Microbial oil, method for producing microbial oil, concentrated microbial oil, and method for producing concentrated microbial oil

Abstract

A microbial oil, comprising: a specific amount of at least one polyunsaturated fatty acid with a carbon number of 20 or more in the form of fatty acid alkyl esters and / or free fatty acids, and a specific amount of thermally generated fatty acids with a carbon number of 16 to 22 in the form of fatty acid alkyl esters and / or free fatty acids. A method for producing microbial oil, comprising: preparing a feedstock oil containing at least one polyunsaturated fatty acid with a carbon number of 20 or more in the form of alkyl esters and / or free fatty acids obtained from microbial biomass; performing precision distillation based on specific conditions on the feedstock oil; and obtaining the above-mentioned microbial oil. Concentrated microbial oil obtained by the production method and a method for producing the same. A therapeutic or preventive agent for inflammatory diseases containing the microbial oil or concentrated microbial oil.

Description

[0001] The present application is a divisional application of the Chinese invention patent application with application number 201480066446.2, application date December 4, 2014, and invention name “Microbial oil, method for producing microbial oil, concentrated microbial oil, and method for producing concentrated microbial oil”. Technical Field

[0002] The present invention relates to a microbial oil, a method for producing the microbial oil, concentrated microbial oil and a method for producing the concentrated microbial oil. Background Art

[0003] In microbial oil, there are long-chain polyunsaturated fatty acids with carbon number of more than 20, such as eicosadienoic acid, dihomo-γ-linolenic acid (DGLA), eicosatetraenoic acid, arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosatetraenoic acid, eicosapentaenoic acid and docosahexaenoic acid (DHA). Pharmaceuticals, health foods, cosmetics, etc. that utilize these long-chain polyunsaturated fatty acids as functional ingredients have attracted much attention, and their uses have also been studied. Along with this, it is required to produce polyunsaturated fatty acids in large quantities at high concentrations.

[0004] In microbial oil, except long-chain polyunsaturated fatty acids, contain short-chain fatty acids, saturated fatty acids, phospholipids, sterols, glycerides, ceramides, sphingomyelins, terpenoids, flavonoids, tocopherols and other diverse and unique oily components. These compositions sometimes bring into play inherent functions. For example short-chain fatty acids sometimes become the reason for the unique stink of microbial oil, and its unique stink is sometimes not preferred aspect the performance of the function required by specific long-chain polyunsaturated fatty acids. When the specific long-chain polyunsaturated fatty acids contained in the microbial oil are concentrated or refined, high performance liquid chromatography, liquid-liquid distribution, urea can be used to add the like sometimes.

[0005] For example, Patent Document 1 discloses a method for reducing the amount of sterols in a sterol-containing microbial oil composition by distilling a microbial oil obtained from a genetically modified microorganism that produces linoleic acid, DGLA, DHA, EPA, etc. at least once under short-path distillation conditions.

[0006] Patent Document 2 discloses a method comprising: in the preparation of edible marine oil containing deodorized and stabilized microbial oil, subjecting the oil to countercurrent steam distillation (CCSD) in a thin film column containing regular packing, and adding an antioxidant as desired.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application No. 2014-510166

[0010] Patent Document 2: Japanese Patent Application No. 2010-526896 Summary of the invention

[0011] Problems to be solved by the invention

[0012] The purpose of the method in the past is to separate the specific composition of a part beyond the lipid acid with the specific lipid acid as the purpose of concentrating or refining, and in the method in the past, it is difficult to concentrate or refine the long-chain polyunsaturated fatty acids as the purpose of concentrating or refining highly.Using microbial oil as the situation of raw material, because of the influence of various other compositions contained in the raw material etc., can not fully concentrate or concentrate or refine the long-chain polyunsaturated fatty acids as target.Also can utilize distillation technology to concentrate or refine, in microbial oil, be used for the long-chain polyunsaturated fatty acids as the target to concentrate or refine the distillation technology also not fully established.

[0013] The object of the present invention is to provide a microbial oil useful for effectively obtaining a refined microbial oil containing a target polyunsaturated fatty acid at a high ratio and a method for producing the same, a concentrated microbial oil containing a polyunsaturated fatty acid at a high content ratio and a method for producing the same, and various uses of the microbial oil and the concentrated microbial oil.

[0014] Technical means to solve the problem

[0015] In each aspect of the present invention, the following microbial oil, method for producing microbial oil, concentrated microbial oil, method for producing concentrated microbial oil, microbial oil, and uses of concentrated microbial oil are provided.

[0016] <1> A microbial oil comprising: at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids, with a content of 50% by weight or more of the total weight of fatty acids in the oil; and

[0017] A thermally generated fatty acid having 16 to 22 carbon atoms and having a content of 3.0% by weight or less based on the total weight of the fatty acids in the oil.

[0018] <2> according to <1> The microbial oil, wherein the content of the polyunsaturated fatty acids is 80% to 98% by weight of the total weight of the fatty acids in the oil.

[0019] <3> according to <1> or <2> The microbial oil, wherein the content of the thermally generated fatty acid is 0.0001 wt % to 3.0 wt % of the total weight of the fatty acids in the oil.

[0020] <4> according to <1> ~ <3> The microbial oil according to any one of the preceding claims, wherein the total content of the saturated fatty acid having 22 carbon atoms and the saturated fatty acid having 24 carbon atoms is 6.0% by weight or less of the total weight of the fatty acids in the oil.

[0021] <5> according to <1> ~ <4> The microbial oil according to any one of the preceding claims, wherein the total content of the saturated fatty acid having 22 carbon atoms and the saturated fatty acid having 24 carbon atoms is 10 / 100 or less of the content of the polyunsaturated fatty acid.

[0022] <6> according to <1> ~ <5> The microbial oil according to any one of the preceding claims, wherein the content of saturated fatty acids having 24 carbon atoms is 3.0% by weight or less based on the total weight of fatty acids in the oil.

[0023] <7> according to <1> ~ <6> The microbial oil according to any one of the preceding claims, wherein the content of saturated fatty acids having 24 carbon atoms is 4 / 100 or less of the content of the polyunsaturated fatty acids.

[0024] <8> according to <1> ~ <7> The microbial oil according to any one of the preceding claims, wherein the content of other saturated or unsaturated fatty acids is 10.0% by weight or less of the total weight of the fatty acids in the oil,

[0025] The other saturated or unsaturated fatty acids, when used as an indicator related to separation using liquid chromatography and the separation number calculated from the number of carbon atoms and the number of double bonds of the fatty acid, have a separation number greater than -2 and less than +2 compared to the separation number of the polyunsaturated fatty acid, and have a carbon atom number different from the carbon atom number of the polyunsaturated fatty acid.

[0026] <9> according to <8> The microbial oil, wherein the content of the other saturated or unsaturated fatty acids is less than 15 / 100 of the content of the polyunsaturated fatty acids.

[0027] <10> according to <1> ~ <9> The microbial oil described in any one of the above, wherein the polyunsaturated fatty acid is at least one selected from the group consisting of eicosadienoic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid and docosahexaenoic acid.

[0028] <11> according to <8> ~ <10> The microbial oil described in any one of the preceding claims, wherein the other saturated or unsaturated fatty acids contain at least one selected from the group consisting of a saturated fatty acid having 18 carbon atoms, a monounsaturated fatty acid having 18 carbon atoms, a diunsaturated fatty acid having 18 carbon atoms, a triunsaturated fatty acid having 18 carbon atoms, and a tetraunsaturated fatty acid having 18 carbon atoms.

[0029] <12> according to <1> ~ <11> The microbial oil described in any one of the preceding claims, wherein the polyunsaturated fatty acid is dihomo-γ-linolenic acid, and the thermogenic fatty acid is a thermogenic fatty acid having 20 carbon atoms.

[0030] <13> according to <12> The microbial oil, wherein, when the retention time of dihomo-γ-linolenic acid ethyl ester is set to 1 in a gas chromatography analysis of the thermally generated fatty acid ethyl ester using the following conditions, the thermally generated fatty acid contains at least one of a first substance having a retention time as a peak appearing in the range of 1.001 to 1.011 and a second substance having a retention time as a peak appearing in the range of 1.013 to 1.027,

[0031] Apparatus: 6890N Network GC system, Agilent Technologies Inc.;

[0032] Column: DB-WAX, length 30 m × inner diameter 0.25 mm × film thickness 0.25 μm, Agilent Technologies;

[0033] Column temperature conditions: 60°C for 2.5 minutes → temperature rise 20°C / minute → 180°C → temperature rise 2°C / minute → 230°C for 15 minutes;

[0034] Inlet temperature conditions: 210°C, no split, sampling time at the split outlet 1.5 minutes, purge flow 40 mL / min;

[0035] Injection volume conditions: 1 μL, sample concentration 1 mg / mL or less;

[0036] Detector: FID;

[0037] Detector temperature: 280°C;

[0038] Carrier gas conditions: helium, linear velocity 24 cm / min.

[0039] <14> according to <13> In the microbial oil, the polyunsaturated fatty acid is dihomo-γ-linolenic acid, and the total content of the first substance and the second substance is 0.001 wt % to 2.8 wt % of the total weight of the fatty acids in the oil.

[0040] <15> according to <10> ~ <14> The microbial oil according to any one of the preceding claims, wherein the content of the monounsaturated fatty acid having 18 carbon atoms is 7.0% by weight or less based on the total weight of the fatty acids in the oil.

[0041] <16> according to <10> ~ <15> The microbial oil according to any one of the preceding claims, wherein the content of the monounsaturated fatty acid having 18 carbon atoms is 10 / 100 or less of the content of the polyunsaturated fatty acid.

[0042] <17> according to <10> ~ <16> The microbial oil according to any one of the preceding claims, wherein the content of the diunsaturated fatty acid having 18 carbon atoms is 7 / 100 or less of the content of the polyunsaturated fatty acid.

[0043] <18> according to <10> ~ <17> The microbial oil according to any one of the preceding claims, wherein the total content of the monounsaturated fatty acid having 18 carbon atoms and the diunsaturated fatty acid having 18 carbon atoms is 15 / 100 or less of the content of the polyunsaturated fatty acid.

[0044] <19> according to <10> ~ <18> The microbial oil according to any one of the preceding claims, wherein the content of saturated fatty acids having 18 carbon atoms is 11 / 100 or less of the content of the polyunsaturated fatty acids.

[0045] <20> A method for producing microbial oil, comprising:

[0046] preparing a feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl esters and / or free fatty acids obtained from microbial biomass; and

[0047] The feedstock oil is refined by precision distillation under conditions including a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa.

[0048] <21> A method for producing microbial oil, comprising:

[0049] A feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl esters and / or free fatty acids obtained from microbial biomass is prepared;

[0050] The crude oil is subjected to precision distillation using a distillation tower containing regular packing under conditions including a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa; and

[0051] The microbial oil according to any one of claims 1 to 19 is obtained.

[0052] <22> A method for producing microbial oil, comprising:

[0053] A feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl esters and / or free fatty acids obtained from microbial biomass is prepared;

[0054] The feedstock oil is subjected to precision distillation using a distillation tower containing a regular packing, based on the following conditions, the conditions including a tower bottom temperature and a minimum pressure in the distillation tower corresponding to the type of the polyunsaturated fatty acid as a target, and conditions that can obtain a microbial oil containing thermally generated fatty acids having 16 to 22 carbon atoms at a content of 3.0% by weight or less of the total weight of the fatty acids in the oil; and

[0055] get <1> ~ <19> The microbial oil according to any one of claims 1 to 5.

[0056] <23> according to <22> The production method is characterized in that the precision distillation is carried out at a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa.

[0057] <24> according to <20> ~ <23> The production method according to any one of the preceding claims, wherein the precision distillation comprises a plurality of precision distillations performed under conditions of different column bottom temperatures and minimum pressures in the distillation column.

[0058] <25> according to <24> The production method, wherein the precision distillation includes low-temperature precision distillation based on a tower bottom temperature of 160°C to 220°C and a minimum pressure in the distillation tower of 0.1Pa to 30Pa, and high-temperature precision distillation based on a tower bottom temperature of 170°C to 230°C and a minimum pressure in the distillation tower of 0.1Pa to 30Pa.

[0059] <26> according to <25> In the production method, the tower bottom temperature in the high-temperature precision distillation is 3° C. to 20° C. higher than the tower bottom temperature in the low-temperature precision distillation.

[0060] <27> according to <21> ~ <26> The method of any one of the above, wherein the specific surface area per unit of regular filler is 125 m 2 / m 3 ~1700m 2 / m 3 .

[0061] <28> A concentrated microbial oil, wherein the content of polyunsaturated fatty acids with more than 20 carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids is 90% to 98% by weight of the total weight of fatty acids in the oil; the content of thermally generated fatty acids with 16 to 22 carbon atoms is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil; the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is less than 1.0% by weight of the total weight of fatty acids in the oil; and the content of monounsaturated fatty acids with 18 carbon atoms is less than 5.0% by weight of the total weight of fatty acids in the oil.

[0062] <29> A concentrated microbial oil, wherein the content of dihomo-γ-linolenic acid in the form of fatty acid alkyl esters and / or free fatty acids is 90% to 98% by weight of the total weight of fatty acids in the oil; the content of thermally generated fatty acids with 16 to 22 carbon atoms is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil; the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is less than 1.0% by weight of the total weight of fatty acids in the oil; and the content of monounsaturated fatty acids with 18 carbon atoms is less than 5.0% by weight of the total weight of fatty acids in the oil.

[0063] <30> A method for preparing concentrated microbial oil, comprising:

[0064] use <20> ~ <27> The production method described in any one of the above can obtain a microbial oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids as a target;

[0065] The obtained microbial oil was concentrated using reverse phase column chromatography.

[0066] <31> A sort of <1> ~ <19> Any of the microbial oils or <28> or <29> The concentrated microbial oil is used in food, supplements, medicines, cosmetics or feed.

[0067] <32> A sort of <1> ~ <19> Any of the microbial oils or <28> or <29> The concentrated microbial oil is used in a method for producing food, supplements, medicines, cosmetics or feed.

[0068] <33> A pharmaceutical product comprising <1> ~ <19> Any of the microbial oils or <1> or <29> The concentrated microbial oil.

[0069] <34> An agent for preventing or treating inflammatory diseases, comprising <1> ~ <19> Any of the microbial oils or <28> or <29> The concentrated microbial oil.

[0070] <35> according to <34> The inflammatory disease prevention or treatment agent is an anti-allergic agent or an anti-inflammatory agent.

[0071] <36> according to <34> or <35> The inflammatory disease prevention or treatment agent, wherein the inflammatory disease is an inflammatory disease of the skin selected from at least one of the group consisting of macules, urticaria, blisters, swellings and eczema, or an inflammatory disease of the skin caused by at least one of the group consisting of exposure to radiation, autoimmune diseases and uremic pruritus.

[0072] <37> according to <34> or <35> The inflammatory disease prevention or treatment agent, wherein the inflammatory disease is at least one selected from the group consisting of atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, photocontact dermatitis, systemic contact dermatitis, rheumatism, psoriasis and lupus.

[0073] <38> A method for preventing, treating or alleviating inflammatory diseases, comprising: <34> ~ <37> Any one of the agents for preventing or treating inflammatory diseases is administered to a subject suffering from an inflammatory disease or a subject at risk of suffering from the disease.

[0074] <39> according to <37> The method for preventing, treating or alleviating inflammatory diseases, wherein the administration is oral administration or local administration.

[0075] <40> A microbial oil, which is <20> ~ <27> It can be obtained by any of the production methods described above.

[0076] <41> A concentrated microbial oil, which is <30> The above-mentioned manufacturing method is obtained.

[0077] Effects of the Invention

[0078] According to the present invention, there can be provided a microbial oil useful for effectively obtaining a refined microbial oil containing a target polyunsaturated fatty acid at a high ratio and a method for producing the same, a concentrated microbial oil containing a polyunsaturated fatty acid at a high content ratio and a method for producing the same, and various uses of the microbial oil and the concentrated microbial oil. DETAILED DESCRIPTION

[0079] A method for producing microbial oil according to one embodiment of the present invention comprises: preparing a feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl esters and / or free fatty acids obtained from microbial biomass; and refining the feedstock oil by precision distillation under conditions including a tower bottom temperature of 160°C to 230°C and a minimum pressure in a distillation tower of 0.1Pa to 30Pa.

[0080] The present invention is based on the following finding: by refining a feedstock oil containing at least one polyunsaturated fatty acid with a carbon number of 20 or more in the form of alkyl esters and / or free fatty acids obtained from microbial biomass using precision distillation under specific conditions, a microbial oil containing at least one polyunsaturated fatty acid with a carbon number of 20 or more in the form of alkyl esters and / or free fatty acids as the target at a high content can be obtained.

[0081] In this specification, unless otherwise specified, at least one polyunsaturated fatty acid having more than 20 carbon atoms in the form of alkyl esters and / or free fatty acids is sometimes referred to as a target LC-PUFA. In addition, in this specification, unless otherwise specified, each form of saturated or unsaturated fatty acids in the form of fatty acid alkyl esters or free fatty acids contained in the feedstock oil obtained from microbial biomass is sometimes omitted. For example, unsaturated fatty acids having more than 20 carbon atoms in the form of fatty acid alkyl esters and unsaturated fatty acids having more than 20 carbon atoms in the form of free fatty acids are both referred to as "unsaturated fatty acids having more than 20 carbon atoms", and saturated fatty acids having 22 carbon atoms in the form of fatty acid alkyl esters and saturated fatty acids having 22 carbon atoms in the form of free fatty acids are both referred to as "saturated fatty acids having 22 carbon atoms".

[0082] That is, in the purification of raw oil obtained from microbial biomass, simple distillation such as molecular distillation can be used in the past, but simple distillation only separates fatty acids by heating, and cannot accurately separate specific polyunsaturated fatty acids that are targets from fatty acids that are not targets.

[0083] In the present invention, when purifying the target LC-PUFA from the raw material oil obtained from the microbial biomass, the purification is performed by precision distillation under specific temperature and pressure conditions, so the target polyunsaturated fatty acids can be purified with better accuracy and at a high content.

[0084] In addition, the method for producing microbial oil in another embodiment of the present invention comprises: preparing a raw material oil containing at least one polyunsaturated fatty acid with a carbon number of 20 or more in the form of alkyl esters and / or free fatty acids obtained from microbial biomass; using a distillation tower containing regular fillers to perform precision distillation on the raw material oil under conditions including a tower bottom temperature of 160°C to 230°C and a minimum pressure in the distillation tower of 0.1Pa to 30Pa, and obtaining a specific microbial oil in one embodiment of the present invention described later.

[0085] In addition, a method for producing microbial oil according to another embodiment of the present invention comprises: preparing a raw material oil containing at least one polyunsaturated fatty acid with a carbon number of 20 or more in the form of alkyl esters and / or free fatty acids obtained from microbial biomass; using a distillation tower containing a regular filler to perform precision distillation on the raw material oil based on the conditions described below, the conditions being a tower bottom temperature and a minimum pressure in the distillation tower corresponding to the type of the polyunsaturated fatty acid as a target, and conditions that can obtain a microbial oil containing thermally generated fatty acids with a carbon number of 16 to 22 at a content of less than 3.0 weight % of the total weight of the fatty acids in the oil; and obtaining the microbial oil according to one embodiment of the present invention described below.

[0086] The microbial oil of one embodiment of the present invention is a microbial oil containing at least one polyunsaturated fatty acid with 20 carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids at a content of 50% by weight or more of the total weight of the fatty acids in the oil, and a thermally generated fatty acid with 16 to 22 carbon atoms at a content of 3.0% by weight or less of the total weight of the fatty acids in the oil.

[0087] In order to purify specific fatty acids from various components contained in the raw material oil with higher precision through precision distillation, it can be advantageous to carry out the process under more stringent conditions, such as higher temperature conditions. For example, by increasing the bottom temperature and increasing the amount of steam, the reflux ratio (reflux amount / fraction extraction amount) can be increased, thereby improving the separation of each fatty acid in the precision distillation. In addition, the raw material oil from microbial biomass has the following tendency: the content of long-chain saturated fatty acids with a higher melting point than the target LC-PUFA, such as saturated fatty acids with 22 carbon atoms or saturated fatty acids with 24 carbon atoms, is higher than that of the raw material oil obtained from commonly known fish oil, vegetable oil, etc. It was found that the long-chain saturated fatty acids in this microbial oil have a high molecular weight and a high boiling point compared to the target LC-PUFA, and the saturated vapor pressure at the same temperature is also low. Therefore, it was found that when the raw material oil from the microbial oil containing more of these long-chain saturated fatty acids is distilled, it requires a high distillation temperature, i.e., a bottom temperature, compared to the raw material oil from fish oil, etc. That is, in order to obtain a microbial oil having a high content of polyunsaturated fatty acids with a carbon number of 20 or more and a low content of long-chain saturated fatty acids with a higher melting point than these, the distillation of the raw material oil from microbial biomass requires more stringent conditions, such as higher temperature conditions, than the raw material oils from commonly known fish oils, vegetable oils, etc. On the other hand, it was found that when distillation is performed under higher temperature conditions, fatty acid components that were not generated before distillation, so-called thermogenic fatty acids, are generated in the microbial oil. It is believed that the thermogenic fatty acids generated in the microbial oil contain substances generated from the target LC-PUFA due to the influence of overheating, and it was found that the content of the target LC-PUFA tends to decrease as the content of the thermogenic fatty acids increases. It was found that the thermogenic fatty acids generated from the target LC-PUFA in the microbial oil may not be effectively separated from the target LC-PUFA even if reverse phase column chromatography is used, which becomes the cause of the reduction in the content of the target LC-PUFA in the concentrated microbial oil and the reduction in the yield. This revealed that purification of microbial oil with a reduced content of target LC-PUFA by reverse phase column chromatography is ineffective.

[0088] The present invention focuses on such thermogenic fatty acids, and unexpectedly obtains the relationship between the precision of purification of target LC-PUFA and the increase in the content of thermogenic fatty acids with 16 to 22 carbon atoms based on the following insight: by using a distillation column containing a regular packing for precision distillation, or by performing precision distillation in a manner that contains thermogenic fatty acids with 16 to 22 carbon atoms to a certain extent, the target LC-PUFA with a high content can be purified simply and efficiently. In addition, the present invention focuses on such thermogenic fatty acids with 16 to 22 carbon atoms, and unexpectedly obtains the insight that in order to more efficiently obtain a microbial oil containing a target LC-PUFA with a high content, a microbial oil containing thermogenic fatty acids with 16 to 22 carbon atoms to a certain extent is advantageous. In this specification, unless otherwise specified, thermogenic fatty acids with 16 to 22 carbon atoms are sometimes referred to as "thermally produced fatty acids".

[0089] The microbial oil obtained by the production method of the present invention and the microbial oil of the present invention are further subjected to a specific concentration method, such as reverse phase column chromatography, thereby obtaining a concentrated microbial oil containing the target LC-PUFA at a high content rate.

[0090] That is, the concentrated microbial oil of another embodiment of the present invention is the following concentrated microbial oil: the content of polyunsaturated fatty acids with more than 20 carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids is 90% to 98% by weight of the total weight of the fatty acids in the oil, the content of thermally generated fatty acids with 16 to 22 carbon atoms is 0.0001% to 3.0% by weight of the total weight of the fatty acids in the oil, the total content of saturated fatty acids with 24 carbon atoms and 22 carbon atoms is less than 1.0% by weight of the total weight of the fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms is less than 5.0% by weight of the total weight of the fatty acids in the oil.

[0091] In addition, the method for producing concentrated microbial oil according to another embodiment of the present invention comprises: using any one of the methods for producing microbial oil according to another embodiment of the present invention to obtain a microbial oil containing at least one polyunsaturated fatty acid with 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids as a target; and concentrating the obtained microbial oil using reverse phase column chromatography.

[0092] As long as it is a concentrated microbial oil or microbial oil in the form of the present invention, it contains or can contain the target LC-PUFA at a high content rate, so it is useful in the fields of food, supplements, pharmaceuticals, cosmetics, feed, etc., for example, in inflammatory disease prevention or treatment agents, and in methods for preventing, treating or alleviating inflammatory diseases. In addition, as long as it is a method for producing concentrated microbial oil in the form of the present invention, the method for producing microbial oil containing a high content rate of the target LC-PUFA of the present invention is used, so concentrated microbial oil can be effectively provided.

[0093] The term "step" in this specification refers not only to an independent step but also to a step that cannot be clearly distinguished from other steps as long as the intended purpose of the step can be achieved.

[0094] In the present specification, the numerical range expressed by "to" means a range including the numerical values ​​described before and after the "to" as the minimum value and the maximum value, respectively.

[0095] In this specification, when the mixture contains a plurality of substances belonging to each component, the amount of each component in the mixture refers to the total amount of the plurality of substances present in the mixture unless otherwise specified.

[0096] In this specification, when a plurality of substances belonging to each component are present in the mixture, the content rate of each component in the mixture refers to the total content rate of the plurality of substances present in the mixture unless otherwise specified.

[0097] In the present invention, "microbial oil" refers to a mixture of organic substances obtained from microbial biomass and insoluble in water at normal temperature and pressure. Microbial oil contains oily components such as saturated or unsaturated fatty acids, phospholipids, sterols, glycerol, ceramide, sphingomyelin, terpenoids, flavonoids, tocopherol, and saturated or unsaturated fatty acids sometimes exist as constituent fatty acids in other oily components.

[0098] In the present invention, "fatty acid" refers to free saturated or unsaturated fatty acids, fatty acids contained in saturated or unsaturated fatty acid alkyl esters, triglycerides, diglycerides, monoglycerides, phospholipids, sterol esters, etc., which can be called constituent fatty acids in other words.

[0099] In this manual, unless otherwise specified, the form of the compound containing fatty acid is sometimes omitted. As the form of the compound containing fatty acid, free fatty acid form, fatty acid alkyl ester form, glyceride form, phospholipid form, sterol ester form, etc. can be listed. The compound containing the same fatty acid can be contained in a single form in the microbial oil, and can also be contained as a mixture of more than two forms.

[0100] In addition, when recording fatty acids, numerical expressions are sometimes adopted in which the number of carbon atoms, the number of double bonds, and the location of double bonds are simply expressed using numbers and letters, respectively. For example, a saturated fatty acid with 20 carbon atoms is recorded as "C20:0", a monounsaturated fatty acid with 18 carbon atoms is recorded as "C18:1", etc., dihomo-γ-linolenic acid is recorded as "C20:3, n-6", etc., and arachidonic acid can be recorded as "C20:4, n-6", etc. This method is well known to those skilled in the art, and the fatty acids recorded according to this method can be easily specified by those skilled in the art.

[0101] The total containing ratio of the lipid acid in the microbial oil can be set as the gross weight of the microbial oil for example more than 80 % by weight, more than 90 % by weight, more than 95 % by weight or more than 98 % by weight.As for being present in other components in the microbial oil and not containing the compound of lipid acid or the partial structure beyond the lipid acid of the compound that contains lipid acid, can enumerate: the glycerine skeleton partial structure of glycerine, sterol, hydrocarbon, terpenoid, flavonoid, tocopherol, glyceride, the phosphate skeleton partial structure of phosphoric acid, the sphingosine skeleton partial structure etc.

[0102] In this specification, a mixture of compounds extracted only from microbial cells may be referred to as crude microbial oil.

[0103] The fatty acid alkyl esters or free fatty acids in the present invention refer to a mixture of fatty acid alkyl esters or a mixture of free fatty acids obtained by subjecting crude oil obtained from microbial biomass to hydrolysis, alkyl esterification or the like, unless the specific fatty acid species are specified.

[0104] The content of fatty acids relative to the total weight of fatty acids in the oil of the present invention is determined based on the fatty acid composition. The fatty acid composition can be obtained according to a conventional method. Specifically, the oil to be measured is esterified using a lower alcohol and a catalyst to obtain fatty acid lower alkyl esters. Then, the obtained fatty acid lower alkyl esters are analyzed using a gas chromatograph with a hydrogen flame ionization detector (FID). The peaks corresponding to each fatty acid in the obtained gas chromatography chart are identified, and the peak area of ​​each fatty acid is obtained using the Agilent ChemStation integration algorithm (revised version C.01.03

[37] , Agilent Technologies). The percentage of each peak area relative to the sum of the peak areas of the fatty acids is set as the fatty acid composition. The area % obtained using the above-mentioned determination method is set to be the same as the weight % of each fatty acid in the sample. Refer to the 2013 edition of the benchmark oil and fat analysis test method established by the Japan Oil Chemistry Society (JOCS), 2.4.2.1-2013 Fatty acid composition (FID constant temperature gas chromatography) and 2.4.2.2-2013 Fatty acid composition (FID elevated temperature gas chromatography).

[0105] When microbial oil contains the fatty acid beyond the fatty acid of fatty acid alkyl ester form and free fatty acid form, after the fatty acid beyond the fatty acid of fatty acid alkyl ester form and the fatty acid of free fatty acid form is separated from microbial oil, the fatty acid composition that becomes measurement object is measured.As the method that makes the fatty acid separation beyond the fatty acid of fatty acid alkyl ester form and free fatty acid form from microbial oil, for example, can refer to the methods such as disclosed thin-layer chromatography in The Journal of Biological Chemistry 1958,233:311-320., The Lipid Handbook with CD-ROM Third Edition CRC Press Taylor&Francis Group (2007).

[0106] Hereinafter, each aspect of the present invention will be described.

[0107] (1) Microbial oil

[0108] The microbial oil in one embodiment of the present invention contains at least one polyunsaturated fatty acid with 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids, with a content of 50% by weight or more of the total weight of the fatty acids in the oil, and a thermally generated fatty acid with 16 to 22 carbon atoms, with a content of 3.0% by weight or less of the total weight of the fatty acids in the oil.

[0109] As described above, the microbial oil may be obtained from microbial biomass. As the microorganism, any microorganism may be lipid-producing, and examples thereof include algae and fungi.

[0110] Examples of the algae include the genus Labyrinthulamycota.

[0111] As fungi, at least one selected from the group consisting of the genus Mortierella, the genus Conidiobolus, the genus Pythium, the genus Phytophthora, the genus Penicillium, the genus Cladosporium, the genus Mucor, the genus Fusarium, the genus Aspergillus, the genus Rhodotorula, the genus Entomophthora, the genus Echinosporangium, and the genus Saprolegnia can be cited. Among them, microorganisms belonging to the genus Mortierella are more preferred. Examples of the microorganism belonging to the genus Mortierella include microorganisms belonging to the subgenus Mortierella, such as Mortierella elongata, Mortierella exigua, Mortierella hygrophila, and Mortierella alpina.

[0112] The polyunsaturated fatty acid having 20 or more carbon atoms in the present invention includes a divalent or more, preferably a trivalent or more unsaturated fatty acid. The carbon number of the polyunsaturated fatty acid refers to the carbon number of the constituent fatty acid. As polyunsaturated fatty acids having 20 or more carbon atoms, for example, polyunsaturated fatty acids having 20 or more carbon atoms but less than 22 carbon atoms can be listed, specifically, eicosadienoic acid (C20: 2, n-9), dihomo-γ-linolenic acid (C20: 3, n-6), eicosatrienoic acid (C20: 3, n-9), eicosatetraenoic acid (C20: 4, n-3), arachidonic acid (C20: 4, n-6), eicosapentaenoic acid (C20: 5, n-3), docosatetraenoic acid (C22: 4, n-6), docosapentaenoic acid (C22: 5, n-3), docosapentaenoic acid (C22: 5, n-6), and docosahexaenoic acid (C22: 6, n-3), etc. The microbial oil may contain at least one of these polyunsaturated fatty acids, may contain two or more of them in combination, may contain one selected from these polyunsaturated fatty acids, and may not contain other polyunsaturated fatty acids. In addition, as the target LC-PUFA, the microbial oil only needs to contain at least one of the polyunsaturated fatty acids having a carbon number of 20 or more and 22 or less, and may not contain one or more of the specific ones. For example, the microbial oil may not contain at least one selected from the group consisting of eicosadienoic acid (C20: 2, n-9), dihomo-γ-linolenic acid (C20: 3, n-6), eicosatrienoic acid (C20: 3, n-9), eicosatetraenoic acid (C20: 4, n-3), arachidonic acid (C20: 4, n-6), eicosapentaenoic acid (C20: 5, n-3), docosatetraenoic acid (C22: 4, n-6), docosapentaenoic acid (C22: 5, n-3), docosapentaenoic acid (C22: 5, n-6) and docosahexaenoic acid (C22: 6, n-3). Here, “containing no polyunsaturated fatty acids” means that the content of the polyunsaturated fatty acids in question is less than 5% by weight of the total weight of fatty acids in the oil or is 0.

[0113] The alkyl group in the polyunsaturated fatty acid in the form of fatty acid alkyl esters preferably has 1 to 3 carbon atoms, such as methyl, ethyl or propyl. The polyunsaturated fatty acid in the form of alkyl esters is particularly preferably in the form of ethyl ester or methyl ester.

[0114] The content of the target LC-PUFA in the microbial oil, i.e., a polyunsaturated fatty acid having 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids, is 50% by weight or more of the total weight of the fatty acids in the oil. When the content of the target LC-PUFA is less than 50% by weight, it is not possible to effectively obtain a refined microbial oil containing a high content of the target LC-PUFA. As described above, the content of the target LC-PUFA in the microbial oil is set to a value obtained by analyzing the fatty acid composition of the microbial oil.

[0115] From the viewpoint of more effectively achieving the purification of the target LC-PUFA, the content of the target LC-PUFA in the microbial oil is preferably 60% by weight or more, more preferably 70% by weight or more, further preferably 80% by weight or more, further more preferably 85% by weight or more, particularly preferably 90% by weight or more, further particularly preferably 95% by weight or more, and most preferably 98% by weight, based on the total weight of the fatty acids in the oil. In addition, the content of the target LC-PUFA in the microbial oil may be 50% to 98% by weight, 60% to 98% by weight, 70% to 98% by weight, 80% to 98% by weight, 85% to 98% by weight, 90% to 98% by weight, or 95% to 98% by weight, based on the total weight of the fatty acids in the oil.

[0116] The microbial oil of the present invention contains thermally generated fatty acids having 16 to 22 carbon atoms at a content of 3.0% by weight or less based on the total weight of the fatty acids in the oil.

[0117] As described above, the thermogenic fatty acid is a fatty acid having 16 to 22 carbon atoms that is generated based on the presence of the target LC-PUFA by the heat of a high-temperature treatment such as distillation. That is, the thermogenic fatty acid is considered to be a fatty acid that is generated by the heat of a high-temperature treatment such as distillation, the decomposition, isomerization, etc. of the target LC-PUFA, but the present invention is not limited to this theory. The form of the thermogenic fatty acid is not particularly limited, and is not limited to the fatty acid alkyl ester form or the free fatty acid form.

[0118] The amount and type of thermally generated fatty acids contained in the microbial oil vary depending on the conditions of precision distillation, the type of target LC-PUFA contained in the microbial oil, and the like.

[0119] As an example of thermogenic fatty acids, it is considered to be the trans isomer of the target LC-PUFA (see Journal of the American Oil Chemists' Society, Vol. 66, No. 12, pp. 1822-1830 (1989)). That is, the carbon double bond part contained in the target LC-PUFA is usually cis, while the thermogenic fatty acids are considered to be fatty acids in which part or all of the carbon double bond part is changed to trans, fatty acids having conjugated double bonds by changing the position of the double bond, etc.

[0120] Examples of thermally generated fatty acids include the following fatty acids. The microbial oil may contain any one or a combination of two or more of the following compounds:

[0121] 8Z,11E-eicosatrienoic acid, 8E,11Z-eicosadienoic acid, 8E,11E-eicosadienoic acid, 8Z,11Z,14E-eicosatrienoic acid, 8Z,11E,14Z-eicosatrienoic acid, 8E,11Z,14Z-eicosatrienoic acid, 8Z,11E,14E-eicosatrienoic acid, 8E,11Z,14E-eicosatrienoic acid, 8E,11E,14Z-eicosatrienoic acid, 8E,11E,14E-eicosatrienoic acid, 5Z,8Z,11E-eicosatrienoic acid, 5Z,8E,11Z-eicosatrienoic acid, 5E,8Z,11Z-eicosatrienoic acid, 5Z,8E,11E-eicosatrienoic acid, 5E,8Z,11E-eicosatrienoic acid trienoic acid, 5E,8E,11Z-eicosatrienoic acid, 5E,8E,11E-eicosatrienoic acid, 8Z,11Z,14Z,17E-eicosatrienoic acid, 8Z,11Z,14E,17Z-eicosatrienoic acid, 8Z,11E,14Z,17Z-eicosatrienoic acid, 8E,11Z,14Z,17E-eicosatrienoic acid, 8Z,11E,14Z,17E-eicosatrienoic acid, 8Z,11E,14Z,17E-eicosatrienoic acid, 8Z,11E,14Z,17E-eicosatrienoic acid, 8Z,11E,14Z,17E-eicosatrienoic acid, 8Z,11E,14Z,17E-eicosatrienoic acid, 8Z,11E,14Z,17E-eicosatrienoic acid, Z-eicosatetraenoic acid, 8E,11E,14E,17Z-eicosatetraenoic acid, 8E,11E,14Z,17E-eicosatetraenoic acid, 8E,11Z,14E,17E-eicosatetraenoic acid, 8Z,11E,14E,17E-eicosatetraenoic acid, 8E,11E,14E,17E-eicosatetraenoic acid, 5Z,8Z,11Z,14E-eicosatetraenoic acid, 5Z,8Z,11E,14Z-eicosatetraenoic acid, 5Z,8E,11Z,14Z-eicosatetraenoic acid, 5E,8Z,11Z,14E-eicosatetraenoic acid, 5Z,8E,11Z,14E-eicosatetraenoic acid, 5Z,8 Z,11E,14E-eicosatetraenoic acid, 5E,8Z,11E,14Z-eicosatetraenoic acid, 5Z,8E,11E,14Z-eicosatetraenoic acid, 5E,8E,11Z,14Z-eicosatetraenoic acid, 5E,8E,11E,14Z-eicosatetraenoic acid, 5E,8E,11Z,14E-eicosatetraenoic acid, 5E,8Z,11E,14E-eicosatetraenoic acid, 5E,8E,11E,14E-eicosatetraenoic acid, 5Z,8Z,11E,14E-eicosatetraenoic acid, 5E,8E,11E,14E-eicosatetraenoic acid, 5Z,8Z,11Z,14Z,17E-eicosapentaenoic acid, 5Z,8Z,11E,14E,17Z-eicosapentaenoic acid, 5Z,8Z,11E,14Z,17Z-eicosapentaenoic acid, 5Z,8E,11Z,14Z,17Z-eicosapentaenoic acid, 5E,8Z,11Z,14Z,17Z-eicosapentaenoic acid, 5E,8Z,11Z,14Z,17E-eicosapentaenoic acid, 5Z,8E,11Z,14Z,17E-eicosapentaenoic acid, 5Z,8Z,11E,14Z,17E-eicosapentaenoic acid, 5Z,8Z,11Z,14E,17E-eicosapentaenoic acid, 5Z,8Z,11Z,14E,17E-eicosapentaenoic acid, 5Z,8Z,11Z,14E,17Z-eicosapentaenoic acid, 5Z,8Z,11E,14E,17Z ,17Z-eicosapentaenoic acid, 5Z,8E,11E,14Z,17Z-eicosapentaenoic acid, 5E,8E,11Z,14Z,17Z-eicosapentaenoic acid, 5Z,8E,11E,14E,17Z-eicosapentaenoic acid, 5E,8Z,11E,14E,17Z-eicosapentaenoic acid, 5E,8E,11Z,14E,17Z-eicosapentaenoic acid, 5E,8E,11E,14Z,17Z-eicosapentaenoic acid, 5E,8E,11E,14Z,17Z-eicosapentaenoic acid, 5Z,8E,11E,14Z,17E-eicosapentaenoic acid, 5E,8E,11Z,14Z,17E-eicosapentaenoic acid, 5Z,8E,11Z,14 E,17E-eicosapentaenoic acid, 5E,8Z,11Z,14E,17E-eicosapentaenoic acid, 5Z,8Z,11E,14E,17E-eicosapentaenoic acid, 5E,8E,11E,14E,17Z-eicosapentaenoic acid, 5E,8E,11E,14E,17E-eicosapentaenoic acid, 5E,8E,11Z,14E,17E-eicosapentaenoic acid, 5E,8Z,11E,14E,17E-eicosapentaenoic acid, 5E,8E,11E,14E,17E-eicosapentaenoic acid, 5Z,8E,11E,14E,17E-eicosapentaenoic acid, 5E,8E,11E,14E,17E-eicosapentaenoic acid, 7Z,10Z,13Z,16Z,19E-docosapentaenoic acid, 7Z,10Z,13 Z,16E,19Z-docosapentaenoic acid, 7Z,10Z,13E,16Z,19Z-docosapentaenoic acid, 7Z,10E,13Z,16Z,19Z-docosapentaenoic acid, 7E,10Z,13Z,16Z,19Z-docosapentaenoic acid, 7E,10Z,13Z,16Z,19E-docosapentaenoic acid, 7Z,10E,13Z,16Z,19E-docosapentaenoic acid, 7Z,10Z,13E,16Z,19E-docosapentaenoic acid, 7Z,10Z,13Z,16E,19E-docosapentaenoic acid, 7Z,10Z,13Z,16E,19E-docosapentaenoic acid, 7Z,10Z,13Z,16E,19E-docosapentaenoic acid, 7Z,10Z,13Z,16E,19E-docosapentaenoic acid, 7Z,10E,13Z,16E,1919Z-docosapentaenoic acid, 7Z,10Z,13E,16E,19Z-docosapentaenoic acid, 7E,10Z,13E,16Z,19Z-docosapentaenoic acid, 7Z,10E,13E,16Z,19Z-docosapentaenoic acid, 7E,10E,13Z,16Z,19Z-docosapentaenoic acid, 7Z,10E,13E,16E,19Z-docosapentaenoic acid, 7E,10Z,13E,16E,19Z-docosapentaenoic acid, 7E,10E,13Z,16E,19Z-docosapentaenoic acid, 7E,10E,13Z,16E,19Z-docosapentaenoic acid, 7E,10E,13E,16Z,19Z-docosapentaenoic acid, 7Z,10E,13E,16Z,19Z-docosapentaenoic acid Docosapentaenoic acid, 7E,10Z,13E,16Z,19E-docosapentaenoic acid, 7E,10E,13Z,16Z,19E-docosapentaenoic acid, 7Z,10E,13Z,16E,19E-docosapentaenoic acid, 7E,10Z,13Z,16E,19E-docosapentaenoic acid, 7Z,10Z,13E,16E,19E-docosapentaenoic acid, 7E,10E,13E,16E,19E-docosapentaenoic acid, 7E,10E,13E,16E,19E-docosapentaenoic acid, 7E,10E,13E,16E,19E-docosapentaenoic acid, 7E,10E,13Z,16E,19E-docosapentaenoic acid, 7E,10E,13Z,16E,19E-docosapentaenoic acid, Enoic acid, 7Z,10E,13E,16E,19E-docosapentaenoic acid, 7E,10E,13E,16E,19E-docosapentaenoic acid, 4Z,7Z,10Z,13Z,16Z,19E-docosahexaenoic acid, 4Z,7Z,10Z,13Z,16E,19Z-docosahexaenoic acid, 4Z,7Z,10E,13Z,16Z,19Z-docosahexaenoic acid, 4Z,7Z,10E,13Z,16Z,19Z-docosahexaenoic acid, 4Z,7E,10Z,13Z,16Z,19Z-docosahexaenoic acid, 4E,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid, 4E,7Z,10Z, 13Z,16Z,19E-docosahexaenoic acid, 4Z,7E,10Z,13Z,16Z,19E-docosahexaenoic acid, 4Z,7Z,10E,13Z,16Z,19E-docosahexaenoic acid, 4Z,7Z,10Z,13E,16Z,19E-docosahexaenoic acid, 4Z,7Z,10Z,13Z,16E,19E-docosahexaenoic acid, 4E,7Z,10Z,13Z,16E,19Z-docosahexaenoic acid, 4Z,7E,10Z,13Z,16E,19Z-docosahexaenoic acid, 4Z,7Z,10E,13Z,16E,19Z-docosahexaenoic acid, 4Z,7Z,10E,13Z,16E,19Z19Z-docosahexaenoic acid, 4E,7Z,10Z,13E,16Z,19Z-docosahexaenoic acid, 4Z,7E,10Z,13E,16Z,19Z-docosahexaenoic acid, 4Z,7Z,10E,13E,16Z,19Z-docosahexaenoic acid, 4E,7Z,10E,13Z,16Z,19Z-docosahexaenoic acid, 4E,7E,10E,13Z,16Z,19Z-docosahexaenoic acid, 4E,7E,10Z,13Z,16Z,19Z-docosahexaenoic acid, 4E,7E,10E,13Z,16Z,19Z-docosahexaenoic acid, 4E,7E,10Z,13E,16Z,19Z-docosahexaenoic acid Docosahexaenoic acid, 4E,7E,10Z,13Z,16E,19Z-docosahexaenoic acid, 4E,7E,10Z,13Z,16E,19E-docosahexaenoic acid, 4E,7Z,10E,13E,16Z,19Z-docosahexaenoic acid, 4E,7Z,10E,13Z,16E,19Z-docosahexaenoic acid, 4E,7Z,10E,13Z,16E,19E-docosahexaenoic acid, 4E,7Z,10Z,13E,16E,19E-docosahexaenoic acid, 4E,7Z,10Z,13E,16E,19E-docosahexaenoic acid, 4E,7Z,10Z,13Z,16E,19E-docosahexaenoic acid, 4E,7Z,10Z,13Z,16E,19E-docosahexaenoic acid, 7E,10E,13E,16Z,19Z-docosahexaenoic acid, 4Z,7E,10E,13Z,16E,19Z-docosahexaenoic acid, 4Z,7E,10E,13Z,16Z,19E-docosahexaenoic acid, 4Z,7E,10Z,13E,16E,19Z-docosahexaenoic acid, 4Z,7E,10Z,13E,16E,19E-docosahexaenoic acid, 4Z,7E,10Z,13E,16E,19E-docosahexaenoic acid, 4Z,7Z,10E,13E,16E,19E-docosahexaenoic acid, 4Z,7Z,10E,13E,16E,19E-docosahexaenoic acid, 4Z,7Z,10E,13E,16Z,19E-docosahexaenoic acid, 13Z,16E,19E-docosahexaenoic acid, 4Z,7Z,10Z,13E,16E,19E-docosahexaenoic acid, 4Z,7Z,10E,13E,16E,19E-docosahexaenoic acid, 4Z,7E,10Z,13E,16E,19E-docosahexaenoic acid, 4Z,7E,10E,13Z,16E,19E-docosahexaenoic acid, 4Z,7E,10E,13E,16E,19E-docosahexaenoic acid, 4Z,7E,10E,13E,16E,19E-docosahexaenoic acid, 4Z,7E,10E,13E,16E,19E-docosahexaenoic acid, 4E,7Z,10Z,13E,16E,19E-docosahexaenoic acid, 4E,7Z,10E,13Z,16E,19E-docosahexaenoic acid, 4E,7Z,10E,13E,16Z,19E-docosahexaenoic acid, 4E,7Z,10E,13E,16E,19Z-docosahexaenoic acid, 4E,7E,10Z,13Z,16E,19E-docosahexaenoic acid, 4E,7E,10Z,13E,16Z,19E-docosahexaenoic acid, 4E,7E,10E,13Z,16E,19Z-docosahexaenoic acid, 4E,7E,10E,13Z,16E,19E-docosahexaenoic acid, 4E,7E,10E,13Z,16E,19Z-docosahexaenoic acid Enoic acid, 4Z,7E,10E,13E,16E,19E-docosahexaenoic acid, 4E,7Z,10E,13E,16E,19E-docosahexaenoic acid, 4E,7E,10Z,13E,16E,19E-docosahexaenoic acid, 4E,7E,10E,13Z,16E,19E-docosahexaenoic acid, 4E,7E,10E,13E,16E,19E-docosahexaenoic acid, 4E,7E,10E,13E,16E,19Z-docosahexaenoic acid, 4E,7E,10E,13E,16E,19E-docosahexaenoic acid, 7Z,10Z,13E,16E-docosatetraenoic acid, 7Z,10Z,13E,16E -Docosatatetraenoic acid, 7Z,10E,13Z,16Z-docosatatetraenoic acid, 7E,10Z,13Z,16Z-docosatatetraenoic acid, 7E,10Z,13Z,16E-docosatatetraenoic acid, 7Z,10E,13Z,16E-docosatatetraenoic acid, 7Z,10E,13Z,16E-docosatatetraenoic acid, 7E,10Z,13E,16E-docosatatetraenoic acid, 7Z,10E,13E,16Z-docosatatetraenoic acid, 7E,10E,13Z,16Z-docosatatetraenoic acid, 7E,10E,13Z,16Z-docosatatetraenoic acid, 7E,10E,13Z,16E-docosatatetraenoic acid, 7E,10E,13Z, 16E-docosatetraenoic acid, 7E,10E,13E,16Z-docosatetraenoic acid, 7E,10E,13E,16E-docosatetraenoic acid, 4Z,7Z,10Z,13Z,16E-docosapentaenoic acid, 4Z,7Z,10Z,13E,16Z-docosapentaenoic acid, 4Z,7Z,10E,13Z,16Z-docosapentaenoic acid, 4Z,7E,10Z,13Z,16Z-docosapentaenoic acid, 4E,7Z,10Z,13Z,16Z-docosapentaenoic acid, 4E,7Z,10Z,13Z,16E-docosapentaenoic acid, 4Z,7E,10Z,13Z,16E-docosapentaenoic acid, 4Z,7Z,10E,13Z,16E-docosapentaenoic acid, 4Z,7Z,10Z,13E,16E-docosapentaenoic acid, 4E,7Z,10Z,13E,16Z-docosapentaenoic acid, 4Z,7E,10Z,13E,16Z-docosapentaenoic acid, 4Z,7Z,10E,13E,16Z-docosapentaenoic acid, 4E,7Z,10E,13Z,16Z-docosapentaenoic acid, 4Z,7E,1 0E,13Z,16Z-docosapentaenoic acid, 4E,7E,10Z,13Z,16Z-docosapentaenoic acid, 4E,7E,10E,13Z,16Z-docosapentaenoic acid, 4E,7E,10Z,13E,16Z-docosapentaenoic acid, 4E,7E,10Z,13E,16Z-docosapentaenoic acid, 4E,7E,10Z,13Z,16E-docosapentaenoic acid, 4E,7Z,10E,13E,16Z-docosapentaenoic acid, 4E,7Z ,10E,13Z,16E-docosapentaenoic acid, 4E,7Z,10Z,13E,16E-docosapentaenoic acid, 4Z,7E,10E,13E,16E-docosapentaenoic acid, 4Z,7E,10E,13Z,16E-docosapentaenoic acid, 4Z,7E,10Z,13E,16E-docosapentaenoic acid, 4Z,7Z,10E,13E,16E-docosapentaenoic acid, 4Z ,7E,10E,13E,16E-docosapentaenoic acid, 4E,7Z,10E,13E,16E-docosapentaenoic acid, 4E,7E,10Z,13E,16E-docosapentaenoic acid, 4E,7E,10E,13Z,16E-docosapentaenoic acid, 4E,7E,10E,13E,16Z-docosapentaenoic acid, 4E,7E,10E,13E,16E-docosapentaenoic acid. ,

[0122] When the content of thermally generated fatty acids in the microbial oil exceeds 3.0 wt%, it is not possible to effectively obtain a microbial oil containing the target LC-PUFA at a high content. Since the microbial oil containing thermally generated fatty acids is obtained through a heating process including distillation, the microbial oil can contain the thermally generated fatty acids at a content of 0.0001 wt% to 3.0 wt%, 0.001 wt% to 3.0 wt%, or 0.01 wt% to 3.0 wt%.

[0123] From the viewpoint of efficiently obtaining concentrated microbial oil containing a high content of target LC-PUFA using reverse phase column chromatography, the content of thermally generated fatty acids in the microbial oil can be set to 0.001% to 2.8% by weight, 0.01% to 2.8% by weight, 0.1% to 2.8% by weight, 0.1% to 2.5% by weight, 0.1% to 2.0% by weight, 0.1% to 1.5% by weight, 0.1% to 1.0% by weight, or 0.1% to 0.7% by weight of the total weight of the fatty acids in the oil.

[0124] Thermogeneous fatty acids are fatty acids with 16 to 20 carbon atoms that can be detected after the treatment of precision distillation and cannot be detected before the treatment. Therefore, for example, various chromatographic methods can be used to analyze and compare the fatty acid composition before and after the distillation treatment, and the fatty acid having a peak that appears after the treatment can be identified. As a chromatographic method, gas chromatography can be used in particular from the viewpoint of high analytical ability or detection sensitivity and relatively simple operation. In the case of specifying thermogenetic fatty acids with higher accuracy, for example, silver-ion solid phase extraction using silver ion chromatography can be used to analyze and identify the components from microbial biomass that overlap with the thermogenetic fatty acids.

[0125] For example, when the target LC-PUFA is dihomo-γ-linolenic acid (DGLA), the thermogenic fatty acid can be set to a thermogenic fatty acid with 20 carbon atoms. As a thermogenic fatty acid with 20 carbon atoms, for example, a thermogenic fatty acid with 20 carbon atoms and one or more fatty acids (hereinafter referred to as compound A) having a retention time as a peak appearing in the range of 1.001 to 1.011 when the retention time of dihomo-γ-linolenic acid ethyl ester (Dihomo-γ-linolenic acid) is set to 1 in gas chromatography analysis, and one or more fatty acids (hereinafter referred to as compound B) having a retention time as a peak appearing in the range of 1.013 to 1.027. Compound A and compound B can be a group of one or more compounds, or they can be single compounds respectively. As a thermogenic fatty acid, it can be either compound A or compound B, or both. The conditions of gas chromatography when compound A and compound B are specified as thermogenic fatty acids are as follows:

[0126] [Gas chromatography analysis conditions]

[0127] GC device: 6890N Network GC system (Agilent Technologies)

[0128] Column: DB-WAX (Agilent Technologies)

[0129] 30m×0.25mmID、0.25μm film thickness

[0130] Column temperature conditions: 60°C for 2.5 minutes → temperature rise 20°C / minute → 180°C → temperature rise 2°C / minute → 230°C for 15 minutes

[0131] Inlet temperature conditions: 210°C, no split, sampling time at split outlet 1.5 minutes, purge flow 40 mL / min

[0132] Injection volume conditions: 1 μL, sample concentration 1 mg / mL or less

[0133] Carrier gas conditions: helium, linear velocity 24 cm / min

[0134] Detector: FID

[0135] Detector temperature: 280°C

[0136] From the perspective of the purification efficiency of DGLA, the content of compound A and compound B in the microbial oil, which are thermally generated fatty acids when the target LC-PUFA is set to DGLA, can be set to 0.001% by weight to 2.8% by weight, 0.1% by weight to 2.8% by weight, 0.1% by weight to 2.5% by weight, 0.1% by weight to 2.0% by weight, 0.1% by weight to 1.5% by weight, 0.1% by weight to 1.0% by weight, or 0.1% by weight to 0.7% by weight of the total weight of the fatty acids in the oil.

[0137] The microbial oil of the present invention is preferably a composition having a low content of at least one specific fatty acid that should be separated from the target LC-PUFA by precision distillation. In this specification, unless otherwise specified, the fatty acid that should be separated from the target LC-PUFA in the refining process is referred to as the separation target fatty acid. There is no particular limitation on the separation target fatty acid as long as it is a fatty acid other than the target LC-PUFA, and there is no particular limitation on the form of the separation target fatty acid, which may be a fatty acid alkyl ester, a free fatty acid, etc.

[0138] As the separation target fatty acids, saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms can be listed. The content of saturated fatty acids with 22 carbon atoms and 24 carbon atoms in crude oil obtained from microbial biomass generally tends to be higher than that of fish oil or animal and vegetable oils. In addition, saturated fatty acids with 22 carbon atoms and 24 carbon atoms are high-melting-point long-chain fatty acids with a higher melting point than the target LC-PUFA. The reduction in the content of saturated fatty acids with 22 carbon atoms and 24 carbon atoms can suppress the clogging of the pipeline in the column chromatography treatment, and reverse phase column chromatography can be implemented. In addition, compared with the target LC-PUFA, the retention time of saturated fatty acids with 22 carbon atoms and 24 carbon atoms in reverse phase column chromatography is long, which can be the main reason for the increase in the time required for chromatography. Therefore, from the perspective of the refining efficiency per hour, it is also preferred to reduce the content of these saturated fatty acids.

[0139] When a saturated fatty acid with 22 carbon atoms and a saturated fatty acid with 24 carbon atoms are present separately, the total content of the saturated fatty acid with 22 carbon atoms and the saturated fatty acid with 24 carbon atoms is the total content of both of them. When only one of them is present, it refers to the content of only the one that exists.

[0140] From the viewpoint of suppressing the clogging of the pipe in the column chromatography method and the viewpoint of the purification efficiency of the target LC-PUFA, the total content of the saturated fatty acids with 22 carbon atoms and the saturated fatty acids with 24 carbon atoms in the microbial oil is more preferably 6.0% by weight or less, more preferably 1.8% by weight or less, and even more preferably 0.1% by weight or less of the total weight of the fatty acids in the oil. From the viewpoint of suppressing the clogging of the pipe in the column chromatography method and the viewpoint of the purification efficiency of the target LC-PUFA, the total content of the saturated fatty acids with 22 carbon atoms and the saturated fatty acids with 24 carbon atoms in the microbial oil is preferably 10 / 100 or less of the content of the target LC-PUFA, more preferably 3 / 100 or less, and even more preferably 0.1 / 100 or less. From the viewpoint of suppressing clogging of the pipeline in column chromatography and the viewpoint of the purification efficiency of the target LC-PUFA, the total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms in the microbial oil is preferably 6.0 wt% or less, more preferably 1.0 wt% or less, and even more preferably 0.1 wt% or less, relative to the total weight of the microbial oil.

[0141] In addition, from the viewpoint of suppressing the clogging of the pipeline in the column chromatography method and the viewpoint of the refining efficiency of the target LC-PUFA, the content of saturated fatty acids with 24 carbon atoms in the microbial oil is preferably 3.0% by weight or less of the total weight of the fatty acids in the oil, further preferably 1.0% by weight or less, and further preferably 0.1% by weight or less. From the viewpoint of suppressing the clogging of the pipeline in the column chromatography method and the viewpoint of the refining efficiency of the target LC-PUFA, the content of saturated fatty acids with 24 carbon atoms in the microbial oil is preferably 4 / 100 or less of the content of the target LC-PUFA, more preferably 1.4 / 100 or less, and further preferably 0.1 / 100 or less. From the viewpoint of suppressing the clogging of the pipeline in the column chromatography method and the viewpoint of the refining efficiency of the target LC-PUFA, the content of saturated fatty acids with 24 carbon atoms in the microbial oil is preferably 3.0% by weight or less, more preferably 1.0% by weight or less, and further preferably 0.1% by weight or less relative to the total weight of the microbial oil.

[0142] As other separation target fatty acids, saturated or unsaturated fatty acids can be cited, which, when using the separation number obtained by the carbon number and double bond number of the fatty acid as an index related to the separation using liquid chromatography, have a separation number of more than -2 and less than +2 compared to the separation number of the polyunsaturated fatty acid, and have a carbon number different from the carbon number of the polyunsaturated fatty acid. Hereinafter, such other separation target fatty acids are referred to as separation target fatty acids with a PN difference of more than -2 and less than +2.

[0143] When the PN of one of the two fatty acids being compared is 2 less than the other PN, that is, -2, 1 less than the other PN, the same number, that is, 0, 1 more than the other PN, that is, +1, or 2 more than the other PN, the difference in the elution time of the two fatty acids being compared cannot be said to be sufficient when separating them using liquid chromatography, and it can be considered that separation by liquid chromatography is difficult. Therefore, in terms of the purification efficiency of the target LC-PUFA with a high content, it is preferable to reduce the content of the separation target fatty acid having a PN difference of -2 or more and 2 or less.

[0144] The partition number (PN) is sometimes referred to as the equivalent carbon number (ECN). The partition number is an index empirically obtained from the definition of the separation factor that affects the elution time in the analysis of molecular species by reversed-phase high performance liquid chromatography, and is represented by the following formula (I).

[0145] PN = [number of carbon atoms] - 2 x [number of double bonds] (I)

[0146] In formula (I), the number of carbon atoms refers to the number of carbon atoms of the fatty acid. However, in the present invention, the number of carbon atoms in formula (I) refers to the number of carbon atoms of the fatty acid in the case of free fatty acid form, which is an integer inherent to each fatty acid. In this specification, the separation number is referred to as PN.

[0147] For example, in the case of DGLA, that is, C20:3, PN=20-2×3=14.

[0148] The separation target fatty acid having a PN difference of -2 or more and 2 or less is a saturated or unsaturated fatty acid having a carbon number different from that of the target LC-PUFA, that is, a carbon number greater or less than that of the target LC-PUFA, and can be, for example, a saturated or unsaturated fatty acid having a carbon number less than that of the target LC-PUFA. The separation target fatty acid having a PN difference of -2 or more and 2 or less can be at least one selected from the group consisting of a saturated fatty acid having 18 carbon atoms, a monounsaturated fatty acid having 18 carbon atoms, a diunsaturated fatty acid having 18 carbon atoms, a triunsaturated fatty acid having 18 carbon atoms, and a tetraunsaturated fatty acid having 18 carbon atoms.

[0149] Examples of the combination of the target LC-PUFA in the microbial oil and the separation target fatty acid include the following combinations.

[0150] [Table 1]

[0151]

[0152] From the viewpoint of effectively obtaining a high content of the target LC-PUFA, the total content of the separated target fatty acids having a PN difference of -2 or more and 2 or less in the microbial oil for separating the target fatty acids is, for example, more preferably 10.0% by weight or less of the total weight of the fatty acids in the oil, further preferably 4.0% by weight or less, and further more preferably 0.7% by weight or less. In the microbial oil, from the viewpoint of effectively obtaining the target LC-PUFA, the total content of the separated target fatty acids having a PN difference of -2 or more and 2 or less is preferably 15 / 100 or less of the content of the target LC-PUFA, more preferably 5 / 100 or less, and further preferably 1 / 100 or less. In the microbial oil, from the viewpoint of effectively obtaining the target LC-PUFA, the total content of the separated target fatty acids having a PN difference of -2 or more and 2 or less is preferably 10.0% by weight or less, more preferably 4.0% by weight or less, and further more preferably 0.7% by weight or less relative to the total weight of the microbial oil.

[0153] For example, when the target LC-PUFA is a PN16 fatty acid, i.e., eicosadienoic acid, the total content of separated target fatty acids having a PN difference of -2 or more and 2 or less, such as C18:0 and C18:1 in the microbial oil, is more preferably 10.0 wt% or less, further preferably 4.0 wt% or less, and further preferably 0.7 wt% or less of the total weight of the fatty acids in the oil; in addition, it is preferably 15 / 100 or less, more preferably 5 / 100 or less, and further preferably 1 / 100 or less of the content of the target LC-PUFA; in addition, relative to the total weight of the microbial oil, it is preferably 10.0 wt% or less, more preferably 4.0 wt% or less, and further preferably 0.7 wt% or less.

[0154] When the target LC-PUFA is a PN14 fatty acid, i.e., DGLA, eicosatrienoic acid or docosatetraenoic acid, the total content of separated target fatty acids having a PN difference of -2 or more and 2 or less, such as C18:1, C18:2, etc. in the microbial oil is more preferably 10.0 wt% or less, further preferably 4.0 wt% or less, and further more preferably 0.7 wt% or less of the total weight of the fatty acids in the oil; in addition, it is preferably 15 / 100 or less, further preferably 5 / 100 or less, and further preferably 1 / 100 or less of the content of the target LC-PUFA; in addition, relative to the total weight of the microbial oil, it is preferably 10.0 wt% or less, more preferably 4.0 wt% or less, and further more preferably 0.7 wt% or less.

[0155] When the target LC-PUFA is a PN12 fatty acid, i.e., eicosatetraenoic acid, arachidonic acid, or docosapentaenoic acid, the total content of separated target fatty acids having a PN difference of -2 or more and 2 or less, such as C18:2 and C18:3 in the microbial oil is more preferably 10.0 wt% or less, further preferably 4.0 wt% or less, and further more preferably 0.7 wt% or less of the total weight of the fatty acids in the oil; in addition, it is preferably 15 / 100 or less, more preferably 5 / 100 or less, and further preferably 1 / 100 or less of the content of the target LC-PUFA; in addition, relative to the total weight of the microbial oil, it is preferably 10.0 wt% or less, more preferably 4.0 wt% or less, and further more preferably 0.7 wt% or less.

[0156] When the target LC-PUFA is a PN10 fatty acid, i.e., eicosapentaenoic acid or docosahexaenoic acid, the total content of separated target fatty acids having a PN difference of -2 or more and 2 or less, such as C18:3 and C18:4 in the microbial oil is more preferably 10.0 wt% or less, further preferably 4.0 wt% or less, and further more preferably 0.7 wt% or less of the total weight of the fatty acids in the oil; in addition, it is preferably 15 / 100 or less, more preferably 5 / 100 or less, and further preferably 1 / 100 or less of the content of the target LC-PUFA; in addition, relative to the total weight of the microbial oil, it is preferably 10.0 wt% or less, more preferably 4.0 wt% or less, and further more preferably 0.7 wt% or less.

[0157] In addition, from the viewpoint of efficiently obtaining a high content of target LC-PUFA such as eicosadienoic acid, DGLA, eicosatrienoic acid, and eicosatetraenoic acid by reverse phase column chromatography, it is preferred that the content of monounsaturated fatty acids having 18 carbon atoms in the microbial oil is low. When eicosadienoic acid, DGLA, eicosatrienoic acid, or eicosatetraenoic acid is used as the target LC-PUFA, the PN of the monounsaturated fatty acid having 18 carbon atoms is 2 more than that of the target LC-PUFA. For example, in the microbial oil, from the viewpoint of the purification efficiency of the target LC-PUFA, the content of monounsaturated fatty acids having 18 carbon atoms is more preferably 7.0% by weight or less, further preferably 1.5% by weight or less, and further more preferably 0.4% by weight or less, based on the total weight of the fatty acids in the oil. In the microbial oil, from the viewpoint of the refining efficiency of the target LC-PUFA, the content of monounsaturated fatty acids with 18 carbon atoms is preferably 10 / 100 or less of the content of the target LC-PUFA, more preferably 2 / 100 or less, and further preferably 0.5 / 100 or less. In the microbial oil, from the viewpoint of the refining efficiency of the target LC-PUFA, the content of monounsaturated fatty acids with 18 carbon atoms is preferably 7.0% by weight or less, more preferably 1.5% by weight or less, and further preferably 0.4% by weight or less relative to the total weight of the microbial oil.

[0158] In addition, from the viewpoint of efficiently obtaining a high content of target LC-PUFA such as DGLA, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, docosatetraenoic acid, and docosapentaenoic acid by reverse phase column chromatography, it is preferred that the content of diunsaturated fatty acids having 18 carbon atoms in the microbial oil is particularly low. When DGLA, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, docosatetraenoic acid, or docosapentaenoic acid is set as the target LC-PUFA, the PN of the diunsaturated fatty acid having 18 carbon atoms is equal to that of the target LC-PUFA. For example, in the microbial oil, from the viewpoint of the purification efficiency of the target LC-PUFA, the content of diunsaturated fatty acids having 18 carbon atoms is more preferably 5.0% by weight or less, more preferably 0.7% by weight or less, and even more preferably 0.4% by weight or less, based on the total weight of the fatty acids in the oil. In the microbial oil, from the viewpoint of the purification efficiency of the target LC-PUFA, the content of diunsaturated fatty acids having 18 carbon atoms is preferably 7 / 100 or less of the content of the target LC-PUFA, more preferably 1 / 100 or less, and further preferably 0.5 / 100 or less. In the microbial oil, the content of diunsaturated fatty acids having 18 carbon atoms is preferably 5.0% by weight or less, more preferably 0.7% by weight or less, and further preferably 0.4% by weight or less relative to the total weight of the microbial oil.

[0159] In addition, from the viewpoint of efficiently obtaining a high content of target LC-PUFA such as DGLA, eicosatrienoic acid, and docosatetraenoic acid by reverse phase column chromatography, it is preferred that the content of triunsaturated fatty acids having 18 carbon atoms is low in the microbial oil. When DGLA, eicosatrienoic acid, or docosatetraenoic acid is used as the target LC-PUFA, the PN of the triunsaturated fatty acid having 18 carbon atoms is 2 less than that of the target LC-PUFA. For example, in the microbial oil, from the viewpoint of the purification efficiency of the target LC-PUFA, the content of triunsaturated fatty acids having 18 carbon atoms is more preferably 7.0% by weight or less, further preferably 1.5% by weight or less, and further more preferably 0.4% by weight or less, based on the total weight of the fatty acids in the oil. In the microbial oil, from the viewpoint of the refining efficiency of the target LC-PUFA, the content of triunsaturated fatty acids having 18 carbon atoms is preferably 10 / 100 or less of the content of the target LC-PUFA, more preferably 2 / 100 or less, and further preferably 0.5 / 100 or less. In the microbial oil, from the viewpoint of the refining efficiency of the target LC-PUFA, the content of triunsaturated fatty acids having 18 carbon atoms is preferably 7.0% by weight or less, more preferably 1.5% by weight or less, and further preferably 0.4% by weight or less relative to the total weight of the microbial oil.

[0160] From the viewpoint of effectively obtaining a high content of target LC-PUFAs such as DGLA, eicosatrienoic acid, and docosatetraenoic acid by reverse phase column chromatography, it is preferred that the total content of monounsaturated fatty acids having 18 carbon atoms and diunsaturated fatty acids having 18 carbon atoms in the microbial oil is low. For example, in the microbial oil, from the viewpoint of effectively obtaining the target LC-PUFA, the total content of monounsaturated fatty acids having 18 carbon atoms and diunsaturated fatty acids having 18 carbon atoms is more preferably 10.0% by weight or less of the total weight of the fatty acids in the oil, further preferably 4.0% by weight or less, and further preferably 0.7% by weight or less. In the microbial oil, from the viewpoint of effectively obtaining the target LC-PUFA, the total content of monounsaturated fatty acids having 18 carbon atoms and diunsaturated fatty acids having 18 carbon atoms is preferably 15 / 100 or less of the content of the target LC-PUFA, more preferably 5 / 100 or less, and further preferably 1 / 100 or less. In the microbial oil, from the viewpoint of effectively obtaining the target LC-PUFA, the total content of the monounsaturated fatty acid having 18 carbon atoms and the diunsaturated fatty acid having 18 carbon atoms is preferably 10.0 wt % or less, more preferably 4.0 wt % or less, and even more preferably 0.7 wt % or less, relative to the total weight of the microbial oil.

[0161] From the viewpoint of the melting point of the microbial oil, the ease of crystallization, and the time productivity in the column chromatography, the microbial oil of the present invention preferably has a low content of saturated fatty acids with 18 carbon atoms. In addition, when eicosadienoic acid is set as the target LC-PUFA, saturated fatty acids with 18 carbon atoms also belong to the separation target fatty acids with a PN difference of more than -2 and less than 2. From the viewpoint of the melting point of the microbial oil, the ease of crystallization, and the time productivity in the column chromatography, in the microbial oil, the content of saturated fatty acids with 18 carbon atoms is more preferably less than 7.0% by weight of the total weight of fatty acids in the oil, further preferably less than 3.0% by weight, and further more preferably less than 1.5% by weight. In the microbial oil, the content of saturated fatty acids with 18 carbon atoms is preferably less than 11 / 100 of the content of the target LC-PUFA, more preferably less than 4 / 100, and further preferably less than 2 / 100. From the viewpoint of the melting point of the microbial oil, the ease of crystallization, and the time productivity in column chromatography, the content of saturated fatty acids having 18 carbon atoms in the microbial oil is preferably 7.0 wt % or less, more preferably 3.0 wt % or less, and even more preferably 1.5 wt % or less, relative to the total weight of the microbial oil.

[0162] The above-mentioned various contents of the separation target fatty acids are respectively independent embodiments, and therefore a preferred embodiment of the microbial oil may be an embodiment combining two or more arbitrary preferred contents of the separation target fatty acids.

[0163] When the target LC-PUFA in the microbial oil is set to eicosadienoic acid, DGLA, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid or docosahexaenoic acid, the total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms in the above-mentioned microbial oil includes a preferred range, and can be set to the same range as the above-mentioned range, or can be a range in which they are arbitrarily combined, and the corresponding content of monounsaturated fatty acids with 18 carbon atoms, the content of diunsaturated fatty acids with 18 carbon atoms, the total content of monounsaturated fatty acids with 18 carbon atoms and diunsaturated fatty acids with 18 carbon atoms, and the content of saturated fatty acids with 18 carbon atoms can also be set to arbitrarily combined contents.

[0164] From the viewpoint of the processing efficiency by reverse phase column chromatography or the heat resistance of the filler, the melting point of the microbial oil is preferably 40° C. or less, preferably 30° C. or less. The melting point of the microbial oil is set to the transparent melting point measured by the method described in the Standard Oil and Fats Analysis Test Method 2013 Edition 3.2.2.1-2013 established by the Japan Oil Chemistry Society (JOCS).

[0165] (2) Method for producing microbial oil

[0166] The methods for producing microbial oil according to other aspects of the present invention all include purification by precise distillation, and obtaining a specific microbial oil after precise distillation.

[0167] That is, the method for producing the first microorganism in another embodiment of the present invention includes: a feedstock oil supply step of preparing a feedstock oil containing a target LC-PUFA obtained from microbial biomass; and a first precision distillation step of refining the feedstock oil by precision distillation according to conditions including a bottom temperature of 160°C to 230°C and a minimum pressure in a distillation tower of 0.1Pa to 30Pa. After the first precision distillation step, a microbial oil containing a specific polyunsaturated fatty acid can be obtained. Here, the specific polyunsaturated fatty acid obtained includes the specific microbial oil in one embodiment of the present invention, but is not limited thereto.

[0168] The method for producing the second microbial oil in a further embodiment of the present invention comprises: the feedstock oil supply step; a second precision distillation step of performing precision distillation on the feedstock oil using a distillation tower containing a regular filler according to conditions including a tower bottom temperature of 160°C to 230°C and a minimum pressure in the distillation tower of 0.1Pa to 30Pa; and a microbial oil recovery step of obtaining the specific microbial oil in one embodiment of the present invention.

[0169] The third method for producing microbial oil in a further embodiment of the present invention comprises: the raw oil supply step; a third precision distillation step of performing precision distillation on the raw oil using a distillation tower containing a regular filler, according to conditions including a tower bottom temperature corresponding to the type of the polyunsaturated fatty acid as a target and a minimum pressure in the distillation tower, so as to obtain microbial oil containing thermally generated fatty acids having a content of less than 3.0 weight % of the total weight of fatty acids in the oil; and the microbial oil recovery step.

[0170] In the feedstock oil supply step in the first to third production methods, the feedstock oil is obtained through the following steps: a step of culturing a known microorganism that is a lipid-producing microorganism that can produce the target LC-PUFA in a culture medium to obtain a microbial biomass containing fatty acids; a crude oil separation step of separating a crude oil that is a mixture of fatty acids from the obtained microbial biomass; a triacylglycerol concentrate production step of obtaining a triacylglycerol concentrate by performing a degumming step, a deacidification step, a decolorization step, and a deodorization step on the crude oil to remove substances other than the target substances such as phospholipids and sterols; and a processing step of performing processes such as hydrolysis and alkyl esterification on the triacylglycerol concentrate.

[0171] As lipid-producing microorganisms, the above-mentioned microorganisms can be cited. In addition, the cultivation of lipid-producing microorganisms can be carried out under conditions known to those skilled in the art. For example, when the target LC-PUFA is set to DGLA, DGLA from microorganisms described in Japanese Patent Laid-Open No. 5-091887 can be cited.

[0172] Japanese Patent Application Laid-Open No. 5-091887 discloses a method for producing DGLA by culturing a mutant strain of Mortierella alpina SAM1860 (Micro-industry Research Institute No. 3589) having reduced or missing Δ5 unsaturase activity in the presence of a Δ5 unsaturase inhibitor. Examples of the Δ5 unsaturase inhibitor include 2-amino-N-(3-chlorophenyl)benzamide, dioxabicyclo[3.3.0]octane derivatives, piperonyl butoxide, and curcumin. Among them, examples of dioxabicyclo[3.3.0]octane derivatives include sesamin, sesaminol, episesamin, episesaminol, sesamolin, 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, 2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, and 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane.

[0173] There is no particular limitation on the culture vessel used in the culture. Generally, any device used in the culture of microorganisms can be used. For example, a culture vessel capable of liquid culture on a scale of 1L to 50L can be appropriately selected according to the scale of the culture. For example, when liquid culture is performed on a scale of 1L to 50L, in order to obtain the target LC-PUFA at a higher concentration, a stirring culture vessel is preferably used as the culture vessel. As the stirring culture vessel, a stirring culture vessel having at least one turbine disc type stirring wing is preferred, and a stirring culture vessel having two turbine disc type stirring wing is more preferred.

[0174] In the crude oil separation step, the crude oil containing the lipid produced in the production step is separated from the microbial cells. The separation of the microbial cells and the collection of the crude oil can be carried out using a separation method and an extraction method according to the culture form.

[0175] When a liquid culture medium is used, the cultured bacterial cells are obtained by conventional solid-liquid separation methods such as centrifugation and filtration. When culture is carried out in a solid culture medium, the bacterial cells and the culture medium are not separated, and the solid culture medium and the bacterial cells can be crushed with a homogenizer or the like, and crude oil can be directly taken from the resulting crushed product.

[0176] The extraction of crude oil may include extracting the separated dried cells preferably with supercritical carbon dioxide or with an organic solvent under a nitrogen stream. As organic solvents, ethers such as dimethyl ether and diethyl ether; hydrocarbons with a carbon number of 10 or less such as petroleum ether, hexane, and heptane; alcohols such as methanol and ethanol; chloroform; methylene chloride; fatty acids such as octanic acid or their alkyl esters; fats and oils such as vegetable oils, etc. may be used. In addition, good extraction results may be obtained by using alternating extractions with methanol and petroleum ether or extractions with a solvent of a one-layer system of chloroform-methanol-water. By distilling off the organic solvent from the extract under reduced pressure, a crude oil containing a high concentration of fatty acids may be obtained. When extracting triglycerides, hexane is most commonly used.

[0177] In addition, the above method can be replaced by using wet cells for extraction. The crude oil from the wet cells can be extracted by squeezing the wet cells, or a mixed solvent consisting of a solvent that is miscible with water, such as methanol or ethanol, or a solvent that is miscible with water and water and / or other solvents can be used. The other steps are the same as above.

[0178] In the triglyceride concentrate production step, the crude oil collected is degummed, deacidified, decolorized, and deodorized by a method known to those skilled in the art by a method used for refining vegetable oils, fish oils, etc. For example, the degumming treatment is carried out by a water washing treatment, the deacidification treatment is carried out by a distillation treatment, the decolorization treatment is carried out by a decolorization treatment using activated clay, activated carbon, silica gel, etc., and the deodorization treatment is carried out by steam distillation.

[0179] In the processing step, the triacylglycerol concentrate is subjected to processing treatments such as esterification treatment using a catalyst and hydrolysis treatment. The alkyl esterification treatment and hydrolysis treatment can be carried out under conditions known to those skilled in the art.

[0180] For example, to obtain fatty acid methyl ester, a triglyceride concentrate is treated with anhydrous methanol-hydrochloric acid 5% to 10%, BF3-methanol 10% to 50%, etc. at room temperature for 1 to 24 hours. To obtain fatty acid ethyl ester, fats and oils are treated with 1% to 20% sulfuric acid ethanol at 25°C to 100°C for 15 minutes to 60 minutes. Methyl ester or ethyl ester can be extracted from the reaction solution with an organic solvent such as hexane, diethyl ether, ethyl acetate, etc. By drying the extract with anhydrous sodium sulfate, etc. and distilling off the organic solvent, a composition containing fatty acid alkyl ester as the main component can be obtained.

[0181] The first to third microbial oil production methods include first to third precision distillation steps of performing precision distillation according to specific conditions on the raw material oil obtained in the raw material oil supply step, respectively. By performing the first to third precision distillation steps, in the microbial oil production method of the present invention, a specific microbial oil as a target, for example, a desired microbial oil containing a target LC-PUFA can be effectively obtained.

[0182] In the first precision distillation step in the first method for producing microbial oil, the feedstock oil is refined by precision distillation under conditions including a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa. As long as the precision distillation is performed under the tower bottom temperature and the minimum pressure in the distillation tower within this range, the desired specific unsaturated fatty acids, such as target LC-PUFA, can be obtained with good accuracy and efficiency.

[0183] Precision distillation is a technology that returns a portion of the vapor generated under heating conditions to the distillation tower as reflux liquid, and utilizes the gas-liquid equilibrium between the vapor rising in the tower and the liquid sample to separate the components with high precision. This technology is well known to those skilled in the art.

[0184] The tower bottom temperature refers to the temperature of the sample at the bottom of the distillation tower. When the tower bottom temperature is lower than 160°C, fatty acids other than the target fatty acids, such as unsaturated fatty acids with 18 carbon atoms, other than the target LC-PUFA cannot be fully separated. On the other hand, when the tower bottom temperature exceeds 230°C, the content of fatty acids and the like generated by heat increases even with precise distillation, and there is a tendency that microbial oil containing a high content of the target LC-PUFA cannot be effectively obtained. From the viewpoint of separation efficiency, the tower bottom temperature is more preferably 160°C to 210°C, and further preferably 160°C to 200°C.

[0185] The temperature at the top of the tower is not particularly limited, and can be, for example, 80°C to 160°C, more preferably 90°C to 140°C.

[0186] The minimum pressure in a distillation tower generally corresponds to the pressure at the top of the distillation tower. In the case of a general distillation tower equipped with a condenser and a vacuum pump at the top of the tower, the pressure from the condenser at the top of the tower that liquefies the rising vapor, that is, the fraction, to the vacuum pump shows the lowest pressure in the distillation tower. When the minimum pressure in the distillation tower is higher than 30 Pa, the bottom temperature rises in order to generate the vapor required for precision distillation, and as a result, there is a tendency for the content of thermally generated fatty acids to increase. In addition, generally speaking, pressure loss is generated due to the packing or piping contained in the distillation tower, so the minimum pressure in the distillation tower can be set to 0.1 Pa or less. From the viewpoint of suppressing the generation of thermally generated fatty acids, the minimum pressure in the distillation tower is more preferably 0.1 Pa to 20 Pa.

[0187] In the second precision distillation step in the second method for producing microbial oil, a distillation tower containing a regular filler is used to perform precision distillation with respect to the raw oil under conditions including a tower bottom temperature of 160°C to 230°C and a minimum pressure in the distillation tower of 0.1Pa to 30Pa. In the second precision distillation step, precision distillation is performed using a distillation tower containing a regular filler, so that gas-liquid exchange can be achieved with very little pressure loss, thereby allowing precision distillation to be performed relatively smoothly under conditions including the same tower bottom temperature and minimum pressure in the distillation tower. By easing the heating conditions relative to the raw oil through this relatively smooth precision distillation, the generation of thermally generated fatty acids is effectively suppressed, and microbial oil containing the target LC-PUFA at a high content rate can be more effectively obtained.

[0188] Structured packing is a well-known packing suitable for distillation, and is formed by multiple layers that are interconnected in a regularly repeated geometric relationship. The structured packing is not particularly limited in material as long as it has a unique repeated shape, and can be made of metal such as stainless steel, aluminum, nickel, copper, Hastelloy, Monel alloy, etc.; resin such as polypropylene; ceramic; carbon such as carbon steel, carbon fiber, etc., and can be appropriately selected according to the heating conditions and pressure conditions of distillation.

[0189] From the viewpoint of effectively suppressing the production of thermally generated fatty acids and more effectively obtaining microbial oil containing the target LC-PUFA at a high content rate, the specific surface area per unit of the regular filler is preferably 125 m 2 / m 3 ~1700m 2 / m 3 , preferably 125m 2 / m3~900m 2 / m 3 , more preferably 200m 2 / m3 ~800m 2 / m 3 .

[0190] Preferred regular packings include, for example, Mellapak, Mellapak Plus, plastic Mellapak, Mellagrid, BX / CY packing, BX Plus, plastic BX (Gauze packing), mellacarbon, DX / EX packing, Meladeul, Sulzer·LabPacking EX, Nutter grid, and Kuehne Rombopak manufactured by Sulzer Chemtech.

[0191] The tower bottom temperature in the second precision distillation refers to the temperature of the bottom of the distillation tower. When the tower bottom temperature is lower than 160°C, fatty acids other than the target LC-PUFA such as unsaturated fatty acids with 18 carbon atoms cannot be fully separated. On the other hand, when the tower bottom temperature exceeds 230°C, even with precision distillation, the content of thermally generated fatty acids and the like increases, and microbial oil containing a high content of the target LC-PUFA cannot be effectively obtained. From the viewpoint of separation efficiency, the tower bottom temperature is more preferably 160°C to 210°C, and further preferably 160°C to 200°C.

[0192] The temperature at the top of the column in the second precise distillation is not particularly limited, and can be, for example, 80°C to 160°C, more preferably 90°C to 140°C.

[0193] The minimum pressure in the distillation tower in the second precision distillation generally corresponds to the pressure at the top of the distillation tower. In the case of a general distillation tower equipped with a condenser (condenser) and a vacuum pump at the top of the tower, the pressure from the condenser at the top of the tower that liquefies the rising vapor, that is, the fraction, to the vacuum pump shows the lowest pressure in the distillation tower. When the minimum pressure in the distillation tower is higher than 30 Pa, the bottom temperature rises in order to generate the vapor required for precision distillation, and as a result, there is a tendency for the content of thermally generated fatty acids to increase. In addition, in general, pressure loss is generated due to the packing or piping contained in the distillation tower, so the minimum pressure in the distillation tower can be set to 0.1 Pa or less. From the viewpoint of suppressing the generation of thermally generated fatty acids, the minimum pressure in the distillation tower is more preferably 0.1 Pa to 20 Pa.

[0194] In the third precision distillation step in the third method for producing microbial oil, a distillation tower containing a regular filler is used relative to the raw oil, and precision distillation is performed according to the conditions that the bottom temperature of the tower and the minimum pressure in the distillation tower corresponding to the type of the polyunsaturated fatty acid as the target can obtain a microbial oil containing a content of 3.0 weight % or less of the total weight of the fatty acids in the oil. In the third precision distillation, the bottom temperature of the tower and the minimum pressure in the distillation tower corresponding to the type of the target LC-PUFA meet the conditions that the microbial oil containing a content of 3.0 weight % or less of the total weight of the fatty acids in the oil can be obtained. The bottom temperature of the tower and the minimum pressure in the distillation tower corresponding to the type of the target LC-PUFA can be optimized based on the content of the thermally generated fatty acids, and those skilled in the art can appropriately set them according to the type, size, shape, etc. of the distillation tower used, the type of regular filler contained in the distillation tower, the filling height, and other conditions.

[0195] From the viewpoint of suppressing the purification efficiency of the target LC-PUFA and the production of thermally generated fatty acids, the third precision distillation preferably has a bottom temperature of 160°C to 230°C and a minimum pressure in the distillation tower of 0.1Pa to 30Pa. The bottom temperature in the third precision distillation is more preferably 160°C to 210°C, and even more preferably 160°C to 200°C. From the viewpoint of suppressing the production of thermally generated fatty acids, the minimum pressure in the distillation tower in the third precision distillation is even more preferably 0.1Pa to 20Pa. The minimum pressure in the distillation tower in the third precision distillation generally corresponds to the pressure at the top of the distillation tower. In the case of a general distillation tower equipped with a condenser (condenser) and a vacuum pump at the top of the tower, the pressure from the condenser at the top of the tower that liquefies the rising vapor (fraction) to the vacuum pump shows the lowest pressure in the distillation tower. The temperature at the top of the tower is not particularly limited, and can be, for example, 80°C to 160°C, and more preferably 90°C to 140°C.

[0196] In addition, the conditions of the precision distillation in the first to third precision distillation steps are not limited to the above. For example, when distillation is used, as a distillation step, in terms of suppressing the modification of oil due to heat and improving the distillation efficiency, it is preferred to perform distillation under the conditions of reducing the pressure of the upper part of the distillation tower to 10 mmHg (1333 Pa) or less and setting the tower bottom temperature to 165°C to 210°C, preferably 170°C to 195°C. The lower the pressure of the upper part of the distillation tower, the better, and it is more preferably 0.1 mmHg (13.33 Pa) or less. There is no particular limitation on the temperature of the upper part of the tower, and for example, it can be set to 160°C or less.

[0197] In addition, each of the first to third precision distillation steps may include multiple precision distillations under different conditions of the column bottom temperature and the minimum pressure in the distillation column. Thus, different separation target fatty acids can be effectively separated in each precision distillation. As the conditions of different column bottom temperatures and minimum pressures in the distillation column, for example, a combination of two or more stages of precision distillations with different column bottom temperatures can be cited.

[0198] For example, as a combination of precision distillation steps based on different bottom temperatures and minimum pressures in the distillation tower, the first to third precision distillation steps may include: a low-temperature precision distillation step with a bottom temperature of 160°C to 220°C and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa; and a high-temperature precision distillation step with a bottom temperature of 170°C to 230°C and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa.

[0199] By performing the low-temperature precision distillation step, fatty acid components with a relatively small molecular weight compared to the target LC-PUFA, such as fatty acid components with a carbon number of 18 or less, can be removed as a primary fraction, and a microbial oil containing the target LC-PUFA can be obtained as a residue. The tower bottom temperature in the low-temperature precision distillation step is preferably 160° C. to 200° C., more preferably 160° C. to 190° C.

[0200] By going through a high-temperature precision distillation process, the content of at least one of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms, which may cause pipe clogging, can be reduced, and pipe clogging can be suppressed during purification using reverse phase column chromatography. As a result, a microbial oil containing the target LC-PUFA at a high content can be effectively obtained. The content of saturated fatty acids with 22 or 24 carbon atoms in the residue in the high-temperature precision distillation process is increased compared to the low-temperature precision distillation process. From the viewpoint of removing saturated fatty acids with 22 or 24 carbon atoms whose content has increased and suppressing the formation of thermally generated fatty acids, the tower bottom temperature in the high-temperature precision distillation process is preferably 170°C to 210°C.

[0201] Regarding the temperature difference between the bottom temperature of the low-temperature precision distillation step and the high-temperature precision distillation step, from the viewpoint of the necessity of generating vapor from the residue with a high content of saturated fatty acids with 22 carbon atoms and 24 carbon atoms in the high-temperature precision distillation and suppressing the thermal generation of fatty acids, the bottom temperature of the high-temperature precision distillation step is preferably 3°C to 20°C higher than the bottom temperature of the low-temperature precision distillation step, and more preferably 3°C to 10°C higher.

[0202] In either the low-temperature precision distillation step or the high-temperature precision distillation step, from the viewpoint of suppressing the generation of thermally generated fatty acids, the minimum pressure in the distillation column is more preferably 0.1 Pa to 20 Pa, and even more preferably 0.1 Pa to 10 Pa. The temperature at the top of the column is not particularly limited, and can be, for example, 160° C. or less.

[0203] The appropriate heating time can be set by a person skilled in the art according to the charge amount of the raw material composition for distillation and the description of the Examples in this specification.

[0204] Either the low-temperature precision distillation step or the high-temperature precision distillation step may be performed first. For example, by performing the high-temperature precision distillation step after the low-temperature precision distillation step, fatty acid components having a molecular weight larger than the target LC-PUFA as a residue can be removed, and a microbial oil can be obtained as a fraction from which both fatty acid components having a molecular weight smaller than the target LC-PUFA and fatty acid fragments having a molecular weight larger than the target LC-PUFA are removed.

[0205] In the microbial recovery step in the second and third precision distillation steps, a microbial oil containing the target LC-PUFA at a high content rate as a fraction obtained by the precision distillation step can be recovered. This microbial oil is a concentrated microbial oil of the target LC-PUFA, and is useful for efficiently obtaining the target LC-PUFA in the form of free fatty acids and / or their alkyl esters using reverse phase column chromatography.

[0206] (3) Concentrated microbial oil

[0207] As one embodiment of the concentrated microbial oil of the present invention, the content of its target LC-PUFA is 90% to 98% by weight of the total weight of fatty acids in the oil, the content of thermally generated fatty acids is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil, the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is less than 1.0% by weight of the total weight of fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms is less than 5.0% by weight of the total weight of fatty acids in the oil.

[0208] For example, as an example of concentrated microbial oil, its DGLA content is 90% to 98% by weight of the total weight of fatty acids in the oil, the content of thermally generated fatty acids is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil, the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is less than 1.0% by weight of the total weight of fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms is less than 5.0% by weight of the total weight of fatty acids in the oil.

[0209] As the concentrated microbial oil, it is preferred that the content of the target LC-PUFA is 90% to 98% by weight, 95% to 98% by weight, 96% to 98% by weight, or 97% to 98% by weight of the total weight of the fatty acids in the oil, and the content of the thermally generated fatty acids is 0.01% to 3.0% by weight, 0.1% to 3.0% by weight, 0.1% to 2.8% by weight, 0.1% to 2.5% by weight, 0.1% to 3.0% by weight, 0.1% to 2.8% by weight, 0.1% to 2.5% by weight, or 0.1% to 3.0% by weight of the total weight of the fatty acids in the oil. 2.0 wt %, 0.1 wt % to 1.5 wt %, 0.1 wt % to 1.0 wt %, or 0.1 wt % to 0.7 wt %, the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is 1.0 wt % or less, 0.2 wt % or less, or 0 wt % of the total weight of the fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms is 5.0 wt % or less, 2.0 wt % or less, or 0 wt % of the total weight of the fatty acids in the oil.

[0210] In addition, as other preferred concentrated microbial oils, the content of DGLA is preferably 90% to 98% by weight, 95% to 98% by weight, 96% to 98% by weight, or 97% to 98% by weight of the total weight of the fatty acids in the oil, the content of thermally generated fatty acids is 0.1% to 3.0% by weight, 0.1% to 2.8% by weight, 0.1% to 2.5% by weight, 0.1% to 2.0% by weight, 0.1% to 1.5% by weight, 0.1% to 1.0% by weight, or 0.1% to 0.7% by weight of the total weight of the fatty acids in the oil, the total content of saturated fatty acids having 24 carbon atoms and saturated fatty acids having 22 carbon atoms is 1.0% or less, 0.2% or less, or 0% by weight of the total weight of the fatty acids in the oil, and the content of monounsaturated fatty acids having 18 carbon atoms is 5.0% or less, 2.0% or less, or 0% by weight of the total weight of the fatty acids in the oil.

[0211] In addition, as other preferred concentrated microbial oils, it is preferred that the content of eicosadienoic acid, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid, or docosahexaenoic acid is 90% to 98% by weight, 95% to 98% by weight, 96% to 98% by weight, or 97% to 98% by weight of the total weight of the fatty acids in the oil, and the content of the thermally generated fatty acids is 0.1% to 3.0% by weight, 0.1% to 2.8% by weight, or 0.1% to 98% by weight of the total weight of the fatty acids in the oil. % to 2.5% by weight, 0.1% to 2.0% by weight, 0.1% to 1.5% by weight, 0.1% to 1.0% by weight, or 0.1% to 0.7% by weight, the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is 1.0% by weight or less, 0.2% by weight or less, or 0% by weight of the total weight of the fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms (C18:1) is 5.0% by weight or less, 2.0% by weight or less, or 0% by weight of the total weight of the fatty acids in the oil.

[0212] These are concentrated in microbial oil and contain target LC-PUFAs, such as eicosadienoic acid, DGLA, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid, or docosahexaenoic acid, at a high content rate. Therefore, they are very useful for applying target LC-PUFAs, such as DGLA, to the required uses at a high content rate and with good productivity.

[0213] (4) Method for producing concentrated microbial oil

[0214] A method for producing concentrated microbial oil according to one embodiment of the present invention comprises: obtaining a microbial oil containing a target LC-PUFA by any of the above-mentioned production methods; and concentrating the obtained microbial oil using reverse phase column chromatography.

[0215] In the microbial oil obtained by the method for producing microbial oil according to the present invention, the content of target LC-PUFA is high, and the content of fatty acids that are difficult to separate from the target LC-PUFA by reverse phase column chromatography is low, so the target LC-PUFA can be obtained sufficiently and efficiently at a high content.

[0216] As the reverse phase column chromatography used in the concentration treatment, the reverse phase column chromatography known in the industry can be cited, and particularly preferably, the high performance liquid chromatography (HPLC) using a substrate modified with octadecylsilyl (ODS) as the stationary phase can be cited. As the reverse phase distribution column, for example, the YMC Pack ODS-AQ-HG column (YMC Co., Ltd.) can be cited.

[0217] Examples of HPLC conditions suitable for the concentration treatment include the following conditions.

[0218] Column: YMC pack (YMC Co., Ltd.)

[0219] Pump: 1200 Series G1361A PrepPump (Agilent Technologies, Inc.)

[0220] Column temperature: about 21°C

[0221] Mobile phase: methanol 17.5 mL / min

[0222] Sample conditions: loading amount 2.4 g, i.e. raw material loading rate relative to adsorbent 3 wt%

[0223] By using the microbial oil of the embodiment of the present invention, the recovery rate of the target LC-PUFA in reverse phase column chromatography can be preferably 50% or more, more preferably 80% or more, and even more preferably 90% or more.

[0224] The microbial oil and concentrated microbial oil of the present invention, and the microbial oil and concentrated microbial oil obtained by the production method of the present invention are substances that can contain the target LC-PUFA at a high content rate, or substances that contain the target LC-PUFA at a high content rate, and do not contain components that may be mixed by using other separation means other than precise distillation. Examples of components that may be mixed by using other separation means other than precise distillation include metals such as silver, a large amount of urea, and the like.

[0225] Therefore, the microbial oil and concentrated microbial oil of the present invention are very useful for applying the target LC-PUFA, such as DGLA, to the required application with high content and good productivity. Such applications include, for example, applications in foods, supplements, pharmaceuticals, cosmetics, feeds, etc., applications in their production methods, and in particular pharmaceuticals containing the microbial oil and concentrated microbial oil containing the target LC-PUFA as an active ingredient. For example, in the case of DGLA, it is particularly preferably used for applications aimed at the functionality of DGLA, such as anti-inflammatory, anti-allergic, and the like.

[0226] (5) Inflammatory disease prevention or treatment agent

[0227] The microbial oil or concentrated microbial oil of the present invention contains the target LC-PUFA, such as DGLA, as an active ingredient based on the functionality of the target LC-PUFA, such as DGLA, so it can be contained in an inflammatory disease prevention or treatment agent. That is, the inflammatory disease prevention or treatment agent of one embodiment of the present invention contains the microbial oil or concentrated microbial oil of another embodiment of the present invention as an active ingredient. The inflammatory disease prevention or treatment agent can be set as an anti-inflammatory agent, an antiallergic agent, etc.

[0228] Inflammatory diseases include inflammation of the skin. The inflammation of the skin may be inflammation of at least one selected from the group consisting of macules, urticaria, blisters, swellings and eczema, or may be inflammation of the skin caused by at least one selected from the group consisting of exposure to radiation, autoimmune diseases and uremic pruritus.

[0229] In particular, the skin inflammation may be skin inflammation associated with atopic eczema, contact dermatitis, psoriasis, or uremic pruritus, or skin inflammation caused by these symptoms.

[0230] The term "eczema" is applicable to the state of a wide range of skin with a variety of etiologies. In general, eczema is classified according to inflammation of the epidermis. As the general symptoms associated with eczema, there can be listed: dry, recurrent skin rashes, redness, skin edema (swelling), itching, scab formation (crusting), peeling, blister formation, cracking, exudation and bleeding. As eczema, there can be listed: atopic eczema (atopic dermatitis), contact dermatitis, dry eczema, seborrheic dermatitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpeticum, neurodermatitis and autoeczematosis. Eczema is representatively atopic eczema or contact dermatitis.

[0231] Atopic eczema is further aggravated mainly by contact with or ingestion of allergens, and examples of such allergens include animal hair and scalp, food allergens (eg, tree fruits or shells and crustaceans), or drugs (eg, penicillin).

[0232] Examples of contact dermatitis include allergic contact dermatitis, irritant contact dermatitis, and photocontact dermatitis. Photocontact dermatitis includes phototoxic contact dermatitis and photoallergic contact dermatitis.

[0233] The above-mentioned inflammation of the skin can be inflammation of the skin caused by exposure of the skin to electromagnetic radiation. Among them, for example, exposure to sunlight, heat, X-rays, or radioactive substances can be mentioned. Therefore, in this embodiment, the compound of the present invention is used to treat sunburn.

[0234] Examples of the electromagnetic radiation include radio waves, microwaves, terahertz waves, infrared rays, visible rays, ultraviolet rays, X-rays, and gamma rays. The electromagnetic radiation is preferably infrared rays, visible rays, ultraviolet rays, X-rays, and gamma rays, and more preferably ultraviolet rays, X-rays, and gamma rays.

[0235] Autoimmune diseases may involve an autoimmune response to the skin. Examples of such autoimmune diseases are lupus and psoriasis.

[0236] Uremic pruritus is a skin disorder associated with chronic renal failure. It also frequently affects patients undergoing dialysis treatment.

[0237] Depending on the circumstances, the microbial oil or concentrated microbial oil of another embodiment of the present invention and a corticosteroid or any other therapeutic agent for the above-mentioned medical use may be administered simultaneously.

[0238] In another embodiment, the inflammatory disease may be at least one selected from the group consisting of atopic dermatitis, allergic contact dermatitis, primary irritant contact dermatitis, photocontact dermatitis, systemic contact dermatitis, rheumatism, psoriasis, and lupus.

[0239] Inflammatory disease prevention or treatment agents can be administered to subjects suffering from inflammatory diseases or at risk of illness. As a mode of administration, oral administration or topical administration can be used. It should be noted that inflammatory disease treatment agents refer to medicines used to suppress or alleviate the progress of symptoms caused by inflammatory diseases when symptoms caused by inflammatory diseases are found. On the other hand, inflammatory disease prevention agents refer to medicines used to suppress the onset of symptoms caused by inflammatory diseases in order to pre-administer the drugs in advance. However, these terms are used in combination according to the period of use or the symptoms during use, and are not interpreted in a limiting manner.

[0240] Another aspect of the present invention provides a method for preventing, treating or alleviating an inflammatory disease, comprising administering the above-mentioned inflammatory disease prevention or treatment agent to a subject suffering from an inflammatory disease or a subject at risk of the disease. The administration method may be oral administration or topical administration.

[0241] The microbial oil of the present invention may contain each component at a content rate based on area % according to column chromatography analysis. That is, each embodiment of the present invention further provides the following microbial oil, a method for producing a microbial oil, a concentrated microbial oil, and a method for producing a concentrated microbial oil.

[0242] <1> A microbial oil comprising: at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids, having a content of 50% by area or more of the total area of ​​fatty acids in the oil as measured by gas chromatography;

[0243] Thermally generated fatty acids having 16 to 22 carbon atoms having a content of 3.0 area % or less of the total area of ​​fatty acids in the oil as measured by gas chromatography.

[0244] <2> The microbial oil according to <1>, wherein the content of the polyunsaturated fatty acid is 80 area % to 98 area % of the total area of ​​the fatty acids in the oil as measured by gas chromatography.

[0245] <3> The microbial oil according to <1> or <2>, wherein the content of the thermally generated fatty acid is 0.0001 area % to 3.0 area % of the total area of ​​fatty acids in the oil as measured by gas chromatography.

[0246] <4> The microbial oil according to any one of <1> to <3>, wherein the total content of saturated fatty acids having 22 carbon atoms and saturated fatty acids having 24 carbon atoms is 6.0 area % or less of the total area of ​​fatty acids in the oil measured by gas chromatography.

[0247] <5> The microbial oil according to any one of <1> to <4>, wherein the total content of the saturated fatty acid having 22 carbon atoms and the saturated fatty acid having 24 carbon atoms is 10 / 100 or less of the content of the polyunsaturated fatty acid.

[0248] <6> The microbial oil according to any one of <1> to <5>, wherein the content of saturated fatty acids having 24 carbon atoms is 3.0 area % or less based on the total area of ​​fatty acids in the oil as measured by gas chromatography.

[0249] <7> The microbial oil according to any one of <1> to <6>, wherein the content of the saturated fatty acid having 24 carbon atoms is 4 / 100 or less of the content of the polyunsaturated fatty acid.

[0250] <8> According to any one of <1> to <7>, the content of other saturated or unsaturated fatty acids is less than 10.0 area % of the total area of ​​fatty acids in the oil when measured by gas chromatography; when the other saturated or unsaturated fatty acids are used as an indicator related to separation by liquid chromatography and the separation number calculated from the number of carbon atoms and double bonds of the fatty acids, they have a separation number of greater than -2 and less than +2 compared to the separation number of the polyunsaturated fatty acids, and have a carbon atom number different from that of the polyunsaturated fatty acids.

[0251] <9> The microbial oil according to <8>, wherein the content of the other saturated or unsaturated fatty acids is 15 / 100 or less of the content of the polyunsaturated fatty acids.

[0252] <10> The microbial oil according to any one of <1> to <9>, wherein the polyunsaturated fatty acid is at least one selected from the group consisting of eicosadienoic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid and docosahexaenoic acid.

[0253] <11> The microbial oil according to any one of <8> to <10>, wherein the other saturated or unsaturated fatty acids contain at least one selected from the group consisting of saturated fatty acids having 18 carbon atoms, monounsaturated fatty acids having 18 carbon atoms, diunsaturated fatty acids having 18 carbon atoms, triunsaturated fatty acids having 18 carbon atoms and tetraunsaturated fatty acids having 18 carbon atoms.

[0254] <12> The microbial oil according to any one of <1> to <11>, wherein the polyunsaturated fatty acid is dihomo-γ-linolenic acid, and the thermogenic fatty acid is a thermogenic fatty acid having 20 carbon atoms.

[0255] <13> The microbial oil according to <12>, wherein, when the retention time of dihomo-γ-linolenic acid ethyl ester is set to 1 in a gas chromatography analysis of the thermally generated fatty acid ethyl ester under the following conditions, the thermally generated fatty acid contains at least one of a first substance having a retention time as a peak appearing in the range of 1.001 to 1.011 and a second substance having a retention time as a peak appearing in the range of 1.013 to 1.027,

[0256] Apparatus: 6890N Network GC system, Agilent Technologies Inc.;

[0257] Column: DB-WAX, length 30 m × inner diameter 0.25 mm × film thickness 0.25 μm, Agilent Technologies;

[0258] Column temperature conditions: 60°C for 2.5 minutes → temperature rise 20°C / minute → 180°C → temperature rise 2°C / minute → 230°C for 15 minutes;

[0259] Inlet temperature conditions: 210°C, no split, sampling time at the split outlet 1.5 minutes, purge flow 40 mL / min;

[0260] Injection volume conditions: 1 μL, sample concentration 1 mg / mL or less;

[0261] Detector: FID;

[0262] Detector temperature: 280°C;

[0263] Carrier gas conditions: helium, linear velocity 24 cm / min.

[0264] <14> The microbial oil according to <13>, wherein the polyunsaturated fatty acid is dihomo-γ-linolenic acid, and the total content of the first substance and the second substance is 0.001 area % to 2.8 area % of the total area of ​​the fatty acids in the oil when measured by gas chromatography.

[0265] <15> The microbial oil according to any one of <10> to <14>, wherein the content of the monounsaturated fatty acid having 18 carbon atoms is 7.0 area % or less based on the total area of ​​the fatty acids in the oil as measured by gas chromatography.

[0266] <16> The microbial oil according to any one of <10> to <15>, wherein the content of the monounsaturated fatty acid having 18 carbon atoms is 10 / 100 or less of the content of the polyunsaturated fatty acid.

[0267] <17> The microbial oil according to any one of <10> to <16>, wherein the content of the diunsaturated fatty acid having 18 carbon atoms is 7 / 100 or less of the content of the polyunsaturated fatty acid.

[0268] <18> The microbial oil according to any one of <10> to <17>, wherein the total content of the monounsaturated fatty acid having 18 carbon atoms and the diunsaturated fatty acid having 18 carbon atoms is 15 / 100 or less of the content of the polyunsaturated fatty acid.

[0269] <19> The microbial oil according to any one of <10> to <18>, wherein the content of the saturated fatty acid having 18 carbon atoms is 11 / 100 or less of the content of the polyunsaturated fatty acid.

[0270] <20> A method for producing microbial oil, comprising:

[0271] preparing a feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl esters and / or free fatty acids obtained from microbial biomass; and

[0272] The feedstock oil is purified by precision distillation under conditions including a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa.

[0273] <21> A method for producing microbial oil, comprising:

[0274] A feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl esters and / or free fatty acids obtained from microbial biomass is prepared;

[0275] Using a distillation tower containing regular packing, the crude oil is subjected to precision distillation under conditions including a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa; and

[0276] The microbial oil described in any one of <1> to <19> is obtained.

[0277] <22> A method for producing microbial oil, comprising:

[0278] A feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl esters and / or free fatty acids obtained from microbial biomass is prepared;

[0279] With respect to the feedstock oil, precision distillation is performed using a distillation tower containing a regular packing under the conditions described below, the conditions including a tower bottom temperature and a minimum pressure in the distillation tower corresponding to the type of the polyunsaturated fatty acid as a target, and conditions that can obtain a microbial oil containing thermally generated fatty acids with carbon atoms of 16 to 22 at a content of 3.0 area % or less based on the total area of ​​fatty acids in the oil measured by gas chromatography; and

[0280] The microbial oil described in any one of <1> to <19> is obtained.

[0281] <23> The production method according to <22>, wherein the precision distillation is performed at a column bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation column of 0.1 Pa to 30 Pa.

[0282] <24> The production method according to any one of <20> to <23>, wherein the precision distillation includes a plurality of precision distillations performed under conditions of different bottom temperature and top pressure.

[0283] <25> The production method according to <24>, wherein the precision distillation includes low-temperature precision distillation based on a bottom temperature of 160°C to 220°C and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa, and high-temperature precision distillation based on a bottom temperature of 170°C to 230°C and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa.

[0284] <26> The production method according to <25>, wherein a bottom temperature in the high-temperature precision distillation is 3° C. to 20° C. higher than a bottom temperature in the low-temperature precision distillation.

[0285] <27> The production method according to any one of <21> to <26>, wherein the specific surface area per unit of the regular packing is 125 m 2 / m 3 ~1700m 2 / m 3 .

[0286] <28> A concentrated microbial oil, wherein the content of polyunsaturated fatty acids having 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids is 90% to 98% by area of ​​the total area of ​​fatty acids in the oil as measured by gas chromatography.

[0287] The content of thermally generated fatty acids having 16 to 22 carbon atoms is 0.0001 area % to 3.0 area % of the total area of ​​fatty acids in the oil as measured by gas chromatography.

[0288] The total content of saturated fatty acids having 24 carbon atoms and saturated fatty acids having 22 carbon atoms is 1.0 area % or less of the total area of ​​fatty acids in the oil as measured by gas chromatography,

[0289] The content of the monounsaturated fatty acid having 18 carbon atoms is 5.0 area % or less based on the total area of ​​the fatty acids in the oil as measured by gas chromatography.

[0290] <29> A concentrated microbial oil, wherein the content of dihomo-γ-linolenic acid in the form of fatty acid alkyl esters and / or free fatty acids is 90% to 98% by area of ​​the total area of ​​fatty acids in the oil as measured by gas chromatography.

[0291] The content of thermally generated fatty acids having 16 to 22 carbon atoms is 0.0001 area % to 3.0 area % of the total area of ​​fatty acids in the oil as measured by gas chromatography.

[0292] The total content of saturated fatty acids having 24 carbon atoms and saturated fatty acids having 22 carbon atoms is 1.0 area % or less of the total area of ​​fatty acids in the oil as measured by gas chromatography,

[0293] The content of the monounsaturated fatty acid having 18 carbon atoms is 5.0 area % or less based on the total area of ​​the fatty acids in the oil as measured by gas chromatography.

[0294] <30> A method for producing concentrated microbial oil, comprising:

[0295] Using the production method described in any one of <20> to <27>, a microbial oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of fatty acid alkyl esters and / or free fatty acids as a target is obtained;

[0296] The obtained microbial oil was concentrated using reverse phase column chromatography.

[0297] <31> Use of the microbial oil described in any one of <1> to <19> or the concentrated microbial oil described in <28> or <29> in food, supplements, medicines, cosmetics or feed.

[0298] <32> Use of the microbial oil described in any one of <1> to <19> or the concentrated microbial oil described in <28> or <29> in a method for producing a food, a supplement, a medicine, a cosmetic or a feed.

[0299] <33> A pharmaceutical product comprising the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29>.

[0300] <34> A preventive or therapeutic agent for inflammatory diseases, comprising the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29>.

[0301] <35> The agent for preventing or treating an inflammatory disease according to <34>, which is an antiallergic agent or an anti-inflammatory agent.

[0302] <36> An agent for preventing or treating an inflammatory disease according to <34> or <35>, wherein the inflammatory disease is an inflammatory disease of the skin of at least one type selected from the group consisting of macules, urticaria, blisters, swellings and eczema, or an inflammatory disease of the skin caused by at least one selected from the group consisting of exposure to radiation, autoimmune diseases and uremic pruritus.

[0303] <37> The agent for preventing or treating an inflammatory disease according to <34> or <35>, wherein the inflammatory disease of the skin is at least one selected from the group consisting of atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, photocontact dermatitis, systemic contact dermatitis, rheumatism, psoriasis and lupus.

[0304] <38> A method for preventing, treating or alleviating an inflammatory disease, comprising administering the agent for preventing or treating an inflammatory disease according to any one of <34> to <37> to a subject suffering from the inflammatory disease or a subject at risk of suffering from the inflammatory disease.

[0305] <39> The method for preventing, treating or alleviating an inflammatory disease according to <38>, wherein the administration is oral administration or topical administration.

[0306] <40> A microbial oil obtained by the production method according to any one of <20> to <27>.

[0307] <41> A concentrated microbial oil obtained by the production method described in <30>.

[0308] As described above, in the present invention, the content of each component of microbial oil and concentrated microbial oil is set to be the same in the area % based on the measurement using gas chromatography and in the weight %; therefore, in the records related to the content of each component of microbial oil and concentrated microbial oil recorded in area % based on the measurement using gas chromatography, each numerical value recorded in weight % is directly rewritten as a numerical value recorded in area %, and the entire text is directly applicable.

[0309] In addition, in this specification, each technical feature of the invention described in an embodiment related to each aspect of the invention can be arbitrarily combined to form a new embodiment, and such a new embodiment should also be understood to be included in each aspect of the present invention.

[0310] Example

[0311] The present invention is described in detail below by way of examples. However, the present invention is not limited to these examples. It should be noted that, unless otherwise specified, "%" is a mass basis.

[0312] In the examples and comparative examples in the following sections, the target LC-PUFA is DGLA in the form of ethyl ester, but the present invention is not limited to this. DGLA in the form of free fatty acids may be set as the target LC-PUFA, or other fatty acids in the form of alkyl esters or free fatty acids may be set as the target LC-PUFA.

[0313] The ethyl esterification rate of the alkyl esterification method used in the following embodiment and comparative example is empirically determined to be 95%~100%. Therefore, in the item of the present embodiment, the raw material ethyl ester obtained is inferred to be the major part of the saturated or unsaturated fatty acids contained as the fatty acid ethyl ester form. Therefore, in the following comparative example and embodiment, the saturated or unsaturated fatty acids contained in the microbial oil are all recorded as the saturated or unsaturated fatty acids in the ethyl ester form.

[0314] In addition, hereinafter, DGLA ethyl ester is recorded simply as "DGLA", the monovalent unsaturated fatty acid ethyl ester having 18 carbon atoms is recorded simply as "C18:1", the divalent unsaturated fatty acid ethyl ester having 18 carbon atoms is recorded simply as "C18:2", the saturated fatty acid ethyl ester having 22 carbon atoms is recorded simply as "C22:0", and the saturated fatty acid ethyl ester having 24 carbon atoms is recorded simply as "C24:0".

[0315] [Comparative Example 1]

[0316] The microbial oil 1 from the genus Mortierella containing 37.2% by weight of DGLA in the fatty acid composition is ethylated with an alkali catalyst according to a conventional method to prepare the raw material ethyl ester 1. That is, 14g of 20% by weight sodium ethoxide-ethanol solution and 40mL of ethanol are added to 120g of the microbial oil 1, and the mixture is heated in an oil bath for 2 hours while being refluxed. Thereafter, the reaction solution is air-cooled to below 40°C, and then, it is transferred to a separating funnel. 400mL of hexane is added to the reaction solution transferred to the separating funnel, and then, purified water is added and washed repeatedly. After the washing liquid becomes neutral, it is washed once with saturated brine and the hexane layer is recovered. Anhydrous sodium sulfate is added to the recovered hexane layer to dehydrate it, and the solvent is removed by evaporator and vacuum suction to obtain the raw material ethyl ester 1.

[0317] In the raw material ethyl ester 1, the DGLA content, i.e., the content of DGLA relative to the obtained raw material ethyl ester, was 37.2% by weight, the weight ratio of C18:1 to DGLA, i.e., C18:1 / DGLA was 23.5 / 100, and the weight ratio of C18:2 to DGLA, i.e., C18:2 / DGLA was 17.8 / 100.

[0318] The raw material ethyl ester 1 was not subjected to distillation treatment but was subjected to HPLC under the following conditions, and the DGLA elution fraction was fractionated.

[0319] In HPLC, the raw material ethyl ester 1 is put into the device and the treatment is started. The eluate is fractionated until all the fatty acids contained in the raw material ethyl ester 1 are completely eluted. For each obtained fraction, 1 mL is accurately taken, and then the solvent is removed by an evaporator. The fraction after the solvent is removed is accurately dissolved in 1 mL of 1.0 mg / mL hexane solution of tricosanoic acid methyl ester, i.e., C23:0 methyl ester, as an internal standard, and is used as a measurement sample and subjected to gas chromatography (GC) under the conditions shown below.

[0320] The amount and fatty acid composition of the fatty acids contained in the sample were obtained by the peak area of ​​each fatty acid obtained in the GC based on the following formula (II), and the DGLA content, i.e., the content and recovery of DGLA relative to the obtained components were further calculated. The recovery of DGLA was calculated by calculating the total amount of DGLA in the recovered components relative to the total amount of DGLA in the fractionated HPLC eluent. Hereinafter, the recovery was calculated using the same method.

[0321] Amount of fatty acids contained in the component [mg] =

[0322] (Peak area of ​​each fatty acid × component volume [mL]) / (Peak area of ​​C23:0 methyl ester) × amount of internal standard added 1.0 mg... (II)

[0323] The results are shown in Table 2. As shown in Table 2, the DGLA content was 91.1% by weight and the DGLA recovery was 8.1%. In Table 2, the values ​​in the column "microbial oil" of Comparative Example 1 are the values ​​of the raw material ethyl ester 1. The content and weight ratio in Table 2 all represent the content and weight ratio based on the fatty acid composition. The same applies below.

[0324] HPLC conditions

[0325] Column: YMC pack ODS-AQ-HG 20 mmφ×1000 mm (YMC Corporation), two columns with a length of 500 mm were connected in series.

[0326] Pump: 1200 Series G1361A PrepPump (Agilent Technologies, Inc.)

[0327] Column temperature: 40°C

[0328] Mobile phase: methanol 35mL / min

[0329] Sample conditions: Loading amount 2.4 g, raw material loading rate relative to filler 3 wt%

[0330] GC conditions

[0331] Device: 6890N Network GC system (Agilent Technologies, Inc.)

[0332] Column: DB-WAX, length 30 m × inner diameter 0.25 mm × film thickness 0.25 μm (Agilent Technologies, Inc.)

[0333] Column temperature conditions: 180°C → temperature rise 3°C / min → 230°C for 30 min

[0334] Inlet temperature: 250℃

[0335] Detector: FID

[0336] Detector temperature: 250°C

[0337] Carrier gas conditions: Helium linear velocity 30 cm / min

[0338] Split conditions: split ratio 1:30, injection volume 1μL, sample concentration 9mg / mL

[0339] [Comparative Example 2]

[0340] The raw material ethyl ester 1 used in Comparative Example 1 was subjected to short path distillation (SPD) under the following conditions to remove fatty acid components having 18 or less carbon atoms.

[0341] The SPD device used was KDL-5 (UIC GmH). 160.7 g of the raw material was fed at 300 mL / h under vacuum conditions of 40°C raw material temperature, 100°C heat medium temperature at the evaporation surface inlet, 87°C heat medium temperature at the outlet, 30°C internal condenser temperature, and 0.001 mbar (i.e., 0.133 mPa) pump pressure, and removed the fractions containing more components below C18 to obtain 65.9 g of residue. DGLA was concentrated and contained in the residue.

[0342] The obtained residue was subjected to HPLC under the following conditions to fractionate the DGLA elution fraction. The DGLA elution fraction corresponds to the concentrated microbial oil.

[0343] Regarding the obtained fraction after SPD treatment and the DGLA eluted fraction after HPLC treatment, the amount of fatty acids contained and the fatty acid composition were determined by gas chromatography in the same manner as in Comparative Example 1, and the DGLA content, that is, the content and recovery rate of DGLA relative to the obtained fraction were further calculated. The results are shown in Table 2. As shown in Table 2, the DGLA content in the fraction after SPD treatment was 40.9% by weight, the DGLA content in the DGLA eluted fraction was 94.4% by weight, and the DGLA recovery rate was 5.1%.

[0344] HPLC conditions

[0345] Column: YMC pack (YMC Co., Ltd.), connect 2 columns of 500mm in length in series.

[0346] Pump: 1200 Series G1361A PrepPump (Agilent Technologies, Inc.)

[0347] Column temperature: 40°C

[0348] Mobile phase: methanol 12mL / min

[0349] Sample conditions: Loading amount 2.4 g, i.e. raw material loading rate relative to filler 1.5 wt %

[0350] [Example 1]

[0351] The microbial oil 2 from the genus Mortierella containing DGLA 32.8% by weight in the fatty acid composition is ethylated with an alkali catalyst using a conventional method to prepare the raw material ethyl ester 2. That is, 14g of 20% by weight sodium ethoxide-ethanol solution and 40mL of ethanol are added to 120g of the microbial oil 2, and the mixture is heated in an oil bath for 2 hours while being refluxed. Thereafter, the reaction solution is air-cooled to below 40°C, and then, it is transferred to a separating funnel. 400mL of hexane is added to the reaction solution transferred to the separating funnel, and then, purified water is added and washed repeatedly. After the washing liquid becomes neutral, it is washed once with saturated brine to reclaim the hexane layer. Anhydrous sodium sulfate is added to the recovered hexane layer to dehydrate it, and the solvent is removed by evaporator and vacuum suction to obtain the raw material ethyl ester 2.

[0352] For the raw material ethyl ester 2, the DGLA content, i.e., the content of DGLA relative to the obtained raw material ethyl ester, was 32.8% by weight, the weight ratio of C18:1 to DGLA, i.e., C18:1 / DGLA was 26.1 / 100, and the weight ratio of C18:2 to DGLA, i.e., C18:2 / DGLA was 17.2 / 100.

[0353] The raw material ethyl ester 2 was used as a sample and subjected to precision distillation including the following low-temperature precision distillation step and high-temperature precision distillation step.

[0354] In the low-temperature precision distillation process, 100 g of the raw material ethyl ester 2 was subjected to the following precision distillation. A fractionating tube with a vacuum jacket (Kiriyama Glass) was used in the fractionating tube, and 5 Sulzer·Lab PackingEX (Sulzer Chemtech) were used as the internal filler. The diameter of the fractionating tube with a vacuum jacket was 25 mm, and the size of one unit of Sulzer·Lab PackingEX was 25 mm×50 mm. The liquid temperature in the bottom kettle, i.e., the tower bottom temperature, was set to 185°C, the tower top vapor temperature, i.e., the tower top temperature, was set to 135°C, and the pressure before the vacuum pump, i.e., the minimum pressure in the distillation tower, i.e., the vacuum degree, was set to 30 Pa for precision distillation. In the low-temperature precision distillation process, the components below C18 were removed as the primary distillate to obtain 40 g of the primary distillation residue. The primary distillation residue was confirmed by the same gas chromatography as in Comparative Example 1. As a result, DGLA ethyl ester was concentrated and contained in the primary distillation residue.

[0355] In addition, in the chromatographic column of the primary distillation residue, between the peak representing C20:3,n-6 (DGLA) and the peak representing C20:4,n-6, compound A, which is not usually seen in the chromatographic column using crude oil as a sample, appears (see Table 3). In addition, near the peak representing C20:4n-6, compound B, which is not usually seen in the chromatographic column using crude oil as a sample, appears. Compound A and compound B can be considered to be compounds formed by distillation treatment and are set as thermally generated fatty acids. The content of compound A and compound B is shown in Tables 2 and 3. It should be noted that the content in Table 3 represents the content based on the fatty acid composition.

[0356] It should be noted that, in the case where the thermally generated fatty acid peak overlaps with the fatty acid contained in the crude oil under the GC conditions used in Comparative Example 1, in the separation and quantification of compound A and compound B, after removing the fatty acid originally contained in the crude oil by silver-ions solid phase extraction, the crude oil was subjected to gas chromatography under the following conditions, and when the retention time of dihomo-γ-linolenic acid ethyl ester was set to 1, the compound having a peak appearing in the range of 1.001 to 1.009, that is, the retention time indicated by peak A was identified as compound A. In addition, similarly, the compound having a peak appearing in the range of 1.013 to 1.024, the retention time indicated by peak B was identified as compound B. Thereafter, the relative ratio of DGLA to compound A or compound B was determined, and the weight % of DGLA, compound A, and compound B was calculated.

[0357] Device: 6890N Network GC system (Agilent Technologies, Inc.)

[0358] Column: DB-WAX, length 30 m × inner diameter 0.25 mm × film thickness 0.25 μm (Agilent Technologies, Inc.)

[0359] Column temperature conditions: 60°C for 2.5 minutes → temperature rise 20°C / minute → 180°C → temperature rise 2°C / minute → 230°C for 15 minutes

[0360] Inlet temperature conditions: 210°C, no split, sampling time at split outlet 1.5 minutes, purge flow 40 mL / min

[0361] Injection volume conditions: 1 μL, sample concentration 1 mg / mL or less

[0362] Detector: FID

[0363] Detector temperature: 280°C

[0364] Carrier gas conditions: helium, linear velocity 24 cm / min

[0365] Thereafter, in the high-temperature precision distillation step, 32 g of the residue obtained in the initial distillation step at low temperature precision distillation step was extracted and subjected to the following precision distillation. A fractionation tube with a vacuum jacket (Kiriyama Glass) was used as the fractionation tube, and two Sulzer Lab Packing EX (Sulzer Chemtech) were used as the internal filler. The diameter of the fractionation tube with a vacuum jacket was 25 mm, and the size of one unit of Sulzer Lab Packing EX was 25 mm × 50 mm. The liquid temperature in the bottom kettle, i.e., the bottom temperature of the tower, was set to 195°C, the vapor temperature at the top of the tower, i.e., the top temperature of the tower, was set to 150°C, and the pressure before the vacuum pump, i.e., the minimum pressure in the distillation tower, i.e., the vacuum degree, was set to 30 Pa, and precision distillation was performed. In the high-temperature precision distillation step, the components above C22 were removed as residues, i.e., residuals, to obtain 19 g of the main fraction. In the main fraction, DGLA was further concentrated.

[0366] The obtained main fraction was subjected to HPLC under the following conditions to fractionate the DGLA elution fraction. The DGLA elution fraction corresponds to the concentrated microbial oil.

[0367] The amount of fatty acids and the fatty acid composition of the main distillate fraction and the DGLA elution fraction obtained after the high-temperature precision distillation step were determined by gas chromatography (GC) in the same manner as in Comparative Example 1, and the DGLA content, that is, the content and recovery rate of DGLA relative to the obtained fractions were further calculated. The results are shown in Tables 2 and 3. As shown in Table 2, the DGLA content in the main distillate fraction after the high-temperature precision distillation step was 91.9% by weight, the DGLA content in the DGLA elution fraction was 96.4% by weight, and the DGLA recovery rate was 100.0%, and DGLA was obtained with very high refining efficiency.

[0368] HPLC conditions

[0369] Column: YMC pack (YMC Co., Ltd.)

[0370] Pump: 1200 Series G1361A PrepPump (Agilent Technologies, Inc.)

[0371] Column temperature: about 21°C

[0372] Mobile phase: methanol 17.5 mL / min

[0373] Sample conditions: loading amount 2.4 g, i.e. raw material loading rate relative to adsorbent 3 wt%

[0374] [Example 2]

[0375] The raw material ethyl ester 2 used in Example 1 was used as a sample and subjected to precision distillation including the following low-temperature precision distillation step and high-temperature precision distillation step.

[0376] In the low-temperature precision distillation process, 100 g of the raw material ethyl ester 2 was subjected to the following precision distillation. A fractionating tube with a vacuum jacket (Kiriyama Glass) was used as the fractionating tube, and two Sulzer Lab Packing EX (Sulzer Chemtech) were used as the internal filler. The diameter of the fractionating tube with a vacuum jacket was 25 mm, and the size of one unit of Sulzer Lab Packing EX was 25 mm×50 mm. The liquid temperature in the bottom kettle, i.e., the tower bottom temperature, was set to 180°C, the tower top vapor temperature, i.e., the tower top temperature, was set to 140°C, and the pressure before the vacuum pump, i.e., the minimum pressure in the distillation tower, i.e., the vacuum degree, was set to 20 Pa for precision distillation. In the low-temperature precision distillation process, components below C18 were removed as the primary distillate to obtain 48 g of the primary distillation residue. The primary distillation residue was confirmed by the same gas chromatography as in Comparative Example 1. As a result, DGLA was concentrated and contained in the primary distillation residue. In addition, in the chromatographic column where Compound A and Compound B appeared in the primary distillation extraction residue, the content ratios of Compound A and Compound B are shown in Table 2.

[0377] Thereafter, in the high-temperature precision distillation step, 45 g of the residue obtained in the initial distillation step at the low-temperature precision distillation step was extracted and subjected to the following precision distillation. A fractionation tube with a vacuum jacket (Kiriyama Glass) was used as the fractionation tube, and two Sulzer Lab Packing EX (Sulzer Chemtech) were used as the internal filler. The diameter of the fractionation tube with a vacuum jacket was 25 mm, and the size of one unit of Sulzer Lab Packing EX was 25 mm × 50 mm. The liquid temperature in the bottom kettle, i.e., the bottom temperature of the tower, was set to 185°C, the vapor temperature at the top of the tower, i.e., the top temperature of the tower, was set to 145°C, and the pressure before the vacuum pump, i.e., the minimum pressure in the distillation tower, i.e., the vacuum degree, was set to 20 Pa, and precision distillation was performed. In the second precision distillation step, the components above C22 were removed as residues, i.e., residuals, to obtain 28 g of a main fraction. In the main fraction, it is estimated that DGLA is further concentrated.

[0378] The obtained main fraction was subjected to HPLC under the following conditions to fractionate the DGLA elution fraction. The DGLA elution fraction corresponds to the concentrated microbial oil.

[0379] Regarding the main distillate fraction and the DGLA elution fraction obtained after the high-temperature precision distillation process, the amount of fatty acids contained and the fatty acid composition were determined by gas chromatography (GC) in the same manner as in Comparative Example 1, and the DGLA content, that is, the content and recovery rate of DGLA relative to the obtained fractions were further calculated. The results are shown in Tables 2 and 4. As shown in Table 2, the DGLA content in the main distillate fraction after the high-temperature precision distillation process was 75.0% by weight, the DGLA content in the DGLA elution fraction was 95.1% by weight, and the DGLA recovery rate was 61.7%, and DGLA was obtained with high refining efficiency.

[0380] In addition, similarly to Example 1, in the chromatographic column of the main fraction, compound A representing peak A and compound B representing peak B appear (see Table 4). Compound A and compound B can be considered to be thermally generated fatty acids formed by distillation. The content of compound A and compound B is shown in Table 2 and Table 4. It should be noted that the content in Table 4 represents the content based on the fatty acid composition.

[0381] HPLC conditions

[0382] Column: YMC pack (YMC Co., Ltd.) Columns with a length of 500 mm2 are connected in series.

[0383] Pump: 1200 Series G1361A Prep Pump (Agilent Technologies, Inc.)

[0384] Column temperature: about 21°C

[0385] Mobile phase: methanol, 12 mL / min Sample conditions: Loading amount 2.4 g, i.e., raw material loading rate relative to adsorbent 1.5 wt %

[0386] [Table 2]

[0387]

[0388] [Table 3]

[0389]

[0390] [Table 4]

[0391]

[0392] As shown in Tables 2 to 4, it is known that microbial oils containing 50% by weight or more of DGLA and 0.0001% by weight or more of thermally generated fatty acids are very useful in effectively obtaining high-concentration DGLA when DGLA is obtained using reverse phase column chromatography. Such microbial oils can be obtained by a manufacturing method comprising a precision distillation process under specific conditions or a manufacturing method comprising a rectification process using a distillation tower containing a regular filler.

[0393] Thus, according to the present invention, a microbial oil containing a high content of DGLA can be efficiently obtained, and a concentrated microbial oil containing a high content of DGLA can be efficiently obtained.

[0394] Therefore, according to the present invention, microbial oil and concentrated microbial oil containing a high content of target LC-PUFA can be effectively provided, and a production method useful for efficiently obtaining such microbial oil and concentrated microbial oil, as well as various uses of the microbial oil and concentrated microbial oil can be provided.

[0395] The disclosure of Japanese Patent Application No. 2013-251401 filed on December 4, 2013 is incorporated herein by reference in its entirety.

[0396] All documents, patent applications, and technical specifications described in this specification are incorporated herein by reference to the same extent as if each document, patent application, and technical specification were specifically and individually described.

Claims

1. A microbial oil obtained by the following production method: a raw material oil containing dihomo-γ-linolenic acid in the form of alkyl esters and / or free fatty acids obtained from microbial biomass is subjected to precision distillation using a distillation tower containing regular packing under conditions including a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa, The microbial oil contains: dihomo-γ-linolenic acid in the form of fatty acid alkyl esters and / or free fatty acids, having a content of 50% by weight or more of the total weight of the fatty acids in the microbial oil; a thermally generated fatty acid having 20 carbon atoms and having a content of 0.0001% by weight to 3.0% by weight of the total weight of the fatty acids in the microbial oil; A saturated fatty acid with 22 carbon atoms and a saturated fatty acid with 24 carbon atoms, the total weight content of which is 3 / 100 or less of the weight content of dihomo-γ-linolenic acid in the fatty acids in the microbial oil, and The total weight content of C18:1 fatty acid and C18:2 fatty acid in the fatty acids in the microbial oil is less than 15 / 100 of the weight content of dihomo-γ-linolenic acid, in, The weight content of the C18:1 fatty acid in the fatty acids in the microbial oil is less than 10 / 100 of the weight content of dihomo-γ-linolenic acid, The thermally generated fatty acids are generated during the precision distillation.

2. A method for producing a microbial oil, comprising: preparing a raw material oil containing dihomo-γ-linolenic acid in the form of alkyl esters and / or free fatty acids obtained from microbial biomass; Using a distillation tower containing regular packing, the crude oil is subjected to precision distillation under conditions including a tower bottom temperature of 160° C. to 230° C. and a minimum pressure in the distillation tower of 0.1 Pa to 30 Pa; and The microbial oil according to claim 1 is obtained.

3. The concentrated microbial oil obtained by concentrating the microbial oil according to claim 1, characterized in that: The content of dihomo-γ-linolenic acid in the form of fatty acid alkyl esters and / or free fatty acids is 90% to 98% by weight of the total weight of the fatty acids in the concentrated microbial oil. The content of the thermally generated fatty acid having 20 carbon atoms is 0.0001 wt% to 3.0 wt% of the total weight of the fatty acids in the concentrated microbial oil. The total content of the saturated fatty acid having 24 carbon atoms and the saturated fatty acid having 22 carbon atoms is 1.0% by weight or less of the total weight of the fatty acids in the concentrated microbial oil, The content of C18:1 fatty acid is 5.0 wt % or less based on the total weight of fatty acids in the concentrated microbial oil.

4. The method for producing concentrated microbial oil according to claim 3, comprising: Using the production method of claim 2, a microbial oil containing dihomo-γ-linolenic acid in the form of fatty acid alkyl esters and / or free fatty acids as a target is obtained; The obtained microbial oil was concentrated using reverse phase column chromatography.

5. Use of the microbial oil according to claim 1 or the concentrated microbial oil according to claim 3 in the manufacture of food, supplements, medicines, cosmetics or feed.

Images