Method for extracting fatty acid from euglena oil

Abstract

The present invention provides a method for extracting a fatty acid from Euglena oil which contains a wax ester having a group derived from an alcohol having 8-18 carbon atoms, said method comprising: a step for adding an alkaline solution containing ethanol to Euglena oil and saponifying and decomposing a wax ester into fatty acid soap and an alcohol having 8-18 carbon atoms; a step for adding water and oil / fat for extraction and separating and removing the alcohol having 8-18 carbon atoms; a step for adding an acid and decomposing the fatty acid soap into a fatty acid and a salt; and a step for adding one or two selected from the group consisting of water and hexane as an extraction solvent and separating and recovering the fatty acid from an ethanol / water layer, wherein the oil / fat for extraction contains a triglyceride containing a fatty acid residue having 13 or more carbon atoms in an amount of not less than 60 mass%.

Description

Method for extracting fatty acids from Euglena oil

[0001] The present invention relates to a method for extracting fatty acids from Euglena oil.

[0002] Methods for extracting components such as fatty acids or alcohols from natural components including wax esters are disclosed in Patent Documents 1 and 2.

[0003] Patent Document 1 discloses a method for obtaining a mixture of high molecular weight primary fatty acids containing fatty acids having 24 to 38 carbon atoms from sugarcane wax, either as free acids or in the form of their salts, by homogeneously saponifying the sugarcane wax with a concentrated solution of alkali and alkaline earth hydroxides, extracting these components using a solid-liquid extraction system to separate the mixture from the remaining components, and using an organic solvent selected from the group consisting of ketones having 3 to 8 carbon atoms, alcohols having 1 to 5 carbon atoms, hydrocarbons having 5 to 8 carbon atoms, haloforms, and aromatic compounds, and purifying the mixture (claim 8). It is stated that this method can provide a novel natural mixture of high molecular weight primary fatty acids having 24 to 38 carbon atoms.

[0004] Patent Document 2 discloses a method for producing a higher alcohol, which comprises saponifying a natural wax containing a higher alcohol having 20 to 36 carbon atoms into a long-chain fatty acid soap and a higher alcohol, and extracting and fractionating the higher alcohol having 20 to 36 carbon atoms with a solvent, wherein the higher alcohol is extracted at a temperature of 55°C or higher using a fat or oil having a melting point of 30°C or lower as the solvent, and then washing with hot water at a temperature of 55°C or higher to separate and remove the long-chain fatty acid soap, thereby obtaining the higher alcohol. The document states that this production method provides a method for producing a higher alcohol that is safe and preferable in terms of production and hygiene, and a method for producing a composition of fat or oil and a higher alcohol in a simple manner and with a high yield.

[0005] JP 2012-067119 A JP 2004-217633 A

[0006] The present invention provides a method for efficiently obtaining saturated fatty acids having 13 to 15 carbon atoms from Euglena oil, even when no organic solvent is used in steps subsequent to saponification.

[0007] The present inventors have found that by using Euglena oil as a raw material oil and fat and an oil containing 60% or more of fatty acids having 13 or more carbon atoms as an oil for extraction, saturated fatty acids having 13 to 15 carbon atoms can be efficiently obtained even when no organic solvent is used in the steps subsequent to saponification and hydrolysis.

[0008] The present invention provides the following method for extracting fatty acids from Euglena oil.

[0009] [1] A method for extracting fatty acids from Euglena oil containing wax esters, which are esters having a group derived from an alcohol having from 8 to 18 carbon atoms, comprising: a step of adding an alkaline solution containing ethanol to the Euglena oil and saponifying the wax ester into a fatty acid soap and the alcohol having from 8 to 18 carbon atoms; a step of adding water and an oil for extraction after the saponifying step and separating and removing the alcohol having from 8 to 18 carbon atoms; a step of adding an acid after the separating and removing step to decompose the fatty acid soap into fatty acids and a salt; and a step of adding one or two solvents selected from the group consisting of water and hexane as an extraction solvent and separating and recovering the fatty acids from an ethanol / aqueous layer containing the ethanol and the water, wherein the oil for extraction contains triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms. [2] The method according to [1], wherein the melting point of the triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms, as measured in accordance with Standard Methods for the Analysis of Fats, Oils, and Related Materials, 3.2.2.2-2013, is 10°C or lower. [3] The method according to [1] or [2], wherein the content of fatty acids having 12 or less carbon atoms in the triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is 0.1% by mass or lower. [4] The method according to any one of [1] to [3], wherein the iodine value of the triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms, as measured in accordance with Standard Methods for the Analysis of Fats, Oils, and Related Materials, 2.3.4.1-2013, is 65 or higher and 200 or lower. [5] The method according to any one of [1] to [4], wherein in the step of separating and removing alcohols having from 8 to 18 carbon atoms, the amount of triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms added is from 3 to 50 parts by mass per part by mass of the Euglena oil. [6] The method according to any one of [1] to [5], wherein the ethanol concentration of the ethanol / water layer in the step of separating and recovering is 40% (w / w) or less. [7] The method according to any one of [1] to [6], wherein the reaction temperature in the step of saponification is 40°C or higher. [8] The method according to any one of [1] to [7], wherein the step of separating and removing includes a step of stirring at 40°C or higher.[9] The method according to any one of [1] to [8], wherein the Euglena oil further contains triglycerides.

[10] The method according to any one of [1] to [9], wherein the step of decomposing the fatty acid soap into fatty acids and salts comprises a step of lowering the pH of the ethanol / water layer to 3 or less.

[0010] According to the present invention, a method for efficiently obtaining saturated fatty acids having 13 to 15 carbon atoms from Euglena oil can be provided, even when no organic solvent is used in the steps subsequent to saponification and decomposition.

[0011] Hereinafter, embodiments of the present invention will be described with specific examples of each component. In this specification, "A to B" indicating a numerical range means A or more and B or less unless otherwise specified. In addition, when an upper limit and a lower limit of a numerical range are indicated in this specification, the upper limit and the lower limit can be appropriately combined, and the resulting numerical range is also considered to be disclosed.

[0012] (Method for Extracting Fatty Acids from Euglena Oil) The method for extracting fatty acids from Euglena oil according to this embodiment is a method for extracting fatty acids from Euglena oil containing wax esters, which are esters having a group derived from an alcohol having from 8 to 18 carbon atoms. The method includes the following steps: adding an alkaline solution containing ethanol to Euglena oil to saponify the wax esters into a fatty acid soap and an alcohol having from 8 to 18 carbon atoms (Step 1); adding water and an oil for extraction after the saponification step and separating and removing the alcohol having from 8 to 18 carbon atoms (Step 2); adding an acid after the separation and removal step to decompose the fatty acid soap into fatty acids and a salt (Step 3); and adding one or two solvents selected from the group consisting of water and hexane as an extraction solvent and separating and recovering fatty acids from an ethanol / aqueous layer containing ethanol and water (Step 4). The oil for extraction contains triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms.

[0013] In this embodiment, the inventors discovered that saturated fatty acids having 13 to 15 carbon atoms can be efficiently obtained by a method including the above steps 1 to 4, using Euglena oil as the raw oil and fat, and using an oil containing 60% or more fatty acids having 13 or more carbon atoms as the extraction oil. Patent Document 2 describes a method for producing higher alcohols in which medium-chain triglycerides (MCTs) are used as the extraction oil to extract higher alcohols. However, when MCTs are used as the extraction oil and alcohols having 8 to 18 carbon atoms are separated and removed as described in Patent Document 2, the resulting extracted oil is contaminated with fatty acids having 8 to 12 carbon atoms derived from the extraction oil, and saturated fatty acids having 13 to 15 carbon atoms derived from Euglena oil cannot be extracted at a high concentration in the extracted oil. Further investigation by the inventors revealed that the content of fatty acid residues having 13 or more carbon atoms in the extraction oil contributes to the proportion of saturated fatty acids having 13 to 15 carbon atoms derived from Euglena oil in the extracted oil. In this embodiment, saturated fatty acids having 13 to 15 carbon atoms can be efficiently obtained from Euglena oil by controlling the amount of fatty acids having 13 or more carbon atoms in the oil or fat for extraction. Furthermore, in this embodiment, saturated fatty acids having 13 to 15 carbon atoms can be efficiently obtained in the steps subsequent to the saponification decomposition in step 1, for example, even when an organic solvent such as hexane is not used as the extraction solvent in step 4. This reduces the environmental impact of the entire method and allows the method to be carried out under milder conditions.

[0014] [Euglena Oil] In this embodiment, Euglena refers to a type of microalgae classified in the genus Euglena, also known as Euglena. Euglena species are widely distributed in freshwater environments such as ponds, swamps, and rice paddies. They are spindle-shaped, single-celled organisms with chloroplasts within their cells. In this embodiment, Euglena oil refers to an oily component extracted from at least one of Euglena cells and the culture medium in which Euglena is cultured. Euglena stores wax esters within its cells, and Euglena oil preferably contains wax esters. Wax esters are compounds formed by ester-bonding a fatty acid having 10 or more carbon atoms with an alcohol having 8 to 18 carbon atoms. The fatty acid constituting the wax ester preferably contains one or more species selected from the group consisting of saturated fatty acids having 13 to 15 carbon atoms.

[0015] The Euglena oil of this embodiment may further contain triglycerides. The method for extracting fatty acids from Euglena oil according to this embodiment may preferably be a method for extracting fatty acids derived from wax esters and triglycerides contained in Euglena oil.

[0016] [Step 1] Step 1 is a step in which an alkaline solution containing ethanol is added to Euglena oil to saponify and decompose the wax ester into a fatty acid soap and an alcohol having 8 to 18 carbon atoms.

[0017] The alkali contained in the alkaline solution may be one or two selected from the group consisting of potassium hydroxide and sodium hydroxide, and preferably contains potassium hydroxide from the viewpoint of further improving the efficiency of the saponification decomposition reaction. When potassium hydroxide is used as the alkali, the fatty acid soap obtained is a fatty acid potassium salt. The alkaline solution of this embodiment contains ethanol as described above. That is, the alkaline solution contains ethanol as a solvent. The inclusion of ethanol in the alkaline solution can improve the saponification efficiency. This is also preferable in that in Step 3 (decomposition step) described below, the fatty acid obtained by decomposing the fatty acid soap can be dissolved in the ethanol / water layer, and in Step 4 (separation and recovery step) described below, an extraction solvent can be added to the ethanol / water layer to separate and recover the fatty acid.

[0018] The concentration of the alkaline solution is preferably 3 (w / v)% or more, more preferably 4 (w / v)% or more, and even more preferably 5 (w / v)% or more, from the viewpoint of further improving the decomposition efficiency when saponifying wax esters into fatty acids and alcohols. Furthermore, from the viewpoint of further suppressing solidification of soap during saponification and the resulting decrease in workability, the concentration of the alkaline solution is preferably 10 (w / v)% or less, more preferably 8 (w / v)% or less, and even more preferably 7 (w / v)% or less. Among these, it is more preferable to use, for example, a 6 (w / v)% potassium hydroxide / ethanol solution as the alkaline solution containing ethanol.

[0019] In step 1, the amount of alkaline solution added is, for example, a moderate excess relative to the saponification value of Euglena oil, preferably about 11 to 13 times the saponification value, and more preferably about 12 times the saponification value. The saponification value is measured according to Standard Methods for the Analysis of Fats, Oils, and Related Materials 2.3.2.2-2013 and represents the amount of alkali (mg) required to saponify 1 g of oil. The saponification value of Euglena oil is approximately 150 mg. Therefore, for example, when saponifying 1 g of Euglena oil using a 6 (w / v)% potassium hydroxide / ethanol solution, the amount of potassium hydroxide / ethanol solution added is preferably 30 mL, which is 12 times the 2.5 mL calculated based on the saponification value. Adding an alkaline solution in an amount approximately 12 times the saponification value allows for more thorough saponification of Euglena oil and also allows for a more appropriate range for the amount of water added as an extraction solvent in step 4, described below.

[0020] In step 1, the reaction temperature when an alkaline solution containing ethanol is added to Euglena oil to saponify and decompose the wax esters is preferably 40°C or higher, more preferably 45°C or higher, even more preferably 50°C or higher, even more preferably 55°C or higher, and preferably 70°C or lower, more preferably 65°C or lower. When the reaction temperature in step 1 is at or above the above-mentioned lower limit, Euglena oil and the alkaline solution can be reacted at a temperature higher than the melting point of Euglena oil, which is about 38°C. This allows the saponification reaction to proceed in a state in which Euglena oil is fully dissolved, and allows the saponification and decomposition of the wax esters in Euglena oil to proceed more efficiently and sufficiently. Furthermore, when the reaction temperature in step 1 is at or below the above-mentioned upper limit, the temperature is kept below the boiling point of ethanol, which further suppresses the oxidation of the wax esters and fatty acids in Euglena oil. In Step 1, the reaction time when the ethanol-containing alkaline solution is added to Euglena oil to saponify and decompose the wax ester is preferably 30 minutes or more, more preferably 60 minutes or more, and even more preferably 90 minutes or more in order to allow the reaction to proceed more sufficiently, and is preferably 200 minutes or less, more preferably 150 minutes or less in order to further improve productivity. In Step 1, the stirring speed when the ethanol-containing alkaline solution is added to Euglena oil to saponify and decompose the wax ester is preferably 100 rpm or more, more preferably 200 rpm or more, and preferably 800 rpm or less, more preferably 700 rpm or less.

[0021] Furthermore, when the Euglena oil of this embodiment contains triglycerides, step 1 can be a step of saponifying and decomposing wax esters and triglycerides in the Euglena oil. The triglycerides can be saponified into fatty acid soaps and glycerin in step 1. In steps 1 and after, the fatty acid soaps derived from triglycerides behave in the same manner as the fatty acid soaps derived from wax esters. Therefore, the method for extracting fatty acids from Euglena oil of this embodiment can extract both fatty acids derived from wax esters and fatty acids derived from triglycerides.

[0022] [Step 2] Step 2 is a step in which water and the extracting oil are added to the saponification solution obtained in Step 1 (saponification decomposition step) and the alcohol having 8 to 18 carbon atoms is separated and removed. Adding water to the saponification solution in Step 2 reduces the solubility of the extracting oil in ethanol, thereby enabling smooth separation. Furthermore, when an organic solvent such as hexane is used in Step 2, the alcohol saponified in Step 1 can be dissolved in the organic solvent and stably separated. Furthermore, when the extracting solvent in Step 4 (separation and recovery step) described below contains hexane, fatty acids can be dissolved and separated in hexane. Here, the ethanol concentration of the ethanol / water layer that can be separated from hexane is specifically 80 (w / w)% or less. In Step 2, adding the extracting oil to the saponification solution dissolves the alcohol having 8 to 18 carbon atoms in the saponification solution, allowing the alcohol having 8 to 18 carbon atoms to be separated and removed. Furthermore, since alcohols having 8 to 18 carbon atoms dissolve in fats and oils, the alcohols having 8 to 18 carbon atoms can be separated and removed by using an oil or fat for extraction as an alcohol extraction solvent without using an organic solvent such as hexane. Step 2 may be performed once or may be repeated two or more times. By repeating this step as necessary, the efficiency of alcohol removal can be further improved.

[0023] (Oil and fat for extraction) The oil and fat for extraction used in step 2 contains triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms. This allows saturated fatty acids having 13 to 15 carbon atoms to be efficiently obtained from Euglena oil. In triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms, the content of fatty acid residues having 13 or more carbon atoms is 60% by mass or more, preferably 65% ​​by mass or more, more preferably 70% by mass or more, even more preferably 80% by mass or more, and even more preferably 90% by mass or more. There is no upper limit to the content of fatty acid residues having 13 or more carbon atoms in the triglycerides in the oil and fat for extraction, but it may be, for example, 100% by mass or less, and preferably 100% by mass.

[0024] In triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms, the content of fatty acids having 12 or less carbon atoms is preferably 0.1% by mass or less, more preferably 0.05% by mass or less, even more preferably 0.01% by mass or less, and specifically 0% by mass or more. When the content of fatty acids having 12 or less carbon atoms in triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is equal to or less than the above upper limit, the resulting extracted oil is further prevented from being contaminated with fatty acids having 12 or less carbon atoms derived from the oil or fat used for extraction, and saturated fatty acids having 13 to 15 carbon atoms can be obtained from Euglena oil more efficiently, while the generation of a soapy odor derived from fatty acids having 12 or less carbon atoms derived from the oil or fat used for extraction can be further suppressed.

[0025] Furthermore, from the viewpoint of further improving the efficiency of separation and removal of alcohols having from 8 to 18 carbon atoms, the content of triglycerides in the oil for extraction containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is preferably 50% by mass or more, more preferably 70% by mass or more, even more preferably 90% by mass or more, even more preferably 95% by mass or more, and even more preferably 99% by mass or more, relative to the total oil for extraction. It may be, for example, 100% by mass or less, and is preferably 100% by mass.

[0026] The melting point of triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is preferably 10°C or lower, more preferably 5°C or lower, even more preferably 0°C or lower, and even more preferably -5°C or lower, and may be, for example, -30°C or higher, or even -20°C or higher. When the melting point of triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is below the above upper limit, the oil for extraction can remain liquid at temperatures above room temperature in the separation and removal step, making it easier for alcohols having 8 to 18 carbon atoms to dissolve in the oil for extraction, and thus further improving the efficiency of separation and removal of alcohols having 8 to 18 carbon atoms. The melting point represents the slip melting point and is measured in accordance with Standard Methods for the Analysis of Fats, Oils, and Related Materials 3.2.2.2-2013.

[0027] The iodine value of triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is preferably 65 or more, more preferably 75 or more, even more preferably 90 or more, even more preferably 100 or more, even more preferably 110 or more, and even more preferably 120 or more, and preferably 200 or less, more preferably 180 or less, even more preferably 160 or less, and even more preferably 150 or less. When the iodine value of triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is above the above lower limit, the oil for extraction can be maintained in a liquid state at temperatures above room temperature in the separation and removal step, and alcohols having 8 to 18 carbon atoms are more easily dissolved in the oil for extraction, thereby further improving the efficiency of separation and removal of alcohols having 8 to 18 carbon atoms. Furthermore, when the iodine value of triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is below the above upper limit, the storage stability of the oil for extraction is further improved, and oxidation of fatty acids in the resulting extracted oil can be further suppressed. The iodine value is measured in accordance with the Standard Method for Analysis of Fats, Oils and Related Materials 2.3.4.1-2013, and its unit is Ig / 100g of fats and oils.

[0028] The oils and fats containing triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms preferably include one or more selected from soybean oil, rapeseed oil (including high-oleic rapeseed oil), palm oil, rice oil, corn oil, cottonseed oil, safflower oil, sunflower oil, olive oil, linseed oil, sesame oil, peanut oil, and fish oil, and among these, from the viewpoint of more efficiently obtaining saturated fatty acids having 13 to 15 carbon atoms from Euglena oil, more preferably one or more selected from soybean oil and rapeseed oil, and even more preferably soybean oil. By using these oils and fats, the above-mentioned conditions for melting point, content of fatty acids having 12 or less carbon atoms, and iodine value for triglycerides containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms can be satisfied.

[0029] In step 2, the amount of triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms added is preferably 3 parts by mass or more, more preferably 5 parts by mass or more, even more preferably 8 parts by mass or more, even more preferably 10 parts by mass or more, and even more preferably 20 parts by mass or more, relative to 1 part by mass of Euglena oil, and is preferably 50 parts by mass or less, more preferably 45 parts by mass or less, even more preferably 40 parts by mass or less, and even more preferably 35 parts by mass or less. When the amount of triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is equal to or greater than the above-mentioned lower limit, alcohols having 8 to 18 carbon atoms can be more fully dissolved in the oil for extraction, the separation and removal rate of alcohols having 8 to 18 carbon atoms is further improved, and saturated fatty acids having 13 to 15 carbon atoms can be more efficiently obtained from Euglena oil. When the amount of triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms is equal to or less than the above-mentioned upper limit, the amount of triglyceride used can be reduced, and the efficiency of the fatty acid extraction method can be further improved.

[0030] Step 2 preferably includes a stirring step at 40°C or higher. More specifically, it is preferable to add water and the oil / fat for extraction to the saponification solution obtained in Step 1, and then stir the mixture at 40°C or higher. Stirring at 40°C or higher in Step 2 facilitates separation of the oil / fat for extraction from the ethanol / water layer after stirring, thereby further preventing the resulting extracted oil from being contaminated with fatty acids derived from the oil / fat for extraction, and ultimately allows saturated fatty acids having 13 to 15 carbon atoms to be obtained more efficiently from Euglena oil. The stirring temperature is, for example, 85°C or lower, and preferably 70°C or lower from the viewpoint of preventing evaporation of ethanol.

[0031] In step 2, water and the oil for extraction are added and stirred, and then the mixture is separated into an ethanol / water layer containing a fatty acid soap and an oil for extraction layer containing an alcohol having a carbon number of 8 to 18. The separation of the ethanol / water layer and the oil for extraction layer can be carried out using, for example, a separatory funnel.

[0032] [Step 3] Step 3 is a step of adding an acid to the ethanol / aqueous layer containing the fatty acid soap obtained in step 2 (the separation and removal step) to decompose the fatty acid soap into fatty acids and salts. Step 3 preferably includes a step of adding an acid to the ethanol / aqueous layer containing the fatty acid soap obtained in step 2 to lower the pH of the ethanol / aqueous layer to 3 or less. The pH of the ethanol / aqueous layer after the acid addition is preferably 3 or less, more preferably 2 or less, and even more preferably 1 or less. By adjusting the pH of the ethanol / aqueous layer to the above-mentioned upper limit or less, the fatty acid soap can be more reliably decomposed into fatty acids and salts. Here, the salt refers to a salt formed by combining an alkali derived from the fatty acid soap with an acid. Step 3 can be completed, for example, by adding an acid little by little to the ethanol / aqueous layer and stirring while measuring the pH of the ethanol / aqueous layer using a pH meter or pH test paper, and then ending when the pH reaches a predetermined value or less.

[0033] The acid added in step 3 may be, for example, one or more selected from the group consisting of hydrochloric acid, sulfuric acid, and nitric acid, and from the viewpoint of further improving the decomposition efficiency, it is more preferable to use sulfuric acid. For example, sulfuric acid having a normality of 20 to 30 N may be used as the acid.

[0034] [Step 4] Step 4 is a step in which one or two solvents selected from the group consisting of water and hexane are added as an extraction solvent to the ethanol / water layer to which the acid was added in Step 3 (decomposition step), and fatty acids are separated and recovered from the ethanol / water layer containing ethanol and water. The extraction solvent is preferably one or two solvents selected from the group consisting of water and hexane. The water added as the extraction solvent in Step 4 is different from the water added in Step 2. When water is added as the extraction solvent, the ethanol concentration of the ethanol / water layer (described below) is reduced by adding water, thereby reducing the solubility of fatty acids in the ethanol / water layer, thereby separating the fatty acid layer and extracting the fatty acids. On the other hand, when hexane is added as the extraction solvent, the mixture is separated into an ethanol / water layer and a hexane layer containing the fatty acids. The hexane layer is recovered, and the fatty acids can be recovered by distilling off the hexane. Distillation of the hexane can be performed, for example, using an evaporator. From an environmentally friendly perspective, it is preferable that the extraction solvent contains water. On the other hand, from the viewpoint of more efficiently extracting saturated fatty acids having 13 to 15 carbon atoms from Euglena oil, it is preferable that the extraction solvent contains hexane.

[0035] In step 4, the ethanol concentration of the ethanol / aqueous layer is preferably 40 (w / w)% or less, more preferably 35 (w / w)% or less, even more preferably 30 (w / w)% or less, even more preferably 20 (w / w)% or less, and even more preferably 15 (w / w)% or less. By setting the ethanol concentration of the ethanol / aqueous layer at or below the upper limit, fatty acids are unable to dissolve in the ethanol / aqueous layer and can be separated as a fatty acid layer. In other words, it becomes possible to extract fatty acids with water without using hexane. The ethanol concentration of the ethanol / aqueous layer represents the ethanol concentration after the addition of an extraction solvent. The ethanol / aqueous layer in step 4 contains the ethanol in the alkaline solution added in step 1 and the water added in step 2. Therefore, when water is added as an extraction solvent in step 4, it is preferable to determine the amount of water added as an extraction solvent so that the ethanol concentration of the ethanol / aqueous layer as a whole is at or below the upper limit, taking into account the amount of ethanol in the alkaline solution added in step 1 and the amount of water added in step 2.

[0036] Furthermore, when the Euglena oil of this embodiment contains triglycerides, both the fatty acids derived from the wax esters and the fatty acids derived from the triglycerides can be separated and recovered in step 4.

[0037] Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various other configurations may be adopted. Furthermore, the present invention is not limited to the above-described embodiments, and modifications and improvements within the scope of achieving the object of the present invention are included in the present invention.

[0038] Examples of the present invention will be described below, but the scope of the present invention is not limited to these examples.

[0039] The following oils and fats were used for extraction. Soybean oil: manufactured by J-Oil Mills Co., Ltd., content of fatty acids with 13 or more carbon atoms: 100% by mass, content of fatty acids with 12 or less carbon atoms: 0% by mass, melting point: -8°C, iodine value: 130 Rapeseed oil: manufactured by J-Oil Mills Co., Ltd., content of fatty acids with 13 or more carbon atoms: 100% by mass, content of fatty acids with 12 or less carbon atoms: 0% by mass, melting point: -15°C, iodine value: 112 MCT: manufactured by Kao Corporation, product name: MT-60, content of fatty acids with 13 or more carbon atoms: 0.4% by mass, content of fatty acids with 12 or less carbon atoms: 99.6% by mass, melting point: -10°C, iodine value: 1 or less

[0040] [Melting Point] The melting point (°C) of each oil for extraction was measured in accordance with the Standard Fats and Oils Analysis Test Method 3.2.2.2-2013.

[0041] [Iodine Value] The iodine value (Ig / 100g of fats and oils) of each oil and fat for extraction was measured in accordance with the Standard Method for Analysis of Fats and Oils 2.3.4.1-2013.

[0042] [Production of Euglena Oil] Euglena oil as a raw material oil was produced as follows. (Culturing Euglena) Euglena gracilis Z strain was used as Euglena. The culture medium was prepared by autoclaving Hutner medium and Koren-Hutner medium prepared based on the description in "Euglena Physiology and Biochemistry" (edited by Kitaoka Shozaburo, published by Academic Press Center). Euglena was cultured as follows. (Culturing step 1: Culturing Euglena) 880 mL of the prepared medium was placed in a 1 L shake flask, and Euglena seed algae were inoculated to an initial concentration of approximately 0.05 g / L. The culture was cultured with shaking for 9 days in an artificial climate chamber (GROWTH CABINET, manufactured by SANYO) set at 29°C. The light irradiation intensity was approximately 100 μmol / (m ２ The incubation time was 12 hours per day (s), and the light exposure time was 12 hours per day. It was confirmed that the nitrogen source in the medium was consumed during the 9-day culture period. (Cultivation step 2: anaerobic treatment) After the 9-day culture period, the shake flask was sealed and left to stand in a light-shielded chamber set at 29°C, thereby placing the cells under anaerobic conditions. The sample that had been subjected to anaerobic treatment for 48 hours was used as a Euglena culture solution and subjected to the following extraction and purification steps.

[0043] (Extraction and Purification of Euglena Oil) A deodorized Euglena oil was obtained through the following steps. 1 (Extraction Step): The Euglena culture solution obtained above was centrifuged (3000 rpm, room temperature, 5 minutes), and the supernatant was removed. The resulting precipitate was dried in a dryer at 105°C for 24 hours to obtain dried Euglena. 10 mL of hexane was added to 10 g of dried Euglena and the mixture was stirred thoroughly. The hexane was removed from the resulting hexane solution using an evaporator to obtain a crude extracted oil. 2 (Degumming Step): 100 mL of water and 3 g of a degumming agent (phosphoric acid) were added to 3 g of the obtained crude extracted oil, and the mixture was stirred at 80°C and 100 rpm for 120 minutes. The gums were transferred to the aqueous layer, and the aqueous layer was removed to obtain a degummed oil. 3 (Deoxidation step): 100 mL of water and 1.25 g of sodium hydroxide were added to the degummed oil and stirred at 80°C, 100 rpm, and 120 minutes. The free fatty acids and soap were transferred to the aqueous layer, and the aqueous layer was removed to obtain the deoxidized oil. 4 (Bleaching step): Activated clay (product name: GSF) was added to 1,000 g of deoxidized oil at a concentration of 2% by mass relative to the oil, and the mixture was stirred at 80°C, 300 rpm, and 30 minutes. The clay was then removed by filtration at 60°C using a funnel and filter paper (Advantec, No. 2) to obtain a bleached oil. 5 (Deodorization step): 1,000 g of the bleached oil was steam distilled at 150°C, with a steam injection rate of 2% relative to the oil, and for 50 minutes to obtain a deodorized oil. This was used as Euglena oil (raw oil). The saponification value of the resulting Euglena oil was 150 mg. The fatty acid composition of the obtained Euglena oil and the carbon chain composition derived from C13-16 alcohols in the higher alcohols in the Euglena oil are shown in Table 1. These were measured according to the method described below in "Analysis of Fatty Acids and Alcohols."

[0044] <Analysis of wax esters and triglycerides in Euglena oil> [Thin layer chromatography] The obtained Euglena oil was analyzed using thin layer chromatography. As a result, it was confirmed that wax esters and triglycerides were contained (results not shown). Thin layer chromatography was performed under the following conditions: Plate: HPTLC Silica gel 60 10 cm x 10 cm (Merck) Developing solvent (mobile phase): hexane / diethyl ether / acetic acid = 80 / 20 / 1 Sample: Euglena oil 1 was dissolved in hexane to a concentration of 20 mg / mL, injected, and developed.

[0045] [Extraction of Fatty Acids from Euglena Oil] (Example 1) (Step 1: Saponification and Decomposition Step) 1 g of Euglena oil and 30 mL of a 6 (w / v)% potassium hydroxide / ethanol solution were added to a 300 mL screw-type Erlenmeyer flask. The mixture was heated to 60°C in a water bath and stirred with a stirrer at 300 rpm for 2 hours to carry out a saponification reaction, yielding a saponified solution. (Step 2: Separation and Removal Step) 50 mL of water and 30 g of soybean oil as an extraction oil were added to the saponified solution obtained in Step 1 above. The mixture was heated to 80°C (separation temperature) in a water bath and stirred with a stirrer at 300 rpm for 10 minutes. Before cooling to room temperature, the resulting solution was transferred to a separatory funnel and allowed to stand for 10 minutes. The solution separated into an upper layer (alcohol / soybean oil layer) and a lower layer (fatty acid potassium salt / aqueous ethanol layer), and the lower layer was recovered. (Step 3: Decomposition Step) 1 mL of 24N sulfuric acid was added to the lower layer (fatty acid potassium salt / ethanol aqueous solution layer) obtained in Step 2 above, using pH test paper (manufactured by AS ONE Corporation), to adjust the pH of the lower layer to 1. (Step 4: Separation and Recovery Step) 200 mL of hexane was added as an extraction solvent to the liquid obtained in Step 3 above, and after thorough stirring, the entire amount of the resulting liquid was transferred to a separatory funnel, and the upper layer (fatty acid layer) was recovered. The hexane was removed from the recovered upper layer using an evaporator to obtain an extract. The ethanol concentration (w / w) % of the lower layer (fatty acid potassium salt / ethanol aqueous solution layer) after addition of the extraction solvent is shown in Table 1 ("Step 4: Ethanol concentration during fatty acid separation and recovery" in Table 1). The recovered upper layer (fatty acid layer) also contains alcohols contained in the wax esters of Euglena oil. For this reason, the recovered upper layer (fatty acid layer) is referred to as the "extracted oil."

[0046] (Comparative Examples 1 and 2 and Examples 2 to 5) Extracted oils were obtained from Euglena oil in the same manner as in Example 1, except that the type and amount of fats and oils for extraction, the type and amount of extraction solvent, and the separation temperature were as shown in Table 1. (Control Example 1) Extracted oils were obtained from Euglena oil in the same manner as in Example 1, except that hexane was used instead of fats and oils for extraction.

[0047] [Analysis of Fatty Acids and Alcohols] Euglena oil, each oil for extraction, and each example of the extracted oil obtained from Euglena oil were subjected to alcohol composition analysis and fatty acid composition analysis as follows in accordance with Standard Methods for Analysis of Fats, Oils, and Related Materials 2.4.1.2-2013. Euglena oil, soybean oil, rapeseed oil, and each example of the extracted oil obtained from Euglena oil were each dissolved in hexane and converted into fatty acid methyl esters using the boron trifluoride methanol method. The fatty acid composition of the obtained fatty acid methyl esters was analyzed using gas chromatography (GC) under the following conditions, and the content (mass%) of saturated fatty acids having 13 to 15 carbon atoms in the entire extracted oil was calculated. The total saturated fatty acids having 13 to 15 carbon atoms and saturated fatty acids having 13 to 15 carbon atoms in Euglena oil and each example of the extracted oil obtained from Euglena oil are shown in Table 1 ("Fatty Acid Composition" in Table 1, mg / g). In addition, the content of alcohols having 8 to 18 carbon atoms in the entire extracted oil ("Total alcohol" in Table 1, mg / g) was calculated. The results are shown in Table 1. In the table, for example, "C14:0" represents a fatty acid with 14 carbon atoms and 0 unsaturated bonds. GC apparatus: Product name GC2010 (manufactured by Shimadzu Corporation) Column: SP-2560 (100 m x 0.25 mm x 0.2 μm) (manufactured by Supelco) Injection port temperature: 250°C Carrier gas: Helium (29.1 mL / min) Split ratio: 25:1 Column temperature: 180°C 55 min → (8°C / min) → 220°C 5 min Detector: Hydrogen flame ionization detector (260°C)

[0048]

[0049] As can be seen from Table 1, in Examples 1 to 5, the total content of saturated fatty acids having a carbon number of 13 or more and 15 or less in the extracted oil obtained was higher than in Comparative Examples 1 and 2. In addition, in Examples 1 and 2, the total content of saturated fatty acids having a carbon number of 13 or more and 15 or less in the extracted oil obtained was equal to or greater than that of Control Example 1.

[0050] This application claims priority based on Japanese Patent Application No. 2024-011701, filed January 30, 2024, the disclosure of which is incorporated herein by reference in its entirety.

Claims

1. A method for extracting fatty acids from Euglena oil containing wax esters, which are esters having a group derived from an alcohol having from 8 to 18 carbon atoms, comprising the steps of: adding an alkaline solution containing ethanol to the Euglena oil and saponifying the wax ester into a fatty acid soap and the alcohol having from 8 to 18 carbon atoms; adding water and an oil for extraction after the saponifying step and separating and removing the alcohol having from 8 to 18 carbon atoms; adding an acid after the separating and removing step to decompose the fatty acid soap into fatty acids and a salt; and adding one or two solvents selected from the group consisting of water and hexane as an extraction solvent and separating and recovering the fatty acids from an ethanol / aqueous layer containing the ethanol and the water, wherein the oil for extraction contains triglycerides containing at least 60% by mass of fatty acid residues having 13 or more carbon atoms.

2. The method according to claim 1, wherein the melting point of the triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms, as measured in accordance with Standard Methods for the Analysis of Fats, Oils, and Related Materials 3.2.2.2-2013, is 10°C or lower.

3. The method according to claim 1 or 2, wherein the triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms has a content of fatty acids having 12 or less carbon atoms of 0.1% by mass or less.

4. The method according to claim 1 or 2, wherein the triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms has an iodine value of 65 or more and 200 or less, as measured in accordance with Standard Methods for the Analysis of Fats, Oils, and Related Materials 2.3.4.1-2013.

5. The method according to claim 1 or 2, wherein in the step of separating and removing alcohols having 8 to 18 carbon atoms, the amount of triglyceride containing 60% by mass or more of fatty acid residues having 13 or more carbon atoms added is 3 to 50 parts by mass per part by mass of the Euglena oil.

6. The method according to claim 1 or 2, wherein the ethanol concentration of the ethanol / water layer in the step of separating and recovering is 40 (w / w)% or less.

7. The method according to claim 1 or 2, wherein the reaction temperature in the saponification decomposition step is 40°C or higher.

8. The method according to claim 1 or 2, wherein the step of separating and removing comprises a step of stirring at 40°C or higher.

9. The method according to claim 1 or 2, wherein the Euglena oil further contains triglycerides.

10. The method of claim 1 or 2, wherein said step of decomposing said fatty acid soaps into fatty acids and salts comprises lowering the pH of said ethanol / water layer to 3 or less.

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