JP2021195471A - Method to reduce 3-MCPD content in fats and oils - Google Patents

Abstract

[Problem] The present invention aims to provide a method for producing a composition containing triglyceride as a main component, in which the content of 3-MCPDE is sufficiently reduced by removing 3-MCPDE once produced from the oil. [Solution] A method for producing a composition containing triglyceride, comprising the steps of: (1) reacting a raw oil containing triglyceride with partial glyceride lipase in the presence of water to hydrolyze 3-MCPD fatty acid esters in the raw oil into 3-MCPD and fatty acids; and (2) removing the aqueous layer after step (1), in which the concentration of 3-MCPD obtained when the raw oil is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is 0.3 ppm or more, and the concentration of 3-MCPD obtained when the oil layer after step (2) is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is lower than the concentration obtained for the raw oil. [Selected Figure] None

Description

The present invention relates to a composition containing triglyceride, a method for producing the same, and the like.

Numerous studies have been conducted on the function of fatty acids in fats and oils in fields such as foods and pharmaceuticals. For example, eicosapentaenoic acid (C20: 5, EPA) and docosahexaenoic acid (C22: 6, DHA), which are constituents of fish oil, have anti-arteriosclerosis effect, brain function improving effect, visual function improving effect, antitumor effect, and anti. Inflammatory effects have been reported. Fish oil is a fat and oil rich in EPA and DHA, and its intake is widely recommended.

3-Chloropropane-1,2-diol (3-MCPD), which is a kind of chloropropanols, is a compound suspected to be carcinogenic. It has been found that 3-MCPD fatty acid ester (3-MCPDE), in which one or two fatty acids are ester-bonded, is present in a trace amount in refined cooking oil. 3-MCPDE is believed to be produced from lipids and chloride ions in the process of high temperature treatment in the refining process of cooking oil. It is known that chloropropanols and fatty acid esters thereof are contained in relatively large amounts in palm-based fats and oils.

Regulations have been set on the content of 3-MCPD in foods in each country such as the EU. In fats and oils, it is known that 3-MCPDE is produced by using diacylglycerol (DAG) or monoacylglycerol (MAG) as a substrate (Non-Patent Document 1). Since the existence of 3-MCPD and its fatty acid esters has been pointed out for DAG-rich fats and oils such as palm-based fats and oils, attempts have been made to reduce the content in the fats and oils by various methods. The European Vegetable Oil Business Association (FEDIOL) has published a list of technologies and their characteristics that can be used to reduce 3-MCPDE in the process of producing fats and oils (Non-Patent Document 2). Among them, it is disclosed that the concentration of 3-MCPDE in fats and oils can be reduced by adsorbent treatment, lowering of deodorizing temperature, shortening of treatment time at high temperature, and the like.

As a method for reducing the content of 3-MCPD and its fatty acid ester in fats and oils, a method of suppressing their production and a method of removing 3-MCPD and its fatty acid ester produced in the process of producing fats and oils are known. Has been done. The former is a method of suppressing the formation of 3-MCPDE in fish oil by a combination of alkali treatment, white clay treatment, and deodorization treatment (Patent Document 1), and the deodorization step of fats and oils is performed in a temperature range of 190 to 230 ° C. A method for producing an edible oil / fat with a reduced amount of a substance that produces 3-MCPD (Patent Document 2), which is treated with an adsorbent and / or an alkali before the treatment in which the glyceride oil / fat is heated to 100 ° C. or higher. A method for suppressing the production of chloropropanols and the like in glyceride fats and oils (Patent Document 3), which is characterized by the above, is known. The latter includes a method for producing an edible oil / fat with a reduced amount of substances that produce 3-MCPD, which comprises adding an adsorbent to a refined oil / fat that has undergone a deodorization step, treating the mixture, and then filtering the oil / fat (Patent Document 4). It has been known.

JP 2015-105354 JP-A-2011-147436 International release 2010/126136 JP-A-2011-147435

Eur. J. Lipid Sci. Technol. 114, 1268-1273 (2012) Review of mitigation measures for 3-MCPD resonator and Glycidyl esters, FEDIOL, Ref. 15SAF108, 24 June 2015

Biological oils such as fish oil usually only remove environmental pollutants, cholesterol, free fatty acids, colored or odor-causing small molecules from crude oil (crude oil) and meet certain standards. It is provided as a product for foods, pharmaceuticals, cosmetics, etc. As a result of heat treatment at a temperature exceeding 100 ° C. in the process, 3-MCPDE is produced. Free 3-MCPD can be relatively easily separated from triglyceride by washing with water, whereas 3-MCPDE once produced in refined oil produced from fish oil or the like has low water solubility and is therefore removed by washing with water. It is difficult to do. Further, 3-MCPDE cannot be easily removed even in a general oil refining step other than washing with water because the hydroxyl group of 3-MCPD is bonded to a fatty acid. The present invention provides a means for removing 3-MCPDE once produced from oil, and a method for producing a composition containing triglyceride as a main component, wherein the content of 3-MCPDE is sufficiently reduced. With the goal.

As a result of diligent research to achieve the above object, the present inventors treated the heat-treated oil with partial glyceride lipase to convert 3-MCPDE into water-soluble 3-MCPD and fatty acid. It was found that 3-MCPD can be removed by hydrolysis and subsequent washing with water. In addition, this treatment did not show a decrease in the ratio of triglyceride in the glyceride contained in the oil.
The present inventors have further studied based on such findings, and have completed the present invention.
That is, the present invention is as follows.

[1] A method for producing a composition containing triglyceride.
(1) A step of reacting a raw material oil containing triglyceride with a partial glyceride lipase in the presence of water to hydrolyze 3-MCPD fatty acid esters in the raw material oil into 3-MCPD and fatty acid, and (2). After step (1), the step of removing the aqueous layer,
Including
The value obtained as the concentration of 3-MCPD generated when the raw material oil is analyzed by the official method Cd29b-13 assay A of the American Society of Oil Chemistry is 0.3 ppm or more.
The above method, wherein the value obtained as the concentration of 3-MCPD generated when the oil layer after the step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is lower than the value obtained for the feedstock oil.
[2] The method according to [1], wherein the raw material oil containing triglyceride is a heating oil.
[3] The method according to [1] or [2], wherein the raw material oil containing triglyceride is fish oil.
[4] The numerical value obtained as the concentration of 3-MCPD generated when the oil reservoir after the step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 10% or more than the numerical value obtained for the raw material oil. Low, the method according to any one of [1] to [3].
[5] The method according to any one of [1] to [4], wherein the partial glyceride lipase is a lipase derived from Penicillium camemberti.
[6] The method according to any one of [1] to [5], wherein the amount of the partial glyceride lipase used in the step (1) is 200 to 1,600 units with respect to 1 g of the raw material oil.
[7] The method according to any one of [1] to [6], wherein the hydrolysis reaction of the step (1) is carried out at 10 to 50 ° C.
[8] The amount of water added to the reaction system of the hydrolysis reaction of the step (1) is 0.10 to 2.5 g with respect to 1 g of the raw material oil, according to any one of [1] to [7]. Method.
[9] A composition containing triglyceride as a main component, wherein the composition contains triglyceride as a main component.
The proportion of triglyceride in the glyceride fraction in the composition is 90.0-99.9 area%.
The numerical value obtained as the concentration of 3-MCPD generated when analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 0.10 ppm to 2.80 ppm, and satisfies at least one of the following a to e. Composition:
a) The value obtained as the concentration of 2-MCPD generated when the composition is analyzed by the American Chemical Society official method Cd29b-13 assay A is 0.50 ppm or less.
b) The concentration of cholesterol ester is 0.030 to 0.20% by weight.
c) The concentration of di- (2-ethylhexyl) phthalate (DEHP) is 14.20-15.60 ppb,
d) The concentration of diisononyl phthalate (DINP) is 9.7 ppb to 11.7 ppb, and e) the acid value (AV) is 1.5 to 37.2.
[10] The composition according to [9], which is a heating oil.
[11] The composition according to [10], which is heated fish oil.
[12] The triglyceride is contained in an amount of 50% by weight or more, 60% by weight or more, 70% by weight or more, 80% by weight or more, 90% by weight or more, or 95% by weight or more based on the total weight of the composition [9] to [ 11] The composition according to any one of.
[13] The proportion of triglyceride in the glyceride fraction in the composition is 95.0-99.9 area%, 96.0-99.9 area%, 97.0-area%, or 99.0- The composition according to any one of [9] to [12], which is 99.9 area%.
[14] The values obtained as the concentration of 3-MCPD generated when analyzed by the American Oil Chemistry Society official method Cd29b-13 assay A are 0.16 to 0.30 ppm, 1.17 to 2.10 ppm, 1.69 to 2.74 ppm, or 1.72 to 2.21. The composition according to any one of [9] to [13], which is ppm.
[15] The numerical values obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the American Society of Oil Chemistry, are 0.50 ppm or less, 0.40 ppm or less, 0.36 ppm or less, 0.27 ppm or less, 0.10 ppm (lower limit of quantification). ) Is less than the composition according to any one of [9] to [14].
[16] The numerical value obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the American Society of Oil Chemistry, is 0.10 ppm (lower limit of quantification) or more, 0.15 ppm or more, 0.17 ppm or more, 0.19 ppm or more, or The composition according to any one of [9] to [14], which is 0.21 ppm or more.
[17] The values obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the American Society of Oil Chemistry, are 0.10ppm to 0.50ppm, 0.10ppm to 0.50ppm, 0.10 to 0.27ppm, 0.19 to 0.50ppm, Or the composition according to any one of [9] to [14], which is 0.21 to 0.36 ppm.
[18] The composition according to any one of [9] to [17], wherein the concentration of cholesterol ester is 0.030 to 0.20% by weight, 0.030 to 0.15% by weight, 0.030 to 0.070% by weight, or 0.030 to 0.050% by weight. thing.
[19] The composition according to any one of [9] to [18], wherein the concentration of di- (2-ethylhexyl) phthalate (DEHP) is 14.20 to 15.60 ppb.
[20] The composition according to any one of [9] to [19], wherein the concentration of diisononyl phthalate (DINP) is 9.7 ppb to 11.7 ppb.
[21] Acid value is 1.5 to 37.2, 2.0 to 37.2, 2.5 to 37.2, 3.0 to 37.2, 3.5 to 37.2, 4.0 to 37.2, 4.5 to 37.2, 5.0 to 37.2, 5.5 to 37.2, 10 to 37.2, or 20 to 20. The composition according to any one of [9] to [20], which is 37.2.
[22] One of [9] to [21], wherein the constituent fatty acids contain 10 area% or more, 15 area% or more, 20 area% or more, 25 area% or more, or 30 area% or more. The composition described.
[23] The composition according to any one of [9] to [22], which is an edible oil.
[24] The composition according to any one of [9] to [22], which is a raw material for foods, supplements, pharmaceuticals, or cosmetics.

INDUSTRIAL APPLICABILITY According to the present invention, the content of 3-MCPDE produced by heat treatment of oil can be reduced without reducing the ratio of triglyceride in the glyceride contained in the oil, and safer cooking oil and the like can be stably produced. Can be manufactured.

In the present specification, when expressing fatty acids, a numerical expression that simply expresses the number of carbon atoms, the number of double bonds, and the position of double bonds by combining numbers and alphabets may be used. For example, a saturated fatty acid having 20 carbon atoms is described as "C20: 0", a monounsaturated fatty acid having 18 carbon atoms is described as "C18: 1", eicosapentaenoic acid is described as "C20: 5 n-3", etc. Can be written as. "N-" indicates the position of the first double bond counting from the methyl end of the fatty acid. For example, in the case of "n-3", the position of the first double bond counting from the methyl end of the fatty acid is 3. Indicates that it is between the 4th carbon atom and the 4th carbon atom. This method is well known to those skilled in the art, and the fatty acids described according to this method can be easily specified by those skilled in the art.

As used herein, the term "highly unsaturated fatty acid" means a fatty acid having 18 or more carbon atoms and 3 or more double bonds. The highly unsaturated fatty acid can be, for example, a fatty acid having 20 or more carbon atoms and 3 or more or 4 or more double bonds, or a fatty acid having 20 or more carbon atoms and 5 or more double bonds. Examples of highly unsaturated fatty acids include α-linolenic acid (18: 3 n-3), γ-linolenic acid (18: 3 n-6), dihomo-γ-linolenic acid (20: 3 n-6), and arachidone. Examples include acid (20: 4 n-6), eicosapentaenoic acid (20: 5 n-3), docosapentaenoic acid (22: 5 n-6), and docosahexaenoic acid (22: 6 n-3).

In the present specification, "crude oil" means a mixture of lipids in a state extracted from living organisms, and may be referred to as crude oil. In the present specification, the term "refined oil" refers to crude oil obtained by performing a refining step of at least one fat or oil selected from the group consisting of a degumming step, a deoxidizing step, a decoloring step, and a deodorizing step. It means oil that has been refined into crude oil to remove substances other than the target substances such as lipids and sterols. Those skilled in the art can distinguish between refined oils and crude oils by routine analysis.

As used herein, the generic term (eg, highly unsaturated fatty acids) does not rule out the possibility of the presence of multiple components unless the context clearly indicates otherwise. Therefore, the generic term usually means that at least one component is present.

<Method for producing a composition containing triglyceride>
The present invention is a method for producing a composition containing triglyceride.
(1) A step of reacting a raw material oil containing triglyceride with a partial glyceride lipase in the presence of water to hydrolyze 3-MCPD fatty acid esters in the raw material oil into 3-MCPD and fatty acid, and (2). After step (1), the step of removing the aqueous layer,
Including
The value obtained as the concentration of 3-MCPD generated when the raw material oil is analyzed by the official method Cd29b-13 assay A of the American Society of Oil Chemistry is 0.3 ppm or more.
Regarding the above method, the numerical value obtained as the concentration of 3-MCPD generated when the oil reservoir after the step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is lower than the numerical value obtained for the feedstock oil. Hereinafter, it is also referred to as the method of the present invention).

The raw material oil in the method of the present invention is not particularly limited as long as it is an oil containing triglyceride as a main component, and may be, for example, aquatic animal oil, microbial oil, or vegetable oil. In the present specification, when triglyceride is contained as a main component, the content of triglyceride in the raw material oil is 50% by weight or more, for example, 60% by weight or more, 70% by weight or more, 80% by weight or more of the total weight of the raw material oil. , 90% by weight or more, or 95% by weight or more. Specific examples of raw material oils include sardine oil, tuna oil, bonito oil, menhayden oil, cod liver oil, herring oil, capellin oil, fish oil such as salmon oil, marine animal oil derived from shellfish such as oyster, egoma, and flaxseed. , Soybeans, vegetable oils derived from rapeseed, yeasts such as Yarrowia, Mortierella, Penicillium, Aspergillus, Rhodotorula, Fusarium, etc. Examples thereof include filamentous fungi to which it belongs, algae such as Euglena, and oils derived from lipid-producing microorganisms such as stramenopile. The feedstock oil may be an oil derived from a genetically modified microorganism into which a gene such as a genetically modified mutant Δ9 erongase gene has been introduced. In addition, oils derived from genetically modified plants from oil seed plants such as Brassica species, sunflowers, corn, cotton, flax, and safflower into which genes such as the mutant Δ9 erongase gene have been introduced by recombinant technology can also be used as raw material oils. .. As the genetically modified vegetable oil, the genetically modified microbial oil and the like, those described in WO2012 / 027698, WO2010 / 033753 and the like can be exemplified. It is preferable to select a raw material oil containing a certain amount or more of highly unsaturated fatty acids among the constituent fatty acids, and those containing 10 area% or more, 15 area% or more, 20 area% or more, 25 area% or more, or 30 area% or more. You can choose. In one embodiment, the feedstock oil in the method of the invention may be fish oil, preferably tuna oil or bonito oil.

The feedstock oil contains a 3-MCPD forming material. In the present specification, the 3-MCPD-forming substance means free 3-MCPD and its fatty acid ester (3-MCPDE), and its content in the sample is the AOCS official method. It is represented by a numerical value obtained as a concentration of 3-MCPD by Cd29b-13 assay A. The numerical value obtained as the concentration of 3-MCPD generated when the raw material oil is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 0.3 ppm or more, for example, 0.31 ppm or more, 0.32 ppm or more, 0. .33 ppm or more, 0.34 ppm or more, 0.35 ppm or more, 0.4 ppm or more, 0.45 ppm or more, 0.5 ppm or more, 1 ppm or more, 1.5 ppm or more, 2 ppm or more, 2.5 ppm or more, or 3 ppm or more. obtain.

In the present specification, the analysis by the American Petrochemical Society official method Cd29b-13 assay A is performed by the following procedure well known to those skilled in the art.
Add 100 μL of 100 mg of sample to 3-MCPD-d5-dipalmitate standard solution (diluted with toluene to make 5 ppm 3-MCPD-d5) and 600 μL of diethyl ether, stir and mix until completely dissolved, and then -22. Cool at ° C to -25 ° C for about 15 minutes. Then, 350 μL of a sodium hydroxide-methanol solution (0.25 g of sodium hydroxide dissolved in 100 mL of methanol) is added, and the mixture is thoroughly stirred and then reacted at -22 ° C to -25 ° C for 16 hours or more. After stopping the reaction by adding 600 μL of an acidic sodium bromide solution (600 g of sodium bromide dissolved in 1 L of purified water and 3 mL of 85% phosphoric acid added) at the same temperature, the organic layer separated into two layers becomes about 100 μL. Spray with nitrogen to concentrate. Then, 600 μL of hexane is added, the mixture is vigorously stirred, and the mixture is allowed to stand for 5 to 10 minutes to remove the organic layer twice. 600 μL of a mixed solution of diethyl ether: ethyl acetate (3: 2, V / V) is added to the remaining aqueous layer, and the mixture is vigorously stirred, and the recovery of the organic layer is repeated 3 times. The collected three organic layers are combined and dehydrated with anhydrous sodium sulfate. The organic layer after dehydration is concentrated to 200 μL by spraying nitrogen, 20 μL of a saturated phenylboronic acid-diethyl ether solution is added, and the mixture is vigorously stirred for 10 seconds, and then nitrogen is sprayed to completely remove the solvent. 200 μL of isooctane is added thereto and stirred vigorously for 10 seconds, and the obtained solution is used as a GC-MS sample.
The calibration curve for 3-MCPD quantification is a 3-MCPD standard solution prepared by dissolving 3-MCPD-dipalmitate in toluene so that the 3-MCPD concentration is 0 ppm, 0.5 ppm, 1 ppm, or 5 ppm. It is prepared by analyzing in the same way as the sample of.
The analysis conditions for GC-MS are as follows.
GC-MS conditions
GC: GC-2010 and GCMS-QP2010 (Shimadzu Corporation)
Column: DB-5ms (Agilent Technologies)
30 mx 0.25 mm ID, 0.25 μm film thickness
Carrier gas: helium, 1.2 mL / min
Injection port: 250 ° C, 1 μL, splitless sampling time 1 minute Column temperature: 85 ° C, 0.5 minute retention → 6 ° C / min → 150 ° C, 5 minute retention → 12 ° C / min → 180 ° C → 25 ° C / min → 280 ℃, hold for 7 minutes Ionization temperature: 200 ℃
Interface temperature: 200 ° C
Ionization method: EI, SIM monitoring m / z is as follows
3-MCPD-d5: m / z = 149, 150, 201, 203
3-MCPD: m / z = 146, 147, 196, 198
For quantification, m / z = 150 of 3-MCPD-d5 and m / z = 147 of 3-MCPD are used, and the others are used to confirm the target substance.

In the above analytical method, both the free form and the ester form of 3-MCPD in the sample are detected without distinction as 3-MCPD. Therefore, the measured value of the above analysis method is the total content (ppm (mg / kg)) of the 3-MCPD free form originally present in the sample and the 3-MCPD free form that can be formed from the ester form. Represents.

The raw material oil in the method of the present invention may be crude oil or refined oil. 3-MCPD or 3-MCPDE is usually contained only slightly or hardly in crude oil, and 3-MCPDE is produced in the process of refining. Therefore, in a preferred embodiment, the feedstock in the method of the invention can be refined oil.

Alternatively, when the raw material oil in the method of the present invention is crude oil, a step of refining the crude oil may be included before the step (1). The purification step may be at least one selected from the group consisting of a degumming step, a deoxidizing step, a decoloring step, and a deodorizing step. Each of these steps can be performed by a method known in the art.

3-MCPDE can be produced by heat treatment at temperatures above 100 ° C. In the present specification, oils that have been heat-treated at temperatures exceeding 100 ° C., such as 110 ° C. or higher, 120 ° C. or higher, 130 ° C. or higher, 140 ° C. or higher, 160 ° C. or higher, 180 ° C. or higher, or 200 ° C. or higher, are used. It is called heating oil. The raw material oil in the method of the present invention may be a heated oil containing triglyceride, for example, an oil that has been heat-treated at a temperature of 160 ° C. or higher for 15 minutes or longer, or an oil that has been heat-treated at a temperature of 120 ° C. or higher for 5 hours or longer. Can be oil that has received. Examples of the heat treatment include distillation such as short-stroke distillation, molecular distillation, and steam distillation. In one embodiment, the feedstock oil can be the oil after distillation.

Alternatively, when the raw material oil in the method of the present invention is not a heating oil, the raw material oil containing triglyceride is charged at 100 ° C. or higher, for example, 110 ° C. or higher, 120 ° C. or higher, 130 ° C. or higher, 140 ° C. or higher before the step (1). , 160 ° C. or higher, 180 ° C. or higher, or 200 ° C. or higher may be further included. The heating time can be 15 minutes or longer, for example 30 minutes or longer, 45 minutes or longer, 1 hour or longer, 2 hours or longer, 3 hours or longer, 4 hours or longer, or 5 hours or longer.

In the method of the present invention, the partial glyceride lipase refers to a lipase having higher substrate selectivity than monoglyceride and / or diglyceride as compared with triglyceride. Partial glyceride lipase includes monoglyceride lipase or diglyceride lipase derived from animal organs such as rat small intestine and pig adipose tissue, and monoglyceride lipase derived from Bacillus sp. H-257 (J. Biochem., 127, 419-425, 2000). ), Pseudomonas sp. LP7315-derived monoglyceride lipase (J. Biosci. Bioeng., 91 (1), 27-32, 2001), Penicillium cyclopium-derived lipase (J. Biochem., 87 (1)) ), 205-211, 1980), Penicillium camemberti U-150-derived lipase (J. Ferment. Bioeng., 72 (3), 162-167, 1991). In one embodiment, the partial glyceride lipase in the present invention is a lipase derived from a penicillium genus microorganism, preferably a lipase derived from penicillium camemberti, more preferably lipase GS "Amano" 250G (manufactured by Amano Enzyme Co., Ltd.) or Lipase G Amano 50 (manufactured by Amano Enzyme Co., Ltd.).

The amount of partial glyceride lipase used is not particularly limited, but it reacts with 10 units (that is, 10 unit / g oil) or more per 1 g of raw material oil, and 30 unit / g oil or more considering the practicality considering the reaction rate. It is preferable to add it to the system, for example, 50 unit / g oil or more, 100 unit / g oil or more, 200 unit / g oil or more, 400 unit / g oil or more, 800 unit / g oil or more, or 1,600 unit / g oil. The above can be added to the reaction system. For example, the amount of lipase used is 10 to 2,000 unit / g oil, 50 to 1,600 unit / g oil, 100 to 1,600 unit / g oil, 200 to 1,600 unit / g oil, or 200 to 800 unit / g oil. obtain. As for immobilized lipase, since it can be used repeatedly, instead of adding the required amount as in the case of non-immobilized lipase, add an excessive amount of immobilized lipase to the reaction system, recover it after the reaction, and use it repeatedly. Can be done. Therefore, at least the amount used for 1 g of the above raw material oil may be added to the reaction system, and a reasonable amount may be added depending on whether it is used in batch processing, column processing, or how many times it is used repeatedly. May be changed to. For example, an immobilized lipase of 3 to 30% (w / w), 4 to 25% (w / w), more preferably 5 to 20% (w / w) with respect to the feedstock oil can be used.

Here, one unit of partial glyceride lipase is measured by the LV emulsification method, and the amount of enzyme that causes an increase of 1 micromol of fatty acid per minute is defined as 1 unit (unit). In the LV emulsification method, lipase is allowed to act on an emulsified solution of vinyl laurate, the reaction is stopped with a mixed solvent of ethanol / acetone, the produced fatty acid is neutralized with sodium hydroxide, and the remaining sodium hydroxide is titrated with hydrochloric acid. Therefore, it is a method of quantifying the produced fatty acid to obtain the number of units.

In the step (1) of the method of the present invention, the raw material oil containing triglyceride is reacted with partial glyceride lipase in the presence of water. The hydrolysis reaction by lipase should be carried out in the presence of a sufficient amount of water for the hydrolytic activity of lipase to be exhibited. The amount of water added is, for example, 0.10 to 2.5 g, 0.15 to 2.5 g, 0.15 to 2.0 g, or 0.15 to 1.5 g, preferably 0.15 to 0.70 g per 1 g of raw material oil.

Hydrolysis is preferably carried out in an atmosphere of an inert gas such as dry nitrogen in order to suppress deterioration of fatty acids and deactivation of lipase. Further, the raw material oil may contain an antioxidant such as tocopherol, ascorbic acid, and t-butylhydroquinone.

The reaction temperature in hydrolysis is not particularly limited as long as the lipase exhibits activity, and may be, for example, 10 to 50 ° C., 20 to 50 ° C., preferably about 20 ° C. to 40 ° C. Considering that the lipase reaction rate itself becomes too slow and the viscosity of fats and oils becomes high at 10 ° C or lower, the reaction temperature is most preferably about 30 ° C ± 5 ° C. In the case of a large-scale reaction, the temperature in the reaction vessel may be set to be 10 to 50 ° C on average, and the reaction may be carried out while keeping the change in the reaction temperature within the range of about ± 5 ° C. The hydrolysis reaction can be carried out under a fluid state by mechanical stirring or blowing of an inert gas or the like.

Hydrolysis is carried out until the hydrolysis of 3-MCPDE proceeds to the desired degree. The reaction conditions may differ depending on the raw material oil, the amount of enzyme, and the like. For example, when the amount of partial glyceride lipase used is large, the reaction time may be shortened, and when the amount of partial glyceride lipase used is small, the reaction time may be lengthened. In one embodiment, the reaction time can be, for example, 5 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more, 48 hours or less, 36 hours or less, or 24 hours or less.

In the step (2), the aqueous layer of the reaction solution of the step (1) is removed. Since the 3-MCPD produced in the step (1) is contained in the aqueous layer, a composition containing triglyceride as a main component, in which the content of the 3-MCPD forming substance is reduced, is obtained as the oil layer by the step (2). Be done. The water layer can be removed by a known method. For example, after the hydrolysis reaction time is completed, the lipase can be inactivated by heat treatment, the aqueous layer and the oil layer can be separated by centrifugation, and the aqueous layer can be removed.

After removing the aqueous layer, it may be washed with water one or more times. Washing with water can be performed by a known method. For example, distilled water is added to the oil layer and shaken, and then the water layer and the oil layer are separated by centrifugation, and the water layer is removed.

The obtained oil layer may be further dehydrated. The dehydration treatment can be carried out by a known method. For example, dehydration can be achieved by adding an azeotropic agent such as ethanol to the oil layer and performing distillation such as vacuum distillation.

The content of the 3-MCPD-forming substance in the oil layer after the step (2) is lower than the content in the raw material oil. In a preferred embodiment, the value obtained as the concentration of 3-MCPD produced when the oil reservoir after step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 10% higher than the value obtained for feedstock oil. The above, for example, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more lower.

The content of the 3-MCPD-forming substance in the oil layer after the step (2) may vary depending on the content of the 3-MCPD-forming substance in the raw material oil. For example, if the value obtained as the concentration of 3-MCPD generated when the raw material oil is analyzed by the official method Cd29b-13 assay A of the American Society of Oil Chemistry is about 3.2 ppm, the oil layer after step (2) is American oil chemistry. The numerical values obtained as the concentration of 3-MCPD generated when analyzed by the official method Cd29b-13 assay A are 2.9 ppm or less, for example, 2.7 ppm or less, 2.6 ppm or less, 2.5 ppm or less, 2.4 ppm or less, 2.3 ppm or less. , 2.2 ppm or less, 2.1 ppm or less, 2.0 ppm or less, 1.9 ppm or less, 1.8 ppm or less, or 1.7 ppm or less. When the value obtained as the concentration of 3-MCPD generated when the raw material oil is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is about 2.4 ppm, the oil layer after step (2) is officially approved by the American Petrochemical Society. The values obtained as the concentration of 3-MCPD produced by the method Cd29b-13 assay A are 2.1 ppm or less, for example, 2.0 ppm or less, 1.9 ppm or less, 1.8 ppm or less, 1.7 ppm or less, 1.6 ppm or less, 1.5. It can be less than ppm, less than 1.4ppm, less than 1.3ppm, less than 1.2ppm, or less than 1.1ppm. When the value obtained as the concentration of 3-MCPD generated when the raw material oil is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is about 0.34 ppm, the oil layer after step (2) is officially approved by the American Petrochemical Society. The values obtained as the concentration of 3-MCPD generated by the method Cd29b-13 assay A are 0.31 ppm or less, for example, 0.30 ppm or less, 0.29 ppm or less, 0.27 ppm or less, 0.25 ppm or less, 0.23 ppm or less, 0.21. It can be less than ppm, less than 0.19ppm, less than 0.17ppm, or less than 0.16ppm.

In one aspect, the method of the invention can further reduce the content of 2-MCPD-forming material in the feedstock oil. In the present specification, the 2-MCPD-forming substance means free 2-MCPD and its fatty acid ester (2-MCPDE), and the content thereof is 2-according to the American Society of Petrochemicals official method Cd29b-13 assay A. It is expressed as a numerical value obtained as the concentration of MCPD. Analysis by the American Petrochemical Society official method Cd29b-13 assay A is performed by the following procedure well known to those skilled in the art.
Add 100 μL of 100 mg of sample to 2-MCPD-d5-dipalmitate standard solution (diluted with toluene to make 5 ppm 2-MCPD-d5) and 600 μL of diethyl ether, stir and mix until completely dissolved, and then -22. Cool at ° C to -25 ° C for about 15 minutes. Then, 350 μL of a sodium hydroxide-methanol solution (0.25 g of sodium hydroxide dissolved in 100 mL of methanol) is added, and the mixture is thoroughly stirred and then reacted at -22 ° C to -25 ° C for 16 hours or more. After stopping the reaction by adding 600 μL of an acidic sodium bromide solution (600 g of sodium bromide dissolved in 1 L of purified water and 3 mL of 85% phosphoric acid added) at the same temperature, the organic layer separated into two layers becomes about 100 μL. Spray with nitrogen to concentrate. Then, 600 μL of hexane is added, the mixture is vigorously stirred, and the mixture is allowed to stand for 5 to 10 minutes to remove the organic layer twice. 600 μL of a mixed solution of diethyl ether: ethyl acetate (3: 2, V / V) is added to the remaining aqueous layer, and the mixture is vigorously stirred, and the recovery of the organic layer is repeated 3 times. The collected three organic layers are combined and dehydrated with anhydrous sodium sulfate. The organic layer after dehydration is concentrated to 200 μL by spraying nitrogen, 20 μL of a saturated phenylboronic acid-diethyl ether solution is added, and the mixture is vigorously stirred for 10 seconds, and then nitrogen is sprayed to completely remove the solvent. 200 μL of isooctane is added thereto and stirred vigorously for 10 seconds, and the obtained solution is used as a GC-MS sample.
The calibration curve for 2-MCPD quantification is a 2-MCPD standard solution prepared by dissolving 2-MCPD-dipalmitate in toluene so that the 2-MCPD concentration is 0 ppm, 0.5 ppm, 1 ppm, or 5 ppm. It is prepared by analyzing in the same way as the sample of.
The analysis conditions for GC-MS are as follows.
GC-MS conditions
GC: GC-2010 and GCMS-QP2010 (Shimadzu Corporation)
Column: DB-5ms (Agilent Technologies)
30 mx 0.25 mm ID, 0.25 μm film thickness
Carrier gas: helium, 1.2 mL / min
Injection port: 250 ° C, 1 μL, splitless sampling time 1 minute Column temperature: 85 ° C, 0.5 minute retention → 6 ° C / min → 150 ° C, 5 minute retention → 12 ° C / min → 180 ° C → 25 ° C / min → 280 ℃, hold for 7 minutes Ionization temperature: 200 ℃
Interface temperature: 200 ° C
Ionization method: EI, SIM; m / z to be monitored is as follows
2-MCPD-d5: m / z = 201, 203
2-MCPD: m / z = 196, 198
For quantification, m / z = 201 of 2-MCPD-d5 and m / z = 196 of 3-MCPD are used, and the others are used to confirm the target substance.

In the above analytical method, both the free form and the ester form of 2-MCPD in the sample are detected without distinction as 2-MCPD. Therefore, the measured value of the above analysis method is the total content (ppm (mg / kg)) of the 2-MCPD free form originally present in the sample and the 2-MCPD free form that can be formed from the ester form. Represents.

The numerical value obtained as the concentration of 2-MCPD generated when the oil reservoir after the step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A may be lower than the numerical value obtained for the feedstock oil. In a preferred embodiment, the value obtained as the concentration of 2-MCPD produced when the oil reservoir after step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 10% higher than the value obtained for feedstock oil. Above, for example, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, or 60% or more lower.

The content of the 2-MCPD-forming substance in the oil layer after the step (2) may vary depending on the content of the 2-MCPD-forming substance in the raw material oil. For example, if the value obtained as the concentration of 2-MCPD generated when the raw material oil is analyzed by the official method Cd29b-13 assay A of the American Society of Oil Chemistry is about 0.3 ppm, the oil layer after step (2) is American oil chemistry. The values obtained as the concentration of 2-MCPD generated when analyzed by the official method Cd29b-13 assay A are 0.27 ppm or less, for example, 0.24 ppm or less, 0.21 ppm or less, 0.18 ppm or less, 0.16 ppm or less, 0.14 ppm or less. , 0.12 ppm or less, or 0.10 ppm or less. When the value obtained as the concentration of 2-MCPD generated when the raw material oil is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is about 0.6 ppm, the oil layer after step (2) is officially approved by the American Petrochemical Society. The value obtained as the concentration of 2-MCPD produced by the method Cd29b-13 assay A is 0.54 ppm or less, for example, 0.48 ppm or less, 0.42 ppm or less, 0.38 ppm or less, 0.30 ppm or less, 0.24 ppm or less, or It can be 0.20 ppm or less.

Biological oils such as fish oil and microbial oil contain cholesterol in addition to triglycerides. Partial glyceride lipase treatment or washing with water on cholesterol-containing oils does not completely remove cholesterol. Therefore, in certain embodiments herein, the composition produced by the method of the invention contains cholesterol. The cholesterol concentration in the composition is similar to the concentration in the feedstock oil.

Cholesterol is a compound having a steroid skeleton represented by the molecular formula C ₂₇ H ₄₆ O, and exists as a free form or an ester form in natural products. The ester form is acylcholesterol in which a fatty acid is bound to a portion of a hydroxy group (OH group). In the present invention, the total cholesterol content (or the concentration of total cholesterol) means the total content of the free form and the ester form. The total cholesterol content is measured by the following method.
Add 1 mL of 0.1 g / L 5α-cholestane / ethanol solution as an internal standard to about 0.1 g of the sample, add 1 mL of 2 mol / L potassium hydroxide / hydrous ethanol solution, and heat at 100 ° C. for 10 minutes. After cooling, 3 mL of petroleum ether and 3 mL of saturated ammonium sulfate are added, and after stirring and standing, the upper layer is recovered and measured by gas chromatography under the following measurement conditions. In order to determine the relative sensitivity of 5α-cholestane and cholesterol, a hexane solution containing 0.25 mg / L of 5α-cholestane and 0.25 mg / L of cholesterol is analyzed by gas chromatography. Calculate the total cholesterol content from the sample volume and each area value.
Gas chromatography analysis conditions Model: Agilent 6890 GC system (Agilent)
Column: DB-1 J & W 123-1012
Column temperature: 270 ° C
Injection temperature: 300 ° C
Injection method: Split Split ratio: 50: 1
Detector temperature: 300 ° C
Detector: FID
Carrier gas: Helium (39.3 kPa, constant pressure)

The amount of free cholesterol and cholesterol ester is measured by the following method.
Mes up about 1 g of the sample with hexane to 10 mL and sample 0.5 mL. This is charged into a silica gel cartridge (Waters, Sep-pack® Plus Silica Cartridge 55-105 μm), hexane: diethyl ether = 9: 1 (v / v) 5 mL is poured, and the eluate is cholesterol ester. It is a fraction. Further, 5 mL of diethyl ether is poured, and the eluate is used as a cholesterol fraction. After evaporating the solvent under a nitrogen stream, perform the same operation as the total cholesterol content measurement, and calculate the cholesterol ester amount and free cholesterol content from the sample amount and area value.

The concentration of free cholesterol, the concentration of cholesterol ester, and the concentration of total cholesterol in the composition produced by the method of the present invention are all similar to those of the feedstock oil. The concentration of free cholesterol and the concentration of total cholesterol in the feedstock oil can vary depending on the pretreatment (eg, degumming, deoxidizing, decolorizing, and deodorizing), but the concentration of cholesterol ester is relatively variable. .. In the production of the composition of the present invention, reaction conditions may be selected so as to reduce the fluctuation of the cholesterol ester concentration. By using a partial glyceride lipase that does not catalyze the hydrolysis of the cholesterol ester, it is possible to adjust the concentration of the cholesterol ester so that it does not fluctuate significantly even if the raw material oil is reacted with the partial glyceride lipase. In one embodiment, when the raw material oil is fish oil, the concentration of cholesterol ester in the composition produced by the method of the present invention may vary depending on the fish species, but may vary from 0.030 to 0.20% by weight, for example 0.030 to 0.15% by weight. , 0.030 to 0.070% by weight, or 0.030 to 0.050% by weight.

In certain embodiments herein, the composition produced by the method of the invention comprises phthalates. Phthalate esters are chemical substances widely used as plasticizers for plastic products such as polyvinyl chloride, and are suspected to have endocrine disrupting effects. Phthalate esters are easily soluble in oil and can also be included in the feedstock in the method of the invention. Phthalate esters are quantified by dissolving the sample in dichloromethane, mixing with an internal standard, then separating the phthalates from the fat / oil by liquid chromatography and analyzing by GC-MS / MS. .. The concentration of phthalates in the composition produced by the method of the present invention is similar to that of raw material oil, except for di- (2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DINP). The concentration of DEHP in the composition produced by the method of the present invention is lower than the concentration in the feedstock oil, for example, if the feedstock oil is fish oil, it can be 14.20 to 15.60 ppb. The concentration of DINP in the composition produced by the method of the present invention is lower than the concentration in the feedstock oil, for example, when the feedstock oil is fish oil, it can be 9.7 ppb to 11.7 ppb.

The acid value (AV) of the composition produced by the method of the present invention may vary depending on the acid value of the feedstock oil, and may be, for example, 1.5 to 37.2. For example, when the acid value of the raw material oil is about 0.08, the acid value in the oil layer after the step (2) can be 2.0 or more, for example, 3.0 or more, 5.0 or more, 10 or more, or 20 or more. When the acid value of the raw material oil is about 0.20, the acid value in the oil layer after the step (2) can be 1.8 or more, for example, 2.0 or more, 2.4 or more, 3.0 or more, 4.0 or more, or 5.0 or more. When the acid value of the raw material oil is about 0.34, the acid value in the oil layer after step (2) is 2.0 or more, for example, 2.5 or more, 3.0 or more, 3.5 or more, 4.0 or more, 4.5 or more, 5.0 or more, or 5.5 or more. possible.

Since the method of the present invention does not cause decomposition that changes the fatty acid composition before and after the lipase reaction, it does not significantly change the fatty acid composition of the raw material oil. The fatty acid composition of the composition produced by the method of the present invention comprises a certain amount or more of highly unsaturated fatty acids in the constituent fatty acids, for example, 10 area% or more, 15 area% or more, 20 area% or more, 25 area% or more. Or it may contain 30 area% or more. In the present specification, the fatty acid composition is measured by the following method.
Add 1 mL of 1N sodium ethylate / ethanol solution to 40 μL of the sample, stir for about 30 seconds, and leave it for 2 minutes. Then, 1 mL of 1N hydrochloric acid is added for neutralization, 2 mL of hexane and 3 mL of saturated aqueous ammonia sulfate are added, and the mixture is stirred and allowed to stand, and then the upper layer is subjected to gas chromatography for measurement. The value of fatty acid composition is shown in area%. The analysis conditions for gas chromatography are as follows.
Gas chromatography analysis conditions Model: Agilent 6850 GC system (Agilent)
Column: DB-WAX J & W 123-7032
Column temperature: 200 ° C
Injection temperature: 300 ° C
Injection method: Split Split ratio: 30: 1
Detector temperature: 300 ° C
Detector: FID
Carrier gas: helium (3.5 mL / min, constant flow)

<Composition containing triglyceride as a main component>
The present invention is a composition containing triglyceride as a main component.
The proportion of triglyceride in the glyceride fraction in the composition is 90.0-99.9 area%.
The numerical value obtained as the concentration of 3-MCPD generated when analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 0.10 ppm to 2.80 ppm, and satisfies at least one of the following a to e. Composition:
a) The value obtained as the concentration of 2-MCPD for the composition by the method of Cd29b-13 assay A, which is the official method of the American Society of Oil Chemistry, is 0.50 ppm or less.
b) The concentration of cholesterol ester is 0.030 to 0.20% by weight.
c) The concentration of di- (2-ethylhexyl) phthalate (DEHP) is 14.20-15.60 ppb,
d) The concentration of diisononyl phthalate (DINP) is 9.7 ppb to 11.7 ppb, and e) the acid value (AV) is 1.5 to 37.2 (hereinafter, also referred to as the composition of the present invention).

The composition of the present invention can be produced by the method of the present invention.

In the composition of the present invention, the inclusion of triglyceride as a main component means that the triglyceride is 50% by weight or more, for example, 60% by weight or more, 70% by weight or more, 80% by weight or more, 90% by weight of the total weight of the composition of the present invention. % Or more, or 95% by weight or more.

The proportion of triglyceride in the glyceride fraction in the composition of the present invention is 90.0 to 99.9 area%, for example 95.0 to 99.9 area%, 96.0-99.9 area%, etc. It can be 97.0 to 99.9 area%, or 99.0 to 99.9 area%.
The glyceride composition (that is, the ratio of monoglyceride, diglyceride, and triglyceride in the glyceride contained in the sample) is measured by the following procedure by the TLC / FID method.
40 μL of the sample is dissolved in 1 mL of hexane, and 0.4 μL or 0.6 μL is loaded on the rod-shaped thin layer. A mixed solvent of chloroform and methanol (chloroform: methanol = 95: 5 (volume ratio)) is used as a developing solvent for 2 minutes, the rod-shaped thin layer is dried, and then a mixed solution of hexane, diethyl ether and acetic acid (hexane: diethyl ether:). Acetic acid = 70: 30: 0.1 (volume ratio)) is used as a developing solvent for development for 35 minutes. This is analyzed by thin layer chromatography / hydrogen flame ionization detector. After removing the peak derived from free fatty acid from the analysis result, the ratio of each glyceride is shown in area%.

When the composition of the present invention is analyzed by the American Petrochemical Society official method Cd29b-13 assay A, the values obtained as the concentration of 3-MCPD are 0.10 ppm to 2.80 ppm, for example, 0.16 to 0.30 ppm, 1.17 to 2.10 ppm. , 1.69 to 2.74 ppm, or 1.72 to 2.21 ppm. Such characteristics can be brought about by treating the feedstock oil with a partial glyceride lipase.

The composition of the present invention comprises at least one of the above a to e, for example any one (ie, a; b; c; d; or e), or any two (ie, a and b; a and c). A and d; a and e; b and c; b and d; b and e; c and d; c and e; or d and e), any three (ie, a, b, and c; a) , B, and d; a, b, and e; a, c, and d; a, c, and e; a, d, and e; b, c, and d; b, c, and e; b, d and e; or c, d, and e), or any four (ie, a, b, c, and d; a, b, c, and e; a, b, d, and e; a. , C, d, and e; or b, c, d, and e), preferably all of a to e above. Each of these characteristics can be brought about by treating the feedstock with a partial glyceride lipase.

In one aspect relating to a, the numerical value obtained as the concentration of 2-MCPD for the composition of the present invention by the method of the American Petrochemical Society official method Cd29b-13 assay A is 0.50 ppm or less, for example 0.40 ppm or less, 0.36 ppm or less, It can be 0.27 ppm or less and less than 0.10 ppm (lower limit of quantification). In addition, the numerical value obtained as the concentration of 2-MCPD for the composition of the present invention by the method of Cd29b-13 assay A, which is the official method of the American Society of Oil Chemistry, is 0.10 ppm (lower limit of quantification) or more, for example, 0.15 ppm or more, 0.17 ppm or more. , 0.19 ppm or more, or 0.21 ppm or more. For the composition of the present invention, the numerical values obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the American Society of Oil Chemistry, are 0.10ppm to 0.50ppm, 0.10ppm to 0.50ppm, 0.10 to 0.27ppm, 0.19 to. It can be 0.50 ppm, or 0.21 to 0.36 ppm.

In one aspect of b, the concentration of free cholesterol, the concentration of cholesterol ester, and the concentration of total cholesterol in the composition of the present invention may be comparable to that of feedstock oil. The concentration of cholesterol ester in the composition of the present invention may vary from 0.030 to 0.20% by weight, for example 0.030 to 0.15% by weight, 0.030 to 0.070% by weight, or 0.030 to 0.050% by weight, depending on the type of feedstock. ..

In one aspect with respect to c, the composition of the invention may contain phthalates comparable to feedstock oil, with the exception of di- (2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DINP). The concentration of DEHP in the composition of the present invention is lower than that in the feedstock oil and can be, for example, 14.20 to 15.60 ppb.

In one aspect with respect to d, the concentration of DINP in the composition of the present invention may be lower than the concentration in the feedstock oil, for example 9.7 ppb to 11.7 ppb.

In one aspect with respect to e, the acid value (AV) of the composition of the invention can be 1.5-37.2. The acid value of the composition of the present invention is higher than the acid value of the raw material oil, for example, 1.5 or more, 2.0 or more, 2.5 or more, 3.0 or more, 3.5 or more, 4.0 or more, 4.5 or more, 5.0 or more, 5.5 or more, 10 or more, Or it can be 20 or more. The acid value of the composition of the present invention is 2.0 to 37.2, 2.5 to 37.2, 3.0 to 37.2, 3.5 to 37.2, 4.0 to 37.2, 4.5 to 37.2, 5.0 to 37.2, 5.5 to 37.2, 10 to 37.2, or 20 to 37.2. Can be.

In a particular embodiment, the composition of the present invention has a value of 1.17 to 2.10 ppm as the concentration of 3-MCPD produced when analyzed by the American Oil Chemistry Society official method Cd29b-13 assay A, and American oil. The numerical value obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the Society of Chemistry, is 0.10 to 0.27 ppm, and the acid value is 1.8 to 5.8.

In a specific embodiment, the composition of the present invention has a value obtained as the concentration of 3-MCPD generated when analyzed by the American Oil Chemistry Society official method Cd29b-13 assay A, which is 0.16 to 0.30 ppm, and is an American oil. The value obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the Society of Chemistry, is less than 0.10 ppm (less than the lower limit of quantification), and the acid value is 2.0 to 30.

In a specific embodiment, the composition of the present invention has a value of 1.69 to 2.74 ppm obtained as the concentration of 3-MCPD generated when analyzed by the American Oil Chemistry Society official method Cd29b-13 assay A, and the American oil. The numerical value obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the Society of Chemistry, is 0.19 to 0.50 ppm, and the acid value is 2.3 to 5.7.

In a particular embodiment, the composition of the present invention has a value of 1.72 to 2.21 ppm as the concentration of 3-MCPD produced when analyzed by the American Oil Chemistry Society official method Cd29b-13 assay A, which is American oil. The numerical value obtained as the concentration of 2-MCPD by the method of Cd29b-13 assay A, which is the official method of the Society of Chemistry, is 0.21 to 0.36 ppm, and the acid value is 2.6 to 5.1.

The composition of the present invention may contain a certain amount or more of highly unsaturated fatty acids, for example, 10 area% or more, 15 area% or more, 20 area% or more, 25 area% or more, or 30 area% or more in the constituent fatty acids.

In one embodiment, the composition of the invention can be a heated oil. 3-MCPDE can be produced by heat treating the oil at temperatures above 100 ° C. Therefore, although the heated oil usually contains 3-MCPDE, it can be efficiently removed by the method of the present invention. Therefore, in the composition of the present invention, the American Petrochemical Society official method Cd29b-13 assay A is used. The numerical value obtained as the concentration of 3-MCPD generated in the analysis is reduced as compared with the numerical value obtained for the feedstock oil.

In a preferred embodiment, the composition of the invention can be heated fish oil. When refining oil is prepared from crude oil for fish oil, heat treatment at a temperature exceeding 100 ° C. is required in a deoxidizing step, a deodorizing step, and the like. Therefore, the heated fish oil usually contains 3-MCPDE, but since this can be efficiently removed by the method of the present invention, the composition of the present invention is prepared by the American Society of Oil Chemistry official method Cd29b-13 assay A. The numerical value obtained as the concentration of 3-MCPD generated in the analysis is reduced as compared with the numerical value obtained for the feedstock oil.

The composition of the present invention is useful as an edible oil or as a raw material for foods, supplements, pharmaceuticals, cosmetics and the like.

Examples of the present invention will be described below, but the present invention is not limited thereto.
In the examples, ppm and ppb are, unless otherwise specified, weight ppm (ie, mg / kg) and weight ppb (ie, μg / kg), respectively.
In the examples,% for fatty acid composition and glyceride composition is area%. Here, the area% is the ratio of the peak area of each component to the total peak area in the chart obtained by analyzing the mixture of each component using gas chromatography or thin layer chromatography / hydrogen flame ionization detector (TLC / FID). It shows the content ratio of each component. The fatty acid composition was calculated from the results analyzed by gas chromatography by the method shown in Examples, and the glyceride composition was calculated from the results analyzed using TLC / FID.
In the examples,% for cholesterol content is% by weight.
In the examples, the measured values of 3-MCPD, 2-MCPD, and glycidol mean the measured values by the American Oil Chemistry Society official method (AOCS official method) Cd29b-13 assay A.

<Measurement of fatty acid composition>
The fatty acid composition was measured by gas chromatography after ethyl esterifying the oil. That is, 1 mL of a 1N sodium ethylate / ethanol solution was added to 40 μL of oil, and the mixture was stirred for about 30 seconds and left for 2 minutes. Then, 1 mL of 1N hydrochloric acid was added for neutralization, 2 mL of hexane and 3 mL of a saturated aqueous ammonia sulfate solution were added, and the mixture was stirred and allowed to stand, and then the upper layer was subjected to gas chromatography for measurement. The value of fatty acid composition is shown in area%.

Gas chromatography analysis conditions Model: Agilent 6850 GC system (Agilent)
Column: DB-WAX J & W 123-7032
Column temperature: 200 ° C
Injection temperature: 300 ° C
Injection method: Split Split ratio: 30: 1
Detector temperature: 300 ° C
Detector: FID
Carrier gas: helium (3.5 mL / min, constant flow)

<Measurement of glyceride composition>
The glyceride composition was measured by thin layer chromatography / hydrogen flame ionization detector (TLC / FID, Iatroscan, Mitsubishi Chemical Yatron Co., Ltd.). 40 μL of oil was dissolved in 1 mL of hexane and 0.4 μL or 0.6 μL was loaded on Chromarod®. A mixed solvent of chloroform and methanol (chloroform: methanol = 95: 5 (volume ratio)) is used as a developing solvent for 2 minutes, and after the rod is dried, a mixed solution of hexane, diethyl ether and acetic acid (hexane: diethyl ether: acetic acid =). It was developed for 35 minutes using 70:30: 0.1 (volume ratio) as a developing solvent. This was analyzed by Iatroscan. After removing the peak derived from free fatty acid from the analysis result, the ratio of each glyceride is shown by area%.

<Analysis of 3-MCPD, 2-MCPD, and glycidol>
Analysis of 3-MCPD, 2-MCPD, and glycidol was outsourced to SGS Germany GmbH. Analysis was performed according to AOCS Cd 29b-13.

<Analysis of phthalates>
Analysis of phthalates was outsourced to SGS Germany GmbH. The sample was dissolved in dichloromethane, mixed with an internal standard, and then analyzed by LC-GC-MS / MS. In LC, fat / oil was separated from phthalates by size exclusion chromatography, and phthalates were analyzed and quantified by GC-MS / MS.

<Measurement of acid value (AV)>
The acid value was measured by the following method. A mixed solvent of ethanol and diethyl ether (1: 1, v / v) was prepared, a few drops of phenolphthalein were added, and then a 0.1 N sodium hydroxide / ethanol solution was added until the color developed. About 1 g of the sample oil was precisely weighed in a glass container, and 30 to 40 mL of the mixed solvent was added to dissolve the sample oil. A 0.1 N sodium hydroxide / ethanol solution was added dropwise to this, and neutralization titration was performed. The formula was calculated from the weight of the sample oil and the volume of the 0.1 N sodium hydroxide / ethanol solution used until color development.
AV = Titration (mL) x 5.611 / Sample weight (g)

<Measurement of total cholesterol content, free cholesterol content, and cholesterol ester content>
The total cholesterol content, free cholesterol content, and cholesterol ester content of the oil were measured by gas chromatography.
The total cholesterol content was measured by the following method.
To about 0.1 g of oil, 1 mL of 0.1 g / L 5α-cholestane / ethanol solution was added as an internal standard, 1 mL of 2 mol / L potassium hydroxide / hydrous ethanol solution was added, and then the mixture was heated at 100 ° C. for 10 minutes. After cooling, 3 mL of petroleum ether and 3 mL of saturated ammonium sulfate were added, shaken, allowed to stand, and the upper layer was recovered and analyzed by gas chromatography. To determine the relative sensitivity of 5α-cholestane and cholesterol, a hexane solution with 5α-cholestane of 0.25 g / L and cholesterol of 0.25 g / L was analyzed by gas chromatography. The total cholesterol content was calculated from the sample volume and each area value.
Gas chromatography analysis conditions Model: Agilent 6890 GC system (Agilent)
Column: DB-1 J & W 123-1012
Column temperature: 270 ° C
Injection temperature: 300 ° C
Injection method: Split Split ratio: 50: 1
Detector temperature: 300 ° C
Detector: FID
Carrier gas: Helium (39.3 kPa, constant pressure)

The amount of free cholesterol and cholesterol ester was measured by the following method.
About 1 g of oil was dispensed with hexane to 10 mL and 0.5 mL was sampled. This is charged into a silica gel cartridge (Waters, Sep-pack® Plus Silica Cartridge 55-105 μm), hexane: diethyl ether = 9: 1 (v / v) 5 mL is poured, and the eluate is cholesterol ester. It was a fraction. Further, 5 mL of diethyl ether was poured, and the eluate was used as a cholesterol fraction. After evaporating the solvent under a nitrogen stream, the same operation as the total cholesterol content measurement was performed, and the free cholesterol content and the cholesterol ester content were calculated from the sample amount and the area value.

<Example 1> Production of 3-MCPD fatty acid ester by heat treatment The process of refining fish oil includes short-stroke distillation (SPD), wintering, decolorization, deodorization, etc., where heating at a temperature exceeding 100 ° C. Processing is done. In this example, the formation behavior of 3-MCPD fatty acid ester by heat treatment of fish oil was investigated.

(1) Method 3-MCPD was measured after heating sardine crude oil (crude fish oil; 3-MCPD measured value: 0.20 ppm) at 120 to 230 ° C. for 15 minutes or 5 hours under vacuum.

(2) Results Table 1 shows the measurement results of 3-MCPD. At any heating time, the measured values of 3-MCPD increased in a temperature-dependent manner. It can be said that the higher the temperature, the easier it is to form the 3-MCPD fatty acid ester.

<Example 2> Reduction of 3-MCPD fatty acid ester produced in the purification step [Test 1]
Fish oil 1 (distilled deoxidized tuna oil, Nippon Suisan Kaisha Co., Ltd., AV 0.20) was treated with lipase GS and washed with water by the following method, and then 3-MCPD and the like were measured.
Fish oil 1 was prepared by washing tuna crude oil with warm water at 85 ° C, dehydrating it, and then performing short-stroke distillation using a short-stroke distillation ^{apparatus (SPD) KD450 (manufactured by UIC GmbH, distillation surface 4.5 m 2).} .. Short-stroke distillation was performed at a main unit temperature of 262 ° C.
In a 50 mL glass container, 15 g of fish oil, 10 mL of distilled water, 250 G of Lipase GS "Amano" (274000 unit / g, Amano Enzyme Co., Ltd.) 50 unit / g oil (2.7 mg), 100 unit / g Oil (5.5 mg), 200 unit / g oil (11.0 mg), 400 unit / g oil (21.9 mg), or 800 unit / g oil (43.8 mg) was added, and the mixture was stirred at 20 ° C. with a stirrer for 20 hours. After completion of the reaction, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. After adding 10 mL of distilled water to the obtained upper layer and shaking vigorously, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above operation was performed twice, and the obtained two samples were combined for analysis of 3-MCPD, 2-MCPD, and glycidol, acid value analysis, fatty acid composition analysis, and glyceride composition analysis. .. In the examples, "3-MCPDs reduction rate" indicates the rate of decrease in the 3-MCPD measured value of the fish oil after the lipase treatment as a percentage based on the 3-MCPD measured value of the fish oil before the lipase treatment. It is a thing.

The results are shown in Tables 2 and 3. Treatment with Lipase GS and washing with water reduced 3-MCPD measurements. At any lipase GS concentration of 50-800 unit / g oil, 3-MCPD measurements were more than 10% lower than fish oil 1. In addition, as the amount of lipase GS added increased, the reduction rate of the 3-MCPD measured value increased. In addition, the ratio of triglyceride in the glyceride contained in the oil treated with Lipase GS did not decrease as compared with fish oil 1.

[Test 2]
Fish oil 5 (refined bonito oil, Nippon Suisan Kaisha Co., Ltd., AV 0.08) was treated with different concentrations of lipase GS and washed with water by the following method, and then 3-MCPD and the like were measured.
Fish oil 5 was prepared by the following method. Alkaline deoxidation was performed on bonito crude oil. Then, the color was decolorized by a column, the solvent was removed, and then steam distillation was performed. Further short-stroke distillation was performed, steam distillation was performed again, and then an antioxidant was added.
15 g of fish oil 5 in a 50 mL glass container, 10 mL of distilled water, 100 unit / g oil (5.5 mg), 200 unit / g oil of Lipase GS "Amano" 250 G (274000 unit / g, Amano Enzyme Co., Ltd.) (11.0 mg), 400 unit / g oil (21.9 mg), 800 unit / g oil (43.8 mg), or 1600 unit / g oil (87.6 mg) was added, and the mixture was stirred at 30 ° C. for 20 hours with a stirrer. After completion of the reaction, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. After adding 10 mL of distilled water to the obtained upper layer and shaking vigorously, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above operation was performed twice, and the obtained two samples were combined for analysis of 3-MCPD, 2-MCPD, and glycidol, acid value analysis, fatty acid composition analysis, and glyceride composition analysis. ..

The results are shown in Tables 4 and 5. Even for fish oil having a lower 3-MCPD measured value than the fish oil of Test 1, the 3-MCPD measured value decreased by treatment with Lipase GS and washing with water. In addition, as the amount of lipase GS added increased, the reduction rate of the 3-MCPD measured value increased. In addition, the ratio of triglyceride in the glyceride contained in the oil treated with Lipase GS did not decrease compared with that of fish oil 5.

[Test 3]
Fish oil 2 (distilled deoxidized tuna oil, Nippon Suisan Kaisha Co., Ltd., AV 0.34) was treated with lipase GS at different temperatures and washed with water by the following method, and then 3-MCPD and the like were measured.
Fish oil 2 was prepared in the same manner as fish oil 1.
Add 15 g of fish oil 2, 10 mL of distilled water, and 400 unit / g oil (21.9 mg) of Lipase GS "Amano" 250 G (274000 unit / g) to a 50 mL glass container, and add 20 ° C, 30 ° C, 40 ° C, Alternatively, the mixture was stirred with a stirrer at 50 ° C. for 20 hours. After completion of the reaction, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. After adding 10 mL of distilled water to the obtained upper layer and shaking vigorously, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above operation was performed twice, and the obtained two samples were combined for analysis of 3-MCPD, 2-MCPD, and glycidol, acid value analysis, fatty acid composition analysis, and glyceride composition analysis. ..

The results are shown in Tables 6 and 7. When Lipase GS was used, the 3-MCPD measurements decreased at any temperature between 20 ° C and 50 ° C. The reduction rate of the 3-MCPD measured value was the highest at 30 ° C, and an upward tendency was observed up to 30 ° C, and a downward tendency was observed above 30 ° C.

[Test 4]
For fish oil 2, after lipase GS treatment and washing with different amounts of distilled water added by the following method, 3-MCPD and the like were measured.
Add 15 g of fish oil 2 to a 50 mL glass container, 2.5 mL, 5 mL, 10 mL, 20 mL of distilled water, and 400 unit / g oil (21.9 mg) of Lipase GS "Amano" 250 G, and magne at 30 ° C. The mixture was stirred with a tick stirrer for 20 hours. After completion of the reaction, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. After adding 10 mL of distilled water to the obtained upper layer and shaking vigorously, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above was repeated, and the sample oils for two times were totaled and used for analysis.

The results are shown in Tables 8 and 9. Under any condition in which 2.5 to 20 mL of distilled water was added to 15 g of the raw material oil, the 3-MCPD measured value decreased. The reduction rate of the 3-MCPD measured value was the highest under the condition that 10 mL of distilled water was added to 15 g of the raw material oil, and an upward tendency was observed up to 10 mL and a downward tendency was observed when it exceeded 10 mL.

[Test 5]
Fish oil 2 and fish oil 3 (water-washed tuna oil, Nippon Suisan Kaisha Co., Ltd., AV 1.26) were subjected to lipase treatment and water-washing by the following methods, and then the cholesterol content and the like were analyzed.
Fish oil 3 was prepared by washing the crude tuna oil with warm water at 85 ° C. and then dehydrating it.
6 g of fish oil 2 or fish oil 3 in a 50 mL glass container, 4 mL of distilled water, 400 unit / g of lipase GS "Amano" 250 G oil (8.8 mg) or lipase OF360 (384000 unit / g, Meishu Sangyo Co., Ltd.) ) Was added at 400 unit / g oil (6.3 mg), and the mixture was stirred at 30 ° C. with a magnetic stirrer for 20 hours. After completion of the reaction, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation and subjected to analysis of cholesterol content, acid value, fatty acid composition, and glyceride composition.

The results are shown in Tables 10-13. There were no significant changes in cholesterol content and glyceride composition between the oil after lipase GS treatment and the feedstock oil.

[Test 6]
Fish oil 4 (solvent deoxidized bonito oil, Nippon Suisan Kaisha Co., Ltd., AV 0.35) was lipase-treated and washed with water by the following method, and then phthalates and the like were analyzed.
Fish oil 4 was prepared by the following method. After heating the bonito crude oil to 95 ° C., 10% volume of 50% NaOH was added to the bonito crude oil and stirred. After centrifuging and removing the aqueous layer, warm water at 85 ° C. was added and stirred. Centrifugation was performed again to remove the aqueous layer. 2% volume of silica was added to the oil layer, and the mixture was stirred and then dehydrated, and the silica was removed by a filter.
15 g of fish oil 4 (solvent deoxidized bonito oil, Nippon Fisheries Co., Ltd., AV 0.35), 10 mL of distilled water, 400 unit / g of lipase GS "Amano" 250 G oil (21.9 mg) or lipase OF360 in a 50 mL glass container. (384000 unit / g, Meisho Sangyo Co., Ltd.) was added in 400 unit / g oil (15.6 mg), and the mixture was stirred at 30 ° C. with a magnetic stirrer for 20 hours. After completion of the reaction, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. After adding 10 mL of distilled water to the obtained upper layer and shaking vigorously, the mixture was heated in a constant temperature bath at 90 ° C. for 10 minutes and centrifuged (1900 g, 10 minutes) to recover the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above operation was performed twice, and the obtained two samples were combined for analysis of phthalates, acid value, fatty acid composition, and glyceride composition.

The results are shown in Tables 14 and 15. Regarding the content of phthalates, no significant change was observed between the oil after the lipase GS treatment and the feedstock oil.

INDUSTRIAL APPLICABILITY According to the present invention, the content of 3-MCPDE produced by heat treatment of oil can be reduced without reducing the ratio of triglyceride in the glyceride contained in the oil, and safer cooking oil and the like can be stably produced. Can be manufactured.

Claims (11)

A method for producing a composition containing triglyceride.
(1) A step of reacting a raw material oil containing triglyceride with a partial glyceride lipase in the presence of water to hydrolyze 3-MCPD fatty acid esters in the raw material oil into 3-MCPD and fatty acid, and (2). After step (1), the step of removing the aqueous layer,
Including
The value obtained as the concentration of 3-MCPD generated when the raw material oil is analyzed by the official method Cd29b-13 assay A of the American Society of Oil Chemistry is 0.3 ppm or more.
The above method, wherein the value obtained as the concentration of 3-MCPD generated when the oil layer after the step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is lower than the value obtained for the feedstock oil. The method according to claim 1, wherein the raw material oil containing triglyceride is a heated oil. The method according to claim 1 or 2, wherein the raw material oil containing triglyceride is fish oil. Claimed that the value obtained as the concentration of 3-MCPD generated when the oil layer after the step (2) is analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 10% or more lower than the value obtained for the feedstock oil. Item 1. The method according to any one of Items 1 to 3. The method according to any one of claims 1 to 4, wherein the partial glyceride lipase is a lipase derived from Penicillium camemberti. The method according to any one of claims 1 to 5, wherein the amount of the partial glyceride lipase used in the step (1) is 200 to 1,600 units with respect to 1 g of the raw material oil. The method according to any one of claims 1 to 6, wherein the hydrolysis reaction of the step (1) is carried out at 10 to 50 ° C. The method according to any one of claims 1 to 7, wherein the amount of water added to the reaction system of the hydrolysis reaction in the step (1) is 0.10 to 2.5 g with respect to 1 g of the raw material oil. A composition containing triglyceride as a main component,
The proportion of triglyceride in the glyceride fraction in the composition is 90.0-99.9 area%.
The numerical value obtained as the concentration of 3-MCPD generated when analyzed by the American Petrochemical Society official method Cd29b-13 assay A is 0.10 ppm to 2.80 ppm, and satisfies at least one of the following a to e. Composition:
a) The value obtained as the concentration of 2-MCPD generated when the composition is analyzed by the American Chemical Society official method Cd29b-13 assay A is 0.50 ppm or less.
b) The concentration of cholesterol ester is 0.030 to 0.20% by weight.
c) The concentration of di- (2-ethylhexyl) phthalate (DEHP) is 14.20-15.60 ppb,
d) The concentration of diisononyl phthalate (DINP) is 9.7 ppb to 11.7 ppb, and e) the acid value (AV) is 1.5 to 37.2. The composition according to claim 9, which is a heating oil. The composition according to claim 10, which is heated fish oil.