが好ましい。また、接触時間は１０分間以上接触させることが好ましく、３０分間以上接触させることがより好ましい。接触時には攪拌するのがより好ましい。ここで、反応に使用する脂肪酸アルキルエステルの全量の３～１００質量％をリパーゼと接触させることが好ましい。１つの反応容器中で脂肪酸アルキルエステルとリパーゼを接触させる場合、脂肪酸アルキルエステルの全量をリパーゼと接触しても問題はないが、使用する脂肪酸アルキルエステルの全量の５～５０質量％の脂肪酸アルキルエステルとリパーゼを混合するのが好ましく、より好ましくは、使用する脂肪酸アルキルエステルの全量の５～２０質量％を混合する。 In the present invention, it is important that the lipase is in contact with a specific fatty acid alkyl ester. Therefore, a fatty acid alkyl ester that is an ester of a fatty acid having 16 to 22 carbon atoms and an alcohol having 1 to 3 carbon atoms is mixed with the lipase so that the entire lipase is covered with the fatty acid alkyl ester. It is preferable that the fatty acid alkyl ester in the oil-soluble component in the mixed state when the fatty acid alkyl ester and the lipase are mixed is highly purified. Especially when the lipase is repeatedly used in the reaction, a reaction product or the like is mixed and the fatty acid alkyl ester is mixed. The ester concentration decreases. Therefore, it is preferable that components other than fatty acid alkyl ester are 15 mass% or less. More preferably, it is 0 to 10% by mass, and still more preferably 1 to 10% by mass. Examples of oil-soluble components other than fatty acid alkyl esters include free fatty acids and alcohols, further plant oil as a raw material for transesterification, and glycerides generated by transesterification.
When mixing the fatty acid alkyl ester and the lipase, the lipase and the fatty acid are used at a temperature of 20 to 80 ° C., more preferably 30 to 70 ° C., and even more preferably 40 to 60 ° C. so that the fatty acid alkyl ester can be used in a liquid state. It is preferable to contact the alkyl ester. Further, the contact time is preferably 10 minutes or longer, more preferably 30 minutes or longer. It is more preferable to stir at the time of contact. Here, it is preferable that 3 to 100% by mass of the total amount of the fatty acid alkyl ester used in the reaction is brought into contact with the lipase. When the fatty acid alkyl ester and the lipase are contacted in one reaction vessel, there is no problem if the total amount of the fatty acid alkyl ester is contacted with the lipase, but the fatty acid alkyl ester is 5 to 50% by mass of the total amount of the fatty acid alkyl ester used. And lipase are preferably mixed, and more preferably 5 to 20% by mass of the total amount of fatty acid alkyl ester used is mixed.
　本発明では、このようにして混合した脂肪酸アルキルエステルとリパーゼを、混合状態を維持したまま、３０～７０℃で、３０分以上接触させる。３０℃以上で接触させることで、脂肪酸アルキルエステルが適度な粘度を有し、十分に接触させることができる。また、７０℃以下で接触させることで、熱によるリパーゼの失活を防ぐことができる。また、３０分以上接触させることで十分な効果が得られるが、脂肪酸アルキルエステルと接触したまま長時間保存しても何ら問題はない。より好ましい接触条件は４０～６０℃で３０分以上である。
　次いで、この混合物を、残りの脂肪酸アルキルエステルと植物油／又は植物油に加えて混合し、エステル交換反応が生じる温度にして、好ましくは、４０～６０℃に加熱して、エステル交換油を製造するのが好ましい。
　ここで、脂肪酸アルキルエステルとリパーゼの接触時間は、脂肪酸アルキルエステルとリパーゼの混合開始から、反応基質の植物油を添加するまでの時間である。
　上記脂肪酸アルキルエステルとリパーゼの混合物を、残りの脂肪酸アルキルエステルと植物油に加える場合には、予め、残りの脂肪酸アルキルエステルと植物油を３０～１００℃の温度で均一になるまで混合しておくのが好ましく、この温度に保持した状態で、脂肪酸アルキルエステルとリパーゼの混合物を加えるのが好ましい。
　上記混合は、通常使用される攪拌手段、例えば、攪拌羽根などを用いて行うことができる。 In the present invention, the fatty acid alkyl ester and lipase thus mixed are brought into contact at 30 to 70 ° C. for 30 minutes or longer while maintaining the mixed state. By contacting at 30 ° C. or higher, the fatty acid alkyl ester has an appropriate viscosity and can be sufficiently contacted. Moreover, the deactivation of the lipase by a heat | fever can be prevented by making it contact at 70 degrees C or less. Moreover, although sufficient effect is acquired by making it contact for 30 minutes or more, even if it preserve | saves for a long time, contacting with a fatty-acid alkylester, there is no problem. More preferable contact conditions are 40 to 60 ° C. and 30 minutes or more.
This mixture is then added to and mixed with the remaining fatty acid alkyl ester and the vegetable oil / or vegetable oil, brought to a temperature at which the transesterification reaction occurs, and preferably heated to 40-60 ° C. to produce the transesterified oil. Is preferred.
Here, the contact time of the fatty acid alkyl ester and the lipase is the time from the start of mixing the fatty acid alkyl ester and the lipase until the vegetable oil of the reaction substrate is added.
When the mixture of fatty acid alkyl ester and lipase is added to the remaining fatty acid alkyl ester and vegetable oil, the remaining fatty acid alkyl ester and vegetable oil are mixed in advance at a temperature of 30 to 100 ° C. until uniform. Preferably, a mixture of fatty acid alkyl ester and lipase is added while maintaining this temperature.
The above mixing can be performed using a commonly used stirring means such as a stirring blade.
　本発明で行うエステル交換反応は、脂肪酸アルキルエステルと植物油の原料１００質量部当り、粉末リパーゼを０．０１～１０質量部（好ましくは０．０１～２質量部、より好ましくは０．１～１．５質量部）添加し、３０～１００℃の温度（好ましくは３５～８０℃、より好ましくは４０～６０℃）で、０．１～５０時間（好ましくは０．５～３０時間、より好ましくは１～２０時間）エステル交換反応を行うのが好ましい。反応はバッチ式で行うのが好ましい。
　尚、エステル交換反応用の原料（脂肪酸アルキルエステルと植物油）としては、水分含量が１００～２０００ｐｐｍのものを用いるのが好ましい。
　エステル交換反応後、未反応脂肪酸アルキル及び生成した脂肪酸アルキルエステルを除去するのが好ましい。除去の方法としては、蒸留法を用いるのが好ましい。
　このようにエステル交換油が得られるが、必要に応じて、脱溶剤、脂肪酸除去、脂肪酸低級アルコールエステル除去、脱色、脱臭等の通常の油脂の精製及び分別などの加工を行ってもよい。
　分別は精製の前でも後でもよい。分別には、溶剤をもちいても用いなくてもかまわない。溶剤分別で使用する溶剤としては、アセトン、ヘキサン、エタノール、含水エタノールなどが上げられるが、アセトン、ヘキサンが好ましい。ここで、エステル交換反応物１００質量部当り、溶剤を５０～１０００質量部添加し、分別を行なうのが好ましい。
　このようにして得たエステル交換油は、チョコレート用油脂、マーガリン用油脂及びショートニング用油脂などして広くもちいることができる。
　次に本発明を実施例により詳細に説明する。 In the transesterification reaction carried out in the present invention, 0.01 to 10 parts by mass (preferably 0.01 to 2 parts by mass, more preferably 0.1 to 1 part by mass) of powder lipase per 100 parts by mass of the fatty acid alkyl ester and the vegetable oil raw material. 0.5 parts by mass) at a temperature of 30 to 100 ° C. (preferably 35 to 80 ° C., more preferably 40 to 60 ° C.) for 0.1 to 50 hours (preferably 0.5 to 30 hours, more preferably Is preferably 1 to 20 hours). The reaction is preferably carried out batchwise.
In addition, as the raw materials for the transesterification reaction (fatty acid alkyl ester and vegetable oil), those having a water content of 100 to 2000 ppm are preferably used.
After the transesterification reaction, it is preferable to remove the unreacted fatty acid alkyl and the produced fatty acid alkyl ester. As a removal method, it is preferable to use a distillation method.
Thus, transesterified oil can be obtained, but if necessary, processing such as purification and fractionation of ordinary fats and oils such as solvent removal, fatty acid removal, fatty acid lower alcohol ester removal, decolorization, and deodorization may be performed.
Fractionation may be before or after purification. The separation may or may not use a solvent. As the solvent used in the solvent fractionation, acetone, hexane, ethanol, hydrous ethanol and the like can be mentioned, and acetone and hexane are preferable. Here, it is preferable to add 50 to 1000 parts by mass of the solvent per 100 parts by mass of the transesterification reaction product and perform fractionation.
The transesterified oil thus obtained can be widely used as fats and oils for chocolate, fats and oils for margarine, and fats and oils for shortening.
EXAMPLES Next, an Example demonstrates this invention in detail.
　本発明で使用した基質及び酵素反応を次に示す。
基質
　ステアリン酸エチル（株式会社井上香料製造所製：純度９９質量％）及びハイオレイックヒマワリ油（日清オイリオグループ株式会社製）を６：４の比率（質量比）で混ぜ合わせ、油中の水分を３００ｐｐｍに合わせたものを基質とした。
リパーゼ酵素組成物
　天野エンザイム株式会社の商品：リパーゼＤＦ　“Ａｍａｎｏ”　１５－Ｋ　（リパーゼＤともいう）の酵素溶液（１５００００Ｕ／ｍｌ）に脱臭全脂大豆粉末（商品名：アルファプラスＨＳ－６００、日清オイリオグループ株式会社製）１０ｗｔ％水溶液を攪拌しながら３倍量加え、０．５Ｎ　ＮａＯＨ溶液でｐＨ７．８に調整後、噴霧乾燥（東京理科器械（株）社、ＳＤ－１０００型）を行い、粉末リパーゼを得た。
　この粉末リパーゼにろ過助剤としてセルロースパウダーを加えたものを、リパーゼ酵素組成物として使用した。
ＸＯＸ量
　エステル交換反応開始後、一時間後に７．５ｕｌのサンプリングを行い、ヘキサン１．５ｍｌで希釈後、リパーゼ酵素組成物をろ過した。ろ過溶液をガスクロマトグラフィー（ＧＣ）用サンプルとした。ＧＣ（カラム：６５ＴＧ）で分析し、ＰＯＳおよびＳＯＳの面積エリアの合計パーセントをＸＯＸ量とした。ＧＣ条件は、カラム温度：３５０℃、昇温：１℃／分、最終温度：３６５℃である。
　ここで、ＰＯＳは、トリグリセリドを構成している脂肪酸残基が、パルミチン酸残基、オレイン酸残基、ステアリン酸残基であることを示し、ＳＯＳは、トリグリセリドを構成している脂肪酸残基が、ステアリン酸残基、オレイン酸残基、ステアリン酸残基であることを示す。又、ＸＯＸは、トリグリセリドを構成している２位の脂肪酸残基がオレイン酸残基、１位及び３位の脂肪酸残基が飽和脂肪酸残基であることを示す。 The substrate and enzyme reaction used in the present invention are shown below.
The substrate ethyl stearate (manufactured by Inoue Fragrance Co., Ltd .: purity 99% by mass) and high oleic sunflower oil (manufactured by Nisshin Oillio Group Co., Ltd.) were mixed at a ratio (mass ratio) of 6: 4. A substrate whose water content was adjusted to 300 ppm was used as a substrate.
Lipase enzyme composition Amano Enzyme Co., Ltd. product: Lipase DF “Amano” 15-K (also called Lipase D) enzyme solution (150,000 U / ml) in deodorized whole fat soybean powder (trade name: Alpha Plus HS-600, JP (Seiyo Rio Group Co., Ltd.) Add 3 times volume of 10wt% aqueous solution with stirring, adjust to pH 7.8 with 0.5N NaOH solution, and perform spray drying (Tokyo Science Instruments Co., Ltd., SD-1000 type) A powder lipase was obtained.
What added cellulose powder as a filter aid to this powder lipase was used as a lipase enzyme composition.
After the start of the XOX transesterification reaction, 7.5 ul of sampling was performed one hour later, diluted with 1.5 ml of hexane, and the lipase enzyme composition was filtered. The filtered solution was used as a sample for gas chromatography (GC). Analyzed by GC (column: 65TG), the total percentage of the area area of POS and SOS was taken as the amount of XOX. The GC conditions are column temperature: 350 ° C., temperature increase: 1 ° C./min, and final temperature: 365 ° C.
Here, POS indicates that the fatty acid residue constituting the triglyceride is a palmitic acid residue, an oleic acid residue, or a stearic acid residue. SOS indicates that the fatty acid residue constituting the triglyceride is , Stearic acid residue, oleic acid residue, stearic acid residue. XOX indicates that the fatty acid residue at the 2nd position constituting the triglyceride is the oleic acid residue, and the fatty acid residues at the 1st and 3rd positions are saturated fatty acid residues.
実施例１
　リパーゼ酵素組成物をリパーゼ粉末製剤濃度が６．２５ｗｔ％になるように４グラムのステアリン酸エチルに入れ（温度５０℃）、スターラーを用いて攪拌して、リパーゼ粉末製剤全体がステアリン酸エチルで覆われるようにした（ステアリン酸エチル以外の油溶性成分は１％未満であった）。その状態で１時間接触後、これを、ステアリン酸エチルとハイオレイックヒマワリ油が１３：１０の比率（質量比）で均一に混合された５０℃の４６グラムの基質に入れ（リパーゼ粉末製剤の最終濃度０．５ｗｔ％）、基質温度を５０℃に保ちながらスターラーで攪拌しながら１６時間反応させた（最終的に基質のステアリン酸エチルとハイオレイックヒマワリ油が６：４（質量比）になるように調整）。反応後、リパーゼ粉末製剤を除去した。以上の操作をさらに４回繰り返した（リパーゼ粉末製剤全体がステアリン酸エチルで覆われた状態において、ステアリン酸エチル以外の油溶性成分は６～１０％であった）。下記の比較例１の反応１回目の１時間後のＸＯＸ量から０時間後のＸＯＸ量を差し引いた値を１００とし、以降の反応の１時間後のＸＯＸ量から０時間後のＸＯＸ量を差し引いた値を相対値として表した。 Example 1
The lipase enzyme composition is placed in 4 grams of ethyl stearate so that the lipase powder formulation concentration is 6.25 wt% (temperature 50 ° C.), stirred using a stirrer, and the entire lipase powder formulation is covered with ethyl stearate. (Oil-soluble components other than ethyl stearate were less than 1%). After contact for 1 hour in this state, this was put into 46 g of a substrate at 50 ° C. in which ethyl stearate and high oleic sunflower oil were uniformly mixed at a ratio (mass ratio) of 13:10 (of the lipase powder formulation). The final concentration was 0.5 wt%, and the reaction was performed for 16 hours while stirring with a stirrer while maintaining the substrate temperature at 50 ° C. (finally the ethyl stearate of the substrate and high oleic sunflower oil were 6: 4 (mass ratio)) Adjusted to be). After the reaction, the lipase powder formulation was removed. The above operation was further repeated 4 times (in the state where the entire lipase powder formulation was covered with ethyl stearate, the oil-soluble component other than ethyl stearate was 6 to 10%). The value obtained by subtracting the XOX amount after 0 hour from the XOX amount after 1 hour of the first reaction of Comparative Example 1 below as 100 is taken as 100, and the XOX amount after 0 hour is subtracted from the XOX amount after 1 hour of the subsequent reaction. Values were expressed as relative values.
比較例１
　リパーゼ酵素組成物を、リパーゼ粉末製剤濃度が６．２５ｗｔ％になるように４グラムの基質に入れ攪拌した。その状態で１時間接触後、これを、４６グラムの基質に添加し（リパーゼ粉末製剤の最終濃度０．５ｗｔ％）、基質温度を５０℃に保ちながらスターラーで攪拌しながら１６時間反応させた。反応後、リパーゼ粉末製剤を除去した。以上の操作をさらに４回繰り返した。反応１回目の１時間後のＸＯＸ量から０時間後のＸＯＸ量を差し引いた値を１００とし、以降の反応の１時間後のＸＯＸ量から０時間後のＸＯＸ量を差し引いた値を相対値として表した。 Comparative Example 1
The lipase enzyme composition was stirred in 4 grams of substrate so that the lipase powder formulation concentration was 6.25 wt%. After 1 hour contact in this state, this was added to 46 grams of substrate (final concentration of lipase powder formulation 0.5 wt%) and allowed to react for 16 hours with stirring with a stirrer while maintaining the substrate temperature at 50 ° C. After the reaction, the lipase powder formulation was removed. The above operation was further repeated 4 times. The value obtained by subtracting the amount of XOX after 0 hours from the amount of XOX after 1 hour of the first reaction as 100 is taken as 100, and the value obtained by subtracting the amount of XOX after 0 hours from the amount of XOX after 1 hour of the subsequent reaction as a relative value. expressed.
比較例２
　リパーゼ酵素組成物をリパーゼ粉末製剤濃度が６．２５ｗｔ％になるように４グラムのハイオレイックヒマワリ油に入れ、スターラーを用いて攪拌した。その状態で１時間接触後、これを、ステアリン酸エチルとハイオレイックヒマワリ油が１５：８の比率（質量比）で混合された４６グラムの基質に添加し（リパーゼ粉末製剤の最終濃度０．５ｗｔ％）、基質温度を５０℃に保ちながらスターラーで攪拌しながら１６時間反応させた（最終的に基質のステアリン酸エチルとハイオレイックヒマワリ油が６：４（質量比）になるように調整）。反応後、リパーゼ粉末製剤を除去した。以上の操作をさらに４回繰り返した。比較例１の反応１回目の１時間後のＸＯＸ量から０時間後のＸＯＸ量を差し引いた値を１００とし、以降の反応の１時間後のＸＯＸ量から０時間後のＸＯＸ量を差し引いた値を相対値として表した。
　実施例１及び比較例１と２の結果をまとめて表１に示す。 Comparative Example 2
The lipase enzyme composition was placed in 4 grams of high oleic sunflower oil so that the concentration of the lipase powder preparation was 6.25 wt%, and stirred using a stirrer. After 1 hour of contact in that state, this was added to 46 grams of substrate in which ethyl stearate and high oleic sunflower oil were mixed in a ratio (mass ratio) of 15: 8 (the final concentration of the lipase powder formulation was 0. 1). 5 wt%), and the reaction was continued for 16 hours while stirring with a stirrer while maintaining the substrate temperature at 50 ° C. (finally adjusted to 6: 4 (mass ratio) of the substrate ethyl stearate and high oleic sunflower oil) ). After the reaction, the lipase powder formulation was removed. The above operation was further repeated 4 times. The value obtained by subtracting the XOX amount after 0 hour from the XOX amount after 1 hour of the first reaction of Comparative Example 1 as 100, and the value obtained by subtracting the XOX amount after 0 hour from the XOX amount after 1 hour of the subsequent reaction Was expressed as a relative value.
Table 1 summarizes the results of Example 1 and Comparative Examples 1 and 2.
表１
Figure JPOXMLDOC01-appb-I000001
　表１から明らかなように、ステアリン酸エチルで一時間前処理を行った本発明が最も安定性が高かった。また、ステアリン酸エチルの純度が低い比較例１では、効果が得られなかった。 Table 1
Figure JPOXMLDOC01-appb-I000001
As is apparent from Table 1, the present invention, which was pretreated with ethyl stearate for 1 hour, had the highest stability. In Comparative Example 1 where the purity of ethyl stearate is low, no effect was obtained.

Claims (8)

  1. 　炭素数１６～２２の脂肪酸と炭素数１～３のアルコールとのエステルである脂肪酸アルキルエステルとリパーゼを混合し、混合状態を維持したまま、２０～８０℃で１０分以上接触させ、次いで、この混合物に、残りの脂肪酸アルキルエステルと植物油／又は植物油を混合してエステル交換反応を行うことを特徴とするエステル交換油の製造方法。 A fatty acid alkyl ester, which is an ester of a fatty acid having 16 to 22 carbon atoms and an alcohol having 1 to 3 carbon atoms, and lipase are mixed and kept in a mixed state at 20 to 80 ° C. for 10 minutes or more. A method for producing a transesterified oil, which comprises carrying out a transesterification reaction by mixing a remaining fatty acid alkyl ester and a vegetable oil / or vegetable oil in a mixture.
  2. 　脂肪酸アルキルエステルの全量の３～１００質量％の脂肪酸アルキルエステルとリパーゼを混合し、この混合物に、残りの脂肪酸アルキルエステルと植物油を混合してエステル交換反応を行う請求項１記載の製造方法。 2. The production method according to claim 1, wherein 3 to 100% by mass of the fatty acid alkyl ester of the total amount of the fatty acid alkyl ester and the lipase are mixed, and the remaining fatty acid alkyl ester and the vegetable oil are mixed with this mixture to perform a transesterification reaction.
  3. 　脂肪酸アルキルエステルが、飽和脂肪酸エチルエステル及び／又はメチルエステルである請求項１又は２記載の製造方法。 The method according to claim 1 or 2, wherein the fatty acid alkyl ester is a saturated fatty acid ethyl ester and / or a methyl ester.
  4. 　脂肪酸アルキルエステルが、ステアリン酸エチルである請求項３記載の製造方法。 The method according to claim 3, wherein the fatty acid alkyl ester is ethyl stearate.
  5. 　炭素数１６～２２の脂肪酸と炭素数１～３のアルコールとのエステルである脂肪酸アルキルエステルとリパーゼを混合した時の混合状態における油溶性成分中の脂肪酸アルキルエステル以外の成分が１５質量％以下である請求項１～４のいずれか１項記載の製造方法。 Components other than fatty acid alkyl esters in the oil-soluble component in a mixed state when a fatty acid alkyl ester, which is an ester of a fatty acid having 16 to 22 carbon atoms and an alcohol having 1 to 3 carbon atoms, and lipase are mixed are 15% by mass or less. The production method according to any one of claims 1 to 4.
  6. 　植物油が、２位にオレオイル基を有するトリグリセリドである請求項１～５のいずれか１項記載の製造方法。 The production method according to any one of claims 1 to 5, wherein the vegetable oil is a triglyceride having an oleoyl group at the 2-position.
  7. 　植物油が、ハイオレイックヒマワリ油である請求項６記載の製造方法。 The production method according to claim 6, wherein the vegetable oil is high oleic sunflower oil.
  8. 　リパーゼが粉末リパーゼである請求項１～７のいずれか１項記載の製造方法。 The method according to any one of claims 1 to 7, wherein the lipase is a powder lipase.
PCT/JP2012/069676 2011-08-02 2012-08-02 Method for producing transesterified oil WO2013018859A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2011169042A JP5178888B2 (en) 2011-08-02 2011-08-02 Method for producing transesterified oil
JP2011-169042 2011-08-02

Publications (1)

Publication Number Publication Date
WO2013018859A1 true WO2013018859A1 (en) 2013-02-07

Family

ID=47629376

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/069676 WO2013018859A1 (en) 2011-08-02 2012-08-02 Method for producing transesterified oil

Country Status (2)

Country Link
JP (1) JP5178888B2 (en)
WO (1) WO2013018859A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013049803A (en) * 2011-08-31 2013-03-14 Nisshin Oillio Group Ltd Method for producing interesterified fat and device of the same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018056609A1 (en) * 2016-09-23 2018-03-29 씨제이제일제당 (주) Method for preparing fatty acid ethyl ester

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007132775A1 (en) * 2006-05-11 2007-11-22 The Nisshin Oillio Group, Ltd. Method for recovery of lipase activity
JP2010081834A (en) * 2008-09-30 2010-04-15 Fuji Oil Co Ltd Method for producing hard butter composition
JP2010525834A (en) * 2007-05-09 2010-07-29 トランス バイオディーゼル リミテッド Modified immobilized enzyme with high resistance to hydrophilic substrates in organic media
JP2010209153A (en) * 2009-03-06 2010-09-24 Nisshin Oillio Group Ltd Method for manufacturing oils and fats
WO2010110260A1 (en) * 2009-03-25 2010-09-30 不二製油株式会社 Method for producing hard butter composition

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10110260A (en) * 1996-08-12 1998-04-28 Mitsubishi Plastics Ind Ltd Filament for vacuum deposition

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007132775A1 (en) * 2006-05-11 2007-11-22 The Nisshin Oillio Group, Ltd. Method for recovery of lipase activity
JP2010525834A (en) * 2007-05-09 2010-07-29 トランス バイオディーゼル リミテッド Modified immobilized enzyme with high resistance to hydrophilic substrates in organic media
JP2010081834A (en) * 2008-09-30 2010-04-15 Fuji Oil Co Ltd Method for producing hard butter composition
JP2010209153A (en) * 2009-03-06 2010-09-24 Nisshin Oillio Group Ltd Method for manufacturing oils and fats
WO2010110260A1 (en) * 2009-03-25 2010-09-30 不二製油株式会社 Method for producing hard butter composition

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J AM OIL CHEM SOC, vol. 75, no. 4, 1998, pages 511 - 516 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013049803A (en) * 2011-08-31 2013-03-14 Nisshin Oillio Group Ltd Method for producing interesterified fat and device of the same

Also Published As

Publication number Publication date
JP2013032439A (en) 2013-02-14
JP5178888B2 (en) 2013-04-10

Similar Documents

Publication Publication Date Title
JP5186505B2 (en) Hard butter production method
EP2175030B1 (en) Method for producing diacylglycerol-rich fat and/or oil
JP2008022744A (en) Method for modifying oil and fat
JP5145609B2 (en) Lipase powder formulation, production method and use thereof
JP4394598B2 (en) Lipase powder composition and method for producing esterified product using the same
JP5178888B2 (en) Method for producing transesterified oil
JP2007068426A (en) Lipase powder preparation, method for producing the same and use of the same
JPWO2006077860A1 (en) Method for producing purified lipase
JPWO2005097985A1 (en) 1,3-specific lipase powder, process for its production and use thereof
JP5728335B2 (en) Method and apparatus for producing transesterified oil and fat
WO2011068076A1 (en) Lipase powder preparation and use thereof
JP5782130B2 (en) Process for producing diacylglycerol-enriched oil or fat
JP4694938B2 (en) Method for producing fatty acids
WO2007043631A2 (en) Two-staged process for the preparation of fatty acids from fat or oil comprising one step of enzymatic hydrolysis employing an immobilized lipase and an other step of high temperature and pressure hydrolysis
JP5258941B2 (en) Recovery method of lipase activity
JP2019054738A (en) Production method of fatty acids
JP6990019B2 (en) Method for producing fatty acids
JP2008253196A (en) Method for producing fatty acids
JP6069722B2 (en) Method for producing transesterified oil and fat

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12819848

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12819848

Country of ref document: EP

Kind code of ref document: A1