Method for preparing microbial oil containing dihomo-γ-linolenic acid

Abstract

The present invention provides a method for preparing a microbial oil containing dihomo-γ-linolenic acid, the method comprising: adding at least two types of Δ5 desaturase inhibitors to a liquid medium; and culturing a microorganism having reduced or lost Δ5 desaturase activity in the liquid medium to produce the microbial oil containing dihomo-γ-linolenic acid. The content of arachidonic acid in the microbial oil containing dihomo-γ-linolenic acid obtained by the preparation method of the present invention is lower than that in the oil obtained by the conventional method.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number: 201480066445.8, the filing date: December 4, 2014, and the invention title "Microbial oil containing dihomo-γ-linolenic acid and microbial biomass containing dihomo-γ-linolenic acid". Technical Field

[0002] The present invention relates to a microbial oil containing dihomo-γ-linolenic acid (hereinafter also referred to as DGLA), a microbial biomass containing dihomo-γ-linolenic acid, and relates to its preparation method and uses. Background Art

[0003] DGLA (8,11,14-eicosatrienoic acid) is one of the fatty acid components in fish oil, seaweed, etc. In microorganisms such as Mortierella alpina, DGLA is known to be produced as a precursor of arachidonic acid (hereinafter also referred to as ARA). However, only a small amount of DGLA is produced in microorganisms containing triglyceride, diglyceride, monoglyceride, phospholipid, and sterol as lipid components. DGLA and ARA are fatty acids with similar chemical properties. Therefore, it is difficult to separate DGLA from ARA.

[0004] Techniques for reducing the amount of ARA produced in microorganisms in order to produce DGLA in an effective manner have been proposed.

[0005] For example, Japanese Patent Application Laid-Open No. (JP-A) H5-091887 discloses a method for preparing DGLA or a lipid containing DGLA, which includes culturing a microorganism having the ability to produce arachidonic acid but having reduced or lost Δ5 desaturation activity to produce DGLA or a lipid containing DGLA, and recovering DGLA or a lipid containing DGLA. JP-A No. H5-091887 also discloses culturing a microorganism having the ability to produce arachidonic acid but having reduced or lost Δ5 desaturation activity in the presence of a Δ5 desaturase inhibitor, such as sesamin or the like.

[0006] In addition, WO 2005 / 083101 discloses a method for preparing a phospholipid containing long-chain polyunsaturated fatty acids, such as arachidonic acid and DGLA, as a constituent component. The method includes a step of extracting phospholipids from defatted cells obtained by extracting triglyceride-containing oil / fat from the cells of a lipid-producing microorganism that produces a lipid containing long-chain polyunsaturated fatty acids as a constituent component.

[0007] Despite these prior art disclosures, due to the technical difficulties in obtaining a product with satisfactory and practical quality, the commercial production of DGLA-rich microbial oils has scarcely emerged to date. SUMMARY OF THE INVENTION

[0008] PROBLEMS TO BE SOLVED BY THE INVENTION

[0009] There is an increasing demand for higher purity oils containing DGLA, and the above-mentioned technologies are no longer able to meet this demand.

[0010] An object of the present invention is to provide microbial oils and microbial biomass that can be used to effectively obtain oils containing dihomo-γ-linolenic acid, wherein the arachidonic acid content of the oils containing dihomo-γ-linolenic acid is lower than that of the oils obtained by conventional methods, and to provide corresponding preparation methods and uses thereof.

[0011] MEANS FOR SOLVING THE PROBLEM

[0012] The present invention provides the following aspects.

[0013] The first aspect is a microbial oil comprising dihomo-γ-linolenic acid as a fatty acid component of the oil, and the microbial oil has a weight ratio of arachidonic acid to dihomo-γ-linolenic acid (arachidonic acid / dihomo-γ-linolenic acid) of less than 1 / 13.

[0014] The weight ratio of arachidonic acid / dihomo-γ-linolenic acid can be less than or equal to 1 / 15, preferably less than or equal to 1 / 20.

[0015] Desirably, the triglyceride content of the microbial oil is greater than or equal to 70% by weight, more preferably greater than or equal to 90% by weight. It may contain, for example, 0.1% by weight - 10% by weight of phospholipids.

[0016] The content of saturated fatty acids in the oil desirably does not exceed 40% by weight.

[0017] The microbial oil can be a crude oil or a refined oil. In the crude oil, preferably the triglyceride content is greater than or equal to 90% by weight. In the crude oil, the weight ratio of arachidonic acid to dihomo-γ-linolenic acid desirably is less than or equal to 1 / 15, or any other value disclosed herein for this ratio.

[0018] When the microbial oil is a refined oil, preferably the triglyceride content is greater than or equal to 90% by weight. In the refined oil, the weight ratio of arachidonic acid to dihomo-γ-linolenic acid desirably is less than or equal to 1 / 20, or any other value disclosed herein for this ratio.

[0019] Another aspect is a lower alcohol ester composition containing dihomo-γ-linolenic acid esters, or a free fatty acid composition containing dihomo-γ-linolenic acid, said lower alcohol ester composition or said free fatty acid composition being produced or obtainable by a method comprising subjecting any of the microbial oils disclosed herein to a transesterification reaction or a hydrolysis reaction, respectively.

[0020] Another aspect is a lower alcohol ester composition derived from a microbial oil and containing dihomo-γ-linolenic acid esters, or a free fatty acid composition derived from a microbial oil and containing dihomo-γ-linolenic acid, wherein the weight ratio of arachidonic acid to dihomo-γ-linolenic acid (arachidonic acid / dihomo-γ-linolenic acid) is less than 1 / 13, or less than any other value disclosed herein for this ratio.

[0021] In the microbial oil, the arachidonic acid content of the lower alcohol ester composition or the free fatty acid composition of any aspect of the present invention is generally less than or equal to 7% by weight and preferably much less, as described below.

[0022] The microbial oil, lower alcohol ester composition or free fatty acid composition of any aspect of the present invention can be provided for use as a medicament, preferably as an anti-allergy or anti-inflammatory agent. Use as such a medicament is another aspect proposed by the present invention. This aspect includes a method for preventing, treating or improving an inflammatory disease or an allergic disease, or a substance for such a method, said method comprising: administering to a subject suffering from or at risk of suffering from an inflammatory disease or an allergic disease a medicament comprising the microbial oil, lower alcohol ester composition or free fatty acid composition of any aspect of the present invention, and preferably purified dihomo-γ-linolenic acid or a lower alcohol ester of dihomo-γ-linolenic acid, or a composition containing it. The medicament can be administered topically or orally, preferably topically. The inflammatory disease or allergic disease can be and is not limited to any one of atopic dermatitis, allergic contact dermatitis (ACD), irritant contact dermatitis (ICD), photo contact dermatitis, systemic contact dermatitis, rheumatism, psoriasis, lupus, etc.

[0023] The microbial oil, lower alcohol ester composition or free fatty acid composition of any aspect of the present invention can generally be used in a method for preparing a food, a dietary supplement, a medicament, a cosmetic or an animal feed.

[0024] Further aspects of the present invention are microbial biomass containing any microbial oil as defined herein in combination with microbial cells, and a liquid medium containing such microbial biomass.

[0025] In such a liquid medium, the content of the microbial biomass, based on the dry weight of the microbial biomass, can be greater than or equal to 2.5 g / L.

[0026] Desirably, the liquid medium contains microbial oil in an amount of 0.4 g / L or higher.

[0027] Another aspect of the present invention provides a method for preparing such microbial oil, and a method for its further processing into useful products.

[0028] One aspect of the method is a method for preparing a microbial oil containing dihomo-γ-linolenic acid, such as any microbial oil disclosed herein, the method comprising:

[0029] adding a Δ5 desaturase inhibitor, especially two or more types of Δ5 desaturase inhibitors, to a liquid medium, and

[0030] culturing a microorganism having reduced or lost Δ5 desaturase activity in the liquid medium to produce a microbial oil containing dihomo-γ-linolenic acid.

[0031] One of at least two types of Δ5 desaturase inhibitors may be an arylbenzamide Δ5 desaturase inhibitor, especially 2-amino-N-(3-chlorophenyl)benzamide.

[0032] One of at least two types of Δ5 desaturase inhibitors, or the Δ5 desaturase inhibitor different from 2-amino-N-(3-chlorophenyl)benzamide, may be a dioxabicyclo[3.3.0]octane derivative represented by formula (I):

[0033]

[0034] wherein R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are each independently a hydrogen atom or an alkyl group having 1-3 carbon atoms; or, R 1 and R 2 , and / or R 4 and R 5 together form a methylene or ethylene group, and n, m and L represent 0 or 1;

[0035] Piperonyl butoxide, curcumin or a compound represented by formula (II):

[0036]

[0037] wherein R 1 represents a lower alkyl group; R 2 represents a hydroxyl group, an alkyl group, an alkoxy group, an alkenyl group or an oxyalkyl group, where in the presence of multiple Rs2 In the case of, said plurality of Rs 2 may be the same or different, and n is an integer from 0 to 5.

[0038] In the case of using a dioxabicyclo[3.3.0]octane derivative, it may be selected from sesamin, sesamolinol, epi-sesamin, epi-sesamolinol, sesamolin, 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, 2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, and 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane.

[0039] In particular, one of at least two types of Δ5 desaturase inhibitors, or said Δ5 desaturase inhibitor different from 2-amino-N-(3-chlorophenyl)benzamide may be sesamin or curcumin.

[0040] Generally, the microorganism used as the source of oil herein is most desirably a microorganism belonging to the genus Mortierella. It may be genetically modified or unmodified. It may be a microorganism that has inhibited the function of producing arachidonic acid by reducing or losing Δ5 desaturase activity, for example, through mutation and / or selection.

[0041] Another aspect of the method is a method for preparing a lower alcohol ester composition or a free fatty acid composition as proposed above from any microbial oil as proposed herein, the method comprising:

[0042] (a) obtaining a mixture of free fatty acids or lower alcohol esters of fatty acids by separate hydrolysis or alcoholysis of said microbial oil, and

[0043] (b) purifying, preferably by rectification, said mixture of free fatty acids or lower alcohol esters to obtain a free fatty acid or lower alcohol ester composition in which the fatty acid has at least 20 carbon atoms.

[0044] The mixture or composition may be further purified, for example, by column chromatography such as reverse phase partition column chromatography. For example, a lower alcohol ester composition or a free fatty acid composition as described may be prepared by reverse phase partition column chromatography, and then the lower alcohol ester or fractionation and purification of dihomo-γ-linolenic acid may be carried out to purify or prepare lower alcohol dihomo-γ-linolenate or free dihomo-γ-linolenic acid.

[0045] Another aspect of the method is a method for preparing a lower alcohol ester composition or a free fatty acid composition containing dihomo-γ-linolenic acid, comprising:

[0046] (a) Preparing a microbial oil containing dihomo-γ-linolenic acid by culturing a microorganism in a liquid medium to produce dihomo-γ-linolenic acid, wherein the microorganism is a microorganism that has inhibited the function of producing arachidonic acid by optionally reducing or losing Δ5 desaturation activity in the liquid medium in the presence of one, two or more types of Δ5 desaturase inhibitors, thereby producing the microbial oil in which the weight ratio of arachidonic acid to dihomo-γ-linolenic acid is less than 1 / 13;

[0047] (b) Obtaining a mixture of fatty acids or lower alcohol esters containing dihomo-γ-linolenic acid by hydrolysis or alcoholysis of the microbial oil, optionally after purifying the oil;

[0048] (c) Purifying the mixture of fatty acids or lower alcohol esters.

[0049] In step (c), the mixture can be purified to obtain a mixture of fatty acids or lower alcohol esters in which the fatty acids have at least 20 carbon atoms, and the method further includes

[0050] (d) Fractionating and purifying dihomo-γ-linolenic acid or a lower alcohol ester of dihomo-γ-linolenic acid from the purified mixture by reverse-phase chromatography.

[0051] The purified dihomo-γ-linolenic acid or a lower alcohol ester of dihomo-γ-linolenic acid, or a composition containing it, can be used in any of the ways described herein, for example, as a medicine or incorporated into a medicine, preferably an anti-allergy medicine or an anti-inflammatory medicine.

[0052] Advantages of the Invention

[0053] According to the present invention, a microbial oil and microbial biomass capable of being used to effectively obtain an oil containing dihomo-γ-linolenic acid can be provided, the arachidonic acid content of the oil containing dihomo-γ-linolenic acid is lower than that of the oil obtained by traditional methods, and its uses can be provided.

[0054] According to the present invention, a method for preparing a lipid containing dihomo-γ-linolenic acid (the arachidonic acid content of the lipid containing dihomo-γ-linolenic acid is lower than that of the oil obtained by traditional methods), a free fatty acid of dihomo-γ-linolenic acid, and a lower alcohol ester of dihomo-γ-linolenic acid (the arachidonic acid content of the free fatty acid of dihomo-γ-linolenic acid and the lower alcohol ester of dihomo-γ-linolenic acid is lower than that of the free fatty acid and lower alcohol ester obtained by traditional methods) can be provided.

[0055] According to other aspects of the present invention, uses of such a lipid containing dihomo-γ-linolenic acid, a free fatty acid of dihomo-γ-linolenic acid, or a lower alcohol ester of dihomo-γ-linolenic acid are provided. Detailed implementation mode

[0056] The microbial oil of the present invention is a microbial oil that contains DGLA as a fatty acid component of the oil and has a content of less than 1 / 13 in terms of the weight ratio of arachidonic acid to DGLA (ARA / DGLA).

[0057] The microorganism of the present invention is a microbial biomass containing microbial oil, and the microbial oil contains DGLA as a fatty acid component of the oil and has a content of less than 1 / 13 in terms of the weight ratio of ARA to DGLA (ARA / DGLA).

[0058] Although the content ratio of DGLA and ARA can be defined as the weight ratio (ARA / DGLA), it can also be expressed as the weight ratio (DGLA / ARA). Since the oil often originates from microorganisms having the function of naturally producing ARA, although this function can be reduced and / or inhibited in culture by mutation or strain selection, there is often at least a trace amount of ARA.

[0059] According to the present invention, there are provided a microbial oil containing DGLA and having a weight ratio of DGLA to ARA (DGLA / ARA) of greater than or equal to 13, and a microbial biomass containing DGLA and having a weight ratio of DGLA to ARA (DGLA / ARA) of greater than or equal to 13. Prior to this, microbial oils and microbial biomasses with a DGLA / ARA (weight ratio) of greater than or equal to 13 were unknown. Therefore, by using the microbial oil of the present invention or by using the microbial biomass of the present invention, it is possible to effectively provide an oil containing DGLA with a higher DGLA purity and a lower ARA content compared to conventional oils.

[0060] The microbial oil of the present invention is obtained by culturing a microorganism that produces lipids containing DGLA in a suitable medium and recovering the obtained lipids from the microbial biomass using a method such as solvent extraction. Generally, lipids include triglycerides, diglycerides, monoglycerides, phospholipids, cholesterol, etc., and the lipids mainly consist of triglycerides. A variety of types of fatty acids are included as the fatty acid components of these lipids. In the microbial biomass and microbial oil of the present invention, among these fatty acid components, the content of DGLA is high and the content of ARA is low.

[0061] In the present invention, the term "crude oil" of microbial oil refers to a lipid mixture simply obtained by extracting these lipids from microbial biomass. The refined oil of microbial oil is the microbial oil obtained by refining such microbial oil to remove phospholipids and cholesterol, thereby increasing the proportion of triglycerides. In this specification, the term "microbial oil" means both crude oil and refined oil, unless otherwise specified. Generally, the concentration of the desired fatty acid can be further increased by converting the desired fatty acid into the free fatty acid form or the lower alcohol ester form by using hydrolysis or esterification reaction with a lower alcohol ester, and then refining the free fatty acid or its lower alcohol ester. It is known that since ARA and DGLA have the same number of carbon atoms, that is, 20 carbon atoms, and the number of double bonds is 4 and 3, the properties of these compounds are similar and it is very difficult to separate DGLA from ARA by the purification process achieved on an actual production scale. The present invention can provide a microbial oil with a low ARA content (in other words, a large difference between the DGLA content and the ARA content) at the stage of crude microbial oil, and thus can significantly increase the ability to obtain a very high DGLA / ARA in the refined form of the product and / or the form downstream of chemical processing.

[0062] In this specification, the term "ARA / DGLA" or the term "DGLA / ARA" is the weight ratio between ARA and DGLA based on the analysis of the composition of fatty acids contained in the oil. The composition of fatty acids can be determined by conventional methods. Specifically, the oil to be analyzed is esterified with a lower alcohol and a catalyst to obtain a fatty acid lower alcohol ester. Thereafter, the obtained fatty acid lower alcohol ester is analyzed by gas chromatography. The peaks corresponding to each fatty acid are identified in the obtained gas chromatogram, and the peak area of each fatty acid is determined, for example, using the Agilent ChemStation integration algorithm (version C.01.03

[37] , Agilent Technologies). The "peak area" represents the ratio of the peak area of each component to the total peak area, that is, the proportion of the content of the component of the peak, as determined by the 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 is determined by gas chromatography, for example, according to the method indicated in the following examples. The lipid composition is determined by TLC / FID. The detailed suitable conditions are indicated in the working examples.

[0063] In this specification, the scope of the term "process" includes not only discontinuous processes, but also processes that cannot be clearly distinguished from another process, as long as the expected effect of the target process can be achieved.

[0064] In this specification, any numerical range expressed using "-" refers to a range including the values before and after the "-" as the minimum and maximum values, respectively.

[0065] In cases where the amounts of types of components that can be included in a composition are indicated herein, when there are multiple substances corresponding to the types of components in the composition, the indicated amount means the total amount of the multiple substances present in the composition, unless specifically stated otherwise.

[0066] In this specification, the term "microorganism" includes both eukaryotic cells and prokaryotic cells, such as, by way of specific examples, bacteria, actinomycetes, cyanobacteria, archaea, fungi, algae, lichens, protozoa, and the like.

[0067] For convenience, the term "oil" is used herein to mean "oil / fat". Further, although the terms "oil" and "oil / fat" are sometimes narrowly defined to designate triglycerides, in this specification these terms are employed to include oils containing triglycerides as the main component and other lipid components such as diglycerides, monoglycerides, phospholipids, cholesterol, and free fatty acids, e.g., crude oil. In fact, the triglyceride content is preferably greater than or equal to 30% by weight, more preferably greater than or equal to 50% by weight, further preferably greater than or equal to 70% by weight, and most preferably greater than or equal to 90% by weight.

[0068] In this specification, the term "crude oil" means oil in the state obtained by extraction from microorganisms and is generally a mixture of the above lipid components. In this specification, the term "refined oil" is used to mean oil obtained after a refining process including a degumming process, a deacidification process, a decolorization process (bleaching process), a deodorization process, etc., in any combination of some or all of these processes for removing substances other than the target substances, such as phospholipids and cholesterol. Those skilled in the art are familiar with these terms and can distinguish crude microbial oil and refined microbial oil by referring to their specific compositions. Specific refining steps remove characteristic subgroups of impurities from the original crude microbial oil, which can generally be known to be characteristic of its microbial origin.

[0069] In this specification, the term "microbial oil" is used broadly to mean any oil obtained from microorganisms and is used in this specification without distinguishing between crude oil and refined oil, unless otherwise noted.

[0070] In the present specification, the expression "microbial biomass containing microbial oil" means the microbial biomass that produces the microbial oil of the present invention, which has microbial oil accumulated in microbial cells or released from microbial cells by culturing microorganisms. Both living microorganisms and dead microorganisms can be included in the microbial biomass. Also included is the dried microbial biomass. The expression "dried microbial biomass" is used to mean the dried product of the microbial biomass that substantially does not contain water and the dried product that contains residual culture components, filter aids, etc. The expression "substantially does not contain water" means a water content at or below an amount that would make it difficult for microorganisms to survive. This amount is usually a water content of less than or equal to 15% by weight, and preferably less than or equal to 10% by weight.

[0071] In the present specification, the expression "liquid medium containing microbial biomass" is the liquid medium in which the above-mentioned "microbial biomass" is cultured, and this refers to the state before separating the microbial biomass from the culture liquid.

[0072] Various aspects of the present invention will be described below.

[0073] (1) Microbial oil

[0074] The microbial oil of the present invention contains DGLA and has a DGLA / ARA (weight ratio) of greater than or equal to 13. The value of DGLA / ARA is preferably as high as possible, more preferably greater than or equal to 15, greater than or equal to 20, or greater than or equal to 30, and still more preferably greater than or equal to 50, still more preferably greater than or equal to 100, and particularly preferably greater than or equal to 200. When the value of DGLA / ARA is less than 13, the relative proportion of ARA relative to DGLA in the microbial oil becomes high, and even if the microbial oil is refined, etc., the resulting ARA content can be close to 10% by weight such that the DGLA purity may not be sufficient to increase by purification. There is no special limitation on the upper limit of DGLA / ARA in the microbial oil, and for example, the value of DGLA / ARA can be set to be less than or equal to 3000.

[0075] The content of DGLA in the microbial oil can be 10% by weight or more, preferably 15% by weight or more, more preferably 20% by weight or more, and still more preferably 25% by weight or more based on the total weight of the microbial oil. The microbial oil can have very little ARA. The content of ARA in the microbial oil can be 0.03% by weight or more, 0.01% by weight or more, 0.001% by weight or more, or 0.0005% by weight or more. The content of ARA in the microbial oil is preferably not more than 10% by weight or not more than 7% by weight.

[0076] In addition, relative to the total amount in the microbial oil, the content of triglycerides in the crude oil is preferably greater than or equal to 70% by weight of the above-mentioned microbial oil, more preferably greater than or equal to 90% by weight. When the content of triglycerides in the microbial oil is greater than or equal to 70% by weight, there is a tendency that the water absorption is not overly low, such that, for example, good fluidity can be obtained. Although there is no particular limitation on the upper limit of the content of triglycerides in the microbial oil, generally the weight content of triglycerides in the microbial oil is less than or equal to 99% by weight. The weight content of triglycerides in the microbial oil can be 100% by weight, that is, the microbial oil can be substantially free of non-triglyceride components. Examples of the fatty acids of the triglycerides constituting the microbial oil are saturated or unsaturated fatty acids having 14 - 26 carbon atoms. Since impurities are removed by known methods, for example, the refined oil can have an increased triglyceride concentration.

[0077] In the fatty acid composition of the crude microbial oil, relative to the total weight of the microbial oil, the microbial oil preferably contains less than or equal to 60% by weight of fatty acids having 18 or fewer carbon atoms. This content is more preferably less than or equal to 55% by weight, and this content is further preferably less than or equal to 50% by weight. It is preferable to have an oil with a low content of fatty acids having 18 or fewer carbon atoms in the crude oil because such an oil can be used as a triglyceride without the need to adjust the fatty acid composition by removing fatty acids having 18 or fewer carbon atoms. Such adjustment generally requires low-yield methods such as fractionation (low-temperature processing), etc.

[0078] The microbial oil preferably has a phospholipid content of less than or equal to 10% by weight relative to the total weight of the oil, especially for the crude microbial oil, and more preferably 5% by weight relative to the total weight of the oil, further preferably less than or equal to 1% by weight. However, phospholipids can be present to a certain extent, such as 0.1 - 10% by weight relative to the total weight of the oil, more preferably 0.5 - 7% by weight, still more preferably 1 - 5% by weight.

[0079] The saturated fatty acid content of the microbial oil is preferably less than or equal to 40% by weight relative to the total weight of the crude bio-oil, and more preferably less than or equal to 35% by weight of the crude microbial oil. Microbial oils with a low content of saturated fatty acids are advantageous for some uses, such as functional dietary supplements.

[0080] It should be understood that the above optional values for the various parameters of the microbial oil can generally be obtained independently and can be freely combined to define a preferred microbial oil.

[0081] (2) Preparation of microbial oil

[0082] Microbial oil can be obtained by a preparation method including the following steps: culturing a microorganism known to be capable of producing lipids (hereinafter referred to as the preparation process), and separating the obtained microbial oil from the microbial biomass (separation process) to prepare microbial oil.

[0083] Lipids containing DGLA can be obtained by a preparation method including the following steps: culturing a microorganism known to be capable of producing lipids (hereinafter referred to as the preparation process), and separating the obtained microbial oil from the microbial biomass (separation process) to prepare lipids containing DGLA.

[0084] The method for preparing lipids containing DGLA or the method for preparing microbial oil according to the present invention can be a method including the following steps: adding two or more types of Δ5 desaturase inhibitors to a liquid medium, and culturing in the liquid medium a microorganism having reduced Δ5 desaturase activity or having lost Δ5 desaturase activity to produce lipids containing dihomo-γ-linolenic acid.

[0085] Optionally, the method for preparing lipids containing DGLA or the method for preparing microbial oil according to the present invention can be a method including the following steps: adding two or more types of Δ5 desaturase inhibitors to a liquid medium; and culturing in the liquid medium a microorganism having reduced Δ5 desaturase activity or having lost Δ5 desaturase activity obtained by mutating a microorganism capable of producing arachidonic acid to produce lipids containing dihomo-γ-linolenic acid.

[0086] In other words, the method for preparing lipids containing DGLA or the method for preparing microbial oil according to the present invention can be the following method: a method for preparing lipids having a reduced arachidonic acid content relative to dihomo-γ-linolenic acid in the lipids by culturing a microorganism having reduced Δ5 desaturase activity or having lost Δ5 desaturase activity obtained by mutating a microorganism capable of producing arachidonic acid to produce lipids containing dihomo-γ-linolenic acid, and the method includes adding, for example, two types of Δ5 desaturase inhibitors to the culture solution of the microorganism.

[0087] Known microorganisms for producing lipids for use in a preparation process are preferably at least one selected from the group consisting of microorganisms belonging to the following genera: Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, Cladosporium, Mucor, Fusarium, Aspergillus, Rhodotorula, Entomophthora, Echinosporangium, and Saprolegnia. The microorganism should be one with the ability to produce DGLA, and more preferably a microorganism belonging to the genus Mortierella.

[0088] The microorganism is further preferably one with reduced Δ5 desaturation activity or having lost Δ5 desaturation activity (hereinafter referred to as "microorganism with low Δ5 desaturation activity"), such as a microorganism with reduced Δ5 desaturation activity or having lost Δ5 desaturation activity relative to the natural state. More preferably, it is a microorganism with reduced Δ5 desaturation activity or having lost Δ5 desaturation activity obtained by mutating a microorganism with ARA-producing function, and even more preferably, it is a microorganism belonging to the genus Mortierella and having reduced Δ5 desaturation activity or having lost Δ5 desaturation activity obtained by mutating or causing mutation in a microorganism with ARA-producing function. The microorganism with ARA-producing function for carrying out the mutation is preferably a microorganism of the genus Mortierella with ARA-producing function.

[0089] Examples of microorganisms of the genus Mortierella with ARA-producing function are microorganisms belonging to the subgenus Mortierella, such as Mortierella elongate, Mortierella exigua, Mortierella hygrophila, and Mortierella alpine. Microorganisms with low Δ5 desaturation activity can be obtained by introducing mutations into microorganisms with ARA-producing function and inducing mutant strains with reduced or lost Δ5 desaturase activity.

[0090] Examples of the mutation process include physical treatments such as by radiation (X-rays, γ-rays, neutron beams, etc.), ultraviolet radiation, and heat treatment.

[0091] In addition, a target mutant strain can be obtained by a mutant strain isolation method, which includes cultivating a microorganism to be targeted for mutation in the presence of a mutagen for a fixed time interval and inoculating it in an agar medium according to standard methods to obtain colonies of the target mutant strain. Examples of mutagens for the mutant strain isolation method include alkylating agents such as nitrogen mustard, methyl methanesulfonate (MMS), N-methyl-N'-nitro-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 dichloride, potassium dichromate, nitrous acid, hydrazine, hydroxylamine, formaldehyde, nitrofurans, etc. In addition, the form of the microorganism to be targeted for mutation can be a growing microbial cell body (hyphae, etc.) or a spore.

[0092] For example, in a microorganism with low Δ5 desaturase activity, the mutant strain Mortierella alpina SAM 1860 (Fermentation Research Institute accession number 3589) induced by mutation in the aforementioned manner can be used as a mutant strain of the genus Mortierella. The preparation of DGLA using SAM 1860 is described in detail in JP-A No. H05-091887. The preparation method is summarized as follows.

[0093] To cultivate a microorganism with low Δ5 desaturase activity, spores or hyphae of the microbial strain or a pre-cultured liquid medium obtained by pre-culturing are used to inoculate a liquid or solid medium, and the microorganism is cultivated.

[0094] In the case of a liquid medium, any of the commonly used carbon sources can be used, including glucose, fructose, xylose, sucrose, maltose, soluble starch, molasses, glycerol, mannitol, etc.; however, the carbon source is not limited to these.

[0095] The nitrogen source can be a natural nitrogen source such as peptone, yeast extract, malt extract, meat extract, casein amino acids, corn steep liquor, and organic nitrogen sources such as urea, and inorganic nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate. In addition, if necessary, inorganic salts such as phosphates, magnesium sulfate, iron sulfate, copper sulfate, and vitamins, etc. can also be used as a source of micronutrients.

[0096] The aqueous medium used as the base material for the liquid medium is basically water, and distilled water or purified water can be used.

[0097] There are no particular limitations on these culture medium components as long as the concentrations of these components do not interfere with the growth of the low Δ5 desaturase activity microorganisms. Generally, for practical purposes, the concentration of the carbon source is 0.1% by weight - 30% by weight, and preferably 1% by weight - 10% by weight, and the concentration of the nitrogen source is 0.01% by weight - 5% by weight, and preferably 0.1% by weight - 2% by weight. Moreover, the culture temperature is 5°C - 40°C, and preferably 20°C - 30°C. The pH of the culture medium is 4 - 10, and preferably 6 - 9. The culture can be aerated stirring culture, shaking culture, or static culture. The culture is usually carried out for 2 days - 15 days. The aeration rate during the aerated stirring culture can be the aeration rate commonly used for such aeration.

[0098] To promote the accumulation of DGLA, components that will be substrates for producing ARA and / or DGLA can be added to the culture medium. Examples of such substrates are hydrocarbons such as tetradecane, hexadecane, octadecane; fatty acids such as myristic acid, palmitic acid, stearic acid; salts of such fatty acids, such as sodium salts and potassium salts; fatty acid esters; oils and fats containing fatty acids as constituent components, such as olive oil, soybean oil, cottonseed oil, and palm oil; and so on. However, the substrates are not limited to these.

[0099] Conventional solid media can be used as the solid medium for culturing low Δ5 desaturase activity microorganisms. Examples of such solid media are agar medium, malt extract agar medium, malt agar medium, Czapek - Dox agar medium, Czapek agar medium, potato - carrot agar medium (PCA), potato - dextrose agar medium (trade name “potato dextrose agar medium”, potato dextrose agar: PDA), Sabouraud agar medium, cornmeal agar medium, etc. The medium can be appropriately selected according to the species of the microorganisms to be cultured. Any of such solid media can be obtained from commercially available products, and the commercially available solid media can be used without any modification and according to the instructions provided. Among such solid media, from the perspective of effectively producing DGLA in low Δ5 desaturase activity microorganisms, the PDA medium is preferred.

[0100] To prepare microbial oil with a higher DGLA / ARA ratio, in the case of a liquid medium, the medium for culturing the microorganisms is preferably a liquid medium containing glucose as the carbon source and yeast extract as the nitrogen source, or in the case of a solid medium, the PDA medium is preferred.

[0101] (3) Cultivation of microorganisms

[0102] To prepare microbial oil with a high DGLA / ARA ratio, a microorganism, preferably a microorganism with low Δ5 desaturation activity, is preferably cultured in the presence of a Δ5 desaturase inhibitor. During the production of fatty acids in microbial cells, the Δ5 desaturase inhibitor inhibits the enzymes in the synthesis pathway for forming ARA. Thus, by using one or more Δ5 desaturase inhibitors and / or selecting a suitable microorganism, such as a mutant ARA-producing cell, according to known principles that is favorable for producing DGLA over producing ARA, the synthesis of ARA in microbial cells can be inhibited, and the accumulation amount of DGLA in microbial cells can be significantly increased.

[0103] Any known Δ5 desaturase inhibitor can be used without any limitation, and the Δ5 desaturase inhibitor can be used in one type or in combination of two or more types. To more effectively obtain microbial oil with a high DGLA / ARA ratio, the present inventors have found that it is preferably to use a combination of two or more Δ5 desaturase inhibitors. Unexpectedly, the present inventors have found that through this combination they can achieve a significant increase in the DGLA / ARA ratio and can prepare a new type of microbial oil with an extremely high DGLA / ARA ratio, which was not foreseeable before.

[0104] In the case of using a combination of two types of Δ5 desaturase inhibitors, 2-amino-N-(3-chlorophenyl)benzamide is preferably selected as the first type of Δ5 desaturase inhibitor. Through the combination of 2-amino-N-(3-chlorophenyl)benzamide with another type of Δ5 desaturase inhibitor, the reduction in the total lipid production amount is inhibited, and the DGLA / ARA ratio can be increased. 2-amino-N-(3-chlorophenyl)benzamide is anthranilicanilide, and arylbenzamides known to have Δ5 desaturase inhibitor activity can be used herein, but it was not previously known to be effectively used in this type of process.

[0105] Examples of the second type of Δ5 desaturase inhibitor can be dioxabicyclo[3.3.0]octane derivatives represented by the following formula (I):

[0106]

[0107] wherein, in formula (I), R 1 , R 2 , R 3 , R 4 , R 5 and R 6 each independently represents a hydrogen atom or an alkyl group having 1 - 3 carbon atoms; optionally, R 1 and R 2 and / or R 4 and R5 together form a methylene or ethylene group; and n, m and L represent 0 or 1;

[0108] piperonyl butoxide, curcumin or a compound represented by the following formula (II):

[0109]

[0110] wherein, in formula (II), R 1 represents a lower alkyl group, such as an alkyl group having 1 - 3 carbon atoms; R 2 represents a hydroxyl group, an alkyl group, an alkoxy group, an alkenyl group or an oxyalkyl group; in the case where there are multiple R 2 the multiple R 2 may be the same or different; and n is an integer in the range of 0 - 5, etc. Such Δ5 desaturase inhibitors can be used alone or in combination.

[0111] Examples of the dioxabicyclo[3.3.0]octane derivatives may be sesamin, sesaminol, epi - sesamin, epi - sesaminol, 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 - hydroxy phenoxy)-3,7 - dioxabicyclo[3.3.0]octane, etc. Such dioxabicyclo[3.3.0]octane derivatives can be used alone or in combination of two or more types. In addition, such dioxabicyclo[3.3.0]octane derivatives can be used in combination with stereoisomers or racemates. Particularly preferably, the dioxabicyclo[3.3.0]octane derivative is at least one selected from the group consisting of sesamin and curcumin. Such dioxabicyclo[3.3.0]octane derivatives can be chemically synthesized products or extracts from natural products.

[0112] Unlike 2-amino-N-(3-chlorophenyl)benzamide added to the culture medium, there is no particular limitation on the way of adding the Δ5 desaturase inhibitor, and such a way can be appropriately selected according to the type and form of the Δ5 desaturase inhibitor employed. For example, the Δ5 desaturase inhibitor may be at least one type selected from sesame oil, peanut oil, and natural extracts such as an extract obtained from sesame oil using an organic solvent that is substantially immiscible with sesame oil, a solvent extract of sesame seeds, an extract of Acanthopoanacis Core (Acanthopanax bark extract), a Paulownia tree extract, a Ginkgo bark extract, a Piper longum extract, an Asiasari radix extract, a tarragon extract, a dill seed extract, a parsley extract, a turmeric extract, and a nutmeg extract. Among them, when the Δ5 desaturase inhibitor is a natural extract, such a natural extract may be added to the culture medium for culturing microorganisms, or alternatively, these natural extracts may be added to the liquid culture medium for culturing microorganisms. Microorganisms can be further cultured using the culture medium containing these Δ5 desaturase inhibitors.

[0113] Among them, in the case of using a combination of two or more types of Δ5 desaturase inhibitors, such a combination may be a combination of 2-amino-N-(3-chlorophenyl)benzamide (or one of the other first-type inhibitors listed above) and at least one type of Δ5 desaturase inhibitor selected from the group consisting of: dioxabicyclo[3.3.0]octane derivatives, piperonyl butoxide, curcumin, and the compound represented by formula (II). From the viewpoint of obtaining microbial oil having a high DGLA / ARA ratio, a combination of 2-amino-N-(3-chlorophenyl)benzamide and a dioxabicyclo[3.3.0]octane derivative is preferred. More preferably, it is a combination of 2-amino-N-(3-chlorophenyl)benzamide and at least one selected from the group consisting of: sesamin, sesamol, epi-sesamin, epi-sesamol, sesamolin, 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, 2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, and 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane.

[0114] Although the added concentration of the Δ5 desaturase inhibitor depends on the type of the Δ5 desaturase inhibitor employed, in the case of a liquid medium, the concentration of the Δ5 desaturase inhibitor added to the liquid medium per day is preferably 0.01 g / L - 1 g / L, more preferably 0.03 g / L - 0.50 g / L. In addition, in the case of a solid medium, the concentration of each Δ5 desaturase inhibitor is preferably 0.0001% by weight - 0.1% by weight, more preferably 0.001% by weight - 0.05% by weight. Among them, in the case where the Δ5 desaturase inhibitor is used in the form of sesame oil or an extract such as a sesame oil extract, considering factors such as the amount of the active components contained in the extract, the final concentration of the total amount of the Δ5 desaturase inhibitor in the liquid medium is, for example, 0.001% by weight - 10% by weight, and preferably 0.5% by weight - 10% by weight. Among them, in the case of using a combination of two or more types of Δ5 desaturase inhibitors, there is no special limitation on the ratio of the amounts of the multiple Δ5 desaturase inhibitors employed, and such a ratio can be appropriately selected according to the type of the Δ5 desaturase inhibitor employed. For example, in the case where 2-amino-N-(3-chlorophenyl)benzamide is used in combination with another Δ5 desaturase inhibitor, the ratio of 2-amino-N-(3-chlorophenyl)benzamide or other benzamide to another Δ5 desaturase inhibitor (in the case of a natural extract, it is the active component in the natural extract) can be 100:1 - 1:100 by weight ratio, preferably 10:1 - 1:10, and more preferably 5:1 - 1:5.

[0115] In the case of adding a Δ5 desaturase inhibitor to the medium, the microbial production of lipids may be affected, and in such a case, in order to reduce the impact on the production, it is preferable to add the Δ5 desaturase inhibitor to the culture solution in multiple aliquots rather than in the whole amount.

[0116] There is no special limitation on the addition time of the Δ5 desaturase inhibitor, and such an addition can be carried out once a day, or can be carried out one to several times a day during the cultivation of the microorganism. In the case of adding the Δ5 desaturase inhibitor at intervals of one to several times a day, these additions can be carried out at equal time intervals, irregular time intervals, or a combination of these intervals. The addition time of the Δ5 desaturase inhibitor can be appropriately selected according to the growth state of the microorganism.

[0117] Among them, in the case where a Δ5 desaturase inhibitor is added to the culture medium during the preparation process, in order to prepare microbial oil having a high DGLA / ARA ratio, for the culture medium used during the preparation process, in the case of a liquid culture medium, it is preferable to use a liquid culture medium using glucose as a carbon source and yeast extract as a nitrogen source, and in the case of a solid culture medium, PDA medium is preferable.

[0118] There are no special restrictions on the culture vessel used in the preparation process, and any culture vessel commonly used for culturing microorganisms can be used. The culture vessel can be appropriately selected according to the scale of the culture.

[0119] For example, in the case of liquid culture on a scale of 1 L - 50 L, a stirred culture vessel is preferably used as the culture vessel to prepare microbial oil having a high DGLA / ARA ratio. The stirred culture vessel preferably has a disk turbine type agitator blade in at least one stage, and the stirred culture vessel more preferably has a disk turbine type agitator blade in two stages. In the case where the stirred culture vessel is equipped with disk turbine type agitator blades in two stages, the distance between the agitator blades closer to the bottom surface can be small so as to effectively agitate the culture solution at the bottom surface of the culture vessel. For example, the placement positions of the upper and lower agitator blades can be appropriately selected. For example, the ratio of "the distance from the bottom of the culture vessel to the lower agitator blade": "the distance between the lower agitator blade and the upper agitator blade": "the distance from the upper agitator blade to the surface of the culture solution" is preferably adjusted to "1": "1 - 3": "1 - 5", preferably "1": "1.5 - 2": "2 - 4". Preferred examples of these ratios are 4:7:15. In the case of culturing microorganisms using a liquid culture medium containing a culture medium containing a Δ5 desaturase inhibitor, a stirred culture vessel is particularly preferred.

[0120] (4) Isolation of microbial biomass from the culture medium and recovery of microbial oil from the microbial biomass

[0121] In the separation process, the microbial oil containing DGLA produced during the preparation process is separated from the microbial biomass. The separation process preferably includes separating the cultured microbial biomass from the culture medium used in the culture (microbial biomass separation process) and recovering the microbial oil containing DGLA from the cultured microbial biomass (recovery process), that is, obtaining crude oil.

[0122] In the microbial biomass separation process and the microbial oil recovery process, extraction methods and separation methods are used according to the culture method so as to recover the microbial oil containing DGLA from the cultured microbial mass.

[0123] In the case of using a liquid culture medium, for example, the microbial oil containing DGLA is recovered from the cultured microbial biomass in the following manner.

[0124] After the cultivation is completed, the cultured microbial biomass is obtained from the liquid medium by using conventional means for solid-liquid separation, such as centrifugation and filtration. The microbial biomass is washed thoroughly with water and then preferably dried. Drying can be carried out by freeze-drying, air-drying, heat-drying, etc.

[0125] In the case of cultivation using a solid medium, a homogenizer or the like can be used to crush the solid medium and the microbial biomass without separating the microbial mass from the medium, and the obtained crushed material can be directly supplied to the recovery process.

[0126] The recovery process can include extraction processing of the dried microbial biomass obtained in the microbial biomass separation process by using an organic solvent, preferably under a nitrogen stream. The organic solvents used include ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleum ether, etc. Optionally, good results can be obtained by alternately extracting with methanol and petroleum ether, or by extraction using a single-layer solvent of chloroform-methanol-water. The organic solvent is distilled off from the extract under reduced pressure to obtain microbial oil containing a high concentration of DGLA. Hexane is most commonly used in the case of recovering triglycerides.

[0127] In addition, as an alternative to the above method, wet microbial biomass can be used for extraction. A water-miscible solvent, such as methanol or ethanol, or a water-miscible mixed solvent containing the solvent and water and / or other solvents is used. The remaining steps of the process are similar to those described above.

[0128] The crude microbial oil recovered can be refined by methods used for refining vegetable oils, fish oils, etc. Examples of the refining processes commonly used for oils / fats are degumming, deacidification, bleaching (decolorization), and deodorization processes. Such processing can be carried out by any method. An example of degumming is water washing treatment. An example of deacidification is distillation treatment. An example of bleaching is bleaching using activated clay, activated carbon, silica gel, etc. An example of deodorization is steam distillation, etc.

[0129] (5) Preparation of lower alcohol esters and free fatty acids of fatty acids from microbial oil

[0130] DGLA, which is included as a fatty acid component of the microbial oil, can be converted into the form of a lower alcohol ester by using a catalyst, or into the form of a free fatty acid by hydrolysis. Compared with the triglyceride as it is, the lower alcohol ester or the free fatty acid can be easily separated from other fatty acids, and DGLA can be concentrated to increase its purity.

[0131] The method for preparing the lower alcohol ester or free fatty acid of dihomo-γ-linolenic acid according to the present invention may include: (a) obtaining the free fatty acid or lower alcohol ester of fatty acid by hydrolysis or alcoholysis of the microbial oil; (b) rectifying the mixture of the free fatty acid or lower alcohol ester of fatty acid to obtain the free fatty acid or lower alcohol ester of fatty acid, wherein the fatty acid has at least 20 carbon atoms; and (c) fractionating and purifying the free fatty acid or lower alcohol ester of dihomo-γ-linolenic acid from the free fatty acid or lower alcohol ester by reversed-phase partition column chromatography, wherein the fatty acid has at least 20 carbon atoms.

[0132] The method for preparing the lower alcohol ester of dihomo-γ-linolenic acid according to the present invention may include: (a) obtaining the lower alcohol ester of fatty acid by alcoholysis of the microbial oil; (b) rectifying the mixture of the lower alcohol ester of fatty acid to obtain the lower alcohol ester of fatty acid, wherein the fatty acid has at least 20 carbon atoms; and (c) fractionating and purifying the lower alcohol ester of dihomo-γ-linolenic acid from the lower alcohol ester by reversed-phase partition column chromatography, wherein the fatty acid has at least 20 carbon atoms.

[0133] The method for preparing the free fatty acid of dihomo-γ-linolenic acid according to the present invention may include: (a) obtaining the free fatty acid by hydrolysis of the microbial oil; (b) rectifying the mixture of the free fatty acid to obtain the free fatty acid having at least 20 carbon atoms; and (c) fractionating and purifying the free dihomo-γ-linolenic acid from the free fatty acid having at least 20 carbon atoms by reversed-phase partition column chromatography.

[0134] Examples of the lower alcohol herein are alcohols having 3 or fewer carbon atoms, particularly ethanol, methanol, etc. Examples of the lower alcohol ester of DGLA are methyl dihomo-γ-linolenate, ethyl dihomo-γ-linolenate, etc.

[0135] For example, the methyl ester of fatty acid is obtained by treating the oil with 5%-10% anhydrous methanol-hydrochloric acid, 10%-50% BF 3 -methanol at room temperature for 1-24 hours. The ethyl ester of fatty acid is obtained by treating the oil with 1%-20% sulfuric acid ethanol, etc. at 25°C-100°C for 15-60 minutes. The methyl ester or ethyl ester can be extracted from the reaction solution using an organic solvent such as hexane, ether or ethyl acetate. The extract is dried using anhydrous sodium sulfate, etc., and then the organic solvent is removed by distillation to obtain a composition containing the fatty acid ester as the main component.

[0136] In addition to the target DGLA lower alcohol ester, other fatty acid lower alcohol esters are contained in the esterification composition obtained by the esterification treatment. In order to separate the DGLA lower alcohol ester from the mixture of these fatty acid lower alcohol esters, distillation, rectification, column chromatography, low-temperature crystallization, urea inclusion method, liquid-liquid countercurrent distribution chromatography, etc. can be used singly or in combination of two or more methods. A combination of distillation or rectification and column chromatography or liquid-liquid countercurrent distribution chromatography is preferably used.

[0137] For these methods, ordinary processes can be used. Reversed-phase partition type (preferably ODS) column chromatography is preferably used as the column chromatography.

[0138] In order to obtain the free fatty acid of DGLA, after the lower alcohol ester of microbial oil is produced in the above manner, the DGLA lower alcohol ester refined to improve the purity can be hydrolyzed to obtain high-purity free DGLA. In order to obtain free DGLA from the DGLA lower alcohol ester, after hydrolysis using an alkali catalyst, an extraction process can be carried out using an organic solvent such as ether, ethyl acetate, etc.

[0139] Optionally, the free fatty acid of DGLA can also be directly obtained from the microbial oil by hydrolysis. For example, the microbial oil undergoes alkali decomposition, for example, using 5% sodium hydroxide to decompose at room temperature for 2-3 hours to obtain a decomposition liquid, and then the free fatty acid of DGLA can be extracted or refined from the decomposition liquid by methods commonly used for extracting or refining fatty acids.

[0140] Using the microbial oil of the present invention as a raw material to prepare the free acid or lower alcohol ester of DGLA obtained by the above method, and thus the free acid or lower alcohol ester of DGLA is a composition with a low ARA content, and ARA is difficult to remove during the refining process. The ARA / DGLA ratio can be made less than 1 / 13, less than 1 / 20 or less than 1 / 30; or further can be made less than 1 / 50, less than 1 / 100, less than 1 / 200, less than 1 / 1,000 or less than 1 / 3,000. That is, the concentration of ARA can be made less than or equal to 7% by weight, less than or equal to 5% by weight, less than or equal to 3% by weight, less than or equal to 2% by weight, less than or equal to 1% by weight, less than or equal to 0.5% by weight, less than or equal to 0.1% by weight or less than or equal to 0.03% by weight. For medical use, DGLA is preferably concentrated to greater than or equal to 90% by weight.

[0141] (6) Microbial biomass containing microbial oil

[0142] The expression "microbial biomass containing microbial oil" refers to the biomass of microorganisms that are cultivated in the above-described manner and produce microbial oil within their cells. The microbial biomass can be the microbial biomass with microbial oil accumulated in the microbial cells or after the oil is released from the microbial cells, as long as the microbial biomass contains the microbial oil of the present invention. Since this microbial biomass contains the microbial oil of the present invention, the microbial biomass contains DGLA as a fatty acid component of the oil and the content of ARA relative to DGLA is greater than or equal to 13, as indicated by the weight ratio (DGLA / ARA). In addition, the triglyceride content of the microbial oil is preferably greater than or equal to 70% by weight, greater than or equal to 80% by weight, or greater than or equal to 90% by weight.

[0143] Optionally, the DGLA / ARA ratio of the microbial oil contained in the microbial biomass of the present invention is preferably greater than or equal to 15, more preferably greater than or equal to 20, further preferably greater than or equal to 30, still further preferably greater than or equal to 50, still further preferably greater than or equal to 100, and particularly preferably greater than or equal to 200.

[0144] The DGLA / ARA ratio in the microbial biomass is the value determined in the above-described manner. Any method can be used to measure DGLA and ARA in the microbial biomass, as long as the method is a method commonly used to measure the relative weights of DGLA and ARA in the microbial biomass, or an equivalent method. For example, the microorganisms can be recovered from the liquid medium during growth and can be esterified by treatment with 5%-10% anhydrous methanol-hydrochloric acid, 10%-50% BF 3 -methanol, 1%-20% sulfuric acid-methanol, 1%-20% sulfuric acid-ethanol, etc. at 25°C - 100°C for 15 - 60 minutes. Then, the fatty acid content (%) in the fatty acids can be analyzed using gas chromatography under conditions of extracting or not extracting the ester form. In the case of esterification, in order to evaluate substances other than free fatty acids, treatment can be carried out using alkoxides such as sodium methoxide, sodium ethoxide, etc. at a concentration of 0.1M - 10M at 25°C - 100°C for 15 - 60 minutes. For the extraction of the ester form after esterification, an organic solvent immiscible with the water-soluble components, such as hexane, can be used.

[0145] In addition, the microorganism is preferably a microorganism that can provide an oil that meets at least one of the various conditions described above for microbial oil, such as triglyceride content, content of fatty acids having less than or equal to 18 carbon atoms, phospholipid content, saturated fatty acid content, etc., and preferably any combination of two or more conditions.

[0146] (7) Liquid culture medium containing microbial biomass containing microbial oil

[0147] The use of "a liquid medium containing microbial biomass containing microbial oil" means the medium before separating the microorganisms grown by the above-described method for preparing microbial oil from the liquid medium. Thus, the liquid medium contains the above-described DGLA and microbial oil with a DGLA / ARA ratio of 13 or more. In order to recover microbial oil from this liquid medium, the liquid medium preferably has a microbial content of 2.5 g / L or more based on the weight of the dried microbial biomass. In addition, the content of microorganisms in the liquid medium containing microorganisms is preferably 5 g / L or more, more preferably 30 g / L or more, and even more preferably 60 g / L or more based on the weight of the dried microbial biomass. Microbial oil with a high DGLA / ARA ratio can be effectively obtained from such a liquid medium.

[0148] In addition, considering the microbial oil in the microbial biomass in the liquid medium, the liquid medium contains oil derived from the above microorganisms, and the oil containing microbial oil contains DGLA and preferably has a content of DGLA-containing oil of 0.4 g / L or more, more preferably 0.8 g / L or more. When the content of the oil containing DGLA from the microbial oil source is 0.4 g / L or more, there is a tendency to obtain advantages such as reducing production costs and improving quality stability.

[0149] Microorganisms grow through cultivation, and DGLA is produced in the microbial cells. Therefore, a liquid medium containing microorganisms can be obtained by recovering the liquid medium containing microorganisms without any modification during the cultivation process. Moreover, since microbial oil containing DGLA is produced inside the microbial cells of the microorganisms during the cultivation process, the liquid medium containing microbial oil can be obtained by recovering the liquid medium containing microorganisms without any modification during the cultivation process, or alternatively, by crushing the microorganisms in the liquid medium, etc. to disrupt the microorganisms and recovering the liquid medium containing microbial oil released into the medium. In addition, the above description also applies to the liquid medium and the medium containing microorganisms in the liquid medium containing microbial oil.

[0150] Application

[0151] According to the present invention, the microorganism, microbial oil, lower alcohol ester, free fatty acid, and culture solution containing the microorganism containing DGLA can each have a lower ARA / DGLA ratio than previously known. Therefore, each is very useful for applications requiring high purity DGLA or preferably a lower ARA content. Examples of such applications are food, dietary supplements, pharmaceuticals, cosmetics, animal feed, and the like. Since the microbial oil containing DGLA has a low ARA content, compared with the microbial oil containing DGLA having a high ARA content, for the same amount of microbial oil and DGLA to be used, the amount of ARA in the microbial oil can be reduced. Therefore, functional applications targeting DGLA are particularly preferred, and examples of such applications are anti-inflammatory applications and anti-allergic applications, especially topical applications, as shown above.

[0152] As described above, a drug comprising a microbial oil, a lower alcohol ester composition, or a free fatty acid composition or a drug consisting of a microbial oil, a lower alcohol ester composition, or a free fatty acid composition can generally be administered topically or orally, preferably topically. The inflammatory disease or allergic disease to be treated, prevented, or alleviated can be, for example but not limited to, any skin inflammation. The skin inflammation can be at least one selected from the group consisting of: rash, urticaria, blisters, and wheals, or can be caused by at least one selected from the group consisting of: eczema, exposure to radiation, autoimmune diseases, and uremic pruritus.

[0153] Specifically, the skin inflammation can be a skin inflammation associated with or caused by atopic dermatitis, contact dermatitis, psoriasis, or uremic pruritus.

[0154] The drug can be used to treat, prevent, or alleviate skin inflammation associated with eczema. The term eczema applies to a wide range of skin conditions with multiple etiologies. Generally, eczema is characterized by inflammation of the epidermis. Common symptoms associated with eczema include dryness, recurrent rash, redness, skin edema (swelling), itching, dryness, crusting, peeling, blistering, cracking, exudation, and bleeding. Eczema includes atopic eczema (atopic dermatitis), contact dermatitis, dry eczema, seborrheic dermatitis, dyshidrosis, discoid eczema, venous eczema, dermatitis herpetiformis, neurodermatitis, and autosensitization eczema. Eczema is typically atopic eczema or contact dermatitis.

[0155] Atopic eczema is mainly exacerbated by contact with or ingestion of allergens, including animal hair and dandruff, food allergens such as nuts or shellfish, and drugs such as penicillin.

[0156] Contact dermatitis includes allergic contact dermatitis, irritant contact dermatitis, and photo-contact dermatitis. Photo-contact dermatitis includes phototoxic contact dermatitis and photoallergic contact dermatitis.

[0157] Skin inflammation can be skin inflammation caused by skin exposure to electromagnetic radiation. This includes, for example, exposure to sunlight, heat, X-rays, or radioactive substances. Thus, the drug can be used to treat sunburn, or for the treatment of sunburn or for treating sunburn.

[0158] Electromagnetic radiation includes radio waves, microwaves, terahertz radiation, infrared radiation, visible light, ultraviolet radiation, X-rays, and gamma rays. The electromagnetic radiation is preferably infrared radiation, visible light, ultraviolet radiation, X-rays, and gamma rays, more preferably ultraviolet radiation, X-rays, and gamma rays.

[0159] Autoimmune diseases can involve an autoimmune response against the skin. Examples of such autoimmune diseases are lupus and psoriasis.

[0160] Uremic pruritus is a skin disorder associated with chronic renal failure. It also often affects patients undergoing dialysis treatment.

[0161] Optionally, the microbial oil, lower alcohol ester composition, or free fatty acid composition herein is used for co-administration with a corticosteroid or other therapeutic agent for any of the above medical uses or for use thereof.

[0162] In other aspects of the present invention, the inflammatory disease can be at least one selected from the group consisting of atopic dermatitis, allergic contact dermatitis (ACD), irritant contact dermatitis (ICD), photo-contact dermatitis, systemic contact dermatitis, rheumatism, psoriasis, lupus, and the like.

[0163] It should be understood that a drug for treating an inflammatory disease / allergic disease is a drug for suppressing or alleviating one or more symptoms when it is found or suspected that the one or more symptoms are caused by an inflammatory disease / allergic disease. On the other hand, a drug for preventing an inflammatory disease / allergic disease is a drug for suppressing the occurrence of one or more symptoms, typically by pre-administration, and the one or more symptoms can be predicted or expected to be caused by an inflammatory disease / allergic disease. However, the terms "drug for treatment" and "drug for prevention" should be understood to consider multiple aspects or general aspects, such as the timing of use and / or the one or more symptoms to be treated / prevented in use, in line with clinical practice, and should not be applied restrictively.

[0164] Examples

[0165] The present invention will be described in detail using working examples. However, the present invention is not limited to these working examples. Unless otherwise specified, "%" in the following working examples means "% by weight".

[0166] Example 1

[0167] Effect of various types of Δ5 desaturase inhibitors on fatty acids produced from microbial biomass: 1

[0168] Five plate media were prepared according to the product instructions of potato dextrose agar medium (commercially available product name; Nissui Pharmaceutical Co., Ltd.), namely, plate medium A without added Δ5 desaturase inhibitor, plate medium B with 0.005% by weight of sesamin added thereto, plate medium C with 0.01% by weight of sesamin added thereto, plate medium D with 0.02% by weight of sesamin added thereto, and plate medium E with 0.01% by weight of sesamin and 0.01% by weight of 2-amino-N-(3-chlorophenyl)benzamide added thereto, the difference being that one or more of the mentioned Δ5 desaturase inhibitors were added to or not added to the potato dextrose agar medium to achieve the listed concentrations. The size of each plate medium was the same, namely, 90 mm in diameter and 5 mm in thickness.

[0169] Each of plate media A - E was inoculated with a 100 μL spore suspension of Mortierella alpina mutant strain SAM1860 and statically cultured at 28 °C for 7 days.

[0170] After the culture was completed, each plate medium containing its microbial biomass was cut into sample wedges approximately 1 cm in size, and the sample wedges were transferred to culture flasks. Then 50 mL of hexane was added to each plate, and the resulting mixture was stirred for 2 minutes to obtain a liquid mixed with an organic solvent. The liquid mixed with the organic solvent was centrifuged (2,000 rpm, 810 G), and the hexane supernatant layer was recovered. Then the solvent was removed by distillation to obtain approximately 40 mg of microbial oil from each of plate media A - E.

[0171] To 0.5 mg of each of microbial oils A - E, 0.10 mL of a 10% (v / v) sulfuric acid ethanol solution was added, and an ethyl esterification reaction was carried out at 80 °C for 30 minutes. To neutralize the reaction solution, 0.18 mL of a 1.0 M sodium hydroxide ethanol solution was added, then 0.05 mL of hexane and 0.30 mL of saturated sodium chloride solution were added and extraction was carried out to obtain ethyl fatty acids A - E respectively. The fatty acid components (%) in the ethyl fatty acid compositions A - E were analyzed by gas chromatography. The analysis conditions for gas chromatography are shown below. The results of gas chromatography are shown in Table 1. In addition, the fatty acid components (%) are based on the area ratio of the gas chromatogram.

[0172] Gas chromatography analysis conditions

[0173] Device type: Agilent 6850 GC system (Agilent Technologies, Inc.)

[0174] Column: DB-WAX (Agilent Technologies, 30 m × 0.25 mm ID, 0.25 μm film thickness) J&W 122-7032

[0175] Oven temperature: 180 °C - 3 °C / min - 230 °C (25 min)

[0176] Injection temperature: 270 °C

[0177] Injection method: Split

[0178] Split ratio: 20:1

[0179] Detector temperature: 270 °C

[0180] Detector: FID

[0181] Carrier gas: Helium (1.0 mL / min, constant flow)

[0182] Table 1

[0183]

[0184] As shown in Table 1, for fatty acid ethyl ester E using microbial oil E as a raw material, the DGLA / ARA ratio is substantially greater than 13, and this ratio is higher than any of microbial oil A, microbial oil B, microbial oil C, and microbial oil D obtained by culturing the known SAM1860 strain in the presence of a type of Δ5 desaturase inhibitor. In addition, 33.8 mg of fatty acid ester E was obtained from microbial oil E.

[0185] Example 2

[0186] Effect of various types of Δ5 desaturase inhibitors on fatty acids produced from microbial biomass: 2

[0187] In a 500 mL Erlenmeyer flask with a corrugated shape, 100 mL of a medium (pH 6.0) containing 2% glucose and 1% yeast extract was added. Then, four types of liquid media were prepared, namely, liquid medium F without a Δ5 desaturase inhibitor, liquid medium G supplemented with 10 mg of sesamin, liquid medium H supplemented with 10 mg of 2-amino-N-(3-chlorophenyl)benzamide, and liquid medium I supplemented with both 10 mg of sesamin and 10 mg of 2-amino-N-(3-chlorophenyl)benzamide. An I-26 stackable shaker (manufactured by New Brunswick Scientific) was used as the shaking culture device.

[0188] After sterilizing liquid media F - I at 121 °C for 15 minutes, 1 mL of a preculture liquid medium of Mortierella alpina mutant SAM 1860 was inoculated into each liquid medium and cultured with shaking at a rotational speed of 200 rpm and a temperature of 28 °C for 15 days. On the 5th and 10th days, 10 mg of sterilized sesamin was added to liquid medium G, 10 mg of sterilized 2-amino-N-(3-chlorophenyl)benzamide was added to liquid medium H, and a combination of 50 mg of sterilized sesamin and 10 mg of 2-amino-N-(3-chlorophenyl)benzamide was added to liquid medium I. After cultivation, the microbial biomass was recovered by centrifugation. After thorough washing with water, the microbial biomass was freeze-dried. 3.2 g of dried microbial biomass (dried microbial biomass F) was obtained from liquid medium F. 2.5 g of dried microbial biomass (dried microbial biomass G) was obtained from liquid medium G. 0.6 g of dried microbial biomass (dried microbial biomass H) was obtained from liquid medium H. 0.6 g of dried microbial biomass (dried microbial biomass I) was obtained from liquid medium I.

[0189] 175 mL of hexane was added to each of the microbial biomasses F - I to extract the liquid, stirred at room temperature for 30 minutes, and then the resulting mixture was filtered to obtain an extract and microbial cells. This operation was repeated three times to obtain a hexane extract. The hexane extract was concentrated under reduced pressure using a rotary evaporator. 613.2 mg of microbial oil, namely microbial oil F, was obtained from the dried microbial biomass F. 328.3 mg of microbial oil, namely microbial oil G, was obtained from the dried microbial biomass G. 77.7 mg of microbial oil, namely microbial oil H, was obtained from the dried microbial biomass H. 105.3 mg of microbial oil, namely microbial oil I, was obtained from the dried microbial biomass I.

[0190] To each of the microbial oils in 0.5 mg of microbial oil F-I, 0.10 mL of 10% (v / v) sulfuric acid ethanol solution was added, and the reaction was carried out at 80 °C for 30 minutes for ethyl esterification. After neutralizing the reaction, 0.18 mL of 1.0 M sodium hydroxide ethanol solution was added to the liquid. Then, 0.05 mL of hexane and 0.30 mL of saturated sodium chloride solution were added for extraction. 533.2 mg of ethyl fatty acid esters, namely ethyl fatty acid esters F, were obtained from microbial oil F. 285.4 mg of ethyl fatty acid esters, namely ethyl fatty acid esters G, were obtained from microbial oil G. 67.6 mg of ethyl fatty acid esters, namely ethyl fatty acid esters H, were obtained from microbial oil H. 91.6 mg of ethyl fatty acid esters, namely ethyl fatty acid esters I, were obtained from microbial oil I. The fatty acid ratios of the obtained ethyl fatty acid esters F-I were analyzed by gas chromatography in the same manner as in Example 1. The results of the gas chromatography are shown in Table 2. In addition, the fatty acid composition (%) was based on the area ratio of the gas chromatogram in the above manner.

[0191] The analysis conditions of the gas chromatography were similar to those of Example 1.

[0192] As shown in Table 2, the DGLA / ARA ratio of ethyl fatty acid esters I using microbial oil I as a raw material was much higher than 13, and this value was higher than any of those in microbial oils F, G, and H, which were produced by culturing the known strain SAM1860 or by culturing SAM 1860 in the presence of a type of Δ5 desaturase inhibitor.

[0193] Table 2

[0194]

[0195] Example 3

[0196] Effect of various types of Δ5 desaturase inhibitors on fatty acids produced from microbial biomass: 3

[0197] A 1-liter (1 L) fermenter equipped with disk turbine agitator paddles in two stages was provided. The position of the agitator paddle in the fermenter was adjusted so that the relationship between the position of the agitator paddle and the liquid level of the contained liquid medium (500 mL) was as follows: "the distance from the bottom of the culture vessel to the lower agitator paddle": "the ratio of the distance from the lower agitator to the upper agitator": "the distance from the upper agitator to the liquid surface of the culture medium" = 4:7:15.

[0198] Place 500 mL of a medium (pH 6.0) containing 2% glucose and 1% yeast extract into each of four 1-L fermenters, and prepare four types of liquid media: liquid medium J without a Δ5 desaturase inhibitor, liquid medium K supplemented with 50 mg of sesamin, liquid medium L supplemented with 50 mg of 2-amino-N-(3-chlorophenyl)benzamide, and liquid medium M supplemented with 50 mg of sesamin and 50 mg of 2-amino-N-(3-chlorophenyl)benzamide.

[0199] After each of the liquid media J - M was sterilized at 120 °C for 20 minutes, 20 mL of a pre-culture liquid medium of Mortierella alpina strain lacking Δ5 desaturase was inoculated into each of the liquid media, and cultured for 12 days at 0.6 v.v.m. aeration and a temperature of 28 °C under aeration and stirring conditions. On the 3rd, 4th, 5th, 6th, 7th, 10th, and 11th days, 50 mg of sterilized sesamin was added to liquid medium K, 50 mg of sterilized 2-amino-N-(3-chlorophenyl)benzamide was added to liquid medium L, and a combination of 50 mg of sterilized sesamin and 50 mg of 2-amino-N-(3-chlorophenyl)benzamide was added to liquid medium M. After cultivation, the microbial biomass was recovered by centrifugation. After washing thoroughly with water, the microbial biomass was freeze-dried. 2.9 g of dry microbial biomass (dry microbial biomass J) was obtained from liquid medium J. 2.9 g of dry microbial biomass (dry microbial biomass K) was obtained from liquid medium K. 1.5 g of dry microbial biomass (dry microbial biomass L) was obtained from liquid medium L. 2.9 g of dry microbial biomass (dry microbial biomass M) was obtained from liquid medium M.

[0200] 175 mL of hexane was added to each of the dry microbial biomass J - M to extract the liquid, stirred at room temperature for 30 minutes, and then the obtained mixture was filtered to obtain an extract and microbial cells. This operation was repeated three times to obtain a hexane extract. The hexane extract was concentrated under reduced pressure using a rotary evaporator. 552.0 mg of microbial oil, namely microbial oil J, was obtained from dry microbial biomass J. 379.5 mg of microbial oil, namely microbial oil K, was obtained from dry microbial biomass K. 195.5 mg of microbial oil, namely microbial oil L, was obtained from dry microbial biomass L. 552.0 mg of microbial oil, namely microbial oil M, was obtained from dry microbial biomass M. The lipid content and type in the obtained microbial oils J - M were analyzed by thin-layer chromatography / flame ionization detector (TLC / FID) method (IATROSCAN (trade name; the same below), Mitsubishi Chemical Medience Corp.).

[0201] Furthermore, 0.10 mL of 10% (v / v) sulfuric acid ethanol solution was added to each of the microbial oils in 0.5 mg of microbial oil J-M, and the reaction was carried out at 80 °C for 30 minutes for ethyl esterification. After the neutralization reaction, 0.18 mL of 1.0 M sodium hydroxide ethanol solution was added to the liquid. Then, 0.05 mL of hexane and 0.30 mL of saturated sodium chloride solution were added for extraction. 480.0 mg of ethyl fatty acid esters, namely ethyl fatty acid ester J, was obtained from microbial oil J. 330.0 mg of ethyl fatty acid esters, namely ethyl fatty acid ester K, was obtained from microbial oil K. 170.0 mg of ethyl fatty acid esters, namely ethyl fatty acid ester L, was obtained from microbial oil L. 480.0 mg of ethyl fatty acid esters, namely ethyl fatty acid ester M, was obtained from microbial oil M. The fatty acid content and type of the obtained ethyl fatty acid esters J-M were analyzed by gas chromatography.

[0202] The results obtained using IATROSCAN and gas chromatography are shown in Table 3.

[0203] The IATROSCAN analysis conditions and gas chromatography analysis conditions are listed below.

[0204] IATROSCAN analysis conditions

[0205] Developing agent:

[0206] 0 - 3 min. CHC1 3 :MeOH = 95:5 (v / v)

[0207] 3 - 23 min. Hexane:diethyl ether:formic acid = 90:10:0.2 (v / v)

[0208] Sample concentration: 10 mg / mL

[0209] Addition amount: 5 μl

[0210] Gas chromatography analysis conditions

[0211] Device type: Agilent 7890 GC system (Agilent Technologies)

[0212] Column: DB-WAX (Agilent Technologies, 30 m × 0.25 mm ID, 0.25 μm film thickness) J&W 122 - 7032

[0213] Column oven: 180 °C - 3 °C / min - 230 °C (25 min)

[0214] Injection temperature: 270 °C

[0215] Injection method: split

[0216] Split ratio: 20:1

[0217] Detector temperature: 270 °C

[0218] Detector: FID

[0219] Carrier gas: helium (1.0 mL / min, constant flow)

[0220] As is clear from Table 3, when the Δ5 desaturase-deficient strain belonging to Mortierella alpina itself (microbial oil J) used in this working example, when cultured in the presence of one type of Δ5 desaturase inhibitor (microbial oils K and L), and when cultured in the presence of two types of Δ5 desaturase inhibitors (microbial oil M), the DGLA / ARA ratio of each oil is greater than or equal to 15.

[0221] Among these microbial oils, although the total fat content in the liquid medium of microbial oil L to which only one type of Δ5 desaturase inhibitor was added decreased; an unexpected result was also obtained, that is, microbial oil M using two types of Δ5 desaturase inhibitors inhibited the decrease in the total fat content in the liquid medium.

[0222] Table 3

[0223]

[0224]

[0225] * The value of each fatty acid is calculated based on the total fatty acid esters (mg).

[0226] * Except for DGLA / ARA, these values refer to the amount (mg) produced per 500 mL of culture medium.

[0227] Example 4

[0228] (1) Refining of fatty acid ethyl ester by rectification

[0229] Each of the fatty acid ethyl ester compositions J, K, and M obtained in Example 3 was subjected to a rectification process under reduced pressure to obtain various fractions (fatty acid ethyl ester fractions J, K, and M), each fraction containing fatty acid ethyl ester J, K, or M derived from fatty acids having 20 or more carbon atoms as the main component.

[0230] The results are shown in Table 4. As is clear from Table 4, by rectifying the ethyl ester in which the DGLA / ARA ratio was controlled to 2836 by adding sesamin and 2-amino-N-(3-chlorophenyl)benzamide in the working example, the obtained fatty acid ester fraction has 20 or more carbon atoms and an extremely low ARA content.

[0231] Table 4

[0232]

[0233] (2) Refining of fatty acid ethyl ester using column chromatography

[0234] The rectified fatty acid ethyl esters J, K, and M (their compositions are shown in Table 4) are subjected to further highly purification using ODS (octadecylsilyl)-HPLC. The separation conditions are listed below.

[0235] Separation conditions

[0236] Column: ODS AQ S-50 12nm (YMC Corp., Ltd.),

[0237] Separation liquid: Methanol

[0238] Flow rate: 25 mL / min

[0239] Column temperature: 40 °C

[0240] Sample size: 1.42 g

[0241] Detector: UV / Vis spectrophotometer and differential refractometer

[0242] Table 5 shows the fatty acid compositions after ODS-HPLC purification of each fatty acid ethyl ester fraction when the HPLC yield is 65%. In addition, the fatty acid compositions (%) are based on the area ratio of the gas chromatogram in the aforementioned manner.

[0243] Table 5

[0244]

[0245] As is clear from Table 5, for each fatty acid ethyl ester fraction (J, K, and M), ODS-HPLC refining can increase the content of DGLA. Specifically, especially for the fatty acid ethyl ester fraction M, a purity of 95% by weight or higher can be obtained.

[0246] On the other hand, compared with the DGLA / ARA ratio of the crude oil before refining, the DGLA / ARA ratio of the fatty acid ethyl ester fraction obtained by further highly purifying DGLA does not change significantly. It should be understood that it is difficult to separate DGLA from ARA during the refining process. In order to obtain a high concentration of DGLA ethyl ester with a low ARA content, therefore, it should be understood that it is effective to increase the DGLA / ARA ratio in the early stage.

[0247] In particular, it is to be understood that culturing a microbial strain while adding a combination of two types of Δ5 desaturase inhibitors (such as sesamin and 2-amino-N-(3-chlorophenyl)benzamide) during the growth of the microorganism is effective for this.

[0248] Therefore, it is to be understood that the present invention provides a microorganism and a microbial oil containing an oil having a high DGLA / ARA ratio, and also provides lower alcohol esters and free fatty acids obtained from such a microorganism and oil.

[0249] The present invention includes the following technical solutions:

[0250] 1. A microbial oil comprising dihomo-γ-linolenic acid as a fatty acid component of the oil, wherein the microbial oil has an arachidonic acid to dihomo-γ-linolenic acid (arachidonic acid / dihomo-γ-linolenic acid) weight ratio of less than 1 / 13.

[0251] 2. The microbial oil according to item 1, wherein the weight ratio is less than or equal to 1 / 15, preferably less than or equal to 1 / 20.

[0252] 3. The microbial oil according to item 1 or 2, wherein the triglyceride content of the microbial oil is greater than or equal to 70% by weight.

[0253] 4. The microbial oil according to item 3, wherein the triglyceride content of the microbial oil is greater than or equal to 90% by weight.

[0254] 5. The microbial oil according to any one of items 1-4, which contains 0.1%-10% by weight of phospholipids.

[0255] 6. The microbial oil according to any one of items 1-5, wherein the saturated fatty acid content of the microbial oil is less than or equal to 40% by weight.

[0256] 7. The microbial oil according to any one of items 1-6, wherein the microbial oil is a crude oil, and preferably, the triglyceride content is greater than or equal to 90% by weight and the weight ratio of arachidonic acid to dihomo-γ-linolenic acid is less than or equal to 1 / 15.

[0257] 8. The microbial oil according to any one of items 1-6, wherein the microbial oil is a refined oil, and preferably, the triglyceride content is greater than or equal to 90% by weight and the weight ratio of arachidonic acid to dihomo-γ-linolenic acid is less than or equal to 1 / 20.

[0258] 9. A lower alcohol ester composition containing dihomo-γ-linolenic acid ester, or a free fatty acid composition containing dihomo-γ-linolenic acid, wherein the lower alcohol ester composition or the free fatty acid composition is produced or obtainable by a method comprising subjecting the microbial oil according to any one of items 1-8 to a transesterification reaction or a hydrolysis reaction.

[0259] 10. A lower alcohol ester composition derived from microbial oil and containing dihomo-γ-linolenic acid ester, or a free fatty acid composition derived from microbial oil and containing dihomo-γ-linolenic acid, wherein the weight ratio of arachidonic acid to dihomo-γ-linolenic acid (arachidonic acid / dihomo-γ-linolenic acid) is less than 1 / 13.

[0260] 11. The microbial oil according to any one of items 1-8, or the lower alcohol ester composition or the free fatty acid composition according to item 9 or 10, wherein the arachidonic acid content is less than or equal to 7% by weight.

[0261] 12. The microbial oil according to any one of items 1-8, or the lower alcohol ester composition or the free fatty acid composition according to item 9 or 10, which is used as a drug, preferably as an anti-allergy drug or an anti-inflammatory drug.

[0262] 13. Use of the microbial oil according to any one of items 1-8, or the lower alcohol ester composition or the free fatty acid composition according to item 9 or 10 in a method for preparing food, dietary supplements, drugs, cosmetics or animal feed.

[0263] 14. A microbial biomass containing the microbial oil according to any one of items 1-6.

[0264] 15. A liquid medium containing the microbial biomass according to item 14.

[0265] 16. The liquid medium according to item 15, wherein the content of the microbial biomass is greater than or equal to 2.5 g / L based on the dry weight of the microbial biomass.

[0266] 17. The liquid medium according to item 15 or 16, wherein the liquid medium contains a content of the microbial oil according to any one of items 1-6 of 0.4 g / L or more.

[0267] 18. A method for preparing a microbial oil containing dihomo-γ-linolenic acid, the method comprising:

[0268] adding at least two types of Δ5 desaturase inhibitors to a liquid medium, and

[0269] Culturing a microorganism having reduced Δ5 desaturase activity or lacking Δ5 desaturase activity in the liquid medium to produce the microbial oil containing dihomo-γ-linolenic acid.

[0270] 19. The method according to item 18, wherein one of the at least two types of Δ5 desaturase inhibitors is 2-amino-N-(3-chlorophenyl)benzamide.

[0271] 20. The method according to item 18 or 19, wherein one of the at least two types of Δ5 desaturase inhibitors, or the Δ5 desaturase inhibitor different from 2-amino-N-(3-chlorophenyl)benzamide is a dioxabicyclo[3.3.0]octane derivative represented by formula (I):

[0272]

[0273] wherein, R 1 、R 2 、R 3 、R 4 、R 5 and R 6 are each independently a hydrogen atom or an alkyl group having 1-3 carbon atoms; or, R 1 and R 2 、and / or R 4 and R 5 together form a methylene or ethylene group, and n, m and L represent 0 or 1;

[0274] Piperonyl butoxide, curcumin or a compound represented by formula (II):

[0275]

[0276] wherein, R 1 represents a lower alkyl group; R 2 represents a hydroxyl group, an alkyl group, an alkoxy group, an alkenyl group or an oxyalkyl group, wherein in the case of the presence of a plurality of R 2 , the plurality of R 2 may be the same or different, and n is an integer from 0 to 5.

[0277] 21. The method according to item 20, wherein the dioxabicyclo[3.3.0]octane derivative is sesamin, sesaminol, epi-sesamin, epi-sesaminol, 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, or 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane.

[0278] 22. The method according to item 20, wherein one of the at least two types of Δ5 desaturase inhibitors, or the Δ5 desaturase inhibitor different from 2-amino-N-(3-chlorophenyl)benzamide is sesamin or curcumin.

[0279] 23. The method according to any one of items 18-22, wherein the microorganism is a microorganism belonging to the genus Mortierella, and is a transgenic and / or non-transgenic microorganism.

[0280] 24. A method for preparing a lower alcohol ester composition or a free fatty acid composition according to item 9 or 10 from the microbial oil according to any one of items 1-8, the method comprising:

[0281] (a) obtaining a mixture of free fatty acids or lower alcohol esters of fatty acids by hydrolysis or alcoholysis of the microbial oil, and

[0282] (b) rectifying the mixture of free fatty acids or lower alcohol esters to obtain a free fatty acid or lower alcohol ester composition in which the fatty acid has at least 20 carbon atoms.

[0283] 25. A method for preparing a lower alcohol dihomo-γ-linolenate or free dihomo-γ-linolenic acid, comprising preparing a lower alcohol ester composition or a free fatty acid composition according to item 24, and then

[0284] (c) fractionating and purifying the lower alcohol ester of dihomo-γ-linolenic acid or dihomo-γ-linolenic acid from the free fatty acid or the lower alcohol ester composition in which the fatty acid has at least 20 carbon atoms by reverse phase distribution column chromatography.

[0285] 26. A method for preparing a lower alcohol ester composition or a free fatty acid composition containing dihomo-γ-linolenic acid, comprising:

[0286] (a) Preparing a microbial oil containing dihomo-γ-linolenic acid by culturing a microorganism in a liquid medium to produce the dihomo-γ-linolenic acid, wherein the microorganism is one that has inhibited the function of producing arachidonic acid by optionally reducing or losing Δ5 desaturation activity in the liquid medium in the presence of one, two or more types of Δ5 desaturase inhibitors, thereby producing the microbial oil in which the weight ratio of arachidonic acid to dihomo-γ-linolenic acid is less than 1 / 13;

[0287] (b) Obtaining a mixture of free fatty acids or lower alcohol esters containing dihomo-γ-linolenic acid by hydrolysis or alcoholysis of the microbial oil, optionally after purifying the oil;

[0288] (c) Purifying the mixture of free fatty acids or lower alcohol esters.

[0289] 27. The method according to item 26, wherein in step (c), the mixture is purified to obtain a mixture of free fatty acids or lower alcohol esters in which the fatty acids have at least 20 carbon atoms, and the method further comprises

[0290] (d) Fractionating and purifying dihomo-γ-linolenic acid or a lower alcohol ester of dihomo-γ-linolenic acid from the purified mixture by reverse-phase chromatography.

[0291] 28. The method according to item 25, 26 or 27, further comprising incorporating the purified dihomo-γ-linolenic acid or a lower alcohol ester of dihomo-γ-linolenic acid into a medicament.

[0292] Industrial applicability

[0293] The microbial oil containing DGLA of the present invention has a low arachidonic acid content. Therefore, the effect of arachidonic acid can become very small while administering a given amount of DGLA. Compositions suitable for applications that do not require arachidonic acid can be provided, for example, compositions for use as anti-allergy drugs and compositions for use as anti-inflammatory drugs.

Claims

1. A method for preparing a microbial oil containing dihomo-γ-linolenic acid, the method comprises: adding at least two types of Δ5 desaturase inhibitors to a liquid medium, and culturing Mortierella alpina SAM 1860 in the liquid medium to produce the microbial oil containing dihomo-γ-linolenic acid, one of the at least two types of Δ5 desaturase inhibitors is 2-amino-N-(3-chlorophenyl)benzamide, and the other is sesamin.

2. A method for preparing a lower alcohol ester composition or free fatty acid composition containing dihomo-γ-linolenic acid, comprises: (a) preparing a microbial oil containing dihomo-γ-linolenic acid by the method according to claim 1; (b) obtaining a mixture of free fatty acids or lower alcohol esters containing dihomo-γ-linolenic acid after hydrolysis or alcoholysis of the microbial oil and purification of the oil; (c) purifying the mixture of free fatty acids or lower alcohol esters.

3. The method according to claim 2, wherein in step (c), the mixture is purified to obtain a mixture of free fatty acids or lower alcohol esters in which the fatty acids have at least 20 carbon atoms, and the method further comprises (d) fractionating and purifying dihomo-γ-linolenic acid or lower alcohol esters of dihomo-γ-linolenic acid from the purified mixture by reverse phase chromatography.

4. The method according to claim 2 or 3, further comprising incorporating the purified dihomo-γ-linolenic acid or lower alcohol esters of dihomo-γ-linolenic acid into a drug.

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