Dihomo-γ-linolenic acid-containing microbial oil with reduced arachidonic acid content

Abstract

To provide an oil that contains dihomo-γ-linolenic acid at a higher purity.SOLUTION: A microbial oil is provided that is characterised by a high content of dihomo-γ-linolenic acid contained therein and / or by a reduced content of undesirable constituent fatty acid(s).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a microbial oil containing dihomo-γ-linolenic acid (hereinafter sometimes referred to as DGLA), a lower alcohol ester composition or a free fatty acid composition obtained from the microbial oil, a microbial cell containing dihomo-γ-linolenic acid, and their uses, their production methods, and microorganisms used for their production. [Background technology]

[0002] DGLA (C20:3,n-6) (8,11,14-eicosatrienoic acid) is one of the constituent fatty acids in fish oil, seaweed, etc. It is known that DGLA is produced as a precursor of arachidonic acid (C20:4,n-6) (5,8,11,14-eicosatetraenoic acid) (hereinafter sometimes referred to as ARA) in microorganisms such as Mortierella alpina. However, the amount of DGLA produced is small in microorganisms that contain triglycerides, diglycerides, monoglycerides, phospholipids, and sterols as lipid components. DGLA and ARA are fatty acids with similar chemical characteristics. For this reason, it is difficult to separate DGLA from ARA.

[0003] In order to efficiently produce DGLA, techniques have been proposed to reduce the amount of ARA produced in microorganisms as much as possible (Non-Patent Documents 1-5, Patent Documents 1-3). For example, Japanese Patent Laid-Open Publication No. 5-091887 discloses a method for producing DGLA or lipids containing DGLA, which includes culturing a microorganism capable of producing ARA but with reduced or absent Δ5 desaturase activity to produce DGLA or lipids containing DGLA, and collecting lipids containing DGLA or DGLA. Patent Document 1 also discloses that ARA can be obtained by culturing a microorganism capable of producing ARA and with reduced or absent Δ5 desaturase activity in the presence of a Δ5 desaturase inhibitor, such as sesamin. Patent Document 2 also discloses that a microbial oil with a high DGLA / ARA ratio can be produced by using two or more Δ5 desaturase inhibitors in combination. In addition, Non-Patent Documents 1 to 5 disclose the production of DGLA-containing oil by introducing a mutation into the Δ5 desaturase gene or disrupting the gene in Mortierella alpina 1S-4 strain. In some of the Δ5 desaturase gene disruption strains in Non-Patent Document 5, ARA is achieved below the detection limit. However, in Table 3 of Non-Patent Document 5, the results are shown only to the first decimal place.

[0004] Furthermore, Patent Document 3 discloses a method for producing phospholipids that contain long-chain highly unsaturated fatty acids such as ARA and DGLA as components.

[0005] Despite these prior art developments, there have been very few commercial productions of microbial oils with enhanced DGLA content due to technical difficulties in achieving product satisfaction and quality of use. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Publication No. 5-091887 [Patent Document 2] Special Publication No. 2017-502114 [Patent Document 3] International Publication No. 2005 / 083101 [Non-patent literature]

[0007] [Non-Patent Document 1] Abe et al., J. Biosci. Bioeng. (2005) vol. 99, No. 3, pp. 296-299 [Non-Patent Document 2] Jareonkitmongkol et al., J. Gneneral Microbiology (1992) vol. 138, pp. 997-1002 [Non-Patent Document 3] Kawashima et al., JAOCS (2000) vol. 77, No. 11, pp. 1135-1138 [Non-Patent Document 4] Jareonkitmongkol et al., Appl. Environ. Microbiol. (1993) vol. 59, No. 12, pp. 4300-4304 [Non-Patent Document 5] Kikukawa et al., J. Biosci. Bioeng.(2016) vol. 122, No. 1, pp. 22-26 Summary of the Invention [Problem to be solved by the invention]

[0008] The object of the present disclosure is to provide a microbial oil and microbial cells that can be used to obtain a dihomo-γ-linolenic acid-containing oil of higher purity than the microbial oil obtained by conventional methods, and to provide a method for producing a microbial oil using the microbial cells. [Means for solving the problem]

[0009] The present disclosure relates to a microbial oil containing dihomo-gamma-linolenic acid (DGLA) at a high purity as a constituent fatty acid of the oil. The microbial oil of the present disclosure is characterized by containing DGLA and / or by reducing undesirable constituent fatty acids. The undesirable constituent fatty acids of the microbial oil of the present disclosure include arachidonic acid (ARA) and / or long-chain saturated fatty acids.

[0010] More specifically, the microbial oils of the present disclosure are characterized by their DGLA content and their ARA content, which may further be characterized by their long chain saturated fatty acid content.

[0011] In another embodiment, the microbial oils of the present disclosure are characterized by their DGLA content and their long chain saturated fatty acid content, which may further be characterized by their ARA content.

[0012] In another embodiment, the microbial oil of the present disclosure comprises DGLA and is characterized by its content of long chain saturated fatty acids and its content of ARA, which may further be characterized by its content of DGLA.

[0013] In another embodiment, the microbial oil of the present disclosure may contain DGLA and be specified by the composition ratio of long-chain saturated fatty acid to DGLA.In this case, it may be further specified by the content of ARA, or further specified by the content of DGLA.

[0014] In another embodiment, the microbial oil of the present disclosure can contain DGLA and be specified by the composition ratio of ARA to DGLA.In this case, it can be further specified by the composition ratio of long chain saturated fatty acid to DGLA, further specified by the content ratio of ARA, or further specified by the content ratio of DGLA.

[0015] In one embodiment of the present disclosure, the long chain saturated fatty acids are C24:0, C22:0, and C20:0, and the content and composition ratio are calculated based on the total amount. In another embodiment, the long chain saturated fatty acids are C24:0 and C22:0, and the content and composition ratio are calculated based on the total amount. In yet another embodiment, the long chain saturated fatty acid is C24:0, and the content and composition ratio of C24:0 can be calculated.

[0016] The DGLA contained in the microbial oil of the present disclosure may be contained in the microbial oil at a content of more than 30% by weight, more than 31% by weight, more than 32% by weight, or more than 34% by weight as a constituent fatty acid of the oil.

[0017] In the microbial oil of the present disclosure, the content of ARA is reduced or substantially absent.In one embodiment, in the microbial oil of the present disclosure, ARA is 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.04% by weight or less, 0.03% by weight or less, or 0.01% by weight or less of the constituent fatty acids of the microbial oil.In another embodiment, in the microbial oil of the present disclosure, the composition ratio of ARA to DGLA may be 1 / 120 or less, 1 / 180 or less, 1 / 360 or less, 1 / 1000 or less, 1 / 2350 or less, or 1 / 3000 or less by weight.

[0018] The term "long-chain saturated fatty acid" refers to a saturated fatty acid having at least 20 carbon atoms, such as at least one fatty acid selected from the group consisting of arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0). One aspect of the present disclosure relates to a microbial oil having a reduced content of long-chain saturated fatty acids.

[0019] Using C24:0 as long chain saturated fatty acid, it can be specified by the content of C24:0 or the composition ratio of C24:0 to DGLA.In one aspect, the microbial oil of the present disclosure relates to a microbial oil having a C24:0 content of 8.5% by weight or less, 7.0% by weight or less, 6.0% by weight or less, 5.5% by weight or less, 5.0% by weight or less, or 4.5% by weight or less.In another aspect, the microbial oil of the present disclosure relates to a microbial oil having a C24:0 composition ratio of 1 / 4.1 or less, 1 / 4.5 or less, 1 / 5.0 or less, 1 / 6.0 or less, 1 / 7.0 or less, or 1 / 7.5 or less.

[0020] As long chain saturated fatty acid, C24:0 and C22:0 are used, and it can be specified by the total content of C24:0 and C22:0 or the total composition ratio of C24:0 and C22:0 to DGLA. In one aspect, the microbial oil of the present disclosure relates to a microbial oil in which the total content of C24:0 and C22:0 is 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, 7.5% by weight or less, 7.0% by weight or less, or 6.5% by weight or less. In another aspect, the microbial oil of the present disclosure relates to a microbial oil in which the total composition ratio of C24:0 and C22:0 to DGLA can be specified, and the composition ratio is 1 / 3.1 or less, 1 / 4.0 or less, 1 / 4.5 or less, or 1 / 5.0 or less.

[0021] As long chain saturated fatty acids, C24:0, C22:0 and C20:0 are used, and it can be specified by the content of the total amount of C24:0, C22:0 and C20:0 or the composition ratio of the total amount of C24:0, C22:0 and C20:0 to DGLA. In one aspect, the microbial oil of the present disclosure relates to a microbial oil having a composition ratio of 12.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less. In another aspect, the microbial oil of the present disclosure relates to a microbial oil having a composition ratio of the total amount of C24:0, C22:0 and C20:0 to DGLA, and the composition ratio is 1 / 3.0 or less, 1 / 3.5 or less, 1 / 4.0 or less, or 1 / 4.5 or less.

[0022] The microbial oils of the present disclosure may be crude or refined oils. In one embodiment, the microbial oils of the present disclosure do not contain a Δ5 desaturase inhibitor.

[0023] Further aspects relate to a lower alcohol ester composition containing dihomo-γ-linolenic acid ester derived from the microbial oil of the present disclosure, or a free fatty acid composition containing DGLA derived from the microbial oil of the present disclosure. The lower alcohol ester composition or free fatty acid composition containing dihomo-γ-linolenic acid produced or capable of being produced from the microbial oil of the present disclosure has the same or similar content or composition ratio of DGLA, arachidonic acid, and long chain unsaturated fatty acid as the content or composition ratio in the original microbial oil.

[0024] The microorganism used in the present disclosure is a filamentous fungus, such as a filamentous fungus of the genus Mortierella, such as Mortierella alpina or a closely related species thereof. The microorganism used in the present disclosure may be a microorganism having a mutation in the Δ5 desaturase (DELTA-5 DESATURASE: hereinafter, sometimes referred to as D5DS) gene. In one embodiment, the microorganism used in the present disclosure is a microorganism with the accession number FERM BP-02778 (strain NSM243-16).

[0025] A further aspect of the present disclosure may relate to a culture of a microorganism used for producing the microbial oil of the present disclosure.The culture of a microorganism may include a microbial cell and a culture medium thereof.The microbial cell containing the microbial oil of the present disclosure and the culture containing the microbial cell of the present disclosure have the same or similar content or composition ratio of DGLA, arachidonic acid, and long-chain unsaturated fatty acid as the content or composition ratio of the microbial oil of the present disclosure.

[0026] The microbial oil, lower alcohol ester composition, free fatty acid composition or culture according to the present disclosure can be used as a pharmaceutical, cosmetic, food, supplement, or animal feed, more specifically, as an antiallergic or anti-inflammatory agent. Thus, the present disclosure also relates to a pharmaceutical composition, cosmetic composition, antiallergic or anti-inflammatory agent comprising the microbial oil, lower alcohol ester composition, free fatty acid composition or culture according to the present disclosure.

[0027] A further aspect of the present disclosure relates to a method for producing a microbial oil. Specifically, the production method includes a step of culturing a microorganism referred to in the present disclosure in a medium. A microbial strain having a mutation in a Δ5 desaturase gene is used as a microorganism for use in the production method of the present disclosure. The mutation introduced into the Δ5 desaturase gene includes a genetic mutation resulting in a mutation of tyrosine (Y) at position 310 of SEQ ID NO: 3 to cysteine ​​(C) (hereinafter referred to as Y310C). In addition, or independently, the gene may be mutated to delete bases at positions 2206 to 2234 of SEQ ID NO: 1. In one aspect, in the method for producing a microbial oil of the present disclosure, a Δ5 desaturase inhibitor is not added to the medium in which the microorganism referred to in the present disclosure is cultured. Therefore, the culture, microbial oil, and / or microbial cells obtained by the culture do not contain the remaining amount of the Δ5 desaturase inhibitor when the Δ5 desaturase inhibitor is added.

[0028] Further process aspects relate to methods of producing the lower alcohol ester compositions or free fatty acid compositions of the present disclosure from the microbial oils of the present disclosure.

[0029] More specifically, the present disclosure may relate to the following inventions: [1] A microbial oil comprising, as constituent fatty acids of the oil, more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid, and substantially no arachidonic acid, or an arachidonic acid content of 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.04% by weight or less, 0.03% by weight or less, or 0.01% by weight or less. [2] The microbial oil according to item 1, wherein the content of C24:0 as a constituent fatty acid of the oil is 8.5% by weight or less, 7.0% by weight or less, 6.0% by weight or less, or 5.5% by weight or less. [3] The microbial oil according to item 1 or 2, wherein the total content of C24:0 and C22:0 as constituent fatty acids of the oil is 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less. [4] The microbial oil according to any one of items 1 to 3, wherein the content of C24:0, C22:0 and C20:0 as constituent fatty acids of the oil is 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less. [5] A microbial oil comprising, as constituent fatty acids of the oil, more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid, and having a C24:0 content of 8.5% by weight or less, 7.0% by weight or less, 6.0% by weight or less, or 5.5% by weight or less. [6] The microbial oil according to item 5, wherein the total content of C24:0 and C22:0 as constituent fatty acids of the oil is 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less. [7] The microbial oil according to item 5 or 6, wherein the content of C24:0, C22:0 and C20:0 as constituent fatty acids of the oil is 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less. [8] A microbial oil comprising, as constituent fatty acids of the oil, more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid, and having a combined content of C24:0 and C22:0 of ​​11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less. [9] The microbial oil according to item 8, wherein the content of C24:0, C22:0 and C20:0 as constituent fatty acids of the oil is 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less.

[10] A microbial oil comprising, as constituent fatty acids of the oil, more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid, and having a C24:0, C22:0, and C20:0 content of 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less.

[11] A microbial oil containing dihomo-gamma-linolenic acid as a constituent fatty acid of the oil, and a weight ratio of arachidonic acid to dihomo-gamma-linolenic acid as a constituent fatty acid of the oil is 1 / 120 or less, 1 / 180 or less, 1 / 360 or less, 1 / 1000 or less, 1 / 2350 or less, or 1 / 3000 or less.

[12] The microbial oil according to item 11, wherein the ratio of C24:0 to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 4.1 or less, 1 / 5.0 or less, or 1 / 6.0 or less by weight.

[13] The microbial oil according to item 11 or 12, wherein the weight ratio of the sum of C24:0 and C22:0 to dihomo-gamma-linolenic acid as constituent fatty acids of the oil is 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less.

[14] The microbial oil according to any one of items 11 to 13, wherein the ratio of long-chain saturated fatty acids to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight.

[15] A microbial oil containing dihomo-gamma-linolenic acid as a constituent fatty acid of the oil, and having a weight ratio of C24:0 to dihomo-gamma-linolenic acid as a constituent fatty acid of the oil of 1 / 4.1 or less, 1 / 5.0 or less, or 1 / 6.0 or less.

[16] The microbial oil according to item 15, wherein the total ratio of C24:0 and C22:0 to dihomo-gamma-linolenic acid as constituent fatty acids of the oil is 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less by weight.

[17] The microbial oil according to item 15 or 16, wherein the total ratio of C24:0, C22:0 and C20:0 to dihomo-gamma-linolenic acid as constituent fatty acids of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight.

[18] A microbial oil containing dihomo-gamma-linolenic acid as a constituent fatty acid of the oil, and having a weight ratio of the sum of C24:0 and C22:0 to the dihomo-gamma-linolenic acid as a constituent fatty acid of the oil of 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less.

[19] The microbial oil according to item 18, wherein the total ratio of C24:0, C22:0 and C20:0 to dihomo-gamma-linolenic acid as constituent fatty acids of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight.

[20] A microbial oil containing dihomo-gamma-linolenic acid as a constituent fatty acid of the oil, and the total constituent ratio of C24:0, C22:0 and C20:0 to the dihomo-gamma-linolenic acid as a constituent fatty acid of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight.

[21] The microbial oil according to any one of items 11 to 20, which is substantially free of arachidonic acid as a constituent fatty acid of the oil, or has an arachidonic acid content of 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.04% by weight or less, 0.03% by weight or less, or 0.01% by weight or less.

[22] The microbial oil according to any one of items 11 to 21, wherein the content of dihomo-γ-linolenic acid is greater than 30% by weight, greater than 32% by weight, or greater than 34% by weight.

[23] The microbial oil according to any one of items 1 to 22, wherein the microbial oil is an oil derived from a filamentous fungus.

[24] The microbial oil according to item 23, wherein the filamentous fungus is a filamentous fungus of the genus Mortierella.

[25] The microbial oil according to any one of items 1 to 24, wherein the microbial oil does not contain a Δ5 desaturase inhibitor.

[26] The microbial oil according to any one of items 1 to 25, wherein the microbial oil is a crude oil.

[27] A pharmaceutical composition comprising the microbial oil according to any one of items 1 to 26.

[28] An antiallergic or anti-inflammatory agent comprising the microbial oil according to any one of items 1 to 26.

[29] A lower alcohol ester composition or free fatty acid composition containing dihomo-γ-linolenic acid, which is produced or can be produced by a method comprising subjecting the microbial oil according to any one of items 1 to 26 to an ester exchange reaction or hydrolysis reaction.

[30] The composition of item 29, comprising more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid.

[31] The composition according to item 29 or 30, which is substantially free of arachidonic acid, or has an arachidonic acid content of 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.04% by weight or less, 0.03% by weight or less, or 0.01% by weight or less.

[32] The composition according to any one of items 29 to 31, wherein the content of C24:0 is 8.5% by weight or less, 7.0% by weight or less, 6.0% by weight or less, or 5.5% by weight or less.

[33] The composition according to any one of items 29 to 32, wherein the total content of C24:0 and C22:0 is 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less.

[34] The composition according to any one of items 29 to 33, wherein the content of C24:0, C22:0, and C20:0 is 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less.

[35] The composition according to any one of items 29 to 34, wherein the ratio of arachidonic acid to dihomo-γ-linolenic acid as constituent fatty acids of the oil is, by weight, 1 / 120 or less, 1 / 180 or less, 1 / 360 or less, 1 / 1000 or less, 1 / 2350 or less, or 1 / 3000 or less.

[36] The composition according to any one of items 29 to 35, wherein the ratio of C24:0 to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 4.1 or less, 1 / 5.0 or less, or 1 / 6.0 or less, by weight.

[37] The composition according to any one of items 29 to 36, wherein the total ratio of C24:0 and C22:0 to dihomo-gamma-linolenic acid as constituent fatty acids of the oil is 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less, by weight.

[38] The composition according to any one of items 29 to 37, wherein the total ratio of C24:0, C22:0 and C20:0 to dihomo-gamma-linolenic acid as constituent fatty acids of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less, by weight.

[39] The composition according to any one of items 29 to 38, which is used as a medicine, for example an antiallergic agent or an anti-inflammatory agent.

[40] Use of the microbial oil according to any one of items 1 to 26 or the composition according to any one of items 29 to 39 in the production of food, supplements, pharmaceuticals, cosmetics, or animal feed.

[41] A microbial cell containing the microbial oil according to any one of items 1 to 26.

[42] A food, supplement, medicine, cosmetic, or animal feed comprising the microbial cell according to Item 41.

[43] A culture comprising the microbial cells according to Item 41.

[44] The culture according to Item 43, comprising the microbial oil at a content of 0.4 g / L or more.

[45] Cultivating a microbial strain having a mutation in a Δ5 desaturase gene in a medium to produce a microbial oil containing dihomo-γ-linolenic acid; A method for producing a dihomo-γ-linolenic acid-containing microbial oil that is substantially free of ARA.

[46] The method according to Item 45, wherein the microbial strain has a genetic mutation resulting in an amino acid substitution of Y310C in SEQ ID NO: 3 of Δ5 desaturase.

[47] The method according to item 45 or 46, wherein the microbial strain has a deletion of bases at positions 2206 to 2234 of SEQ ID NO: 1 in the Δ5 desaturase gene.

[48] ​​The method according to any one of items 45 to 47, wherein the microbial strain is a strain of a microorganism belonging to the genus Mortierella.

[49] The method according to any one of items 45 to 48, wherein the microorganism having the accession number FERM BP-02778 is used as the microbial strain.

[50] Microorganism under accession number FERM BP-02778. Effect of the Invention

[0030] According to the present disclosure, it is possible to provide a microbial oil containing less undesirable constituent fatty acids and high-purity dihomo-gamma-linolenic acid (DGLA) than microbial oils obtained by conventional methods. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows a comparison of the nucleotide sequences of the Δ5 desaturase gene of the Mortierella alpina strain RD056399 and the Δ5 desaturase gene of the Mortierella alpina strain NSM243-16. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] (1) Microbial oil In this disclosure, unless otherwise specified, microbial oil broadly means all lipids obtained from microorganisms, and is used as a term to refer to both crude oil and refined oil without distinction in this disclosure. One example of microbial oil is oil derived from filamentous fungi, and another example is oil derived from filamentous fungi of the genus Mortierella (hereinafter, Mortierella oil). Microbial oil is obtained by culturing a microorganism in an appropriate medium and collecting it from the microbial cell by a method such as solvent extraction. Lipids include triglycerides, diglycerides, monoglycerides, phospholipids, cholesterol, etc., and lipids are mainly composed of triglycerides. Various fatty acids are included as constituent fatty acids of lipids. In the microbial oil of the present disclosure, among those constituent fatty acids, dihomo-gamma-linolenic acid (DGLA) is included as a constituent fatty acid of the oil in high purity. In this disclosure, high purity simply refers to a high content of DGLA, but may also refer to a reduction in undesirable constituent fatty acids. Therefore, the microbial oil of the present disclosure can be specified by the content of DGLA contained in the microbial oil and / or the reduction of undesirable constituent fatty acids. More specifically, the microbial oil of the present disclosure can be specified by the DGLA content and the ARA content, and can be further specified by the content of long-chain saturated fatty acids. In yet another embodiment, the microbial oil of the present disclosure can be specified by the DGLA content and the content of long-chain saturated fatty acids, and can be further specified by the ARA content. In the present disclosure, the content is not limited only to the content rate or the composition ratio to DGLA, and may simply represent the presence of a specific compound.

[0033] For convenience, the term "oil" is used in this disclosure to refer to "fat and oil". In addition, the terms "oil" and "fat and oil" mean triglycerides in the narrow sense, but in this disclosure also include oils that contain triglycerides as the main component and other lipid components such as diglycerides, monoglycerides, phospholipids, cholesterol, free fatty acids, such as crude oils. In practice, the triglyceride content is 30% by weight or more, 50% by weight or more, 70% by weight or more, or 90% by weight or more.

[0034] For convenience, the term "fatty acid" includes not only free saturated or unsaturated fatty acids themselves, but also fatty acids contained as structural units in alkyl esters, triglycerides, diglycerides, monoglycerides, phospholipids, steryl esters, etc., and can be referred to as structural fatty acids. In this disclosure, unless otherwise specified, the form of a compound containing a fatty acid may be omitted. Examples of the form of a compound containing a fatty acid include a free fatty acid form, a fatty acid alkyl ester form, a glyceryl ester form, a phospholipid form, and a steryl ester form. A compound containing the same fatty acid may be contained in an oil in a single form or as a mixture of two or more forms.

[0035] When describing fatty acids, numerical notations are sometimes used that simply represent the number of carbon atoms, the number of double bonds, and the location of the double bonds using numbers and letters, respectively. For example, a saturated fatty acid with 20 carbon atoms is described as "C20:0", and a trivalent unsaturated fatty acid with 20 carbon atoms and three double bonds in the carbon chain is described as "C20:3". For example, behenic acid can be described as "C22:0", arachidonic acid can be described as "C20:4,n-6", etc. "n-" indicates the position of the first double bond counted from the methyl end of the fatty acid, for example, "n-6" indicates that the position of the double bond is the sixth position counted from the methyl end of the fatty acid, and "n-3" indicates that the position of the double bond is the third position counted from the methyl end of the fatty acid. This method is well known to those skilled in the art, and those skilled in the art can easily identify fatty acids described according to this method.

[0036] In this disclosure, the term "process" includes not only independent processes, but also processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. In this disclosure, a numerical range indicated using "~" indicates a range including the numerical values ​​before and after it as the minimum and maximum values, respectively. In this disclosure, the content of each component in a mixture means the total content of the multiple substances present in the mixture when multiple substances corresponding to each component are present in the mixture, unless otherwise specified. In this disclosure, the terms "less than" or "less than" in relation to percentages include 0%, i.e., "not contained", unless the lower limit is specifically specified, or include a range including a value that is undetectable by current means.

[0037] The microbial oil of the present disclosure contains DGLA, and the content of DGLA is more than 30% by weight, more than 31% by weight, more than 32% by weight, or more than 34% by weight as the constituent fatty acid of the oil. The upper limit of the content of dihomo-γ-linolenic acid is not particularly limited, but from the viewpoint of microbial productivity, it is usually 70% by weight or less, or 60% by weight or less.

[0038] The undesirable constituent fatty acids of the microbial oil of the present disclosure include ARA and / or long-chain saturated fatty acids. Although ARA is classified as an essential fatty acid, it is known to cause inflammation, and it is desirable to reduce ARA from this viewpoint. It is desirable to reduce long-chain saturated fatty acids from the viewpoint of increasing the melting point of the oil.

[0039] The content of ARA is reduced in the microbial oil of the present disclosure. In one embodiment, ARA is substantially not contained in the microbial oil of the present disclosure. In yet another embodiment, in the microbial oil of the present disclosure, ARA is 0.3% by weight or less, 0.2% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.04% by weight or less, 0.03% by weight or less, or 0.01% by weight or less of the constituent fatty acids of the microbial oil. No microbial oil (particularly Mortierella oil) containing high purity DGLA (e.g., more than 30% by weight) and substantially no ARA or less than 0.05% by weight has been known so far. Therefore, by using the microbial oil of the present disclosure, it is possible to efficiently provide an oil that contains DGLA at a higher purity than conventional oils and has a low ARA content.

[0040] When C24:0 is used as a long-chain saturated fatty acid, the composition ratio of C24:0 to dihomo-γ-linolenic acid can be used as an index, and the weight ratio is 1 / 4.1 or less, 1 / 4.5 or less, 1 / 5.0 or less, 1 / 6.0 or less, 1 / 7.0 or less, or 1 / 7.5. In yet another aspect, the content of C24:0 is 8.5 wt% or less, 7.0 wt% or less, 6.0 wt% or less, 5.5 wt% or less, 5.0 wt% or less, or 4.5 wt% or less as a constituent fatty acid. In one example, the lower limit of the content of C24:0 is 0 wt%. In another example, the lower limit of the content of C24:0 is usually 1 wt% or more from the viewpoint of using a microorganism.

[0041] When C24:0 and C22:0 are used as long-chain saturated fatty acids, the total composition ratio of C24:0 and C22:0 to dihomo-γ-linolenic acid can be used as an index, and the weight ratio is 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less. In yet another aspect, the total content of C24:0 and C22:0 is 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, 7.5% by weight or less, 7.0% by weight or less, or 6.5% by weight or less as constituent fatty acids. In one example, the lower limit of the total content of C24:0 and C22:0 is 0% by weight. In another example, the lower limit of the total content of C24:0 and C22:0 is usually 1% by weight or more from the viewpoint of using microorganisms.

[0042] When the long chain saturated fatty acid is C24:0, C22:0, and C20:0, the total composition ratio of C24:0, C22:0, and C20:0 to dihomo-γ-linolenic acid can be used as an index, and is 1 / 3.0 or less, 1 / 3.5 or less, 1 / 4.0 or less, or 1 / 4.5 or less by weight. In yet another aspect, the total content of C24:0, C22:0, and C20:0 in the constituent fatty acids is 12.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less. In one example, the lower limit of the total content of C24:0, C22:0, and C20:0 is 0% by weight. As another example, the lower limit of the total content of C24:0, C22:0, and C20:0 is usually 1% by weight or more, from the viewpoint of using microorganisms.

[0043] In the present disclosure, the content ratio of DGLA and undesirable constituent fatty acid may be expressed as a weight ratio (undesirable constituent fatty acid / DGLA), or may be expressed by a weight ratio (DGLA / undesirable constituent fatty acid). As the undesirable constituent fatty acid, ARA and / or long chain saturated fatty acid may be used.

[0044] In the present disclosure, "undesirable constituent fatty acid / DGLA" or "DGLA / undesirable constituent fatty acid" refers to the weight ratio of undesirable constituent fatty acid and DGLA by analysis of the fatty acid composition contained in the oil. The fatty acid composition can be determined according to a conventional method. Specifically, the oil to be measured is esterified using a lower alcohol and a catalyst to obtain a fatty acid lower alcohol ester. The obtained fatty acid lower alcohol ester is then analyzed by gas chromatography. Peaks corresponding to each fatty acid are identified in the obtained gas chromatography, and the peak area of ​​each fatty acid is determined, for example, using the Agilent ChemStation integration algorithm (revision C.01.03

[37] , Agilent Technologies). The "peak area" refers to the ratio of the peak area of ​​each component to the total peak area, that is, the content ratio of the component of that peak determined by an analysis chart obtained by gas chromatography or thin layer chromatography / flame ionization detector (TLC / FID) of an oil having various fatty acids as constituent components. The fatty acid composition was determined by gas chromatography, for example, according to the method shown in the Examples. The lipid composition was determined by TLC / FID. Detailed conditions are shown in the Examples.

[0045] In the present disclosure, a mixture of lipids obtained by simply extracting lipids from microbial cells is referred to as "crude oil" of microbial oil. The microbial oil obtained by purifying this microbial oil to remove phospholipids and cholesterol, thereby increasing the proportion of triglycerides, is a refined microbial oil. In the present disclosure, "microbial oil" means both crude oil and refined oil unless otherwise specified. ARA and DGLA have similar properties as compounds, and it is known that it is extremely difficult to separate DGLA from ARA on a practical production scale. In the present disclosure, the content or composition ratio of DGLA and undesirable constituent fatty acids in the microbial oil can be the same or approximately the same as the content or composition ratio of DGLA and undesirable constituent fatty acids in the lower alcohol ester composition and / or free fatty acid composition obtained from the microbial oil. In particular, the weight ratio of DGLA and ARA as an undesirable constituent fatty acid is equivalent in the microbial oil and the lower alcohol ester composition and / or free fatty acid composition. In the present disclosure, it is possible to provide a microbial oil with a low content of ARA, in other words, a large difference between the contents of ARA and DGLA, at the stage of crude oil, and therefore the ability to obtain very high DGLA / ARA in the refined oil and / or chemically treated product can be significantly improved. The microbial oil of the present disclosure can be produced by culturing the microorganisms specified in the present disclosure. In one embodiment, the production process does not use a medium containing a Δ5 desaturase inhibitor. Thus, in one embodiment, the microbial oil of the present disclosure does not contain a Δ5 desaturase inhibitor.

[0046] The Δ5 desaturase inhibitors include desaturase inhibitors known in the art. The desaturase inhibitors include, for example, dioxabicyclo[3.3.0]octane derivatives. The dioxabicyclo[3.3.0]octane derivatives include, for example, 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, 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane, etc.

[0047] In this disclosure, "refined oil" refers to oil obtained after a refining process that includes a combination of some or all of the following steps to remove non-target substances, such as phospholipids and sterols: degumming, deacidification, bleaching, deodorization, etc.

[0048] In addition, the triglyceride content relative to the total amount of the microbial oil in the crude oil is 70% by weight or more, or 90% by weight or more in the microbial oil. If the triglyceride content in the microbial oil is 70% by weight or more, the hygroscopicity is not too low, and for example, good fluidity can be obtained. There is no particular limit to the upper limit of the triglyceride content in the microbial oil, but generally the weight ratio of triglyceride in the microbial oil is 99% by weight or less. The weight ratio of triglyceride in the microbial oil may be 100% by weight, that is, the microbial oil may not substantially contain components other than glyceride. Examples of fatty acids constituting the triglyceride of the microbial oil include saturated or unsaturated fatty acids having 14 to 26 carbon atoms. In the refined oil, the concentration of triglyceride can be increased by removing impurities, for example, by a known method.

[0049] The fatty acid composition of the crude oil may contain 60% by weight or less, 55% by weight or less, or 50% by weight or less of fatty acids having 18 or less carbon atoms, based on the total weight of the microbial oil. Microbial oils having a low content of fatty acids having 18 or less carbon atoms in the crude oil can be used as triglycerides without the need to adjust the fatty acid composition by removing fatty acids having 18 or less carbon atoms. Such adjustment requires low-yield methods such as wintering (low-temperature processing).

[0050] The phospholipid content in the crude oil is 10% or less, 5% or less, or 1% or less by weight based on the total weight of the microbial oil, however, some phospholipids may be present, for example, 0.1-10%, 0.5-7%, or 1-5% by weight based on the total weight of the microbial oil.

[0051] (2) Microorganisms In the present disclosure, "microorganisms" include both eukaryotes and prokaryotes, and specific examples include bacteria, actinomycetes, cyanobacteria, archaea, fungi, algae, lichens, protozoa, and the like. From the viewpoint of having DGLA producing ability, the microorganism used in the present disclosure is, for example, a filamentous fungus, and more specifically, at least one selected from the group consisting of microorganisms of the genera Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium, and Saprolegnia. The microorganism should be a microorganism capable of producing DGLA, and may be a microorganism belonging to the genus Mortierella, such as Mortierella elongata, Mortierella exigua, Mortierella hygrophila, Mortierella alpina, etc. In one embodiment, the microorganism according to the present disclosure is Mortierella alpina or a closely related species thereof, and an example thereof is the RD056399 strain.

[0052] The microorganism is a microorganism in which Δ5 desaturation activity is reduced or deleted compared to the natural state from the viewpoint of reducing the production amount of ARA (hereinafter referred to as "low Δ5 desaturation activity microorganism"). Mutations can be introduced into any of the above-mentioned microorganisms, and as an example, mutations can be introduced into microorganisms of the genus Mortierella, more specifically, Mortierella alpina or its closely related species. As an example, a mutation is induced in the Δ5 desaturase gene of the RD056399 strain. As an example of the mutation introduced into the Δ5 desaturase gene, a mutation that causes a mutation of tyrosine (Y) at position 310 of SEQ ID NO: 3 to cysteine ​​(C) (hereinafter referred to as Y310C) can be mentioned. In addition, or independently, a gene mutation may be introduced so that a splicing error occurs due to a deletion of bases at positions 2206 to 2234 of SEQ ID NO: 1. The microorganism used in the present disclosure is the microorganism with the accession number FERM BP-02778 (strain NSM243-16).

[0053] Examples of mutation techniques include physical treatments such as radiation (X-rays, gamma rays, neutron rays, etc.), ultraviolet light, and high heat treatment, as well as chemical treatments using compounds that act as mutagens. Examples of mutation sources used in the mutant strain isolation method include alkylating agents such as nitrogen mustard, methyl methanesulfonate (MMS), and N-methyl-N'-nitroso-N-nitrosoguanidine (NTG); base analogs such as 5-bromouracil; antibiotics such as mitomycin C; base synthesis inhibitors such as 6-mercaptopurine; dyes such as proflavine; carcinogens such as 4-nitroquinoline-N-oxide; and manganese chloride, potassium dichromate, nitrous acid, hydrazine, hydroxylamine, formaldehyde, and nitrofuran compounds. The form of the microorganism that is the subject of the mutation technique may be a growing microbial cell (such as a mycelium) or a spore.

[0054] (3) Microbial oil production Microbial oils can be obtained by a manufacturing method that includes producing microbial oil by culturing microorganisms known to produce lipids (hereinafter referred to as the culture / production process), and separating the obtained microbial oil from the microbial cells (hereinafter referred to as the separation process).

[0055] (3-1) Cultivation and production process The method for producing DGLA-containing lipid or microbial oil in the present disclosure comprises the step of culturing the microorganism mentioned in the present disclosure in a medium.By selecting a suitable microorganism that prefers DGLA over ARA production, for example, the mutant ARA-producing strain according to known principles, the synthesis of ARA in the microorganism can be inhibited, and the accumulation of DGLA in the microorganism can be significantly increased.

[0056] The medium used in the production process may be any medium known in the art, particularly a medium suitable for culturing filamentous fungi, particularly microorganisms of the genus Mortierella.

[0057] In the case of liquid media, any of the commonly used carbon sources can be used, including, but not limited to, glucose, fructose, xylose, saccharose, maltose, soluble starch, molasses, glycerol, mannitol, and the like. As the nitrogen source, natural nitrogen sources such as peptone, yeast extract, malt extract, meat extract, casamino acid, corn steep liquor, etc., organic nitrogen sources such as urea, etc., and inorganic nitrogen sources such as sodium nitrate, ammonium nitrate, ammonium sulfate, etc. can be used. In addition, inorganic salts such as phosphate, magnesium sulfate, iron sulfate, copper sulfate, etc., and vitamins can also be used as trace nutrient sources as necessary. When foaming occurs during culture, a foam inhibitor can be used as appropriate. Examples of the foam inhibitor that can be used include soybean oil, Adekanol LG-109, Dow1520US, Basildon86 / 013K, Mazu310R, Hodag K-60, polypropylene glycol, etc. The aqueous medium that can be used as the base material for the liquid medium is essentially water, and distilled water or purified water can be used. In one embodiment, no Δ5 desaturase inhibitor is added to the liquid medium.

[0058] These medium components are not particularly limited as long as they are at concentrations that do not impair the growth of the low Δ5 desaturation activity microorganism. In practice, the concentration of the carbon source is 0.1% by weight to 30% by weight, 1% by weight to 10% by weight, and the concentration of the nitrogen source is 0.01% by weight to 5% by weight, or 0.1% by weight to 2% by weight. The culture temperature is 5° C. to 40° C., 20° C. to 38° C., or 25° C. to 35° C. The pH of the medium is 4 to 10, or 6 to 9. The culture can be aeration culture, shaking culture, or stationary culture. The culture is usually carried out for 2 to 15 days. The aeration amount in aeration culture is the same as that usually used.

[0059] In order to promote the accumulation of DGLA, the substrate for ARA and / or DGLA production can be added to the medium.The substrate for ARA and / or DGLA production can include, but is not limited to, hydrocarbons such as tetradecane, hexadecane, octadecane, etc.; fatty acids such as tetradecanoic acid, hexadecanoic acid, octadecanoic acid, etc.; salts of these fatty acids, such as sodium salts or potassium salts; fatty acid esters; fats and oils that contain fatty acids as components, such as olive oil, soybean oil, cottonseed oil, coconut oil, etc.

[0060] There is no particular limitation on the culture vessel used in the production process, and any culture vessel generally used for culturing microorganisms can be used. The culture vessel can be appropriately selected depending on the scale of the culture.

[0061] For example, in the case of liquid culture at a scale of 1L to 50L, a stirring type culture vessel can be used as the culture vessel in order to produce a microbial oil containing DGLA with higher purity. The stirring type culture vessel has at least one stage of a disc turbine type stirring blade, and in another embodiment, the stirring type culture vessel has two stages of a disc turbine type stirring blade. In the case of a stirring type culture vessel having two stages of a disc turbine type stirring blade, the distance between the stirring blades close to the bottom surface may be short in order to efficiently stir the medium on the bottom surface of the culture vessel. For example, the installation positions of the stirring blades on the upper and lower stages can be appropriately set.

[0062] By carrying out the production process, a culture can be obtained. The culture means anything obtained by culturing, including microbial oil, microbial cells, and culture medium. The "culture containing microbial cells" means a culture in which "microbial cells" are cultured, and the state before the microbial cells are separated from the culture medium. The culture can obtain microbial cells containing microbial oil. The "microbial cells containing microbial oil" means microbial cells (biomass) that accumulate microbial oil in the microbial cells by culturing a microorganism that produces the microbial oil of the present disclosure. Both live and dead cells may be contained in the microbial cells. Dried microbial cells are also included. The expression "dried cells" means a dried product of microbial cells that is substantially free of water, and a dried product that contains residual medium components, filter aids, etc. "Substantially free of water" means a moisture content that is equal to or less than the moisture content that may make it difficult for microorganisms to survive. This amount is generally 15% by weight or less, or 10% by weight or less. According to the present disclosure, microbial oil and microbial cells that contain DGLA while reducing undesirable constituent fatty acids are provided. Among these microbial oils and microbial cells, there have been no known microbial oils or microbial cells that are substantially free of ARA or have an ARA content of less than 0.05% by weight, 0.03% by weight or less, or 0.01% by weight or less. Therefore, by using the microbial oil and microbial cells of the present disclosure, it is possible to efficiently provide an oil that contains DGLA at a higher purity than conventional oils and has a low ARA content.

[0063] (3-2) Separation process In the separation process, the microbial oil containing DGLA produced during the production process is separated from the microbial cells. The separation process includes separating the cultured microbial cells from the medium used for the culture (hereinafter referred to as the microbial cell separation process), and collecting the microbial oil containing DGLA from the cultured microbial cells (hereinafter referred to as the microbial oil collection process), that is, obtaining crude oil. In the microbial cell separation process and the microbial oil collection process, the microbial oil containing DGLA is collected from the cultured microbial cells using a separation method and an extraction method according to the culture form.

[0064] When a liquid medium is used, after the culture is completed, the cultured microbial cells can be separated from the medium by a conventional solid-liquid separation means such as centrifugation and / or filtration. The microbial cells are thoroughly washed with water and then dried, if necessary. Drying can be performed by freeze-drying, air-drying, heat-drying, etc. When the microbial cells are cultured in a solid medium, the solid medium and the microbial cells can be disrupted with a homogenizer or the like without separating the microbial cells from the medium, and the resulting disrupted material can be directly subjected to the microbial oil extraction process.

[0065] The microbial oil collection step may include extracting the dried cells obtained in the microbial cell separation step under nitrogen gas flow using an organic solvent. The organic solvents used include ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, etc. Alternatively, good results can be obtained by alternate extraction with methanol and petroleum ether or extraction with a single solvent system of chloroform-methanol-water. The organic solvent is distilled off from the extract under reduced pressure to obtain a microbial oil containing a high concentration of DGLA. When collecting triglycerides, hexane is most commonly used. Alternatively, extraction can be performed using wet microbial cells. A water-compatible solvent such as methanol or ethanol, or a water-compatible mixed solvent containing this solvent and water and / or other solvents, is used. The other procedures are the same as those described above.

[0066] The collected crude microbial oil can be refined by a method used for refining vegetable oil, fish oil, etc. Examples of the refining process of fats and oils that is usually performed include degumming, deacidification, decolorization, and deodorization. Such treatments may be performed by any method. An example of degumming is a water washing treatment. An example of deacidification treatment is a distillation treatment. An example of decolorization treatment is a treatment using activated clay, activated carbon, silica gel, etc. An example of deodorization is steam distillation, etc.

[0067] (4) Production of lower alcohol esters of fatty acids and free fatty acids from microbial oils DGLA contained as a constituent fatty acid of microbial oil can be converted into a lower alcohol ester type by using a catalyst, or into a free fatty acid type by hydrolysis. Lower alcohol esters or free fatty acids can be more easily separated from other fatty acids than triglycerides, and DGLA can be concentrated and its purity increased.

[0068] The method for producing lower alcohol esters or free fatty acids of dihomo-γ-linolenic acid in the present disclosure includes (a) obtaining free fatty acids or lower alcohol esters by hydrolysis or alcoholysis of a microbial oil, and (b) fractionating the mixture of free fatty acids or lower alcohol esters and obtaining free fatty acids or lower alcohol esters having fatty acids with at least 20 carbon atoms. The mixture or composition further comprises: (c) The method may include fractionating and purifying free fatty acids or lower alcohol esters of dihomo-γ-linolenic acid by reversed-phase column chromatography from free fatty acids or lower alcohol esters having fatty acids with a carbon number of at least 20. For example, lower alcohol dihomo-γ-linolenic acid esters or free dihomo-γ-linolenic acid may be purified or produced by producing a desired lower alcohol ester composition or free fatty acid composition, and fractionating and purifying the lower alcohol esters of dihomo-γ-linolenic acid or fractionating and purifying dihomo-γ-linolenic acid by reversed-phase column chromatography.

[0069] The method for producing a lower alcohol ester of dihomo-γ-linolenic acid in the present disclosure may include: (a) obtaining a lower alcohol ester of a fatty acid by alcoholysis of a microbial oil; (b) fractionating the mixture of lower alcohol esters of fatty acids to obtain a lower alcohol ester of a fatty acid having at least 20 carbon atoms; and (c) fractionating and purifying the lower alcohol ester of dihomo-γ-linolenic acid from the lower alcohol ester of a fatty acid having at least 20 carbon atoms by column chromatography in a reverse phase partition system.

[0070] The method for producing free fatty acids of dihomo-gamma-linolenic acid in the present disclosure may include (a) obtaining free fatty acids by hydrolysis of a microbial oil, (b) fractionating the mixture of free fatty acids to obtain free fatty acids having at least 20 carbon atoms, and (c) fractionating and purifying the free dihomo-gamma-linolenic acid from the free fatty acids having at least 20 carbon atoms by column chromatography in a reverse phase partition system.

[0071] Examples of the lower alcohol here include alcohols having 3 or less carbon atoms, particularly ethanol, methanol, etc. Examples of the lower alcohol esters of DGLA include methyl dihomo-γ-linolenate, ethyl dihomo-γ-linolenate, etc.

[0072] For example, methyl esters of fatty acids can be obtained by treating the microbial oil with 5% to 10% anhydrous methanol-hydrochloric acid, 10% to 50% BF3-methanol, etc. at room temperature for 1 to 24 hours. Ethyl esters of fatty acids can be obtained by treating the microbial oil with 1% to 20% ethanol sulfate, etc. at 25°C to 100°C for 15 to 60 minutes. Methyl esters or ethyl esters can be extracted from the reaction solution using an organic solvent such as hexane, ether, or ethyl acetate. The extract is dried with anhydrous sodium sulfate, etc., and then the organic solvent is distilled off to obtain a composition containing fatty acid esters as a main component.

[0073] In addition to the DGLA lower alcohol ester of interest, the esterification product obtained by esterification process contains other fatty acid lower alcohol esters.To isolate DGLA lower alcohol ester from the mixture of these fatty acid lower alcohol esters, distillation, rectification, column chromatography, low-temperature crystallization, urea clathrate, liquid-liquid countercurrent distribution chromatography, etc., can be used alone or in combination of two or more.The combination of distillation or rectification and column chromatography or liquid-liquid countercurrent distribution chromatography can be used.

[0074] For these methods, conventional methods can be applied. As column chromatography, column chromatography using a reversed phase partition system (ODS as one example) can be used.

[0075] To obtain free fatty acid of DGLA, as mentioned above, after producing low alcohol ester of microbial oil, refine and increase the purity of DGLA low alcohol ester, hydrolyze to obtain high purity free DGLA.To obtain free DGLA from DGLA low alcohol ester, hydrolyze with alkaline catalyst, and then carry out extraction treatment with organic solvent such as ether, ethyl acetate, etc.

[0076] Alternatively, the free fatty acid of DGLA can be obtained directly from microbial oil by hydrolysis.For example, microbial oil is decomposed with alkali, for example, 5% sodium hydroxide, at room temperature for 2-3 hours to obtain decomposition liquid, and the free fatty acid of DGLA can be extracted or purified from this decomposition liquid by the method commonly used for fatty acid extraction or purification.

[0077] The free fatty acid or lower alcohol ester of DGLA obtained by the above method is produced using the microbial oil of the present disclosure as a raw material, and therefore has a low content of ARA, which is difficult to remove in the purification process.These compositions can be produced or obtained by a method comprising subjecting the microbial oil disclosed in the present disclosure to an ester exchange reaction or hydrolysis reaction, respectively.Therefore, the content of dihomo-γ-linolenic acid and / or undesirable constituent fatty acids, particularly ARA, in the lower alcohol ester composition and free fatty acid composition of the present disclosure can be substantially the same as that in the microbial oil of the present disclosure.In this composition, ARA is substantially absent or is less than 0.05% by weight, 0.03% by weight or less, or 0.01% by weight or less.

[0078] (5) Microbial cells containing microbial oil "Microbial cells containing microbial oil" refers to a population (biomass) of microorganisms that produce microbial oil within the cells. Since the microbial cells retain the microbial oil of the present disclosure, the microbial cells have the same or similar fatty acid composition as the microbial oil. Specifically, the microbial cells contain high-purity DGLA, and undesirable constituent fatty acids are reduced, and among them, ARA is substantially absent or is 0.3 wt% or less, 0.2 wt% or less, 0.1 wt% or less, 0.05 wt% or less, 0.04 wt% or less, 0.03 wt% or less, or 0.01 wt% or less. The long-chain unsaturated fatty acids in the microbial cells are also reduced in the same manner as the microbial oil of the present disclosure.

[0079] The ratio of DGLA / undesirable constituent fatty acids in the microbial cells is the value obtained as described above. Any method that is usually used to measure the relative weight of DGLA and ARA in the microbial cells or its equivalent can be applied as the method for measuring DGLA and ARA in the microbial cells. For example, the microorganism can be recovered from the medium during growth, and esterification treatment can be performed at 25°C to 100°C for 15 to 60 minutes using 5% to 10% anhydrous methanol-hydrochloric acid, 10% to 50% BF3-methanol, 1% to 20% methanol sulfuric acid, 1% to 20% ethanol sulfuric acid, etc. Then, with or without extraction of the ester, the fatty acid composition (%) in the fatty acid can be analyzed using gas chromatography. In the case of esterification for evaluating substances other than free fatty acids, treatment with alkoxides such as sodium methoxide and sodium ethoxide at a concentration of 0.1M to 10M at 25°C to 100°C for 15 to 60 minutes can be used. When the ester is extracted after esterification, an organic solvent that is immiscible with the water-soluble component, such as hexane, can be used.

[0080] In addition, the present microorganism is a microorganism capable of providing oil that satisfies at least one, and in some cases any combination of two or more, of the conditions previously described for microbial oil, such as the triglyceride content, the fatty acid content having less than 18 carbon atoms, the phospholipid content, and the saturated fatty acid content.

[0081] (6) A culture containing microbial cells that contains microbial oil. "Culture containing microbial cells containing microbial oil" refers to a culture before the microorganisms grown by the above-mentioned method for producing microbial oil are separated from the medium. Therefore, this culture contains high-purity DGLA. In the present disclosure, the culture has a fatty acid composition that is the same or similar to that of the microbial oil of the present disclosure. Undesirable constituent fatty acids are reduced, and for example, ARA is substantially absent or is 0.3 wt% or less, 0.2 wt% or less, 0.1 wt% or less, 0.05 wt% or less, 0.04 wt% or less, 0.03 wt% or less, or 0.01 wt% or less.

[0082] In addition, considering the microbial oil in the microbial cell in the culture, the culture can contain the above-mentioned microbial oil, and the DGLA-containing oil content is 0.4g / L or more, or 0.8g / L or more.If the content of the oil containing microbial DGLA is 0.4g / L or more, it tends to obtain the advantages of reducing production cost, improving quality stability, etc.

[0083] The microorganism is grown by culturing and produces DGLA in the microbial cell. Therefore, the culture containing the microorganism during the culturing process can be directly collected to obtain a microorganism-containing culture. In addition, since microbial oil containing DGLA is produced in the microbial cell of the microorganism during the culturing process, the culture containing the microorganism during the culturing process can be directly collected, or the microorganism in the culture can be destroyed by crushing or the like, and the culture in which the microbial oil is released can be collected to obtain a microbial oil-containing culture. Furthermore, the above-mentioned matters can be directly applied to the microbial oil-containing culture and the medium contained in the microorganism-containing culture.

[0084] [Application] According to the present disclosure, the microbial oil, lower alcohol ester, free fatty acid, microbial cell, and microbial culture containing DGLA can each have a lower ratio of undesirable constituent fatty acid to DGLA than those previously known. Therefore, the microbial oil, lower alcohol ester, free fatty acid, microbial cell, and microbial culture containing DGLA according to the present disclosure are all extremely useful for applications requiring high purity DGLA or applications requiring reduction of undesirable constituent fatty acid, especially ARA. Examples of such applications include food, supplements, medicines, cosmetics, animal feed, and the like. Thus, the present disclosure relates to a pharmaceutical composition, cosmetic composition, food, supplement, or animal feed containing the microbial oil, lower alcohol ester, free fatty acid, microbial cell, or culture according to the present disclosure. In another aspect, the present disclosure also relates to a microbial oil, lower alcohol ester, free fatty acid, microbial cell, or culture for use as a medicine. The use of the present disclosure as a medicine, cosmetic, food, supplement, or animal feed can be used for the prevention, treatment, or remission of inflammation or allergic disease. In yet another aspect, the present disclosure relates to a method for treating or preventing an inflammatory or allergic disease, comprising administering to a subject a microbial oil, a lower alcohol ester composition, a free fatty acid composition, a microbial cell or a culture according to the present disclosure, The method comprises administering to a subject suffering from or at risk of suffering from an inflammatory or allergic disease, a microbial oil, a lower alcohol ester composition, a free fatty acid composition, a microbial cell or a culture according to the present disclosure.

[0085] As described above, pharmaceuticals or cosmetics that contain or consist of microbial oils, lower alcohol ester compositions, free fatty acid compositions, microbial cells or cultures are usually administered topically or orally. The pharmaceuticals or cosmetics of the present disclosure may be administered topically (e.g., transdermally, intravenously, subcutaneously, intramuscularly, intraperitoneally, or mucosally). The inflammatory disease or allergic disease to be treated, prevented, or ameliorated may include any inflammation of the skin, and examples thereof include, but are not limited to, atopic dermatitis, allergic contact dermatitis (ACD), primary irritant dermatitis (ICD), photocontact dermatitis, systemic contact dermatitis, rheumatism, psoriasis, lupus, atopic eczema, contact dermatitis, psoriasis, dermatitis associated with uremic pruritus, contact dermatitis, dry eczema, seborrheic dermatitis, sweating disorder, discoid eczema, venous eczema, herpetic dermatitis, neurodermatitis, autoimmune disease, and autoeczemaization. The skin inflammation may be inflammation accompanied by at least one symptom selected from the group consisting of rash, eczema, hives, blisters, wheals, redness, skin edema (swelling), itching, dryness, crusting, peeling, blistering, cracking, oozing, and bleeding.

[0086] Skin inflammation can be skin inflammation caused by skin exposure to electromagnetic radiation.Electromagnetic radiation includes radio waves, microwaves, terahertz waves, sunlight (e.g. infrared rays, visible rays, ultraviolet rays), X-rays and gamma rays.Electromagnetic radiation includes infrared rays, visible rays, ultraviolet rays, X-rays and gamma rays, and in one embodiment refers to ultraviolet rays, X-rays and gamma rays.Therefore, the medicine or cosmetic can be used to treat sunburn or for the treatment of sunburn.

[0087] In some cases, the microbial oils, lower alcohol ester compositions, free fatty acid compositions, microbial cells, and microbial-containing cultures of the present disclosure may be administered in combination with any other therapeutic agent (e.g., corticosteroids, etc.) used in the medical applications described above.

[0088] A medicinal product for the treatment of an inflammatory / allergic disease should be understood to mean a medicinal product for suppressing or alleviating one or more symptoms when a symptom due to an inflammatory / allergic disease is found or suspected. On the other hand, a medicinal product for the prevention of an inflammatory / allergic disease is a medicinal product used to suppress the onset of one or more symptoms expected or anticipated due to an inflammatory / allergic disease by administering it in advance. However, the terms "medicinal product for treatment" and "medicinal product for prevention" should take into account complex or general aspects such as the timing of use and / or the symptoms to be treated / prevented when used in accordance with clinical practice, and should not be applied in a restrictive manner. EXAMPLES

[0089] The present disclosure will be described in detail below with reference to examples. However, the present disclosure is not limited thereto. Unless otherwise specified, "%" is based on mass. In the following Examples section, unless otherwise specified, the terms "bacterial cells" and "bacterial liquid" refer to a collection of bacterial cells or bacterial liquid, and correspond to biomass in this disclosure.

[0090] [Example 1] (Obtaining strain RD056399 and obtaining and storing spores) Among the screening strains provided by the National Institute of Technology and Evaluation (NITE) Biotechnology Center, Mortierella alpina ( Mortierella alpina The RD056399 strain, which is a closely related species of the RD056399 strain, was obtained. Based on the results of the 18S rRNA gene sequence and the internal transcribed spacer (ITS) region 1 and 2 gene sequence of the RD056399 strain, M.Alpina ( M. alpina) It was confirmed that.

[0091] The RD056399 strain was cultured at 25°C for 7 days in a slant of Czapek solution agar medium (adjusted to pH 6.0 and sterilized) shown in Table 1 prepared in a test tube. After confirming the growth of mycelium, the test tube was stored in a refrigerator for 30 to 90 days to promote spore formation. Sterile water was added to the test tube and thoroughly stirred to prepare a spore suspension. The spore suspension was appropriately diluted and applied to potato dextrose agar medium (adjusted to pH 6.3 and sterilized) (hereinafter referred to as PDA medium) shown in Table 2, and cultured at 25°C for 3 days. The number of colonies formed on the PDA medium was counted, and the number of spores in the spore suspension was calculated, which was approximately 1 x 10 6 spores / ml. Next, this spore suspension was diluted 100-fold with sterile water. Then, the 100-fold diluted spore suspension, glycerin, and water (water and glycerin were mixed and sterilized in advance) were mixed in a ratio (volume ratio) of "100-fold diluted spore suspension:glycerin:water=1:1:8". 1 ml of this mixture was placed in a 1.2 ml sterile ceramic tube and frozen and stored in an ultra-low temperature freezer at -80°C (hereinafter referred to as frozen storage liquid). When using the RD056399 strain for cultivation, the frozen storage liquid was quickly thawed in a 25°C incubator and inoculated into the medium.

[0092] [Table 1]

[0093] [Table 2]

[0094] [Example 2] (Mutation processing) 0.1 ml of frozen stock of RD056399 strain was applied to PDA medium. The PDA medium to which the frozen stock was applied was irradiated with ultraviolet light using a handy UV lamp SUV-16 (ASONE) to induce genetic mutations so as to achieve a mortality rate of 99% or more. To achieve a mortality rate of 99%, the PDA medium was placed 14 cm away from the SUV-16 and UV was irradiated for 5 to 10 seconds.

[0095] After mutagenesis, the strains were cultured at 28°C for 2 to 5 days. Each colony formed on the PDA medium was picked with a sterile inoculation loop, streaked onto a fresh PDA medium, and cultured at 28°C for 5 days. This process was repeated twice to isolate each mutated strain.

[0096] [Example 3] (Screening of mutated strains and obtaining strain NSM243-16) Each of the mutated strains obtained in Example 2 was collected with a sterile inoculation loop and inoculated into a sterile 500-ml Erlenmeyer flask containing 100 ml of GY21 liquid medium (adjusted to pH 6.3 and sterilized) shown in Table 3. The flask was cultured at 28°C for 5 days with reciprocal shaking at 100 rpm. A thermostatic tank-type shaking incubator TXY-25R-2F (manufactured by Takasaki Scientific Instruments Co., Ltd.) was used for the culture. Then, 100 ml of each of the obtained cultured bacterial cell suspensions was passed through a polyester net with an average mesh size of 0.1 mm, dehydrated, and the cultured bacterial cells were collected. The collected cultured bacterial cells were frozen at -80°C and then subjected to a freeze-dryer VA-140S (manufactured by TAITEC Co., Ltd.) to obtain freeze-dried bacterial cells of the mutated strains.

[0097] Total lipids (microbial oil) were obtained from the freeze-dried cells, and then converted to fatty acid methyl esters. Specifically, total lipids were extracted from the freeze-dried cells with chloroform:methanol (2:1, v / v) according to the method of Folch et al. (J. Biol. Chem. 226:497-509 (1957)). The total lipids obtained were methyl-esterified to obtain fatty acid methyl esters (FAMEs). The obtained FAMEs were subjected to FAME analysis by gas chromatography. The gas chromatograph conditions were set as follows: Column: DB-WAX 0.530 mm x 30 m, film thickness 1.00 μm (Agilent Technologies, Inc.) Carrier gas conditions: Helium 1.0ml / min, separation ratio 100:1 Column temperature conditions: 140℃ for 5 minutes, increase to 240℃ at 4℃ / min, and then 240℃ for 10 minutes Detection: FID Detector temperature: 260℃ ·Inlet temperature: 250℃ ·Injection volume: 1μL

[0098] As a result of FAME analysis, it was possible to confirm multiple mutant strains (hereinafter referred to as DGLA-producing strains) in which the ARA concentration in the total fatty acids was significantly reduced and the DGLA concentration was increased. The obtained DGLA-producing strains were cultured according to the method described in Example 1, and a frozen storage solution was prepared. One of them was named NSM243-16 strain and deposited on September 11, 2018 at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (NITE) (Room 112, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the accession number FERM BP-02778.

[0099] [Table 3]

[0100] [Example 4] (Analysis of the DELTA-5 DESATURASE gene) Total genomic DNA was extracted from the RD0596399 and NSM243-16 strains cultured by the method described in Example 3 using a DNeasy UltraClean Microbial Kit (QIAGEN). According to the report by Abe et al. (J. Biochem. Bioengineer. 99, 296 (2005)), the obtained genomic DNA was used as a template to amplify the DELTA-5 DESATURASE (hereinafter referred to as D5DS) gene by PCR. The nucleotide sequence of the obtained PCR product was determined by the Sanger method, and SEQ ID NO: 1 (D5DS gene sequence of RD0596399 strain) and SEQ ID NO: 2 (D5DS gene sequence of NSM243-16 strain), as well as SEQ ID NO: 3 (D5DS amino acid sequence of RD0596399 strain) and SEQ ID NO: 4 (D5DS amino acid sequence of NSM243-16 strain) were obtained (Table 4). The base sequence shown in SEQ ID NO:1 was searched using the BLAST algorithm by Karlin and Altschul (Pro. Natl. Acad. Sci. 90, 5873 (1993) (http: / / www.ncbi.nlm.nih.gov)). M. alpina It showed 92% identity with the D5DS gene sequence of the 1S-4 strain (Accession number AB188307).

[0101] Next, by comparing the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 shown in FIG. 1, it was confirmed that five point mutations and deletion of bases from 2206 to 2234 occurred in SEQ ID NO: 2. It was confirmed that the point mutation at the 1764th base replaces the 310th tyrosine in the amino acid sequence of the D5DS enzyme (SEQ ID NO: 3) with a cysteine. It was confirmed that the remaining four point mutations exist within an intron. Furthermore, it was confirmed that the deletion of bases from 2206 to 2234 causes a splicing error. It was believed that these mutations caused the D5DS of the NSM243-16 strain to lose its function.

[0102] [Table 4]

[0103] [Example 5] (Production of microbial oil containing dihomo-γ-linolenic acid 1) 0.1 ml of the frozen stock of the NSM243-16 strain was inoculated into a sterile 500 ml Erlenmeyer flask containing 100 ml of the GY21 liquid medium shown in Table 3. The flask was cultured at 28°C for 5 days with reciprocal shaking at 100 rpm. A thermostatic shaking incubator TXY-25R-2F (manufactured by Takasaki Scientific Instruments Co., Ltd.) was used for the culture. The resulting cultured bacterial suspension was used as a preculture solution to be inoculated into a fermentation tank.

[0104] A 5-L fermenter was charged with 2.5 L of the soybean powder liquid medium shown in Table 5 (adjusted to pH 6.0 and sterilized), and inoculated with 1% (v / v) of the preculture solution. The culture was carried out for 2 days under the conditions of a culture temperature of 26-35°C, an agitation speed of 400-800 rpm, an aeration amount of 1.25-3.50 L / min, an internal gauge pressure of 0.05 MPa, and no pH adjustment. Then, 1 L of the feed liquid shown in Table 6 was repeatedly added so that the glucose concentration in the medium was 6% by weight or less. After the entire amount of 1 L of the feed liquid was added, the culture was further carried out until the glucose concentration in the medium was 1 g / L or less. During the culture, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the agitation speed was appropriately increased with an upper limit of 800 rpm. And / or, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the aeration amount was appropriately increased with an upper limit of 3.50 L / min. If a large amount of foaming occurred during the culture, a foam inhibitor (Adekanol LG-109) was appropriately added. The glucose concentration in the medium was measured using Glucose C-II-Test Wako (Wako Pure Chemical Industries, Ltd.).

[0105] 26 days after the start of cultivation in the fermenter, 3.5 L of the cultured bacterial suspension in the fermenter was sterilized using an autoclave, and after cooling, it was passed through a polyester net with an average mesh size of 0.1 mm and dehydrated. The dehydrated bacterial cells were washed twice with 2.5 L of distilled water. The cultured bacterial cells were then passed through a polyester net with an average mesh size of 0.1 mm and dehydrated to recover the cultured bacterial cells. The recovered cultured bacterial cells were dried at 105°C for more than 2 hours to obtain heat-dried bacterial cells.

[0106] Total lipids (microbial oil) were obtained from the obtained heat-dried microbial cells in the same manner as in Example 3, and then converted into fatty acid methyl esters. The obtained FAMEs were subjected to FAME analysis by gas chromatography in the same manner as in Example 3. The fatty acid composition (wt%) of each fatty acid obtained from the NSM243-16 strain is shown in Table 7. In addition, "others" in the table indicates the total concentration of other fatty acids. Furthermore, the composition ratio of each fatty acid calculated from the fatty acid composition is shown in Table 8. The concentrations of each fatty acid in the total fatty acids produced by the NSM243-16 strain were 33.59% by weight for DGLA, 0.00% by weight for ARA, 0.88% by weight for C20:0, 2.05% by weight for C22:0, and 4.26% by weight for C24:0. The weight ratio of C24:0 to DGLA was 1 / 7.9. The total weight ratio of C22:0 and C24:0 to DGLA was 1 / 5.3. The total weight ratio of C24:0, C22:0, and C20:0 to DGLA was 1 / 4.7.

[0107] [Table 5]

[0108] [Table 6]

[0109] [Table 7]

[0110] [Table 8]

[0111] [Example 6] (Production of microbial oil containing dihomo-γ-linolenic acid 2) 0.1 ml of the frozen stock of the NSM243-16 strain was inoculated into a sterile 500 ml Erlenmeyer flask containing 100 ml of the GY21 liquid medium shown in Table 3. The flask was cultured at 28°C for 5 days with reciprocal shaking at 100 rpm. A thermostatic shaking incubator TXY-25R-2F (manufactured by Takasaki Scientific Instruments Co., Ltd.) was used for the culture. The resulting cultured bacterial suspension was used as a preculture solution to be inoculated into a fermentation tank.

[0112] A 5-L fermenter was charged with 2.5 L of the soybean powder liquid medium shown in Table 5 (adjusted to pH 6.0 and sterilized), and inoculated with 1% (v / v) of the preculture solution. The culture was carried out for 2 days under the conditions of a culture temperature of 26°C, an agitation speed of 400 to 800 rpm, an aeration amount of 1.25 to 3.50 L / min, an internal gauge pressure of 0.05 MPa, and no pH adjustment. Then, 1 L of the feed liquid shown in Table 6 was repeatedly added so that the glucose concentration in the medium was 6% by weight or less. After the entire amount of 1 L of the feed liquid was added, the culture was further carried out until the glucose concentration in the medium was 1 g / L or less. During the culture, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the agitation speed was appropriately increased with an upper limit of 800 rpm. And / or, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the aeration amount was appropriately increased with an upper limit of 3.50 L / min. If a large amount of foaming occurred during the culture, a foam inhibitor (Adekanol LG-109) was appropriately added. The glucose concentration in the medium was measured using Glucose C-II-Test Wako (Wako Pure Chemical Industries, Ltd.).

[0113] 20 days after the start of cultivation in the fermenter, 3.5 L of the cultured bacterial suspension in the fermenter was sterilized using an autoclave, and after cooling, it was passed through a polyester net with an average mesh size of 0.1 mm and dehydrated. The dehydrated bacterial cells were washed twice with 2.5 L of distilled water. The cultured bacterial cells were then passed through a polyester net with an average mesh size of 0.1 mm and dehydrated to recover the cultured bacterial cells. The recovered cultured bacterial cells were dried at 105°C for more than 2 hours to obtain heat-dried bacterial cells.

[0114] Total lipids (microbial oil) were obtained from the obtained heat-dried microbial cells in the same manner as in Example 3, and then converted into fatty acid methyl esters. The obtained FAMEs were subjected to FAME analysis by gas chromatography in the same manner as in Example 3. The fatty acid composition (wt%) of each fatty acid obtained from the NSM243-16 strain is shown in Table 9. In addition, "others" in the table indicates the total concentration of other fatty acids. Furthermore, the composition ratio of each fatty acid calculated from the fatty acid composition is shown in Table 10. The concentrations of each fatty acid in the total fatty acids produced by the NSM243-16 strain were 34.01% by weight for DGLA, 0.00% by weight for ARA, 0.81% by weight for C20:0, 1.89% by weight for C22:0, and 4.34% by weight for C24:0. The weight ratio of C24:0 to DGLA was 1 / 7.8. The total weight ratio of C22:0 and C24:0 to DGLA was 1 / 5.5. The total weight ratio of C24:0, C22:0, and C20:0 to DGLA was 1 / 4.8.

[0115] [Table 9]

[0116] [Table 10]

[0117] [Example 7] (Production of microbial oil containing dihomo-γ-linolenic acid 3) 0.1 ml of the frozen stock of the NSM243-16 strain was inoculated into a sterile 500 ml Erlenmeyer flask containing 100 ml of the GY21 liquid medium shown in Table 3. The flask was cultured at 28°C for 5 days with reciprocal shaking at 100 rpm. A thermostatic shaking incubator TXY-25R-2F (manufactured by Takasaki Scientific Instruments Co., Ltd.) was used for the culture. The resulting cultured bacterial suspension was used as a preculture solution to be inoculated into a fermentation tank.

[0118] A 5-L fermenter was charged with 2.5 L of the soybean powder liquid medium shown in Table 5 (adjusted to pH 6.0 and sterilized), and inoculated with 1% (v / v) of the preculture solution. The culture was carried out for 2 days under the conditions of a culture temperature of 26°C, an agitation speed of 400 to 800 rpm, an aeration amount of 1.25 to 3.50 L / min, an internal gauge pressure of 0.05 MPa, and no pH adjustment. Then, 1 L of the feed liquid shown in Table 6 was repeatedly added so that the glucose concentration in the medium was 6% by weight or less. After the entire amount of 1 L of the feed liquid was added, the culture was continued until the glucose concentration in the medium was 1 g / L or less. During the culture, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the agitation speed was appropriately increased with an upper limit of 800 rpm. And / or, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the aeration amount was appropriately increased with an upper limit of 3.50 L / min. At the start of the culture, 2.5 g of soybean oil and 1.0 g of Adekanol LG-109 were added to the medium to prevent foaming, and the culture was started. When a large amount of foaming occurred during the culture, a foaming inhibitor (Adekanol LG-109) was added as needed. The glucose concentration in the medium was measured using Glucose C-II-Test Wako (Wako Pure Chemical Industries, Ltd.).

[0119] Seventeen days after the start of cultivation in the fermenter, 3.5 L of the cultured bacterial suspension in the fermenter was sterilized using an autoclave, and after dissipating heat, it was passed through a polyester net with an average mesh size of 0.1 mm and dehydrated. The dehydrated bacterial cells were washed twice with 2.5 L of distilled water. The cultured bacterial cells were then passed through a polyester net with an average mesh size of 0.1 mm and dehydrated to recover the cultured bacterial cells. The recovered cultured bacterial cells were dried at 105°C for more than 2 hours to obtain heat-dried bacterial cells.

[0120] Total lipids (microbial oil) were obtained from the obtained heat-dried microbial cells in the same manner as in Example 3, and then converted into fatty acid methyl esters. The obtained FAMEs were subjected to FAME analysis by gas chromatography in the same manner as in Example 3. The fatty acid composition (wt%) of each fatty acid obtained from the NSM243-16 strain is shown in Table 11. In addition, "others" in the table indicates the total concentration of other fatty acids. Furthermore, the composition ratio of each fatty acid calculated from the fatty acid composition is shown in Table 12. The concentrations of each fatty acid in the total fatty acids produced by the NSM243-16 strain were 31.27% by weight for DGLA, 0.00% by weight for ARA, 0.77% by weight for C20:0, 1.92% by weight for C22:0, and 5.16% by weight for C24:0. The weight ratio of C24:0 to DGLA was 1 / 6.1. The total weight ratio of C22:0 and C24:0 to DGLA was 1 / 4.4. The total weight ratio of C24:0, C22:0, and C20:0 to DGLA was 1 / 4.0.

[0121] [Table 11]

[0122] [Table 12]

[0123] [Comparative Example] 0.1 ml of the frozen stock of the SAM1860 strain was inoculated into a sterile 500 ml Erlenmeyer flask containing 100 ml of the GY21 liquid medium shown in Table 3. The flask was cultured at 28°C for 5 days with reciprocal shaking at 100 rpm. A thermostatic tank-type shaking incubator TXY-25R-2F (manufactured by Takasaki Scientific Instruments Co., Ltd.) was used for the culture. The resulting cultured cell suspension was used as a preculture solution to be inoculated into the fermentation tank.

[0124] A 5-L fermenter was charged with 2.5 L of the soybean powder liquid medium shown in Table 5 (adjusted to pH 6.0 and sterilized), and inoculated with 1% (v / v) of the preculture solution. The culture was carried out for 2 days under the conditions of a culture temperature of 26°C, an agitation speed of 400 to 800 rpm, an aeration amount of 1.25 to 3.50 L / min, an internal gauge pressure of 0.05 MPa, and no pH adjustment. Then, 1 L of the feed liquid shown in Table 6 was repeatedly added so that the glucose concentration in the medium was 6% by weight or less. After the entire amount of 1 L of the feed liquid was added, the culture was further carried out until the glucose concentration in the medium was 1 g / L or less. During the culture, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the agitation speed was appropriately increased with an upper limit of 800 rpm. And / or, if the dissolved oxygen concentration in the medium fell to 10% or less (dissolved oxygen concentration at the start of culture was taken as 100%), the aeration amount was appropriately increased with an upper limit of 3.50 L / min. If a large amount of foaming occurred during the culture, a foam inhibitor (Adekanol LG-109) was appropriately added. The glucose concentration in the medium was measured using Glucose C-II-Test Wako (Wako Pure Chemical Industries, Ltd.).

[0125] Sixteen days after the start of cultivation in the fermenter, 3.5 L of the cultured bacterial suspension in the fermenter was sterilized using an autoclave, and after cooling, it was passed through a polyester net with an average mesh size of 0.1 mm and dehydrated. The dehydrated bacterial cells were washed twice with 2.5 L of distilled water. The cultured bacterial cells were then passed through a polyester net with an average mesh size of 0.1 mm and dehydrated to recover the cultured bacterial cells. The recovered cultured bacterial cells were dried at 105°C for more than 2 hours to obtain heat-dried bacterial cells.

[0126] Total lipids (microbial oil) were obtained from the obtained heat-dried microbial cells in the same manner as in Example 3, and then converted into fatty acid methyl esters. The obtained FAMEs were subjected to FAME analysis by gas chromatography in the same manner as in Example 3. The fatty acid composition (wt%) of each fatty acid obtained from the SAM1860 strain is shown in Table 13. In addition, "others" in the table indicates the total concentration of other fatty acids. Furthermore, the composition ratio of each fatty acid calculated from the fatty acid composition is shown in Table 14. The concentrations of each fatty acid in the total fatty acids produced by the SAM1860 strain were 35.86% by weight for DGLA, 0.33% by weight for ARA, 0.94% by weight for C20:0, 2.96% by weight for C22:0, and 8.94% by weight for C24:0. The weight ratio of C24:0 to DGLA was 1 / 4.0. The total weight ratio of C22:0 and C24:0 to DGLA was 1 / 3.0. The total weight ratio of C24:0, C22:0, and C20:0 to DGLA was 1 / 2.8.

[0127] [Table 13]

[0128] [Table 14] [Accession number]

[0129] FERM BP-02778

Claims

1. A microbial oil comprising, as a constituent fatty acid of the oil, more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid, and having a C24:0 content of 8.5% by weight or less, 7.0% by weight or less, 6.0% by weight or less, or 5.5% by weight or less.

2. The microbial oil according to claim 1, wherein the total content of C24:0 and C22:0 as constituent fatty acids of the oil is 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less.

3. The microbial oil according to claim 1 or 2, wherein the content of C24:0, C22:0 and C20:0 as constituent fatty acids of the oil is 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less.

4. A microbial oil comprising, as a constituent fatty acid of the oil, more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid, and having a total content of C24:0 and C22:0 of ​​11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, 8.0% by weight or less, or 7.5% by weight or less.

5. The microbial oil according to claim 4, wherein the content of C24:0, C22:0 and C20:0 as constituent fatty acids of the oil is 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less.

6. A microbial oil comprising, as constituent fatty acids of the oil, more than 30% by weight, more than 32% by weight, or more than 34% by weight of dihomo-gamma-linolenic acid, and having a C24:0, C22:0, and C20:0 content of 12.0% by weight or less, 11.0% by weight or less, 10.0% by weight or less, 9.0% by weight or less, or 8.0% by weight or less.

7. A microbial oil comprising dihomo-γ-linolenic acid as a constituent fatty acid of the oil, and a weight ratio of arachidonic acid to dihomo-γ-linolenic acid as a constituent fatty acid of the oil of 1 / 120 or less, 1 / 180 or less, 1 / 360 or less, 1 / 1000 or less, 1 / 2350 or less, or 1 / 3000 or less.

8. The microbial oil according to claim 7, wherein the ratio of C24:0 to dihomo-γ-linolenic acid as a constituent fatty acid of the oil is 1 / 4.1 or less, 1 / 5.0 or less, or 1 / 6.0 or less by weight.

9. The microbial oil according to claim 7 or 8, wherein the total composition ratio of C24: 0 and C22: 0 to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less by weight.

10. The total composition ratio of C24: 0, C22: 0 and C20: 0 to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight. The microbial oil according to any one of claims 7 to 9.

11. A microbial oil comprising dihomo-γ-linolenic acid as a constituent fatty acid of the oil, and a weight ratio of C24:0 to dihomo-γ-linolenic acid as a constituent fatty acid of the oil being 1 / 4.1 or less, 1 / 5.0 or less, or 1 / 6.0 or less.

12. The microbial oil according to claim 11, wherein the total composition ratio of C24: 0 and C22: 0 to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less by weight.

13. The total composition ratio of C24: 0, C22: 0 and C20: 0 to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight. The microbial oil according to claim 11 or 12.

14. A microbial oil comprising dihomo-γ-linolenic acid as a constituent fatty acid of the oil, and a total composition ratio of C24:0 and C22:0 to the dihomo-γ-linolenic acid as a constituent fatty acid of the oil, which is 1 / 3.1 or less, 1 / 4.0 or less, or 1 / 5.0 or less by weight.

15. The microbial oil according to claim 14, wherein the total composition ratio of C24: 0, C22: 0 and C20: 0 to dihomo-γ-linolenic acid as constituent fatty acids of the oil is 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight.

16. A microbial oil comprising dihomo-γ-linolenic acid as a constituent fatty acid of the oil, and a total composition ratio of C24:0, C22:0 and C20:0 to the dihomo-γ-linolenic acid as a constituent fatty acid of the oil, being 1 / 3.0 or less, 1 / 3.9 or less, or 1 / 4.8 or less by weight.

17. Cultivating a microbial strain having a mutation in a Δ5 desaturase gene in a medium to produce a dihomo-γ-linolenic acid-containing microbial oil; The method for producing a dihomo-γ-linolenic acid-containing microbial oil, which is substantially free of ARA, comprises:

18. 18. The method of claim 17, wherein the microbial strain has a genetic mutation resulting in an amino acid substitution of Y310C of SEQ ID NO: 3 in the Δ5 desaturase enzyme.

19. The method according to claim 17 or 18, wherein the microbial strain has a deletion of bases at positions 2206 to 2234 of SEQ ID NO: 1 in the Δ5 desaturase gene.

20. The method according to any one of claims 17 to 19, wherein the microbial strain is a strain of a microorganism belonging to the genus Mortierella.

21. The method according to any one of claims 17 to 20, wherein the microorganism having the accession number FERM BP-02778 is used as the microbial strain.

22. Microorganism with accession number FERM BP-02778.

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