Microbial oil, method for manufacturing microbial oil, concentrated microbial oil, and method for manufacturing concentrated microbial oil
35 claims: 3 independent, 32 dependent
- 1アラキドン酸(ARA)を生産することができる微生物の微生物バイオマス を起源として得られる 、脂肪酸アルキルエステル又は遊離脂肪酸を含んでなる微生物油において、当該微生物油は、脂肪酸アルキルエステル形態及び/又は遊離脂肪酸形態であるアラキドン酸を当該油中の脂肪酸の合計重量の80重量%以上の含有率で含んでなり、ここで、蒸留を含む加熱工程により生成する熱生成脂肪酸の含有率が油中の脂肪酸の合計重量の0.0001重量%~3.0重量%であり、そして前記熱生成脂肪酸は、アラキドン酸の炭素二重結合の一部又はすべてがトランス型に変化したものである。
- 2前記アラキドン酸の含有率が、油中の脂肪酸の合計重量の80重量%~98重量%である請求項1記載の微生物油。
- 3炭素数22の飽和脂肪酸及び炭素数24の飽和脂肪酸の合計含有率が、油中の脂肪酸の合計重量の6.0重量%以下である請求項1又は2記載の微生物油。
- 4炭素数22の飽和脂肪酸及び炭素数24の飽和脂肪酸の合計含有率が、前記アラキドン酸の含有率の10/100以下である請求項1~請求項3のいずれか1項記載の微生物油。
- 5炭素数24の飽和脂肪酸の含有率が、油中の脂肪酸の合計重量の3.0重量%以下である請求項1~請求項4のいずれか1項記載の微生物油。
- 6炭素数24の飽和脂肪酸の含有率が、前記アラキドン酸の含有率の4/100以下である請求項1~請求項5のいずれか1項記載の微生物油。
- 7液体クロマトグラフィーでの分離に関する指標であって、脂肪酸の炭素数及び二重結合数から求められるパーティションナンバーを用いた場合に、アラキドン酸のパーティションナンバーと比べて、2少ない数以上2多い数以下のパーティションナンバーを有し、当該アラキドン酸の炭素数とは異なる炭素数を有する他の飽和又は不飽和脂肪酸の含有率が、油中の脂肪酸の合計重量の10.0重量%以下である請求項1~請求項6のいずれか1項記載の微生物油。
- 8前記他の飽和又は不飽和脂肪酸の含有率が、前記アラキドン酸の含有率の15/100以下である請求項7記載の微生物油。
- 9前記他の飽和又は不飽和脂肪酸が、炭素数18の飽和脂肪酸、炭素数18の一価不飽和脂肪酸、炭素数18の二価不飽和脂肪酸、炭素数18の三価不飽和脂肪酸及び炭素数18の四価不飽和脂肪酸からなる群より選択された少なくとも1つを含む請求項7又は請求項8記載の微生物油。
- 10前記熱生成脂肪酸が、炭素数20の熱生成脂肪酸である請求項1~請求項9のいずれか1項記載の微生物油。
- 11前記熱生成脂肪酸の含有率が、油中の脂肪酸の合計重量の0.001重量%~2.8重量%である請求項1~請求項9のいずれか1項記載の微生物油。
- 12炭素数18の一価不飽和脂肪酸の含有率が、油中の脂肪酸の合計重量の7.0重量%以下である請求項9~請求項11のいずれか1項記載の微生物油。
- 13炭素数18の一価不飽和脂肪酸の含有率が、前記アラキドン酸の含有率の10/100以下である請求項9~請求項12のいずれか1項記載の微生物油。
- 14炭素数18の二価不飽和脂肪酸の含有率が、前記アラキドン酸の含有率の7/100以下である請求項9~請求項13のいずれか1項記載の微生物油。
- 15炭素数18の一価不飽和脂肪酸及び炭素数18の二価不飽和脂肪酸の合計含有率が、前記アラキドン酸の15/100以下である請求項9~請求項14のいずれか1項記載の微生物油。
- 16炭素数18の飽和脂肪酸の含有率が、前記アラキドン酸の含有率の11/100以下である請求項9~請求項15のいずれか1項記載の微生物油。
- 17微生物油の製造方法であって、微生物バイオマスから得られたアルキルエステル形態及び/又は遊離脂肪酸形態のアラキドン酸を含む原料油を用意すること、並びに、前記原料油に対して、160°C~230°Cの塔底温度及び0.1Pa~30Paの蒸留塔内における最低圧力を含む条件によって精密蒸留による精製を行うことにより、脂肪酸の合計重量の80重量%以上の含有率を有する、アラキドン酸を得ること、を含む、当該製造方法。
- 18微生物油の製造方法であって、微生物バイオマスから得られたアルキルエステル形態及び/又は遊離脂肪酸形態のアラキドン酸を含む原料油を用意すること、前記原料油に対して、規則充填物を含む蒸留塔を用いて、160°C~230°Cの塔底温度及び0.1Pa~30Paの蒸留塔内における最低圧力を含む条件による精密蒸留を行うこと、並びに、請求項1~請求項16のいずれか1項記載の微生物油を得ること、を含む、当該製造方法。
- 19微生物油の製造方法であって、微生物バイオマスから得られたアルキルエステル形態及び/又は遊離脂肪酸形態のアラキドン酸を含む原料油を用意すること、前記原料油に対して、規則充填物を含む蒸留塔を用いて、160°C~230°Cの塔底温度及び0.1Pa~30Paの蒸留塔内における最低圧力で、油中の脂肪酸の合計重量の0.0001重量%~3.0重量%の含有率の炭素数16~22の熱生成脂肪酸を含む微生物油が得られ得る条件による精密蒸留を行うこと、並びに、請求項1~請求項16のいずれか1項記載の微生物油を得ること、を含む、当該製造方法。
- 20前記精密蒸留が、互いに異なる塔底温度及び蒸留塔内における最低圧力の条件による複数回の精密蒸留を含む請求項18又は請求項19記載の製造方法。
- 21前記精密蒸留が、160°C~220°Cの塔底温度及び0.1Pa~30Paの蒸留塔内における最低圧力による低温精密蒸留と、170°C~230°Cの塔底温度及び0.1Pa~30Paの蒸留塔内における最低圧力による高温精密蒸留を含む請求項20記載の製造方法。
- 22前記高温精密蒸留における塔底温度が、前記低温精密蒸留の塔底温度よりも3°C~20°C高い請求項21記載の製造方法。
- 23規則充填物1単位あたりの比表面積が、125m 2 /m 3 ~1700m 2 /m 3 である請求項18~請求項22のいずれか1項記載の製造方法。
- 24脂肪酸アルキルエステル形態及び/又は遊離脂肪酸形態のアラキドン酸の含有率が、油中の脂肪酸の合計重量の90重量%~98重量%であり、熱生成脂肪酸の含有率が、油中の脂肪酸の合計重量の0.0001重量%~3. 0重量%であり、炭素数24の飽和脂肪酸及び炭素数22の飽和脂肪酸の合計含有率が、油中の脂肪酸の合計重量の1.0重量%以下であり、炭素数18の一価不飽和脂肪酸の含有率が、油中の脂肪酸の合計重量の5.0重量%以下である、請求項1に記載の微生物油。
- 25濃縮微生物油の製造方法であって、請求項17~請求項23のいずれか1項記載の製造方法を用いて、アラキドン酸を含有する微生物油を得ること、得られた微生物油に対して、逆相カラムクロマトグラフィーを用いた濃縮処理を行うこと、を含む、当該方法。
- 26請求項1~請求項16のいずれか1項記載の微生物油又は請求項24記載の微生物油の、食品、サプリメント、医薬品、化粧品又は飼料における使用。
- 27請求項1~請求項16のいずれか1項記載の微生物油又は請求項24記載の微生物油の、食品、サプリメント、医薬品、化粧品又は飼料の製造方法における使用。
- 28請求項1~請求項16のいずれか1項記載の微生物油又は請求項24記載の微生物油を含む医薬品。
- 29請求項1~請求項16のいずれか1項記載の微生物油又は請求項24記載の微生物油を含む炎症性疾患予防又は治療剤。
- 30抗アレルギー剤又は抗炎症剤である請求項29記載の炎症性疾患予防又は治療剤。
- 31前記炎症性疾患が、発疹、蕁麻疹、水疱、膨疹及び湿疹からなる群より選択される少なくとも1つの皮膚の炎症性疾患、又は、放射線への曝露、自己免疫疾患及び尿毒症性そう痒からなる群より選択される少なくとも1つにより引き起こされる皮膚の炎症性疾患である請求項29又は請求項30記載の炎症性疾患予防又は治療剤。
- 32前記炎症性疾患が、アトピー性皮膚炎、アレルギー性接触性皮膚炎、刺激性接触皮膚炎、光接触皮膚炎、全身性接触皮膚炎、リウマチ、乾癬及び狼瘡からなる群より選択される少なくとも1つである請求項29又は請求項30記載の炎症性疾患予防又は治療剤。
- 33前記精密蒸留が、互いに異なる塔底温度及び蒸留塔内における最低圧力の条件による複数回の精密蒸留を含む請求項17記載の製造方法。
- 34前記精密蒸留が、160°C~220°Cの塔底温度及び0.1Pa~30Paの蒸留塔内における最低圧力による低温精密蒸留と、170°C~230°Cの塔底温度及び0.1Pa~30Paの蒸留塔内における最低圧力による高温精密蒸留を含む請求項33記載の製造方法。
- 35前記高温精密蒸留における塔底温度が、前記低温精密蒸留の塔底温度よりも3°C~20°C高い請求項34記載の製造方法。
Independent claims35
196 paragraphs, as filed
The present invention relates to a microbial oil, a method for producing a microbial oil, a concentrated microbial oil, and a method for producing a concentrated microbial oil.
Microbial oils include eicosadienoic acid, dihomo-gamma-linolenic acid (DGLA), eicosatetraenoic acid, arachidonic acid (ARA), eicosapentaenoic acid (EPA), docosatetraenoic acid, docosapentaenoic acid and docosahexaenoic acid. Contains long-chain polyunsaturated fatty acids with 20 or more carbon atoms such as (DHA). Medicines, health foods, cosmetics and the like using these long-chain polyunsaturated fatty acids as functional ingredients are attracting attention, and further applications are being investigated. Along with this, it is required to produce a large amount of polyunsaturated fatty acids at a high concentration.
In addition to long-chain polyunsaturated fatty acids, microbial oils contain a wide variety of unique oily components such as short-chain fatty acids, saturated fatty acids, phospholipids, sterols, glycerides, ceramides, sphingolipids, terpenoids, flavonoids, and tocopherols. included. These components may exhibit specific functions. For example, short-chain fatty acids may cause the peculiar odor of microbial oils, and the peculiar odor may not be favorable for exhibiting the functions required of specific long-chain polyunsaturated fatty acids. When concentrating or purifying specific long-chain polyunsaturated fatty acids contained in microbial oils, high-performance liquid chromatography, liquid-liquid partitioning, urea addition, etc. may be used.
For example, in Patent Document 1, microbial oil obtained from genetically modified microorganisms producing linoleic acid, DGLA, DHA, EPA, etc. is distilled at least once under short-path distillation conditions to obtain a sterol-containing microbial oil composition. A method for reducing the amount of sterols in a product is disclosed.
US Pat. No. 5,300,003 discloses that in the preparation of edible marine oil containing deodorized and stabilized microbial oil, the oil is subjected to countercurrent steam distillation (CCSD) in a thin film column containing structured packing, optionally with anti-oxidation. A method is disclosed that includes adding an agent.
<p><patcit num="1"><text>Japanese Patent Publication No. 2014-510166</text></patcit><patcit num="2"><text>Japanese Patent Publication No. 2010-526896</text></patcit></p>
<p>The purpose of the conventional methods is to separate some specific components other than fatty acids and specific fatty acids for the purpose of concentration or purification. Long chain polyunsaturated fatty acids are difficult to concentrate or purify to a high degree. When microbial oil is used as a raw material, the long-chain polyunsaturated fatty acids targeted for concentration or purification cannot be sufficiently concentrated or purified due to the influence of various other components contained in the raw material. Concentration or purification can also be performed by distillation techniques, but distillation techniques for concentrating or purifying the target long-chain polyunsaturated fatty acids in microbial oils are not yet well established.</p><p>The present invention provides a microbial oil useful for efficiently obtaining a refined microbial oil containing a high proportion of target polyunsaturated fatty acids, a method for producing the same, and a high polyunsaturated fatty acid content. It is an object of the present invention to provide a concentrated microbial oil, a method for producing the same, and uses of the microbial oil and the concentrated microbial oil.</p>
<p>Aspects of the present invention provide the following microbial oils, methods for producing microbial oils, concentrated microbial oils, methods for producing concentrated microbial oils, microbial oils and uses of concentrated microbial oils.</p><p><1> At least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of a fatty acid alkyl ester and/or free fatty acid, having a content of 50% by weight or more of the total weight of fatty acids in the oil, and in the oil and a thermogenic fatty acid having 16 to 22 carbon atoms having a content of 3.0% by weight or less of the total weight of the fatty acids.</p><p><2> The microbial oil according to <1>, wherein the polyunsaturated fatty acid content is 80% to 98% by weight of the total weight of fatty acids in the oil.</p><p><3> The microbial oil according to <1> or <2>, wherein the thermogenic fatty acid content is 0.0001% by weight to 3.0% by weight of the total weight of fatty acids in the oil.</p><p><4> Any one of <1> to <3>, wherein the total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms is 6.0% by weight or less of the total weight of fatty acids in the oil. microbial oil.</p><p><5> Any one of <1> to <4> in which the total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms is 10/100 or less of the content of the polyunsaturated fatty acids Microbial oil as described.</p><p><6> The microbial oil according to any one of <1> to <5>, wherein the content of saturated fatty acids with 24 carbon atoms is 3.0% by weight or less of the total weight of fatty acids in the oil.</p><p><7> The microbial oil according to any one of <1> to <6>, wherein the content of saturated fatty acids with 24 carbon atoms is 4/100 or less of the content of polyunsaturated fatty acids.</p><p><8> When using the partition number determined from the carbon number and double bond number of the fatty acid, which is an indicator of separation by liquid chromatography, it is 2 less than the partition number of the polyunsaturated fatty acid The content of other saturated or unsaturated fatty acids with a partition number that is equal to or greater than 2 and equal to or less than the number of carbon atoms different from the number of carbon atoms of the polyunsaturated fatty acid is 10.0 of the total weight of fatty acids in the oil. The microbial oil according to any one of <1> to <7>, which is weight % or less.</p><p><9> The microbial oil according to <8>, wherein the other saturated or unsaturated fatty acid content is 15/100 or less of the polyunsaturated fatty acid content.</p><p><10> The polyunsaturated fatty acid is selected from eicosadienoic acid, dihomo-γ-linolenic acid, mead acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid and docosahexaenoic acid. The microbial oil according to any one of <1> to <9>, which is at least one selected from the group consisting of:</p><p><11> The other saturated or unsaturated fatty acids include saturated fatty acids with 18 carbon atoms, monounsaturated fatty acids with 18 carbon atoms, divalent unsaturated fatty acids with 18 carbon atoms, trivalent unsaturated fatty acids with 18 carbon atoms and The microbial oil according to any one of <8> to <10>, containing at least one selected from the group consisting of tetravalent unsaturated fatty acids having 18 carbon atoms.</p><p><12> The microbial oil according to any one of <1> to <11>, wherein the polyunsaturated fatty acid is dihomo-γ-linolenic acid, and the thermogenic fatty acid is a thermogenic fatty acid having 20 carbon atoms. .</p><p><13> Thermally generated fatty acid is a peak that appears within the range of 1.001 to 1.011 when the retention time of dihomo-γ-ethyl linolenate is set to 1 in gas chromatography analysis under the following conditions for the thermally generated fatty acid ethyl ester and the second substance having a retention time as a peak appearing in the range of 1.013 to 1.027.</p><p>Equipment: 6890N Network GC system (Agilent Technologies Inc.) Column: DB-WAX Length 30m x I.D. 0.25mm x Film thickness 0.25μm (Agilent Technologies Inc.) Column temperature conditions: 60°C 2.5 minutes heating up 20 minutes °C/min 180°C 2°C/min 230°C 15 min Inlet temperature: 210°C, splitless, split vent sampling time 1.5 min, purge flow rate 40 mL/min Injection volume conditions: 1 μL , Sample concentration 1 mg/mL or less Detector: FID detector Temperature: 280°C Carrier gas conditions: Helium, linear velocity 24 cm/min <14> The polyunsaturated fatty acid is dihomo-γ-linolenic acid, The microbial oil according to <13>, wherein the total content of the first substance and the second substance is 0.001% by weight to 2.8% by weight of the total weight of fatty acids in the oil.</p><p><15> The microbial oil according to any one of <10> to <14>, wherein the content of C18 monounsaturated fatty acids is 7.0% by weight or less of the total weight of fatty acids in the oil.</p><p><16> The microbial oil according to any one of <10> to <15>, wherein the content of monounsaturated fatty acids with 18 carbon atoms is 10/100 or less of the content of polyunsaturated fatty acids.</p><p><17> The microbial oil according to any one of <10> to <16>, wherein the content of the divalent unsaturated fatty acid having 18 carbon atoms is 7/100 or less of the content of the polyunsaturated fatty acid.</p><p><18> The total content of monounsaturated fatty acids with 18 carbon atoms and divalent unsaturated fatty acids with 18 carbon atoms is 15/100 or less of the polyunsaturated fatty acid content <10> to <17 > The microbial oil according to any one of >.</p><p><19> The microbial oil according to any one of <10> to <18>, wherein the content of saturated fatty acids with 18 carbon atoms is 11/100 or less of the content of polyunsaturated fatty acids.</p><p><20> A method for producing a microbial oil, comprising preparing a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of an alkyl ester and/or free fatty acid obtained from microbial biomass; and purifying the raw material oil by precision distillation under conditions including a bottom temperature of 160°C to 230°C and a minimum pressure in the distillation column of 0.1Pa to 30Pa. .</p><p><21> A method for producing a microbial oil, comprising preparing a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl ester and/or free fatty acid obtained from microbial biomass; The raw material oil is subjected to precision distillation using a distillation column containing structured packing under conditions including a column bottom temperature of 160°C to 230°C and a minimum pressure in the distillation column of 0.1Pa to 30Pa. and obtaining the microbial oil according to any one of <1> to <19>.</p><p><22> A method for producing a microbial oil, comprising preparing a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl ester and/or free fatty acid obtained from microbial biomass; For the raw material oil, using a distillation column containing structured packing, including the bottom temperature and the minimum pressure in the distillation column according to the type of the target polyunsaturated fatty acid, Performing precision distillation under conditions that can obtain a microbial oil containing a thermogenic fatty acid with 16 to 22 carbon atoms with a content of 3.0% by weight or less of the total weight, and any one of <1> to <19> obtaining the described microbial oil.</p><p><23> The production method according to <22>, wherein the precision distillation is performed at a bottom temperature of 160°C to 230°C and a minimum pressure in the distillation column of 0.1Pa to 30Pa.</p><p><24> The production method according to any one of <20> to <23>, wherein the precision distillation includes a plurality of precision distillations under mutually different conditions of bottom temperature and minimum pressure in the distillation column.</p><p><25> The precision distillation includes low-temperature precision distillation at a bottom temperature of 160 ° C to 220 ° C and a minimum pressure in the distillation column of 0.1 Pa to 30 Pa, and a bottom temperature of 170 ° C to 230 ° C and 0.1 The production method according to <24>, which includes high-temperature precision distillation at a minimum pressure in a distillation column of Pa to 30 Pa.</p><p><26> The production method according to <25>, wherein the bottom temperature in the high-temperature precision distillation is 3°C to 20°C higher than the bottom temperature in the low-temperature precision distillation.</p><p><27> The specific surface area per unit of structured packing is 125m<sup>2</sup>/m<sup>3</sup>~1700m<sup>2</sup>/m<sup>3</sup>The production method according to any one of <21> to <26>.</p><p><28> The content of polyunsaturated fatty acids with 20 or more carbon atoms in the form of fatty acid alkyl esters and/or free fatty acids is 90% to 98% by weight of the total weight of fatty acids in the oil and has 16 carbon atoms The content of ~22 thermogenic fatty acids is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil, and the total content of C24 saturated fatty acids and C22 saturated fatty acids is 1.0% by weight or less of the total weight of fatty acids in the oil, and the content of C18 monounsaturated fatty acids is 5.0% or less by weight of the total weight of fatty acids in the oil.</p><p><29> The content of dihomo-γ-linolenic acid in the form of fatty acid alkyl ester and/or free fatty acid is 90% to 98% by weight of the total weight of fatty acids in the oil, and the heat of 16 to 22 carbon atoms The content of produced fatty acids is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil, and the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is the total weight of fatty acids in the oil. A concentrated microbial oil that is no more than 1.0% by weight and has a C18 monounsaturated fatty acid content of no more than 5.0% by weight of the total weight of fatty acids in the oil.</p><p><30> A method for producing a concentrated microbial oil, wherein at least one target fatty acid alkyl ester form and/or free fatty acid form is produced using the production method according to any one of <20> to <27> A method comprising obtaining a microbial oil containing a polyunsaturated fatty acid having 20 or more carbon atoms, and subjecting the obtained microbial oil to a concentration treatment using reversed-phase column chromatography.</p><p><31> Use of the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29> in food, supplements, pharmaceuticals, cosmetics or feed.</p><p><32> Use of the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29> in a method for producing food, supplements, pharmaceuticals, cosmetics, or feed.</p><p><33> A drug containing the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29>.</p><p><34> An agent for preventing or treating inflammatory diseases, comprising the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29>.</p><p><35> The preventive or therapeutic agent for inflammatory diseases according to <34>, which is an anti-allergic agent or an anti-inflammatory agent.</p><p><36> The inflammatory disease is at least one skin inflammatory disease selected from the group consisting of rash, hives, blisters, wheals and eczema, or exposure to radiation, autoimmune disease and uremic ulcer The preventive or therapeutic agent for inflammatory diseases according to <34> or <35>, which is a skin inflammatory disease caused by at least one selected from the group consisting of itching.</p><p><37> The inflammatory disease is selected from the group consisting of atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, photocontact dermatitis, systemic contact dermatitis, rheumatism, psoriasis and lupus The preventive or therapeutic agent for inflammatory diseases according to <34> or <35>, which is at least one.</p><p><38> Inflammation comprising administering the inflammatory disease preventive or therapeutic agent according to any one of <34> to <37> to a subject suffering from or at risk of suffering from an inflammatory disease Methods of prevention, treatment or amelioration of sexually transmitted diseases.</p><p><39> The method for preventing, treating, or ameliorating an inflammatory disease according to <37>, wherein the administration is oral administration or topical administration.</p><p><40> A microbial oil obtained by the production method according to any one of <20> to <27>.</p><p><41> A concentrated microbial oil obtained by the production method described in <30>.</p>
<p>According to the present invention, a microbial oil useful for efficiently obtaining a refined microbial oil containing a high proportion of target polyunsaturated fatty acids, a method for producing the same, and a high content of polyunsaturated fatty acids A concentrated microbial oil containing and a method for producing the same, and uses of the microbial oil and the concentrated microbial oil can be provided.</p>
A method for producing a microbial oil according to one aspect of the present invention is a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of an alkyl ester and/or free fatty acid obtained from microbial biomass. and performing purification by precision distillation under conditions including a column bottom temperature of 160 ° C to 230 ° C and a minimum pressure in the distillation column of 0.1 Pa to 30 Pa for the raw material oil. include.
The present invention uses precision distillation under specific conditions for feedstock oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl ester and/or free fatty acid obtained from microbial biomass. Based on the finding that purification results in a microbial oil containing a high content of at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of the targeted alkyl ester and/or free fatty acid.
In this specification, at least one polyunsaturated fatty acid having 20 or more carbon atoms in alkyl ester form and/or free fatty acid form may be referred to as target LC-PUFA unless otherwise specified. . In addition, in this specification, unless otherwise specified, individual forms of saturated or unsaturated fatty acids in the form of fatty acid alkyl esters or free fatty acids contained in raw material oil obtained from microbial biomass may be omitted. For example, an unsaturated fatty acid with 20 or more carbon atoms in the form of a fatty acid alkyl ester and an unsaturated fatty acid with 20 or more carbon atoms in the form of a free fatty acid are both referred to as "unsaturated fatty acids with 20 or more carbon atoms". The 22-carbon saturated fatty acid and the free fatty acid form of the 22-carbon saturated fatty acid are both referred to as "22-carbon saturated fatty acid."
That is, simple distillation such as molecular distillation has been used to purify raw material oil obtained from microbial biomass, but simple distillation only separates fatty acids by heating. Unsaturated fatty acids could not be separated from non-targeted fatty acids with good accuracy.
In the present invention, when purifying the target LC-PUFA from the raw material oil obtained from such microbial biomass, purification is performed by precision distillation under specific temperature and pressure conditions. Saturated fatty acids can be purified with higher accuracy and higher content.
In addition, a method for producing a microbial oil according to another aspect of the present invention provides a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of an alkyl ester and/or free fatty acid obtained from microbial biomass. Preparing, for the feedstock oil, using a distillation column containing structured packing, precision measurement under conditions including a bottom temperature of 160 ° C to 230 ° C and a minimum pressure in the distillation column of 0.1 Pa to 30 Pa Distilling and obtaining the particular microbial oil in one aspect of the invention described below.
Further, in a method for producing a microbial oil according to still another aspect of the present invention, a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of alkyl ester and/or free fatty acid obtained from microbial biomass is used. Prepare, for the feedstock oil, using a distillation column containing structured packing, including the bottom temperature and the minimum pressure in the distillation column according to the type of the target polyunsaturated fatty acid, performing precision distillation under conditions under which a microbial oil containing a thermogenic fatty acid with 16 to 22 carbon atoms can be obtained at a content of 3.0% by weight or less of the total weight of the fatty acids in the oil; obtaining a microbial oil.
The microbial oil according to one aspect of the present invention is at least one multivalent fatty acid having 20 or more carbon atoms in the form of a fatty acid alkyl ester and/or in the form of a free fatty acid, having a content of 50% or more by weight of the total weight of fatty acids in the oil. The microbial oil contains unsaturated fatty acids and thermogenic fatty acids with 16 to 22 carbon atoms having a content of 3.0% by weight or less of the total weight of the fatty acids in the oil.
In order to purify specific fatty acids with higher accuracy from various components contained in the raw material oil by precision distillation, it may be advantageous to perform the purification under severer conditions, such as higher temperature conditions. For example, by raising the column bottom temperature to increase the amount of vapor, the reflux ratio (reflux amount/fraction withdrawal amount) can be increased, and the separation of each fatty acid in precision distillation can be improved. In addition, in the raw material oil from microbial biomass, the content of long-chain saturated fatty acids with a higher melting point than the target LC-PUFA, such as saturated fatty acids with 22 carbon atoms or saturated fatty acids with 24 carbon atoms, is generally known. It tends to be higher than raw material oils obtained from vegetable oils and the like. It was found that the long-chain saturated fatty acids in such microbial oils have higher molecular weights, higher boiling points, and lower saturated vapor pressures at the same temperature than the target LC-PUFAs. Therefore, when distilling a raw material oil derived from a microbial oil containing a large amount of these long-chain saturated fatty acids, a higher distillation temperature, that is, a tower bottom temperature, is required compared to a raw material oil derived from a fish oil or the like containing less of these long-chain saturated fatty acids. It was found that That is, in order to obtain a microbial oil having a high content of polyunsaturated fatty acids with 20 or more carbon atoms and a low content of long-chain saturated fatty acids having a higher melting point than these by distilling raw material oil from microbial biomass, Severe conditions, such as higher temperature conditions, are required than those of commonly known raw material oils such as fish oil and vegetable oil. On the other hand, it has been found that when distillation is carried out at higher temperature conditions, fatty acid components that were not produced before distillation, so-called thermogenic fatty acids, are generated in the microbial oil. The thermogenic fatty acids generated in the microbial oil are thought to include those produced from the target LC-PUFA under the influence of excessive heat. , it was found that the target LC-PUFA content tended to decrease. Thermogenic fatty acids produced from target LC-PUFAs in microbial oil tend not to be effectively separated from target LC-PUFAs even using reversed-phase column chromatography, and the inclusion of target LC-PUFAs in concentrated microbial oil rate of It was found to cause a decrease and a decrease in yield. From these results, it was found that purification by reversed-phase column chromatography for microbial oils with reduced target LC-PUFA content was not efficient.
Focusing on such thermogenic fatty acids, the present invention surprisingly found that ordered packings By performing precision distillation using a distillation column containing, or by performing precision distillation so as to contain a certain amount of thermally generated fatty acids having 16 to 22 carbon atoms, the target LC- Based on the knowledge that PUFA can be purified. In addition, the present invention focuses on such thermogenic fatty acids with 16 to 22 carbon atoms, and from the relationship between the accuracy of purification of the target LC-PUFA and the increase in the content of thermogenic fatty acids with 16 to 22 carbon atoms, , Surprisingly, based on the finding that a microbial oil containing a certain amount of thermogenic fatty acids with 16 to 22 carbon atoms is advantageous for more efficiently obtaining a microbial oil containing a high content of the target LC-PUFA. . In the present specification, a C16-C22 thermogenic fatty acid may be simply referred to as a "thermally produced fatty acid" unless otherwise specified.
The microbial oil obtained by the production method of the present invention and the microbial oil of the present invention are further subjected to a specific concentration means such as reversed-phase column chromatography to obtain a concentrated microbial oil containing a high target LC-PUFA content. Obtainable.
That is, in the concentrated microbial oil according to another aspect of the present invention, the content of polyunsaturated fatty acids having 20 or more carbon atoms in the form of fatty acid alkyl esters and/or free fatty acids is 90% by weight of the total weight of fatty acids in the oil. % to 98% by weight, and the content of thermogenic fatty acids with 16 to 22 carbon atoms is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil, and saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms The total saturated fatty acid content of is not more than 1.0% by weight of the total weight of fatty acids in the oil, and the content of C18 monounsaturated fatty acids is not more than 5.0% by weight of the total weight of fatty acids in the oil is a concentrated microbial oil.
Also, a method for producing a concentrated microbial oil according to another aspect of the present invention comprises at least one targeted fatty acid alkyl ester form and/or liberated using any method for producing a microbial oil that is another aspect of the present invention. It includes obtaining a microbial oil containing a polyunsaturated fatty acid having 20 or more carbon atoms in the form of a fatty acid, and subjecting the obtained microbial oil to a concentration treatment using reversed-phase column chromatography.
Concentrated microbial oils or microbial oils according to aspects of the present invention contain or may contain a high content of target LC-PUFAs and are therefore useful in fields such as foods, supplements, pharmaceuticals, cosmetics, feedstuffs, e.g. inflammatory disease prevention. or therapeutic agents, and are useful in methods of prevention, treatment or amelioration of inflammatory diseases. In addition, since the method for producing concentrated microbial oil according to the embodiment of the present invention uses the production method for efficiently obtaining microbial oil containing a high content of target LC-PUFA according to the present invention, the concentrated microbial oil can be efficiently produced. can provide.
In this specification, the term "process" is not only an independent process, but even if it cannot be clearly distinguished from other processes, it is included in this term as long as the intended purpose of the process is achieved. .
In this specification, a numerical range indicated using "~" indicates a range including the numerical values before and after it as the minimum and maximum values, respectively.
As used herein, the amount of each component in the mixture means the total amount of the multiple substances present in the mixture unless otherwise specified when there are multiple substances corresponding to each component in the mixture. .
In the present specification, the content of each component in the mixture refers to the total content of the multiple substances present in the mixture when there are multiple substances corresponding to each component in the mixture, unless otherwise specified. means.
In the present invention, the term "microbial oil" refers to a mixture of organic substances obtained from microbial biomass and insoluble in water under normal temperature and normal pressure. Microbial oils include oil components such as saturated or unsaturated fatty acids, phospholipids, sterols, glycerol, ceramides, sphingolipids, terpenoids, flavonoids, tocopherols, etc. Saturated or unsaturated fatty acids are constituent fatty acids in other oil components. It may exist as
In the present invention, "fatty acid" means fatty acids contained in free saturated or unsaturated fatty acids, saturated or unsaturated fatty acid alkyl esters, triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids, steryl esters, and the like. , can also be referred to as constituent fatty acids.
In the present specification, the form of a compound containing fatty acids may be omitted unless otherwise specified. Examples of the form of the fatty acid-containing compound include a free fatty acid form, a fatty acid alkyl ester form, a glyceryl ester form, a phospholipid form, a steryl ester form, and the like. Compounds containing the same fatty acid may be contained in the microbial oil in a single form or as a mixture of two or more forms.
In addition, when representing fatty acids, numerical expressions are sometimes used in which the number of carbon atoms, the number of double bonds, and the location of the double bonds are expressed in a simplified manner using numbers and alphabets. For example, a saturated fatty acid with 20 carbon atoms is denoted as "C20:0", a monounsaturated fatty acid with 18 carbon atoms is denoted as "C18:1", etc. Dihomo-γ-linolenic acid is denoted as "C20:3, arachidonic acid can be written as "C20:4,n-6" and the like. This method is well known to those skilled in the art, and fatty acids designated according to this method can be readily identified by those skilled in the art.
The total content of fatty acids in the microbial oil can be, for example, 80% or more, 90% or more, 95% or more, or 98% or more by weight of the total weight of the microbial oil. Other components that may be present in the microbial oil and that do not contain fatty acids or substructures other than fatty acids of compounds that contain fatty acids include glycerin, sterols, hydrocarbons, terpenoids, plavonoids, tocopherols, and glyceryl esters. Examples include a glycerin skeleton partial structure, a phosphoric acid skeleton partial structure of phosphoric acid, a sphingosine skeleton partial structure, and the like.
As used herein, a mixture of compounds that have just been extracted from microbial cells may be referred to as a crude microbial oil.
The fatty acid alkyl ester or free fatty acid in the present invention means a fatty acid alkyl ester obtained by subjecting crude oil obtained from microbial biomass to processing such as hydrolysis and alkyl esterification, unless a specific fatty acid type is specified. or a mixture of free fatty acids.
The fatty acid content relative to the total weight of fatty acids in the oil in the present invention is determined based on the fatty acid composition. A fatty acid composition can be calculated|required according to a conventional method. Specifically, the oil to be measured is esterified using a lower alcohol and a catalyst to obtain a fatty acid lower alkyl ester. The resulting fatty acid lower alkyl esters are then analyzed using a gas chromatograph with a flame ionization detector (FID). The peak corresponding to each fatty acid is identified in the resulting gas chromatography chart, and the peak area of each fatty acid is determined using the Agilent ChemStation integration algorithm (Revision C.01.03 [37], Agilent Technologies). The fatty acid composition is defined as the percentage of each peak area with respect to the sum of the fatty acid peak areas. The area % obtained by the measurement method described above is the same as the weight % of each fatty acid in the sample. Established by Japan Oil Chemistry Society (JOCS) Standard fat analysis test method 2013 edition 2.4.2.1-2013 Fatty acid composition (FID constant temperature gas chromatograph method) and 2.4.2.2-2013 See Fatty Acid Composition (FID Temperature Programmed Gas Chromatography).
When the microbial oil contains a fatty acid other than the fatty acid alkyl ester form and the free fatty acid form fatty acid, the fatty acid other than the fatty acid alkyl ester form and the free fatty acid form is separated from the microbial oil, and then the fatty acid to be measured. Measure the composition. Methods for separating fatty acids other than fatty acid alkyl ester forms and free fatty acid forms from microbial oils include, for example, silicic acid column chromatography disclosed in The Journal of Biological Chemistry 1958, 233:311-320. Methods such as thin layer chromatography disclosed in Lipid Handbook with CD-ROM Third Edition CRC Press Taylor & Francis Group (2007) can be referred to.
Each aspect of the present invention will be described below.
(1) Microbial oil The microbial oil in one aspect of the present invention has at least 1 carbon atom in the form of a fatty acid alkyl ester and/or free fatty acid with a content of 50% by weight or more of the total weight of fatty acids in the oil. It contains 20 or more polyunsaturated fatty acids and thermally generated fatty acids with 16 to 22 carbon atoms having a content of 3.0% by weight or less of the total weight of fatty acids in the oil.
The microbial oil may be obtained from microbial biomass as described above. Microorganisms may be any lipid-producing microorganism, including algae and fungi.
Examples of algae include the genus Labyrinthulamycota.
As fungi, Mortierella, Conidiobolus, Pythium, Phytophthora, Penicillium, Cladosporium, Mucor , Fusarium genus, Aspergillus genus, Rhodotorula genus, Entomophthora genus, Echinosporangium genus, and Saprolegnia genus. can be mentioned. Among them, microorganisms belonging to the genus Mortierella are more preferable. Microorganisms belonging to the genus Mortierella include, for example, Mortierella elongata, Mortierella exigua, Mortierella hygrophila , Mortierella alpina, and other microorganisms belonging to the subgenus Mortierella.
The polyunsaturated fatty acids having 20 or more carbon atoms in the present invention include unsaturated fatty acids having a valence of 2 or more, preferably a valence of 3 or more. The number of carbon atoms in the polyunsaturated fatty acid means the number of carbon atoms in the constituent fatty acids. Examples of polyunsaturated fatty acids with 20 or more carbon atoms include polyunsaturated fatty acids with 20 or more and 22 or less carbon atoms, specifically eicosadienoic acid (C20:2,n-9), Dihomo-γ-linolenic acid (C20:3,n-6), mead acid (C20:3,n-9), eicosatetraenoic acid (C20:4,n-3), arachidonic acid (C20:4, n-6), eicosapentaenoic acid (C20:5,n-3), docosatetraenoic acid (C22:4,n-6), docosapentaenoic acid (C22:5,n-3), docosapentaenoic acid (C22:5,n-6) and docosahexaenoic acid (C22:6,n-3). The microbial oil may contain at least one of these polyunsaturated fatty acids, may contain two or more in combination, contains selected one of these polyunsaturated fatty acids, and contains other It may be one that does not contain polyunsaturated fatty acids. In addition, if the microbial oil contains at least one of the polyunsaturated fatty acids having 20 to 22 carbon atoms as the target LC-PUFA, it contains one or more specific ones. It may be nothing. For example, microbial oils include eicosadienoic acid (C20:2,n-9), dihomo-γ-linolenic acid (C20:3,n-6), mead acid (C20:3,n-9), eicosatetraene acid (C20:4,n-3), arachidonic acid (C20:4,n-6), eicosapentaenoic acid (C20:5,n-3), docosatetraenoic acid (C22:4,n-6), docosapentaenoic acid (C22:5,n-3), docosapentaenoic acid (C22:5,n-6) and docosahexaenoic acid (C22:6, At least one selected from the group consisting of n-3) may not be included. Here, the term "not containing polyunsaturated fatty acids" means that the content of the target polyunsaturated fatty acids is less than 5% by weight of the total weight of the fatty acids in the oil, or is zero.
The alkyl group in the polyunsaturated fatty acid in the form of a fatty acid alkyl ester is preferably an alkyl group having 1 to 3 carbon atoms, such as a methyl group, an ethyl group, or a propyl group. As the polyunsaturated fatty acid in the form of alkyl ester, it is particularly preferable to use the polyunsaturated fatty acid in the form of ethyl ester or methyl ester.
The target LC-PUFA in the microbial oil, i.e., the content of polyunsaturated fatty acids with 20 or more carbon atoms in the form of fatty acid alkyl esters and/or free fatty acids, is 50% by weight or more of the total weight of fatty acids in the oil. . If the target LC-PUFA content is less than 50% by weight, it is not possible to efficiently obtain a purified microbial oil containing a high target LC-PUFA content. The target LC-PUFA content in the microbial oil is the value obtained by analyzing the fatty acid composition of the microbial oil, as described above.
The content of the target LC-PUFAs in the microbial oil is preferably 60% by weight or more, preferably 70% by weight or more, of the total weight of the fatty acids in the oil, from the viewpoint of achieving more efficient purification of the target LC-PUFAs. more preferably 80% by weight or more, even more preferably 85% by weight or more, particularly preferably 90% by weight or more, and even more particularly 95% by weight or more Preferably, 98% by weight is most preferred. In addition, the target LC-PUFA content in the microbial oil is 50% to 98% by weight, 60% to 98% by weight, 70% to 98% by weight, 80% to 98% by weight of the total weight of fatty acids in the oil. It may be 98% by weight, 85% to 98% by weight, 90% to 98% by weight, or 95% to 98% by weight.
The microbial oil of the present invention contains thermogenic fatty acids with 16 to 22 carbon atoms at a content of 3.0% by weight or less of the total weight of fatty acids in the oil.
The thermogenic fatty acid is a fatty acid having 16 to 22 carbon atoms that is generated based on the presence in the target LC-PUFA by heat associated with high-temperature treatment such as distillation, as described above. That is, the thermogenic fatty acid is considered to be a fatty acid produced by decomposition, isomerization, etc., of the target LC-PUFA due to heat associated with high-temperature treatment such as distillation, but is not limited to this theory. The form of the thermogenic fatty acid is not particularly limited, and is not limited to fatty acid alkyl ester form or free fatty acid form.
The number and types of thermogenic fatty acids contained in the microbial oil vary depending on the conditions of precision distillation, the type of target LC-PUFA contained in the microbial oil, and the like.
Exemplary thermogenic fatty acids are considered to be the trans isomers of the target LC-PUFAs (see Journal of the American Oil Chemists' Society, Vol.66, No.12, pp.1822-1830 (1989)). That is, while the carbon double bond portion contained in the target LC-PUFA is usually cis-type, the thermogenic fatty acid is one in which part or all of the carbon double bond portion is changed to trans-type. It is considered that the position of the bond is changed to have a conjugated double bond.
Examples of thermogenic fatty acids include the following. The microbial oil can contain any one or a combination of two or more of the following compounds: 8Z,11E-eicosadienoic acid, 8E,11Z-eicosadienoic acid, 8E,11E-eicosadienoic acid, 8Z,11Z,14E- Eicosatrienoic Acid, 8Z,11E,14Z-Eicosatrienoic Acid, 8E,11Z,14Z-Eicosatrienoic Acid, 8Z,11E,14E-Eicosatrienoic Acid, 8E,11Z,14E-Eicosatrienoic Acid, 8E ,11E,14Z-eicosatrienoic acid, 8E,11E,14E-eicosatrienoic acid, 5Z,8Z,11E-eicosatrienoic acid, 5Z,8E,11Z-eicosatrienoic acid, 5E,8Z,11Z-eiko Satrienoic Acid, 5Z,8E,11E-Eicosatrienoic Acid, 5E,8Z,11E-Eicosatrienoic Acid, 5E,8E,11Z-Eicosatrienoic Acid, 5E,8E,11E-Eicosatrienoic Acid, 8Z, 11Z,14Z,17E-Eicosatetraenoic acid, 8Z,11Z,14E,17Z-Eicosatetraenoic acid, 8Z,11E,14Z,17Z-Eicosatetraenoic acid, 8E,11Z,14Z,17Z-Eicosa Tetraenoic acid, 8E,11Z,14Z,17E-eicosatetraenoic acid, 8Z,11E,14Z,17E-eicosatetraenoic acid, 8Z,11Z,14E,17E-eicosatetraenoic acid, 8E,11Z, 14E,17Z-eicosatetraenoic acid, 8Z,11E,14E,17Z-eicosatetraenoic acid, 8E,11E,14Z,17Z-eicosatetraenoic acid, 8E,11E,14E,17Z-eicosatetraene acid, 8E,11E,14Z,17E-eicosatetraenoic acid, 8E,11Z,14E,17E-eicosatetraenoic acid, 8Z,11E,14E,17E-eicosatetraenoic acid, 8E,11E,14E, 17E-eicosatetraenoic acid, 5Z,8Z,11Z,14E-eicosatetraenoic acid, 5Z,8Z,11E,14Z-eicosatetraenoic acid, 5Z,8E,11Z,
If the content of thermogenic fatty acids in the microbial oil exceeds 3.0% by weight, the microbial oil containing the target LC-PUFA at a high content cannot be efficiently obtained. The microbial oil of thermogenic fatty acids is obtained through a heating process including distillation, so the content is 0.0001% to 3.0% by weight, 0.001% to 3.0% by weight, or 0.01% to 3.0% by weight. It can be included in the microbial oil at a percentage content by weight.
The content of thermogenic fatty acids in the microbial oil is 0.001 weight of the total weight of fatty acids in the oil from the viewpoint of efficiently obtaining a concentrated microbial oil containing a high content of target LC-PUFA using reversed-phase column chromatography. %~2.8 wt%, 0.01 wt%~2.8 wt%, 0.1 wt%~2.8 wt%, 0.1 wt%~2.5 wt%, 0.1 wt%~2.0 wt%, 0.1 wt%~1.5 wt%, 0.1 wt%~ It can be 1.0% by weight, or 0.1% to 0.7% by weight.
Thermogenic fatty acids are fatty acids with 16 to 20 carbon atoms that are detectable after the microdistillation treatment but not detected before the treatment. For this reason, for example, various chromatographic analyzes can be used to compare the fatty acid composition before and after distillation treatment, and fatty acids having peaks appearing after treatment can be identified. As the chromatography, among others, gas chromatography can be particularly used from the viewpoint of high analytical ability or detection sensitivity and relatively simple operation. When identifying thermogenic fatty acids with higher accuracy, for example, silver-ion solid phase extraction using silver ion chromatography is used to extract components from microbial biomass that overlap with thermogenic fatty acids. You may analyze and identify after removing.
For example, if the target LC-PUFA is dihomo-γ-linolenic acid (DGLA), the thermogenic fatty acid can be a 20 carbon thermogenic fatty acid. The heat-generated fatty acid having 20 carbon atoms is, for example, a heat-generated fatty acid having 20 carbon atoms, and is in the range of 1.001 to 1.011 when the retention time of dihomo-γ-ethyl linolenate is set to 1 in gas chromatography analysis. 1 or 2 or more fatty acids having a retention time as a peak appearing in (hereinafter referred to as compound A) and 1 or 2 or more fatty acids having a retention time as a peak appearing in the range of 1.013 to 1.027 (hereinafter referred to as compound B) can be mentioned. Compound A and compound B may be a group of one or more compounds, or each may be a single compound. The thermogenic fatty acid may be either compound A or compound B, or both. The gas chromatography conditions for specifying compound A and compound B as thermogenic fatty acids are as follows: [Gas chromatography analysis conditions]
GC equipment: 6890N Network GC system (Agilent Technologies) Column: DB-WAX (Agilent Technologies) 30m×0.25mm ID, 0.25μm film thickness column Temperature conditions: 60°C 2.5 minutes 20°C/minute 180 °C 2°C/min 230°C 15 min Inlet temperature conditions: 210°C, splitless, split vent sampling time 1.5 min, purge flow rate 40 mL/min Injection volume conditions: 1 μL, sample concentration 1 mg/min Carrier gas conditions: Helium, Linear velocity: 24 cm/min Detector: FID detector Temperature: 280°C
When the target LC-PUFA is DGLA, the content of compound A and compound B, which are thermogenic fatty acids, in microbial oil is 0.001% to 2.8% by weight of the total weight of fatty acids in the oil from the viewpoint of DGLA refining efficiency. %, 0.1% to 2.8% by weight, 0.1% to 2.5% by weight, 0.1% to 2.0% by weight, 0.1% to 1.5% by weight, 0.1% to 1.0% by weight, or 0.1% to 0.7% by weight can be
The microbial oil of the present invention is preferably of a composition depleted in at least one specific fatty acid to be separated from the target LC-PUFA by microdistillation. In this specification, fatty acids to be separated from target LC-PUFAs in the purification process are referred to as separated target fatty acids unless otherwise specified. The target fatty acid to be separated is not particularly limited as long as it is a fatty acid other than the target LC-PUFA, and the form of the target fatty acid to be separated is also not particularly limited, and may be a fatty acid alkyl ester, free fatty acid, or the like.
Separation target fatty acids can include 22 carbon saturated fatty acids and 24 carbon saturated fatty acids. The content of C22 saturated fatty acids and C24 saturated fatty acids in crude oil obtained from microbial biomass generally tends to be higher than in fish oils or animal and vegetable oils. In addition, the saturated fatty acid with 22 carbon atoms and the saturated fatty acid with 24 carbon atoms are high-melting long-chain fatty acids having a melting point higher than that of the target LC-PUFA. Reducing the content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms can suppress clogging of pipes in column chromatography treatment and enable reverse-phase column chromatography. In addition, the retention time of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms in reversed-phase column chromatography is longer than that of the target LC-PUFA, which can be a factor in lengthening the time required for chromatography. A reduction in the content of saturated fatty acids is also desirable from the viewpoint of purification efficiency per hour.
The total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms is the total content of both when saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms are present, When only one of them exists, it means the content of only one of them.
The total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms in microbial oil is the total fatty acid content in oil from the viewpoint of suppressing clogging of piping in column chromatography and the viewpoint of purification efficiency of target LC-PUFA. It is more preferably 6.0% by weight or less, still more preferably 1.8% by weight or less, and even more preferably 0.1% by weight or less. The total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms in microbial oil is the content of target LC-PUFA from the viewpoint of suppressing clogging of piping in column chromatography and the viewpoint of purification efficiency of target LC-PUFA. It is preferably 10/100 or less, more preferably 3/100 or less, even more preferably 0.1/100 or less of the ratio. The total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms in microbial oil is 10% of the total weight of microbial oil from the viewpoint of clogging suppression of piping in column chromatography and from the viewpoint of purification efficiency of target LC-PUFA. It is preferably 6.0% by weight or less, more preferably 1.0% by weight or less, and even more preferably 0.1% by weight or less.
In addition, the content of saturated fatty acids with 24 carbon atoms in the microbial oil is 3.0 wt% or less of the total weight of fatty acids in the oil from the viewpoint of suppressing clogging of pipes in column chromatography and the viewpoint of purification efficiency of target LC-PUFA. is more preferably 1.0% by weight or less, and even more preferably 0.1% by weight or less. The content of saturated fatty acids with 24 carbon atoms in the microbial oil is 4/100 or less of the content of the target LC-PUFA, from the viewpoint of suppressing clogging of pipes in column chromatography and from the viewpoint of purification efficiency of the target LC-PUFA. is preferred, 1.4/100 or less is more preferred, and 0.1/100 or less is even more preferred. The content of saturated fatty acids with 24 carbon atoms in the microbial oil is 3.0% by weight or less based on the total weight of the microbial oil, from the viewpoint of suppressing clogging of pipes in column chromatography and from the viewpoint of purification efficiency of the target LC-PUFA. preferably 1.0% by weight or less, and even more preferably 0.1% by weight or less.
As other separation target fatty acids, when using a partition number determined from the number of carbon atoms and the number of double bonds of the fatty acid, which is an index related to separation by liquid chromatography, the partition number of the polyunsaturated fatty acid and A saturated or unsaturated fatty acid having a partition number of 2 or more and 2 or less than the polyunsaturated fatty acid can be exemplified. Such other segregated target fatty acids are hereinafter referred to as segregated target fatty acids having a PN difference of -2 or more and 2 or less.
The PN of one of the two fatty acids to be contrasted is 2 less than the other PN, i.e. -2, 1 less, i.e. -1, the same number, i.e. 0, 1 more, i.e. +1, 2 more That is, in the case of +2, when separation is performed using liquid chromatography, the difference in elution time between the two fatty acids to be compared is not sufficient, and separation by liquid chromatography is difficult. can think. Therefore, reducing the content of the separated target fatty acids having a PN difference of -2 or more and 2 or less is desirable from the viewpoint of purification efficiency of the high-content target LC-PUFA.
A partition number (PN) is sometimes called an Equivalent carbon number (ECN). The partition number is an index obtained empirically from the definition of the separation factor that affects the elution time in relation to the analysis of molecular species by reversed-phase high-performance liquid chromatography, and is an index represented by the following formula (I). be.
PN=[number of carbon atoms]2×[number of double bonds] (I) In formula (I), the number of carbon atoms means the number of carbon atoms in the fatty acid. However, in the present invention, the number of carbon atoms in the formula (I) means the number of carbon atoms of the fatty acid in the free fatty acid form, and is an integer specific to each fatty acid. In this specification, the partition number is called PN.
For example, for DGLA, ie C20:3, PN=202×3=14.
The isolated target fatty acid with a PN difference of -2 or more and 2 or less is saturated or unsaturated with a carbon number different from that of the target LC-PUFA, i.e., a carbon number greater or less than that of the target LC-PUFA. A fatty acid, which can be, for example, a saturated or unsaturated fatty acid with fewer carbons than the target LC-PUFA. Separation target fatty acids with a PN difference of -2 or more and 2 or less include saturated fatty acids with 18 carbon atoms, monounsaturated fatty acids with 18 carbon atoms, divalent unsaturated fatty acids with 18 carbon atoms, and triunsaturated fatty acids with 18 carbon atoms. It can be at least one selected from the group consisting of saturated fatty acids and tetravalent unsaturated fatty acids having 18 carbon atoms.
Combinations of target LC-PUFAs and isolated target fatty acids in microbial oils include, for example:
<tables><img file="JP7169331B2_D0001.tif" /></tables>
The total content of separated target fatty acids having a PN difference of -2 or more and 2 or less in the microbial oil of the separated target fatty acids is, for example, the total weight of the fatty acids in the oil from the viewpoint of efficiently obtaining a high content of target LC-PUFA It is more preferably 10.0% by weight or less, still more preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less. In the microbial oil, the total content of separated target fatty acids with a PN difference of -2 or more and 2 or less should be 15/100 or less of the content of the target LC-PUFA from the viewpoint of efficiently obtaining the target LC-PUFA. It is preferably 5/100 or less, more preferably 1/100 or less. In the microbial oil, the total content of separated target fatty acids with a PN difference of -2 or more and 2 or less should be 10.0 wt% or less with respect to the total weight of the microbial oil from the viewpoint of efficiently obtaining the target LC-PUFA. It is preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less.
For example, if the target LC-PUFA is a PN16 fatty acid, i.e., eicosadienoic acid, the total content of separated target fatty acids with a PN difference of -2 or more and 2 or less such as C18:0, C18:1, etc. in the microbial oil is more preferably 10.0% by weight or less, even more preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less of the total weight of fatty acids in the is preferably 15/100 or less, more preferably 5/100 or less, even more preferably 1/100 or less; and 10.0% by weight or less based on the total weight of the microbial oil. is preferred, 4.0% by weight or less is more preferred, and 0.7% by weight or less is even more preferred.
If the target LC-PUFA is a PN14 fatty acid, i.e. DGLA, mead acid or docosatetraenoic acid, isolate target fatty acids with a PN difference of -2 or more and 2 or less such as C18:1, C18:2, etc. in microbial oils is more preferably 10.0% by weight or less, more preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less of the total weight of fatty acids in the oil; It is preferably 15/100 or less of the target LC-PUFA content, more preferably 5/100 or less, even more preferably 1/100 or less; It is preferably 10.0% by weight or less, more preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less.
Separation of C18:2, C18:3, etc. in microbial oils with a PN difference of -2 or more and 2 or less when the target LC-PUFA is a PN12 fatty acid, i.e. eicosatetraenoic acid, arachidonic acid or docosapentaenoic acid More preferably, the total content of target fatty acids is 10.0% or less, more preferably 4.0% or less, even more preferably 0.7% or less, of the total weight of fatty acids in the oil; In addition, the content of the target LC-PUFA is preferably 15/100 or less, more preferably 5/100 or less, even more preferably 1/100 or less; It is preferably 10.0% by weight or less, more preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less.
When the target LC-PUFA is a fatty acid with PN10, i.e. eicosapentaenoic acid or docosahexaenoic acid, the total content of separated target fatty acids with a PN difference of -2 or more and 2 or less such as C18:3, C18:4, etc. in the microbial oil is , more preferably 10.0% by weight or less, more preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less of the total weight of fatty acids in the oil; The content is preferably 15/100 or less, more preferably 5/100 or less, and even more preferably 1/100 or less; It is preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less.
In addition, from the viewpoint of efficiently obtaining target LC-PUFAs such as eicosadienoic acid, DGLA, mead acid, and eicosatetraenoic acid with high content by reversed-phase column chromatography, microbial oil has a monounsaturated A low fatty acid content is preferred. When the target LC-PUFA is eicosadienoic acid, DGLA, mead acid, or eicosatetraenoic acid, the PN of monounsaturated fatty acids with 18 carbon atoms is 2 more than the target LC-PUFA. For example, in the microbial oil, the content of monounsaturated fatty acids with 18 carbon atoms is more preferably 7.0% by weight or less of the total weight of fatty acids in the oil from the viewpoint of purification efficiency of the target LC-PUFA, It is more preferably 1.5% by weight or less, and even more preferably 0.4% by weight or less. In the microbial oil, the content of monounsaturated fatty acids with 18 carbon atoms is preferably 10/100 or less of the content of the target LC-PUFA from the viewpoint of purification efficiency of the target LC-PUFA, and 2/100 It is more preferably 0.5/100 or less, further preferably 0.5/100 or less. In the microbial oil, the content of monounsaturated fatty acids with 18 carbon atoms is preferably 7.0% by weight or less relative to the total weight of the microbial oil, from the viewpoint of purification efficiency of the target LC-PUFA, and 1.5% by weight. is more preferably 0.4% by weight or less.
In addition, from the viewpoint of efficiently obtaining target LC-PUFAs such as DGLA, mead acid, eicosatetraenoic acid, arachidonic acid, docosatetraenoic acid, and docosapentaneenoic acid with high content by reversed-phase column chromatography, microbial oil Therefore, it is preferable that the content of divalent unsaturated fatty acids having 18 carbon atoms is particularly low. When DGLA, mead acid, eicosatetraenoic acid, arachidonic acid, docosatetraenoic acid, or docosapentaneenoic acid is the target LC-PUFA, the target LC- Equal to PUFA. For example, in the microbial oil, the content of divalent unsaturated fatty acids with 18 carbon atoms is more preferably 5.0% by weight or less of the total weight of fatty acids in the oil from the viewpoint of purification efficiency of the target LC-PUFA, It is more preferably 0.7% by weight or less, and even more preferably 0.4% by weight or less. In the microbial oil, the content of divalent unsaturated fatty acids with 18 carbon atoms is preferably 7/100 or less of the content of the target LC-PUFA from the viewpoint of purification efficiency of the target LC-PUFA, and 1/100 It is more preferably 0.5/100 or less, further preferably 0.5/100 or less. In the microbial oil, the content of C18 diunsaturated fatty acids is preferably 5.0% by weight or less, more preferably 0.7% by weight or less, and 0.4% by weight, relative to the total weight of the microbial oil. More preferably:
In addition, from the viewpoint of efficiently obtaining target LC-PUFA such as DGLA, mead acid, and docosatetraenoic acid with high content by reversed-phase column chromatography, the content of trivalent unsaturated fatty acids with 18 carbon atoms in microbial oil is is preferably low. When DGLA, mead acid, or docosatetraenoic acid is the target LC-PUFA, the PN of trivalent unsaturated fatty acids with 18 carbon atoms is 2 less than the target LC-PUFA. For example, in the microbial oil, the content of trivalent unsaturated fatty acids with 18 carbon atoms is more preferably 7.0 wt% or less of the total weight of fatty acids in the oil from the viewpoint of purification efficiency of the target LC-PUFA, It is more preferably 1.5% by weight or less, and even more preferably 0.4% by weight or less. In the microbial oil, the content of trivalent unsaturated fatty acids with 18 carbon atoms is preferably 10/100 or less of the content of the target LC-PUFA from the viewpoint of purification efficiency of the target LC-PUFA, and 2/100 It is more preferably 0.5/100 or less, further preferably 0.5/100 or less. In the microbial oil, the content of trivalent unsaturated fatty acids with 18 carbon atoms is preferably 7.0% by weight or less based on the total weight of the microbial oil, from the viewpoint of purification efficiency of the target LC-PUFA, and 1.5% by weight. is more preferably 0.4% by weight or less.
From the perspective of efficiently obtaining target LC-PUFAs such as DGLA, mead acid, and docosatetraenoic acid with a high content by reversed-phase column chromatography, microbial oils contain 18-carbon monounsaturated fatty acids and 18-carbon fatty acids. It is preferable that the total content with the divalent unsaturated fatty acid is low. For example, in microbial oil, the total content of monounsaturated fatty acid with 18 carbon atoms and diunsaturated fatty acid with 18 carbon atoms is 18%, from the viewpoint of efficiently obtaining the target LC-PUFA, the total weight of fatty acids in the oil is It is more preferably 10.0% by weight or less, still more preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less. In the microbial oil, the total content of the monounsaturated fatty acid with 18 carbon atoms and the divalent unsaturated fatty acid with 18 carbon atoms is 15% of the target LC-PUFA content from the viewpoint of efficiently obtaining the target LC-PUFA. It is preferably 1/100 or less, more preferably 5/100 or less, and even more preferably 1/100 or less. In the microbial oil, the total content of the monounsaturated fatty acid with 18 carbon atoms and the divalent unsaturated fatty acid with 18 carbon atoms is 10.0 with respect to the total weight of the microbial oil from the viewpoint of efficiently obtaining the target LC-PUFA. It is preferably 4.0% by weight or less, and even more preferably 0.7% by weight or less.
The microbial oil of the present invention preferably has a low saturated fatty acid content of 18 carbon atoms, from the viewpoints of the melting point of the microbial oil, the easiness of crystal precipitation, and the time productivity in column chromatography. In addition, when eicosadienoic acid is used as the target LC-PUFA, saturated fatty acids with 18 carbon atoms also correspond to separation target fatty acids having a PN difference of -2 or more and 2 or less. From the viewpoint of melting point of microbial oil, easiness of crystal precipitation, and time productivity in column chromatography, the content of C18 saturated fatty acid in microbial oil is 7.0% by weight of the total weight of fatty acids in the oil. It is more preferably 3.0% by weight or less, even more preferably 1.5% by weight or less. In the microbial oil, the content of C18 saturated fatty acids is preferably 11/100 or less, more preferably 4/100 or less, and 2/100 or less of the target LC-PUFA content. is more preferred. From the viewpoint of melting point of microbial oil, easiness of crystal precipitation, and time productivity in column chromatography, the content of C18 saturated fatty acid in microbial oil is 7.0% by weight % or less, more preferably 3.0 wt % or less, and even more preferably 1.5 wt % or less.
Since the various contents of the separation target fatty acids described above are independent embodiments, the preferred embodiment of the microbial oil is an embodiment combining two or more of any preferred contents of each separation target fatty acid. good.
The total content of 22-carbon saturated fatty acids and 24-carbon saturated fatty acids in microbial oils, as described above, indicates that the target LC-PUFAs in microbial oils are eicosadienoic acid, DGLA, mead acid, eicosatetraenoic acid, and arachidonic acid. , eicosapentaenoic acid, docosatetraenoic acid, docosapentaneenoic acid, or docosahexaenoic acid, the same range as the above range, including the preferred range, can be used, and any combination of these can be used. Often, the corresponding C18 monounsaturated fatty acid content, C18 diunsaturated fatty acid content, C18 monounsaturated fatty acid and C18 diunsaturated fatty acid, and and the content of C18 saturated fatty acid may be combined arbitrarily.
The melting point of the microbial oil is preferably 40° C. or lower, preferably 30° C. or lower, from the viewpoint of efficiency of treatment by reversed-phase column chromatography or heat resistance of the packed material. The melting point of the microbial oil shall be the clear melting point measured by the method described in the standard oil analysis test method 2013 edition 3.2.2.1-2013 established by the Japan Oil Chemistry Society (JOCS).
(2) Method for producing microbial oil The method for producing microbial oil according to another aspect of the present invention includes performing purification by precision distillation and obtaining a specific microbial oil after precision distillation.
That is, the first method for producing microorganisms in another aspect of the present invention includes a raw oil supply step of preparing raw oil containing target LC-PUFA obtained from microbial biomass, and a 160° It includes a first precision distillation step in which purification is performed by precision distillation under conditions including a column bottom temperature of C to 230°C and a minimum pressure in the distillation column of 0.1 Pa to 30 Pa. After the first precision distillation step, a microbial oil containing specific polyunsaturated fatty acids is obtained. Specific polyunsaturated fatty acids provided herein include, but are not limited to, specific microbial oils in one aspect of the present invention.
A second method for producing a microbial oil in still another embodiment of the present invention comprises the step of supplying the raw material oil, and the raw material oil is heated to 160°C to 230°C using a distillation column containing a structured packing. and a bottom temperature of 0.1 Pa to 30 Pa, and a microbial oil recovery step for obtaining a specific microbial oil in one embodiment of the present invention; including.
A third method for producing a microbial oil according to still another embodiment of the present invention comprises the step of supplying the raw material oil, and the target polyvalent heterogeneous oil by using a distillation column containing a structured packing for the raw material oil. Precision by the conditions under which a microbial oil containing thermally generated fatty acids with a content of 3.0% by weight or less of the total weight of fatty acids in the oil can be obtained, including the bottom temperature and the minimum pressure in the distillation column according to the type of saturated fatty acids A third precision distillation step for distillation and the microbial oil recovery step.
In the raw oil supply step in the first to third production methods, the raw oil is obtained by culturing microorganisms known as lipid-producing microorganisms capable of producing target LC-PUFA in a culture solution to obtain microbial biomass containing fatty acids. , A crude oil separation step for separating crude oil, which is a mixture of fatty acids, from the obtained microbial biomass. , a triacylglycerol concentrate production step to obtain a triacylglycerol concentrate by performing a treatment including a decolorization step and a deodorization step, and a processing step of performing hydrolysis, alkyl esterification, etc. on the triacylglycerol concentrate. obtained by
Examples of lipid-producing microorganisms include those microorganisms described above. In addition, culturing of lipid-producing microorganisms can be performed under conditions known to those skilled in the art. For example, when the target LC-PUFA is DGLA, DGLA derived from microorganisms described in JP-A-5-091887 can be used.
Japanese Patent Application Laid-Open No. 5-091887 describes a mutant strain Mortierella alpina SAM1860 with reduced or deleted Δ5 desaturase activity (Bekko Kenjoyori No. 3589), which is characterized by the presence of a Δ5 desaturase inhibitor. Below, a method of producing DGLA by culturing is disclosed. Δ5 desaturase inhibitors include, for example, 2-amino-N-(3-chlorophenyl)benzamide, dioxabicyclo[3.3.0]octane derivatives, piperonyl butoxide, curcumin and the like. Among these, dioxabicyclo[3.3.0]octane derivatives include sesamin, sesaminol, episesamin, episesaminol, sesamolin, 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy- 4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane, 2,6-bis-(3-methoxy-4-hydroxyphenyl)-3,7-dioxabicyclo[3.3.0]octane , 2-(3,4-methylenedioxyphenyl)-6-(3-methoxy-4-hydroxyphenoxy)-3,7-dioxabicyclo[3.3.0]octane and the like.
The incubator used for culturing is not particularly limited, and any apparatus that is usually used for culturing microorganisms can be applied. It can be selected as appropriate according to the scale of culture. For example, when liquid culture is performed on a scale of 1 L to 50 L, a stirred culture vessel is preferable as the culture vessel in order to obtain the target LC-PUFA at a higher concentration. As the stirred culture vessel, a stirred culture vessel having at least one stage of disk turbine stirring blades is preferred, and a stirring type culture vessel having two stages of disk turbine stirring blades is more preferred.
In the crude oil separation step, crude oil containing lipids produced in the production step is separated from the microbial cells. A separation method and an extraction method according to the culture form can be used for the separation of the microbial cells and the collection of the crude oil.
When a liquid medium is used, cultured cells are obtained by conventional solid-liquid separation means such as centrifugation and filtration. When the cells are cultured on a solid medium, the solid medium and the cells may be crushed with a homogenizer or the like without separating the cells from the medium, and the crude oil may be collected directly from the resulting crushed product.
Collecting the crude oil can include extracting the separated dry cells, preferably with supercritical carbon dioxide or with an organic solvent under a nitrogen stream. Examples of organic solvents include ethers such as dimethyl ether and diethyl ether; hydrocarbons having 10 or less carbon atoms such as petroleum ether, hexane and heptane; alcohols such as methanol and ethanol; chloroform; Fats such as oils and fats can be used. Also, alternate extraction with methanol and petroleum ether or extraction with a one-phase solvent of chloroform-methanol-water can provide good extraction results. By distilling off the organic solvent from the extract under reduced pressure, a crude oil containing a high concentration of fatty acids is obtained. Hexane is most commonly used when harvesting triacylglycerols.
In addition, extraction can be performed using wet cells instead of the above method. Crude oil may be collected from the wet cells by squeezing the wet cells, or by adding a water-compatible solvent such as methanol or ethanol, or a water-compatible solvent and water and Mixed solvents consisting of/or other solvents may be used. Other procedures are the same as above.
In the triacylglycerol concentrate production step, the collected crude oil is subjected to degumming, deacidification, decolorization, and deodorization by methods known to those skilled in the art, using methods used for refining vegetable oils, fish oils, and the like. For example, degumming is performed by washing with water, deoxidation is performed by distillation, decolorization is performed using activated clay, activated carbon, silica gel, etc., and deodorization is performed by steam distillation. will be
In the processing step, the triacylglycerol concentrate is subjected to processing such as esterification using a catalyst and hydrolysis. Alkyl esterification treatment and hydrolysis treatment can be performed under conditions known to those skilled in the art.
For example, to obtain fatty acid methyl esters, triacylglycerol concentrate is mixed with anhydrous methanol-hydrochloric acid 5%-10%, BF<sub>3</sub>- Obtained by treating with 10% to 50% methanol at room temperature for 1 to 24 hours. Ethyl esters of fatty acids can be obtained by treating fats and oils with 1% to 20% sulfuric acid ethanol or the like at 25°C to 100°C for 15 minutes to 60 minutes. A methyl ester or ethyl ester can be extracted from the reaction solution with an organic solvent such as hexane, diethyl ether, or ethyl acetate. The extract is dried with anhydrous sodium sulfate or the like, and the organic solvent is distilled off to obtain a composition containing fatty acid alkyl ester as a main component.
The first to third microbial oil production methods include first to third precision distillation steps, respectively, in which the raw material oil obtained in the raw material oil supply step is subjected to precision distillation under specific conditions. By performing the first to third precision distillation steps, the microbial oil production method of the present invention can efficiently obtain a specific target microbial oil, such as a desired microbial oil containing target LC-PUFA. .
In the first precision distillation step in the first method for producing microbial oil, conditions including a bottom temperature of 160 ° C to 230 ° C and a minimum pressure in the distillation column of 0.1 Pa to 30 Pa are applied to the raw material oil. Purification by precision distillation is performed by Purification by precision distillation at the bottom temperature and the minimum pressure in the distillation column within this range allows the desired specific unsaturated fatty acid, such as the target LC-PUFA, to be obtained accurately and efficiently.
In precision distillation, part of the vapor generated under heating conditions is returned to the distillation column as a reflux liquid, and the vapor rising in the column and the liquid sample are used to create a highly accurate vapor-liquid equilibrium. It is a technique for separating components and is a technique known to those skilled in the art.
Bottoms temperature refers to the temperature of the sample at the bottom within the distillation column. If the column bottom temperature is less than 160°C, fatty acids other than the target fatty acids, such as unsaturated fatty acids having 18 carbon atoms, other than the target LC-PUFA cannot be sufficiently separated. On the other hand, if the column bottom temperature exceeds 230°C, the content of thermally generated fatty acids increases even with precision distillation, and there is a tendency not to efficiently obtain microbial oil containing a high content of target LC-PUFA. . From the viewpoint of separation efficiency, the bottom temperature is more preferably 160°C to 210°C, even more preferably 160°C to 200°C.
The temperature at the top of the column is not particularly limited, and can be, for example, 80°C to 160°C, more preferably 90°C to 140°C.
The lowest pressure in the distillation column generally corresponds to the pressure at the top of the distillation column. In the case of a typical distillation column with a condenser (condenser) and vacuum pump at the top of the column, the pressure from the top condenser, which liquefies the ascending vapor, or fraction, to the vacuum pump is the highest in the column. Indicates low pressure. When the minimum pressure in the distillation column is higher than 30 Pa, the temperature at the bottom of the column rises in order to generate steam necessary for precision distillation, and as a result, the content of thermally generated fatty acids tends to increase. In addition, the minimum pressure in the distillation column can be set to 0.1 Pa or less because pressure loss generally occurs due to packing or piping included in the distillation column. The minimum pressure in the distillation column is more preferably 0.1 Pa to 20 Pa from the viewpoint of suppressing generation of thermally generated fatty acids.
In the second precision distillation step in the second method for producing microbial oil, the raw oil is heated to a bottom temperature of 160 ° C to 230 ° C and 0.1 Pa to 30 Pa using a distillation column containing structured packing. Perform precision distillation under conditions including the lowest pressure in the distillation column. In the second precision distillation step, since precision distillation is performed using a distillation column containing ordered packing, gas-liquid exchange can be achieved with extremely small pressure loss, thereby maintaining the same bottom temperature and the lowest pressure in the distillation column Precision distillation can be performed relatively gently even under conditions including Such relatively mild precision distillation relaxes the heating conditions for the feedstock oil, effectively suppresses the generation of thermogenic fatty acids, and more efficiently obtains a microbial oil containing a high content of target LC-PUFAs. be able to.
Structured packings are known in the art as applied to distillation and are formed of layers that are related to each other in a regularly repeating geometric relationship. There are no particular restrictions on the material of the ordered packing as long as it has a unique repeated shape. Metals such as stainless steel, aluminum, nickel, copper, Hastelloy, and Monel; resins such as polypropylene; and ceramics. ; It may be made of carbon such as carbon steel or carbon fiber, and can be appropriately selected according to the heating conditions and pressure conditions of distillation.
From the viewpoint of effectively suppressing the generation of thermogenic fatty acids and more efficiently obtaining microbial oil containing the target LC-PUFA at a high content rate, the structured packing should have a specific surface area of 125m per unit.<sup>2</sup>/m<sup>3</sup>~1700m<sup>2</sup>/m<sup>3</sup>is preferably 125m<sup>2</sup>/m<sup>3</sup>~900m<sup>2</sup>/m<sup>3</sup>is more preferably 200m<sup>2</sup>/m<sup>3</sup>~800m<sup>2</sup>/m<sup>3</sup>is more preferable.
Preferred structured packings can be mentioned, for example: Mellapak, Mellapak Plus, plastic Mellapak, Melagrid, BX/CY packings from Sulzer Chemtech. (BX/CY packing), BX Plus (BX Plus), plastic BX (Gauze packing), mellacarbon, DX/EX packing (DX/EX packing), Meradur, Sulzer Lab Packing EX, Nutter Grid (Nutter grid), Kuehne Rombopak.
The bottom temperature in the second precision distillation means the temperature at the bottom inside the distillation column. If the column bottom temperature is less than 160°C, fatty acids other than the target LC-PUFA, such as unsaturated fatty acids with 18 carbon atoms, cannot be sufficiently separated. On the other hand, if the column bottom temperature exceeds 230°C, the content of thermally generated fatty acids increases even with precision distillation, and a microbial oil containing a high content of target LC-PUFA cannot be obtained efficiently. From the viewpoint of separation efficiency, the bottom temperature is more preferably 160°C to 210°C, even more preferably 160°C to 200°C.
The temperature at the top of the column in the second precision distillation is not particularly limited, and can be, for example, 80°C to 160°C, more preferably 90°C to 140°C.
The lowest pressure in the distillation column in the second precision distillation generally corresponds to the pressure at the top of the distillation column. In the case of a typical distillation column with a condenser (condenser) and vacuum pump at the top of the column, the pressure from the top condenser, which liquefies the ascending vapor, or fraction, to the vacuum pump is the highest in the column. Indicates low pressure. When the minimum pressure in the distillation column is higher than 30 Pa, the temperature at the bottom of the column rises in order to generate steam necessary for precision distillation, and as a result, the content of thermally generated fatty acids tends to increase. In addition, the minimum pressure in the distillation column can be set to 0.1 Pa or less because pressure loss generally occurs due to packing or piping included in the distillation column. The minimum pressure in the distillation column is more preferably 0.1 Pa to 20 Pa from the viewpoint of suppressing generation of thermally generated fatty acids.
In the third precision distillation step in the third method for producing microbial oil, a distillation column containing a structured packing is used for the raw material oil, and a column corresponding to the type of the target polyunsaturated fatty acid is used. Microdistillation is carried out under conditions, including bottom temperature and minimum pressure in the distillation column, that yield a microbial oil containing thermogenic fatty acids with a content of no more than 3.0% by weight of the total weight of fatty acids in the oil. In the third precision distillation, the bottom temperature and the minimum pressure in the distillation column according to the type of target LC-PUFA are microbial oil containing thermally generated fatty acids with a content of 3.0% by weight or less of the total weight of fatty acids in the oil. satisfies the conditions that can be obtained. The bottom temperature and the minimum pressure in the distillation column according to the type of target LC-PUFA can be optimized based on the content of thermogenic fatty acids, and a person skilled in the art will know the type of distillation column used, The size, shape, etc., can be appropriately set according to the type of structured packing contained in the distillation column, packing height, and other conditions.
From the viewpoint of the purification efficiency of the target LC-PUFA and the suppression of the generation of thermogenic fatty acids, the third precision distillation is the bottom temperature of 160 ° C to 230 ° C and the minimum pressure in the distillation column of 0.1 Pa to 30 Pa. is preferred. The bottom temperature in the third precision distillation is more preferably 160°C to 210°C, even more preferably 160°C to 200°C. The minimum pressure in the distillation column in the third precision distillation is more preferably 0.1 Pa to 20 Pa from the viewpoint of suppressing the generation of thermally generated fatty acids. The lowest pressure in the distillation column in the third precision distillation generally corresponds to the pressure at the top of the distillation column. In the case of a general distillation column equipped with a condenser (condenser) and a vacuum pump at the top of the column, the pressure from the top condenser, which liquefies the rising vapor (fraction), to the vacuum pump is the highest in the distillation column. Indicates low pressure. The temperature at the top of the column is not particularly limited, and can be, for example, 80°C to 160°C, more preferably 90°C to 140°C.
Further, the conditions for precision distillation in the first to third precision distillation steps may not be limited to those described above. For example, when rectification is used, the pressure at the top of the distillation column is reduced to 10 mmHg (1333 Pa) or less, and the bottom temperature is 165°C to 210°C, preferably 170°C to 195°C. °C is preferable from the viewpoint of suppressing thermal denaturation of the oil and increasing the rectification efficiency. The lower the pressure at the top of the distillation column, the better, and more preferably 0.1 mmHg (13.33 Pa) or less. There are no particular restrictions on the temperature of the upper part of the tower, and it can be, for example, 160°C or less.
In addition, each of the first to third precision distillation steps can include a plurality of precision distillations under mutually different bottom temperature and minimum pressure conditions in the distillation column. This allows for effective separation of different separation target fatty acids in each microdistillation. Conditions of different bottom temperatures and minimum pressures in the distillation column include, for example, a combination of two or more stages of precision distillation with different bottom temperatures.
For example, the first to third precision distillation steps are a combination of precision distillation steps with different bottom temperatures and minimum pressures in the distillation column, with a bottom temperature of 160 ° C to 220 ° C and a bottom temperature of 0.1 Pa to 30 Pa. A low-temperature precision distillation step, which is the lowest pressure in the distillation column, and a high-temperature precision distillation step, which is a bottom temperature of 170°C to 230°C and a lowest pressure in the distillation column of 0.1Pa to 30Pa, can be included.
By going through the low-temperature precision distillation process, fatty acid components with relatively smaller molecular weights than the target LC-PUFA, such as fatty acid components with 18 or less carbon atoms, can be removed as the initial distillation, and the target LC-PUFA is left as the residue. A microbial oil containing The bottom temperature in the low temperature precision distillation step is preferably 160°C to 200°C, more preferably 160°C to 190°C.
By going through the high-temperature precision distillation process, it is possible to reduce the content of at least one of the saturated fatty acid with 22 carbon atoms and the saturated fatty acid with 24 carbon atoms, which can cause clogging of pipes. In addition, clogging of pipes can be suppressed. As a result, it is possible to efficiently obtain a microbial oil containing the target LC-PUFA at a high content. The content of saturated fatty acids with 22 or 24 carbon atoms in the residue in the high temperature precision distillation process is increased compared to the low temperature precision distillation process. From the viewpoint of removing saturated fatty acids having 22 or 24 carbon atoms and suppressing the formation of thermally generated fatty acids, the bottom temperature in the high-temperature precision distillation step is preferably 170°C to 210°C.
Regarding the temperature difference between the column bottom temperature in the low-temperature precision distillation process and the high-temperature precision distillation process, the need for steam generation from the residue with a high content of saturated fatty acids with 22 and 24 carbon atoms in the high-temperature precision distillation process, and the heat From the viewpoint of suppression of produced fatty acids, the bottom temperature in the high-temperature precision distillation step is preferably 3°C to 20°C higher than the tower bottom temperature in the low-temperature precision distillation step, and more preferably 3°C to 10°C higher. .
In both the low-temperature precision distillation process and the high-temperature precision distillation process, the minimum pressure in the distillation column is more preferably 0.1 Pa to 20 Pa, more preferably 0.1 Pa to 10 Pa, from the viewpoint of suppressing the production of thermally generated fatty acids. is more preferred. The temperature at the top of the column is not particularly limited, and can be, for example, 160°C or less.
Appropriate heating time can be set by a person skilled in the art according to the charged amount of the raw material composition for distillation based on the description of the examples of the present specification.
Either the low-temperature precision distillation process or the high-temperature precision distillation process can be performed first. For example, by performing a high temperature precision distillation process after a low temperature precision distillation process, it is possible to remove fatty acid components with relatively higher molecular weights as a residue than the target LC-PUFA, and as a fraction, A microbial oil can be obtained that is depleted of both relatively low molecular weight fatty acid components and fatty acid subdivisions of relatively higher molecular weight than the target LC-PUFA.
In the microbial recovery step in the second and third precision distillation steps, microbial oil containing a high content of target LC-PUFA can be recovered as a fraction obtained by the precision distillation step. Such microbial oils are concentrated microbial oils of target LC-PUFAs and are useful for efficiently obtaining target LC-PUFAs as free fatty acid forms and/or their alkyl ester forms using reversed-phase column chromatography. be.
(3) Concentrated microbial oil The concentrated microbial oil as one aspect of the present invention has a target LC-PUFA content of 90% to 98% by weight of the total weight of fatty acids in the oil, and contains thermogenic fatty acids. is 0.0001% to 3.0% by weight of the total weight of fatty acids in the oil, and the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is 1.0% by weight of the total weight of fatty acids in the oil. % or less, and the content of C18 monounsaturated fatty acids is 5.0% or less by weight of the total weight of fatty acids in the oil.
For example, as an example of a concentrated microbial oil, the content of DGLA is 90% to 98% by weight of the total weight of fatty acids in the oil, and the content of thermogenic fatty acids is 90% by weight of the total weight of fatty acids in the oil. 0.0001% by weight to 3.0% by weight, the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is 1.0% by weight or less of the total weight of fatty acids in the oil, and monovalent with 18 carbon atoms The content of unsaturated fatty acids is 5.0% by weight or less of the total weight of fatty acids in the oil.
The concentrated microbial oil preferably has a target LC-PUFA content of 90% to 98% by weight, 95% to 98% by weight, 96% to 98% by weight of the total weight of fatty acids in the oil, or 97 wt % to 98 wt %, and the content of thermogenic fatty acids is 0.01 wt % to 3.0 wt %, 0.1 wt % to 3.0 wt %, 0.1 wt % to 2.8 wt %, of the total weight of fatty acids in the oil. C24 saturated fatty acids and The total content of saturated fatty acids with 22 carbon atoms is 1.0% by weight or less, 0.2% by weight or less, or 0% by weight of the total weight of fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms is 5.0% or less, 2.0% or less, or 0% by weight of the total weight of fatty acids in the oil.
Further, other preferred concentrated microbial oils preferably have a DGLA content of 90% to 98% by weight, 95% to 98% by weight, and 96% to 98% by weight of the total weight of fatty acids in the oil. % or 97 wt % to 98 wt %, and the content of thermogenic fatty acids is 0.1 wt % to 3.0 wt %, 0.1 wt % to 2.8 wt %, 0.1 wt % to 2.5 wt % of the total weight of fatty acids in the oil. %, 0.1% to 2.0% by weight, 0.1% to 1.5% by weight, 0.1% to 1.0% by weight, or 0.1% to 0.7% by weight, and a saturated fatty acid having 24 carbon atoms and a saturated fatty acid having 22 carbon atoms is 1.0% by weight or less, 0.2% by weight or less, or 0% by weight of the total weight of fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms is less than the total weight of fatty acids in the oil 5.0% by weight or less, 2.0% by weight or less, or 0% by weight.
Other preferred concentrated microbial oils preferably have a content of eicosadienoic acid, mead acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid, or docosahexaenoic acid. , 90% to 98% by weight, 95% to 98% by weight, 96% to 98% by weight, or 97% to 98% by weight of the total weight of fatty acids in the oil, and the content of thermogenic fatty acids is , 0.1%-3.0% by weight, 0.1%-2.8% by weight, 0.1%-2.5% by weight, 0.1%-2.0% by weight, 0.1%-1.5% by weight, 0.1% by weight of the total weight of fatty acids in the oil % to 1.0% by weight, or 0.1% to 0.7% by weight, and the total content of saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms is 1.0% by weight or less of the total weight of fatty acids in the oil, 0.2 wt% or less, or 0 wt%, and the content of C18 monounsaturated fatty acids (C18:1) is 5.0 wt% or less, 2.0 wt% or less, or 0 wt% of the total weight of fatty acids in the oil %.
These concentrated microbial oils contain high concentrations of target LC-PUFAs such as eicosadienoic acid, DGLA, mead acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid, or docosahexaene. Since it contains an acid, it is extremely useful for applications that require a high content of the target LC-PUFA, such as DGLA, and high productivity.
(4) Method for producing concentrated microbial oil The method for producing concentrated microbial oil according to one aspect of the present invention comprises obtaining a microbial oil containing the target LC-PUFA by any of the above-described production methods, and the resulting microbial oil. It involves subjecting the oil to a concentration treatment using reversed-phase column chromatography.
The microbial oil obtained by the method for producing microbial oil according to the aspect of the present invention has a high target LC-PUFA content and a high content of fatty acids that are difficult to separate from the target LC-PUFA by reversed-phase column chromatography. Since it is low, the target LC-PUFA can be efficiently obtained at a high content rate.
Examples of the reversed-phase column chromatography used for concentration treatment include reversed-phase column chromatography known in the art, particularly high-performance liquid chromatography using a base material modified with an octadecylsilyl group (ODS) as a stationary phase. Graphics (HPLC) are preferred. Examples of reversed-phase partition columns include YMC Pack ODS-AQ-HG columns (YMC Co., Ltd.).
HPLC conditions applied to the concentration treatment include, for example, the following.
Column: YMC pack ODS-AQ-HG 20mmφ×500mm (YMC, Inc.) Pump: 1200 series G1361A Prep Pump (Agilent Technologies, Inc.) Column temperature: Around 21°C Mobile phase: Methanol 17.5mL/min Sample conditions: Load Amount of 2.4 g, i.e., the raw material loading rate is 3% by weight with respect to the adsorbent
By using microbial oils according to embodiments of the present invention, recovery of target LC-PUFAs in reversed-phase column chromatography can be preferably 50% or higher, more preferably 80% or higher, and even more preferably 90% or higher.
Microbial oils and concentrated microbial oils according to aspects of the present invention, and microbial oils and concentrated microbial oils obtained by production methods according to aspects of the present invention, may contain a high content of target LC-PUFAs, or may contain a high content of target LC-PUFAs contains the target LC-PUFAs at 100 rpm and is free of components that may be contaminated by using other separation means than precision distillation. Components that may be mixed in by using separation means other than precision distillation include, for example, metals such as silver and a large amount of urea.
Therefore, microbial oils and concentrated microbial oils according to embodiments of the present invention are extremely useful for applications requiring high levels of target LC-PUFAs, such as DGLA, and high productivity. Such applications include, for example, use in foods, supplements, pharmaceuticals, cosmetics, feeds, etc., and use in production methods thereof. Drugs containing it as an ingredient can be mentioned. For example, in the case of DGLA, it is particularly preferable to use it for uses aiming at the functionality of DGLA, such as anti-inflammatory and anti-allergic uses.
(5) Prophylactic or therapeutic agents for inflammatory diseases The microbial oil or concentrated microbial oil according to embodiments of the present invention contains target LC-PUFA, such as DGLA, as an active ingredient based on the functionality of target LC-PUFA, such as DGLA. , inflammatory disease preventive or therapeutic agents. That is, the preventive or therapeutic agent for inflammatory diseases according to one aspect of the present invention contains the microbial oil or concentrated microbial oil according to another aspect of the present invention as an active ingredient. Inflammatory disease prophylactic or therapeutic agents can be, for example, anti-inflammatory agents, anti-allergic agents, and the like.
Inflammatory diseases include, among others, inflammation of the skin. The skin inflammation may be at least one skin inflammation selected from the group consisting of rashes, hives, blisters, wheals, and eczema, or from exposure to radiation, autoimmune diseases, and uremic pruritus. It may be skin inflammation caused by at least one selected from the group consisting of:
In particular, the skin inflammation may be skin inflammation associated with or caused by atopic eczema, contact dermatitis, psoriasis, or uremic pruritus.
Terminology Eczema applies to a wide range of skin conditions with diverse etiologies. Eczema is generally classified by inflammation of the epidermis. Common symptoms associated with eczema include dryness, recurrent skin rashes, redness, skin edema (swelling), itching, crusting, scaling, blistering, cracking, weeping, and bleeding. . Eczema includes atopic eczema (atopic dermatitis), contact dermatitis, dry eczema, seborrheic dermatitis, sweating disorder, discoid eczema, venous eczema, herpetic dermatitis, neurodermatitis, and autodermatitis. Eczema may be mentioned. Eczema is typically atopic eczema or contact dermatitis.
Atopic eczema is primarily exacerbated by contact with or ingestion of allergens, including animal hair and dander, food allergens (such as nuts or shellfish), or drugs (such as penicillin). be done.
Contact dermatitis includes allergic contact dermatitis, irritant contact dermatitis, and photocontact dermatitis. Photocontact dermatitis includes phototoxic contact dermatitis and photoallergic contact dermatitis.
The skin inflammation may be skin inflammation caused by exposure of the skin to electromagnetic radiation. This includes, for example, exposure to sunlight, heat, X-rays, or radioactive substances. Accordingly, in this embodiment, the compounds of the invention are typically used to treat sunburn.
Electromagnetic radiation includes radio waves, microwaves, terahertz waves, infrared, visible, ultraviolet, X-rays and gamma rays. The electromagnetic radiation is preferably infrared, visible, ultraviolet, X-rays and gamma rays, more preferably ultraviolet, X-rays and gamma rays.
Autoimmune diseases can involve an autoimmune response to the skin. Examples of such autoimmune diseases are lupus and psoriasis.
Uremic pruritus is a skin disorder associated with chronic renal failure. It also frequently affects patients undergoing dialysis treatment.
Optionally, the microbial oil or concentrated microbial oil according to other aspects of the invention and the corticosteroid or any other therapeutic agent used in the pharmaceutical applications described above may be co-administered.
In another aspect, the inflammatory disease is selected from the group consisting of atopic dermatitis, allergic contact dermatitis, primary irritant contact dermatitis, photocontact dermatitis, systemic contact dermatitis, rheumatism, psoriasis and lupus. may be at least one
An inflammatory disease prophylactic or therapeutic agent can be administered to a subject suffering from or at risk of suffering from an inflammatory disease. The mode of administration may be oral administration or topical administration. In addition, the therapeutic agent for inflammatory disease refers to a drug used for suppressing or alleviating the progress of symptoms caused by an inflammatory disease when such symptoms are found. On the other hand, the prophylactic agent for inflammatory disease refers to a drug that is administered in advance to suppress the onset of symptoms due to an inflammatory disease when the onset of symptoms is expected. However, these terms are used in combination depending on the time of use or the symptoms during use, and are not interpreted restrictively.
Another aspect of the present invention is the prevention, treatment or treatment of an inflammatory disease, comprising administering the above-described agent for the prevention or treatment of an inflammatory disease to a subject suffering from or at risk of suffering from an inflammatory disease. A method of remission is provided. The mode of administration may be oral administration or topical administration.
The microbial oil of the present invention may contain each component in terms of area % content according to column chromatography analysis. That is, each aspect of the present invention further provides the following microbial oil, method for producing microbial oil, concentrated microbial oil, and method for producing concentrated microbial oil.
<1> At least one polyvalent with 20 or more carbon atoms in the form of fatty acid alkyl ester and/or free fatty acid, having a content of 50% or more of the total area of fatty acids in the oil as measured by gas chromatography A microbial oil containing unsaturated fatty acids and thermogenic fatty acids having 16 to 22 carbon atoms having a content of 3.0 area % or less of the total area of fatty acids in the oil as determined by gas chromatography.
<2> The microbial oil according to <1>, wherein the polyunsaturated fatty acid content is 80 area % to 98 area % of the total area of fatty acids in the oil as measured by gas chromatography.
<3> The microbial oil according to <1> or <2>, wherein the thermogenic fatty acid content is 0.0001 area % to 3.0 area % of the total area of fatty acids in the oil as measured by gas chromatography.
<4> The total content of saturated fatty acids with 22 carbon atoms and saturated fatty acids with 24 carbon atoms is 6.0 area% or less of the total area of fatty acids in the oil as measured by gas chromatography <1> to <3> The microbial oil according to any one of
<5> Any one of <1> to <4>, wherein the total content of the saturated fatty acid with 22 carbon atoms and the saturated fatty acid with 24 carbon atoms is 10/100 or less of the content of the polyunsaturated fatty acid. of microbial oils.
<6> The microbial oil according to any one of <1> to <5>, wherein the content of saturated fatty acids with 24 carbon atoms is 3.0% or less of the total area of fatty acids in the oil as measured by gas chromatography. .
<7> The microbial oil according to any one of <1> to <6>, wherein the content of saturated fatty acids with 24 carbon atoms is 4/100 or less of the content of polyunsaturated fatty acids.
<8> When using the partition number determined from the carbon number and double bond number of the fatty acid, which is an indicator of separation by liquid chromatography, it is 2 less than the partition number of the polyunsaturated fatty acid The content of other saturated or unsaturated fatty acids having a partition number greater than or equal to 2 and less than or equal to a number and having a carbon number different from that of the polyunsaturated fatty acid is determined by gas chromatography in the oil. The microbial oil according to any one of <1> to <7>, which is 10.0 area % or less of the total area of fatty acids.
<9> The microbial oil according to <8>, wherein the other saturated or unsaturated fatty acid content is 15/100 or less of the polyunsaturated fatty acid content.
<10> The polyunsaturated fatty acid is selected from eicosadienoic acid, dihomo-γ-linolenic acid, mead acid, eicosatetraenoic acid, arachidonic acid, eicosapentaenoic acid, docosatetraenoic acid, docosapentaenoic acid and docosahexaenoic acid. The microbial oil according to any one of <1> to <9>, which is at least one selected from the group consisting of:
<11> The other saturated or unsaturated fatty acids include saturated fatty acids with 18 carbon atoms, monounsaturated fatty acids with 18 carbon atoms, divalent unsaturated fatty acids with 18 carbon atoms, trivalent unsaturated fatty acids with 18 carbon atoms and The microbial oil according to any one of <8> to <10>, containing at least one selected from the group consisting of tetravalent unsaturated fatty acids having 18 carbon atoms.
<12> The microbial oil according to any one of <1> to <11>, wherein the polyunsaturated fatty acid is dihomo-γ-linolenic acid, and the thermogenic fatty acid is a thermogenic fatty acid having 20 carbon atoms. .
<13> Thermally generated fatty acid is a peak that appears within the range of 1.001 to 1.011 when the retention time of dihomo-γ-ethyl linolenate is set to 1 in gas chromatography analysis under the following conditions for the thermally generated fatty acid ethyl ester and the second substance having a retention time as a peak appearing in the range of 1.013 to 1.027.
Equipment: 6890N Network GC system (Agilent Technologies Inc.) Column: DB-WAX Length 30m x I.D. 0.25mm x Film thickness 0.25μm (Agilent Technologies Inc.) Column temperature conditions: 60°C 2.5 minutes heating up 20 minutes °C/min 180°C 2°C/min 230°C 15 min Inlet temperature conditions: 210°C, splitless, split vent sampling time 1.5 min, purge flow rate 40 mL/min Injection volume conditions: 1 μL, sample concentration 1 mg/mL or less Detector: FID detector Temperature: 280°C Carrier gas conditions: Helium, linear velocity 24 cm/min
<14> The polyunsaturated fatty acid is dihomo-γ-linolenic acid, and the total content of the first substance and the second substance is measured by gas chromatography, and the total area of fatty acids in the oil 0.001 area % to 2.8 area % of the microbial oil according to <13>.
<15> Any one of <10> to <14>, wherein the content of monounsaturated fatty acids with 18 carbon atoms is 7.0 area% or less of the total area of fatty acids in the oil as measured by gas chromatography. of microbial oils.
<16> The microbial oil according to any one of <10> to <15>, wherein the content of monounsaturated fatty acids with 18 carbon atoms is 10/100 or less of the content of polyunsaturated fatty acids.
<17> The microbial oil according to any one of <10> to <16>, wherein the content of the divalent unsaturated fatty acid having 18 carbon atoms is 7/100 or less of the content of the polyunsaturated fatty acid.
<18> The total content of monounsaturated fatty acids with 18 carbon atoms and divalent unsaturated fatty acids with 18 carbon atoms is 15/100 or less of the polyunsaturated fatty acid content <10> to <17 The microbial oil according to any one of >.
<19> The microbial oil according to any one of <10> to <18>, wherein the content of saturated fatty acids with 18 carbon atoms is 11/100 or less of the content of polyunsaturated fatty acids.
<20> A method for producing a microbial oil, comprising preparing a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of an alkyl ester and/or free fatty acid obtained from microbial biomass; and purifying the raw material oil by precision distillation under conditions including a bottom temperature of 160°C to 230°C and a minimum pressure in the distillation column of 0.1Pa to 30Pa. .
<21> A method for producing a microbial oil, comprising preparing a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of an alkyl ester and/or free fatty acid obtained from microbial biomass; The raw material oil is subjected to precision distillation using a distillation column containing structured packing under conditions including a column bottom temperature of 160°C to 230°C and a minimum pressure in the distillation column of 0.1Pa to 30Pa. and obtaining the microbial oil according to any one of <1> to <19>.
<22> A method for producing a microbial oil, comprising preparing a raw material oil containing at least one polyunsaturated fatty acid having 20 or more carbon atoms in the form of an alkyl ester and/or free fatty acid obtained from microbial biomass; For the raw material oil, using a distillation column containing structured packing, including the bottom temperature and the minimum pressure in the distillation column according to the type of the target polyunsaturated fatty acid, measurement by gas chromatography and performing precision distillation under conditions under which a microbial oil containing thermogenic fatty acids with 16 to 22 carbon atoms can be obtained with a content of 3.0 area% or less of the total area of fatty acids in the oil, and <1> to < obtaining the microbial oil according to any one of 19>.
<23> The production method according to <22>, wherein the precision distillation is performed at a bottom temperature of 160°C to 230°C and a minimum pressure in the distillation column of 0.1Pa to 30Pa.
<24> The production method according to any one of <20> to <23>, wherein the precision distillation includes a plurality of precision distillations under mutually different bottom temperature and top pressure conditions.
<25> The precision distillation includes low-temperature precision distillation at a bottom temperature of 160 ° C to 220 ° C and a minimum pressure in the distillation column of 0.1 Pa to 30 Pa, and a bottom temperature of 170 ° C to 230 ° C and 0.1 The production method according to <24>, which includes high-temperature precision distillation at a minimum pressure in a distillation column of Pa to 30 Pa.
<26> The production method according to <25>, wherein the bottom temperature in the high-temperature precision distillation is 3°C to 20°C higher than the bottom temperature in the low-temperature precision distillation.
<27> The specific surface area per unit of structured packing is 125m<sup>2</sup>/m<sup>3</sup>~1700m<sup>2</sup>/m<sup>3</sup>The production method according to any one of <21> to <26>.
<28> The content of polyunsaturated fatty acids with 20 or more carbon atoms in the form of fatty acid alkyl esters and/or free fatty acids is 90% to 98% of the total area of fatty acids in the oil as measured by gas chromatography. %, the content of thermogenic fatty acids with 16 to 22 carbon atoms is 0.0001 area % to 3.0 area % of the total area of fatty acids in the oil as measured by gas chromatography, and saturated fatty acids with 24 carbon atoms and The total content of saturated fatty acids with 22 carbon atoms is 1.0 area% or less of the total area of fatty acids in the oil as measured by gas chromatography, and the content of monounsaturated fatty acids with 18 carbon atoms is measured by gas chromatography. A concentrated microbial oil that is less than or equal to 5.0 area % of the total area of fatty acids in the oil, as determined graphically.
<29> The content of dihomo-γ-linolenic acid in the form of fatty acid alkyl ester and/or free fatty acid is 90 area % to 98 area % of the total area of fatty acids in the oil as measured by gas chromatography, The content of thermogenic fatty acids with 16 to 22 carbon atoms is 0.0001 area % to 3.0 area % of the total area of fatty acids in the oil as measured by gas chromatography, and saturated fatty acids with 24 carbon atoms and saturated fatty acids with 22 carbon atoms The total saturated fatty acid content, as determined by gas chromatography, is 1.0% or less of the total area of fatty acids in the oil, and the content of monounsaturated fatty acids with 18 carbon atoms, as determined by gas chromatography, is , a concentrated microbial oil that is no more than 5.0 area % of the total area of fatty acids in the oil.
<30> A method for producing a concentrated microbial oil, wherein at least one carbon in the target fatty acid alkyl ester form and/or free fatty acid form is produced using the production method according to any one of <20> to <27> A method comprising obtaining a microbial oil containing 20 or more polyunsaturated fatty acids, and subjecting the obtained microbial oil to a concentration treatment using reversed-phase column chromatography.
<31> Use of the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29> in food, supplements, pharmaceuticals, cosmetics or feed.
<32> Use of the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29> in a method for producing food, supplements, pharmaceuticals, cosmetics, or feed.
<33> A drug containing the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29>.
<34> An agent for preventing or treating inflammatory diseases, comprising the microbial oil according to any one of <1> to <19> or the concentrated microbial oil according to <28> or <29>.
<35> The preventive or therapeutic agent for inflammatory diseases according to <34>, which is an anti-allergic agent or an anti-inflammatory agent.
<36> The inflammatory disease is at least one skin inflammatory disease selected from the group consisting of rash, hives, blisters, wheals and eczema, or exposure to radiation, autoimmune disease and uremic ulcer The preventive or therapeutic agent for inflammatory diseases according to <34> or <35>, which is a skin inflammatory disease caused by at least one selected from the group consisting of itching.
<37> The skin inflammatory disease is selected from the group consisting of atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, photocontact dermatitis, systemic contact dermatitis, rheumatism, psoriasis and lupus. The preventive or therapeutic agent for inflammatory diseases according to <34> or <35>, which is at least one of
<38> Inflammatory disease, including administering the inflammatory disease prophylactic or therapeutic agent according to any one of <34> to <37> to a subject suffering from or at risk of suffering from an inflammatory disease Disease prevention, treatment or amelioration methods.
<39> The method for preventing, treating, or ameliorating an inflammatory disease according to <38>, wherein the administration is oral administration or topical administration.
<40> A microbial oil obtained by the production method according to any one of <20> to <27>.
<41> A concentrated microbial oil obtained by the production method described in <30>.
As described above, in the present invention, the content of each component of the microbial oil and the concentrated microbial oil is the same in terms of area % based on measurement by gas chromatography and weight %, so Descriptions regarding the content of each component of microbial oil and concentrated microbial oil expressed in area % based on measurement by gas chromatography, each numerical value expressed in weight % is rewritten as it is in area %, and the entire text is Apply as is.
In addition, in this specification, each invention specifying feature described in one embodiment relating to each aspect of the invention may be arbitrarily combined to form a new embodiment. It should be understood that new embodiments may also be encompassed by each aspect of the invention.
<p>EXAMPLES The present invention will be described in detail below with reference to Examples. However, the present invention is by no means limited to them. Unless otherwise specified, "%" is based on mass.</p><p>In the examples and comparative examples in the following sections, the target LC-PUFA is the ethyl ester form of DGLA, but the present invention is not limited to this, and the free fatty acid form of DGLA may be the target LC-PUFA, and the alkyl ester Other fatty acids in either form or free fatty acid form may be targeted LC-PUFAs.</p><p>It has been empirically found that the ethyl esterification rate of the alkyl esterification method used in Examples and Comparative Examples in the following sections is 95% to 100%. For this reason, in the section of this Example, it was presumed that most of the saturated or unsaturated fatty acids contained in the raw material ethyl ester obtained were in the form of fatty acid ethyl ester. Therefore, in the following comparative examples and examples, saturated or unsaturated fatty acids contained in microbial oil are all described as saturated or unsaturated fatty acids in the form of ethyl esters.</p><p>Also, hereinafter, DGLA ethyl ester is simply "DGLA", monounsaturated fatty acid ethyl ester with 18 carbon atoms is simply "C18:1", and divalent unsaturated fatty acid ethyl ester with 18 carbon atoms is simply "C18:2". , the saturated fatty acid ethyl ester having 22 carbon atoms is simply indicated as "C22:0", and the saturated fatty acid ethyl ester having 24 carbon atoms is simply indicated as "C24:0".</p><p>[Comparative example 1]</p><p>A microbial oil 1 derived from a Mortierella microorganism containing 37.2% by weight of DGLA in the fatty acid composition was ethyl-esterified with an alkali catalyst in accordance with a conventional method to prepare raw material ethyl ester 1. Specifically, 14 g of a 20% by weight sodium ethoxide-ethanol solution and 40 mL of ethanol were added to 120 g of the microbial oil 1, and the mixture was heated under reflux for 2 hours in an oil bath. After that, the reaction solution was air-cooled to 40°C or lower, and then transferred to a separating funnel. To the reaction liquid transferred to a separating funnel, 400 mL of hexane was added, and then purified water was added to repeat washing with water. After the washing liquid became neutral, the hexane layer was collected after washing once with a saturated saline solution. Anhydrous sodium sulfate was added to the collected hexane layer for dehydration, and the solvent was removed by an evaporator and vacuum suction to obtain raw material ethyl ester 1.</p><p>In raw material ethyl ester 1, the DGLA content, that is, the content of DGLA in the obtained raw material ethyl ester, was 37.2% by weight, and the weight ratio of C18:1 to DGLA, that is, C18:1/DGLA was 23.5/100, C18 :2 weight ratio, ie C18:2/DGLA was 17.8/100.</p><p>Raw material ethyl ester 1 was subjected to HPLC under the following conditions without distillation to fractionate a DGLA eluted fraction.</p><p>In the HPLC, starting from the start of treatment with the raw material ethyl ester 1 put into the apparatus, the eluate was fractionated until all the fatty acids contained in the raw material ethyl ester 1 were eluted. For each fraction obtained, exactly 1 mL was collected and then the solvent was removed by an evaporator. The fraction after removing the solvent was dissolved in exactly 1 mL of methyl tricosanoate, that is, C23:0 methyl ester 1.0 mg/mL hexane solution as an internal standard, and the measurement sample was analyzed by gas chromatography under the conditions shown below. subjected to graphics (GC).</p><p>From each fatty acid peak area obtained by GC, the amount and fatty acid composition of the fatty acid contained in the measurement sample are obtained based on the following formula (II), and the DGLA content, that is, the DGLA for the obtained fraction The content rate and recovery rate of were calculated. The recovery of DGLA was calculated by calculating the ratio of the total amount of DGLA in the collected fractions to the total amount of DGLA in all fractions of the fractionated HPLC eluate. Hereafter, the recovery rate was calculated by the same method.</p><p>Amount of fatty acid contained in the fraction [mg] = (Peak area of each fatty acid × Fraction volume [mL]) / (Peak area of C23:0 methyl ester) × Amount of internal standard added 1.0 mg (II)</p><p>The results are shown in Table 2. As shown in Table 2, the DGLA content was 91.1% by weight and the DGLA recovery was 8.1%. In Table 2, the numerical values in the "Microbial oil" column of Comparative Example 1 are the numerical values of raw material ethyl ester 1. The contents and weight ratios in Table 2 are both based on the fatty acid composition. Same below.</p><p>- HPLC conditions Column: YMC pack ODS-AQ-HG 20 mmφ x 1000 mm (YMC Co., Ltd.) Note that two columns with a length of 500 mm were connected in series.</p><p>Pump: 1200 series G1361A Prep Pump (Agilent Technologies, Inc.) Column temperature: 40°C Mobile phase: Methanol 35 mL/min Sample conditions: Loading amount 2.4 g, raw material loading rate is 3% by weight of packing material</p><p>GC conditions Equipment: 6890N Network GC system (Agilent Technologies Inc.) Column: DB-WAX Length 30m x inner diameter 0.25mm x film thickness 0.25μm (Agilent Technologies Inc.) Column temperature conditions: 180°C heating 3°C/min 230°C for 30 min Inlet temperature: 250°C Detector: FID detector temperature: 250°C Carrier gas conditions: Helium Linear velocity 30 cm/min Split conditions: Split ratio 1:30, injection volume 1 μL , sample concentration 9mg/mL</p><p>[Comparative example 2]</p><p>Raw material ethyl ester 1 used in Comparative Example 1 was subjected to short path distillation (SPD) under the following conditions to remove a fatty acid fraction having 18 or less carbon atoms.</p><p>The SPD device used was KDL-5 (UIC GmbH). Raw material temperature 40°C, heating medium temperature at inlet of evaporation surface 100°C, heating medium outlet temperature 87°C, internal condenser temperature 30°C, and pressure before pump 0.001 bar, i.e. 0.133 mPa. 160.7 g of the raw material was fed at 300 mL/h, and a fraction containing a large amount of C18 and lower fractions was removed to obtain 65.9 g of the residue. The residue contained concentrated DGLA.</p><p>The resulting residue was subjected to HPLC under the following conditions to fractionate a DGLA eluted fraction. The DGLA elution fraction corresponds to concentrated microbial oil.</p><p>Regarding the obtained fraction after SPD treatment and the DGLA elution fraction after HPLC treatment, the amount of fatty acids contained and the fatty acid composition were determined by gas chromatography in the same manner as in Comparative Example 1, and the DGLA content was determined. That is, the content and recovery of DGLA with respect to the obtained fractions were calculated. The results are shown in Table 2. As shown in Table 2, the DGLA content in the fraction after SPD treatment was 40.9% by weight, the DGLA content in the DGLA-eluted fraction was 94.4% by weight, and the DGLA recovery was 5.1%.</p><p>- HPLC conditions Column: YMC pack ODS-AQ-HG 20 mmφ x 1000 mm (YMC Co., Ltd.), two 500 mm columns were connected in series.</p><p>Pump: 1200 series G1361A Prep Pump (Agilent Technologies, Inc.) Column temperature: 40°C Mobile phase: Methanol 12mL/min Sample conditions: Loading amount 2.4g, that is, raw material loading rate is 1.5% by weight with respect to packing material</p><p>[Example 1]</p><p>A microbial oil 2 derived from a Mortierella microorganism containing 32.8% by weight of DGLA in the fatty acid composition was ethyl-esterified with an alkali catalyst by a conventional method to prepare raw material ethyl ester 2. Specifically, 14 g of a 20% by weight sodium ethoxide-ethanol solution and 40 mL of ethanol were added to 120 g of the microbial oil 2, and the mixture was heated under reflux for 2 hours in an oil bath. After that, the reaction solution was air-cooled to 40°C or lower, and then transferred to a separating funnel. To the reaction liquid transferred to a separating funnel, 400 mL of hexane was added, and then purified water was added to repeat washing with water. After the washing liquid became neutral, the hexane layer was collected after washing once with a saturated saline solution. Anhydrous sodium sulfate was added to the collected hexane layer for dehydration, and the solvent was removed by an evaporator and vacuum suction to obtain raw material ethyl ester 2.</p><p>In raw material ethyl ester 2, the DGLA content, that is, the content of DGLA in the obtained raw material ethyl ester, was 32.8% by weight, the weight ratio of C18:1 to DGLA, that is, C18:1/DGLA was 26.1/100, C18 :2 weight ratio to DGLA (C18:2/DGLA was 17.2/100.</p><p>Raw material ethyl ester 2 was subjected as a sample to precision distillation including the following low-temperature precision distillation process and high-temperature precision distillation process.</p><p>In the low-temperature precision distillation step, 100 g of raw material ethyl ester 2 was subjected to the following precision distillation. A fractionating tube with a vacuum jacket (Kiriyama Glass) was used as the fractionating tube, and five Sulzer Lab Packing EX (Sulzer Chemtech) were used as the internal packing. The diameter of the vacuum jacketed fractionating tube was 25 mm and the size of one unit of Sulzer Labpacking EX was 25 mm x 50 mm. The liquid temperature in the bottom pot, that is, the bottom temperature is 185 ° C, the top vapor temperature, that is, the top temperature is 135 ° C, the pressure before the vacuum pump, that is, the lowest pressure in the distillation column, that is, Precision distillation was performed at a degree of vacuum of 30 Pa. In the low-temperature precision distillation process, the C18 or less fraction was removed as the initial distillation, and 40 g of the initial distillation residue was obtained. When the fatty acid content and fatty acid composition of the initial distillation residue were confirmed using gas chromatography in the same manner as in Comparative Example 1, the initial distillation residue contained concentrated DGLA ethyl ester.</p><p>In addition, in the chromatogram of the initial distillation residue, between the peak indicating C20:3,n-6 (DGLA) and the peak indicating C20:4,n-6, in the chromatogram of crude oil as a sample, Compound A appeared, showing an unusual peak A (see Table 3). Also, in the vicinity of the peak indicating C20:4n-6, compound B appeared that showed peak B, which is not normally seen in the chromatogram of crude oil as a sample. Compound A and compound B can be considered as compounds formed by the distillation process and were thermogenic fatty acids. The contents of compound A and compound B are shown in Tables 2 and 3. The content in Table 3 represents the content based on the fatty acid composition.</p><p>In addition, when the thermally generated fatty acid peak overlaps with the fatty acid contained in the crude oil under the GC conditions used in Comparative Example 1, the separation and quantification of compound A and compound B are performed by a silver ion solid phase extraction method (silver-ion solid phase extraction method). After removing the fatty acids originally contained in the crude oil by extraction), it is subjected to gas chromatography under the following conditions, and when the retention time of dihomo-γ-ethyl linolenate is set to 1, it is within the range of 1.001 to 1.009 The compound with the retention time indicated by the appearing peak, peak A, was identified as compound A. Similarly, a compound having a retention time indicated by peak B, which appears within the range of 1.013 to 1.024, was identified as compound B. After that, the relative ratio of DGLA and compound A or compound B was determined, and the weight % of DGLA, compound A and compound B was calculated.</p><p>Apparatus: 6890N Network GC system (Agilent Technologies Inc.) Column: DB-WAX Length 30m x I.D. 0.25mm x Film thickness 0.25μm (Agilent Technologies Inc.) Column temperature conditions: 60°C for 2.5 minutes heating 20°C/min 180°C 2°C/min 230°C 15 min Inlet temperature conditions: 210°C, splitless, split vent sampling time 1.5 min, purge flow rate 40 mL/min Injection volume conditions : 1 μL, sample concentration 1 mg/mL or less Detector: FID detector Temperature: 280°C Carrier gas conditions: Helium, linear velocity 24 cm/min</p><p>Thereafter, in the high-temperature precision distillation step, 32 g of the initial distillation residue obtained in the low-temperature precision distillation step was subjected to the following precision distillation. A fractionating tube with a vacuum jacket (Kiriyama Glass) was used as the fractionating tube, and two Sulzer Lab Packing EX (Sulzer Chemtech) were used as the internal packing. The diameter of the vacuum jacketed fractionating tube was 25 mm and the size of one unit of Sulzer Labpacking EX was 25 mm x 50 mm. The liquid temperature in the bottom pot, that is, the bottom temperature is 195 ° C, the top vapor temperature, that is, the top temperature is 150 ° C, the pressure before the vacuum pump, that is, the lowest pressure in the distillation column, that is, the vacuum Precision distillation was performed at a temperature of 30 Pa. A hot precision distillation step removed the C22 and above fraction as a residue, ie residue, yielding 19 g of main distillate. DGLA was further concentrated in the main fraction.</p><p>The obtained main fraction was subjected to HPLC under the following conditions to fractionate the DGLA eluted fraction. The DGLA elution fraction corresponds to concentrated microbial oil.</p><p>Regarding the obtained main distillate fraction and the DGLA elution fraction after the high-temperature precision distillation step, gas chromatography (GC) was used in the same manner as in Comparative Example 1 to determine the amount of fatty acids contained and the fatty acid composition. The ratio, that is, the content and recovery of DGLA with respect to the obtained fractions, was calculated. The results are shown in Tables 2 and 3. As shown in Table 2, the DGLA content in the main distillate fraction after the high temperature precision distillation process was 91.9% by weight, the DGLA content in the DGLA elution fraction was 96.4% by weight, and the DGLA recovery was 100.0%. DGLA was obtained with high purification efficiency.</p><p>HPLC conditions Column: YMC pack ODS-AQ-HG 20mmφ×500mm (YMC, Inc.) Pump: 1200 series G1361A Prep Pump (Agilent Technologies, Inc.) Column temperature: Around 21°C Mobile phase: Methanol 17.5mL/min sample Conditions: 2.4g load, i.e. 3wt% raw material load on adsorbent</p><p>[Example 2]</p><p>Using raw material ethyl ester 2 used in Example 1 as a sample, it was subjected to precision distillation including the following low-temperature precision distillation process and high-temperature precision distillation process.</p><p>In the low-temperature precision distillation step, 100 g of raw material ethyl ester 2 was subjected to the following precision distillation. A fractionating tube with a vacuum jacket (Kiriyama Glass) was used as the fractionating tube, and two Sulzer Lab Packing EX (Sulzer Chemtech) were used as the internal packing. The diameter of the vacuum jacketed fractionating tube was 25 mm and the size of one unit of Sulzer Labpacking EX was 25 mm x 50 mm. The liquid temperature in the bottom pot, that is, the bottom temperature is 180 ° C, the top vapor temperature, that is, the top temperature is 140 ° C, the pressure before the vacuum pump, that is, the lowest pressure in the distillation column, that is, Precision distillation was performed at a degree of vacuum of 20 Pa. In the low-temperature precision distillation process, the C18 or lower fraction was removed as the initial distillation to obtain 48 g of the initial distillation residue. When the fatty acid content and fatty acid composition of the initial distillation residue were confirmed using gas chromatography in the same manner as in Comparative Example 1, the initial distillation residue contained concentrated DGLA. In addition, Table 2 shows the content of compound A and compound B in which compound A and compound B appeared in the chromatogram of the initial distillation residue.</p><p>After that, in the high-temperature precision distillation process, 45 g of the initial distillation residue obtained in the low-temperature precision distillation process was subjected to the following precision distillation. A fractionating tube with a vacuum jacket (Kiriyama Glass) was used as the fractionating tube, and two Sulzer Lab Packing EX (Sulzer Chemtech) were used as the internal packing. The diameter of the vacuum jacketed fractionating tube was 25 mm and the size of one unit of Sulzer Labpacking EX was 25 mm x 50 mm. The liquid temperature in the bottom pot, that is, the bottom temperature is 185 ° C, the top vapor temperature, that is, the top temperature is 145 ° C, the pressure before the vacuum pump, that is, the lowest pressure in the distillation column, that is, the vacuum Precision distillation was performed at a temperature of 20 Pa. In a second precision distillation step, the C22 and above fraction was removed as a bottom, ie residue, yielding 28 g of main distillate. It is presumed that DGLA is further concentrated in the main fraction.</p><p>The obtained main fraction was subjected to HPLC under the following conditions to fractionate the DGLA eluted fraction. The DGLA elution fraction corresponds to concentrated microbial oil.</p><p>The main distillate fraction and the DGLA elution fraction obtained after the high-temperature precision distillation process were subjected to gas chromatography (GC) in the same manner as in Comparative Example 1 to determine the amount of fatty acids contained and the fatty acid composition. The ratio, that is, the content and recovery of DGLA with respect to the obtained fractions, was calculated. The results are shown in Tables 2 and 4. As shown in Table 2, the DGLA content in the main distillate fraction after the high temperature precision distillation process was 75.0% by weight, the DGLA content in the DGLA elution fraction was 95.1% by weight, and the DGLA recovery was 61.7%. DGLA was obtained with high purification efficiency.</p><p>Further, as in Example 1, compound A showing peak A and compound B showing peak B appeared in the chromatogram of the main residue (see Table 4). The compounds A and B could be considered thermogenic fatty acids formed by the distillation process. The contents of compound A and compound B are shown in Tables 2 and 4. The content in Table 4 represents the content based on the fatty acid composition.</p><p>- HPLC conditions Column: YMC pack ODS-AQ-HG 20mmφ x 1000mm (YMC Co., Ltd.) Two 500mm columns were connected in series.</p><p>Pump: 1200 series G1361A Prep Pump (Agilent Technologies, Inc.) Column temperature: Around 21°C Mobile phase: Methanol, 12 mL/min Sample conditions: Loading amount: 2.4 g, that is, the raw material loading ratio is 1.5 weight relative to the adsorbent %</p><p><tables><img file="JP7169331B2_D0002.tif" /></tables></p><p><tables><img file="JP7169331B2_D0003.tif" /></tables></p><p><tables><img file="JP7169331B2_D0004.tif" /></tables></p><p>As shown in Tables 2 to 4, microbial oil containing 50% by weight or more of DGLA by precision distillation and 0.0001% by weight or more of thermogenic fatty acids was subjected to reversed-phase column chromatography to obtain DGLA. It was found to be extremely useful in terms of efficiently obtaining high-concentration DGLA when obtaining DGLA. Such a microbial oil could be obtained by a production method including a precision distillation step under specific conditions, or by a production method including a rectification step using a distillation column with structured packing.</p><p>Thus, according to the present invention, it was possible to efficiently obtain a microbial oil containing a high content of DGLA, and to efficiently obtain a concentrated microbial oil containing a high content of DGLA.</p><p>Therefore, according to the present invention, it is possible to efficiently provide a microbial oil and a concentrated microbial oil containing a high target LC-PUFA content, and to efficiently obtain such a microbial oil and a concentrated microbial oil. Useful manufacturing methods and various uses of microbial oils and concentrated microbial oils can be provided.</p><p>The disclosure of Japanese Patent Application No. 2013-251401 filed on December 4, 2013 is incorporated herein by reference in its entirety.</p><p>All publications, patent applications and technical standards mentioned herein are to the same extent as if each individual publication, patent application and technical standard were specifically and individually noted to be incorporated by reference. incorporated herein by reference.</p>
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Numbers
- Publication
- 7169331
- Application
- 191160
Titles2
- Japanese
- 微生物油、微生物油の製造方法、濃縮微生物油及び濃縮微生物油の製造方法
- English
- Microbial oil, method for producing microbial oil, concentrated microbial oil, and method for producing concentrated microbial oil
Classification
- CPC, 35
- A61K31/202
- C12P7/6427
- A01N37/06
- A61K8/36
- A61K8/37
- A61K8/92
- A61K31/232
- A61Q19/00
- C11B1/00
- C11B1/025
- C11C3/10
- C12P7/6436
- C11B3/12
- C11C1/005
- A61K8/361
- C12N1/28
- C12P7/6463
- A23D9/013
- A23D9/02
- C11B3/10
- A23K20/158
- A23L33/115
- A61K8/9706
- A61K8/9728
- A61P17/00
- A61P17/02
- A61P17/04
- A61P17/06
- A61P29/00
- A61P37/06
- A61P37/08
- A61P39/00
- Y02E50/10
- A61K8/99
- A61K36/06
- IPC, 21
- C12P7 64
- A23L33 115
- A23D9 00
- A61K8 92
- A61K8 9728
- A61Q19 00
- A61K8 37
- A61K8 36
- A61K31 20
- A61P29 00
- A61P37 08
- A61P17 00
- A61P17 06
- A61P37 02
- A61P17 04
- C11B3 12
- C11C3 00
- A23K20 158
- A23K10 16
- C12P7 6427
- C12P7 6436
