JP7505925B2 - Method for reducing 3-MCPD content in fats and oils - Google Patents

Description

The present invention relates to a composition containing triglycerides and a method for producing the same.

A great deal of research has been conducted in the fields of food and medicine on the functions of fatty acids in fats and oils. For example, eicosapentaenoic acid (C20:5, EPA) and docosahexaenoic acid (C22:6, DHA), which are components of fish oil, have been reported to have anti-arteriosclerosis effects, brain function improving effects, visual function improving effects, antitumor effects, and anti-inflammatory effects. Fish oil is a fat and oil that is rich in EPA and DHA, and its intake is widely recommended.

3-Chloropropane-1,2-diol (3-MCPD), a type of chloropropanol, is a compound suspected of being carcinogenic. 3-MCPD fatty acid esters (3-MCPDE), in which one or two fatty acids are ester-bonded, have been found to exist in small amounts in refined edible oils. 3-MCPDE is thought to be produced from lipids and chloride ions during high-temperature treatment in the edible oil refining process. It is known that palm oils contain relatively large amounts of chloropropanols and their fatty acid esters.

In the EU and other countries, there are regulations regarding the amount of 3-MCPD contained in food. It is known that in fats and oils, 3-MCPDE is produced from diacylglycerol (DAG) and monoacylglycerol (MAG) as substrates (Non-Patent Document 1). Since the presence of 3-MCPD and its fatty acid esters was pointed out in DAG-rich fats and oils such as palm-based fats, various methods have been attempted to reduce the amount of 3-MCPD in fats and oils. The European vegetable oil trade association (FEDIOL) has published a list of technologies that can be used to reduce 3-MCPDE in the fat and oil manufacturing process and their characteristics (Non-Patent Document 2). It is disclosed that the concentration of 3-MCPDE in fats and oils can be reduced by treatment with adsorbents, lowering the deodorization temperature, and shortening the treatment time at high temperatures.

Known methods for reducing the content of 3-MCPD and its fatty acid esters in fats and oils include a method for suppressing their production and a method for removing 3-MCPD and its fatty acid esters produced during the production process of fats and oils. The former include a method for suppressing the production of 3-MCPDE in fish oil by a combination of alkali treatment, clay treatment, and deodorization treatment (Patent Document 1), a method for producing edible fats and oils in which substances that produce 3-MCPD are reduced, characterized in that the deodorization process of fats and oils is carried out at a temperature range of 190 to 230°C (Patent Document 2), and a method for suppressing the production of chloropropanols and the like in glyceride fats and oils, characterized in that the glyceride fats and oils are treated with an adsorbent and/or alkali before being heated to 100°C or higher (Patent Document 3). The latter include a method for producing edible fats and oils in which substances that produce 3-MCPD are reduced, characterized in that the refined fats and oils that have been subjected to a deodorization process are treated with an adsorbent and then filtered (Patent Document 4).

Patent Publication 2015-105354 Patent Publication 2011-147436 International Publication No. 2010/126136 Patent Publication 2011-147435

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

Oils derived from living organisms, such as fish oil, are usually provided as products for food, medicine, cosmetics, etc. only after environmental pollutants, cholesterol, free fatty acids, small molecules that cause coloring or odor, etc., have been removed from crude oil (crude oil) and the specified standards have been met. During this process, 3-MCPDE is generated as a result of heat treatment at temperatures exceeding 100°C. While free 3-MCPD can be relatively easily separated from triglycerides by washing with water, 3-MCPDE once generated in refined oil produced from fish oil, etc., is difficult to remove by washing with water because of its low water solubility. In addition, 3-MCPDE cannot be easily removed in general oil refining processes other than washing with water, since the hydroxyl group of 3-MCPD is bonded to fatty acids. The present invention aims to provide a means for removing 3-MCPDE once generated from oil, and a method for producing a composition containing triglycerides as a main component, in which the content of 3-MCPDE has been sufficiently reduced by the above.

As a result of intensive research conducted by the present inventors to achieve the above object, they discovered that by treating heat-treated oil with partial glyceride lipase, 3-MCPDE is hydrolyzed into water-soluble 3-MCPD and fatty acids, and the 3-MCPD can be removed by subsequent washing with water. Furthermore, this treatment did not result in a decrease in the proportion of triglycerides in the glycerides contained in the oil.
Based on this finding, the present inventors conducted further research and completed the present invention.
That is, the present invention is as follows.

[1] A method for producing a composition containing triglyceride, comprising the steps of:
(1) reacting a raw oil containing triglycerides with a 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).
Including,
The concentration of 3-MCPD produced when the raw oil is analyzed by the American Oil Chemist's Society official method Cd29b-13 assay A is 0.3 ppm or more.
The method according to claim 2, wherein the concentration of 3-MCPD obtained by analyzing the oil layer after step (2) according to the American Oil Chemists' Society official Cd29b-13 assay A is lower than the concentration obtained for the feedstock oil.
[2] The method according to [1], wherein the raw oil containing triglyceride is a heating oil.
[3] The method according to [1] or [2], wherein the raw material oil containing triglycerides is fish oil.
[4] The method according to any one of [1] to [3], wherein the concentration of 3-MCPD obtained by analyzing the oil layer after step (2) by the American Oil Chemists' Society official Cd29b-13 assay A is 10% or more lower than the concentration obtained for the feedstock oil.
[5] The method according to any one of [1] to [4], wherein the partial glyceride lipase is a lipase derived from Penicillium camembertii.
[6] The method according to any one of [1] to [5], wherein the amount of partial glyceride lipase used in step (1) is 200 to 1,600 units per 1 g of the raw material oil.
[7] The method according to any one of [1] to [6], wherein the hydrolysis reaction in step (1) is carried out at 10 to 50° C.
[8] The method according to any one of [1] to [7], wherein the amount of water added to the reaction system for the hydrolysis reaction in the step (1) is 0.10 to 2.5 g per 1 g of the raw material oil.
[9] A composition containing triglyceride as a main component,
The proportion of triglycerides in a glyceride fraction in the composition is 90.0 to 99.9 area %,
The composition has a concentration of 3-MCPD of 0.10 ppm to 2.80 ppm when analyzed by the American Oil Chemist's Society official Cd29b-13 assay A, and satisfies at least one of the following a to e:
a) when the composition is analyzed by the American Oil Chemist's Society official method Cd29b-13 assay A, the concentration of 2-MCPD obtained 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 to 15.60 ppb;
d) the concentration of diisononyl phthalate (DINP) is between 9.7 ppb and 11.7 ppb; and e) the acid value (AV) is between 1.5 and 37.2.
[10] The composition according to [9], which is a heating oil.
[11] The composition described in [10], which is heated fish oil.
[12] The composition according to any one of [9] to [11], comprising triglycerides 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 of the total weight of the composition.
[13] The composition according to any one of [9] to [12], wherein the proportion of triglycerides in a glyceride fraction in the composition is 95.0 to 99.9 area%, 96.0 to 99.9 area%, 97.0 to 99.9 area%, or 99.0 to 99.9 area%.
[14] The composition according to any one of [9] to [13], wherein the concentration of 3-MCPD generated when analyzed by the American Oil Chemist's Society official Cd29b-13 assay A is 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.
[15] The composition according to any one of [9] to [14], wherein the concentration of 2-MCPD obtained by the American Oil Chemists' Society Cd29b-13 assay A is 0.50 ppm or less, 0.40 ppm or less, 0.36 ppm or less, 0.27 ppm or less, or less than 0.10 ppm (lower limit of quantification).
[16] The composition according to any one of [9] to [14], wherein the concentration of 2-MCPD obtained by the American Oil Chemists' Society Cd29b-13 assay A 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 0.21 ppm or more.
[17] The composition according to any one of [9] to [14], wherein the concentration of 2-MCPD obtained by the American Oil Chemists' Society Cd29b-13 assay A is 0.10 ppm to 0.50 ppm, 0.10 ppm to 0.50 ppm, 0.10 to 0.27 ppm, 0.19 to 0.50 ppm, or 0.21 to 0.36 ppm.
[18] The composition according to any one of [9] to [17], wherein the concentration of the 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.
[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] The composition according to any one of [9] to [20], wherein the 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 37.2.
[22] The composition according to any one of [9] to [21], wherein the constituent fatty acids contain highly unsaturated fatty acids at 10 area % or more, 15 area % or more, 20 area % or more, 25 area % or more, or 30 area % or more.
[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 food, supplements, medicines, or cosmetics.

The present invention makes it possible to reduce the amount of 3-MCPDE produced by heat treatment of oil without decreasing the proportion of triglycerides in the glycerides contained in the oil, and thus to stably produce safer edible oils, etc.

In this specification, when describing fatty acids, numerical notations may be used that combine numbers and letters to simply represent the number of carbon atoms, the number of double bonds, and the position of the double bonds. For example, a saturated fatty acid with 20 carbon atoms may be described as "C20:0", a monounsaturated fatty acid with 18 carbon atoms may be described as "C18:1", and eicosapentaenoic acid may be described as "C20:5 n-3", etc. "n-" indicates the position of the first double bond counting from the methyl end of the fatty acid, for example, "n-3" indicates that the position of the first double bond counting from the methyl end of the fatty acid is between the third and fourth carbon atoms. This method is well known to those skilled in the art, and fatty acids described according to this method can be easily identified by those skilled in the art.

In this specification, "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 may 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), arachidonic 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 this specification, "crude oil" refers to a mixture of lipids and oil extracted from an organism, and is sometimes called crude oil. In this specification, "refined oil" refers to oil that has been subjected to a crude oil refining process in which crude oil is subjected to at least one oil purification process selected from the group consisting of a degumming process, a deacidification process, a bleaching process, and a deodorization process, and substances other than the target substance, such as phospholipids and sterols, are removed. Those skilled in the art can distinguish between refined oil and crude oil by ordinary analysis.

In this specification, a generic term (e.g., highly unsaturated fatty acid) does not exclude the possibility that multiple components are present, unless the context clearly indicates otherwise. Thus, a generic term usually means that at least one component is present.

<Method for producing a composition containing triglyceride>
The present invention provides a method for producing a composition containing triglycerides, comprising the steps of:
(1) reacting a raw oil containing triglycerides with a 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).
Including,
The concentration of 3-MCPD produced when the raw oil is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is 0.3 ppm or more.
The method according to the present invention relates to a method in which the concentration of 3-MCPD obtained by analyzing the oil layer after step (2) by the American Oil Chemists' Society official Cd29b-13 assay A is lower than the concentration obtained for the feedstock oil (hereinafter, also referred to as the method of the present invention).

The raw oil in the method of the present invention is not particularly limited as long as it contains triglyceride as a main component, and may be, for example, aquatic animal oil, microbial oil, or vegetable oil. In this specification, containing triglyceride as a main component means that the content of triglyceride in the raw oil 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 or more, or 95% by weight or more of the total weight of the raw oil. Specific examples of the raw oil include fish oils such as sardine oil, tuna oil, bonito oil, menhaden oil, cod liver oil, herring oil, capelin oil, and salmon oil, marine animal oils derived from crustaceans such as krill, vegetable oils derived from perilla, flax, soybeans, rapeseed, and the like, yeasts such as the Yarrowia genus, filamentous fungi such as those belonging to the Mortierella genus, Penicillium genus, Aspergillus genus, Rhodotorula genus, and Fusarium genus, algae such as Euglena genus, and oils derived from microorganisms that produce lipids such as stramenopiles. The raw oil may be oil derived from a genetically modified microorganism into which a gene such as a genetically modified mutant Δ9 elongase gene has been introduced. In addition, oils derived from genetically modified plants such as Brassica species, sunflower, corn, cotton, flax, and safflower, into which genes such as mutant Δ9 elongase genes have been introduced by recombinant technology, can also be used as raw oils. Examples of genetically modified vegetable oils and genetically modified microbial oils include those described in WO2012/027698 and WO2010/033753. It is preferable to select a raw oil containing a certain amount or more of highly unsaturated fatty acids in the constituent fatty acids, and it is possible to select one containing 10 area% or more, 15 area% or more, 20 area% or more, 25 area% or more, or 30 area% or more. In one embodiment, the raw oil in the method of the present invention is fish oil, and preferably tuna oil or bonito oil.

The raw oil contains 3-MCPD-forming substances. In this specification, 3-MCPD-forming substances means free 3-MCPD and its fatty acid ester (3-MCPDE), and the content of these substances in the sample is expressed as a numerical value obtained as the concentration of 3-MCPD using the American Oil Chemists' Society (AOCS) official method Cd29b-13 assay A. When the feedstock is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A, the resulting concentration of 3-MCPD is 0.3 ppm or more, and may be, 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.

In this specification, the analysis according to the American Oil Chemists' Society Official Method Cd29b-13 Assay A is carried out according to the following procedure which is well known to those skilled in the art.
100 mg of sample is added with 100 μL of 3-MCPD-d5-dipalmitic acid ester standard solution (diluted with toluene to 5 ppm 3-MCPD-d5) and 600 μL of diethyl ether, stirred and mixed until completely dissolved, and then cooled at -22 ° C to -25 ° C for about 15 minutes. Then, 350 μL of sodium hydroxide-methanol solution (0.25 g of sodium hydroxide dissolved in 100 mL of methanol) is added, stirred well, and reacted at -22 ° C to -25 ° C for 16 hours or more. At the same temperature, 600 μL of acidic sodium bromide solution (600 g of sodium bromide dissolved in 1 L of purified water, and 3 mL of 85% phosphoric acid added) is added to stop the reaction, and then nitrogen is blown and concentrated until the organic layer separated into two layers is about 100 μL. Then, 600 μL of hexane is added, stirred vigorously, and left to stand for 5 to 10 minutes to remove the organic layer, which is repeated twice. Add 600 μL of a diethyl ether:ethyl acetate mixture (3:2, V/V) to the remaining aqueous layer and stir vigorously, then collect the organic layer, repeating this process three times. Combine the three collected organic layers and dehydrate them with anhydrous sodium sulfate. After dehydration, concentrate the organic layer to 200 μL by blowing nitrogen, add 20 μL of a saturated phenylboronic acid-diethyl ether solution, and stir vigorously for 10 seconds, then blow nitrogen to completely remove the solvent. Add 200 μL of isooctane to this and stir vigorously for 10 seconds, and use the resulting solution as a GC-MS sample.
A calibration curve for quantifying 3-MCPD is prepared by analyzing 3-MCPD standard solutions prepared by dissolving 3-MCPD dipalmitate in toluene to give 3-MCPD concentrations of 0 ppm, 0.5 ppm, 1 ppm, and 5 ppm in the same manner as for the above samples.
The GC-MS analysis conditions were as follows:
GC-MS conditions
GC: GC-2010 and GCMS-QP2010 (Shimadzu Corporation)
Column: DB-5ms (Agilent Technologies)
30mx 0.25mm ID, 0.25μm film thickness
Carrier gas: Helium, 1.2 mL/min
Injection port: 250°C, 1 μL, splitless Sampling time: 1 min Column temperature: 85°C, hold for 0.5 min → 6°C/min → 150°C, hold for 5 min → 12°C/min → 180°C → 25°C/min → 280°C, hold for 7 min Ionization temperature: 200°C
Interface temperature: 200℃
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 for 3-MCPD-d5 and m/z = 147 for 3-MCPD are used, and the others are used to confirm the target substances.

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

The feedstock oil in the method of the present invention may be a crude oil or a refined oil. 3-MCPD or 3-MCPDE is usually only present in small amounts or almost none in crude oil, and 3-MCPDE is produced during the refining process. Therefore, in a preferred embodiment, the feedstock oil in the method of the present invention may be a refined oil.

Alternatively, when the feed oil in the method of the present invention is a crude oil, a step of refining the crude oil may be included prior to step (1). The refining step may be at least one selected from the group consisting of a degumming step, a deacidification step, a bleaching step, and a deodorization step. Each of these steps may be carried out by a method known in the art.

3-MCPDE may be produced by heat treatment at a temperature exceeding 100°C. In this specification, oil that has been heat-treated at a temperature exceeding 100°C, for example, 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, is referred to as heated oil. The feedstock oil in the method of the present invention may be a heated oil containing triglycerides, for example, an oil that has been heat-treated at a temperature of 160°C or higher for 15 minutes or more, or an oil that has been heat-treated at a temperature of 120°C or higher for 5 hours or more. Examples of heat treatment include distillation such as short-path distillation, molecular distillation, and steam distillation. In one embodiment, the feedstock oil may be an oil after distillation.

Alternatively, when the feedstock oil in the method of the present invention is not a heated oil, the method may further include a step of heating the triglyceride-containing feedstock oil to 100°C or higher, for example, 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, prior to step (1). The heating time may be 15 minutes or more, for example, 30 minutes or more, 45 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, or 5 hours or more.

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

The amount of partial glyceride lipase used is not particularly limited, but it is preferable to add 10 units per 1 g of raw material oil (i.e., 10 units/g oil) or more, and considering practicality in terms of reaction rate, 30 units/g oil or more to the reaction system. For example, 50 units/g oil or more, 100 units/g oil or more, 200 units/g oil or more, 400 units/g oil or more, 800 units/g oil or more, or 1,600 units/g oil or more can be added to the reaction system. For example, the amount of lipase used can be 10 to 2,000 units/g oil, 50 to 1,600 units/g oil, 100 to 1,600 units/g oil, 200 to 1,600 units/g oil, or 200 to 800 units/g oil. Since immobilized lipase can be used repeatedly, instead of adding the required amount as in the case of non-immobilized lipase, an excess amount of immobilized lipase is added to the reaction system, and it can be recovered after the reaction and used repeatedly. Therefore, at least the amount used per 1 g of raw oil may be added to the reaction system, and the amount may be changed to a reasonable amount depending on whether it is used in batch processing or column processing, how many times it is used repeatedly, etc. For example, 3 to 30% (w/w), 4 to 25% (w/w), and more preferably 5 to 20% (w/w) of immobilized lipase can be used relative to the raw oil.

Here, one unit of partial glyceride lipase is measured by the LV emulsification method, and one unit is the amount of enzyme that increases fatty acids by 1 micromole per minute. The LV emulsification method is a method in which lipase is applied to an emulsion of vinyl laurate, the reaction is stopped with an ethanol/acetone mixed solvent, the fatty acids produced are neutralized with sodium hydroxide, and the remaining sodium hydroxide is titrated with hydrochloric acid to quantify the fatty acids produced and determine the number of units.

In step (1) of the method of the present invention, a triglyceride-containing raw oil is reacted with a partial glyceride lipase in the presence of water. The hydrolysis reaction with lipase must be carried out in the presence of a sufficient amount of water to express the hydrolysis activity of the lipase. 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 per 1 g of raw oil, and preferably 0.15 to 0.70 g.

The hydrolysis is preferably carried out under an inert gas atmosphere such as dry nitrogen to prevent deterioration of fatty acids and inactivation of lipase. The raw oil may also contain antioxidants such as tocopherol, ascorbic acid, and t-butyl hydroquinone.

The reaction temperature in hydrolysis is not particularly limited as long as it is a temperature at which lipase is active, and may be, for example, 10 to 50°C or 20 to 50°C, with around 20°C to 40°C being preferred. Considering that the rate of the lipase reaction itself becomes too slow at temperatures below 10°C and the viscosity of the oil increases, a reaction temperature of around 30°C ± 5°C is most preferred. In the case of large-scale reactions, the temperature inside the reaction vessel is set to an average of 10 to 50°C, and the reaction can be carried out while maintaining the change in reaction temperature within a range of around ± 5°C. The hydrolysis reaction can be carried out under a fluidized state by mechanical stirring or blowing in an inert gas or the like.

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

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

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

The oil layer obtained may be further dehydrated. The dehydration may be carried out by a known method. For example, the oil layer may be dehydrated by adding an azeotropic agent such as ethanol to the oil layer and carrying out distillation such as reduced pressure distillation.

The content of 3-MCPD-forming substances in the oil layer after step (2) is lower than the content in the feedstock oil. In a preferred embodiment, the value obtained as the concentration of 3-MCPD when the oil layer after step (2) is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is at least 10% lower, for example, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% lower than the value obtained for the feedstock oil.

The content of 3-MCPD-forming substances in the oil layer after step (2) may vary depending on the content of 3-MCPD-forming substances in the feedstock. For example, if the concentration of 3-MCPD obtained when the feedstock is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is about 3.2 ppm, 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 may be 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 feedstock oil is analyzed according to the American Oil Chemists' Society official Cd29b-13 assay A and the resulting concentration of 3-MCPD is about 2.4 ppm, the oil layer after step (2) can be analyzed according to the American Oil Chemists' Society official Cd29b-13 assay A and the resulting concentration of 3-MCPD can be 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 ppm or less, 1.4 ppm or less, 1.3 ppm or less, 1.2 ppm or less, or 1.1 ppm or less. If the concentration of 3-MCPD obtained by analyzing the feedstock oil according to the American Oil Chemists' Society official method Cd29b-13 assay A is about 0.34 ppm, the concentration of 3-MCPD obtained by analyzing the oil layer after step (2) according to the American Oil Chemists' Society official method Cd29b-13 assay A may be 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 ppm or less, 0.19 ppm or less, 0.17 ppm or less, or 0.16 ppm or less.

In one embodiment, the method of the present invention can further reduce the content of 2-MCPD-forming substances in the feedstock oil. In this specification, 2-MCPD-forming substances refer to free 2-MCPD and its fatty acid esters (2-MCPDE), and the content is expressed as a value obtained as the concentration of 2-MCPD according to the American Oil Chemist's Society official method Cd29b-13 assay A. The analysis according to the American Oil Chemist's Society official method Cd29b-13 assay A is carried out according to the following procedure, which is well known to those skilled in the art.
100 mg of sample is added with 100 μL of 2-MCPD-d5-dipalmitic acid ester standard solution (diluted with toluene to 5 ppm 2-MCPD-d5) and 600 μL of diethyl ether, stirred and mixed until completely dissolved, and then cooled at -22 ° C to -25 ° C for about 15 minutes. Then, 350 μL of sodium hydroxide-methanol solution (0.25 g of sodium hydroxide dissolved in 100 mL of methanol) is added, stirred well, and reacted at -22 ° C to -25 ° C for 16 hours or more. At the same temperature, 600 μL of acidic sodium bromide solution (600 g of sodium bromide dissolved in 1 L of purified water, and 3 mL of 85% phosphoric acid added) is added to stop the reaction, and then nitrogen is blown and concentrated until the organic layer separated into two layers is about 100 μL. Then, 600 μL of hexane is added, stirred vigorously, and left to stand for 5 to 10 minutes to remove the organic layer, which is repeated twice. Add 600 μL of a diethyl ether:ethyl acetate mixture (3:2, V/V) to the remaining aqueous layer and stir vigorously, then collect the organic layer, repeating this process three times. Combine the three collected organic layers and dehydrate them with anhydrous sodium sulfate. After dehydration, concentrate the organic layer to 200 μL by blowing nitrogen, add 20 μL of a saturated phenylboronic acid-diethyl ether solution, and stir vigorously for 10 seconds, then blow nitrogen to completely remove the solvent. Add 200 μL of isooctane to this and stir vigorously for 10 seconds, and use the resulting solution as a GC-MS sample.
A calibration curve for quantifying 2-MCPD is prepared by analyzing 2-MCPD standard solutions prepared by dissolving 2-MCPD dipalmitate in toluene to give 2-MCPD concentrations of 0 ppm, 0.5 ppm, 1 ppm, and 5 ppm in the same manner as for the above samples.
The GC-MS analysis conditions were as follows:
GC-MS conditions
GC: GC-2010 and GCMS-QP2010 (Shimadzu Corporation)
Column: DB-5ms (Agilent Technologies)
30mx 0.25mm ID, 0.25μm film thickness
Carrier gas: Helium, 1.2 mL/min
Injection port: 250°C, 1 μL, splitless Sampling time: 1 min Column temperature: 85°C, hold for 0.5 min → 6°C/min → 150°C, hold for 5 min → 12°C/min → 180°C → 25°C/min → 280°C, hold for 7 min Ionization temperature: 200°C
Interface temperature: 200℃
Ionization method: EI, SIM; m/z monitored are as follows:
2-MCPD-d5: m/z=201, 203
2-MCPD: m/z=196, 198
For quantification, m/z = 201 for 2-MCPD-d5 and m/z = 196 for 3-MCPD are used, and the others are used to confirm the target substances.

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

The value obtained as the concentration of 2-MCPD when the oil layer after step (2) is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A may be lower than the value obtained for the feedstock. In a preferred embodiment, the value obtained as the concentration of 2-MCPD when the oil layer after step (2) is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is 10% or more, e.g., 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 than the value obtained for the feedstock.

The content of 2-MCPD-forming substances in the oil layer after step (2) may vary depending on the content of 2-MCPD-forming substances in the feedstock. For example, if the concentration of 2-MCPD obtained when the feedstock is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is about 0.3 ppm, the concentration of 2-MCPD obtained when the oil layer after step (2) is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A may be 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. If the concentration of 2-MCPD obtained by analyzing the feedstock oil according to the American Oil Chemists' Society official method Cd29b-13 assay A is about 0.6 ppm, the concentration of 2-MCPD obtained by analyzing the oil layer after step (2) according to the American Oil Chemists' Society official method Cd29b-13 assay A can be 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 0.20 ppm or less.

Biologically derived oils such as fish oil and microbial oil contain cholesterol in addition to triglycerides. Even if cholesterol-containing oils are treated with partial glyceride lipase or washed with water, the cholesterol is not completely removed. Therefore, in certain embodiments herein, the composition produced by the method of the present invention contains cholesterol. The cholesterol concentration in the composition is comparable to that in the raw oil.

Cholesterol is a compound having a steroid skeleton represented by the molecular formula C ₂₇ H ₄₆ O, and exists in natural products as a free form or an ester form. An ester form is an acyl cholesterol in which a fatty acid is bound to the hydroxyl 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.
To approximately 0.1 g of sample, add 1 mL of 0.1 g/L 5α-cholestane/ethanol solution as an internal standard, add 1 mL of 2 mol/L potassium hydroxide/aqueous ethanol solution, and heat at 100°C for 10 minutes. After cooling, add 3 mL of petroleum ether and 3 mL of saturated ammonium sulfate, stir, and allow to stand, then collect the upper layer and measure by gas chromatography under the following measurement conditions. To determine the relative sensitivity of 5α-cholestane and cholesterol, analyze a hexane solution containing 0.25 mg/L 5α-cholestane and 0.25 mg/L cholesterol by gas chromatography. Calculate the total cholesterol content from the sample amount and each area value.
Gas chromatographic 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 amounts of free cholesterol and cholesterol esters are measured by the following method.
Approximately 1 g of sample is made up to 10 mL with hexane, and 0.5 mL is sampled. This is charged into a silica gel cartridge (Waters, Sep-pack® Plus Silica Cartridge 55-105 μm), and 5 mL of hexane:diethyl ether = 9:1 (v/v) is poured through, and the eluate is the cholesterol ester fraction. Further, 5 mL of diethyl ether is poured through, and the eluate is the cholesterol fraction. After evaporating the solvent under a nitrogen stream, the same procedure as for the total cholesterol content measurement is performed, and the cholesterol ester content and free cholesterol content are calculated from the sample volume and area value.

The concentrations of free cholesterol, cholesterol esters, and total cholesterol in the composition produced by the method of the present invention are all comparable to those of the raw oil. The concentrations of free cholesterol and total cholesterol in the raw oil may vary depending on pretreatment (e.g., degumming, deacidification, bleaching, and deodorization), but the concentration of cholesterol esters varies relatively little. In producing the composition of the present invention, reaction conditions may be selected that reduce the variation in the concentration of cholesterol esters. By using a partial glyceride lipase that does not catalyze the hydrolysis of cholesterol esters, the concentration of cholesterol esters can be adjusted so that it does not vary significantly even when the partial glyceride lipase is reacted with the raw oil. In one embodiment, when the raw oil is fish oil, the concentration of cholesterol esters in the composition produced by the method of the present invention may vary depending on the fish species, but may be 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 present invention includes phthalate esters. Phthalate esters are chemicals that are widely used as plasticizers for plastic products such as polyvinyl chloride, and are suspected of having endocrine disrupting effects. Phthalate esters are easily soluble in oil and may also be included in the feedstock oil in the method of the present invention. Phthalate esters are quantified by dissolving a sample in dichloromethane, mixing with an internal standard, separating the phthalate esters from the fat/oil by liquid chromatography, and analyzing by GC-MS/MS. The concentration of phthalate esters in the composition produced by the method of the present invention is similar to that of the feedstock 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 that in the feedstock oil, and may be 14.20 to 15.60 ppb when the feedstock oil is fish oil, for example. The concentration of DINP in the composition produced by the method of the present invention is lower than the concentration in the raw oil, and can be, for example, 9.7 ppb to 11.7 ppb when the raw oil is fish oil.

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, but may be, for example, 1.5 to 37.2. For example, when the acid value of the feedstock is about 0.08, the acid value in the oil layer after step (2) may 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 feedstock is about 0.20, the acid value in the oil layer after step (2) may 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 feedstock is about 0.34, the acid value in the oil layer after step (2) may be 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.

The method of the present invention does not cause decomposition that would change the fatty acid composition before and after the lipase reaction, so the fatty acid composition of the raw oil is not significantly changed. The fatty acid composition of the composition produced by the method of the present invention may contain 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 30 area % or more. In this specification, the fatty acid composition is measured by the following method.
1 mL of 1N sodium ethylate/ethanol solution is added to 40 μL of sample, stirred for about 30 seconds, and then left for 2 minutes. Then, 1 mL of 1N hydrochloric acid is added to neutralize, 2 mL of hexane and 3 mL of saturated aqueous ammonium sulfate solution are added, stirred, and allowed to stand, and the upper layer is subjected to gas chromatography for measurement. The fatty acid composition value is shown in area %. The analysis conditions for gas chromatography are as follows.
Gas chromatographic 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 main component>
The present invention relates to a composition containing triglyceride as a main component,
The proportion of triglycerides in a glyceride fraction in the composition is 90.0 to 99.9 area %,
The composition has a concentration of 3-MCPD of 0.10 ppm to 2.80 ppm when analyzed by the American Oil Chemist's Society official Cd29b-13 assay A, and satisfies at least one of the following a to e:
a) the concentration of 2-MCPD in the composition as determined by the American Oil Chemist's Society 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 to 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 present composition, the term "containing triglyceride as a main component" means that the composition contains triglyceride in an amount of 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 or more, or 95% by weight or more of the total weight of the composition.

The proportion of triglycerides in the glyceride fraction in the compositions of the present invention can be from 90.0 to 99.9 area %, for example, from 95.0 to 99.9 area %, from 96.0 to 99.9 area %, from 97.0 to 99.0 area %, or from 99.0 to 99.9 area %.
The glyceride composition (i.e., the proportions of monoglycerides, diglycerides, and triglycerides in the glycerides contained in a sample) is measured by the TLC/FID method according to the following procedure.
40 μL of sample is dissolved in 1 mL of hexane, and 0.4 μL or 0.6 μL is loaded onto the rod-shaped thin layer. A mixed solvent of chloroform and methanol (chloroform:methanol = 95:5 (volume ratio)) is used as the developing solvent for 2 minutes, and after drying the rod-shaped thin layer, a mixed solution of hexane, diethyl ether, and acetic acid (hexane:diethyl ether:acetic acid = 70:30:0.1 (volume ratio)) is used as the developing solvent for 35 minutes. This is analyzed by thin layer chromatography/hydrogen flame ionization detector. After removing the peaks derived from free fatty acids from the analysis results, the proportion of each glyceride is shown in area %.

When the composition of the present invention is analyzed by the American Oil Chemist's Society official method Cd29b-13 assay A, the resulting concentration of 3-MCPD may be 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 may be achieved by treating the feedstock with partial glyceride lipase.

The composition of the present invention satisfies at least one of the above a to e, for example, any one (i.e., a; b; c; d; or e), any two (i.e., 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 (i.e., 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 (i.e., 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), and preferably satisfies all of the above a to e. Each of these characteristics can be achieved by treating the feedstock with partial glyceride lipase.

In one embodiment of a, the concentration of 2-MCPD obtained by the American Oil Chemists' Society Cd29b-13 assay A for the composition of the present invention may be 0.50 ppm or less, for example, 0.40 ppm or less, 0.36 ppm or less, 0.27 ppm or less, or less than 0.10 ppm (lower limit of quantification). The concentration of 2-MCPD obtained by the American Oil Chemists' Society Cd29b-13 assay A for the composition of the present invention may be 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. The concentration of 2-MCPD obtained by the American Oil Chemists' Society official method Cd29b-13 assay A for the composition of the present invention may be 0.10 ppm to 0.50 ppm, 0.10 ppm to 0.50 ppm, 0.10 to 0.27 ppm, 0.19 to 0.50 ppm, or 0.21 to 0.36 ppm.

In one embodiment of b, the concentration of free cholesterol, cholesterol ester, and total cholesterol in the composition of the present invention may be similar to that of the raw oil. The concentration of cholesterol ester in the composition of the present invention may vary depending on the type of raw oil, but may be 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 one embodiment related to c, the composition of the present invention may contain the same amount of phthalates as the feedstock, except for di-(2-ethylhexyl) phthalate (DEHP) and diisononyl phthalate (DINP). The concentration of DEHP in the composition of the present invention may be lower than the concentration in the feedstock, for example, 14.20 to 15.60 ppb.

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

In one embodiment related to e, the acid value (AV) of the composition of the present invention may be 1.5 to 37.2. The acid value of the composition of the present invention may be higher than the acid value of the feedstock, 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 20 or more. The acid value of the composition of the present invention may be 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.

In a specific embodiment, the composition of the present invention has a 3-MCPD concentration of 1.17 to 2.10 ppm when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 assay A, a 2-MCPD concentration of 0.10 to 0.27 ppm when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 assay A, and an acid value of 1.8 to 5.8.

In a specific embodiment, the composition of the present invention has a 3-MCPD concentration of 0.16 to 0.30 ppm when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 Assay A, a 2-MCPD concentration of less than 0.10 ppm (less than the lower limit of quantification) when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 Assay A, and an acid value of 2.0 to 30.

In a specific embodiment, the composition of the present invention has a 3-MCPD concentration of 1.69 to 2.74 ppm when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 assay A, a 2-MCPD concentration of 0.19 to 0.50 ppm when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 assay A, and an acid value of 2.3 to 5.7.

In a specific embodiment, the composition of the present invention has a 3-MCPD concentration of 1.72 to 2.21 ppm when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 assay A, a 2-MCPD concentration of 0.21 to 0.36 ppm when analyzed by the American Oil Chemist's Society Official Method Cd29b-13 assay A, and an acid value of 2.6 to 5.1.

The composition of the present invention may contain 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 30 area% or more.

In one embodiment, the composition of the present invention may be heating oil. 3-MCPDE may be produced by heat-treating oil at a temperature exceeding 100°C. Therefore, heating oil usually contains 3-MCPDE, but this can be efficiently removed by the method of the present invention, and the value obtained as the concentration of 3-MCPD produced when analyzed by the American Oil Chemist's Society official method Cd29b-13 assay A in the composition of the present invention is reduced compared to the value obtained for the raw oil.

In a preferred embodiment, the composition of the present invention may be heated fish oil. When preparing refined fish oil from crude oil, heat treatment at temperatures exceeding 100°C is required in the deacidification and deodorization processes. As a result, heated fish oil usually contains 3-MCPDE, but this can be efficiently removed by the method of the present invention, and therefore, in the composition of the present invention, the value obtained as the concentration of 3-MCPD generated when analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is reduced compared to the value obtained for the raw oil.

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

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

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

Gas chromatographic 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 Iatron Co., Ltd.). 40 μL of oil was dissolved in 1 mL of hexane, and 0.4 μL or 0.6 μL was loaded onto a Chroma Rod (registered trademark). A mixed solvent of chloroform and methanol (chloroform:methanol = 95:5 (volume ratio)) was used as a developing solvent for 2 minutes, and after drying the rod, a mixed solution of hexane, diethyl ether, and acetic acid (hexane:diethyl ether:acetic acid = 70:30:0.1 (volume ratio)) was used as a developing solvent for 35 minutes. This was analyzed by Iatroscan. After removing the peaks derived from free fatty acids from the analysis results, the proportion of each glyceride was shown in area %.

Analysis of 3-MCPD, 2-MCPD, and glycidol
The analyses of 3-MCPD, 2-MCPD, and glycidol were entrusted to SGS Germany GmbH. The analyses were performed in accordance with AOCS Cd 29b-13.

<Analysis of phthalate esters>
Phthalate esters were analyzed by SGS Germany GmbH. Samples were dissolved in dichloromethane and mixed with an internal standard, then analyzed by LC-GC-MS/MS. In the LC, fats/oils were separated from phthalate esters by size exclusion chromatography, and phthalate esters were analyzed and quantified by GC-MS/MS.

<Acid value (AV) measurement>
The acid value was measured by the following method. A mixed solvent of ethanol and diethyl ether (1:1, v/v) was prepared, and after adding a few drops of phenolphthalein, 0.1 N sodium hydroxide/ethanol solution was added until color developed. Approximately 1 g of sample oil was precisely weighed out into a glass container, and 30-40 mL of the mixed solvent was added to dissolve it. 0.1 N sodium hydroxide/ethanol solution was added dropwise to this to perform neutralization titration, and the acid value was calculated using the following formula from the weight of the sample oil and the volume of 0.1 N sodium hydroxide/ethanol solution used until color developed.
AV = Titration volume (mL) x 5.611/sample weight (g)

<Measurement of total cholesterol content, free cholesterol content, and cholesterol ester content>
The total cholesterol, free cholesterol, and cholesterol ester contents of the oils were determined by gas chromatography.
The total cholesterol content was measured by the following method.
Approximately 0.1 g of oil was mixed with 1 mL of 0.1 g/L 5α-cholestane/ethanol solution as an internal standard, and 1 mL of 2 mol/L potassium hydroxide/aqueous ethanol solution was added, followed by heating at 100°C for 10 minutes. After cooling, 3 mL of petroleum ether and 3 mL of saturated ammonium sulfate were added, shaken, and allowed to stand, after which the upper layer was collected and analyzed by gas chromatography. To determine the relative sensitivity of 5α-cholestane and cholesterol, a hexane solution containing 0.25 g/L 5α-cholestane and 0.25 g/L cholesterol was analyzed by gas chromatography. The total cholesterol content was calculated from the sample amount and the respective area values.
Gas chromatographic 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 amounts of free cholesterol and cholesterol esters were measured by the following method.
Approximately 1 g of oil was diluted to 10 mL with hexane, and 0.5 mL was sampled. This was charged into a silica gel cartridge (Waters, Sep-pack Plus Silica Cartridge 55-105 μm), and 5 mL of hexane:diethyl ether = 9:1 (v/v) was poured through, and the eluate was the cholesterol ester fraction. Furthermore, 5 mL of diethyl ether was poured through, and the eluate was the cholesterol fraction. After evaporating the solvent under a nitrogen stream, the same operation as for the total cholesterol content measurement was performed, and the free cholesterol content and cholesterol ester content were calculated from the sample volume and area value.

Example 1: Production of 3-MCPD fatty acid esters by heat treatment The refining process of fish oil includes short path distillation (SPD), wintering, decolorization, deodorization, etc., in which heat treatment is performed at temperatures exceeding 100° C. In this example, the production behavior of 3-MCPD fatty acid esters by heat treatment of fish oil was investigated.

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

(2) Results The measurement results of 3-MCPD are shown in Table 1. Regardless of the heating time, the measured value of 3-MCPD increased in a temperature-dependent manner. It can be said that the higher the temperature, the easier it is to produce 3-MCPD fatty acid esters.

Example 2: Reduction of 3-MCPD fatty acid esters produced during purification process [Test 1]
Fish oil 1 (distilled deacidified tuna oil, Nippon Suisan Co., Ltd., AV 0.20) was treated with lipase GS and washed with water by the following method, and then 3-MCPD and other contents were measured.
Fish oil 1 was prepared by washing tuna crude oil with 85°C hot water, dehydrating it, and then performing short-path distillation using a short-path distillation apparatus (SPD) KD450 (manufactured by UIC GmbH, distillation surface area 4.5 ^m2 ). The short-path distillation was performed at a main apparatus temperature of 262°C.
In a 50 mL glass container, 15 g of fish oil 1, 10 mL of distilled water, and Lipase GS "Amano" 250G (274000 unit/g, Amano Enzyme Co., Ltd.) were added at 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) and stirred at 20°C for 20 hours. After the reaction was completed, the mixture was heated in a constant temperature bath at 90°C for 10 minutes and centrifuged (1900 g, 10 minutes) to collect the upper layer. Distilled water (10 mL) was added to the obtained upper layer, and the mixture was shaken vigorously, heated in a constant temperature bath at 90°C for 10 minutes and centrifuged (1900 g, 10 minutes) to collect the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above procedure was carried out twice, and the two samples obtained were combined and subjected to the analysis of 3-MCPD, 2-MCPD, and glycidol, the analysis of acid value, the analysis of fatty acid composition, and the analysis of glyceride composition. In the examples, the "3-MCPDs reduction rate" is the percentage of the reduction in the 3-MCPD measurement value of the fish oil after lipase treatment, based on the 3-MCPD measurement value of the fish oil before lipase treatment.

The results are shown in Tables 2 and 3. Treatment with Lipase GS and washing with water reduced the measured 3-MCPD value. At all Lipase GS concentrations from 50 to 800 units/g oil, the measured 3-MCPD value was more than 10% lower than that of Fish Oil 1. Furthermore, the reduction rate of the measured 3-MCPD value increased with increasing amounts of Lipase GS added. Furthermore, no reduction was observed in the proportion of triglycerides in the glycerides contained in the oil treated with Lipase GS compared to Fish Oil 1.

[Test 2]
Fish oil 5 (refined bonito oil, Nippon Suisan Co., Ltd., AV 0.08) was treated with Lipase GS at different concentrations and washed with water by the following method, and then 3-MCPD and other contents were measured.
Fish oil 5 was prepared by the following method. Crude bonito oil was deacidified with alkali. It was then decolorized in a column, the solvent was removed, and steam distillation was performed. It was further subjected to short-path distillation and steam distillation again, after which an antioxidant was added.
In a 50 mL glass container, 15 g of fish oil 5, 10 mL of distilled water, and Lipase GS "Amano" 250G (274000 unit/g, Amano Enzyme Co., Ltd.) were added at 100 unit/g oil (5.5 mg), 200 unit/g oil (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) and stirred at 30°C for 20 hours. After the reaction was completed, the mixture was heated in a constant temperature bath at 90°C for 10 minutes and centrifuged (1900 g, 10 minutes) to collect the upper layer. Distilled water (10 mL) was added to the obtained upper layer, and the mixture was shaken vigorously, heated in a constant temperature bath at 90°C for 10 minutes and centrifuged (1900 g, 10 minutes) to collect the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above procedure was carried out twice, and the two samples obtained were combined and subjected to analysis of 3-MCPD, 2-MCPD, and glycidol, analysis of the acid value, analysis of the fatty acid composition, and analysis of the glyceride composition.

The results are shown in Tables 4 and 5. Even for fish oils with lower 3-MCPD measurements than the fish oil in Test 1, treatment with Lipase GS and washing with water reduced the 3-MCPD measurements. Furthermore, the rate of reduction in the 3-MCPD measurements increased with increasing amounts of Lipase GS added. Furthermore, no reduction was observed in the proportion of triglycerides in the glycerides contained in the oil treated with Lipase GS compared to fish oil 5.

[Test 3]
Fish oil 2 (distilled deacidified tuna oil, Nippon Suisan Co., Ltd., AV 0.34) was subjected to lipase GS treatment at different temperatures and water washing by the following method, and then 3-MCPD and other contents were measured.
Fish oil 2 was prepared in a similar manner to fish oil 1.
In a 50 mL glass container, 15 g of fish oil 2, 10 mL of distilled water, and 400 units/g oil (21.9 mg) of Lipase GS "Amano" 250G (274000 units/g) were added, and the mixture was stirred with a stirrer at 20°C, 30°C, 40°C, or 50°C for 20 hours. After the reaction was completed, the mixture was heated in a constant temperature bath at 90°C for 10 minutes, centrifuged (1900 g, 10 minutes), and the upper layer was collected. Distilled water (10 mL) was added to the obtained upper layer, and the mixture was shaken vigorously, heated in a constant temperature bath at 90°C for 10 minutes, centrifuged (1900 g, 10 minutes), and the upper layer was collected. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above procedure was carried out twice, and the two samples obtained were combined and subjected to analysis of 3-MCPD, 2-MCPD, and glycidol, analysis of the acid value, analysis of the fatty acid composition, and analysis of the glyceride composition.

The results are shown in Tables 6 and 7. When Lipase GS was used, the 3-MCPD measurement value decreased at all temperatures between 20°C and 50°C. The reduction rate of the 3-MCPD measurement value was highest at 30°C, and showed an increasing trend up to 30°C, and a decreasing trend above 30°C.

[Test 4]
Fish oil 2 was treated with lipase GS using different amounts of distilled water and washed with water by the following method, and then 3-MCPD and other components were measured.
In a 50 mL glass container, 15 g of fish oil 2, 2.5 mL, 5 mL, 10 mL, and 20 mL of distilled water, and 400 unit/g oil (21.9 mg) of lipase GS "Amano" 250G were added, and the mixture was stirred for 20 hours at 30 °C with a magnetic stirrer. After the reaction was completed, the mixture was heated in a constant temperature bath at 90 °C for 10 minutes, centrifuged (1900 g, 10 minutes), and the upper layer was collected. 10 mL of distilled water was added to the obtained upper layer, and the mixture was vigorously shaken, heated in a constant temperature bath at 90 °C for 10 minutes, centrifuged (1900 g, 10 minutes), and the upper layer was collected. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above procedure was repeated, and the total amount of sample oil from two runs was used for analysis.

The results are shown in Tables 8 and 9. The 3-MCPD measurement value decreased under all conditions in which 2.5 to 20 mL of distilled water was added to 15 g of raw oil. The reduction rate of the 3-MCPD measurement value was highest when 10 mL of distilled water was added to 15 g of raw oil, and an increasing trend was observed up to 10 mL, and a decreasing trend was observed above 10 mL.

[Test 5]
Fish oil 2 and fish oil 3 (washed tuna oil, Nippon Suisan Co., Ltd., AV 1.26) were treated with lipase and washed with water by the following method, and then analyzed for cholesterol content, etc.
Fish oil 3 was prepared by washing tuna crude oil with hot water at 85°C and then dehydrating it.
In a 50 mL glass container, 6 g of fish oil 2 or 3, 4 mL of distilled water, and Lipase GS "Amano" 250G at 400 units/g oil (8.8 mg) or Lipase OF360 (384000 units/g, Meito Sangyo Co., Ltd.) at 400 units/g oil (6.3 mg) were added and stirred with a magnetic stirrer at 30°C for 20 hours. After the reaction was completed, the mixture was heated in a thermostatic bath at 90°C for 10 minutes and centrifuged (1900 g, 10 minutes) to collect 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 to 13. There were no significant changes in cholesterol content or glyceride composition between the oil after lipase GS treatment and the raw oil.

[Test 6]
Fish oil 4 (solvent-deacidified bonito oil, Nippon Suisan Co., Ltd., AV 0.35) was treated with lipase and washed with water by the following method, and then analyzed for phthalate esters, etc.
Fish oil 4 was prepared by the following method. After heating bonito crude oil to 95°C, 10% by volume of 50% NaOH was added to the bonito crude oil and stirred. After centrifuging and removing the water layer, 85°C hot water was added and stirred. Centrifuging was performed again and the water layer was removed. Silica was added by 2% by volume to the oil layer, and after stirring, it was dehydrated and the silica was removed by a filter.
In a 50 mL glass vessel, 15 g of fish oil 4 (solvent-deacidified bonito oil, Nippon Suisan Co., Ltd., AV 0.35), 10 mL of distilled water, and 400 units/g oil (21.9 mg) of lipase GS "Amano" 250G or 400 units/g oil (15.6 mg) of lipase OF360 (384000 units/g, Meito Sangyo Co., Ltd.) were added and stirred for 20 hours at 30°C with a magnetic stirrer. After the reaction was completed, the mixture was heated in a constant temperature bath at 90°C for 10 minutes and centrifuged (1900 g, 10 minutes) to collect the upper layer. Distilled water (10 mL) was added to the obtained upper layer, and the mixture was shaken vigorously, heated in a constant temperature bath at 90°C for 10 minutes and centrifuged (1900 g, 10 minutes) to collect the upper layer. Ethanol was added as an azeotropic solvent, and the mixture was dehydrated by vacuum distillation. The above procedure was carried out twice, and the two samples obtained were combined and subjected to analysis of phthalic acid esters, acid value, fatty acid composition, and glyceride composition.

The results are shown in Tables 14 and 15. There was no significant change in the content of phthalate esters between the oil after lipase GS treatment and the raw oil.

According to the present invention, it is possible to reduce the content of 3-MCPDE produced by heat treatment of oil without decreasing the proportion of triglycerides in the glycerides contained in the oil, and it is possible to stably produce safer edible oils, etc.

Claims (7)

1. A method for producing a triglyceride-containing composition, comprising:
(1) reacting a raw oil containing triglycerides with a 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).
Including,
The concentration of 3-MCPD produced when the raw oil is analyzed by the American Oil Chemists' Society official method Cd29b-13 assay A is 0.3 ppm or more.
The concentration of 3-MCPD obtained by analyzing the oil layer after step (2) according to the American Oil Chemists' Society official method Cd29b-13 assay A is lower than that obtained for the feedstock oil;
The proportion of triglycerides in a glyceride fraction in the composition is 90.0 to 99.9 area %,
The raw oil containing triglycerides is heating oil,
The method according to claim 1, wherein the raw oil containing triglycerides is fish oil. The method according to claim 1, wherein the concentration of 3-MCPD obtained by analyzing the oil layer after step (2) by the American Oil Chemists' Society official method Cd29b-13 assay A is 10% or more lower than the concentration obtained for the feedstock oil. The method according to claim 1 or 2, wherein the partial glyceride lipase is a lipase derived from Penicillium camemberti. The method according to any one of claims 1 to 3, wherein the amount of partial glyceride lipase used in step (1) is 200 to 1,600 units per 1 g of raw material oil. The method according to any one of claims 1 to 4, wherein the hydrolysis reaction in step (1) is carried out at 10 to 50°C. The method according to any one of claims 1 to 5, wherein the amount of water added to the reaction system for the hydrolysis reaction in step (1) is 0.10 to 2.5 g per 1 g of the raw material oil. A composition containing triglyceride as a main component,
The proportion of triglycerides in a glyceride fraction in the composition is 90.0 to 99.9 area %,
The concentration of 3-MCPD obtained by analysis using the American Oil Chemists' Society official method Cd29b-13 assay A is 0.10 ppm to 2.80 ppm.
The composition, wherein the concentration of cholesterol ester is 0.030 to 0.20% by weight, and the composition is heated fish oil.