JPH0216989A - Production of omega6-based unsaturated fatty acid-containing phospholipid - Google Patents
Production of omega6-based unsaturated fatty acid-containing phospholipidInfo
- Publication number
- JPH0216989A JPH0216989A JP16666988A JP16666988A JPH0216989A JP H0216989 A JPH0216989 A JP H0216989A JP 16666988 A JP16666988 A JP 16666988A JP 16666988 A JP16666988 A JP 16666988A JP H0216989 A JPH0216989 A JP H0216989A
- Authority
- JP
- Japan
- Prior art keywords
- unsaturated fatty
- fatty acid
- phospholipid
- conidiobolus
- omega6
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 150000003904 phospholipids Chemical class 0.000 title claims abstract description 40
- 235000021122 unsaturated fatty acids Nutrition 0.000 title claims abstract description 31
- 150000004670 unsaturated fatty acids Chemical class 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 12
- 244000005700 microbiome Species 0.000 claims abstract description 17
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 claims abstract description 16
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 15
- 241001480517 Conidiobolus Species 0.000 claims abstract description 11
- 241000235395 Mucor Species 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 5
- 229960002733 gamolenic acid Drugs 0.000 claims description 14
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 claims description 13
- VZCCETWTMQHEPK-UHFFFAOYSA-N gamma-Linolensaeure Natural products CCCCCC=CCC=CCC=CCCCCC(O)=O VZCCETWTMQHEPK-UHFFFAOYSA-N 0.000 claims description 13
- VZCCETWTMQHEPK-QNEBEIHSSA-N gamma-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCC(O)=O VZCCETWTMQHEPK-QNEBEIHSSA-N 0.000 claims description 13
- 235000020664 gamma-linolenic acid Nutrition 0.000 claims description 13
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 claims description 12
- 150000008104 phosphatidylethanolamines Chemical class 0.000 claims description 12
- 235000021342 arachidonic acid Nutrition 0.000 claims description 6
- 229940114079 arachidonic acid Drugs 0.000 claims description 6
- HOBAELRKJCKHQD-QNEBEIHSSA-N dihomo-γ-linolenic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/CCCCCCC(O)=O HOBAELRKJCKHQD-QNEBEIHSSA-N 0.000 claims description 5
- 239000003921 oil Substances 0.000 abstract description 5
- 238000004440 column chromatography Methods 0.000 abstract description 4
- 239000003925 fat Substances 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 241000142142 Conidiobolus heterosporus Species 0.000 abstract description 3
- 241000306281 Mucor ambiguus Species 0.000 abstract description 3
- 238000005119 centrifugation Methods 0.000 abstract description 3
- 230000004071 biological effect Effects 0.000 abstract 1
- 238000005194 fractionation Methods 0.000 abstract 1
- 238000000746 purification Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 5
- 235000014113 dietary fatty acids Nutrition 0.000 description 5
- 239000000194 fatty acid Substances 0.000 description 5
- 229930195729 fatty acid Natural products 0.000 description 5
- 150000004665 fatty acids Chemical class 0.000 description 5
- 239000008103 glucose Substances 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 235000019198 oils Nutrition 0.000 description 4
- 238000004262 preparative liquid chromatography Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 235000019197 fats Nutrition 0.000 description 3
- 238000010898 silica gel chromatography Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 241000235575 Mortierella Species 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 1
- RYCNUMLMNKHWPZ-SNVBAGLBSA-N 1-acetyl-sn-glycero-3-phosphocholine Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C RYCNUMLMNKHWPZ-SNVBAGLBSA-N 0.000 description 1
- 241001674864 Conidiobolus nanodes Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 235000019485 Safflower oil Nutrition 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- CWRILEGKIAOYKP-SSDOTTSWSA-M [(2r)-3-acetyloxy-2-hydroxypropyl] 2-aminoethyl phosphate Chemical compound CC(=O)OC[C@@H](O)COP([O-])(=O)OCCN CWRILEGKIAOYKP-SSDOTTSWSA-M 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 150000001879 copper Chemical class 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000008344 egg yolk phospholipid Substances 0.000 description 1
- 229940068998 egg yolk phospholipid Drugs 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 235000008524 evening primrose extract Nutrition 0.000 description 1
- 229940089020 evening primrose oil Drugs 0.000 description 1
- 239000010475 evening primrose oil Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 159000000014 iron salts Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 150000004702 methyl esters Chemical class 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229940094443 oxytocics prostaglandins Drugs 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003905 phosphatidylinositols Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000003495 polar organic solvent Substances 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000003813 safflower oil Substances 0.000 description 1
- 235000005713 safflower oil Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008347 soybean phospholipid Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
[産業上の利用分野コ
本発明はω6系不飽和脂肪酸含有リン脂質の製造法に関
し、詳しくは特定の微生物を用いてω6系不飽和脂肪酸
を高含量で含むω6系不飽和脂肪酸含有リン脂質を製造
する方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for producing phospholipids containing ω6 unsaturated fatty acids. The present invention relates to a method for producing phospholipids containing unsaturated fatty acids.
[従来の技術及び発明が解決しようとする課題]リン脂
質は高等動物をはじめ各種生物中に含まれ、主として細
胞膜の構成物として生物中で重要な働きをしている。ま
た、各種生物体内で何らかの刺激により、リン脂質に結
合している不飽和脂肪酸が遊離してプロスタグランジン
等の生理活性物質へと変換される。[Prior Art and Problems to be Solved by the Invention] Phospholipids are contained in various living organisms including higher animals, and play an important role in living organisms mainly as constituents of cell membranes. Moreover, unsaturated fatty acids bound to phospholipids are liberated and converted into physiologically active substances such as prostaglandins due to some stimulus within various living organisms.
現在、工業的規模で生産されているリン脂質は大豆リン
脂質と卵黄リン脂質である。これらは天然界面活性剤で
あり、乳化作用1分散作用、湿潤作用などを利用して食
品、医薬品などの分野で使用されている。しかしながら
、これらのリン脂質は生理活性が十分でないという欠点
があった。Currently, the phospholipids produced on an industrial scale are soybean phospholipids and egg yolk phospholipids. These are natural surfactants and are used in the fields of foods, medicines, etc. due to their emulsifying, dispersing, and wetting effects. However, these phospholipids have the disadvantage that they do not have sufficient physiological activity.
ところで、生理活性作用を持った脂肪酸としてγ−リノ
レン酸、ビス−ホモ−γ−リノレン酸。By the way, γ-linolenic acid and bis-homo-γ-linolenic acid are fatty acids that have physiologically active effects.
アラキドン酸などの不飽和脂肪酸が知られている。この
ような不飽和脂肪酸を含有するリン脂質の製造方法とし
ては、モルティエレラ属に属する微生物を炭水化物を炭
素源とする培地で培養して得た菌体により採取された脂
質からγ−リノレン酸含有ホスファチジルコリンを分m
濃縮する方法(特開昭62−254189号公報)など
が知られている。Unsaturated fatty acids such as arachidonic acid are known. As a method for producing phospholipids containing such unsaturated fatty acids, phospholipids containing γ-linolenic acid are extracted from lipids obtained by culturing microorganisms belonging to the genus Mortierella in a medium using carbohydrates as a carbon source. Minutes of phosphatidylcholine
A method of concentrating (Japanese Unexamined Patent Publication No. 62-254189) is known.
しかし、この方法ではγ−リノレン酸含量が少ないもの
しか得られず、十分な利用が期待できない。However, with this method, only a product with a low content of γ-linolenic acid can be obtained, and sufficient utilization cannot be expected.
[課題を解決するための手段]
そこで、本発明者はω6系の不飽和脂肪酸を高含量で含
むリン脂質を生産する能力を有する微生物について検索
したところ、ムコール属またはコニディオボラス属に属
する微生物が該能力を有していることを見出し、零発咀
を完成した。[Means for Solving the Problems] Therefore, the present inventor searched for microorganisms that have the ability to produce phospholipids containing a high content of ω6 unsaturated fatty acids, and found that microorganisms belonging to the genus Mucor or Conidiobolus were found. He discovered that he possessed this ability and completed Zero Hatsutsui.
すなわち、本発明はムコール腐またはコニディオボラス
属に属し、ω6系不飽和胞肪酸含有胞質生産能を有する
微生物を、炭素源を含む培地に培養し、培養物からω6
系不飽和脂肪酸含有リン脂質を採取することを特徴とす
るω6系不飽和脂肪酸含有リン脂質の製造法を提併する
ものである。That is, the present invention involves culturing a microorganism that belongs to the genus Mucor rot or Conidiobolus and has the ability to produce spores containing ω6-based unsaturated fatty acids in a medium containing a carbon source, and extracts ω6 from the culture.
The present invention provides a method for producing phospholipids containing ω6-based unsaturated fatty acids, which is characterized by collecting phospholipids containing ω-6-based unsaturated fatty acids.
本発明で用いる微生物は、ムコール属またはコニディオ
ボラス属に属し、ω6系不飽和脂肪酸生産能を有する微
生物である。具体的には、ムコール属微生物としてはム
コール・シルシネロイデス1162(FERM P−9
360)などが挙げられ、フニディオボラス属微生物と
してはコニディオボラス・ヘテロスポラス(Coni+
Hobolus heterosporus) ATC
C12941、コニディオボラス・グロプリフェラス(
Conidiobolus globuliferou
s) CB3218764.コニディオボラス・ナノ
デス(Conidiobolus nanodes)C
B5183/62などが挙げられる。The microorganism used in the present invention belongs to the genus Mucor or Conidiobolus and has the ability to produce ω6 unsaturated fatty acids. Specifically, as a microorganism of the genus Mucor, Mucor circinelloides 1162 (FERM P-9
360), and Conidiobolus heterosporus (Coni+
Hobolus heterosporus) ATC
C12941, Conidiobolus glopuriferus (
Conidiobolus globuliferou
s) CB3218764. Conidiobolus nanodes C
Examples include B5183/62.
本発明には上記微生物のほか、これらから誘導される変
異株であって上記のようにω6系不飽和脂肪酸含有脂質
を生産する能力を有するもの等も等しく使用することが
できる。In addition to the above-mentioned microorganisms, mutant strains derived from these microorganisms that have the ability to produce lipids containing ω6 unsaturated fatty acids as described above can also be used in the present invention.
上記微生物を培養するための培地は、該微生物が良く生
育して目的とするリン脂質を生産しうるものであればよ
く、炭素源、窒素源、無機塩類および必要により微生物
の生育に好適なアミノ酸等の成分を含むものが用いられ
る。炭素源としてはグルコース、でんぷん、廃糖蜜等の
糖類や酢酸ソーダなどが使用でき、特にグルコース等の
糖類が好適である。さらに、ムコール属の場合にはリノ
ール酸高含有油脂であるサフラワー油、ヒマワリ油など
を、コニディオボラス属の場合にはγ−リノレン酸含有
油脂である月見草油、ムコール属やモルティエレラ属に
属する糸状菌から抽出された微生物油脂、特に炭素数1
8以上の不飽和脂肪酸含有油脂等を必要に応じて加える
と、目的とするリン脂質の生産量を増大させることがで
きる。The medium for culturing the above-mentioned microorganisms may be one that allows the microorganisms to grow well and produce the desired phospholipid, and contains a carbon source, a nitrogen source, inorganic salts, and, if necessary, amino acids suitable for the growth of the microorganisms. Those containing the following ingredients are used. As the carbon source, sugars such as glucose, starch, blackstrap molasses, and sodium acetate can be used, and sugars such as glucose are particularly preferred. Furthermore, in the case of the Mucor genus, safflower oil, sunflower oil, etc., which are high-linoleic acid-containing oils, are used, and in the case of the Conidiobolus genus, evening primrose oil, which is a γ-linolenic acid-containing fat, and Mucor and Mortierella genus are used. Microbial fats and oils extracted from filamentous fungi, especially those with a carbon number of 1
By adding fats and oils containing unsaturated fatty acids of 8 or more as necessary, the production amount of the desired phospholipid can be increased.
また、窒素源としてはアンモニウム塩、酵母エキス、コ
ーン・ステイープ・リカー、ペプトンなどがあり、無機
塩類としてはマグネシウム塩、カルシウム塩、リン酸塩
、鉄塩、銅塩などがある。Further, nitrogen sources include ammonium salts, yeast extract, corn steep liquor, peptone, etc., and inorganic salts include magnesium salts, calcium salts, phosphates, iron salts, copper salts, etc.
培養にあたり、炭素源は培地に最初から全量を加えても
よいが、培養開始後適当な時期に追加することによって
γ−リノレン酸等のω6系不飽和脂肪酸含有リン脂質の
生産量を増大させることができる。グルコース等の炭素
源を培地に最初から加える場合、初発添加量が多過ぎる
と、微生物の生育に悪影響を及ぼすことがあるので、通
常は10〜250 ghとする。また、炭素源を培養中
に追加する場合、その時期1回数などは適宜決定すれば
よい。たとえば、グルコース等の炭素源の初発濃度を1
00〜zoog#として培養を行い、炭素源の大部分が
消費されたときに50−IQOg/l程度の炭素源を追
加(1回もしくは数回に分けて)することにより菌体の
増殖を高め、結果的にω6系不飽和脂肪酸高含量のリン
脂質の生産量を増すことができる。During culture, the entire amount of carbon source may be added to the medium from the beginning, but it is possible to increase the production amount of phospholipids containing ω6 unsaturated fatty acids such as γ-linolenic acid by adding it at an appropriate time after the start of culture. I can do it. When adding a carbon source such as glucose to the medium from the beginning, if the initial addition amount is too large, it may have an adverse effect on the growth of microorganisms, so the amount is usually set at 10 to 250 gh. Furthermore, when adding a carbon source during culture, the timing and number of times may be determined as appropriate. For example, if the initial concentration of a carbon source such as glucose is
00~zoog#, and when most of the carbon source is consumed, increase the growth of bacterial cells by adding carbon source of about 50-IQOg/l (once or in several times). As a result, the production amount of phospholipids with a high content of ω6 unsaturated fatty acids can be increased.
その他の培養条件、たとえば温度9時間等は使用する微
生物の性質等を考えて目的とするリン脂質の生産量が高
くなるような条件を設定すればよい。通常は20〜32
℃、好ましくは25〜30t、pH3〜7、好ましくは
3.5〜6ニテ60〜120時間、好ましくは70〜1
00時間行えばよい。Other culture conditions, such as temperature for 9 hours, etc., may be set so as to increase the production of the desired phospholipid, taking into consideration the properties of the microorganisms used. Usually 20-32
°C, preferably 25-30t, pH 3-7, preferably 3.5-6 nits, 60-120 hours, preferably 70-1
Just do it for 00 hours.
ω6系不飽和脂肪酸を含有するリン脂質は通常、微生物
菌体中、特に細胞膜に蓄積されるので、遠心分離法等の
常法により培養液を固・液分離し、該脂質を含む菌体を
得る。このようにして得られたリン脂質中のω6系不飽
和脂肪酸含量は38〜56%である。さらに、この菌体
な必要に応じ洗浄、乾燥することもできる。Phospholipids containing ω6 unsaturated fatty acids are usually accumulated in microbial cells, especially in cell membranes, so the culture solution is separated into solid and liquid by a conventional method such as centrifugation, and the microbial cells containing the lipids are separated. obtain. The content of ω6 unsaturated fatty acids in the phospholipids thus obtained is 38 to 56%. Furthermore, the bacterial cells can be washed and dried as necessary.
また、必要により菌体をメタノール、エタノール、ジク
ロロメタン、クロロホルムなどの極性および非極性の有
機溶剤の任意の割合の混合物を用いて脂質抽出を行ない
、分離・精製することもできる。菌体を予めボールミル
で粉砕したのち脂質を抽出したり、有機溶媒中で粉砕し
てリン脂質を抽出することができる。抽出物を脱溶剤し
た後、アセトン分画を行い、アセトンに不溶な物質を集
めれば、リン脂質の粗分−物が得られる。さらに、得ら
れたすべての両分をシリカゲルカラムクロマトグラフィ
ーで再分画すると、ω6系不飽和脂肪酸高含量のリン脂
質組成物が得られる。ただし、ここに示した分別法は1
例であり、本発明のリン脂質組成物の分画法がこれに限
定されるものではない。このようにして得られたリン脂
質中のω6系不飽和脂肪酸含量は80〜90%という高
い値である。Furthermore, if necessary, the bacterial cells can be separated and purified by performing lipid extraction using a mixture of polar and non-polar organic solvents such as methanol, ethanol, dichloromethane, and chloroform in arbitrary proportions. The bacterial cells can be ground in advance in a ball mill and then the lipids extracted, or the cells can be ground in an organic solvent to extract the phospholipids. After removing the solvent from the extract, it is fractionated with acetone and acetone-insoluble substances are collected to obtain a crude phospholipid. Furthermore, all the obtained fractions are re-fractionated by silica gel column chromatography to obtain a phospholipid composition with a high content of ω6 unsaturated fatty acids. However, the separation method shown here is 1
This is an example, and the method of fractionating the phospholipid composition of the present invention is not limited to this. The content of ω6 unsaturated fatty acids in the phospholipids thus obtained is as high as 80 to 90%.
上述の方法により得られるリン脂質組成物は、ホスファ
チジルコリン、ホスファチジルエタノールアミン、リゾ
ホスファチジルコリン、リゾホスファチジルエタノール
アミン、ホスファチジルイノシトール、ホスファチジン
酸などであり、またその構成脂肪酸はγ−リノレン酸、
ビスーポモγ−リノレン酸、アラキドン酸など炭素数1
8個以上で二重結合が3個以上のω6系高度不飽和脂肪
酸である。The phospholipid composition obtained by the above method contains phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, etc., and its constituent fatty acids include γ-linolenic acid,
Bispomo-γ-linolenic acid, arachidonic acid, etc. with 1 carbon number
It is an omega-6 highly unsaturated fatty acid with 8 or more double bonds and 3 or more double bonds.
また、ホスファチジルコリン、ホスファチジルエタノー
ルアミン等はカラムクロマトグラフィー等の常法(特に
、液体クロマトグラフィーが好ましい。)により、ざら
にω6系不飽和脂肪酸を高含量のものとすることができ
る。例えば、ホスファチジルコリンを分取用液体カラム
クロマトグラフィーにより、各ピークごとにフラクショ
ンコレクターに分画し、得られた画分な各々、常法によ
りケン化分解、メチルエステル化した後、ガスクロマト
グラフィーで脂肪酸組成を測定し、γ−リノレン酸濃度
の高いものを目的物とする。ホスファチジルエタノール
アミンについても、同様にしてγ−リノレン酸濃度の高
いものを得ることができる。また、アラキドン酸、ビス
−ホモ−γ−リノレン酸濃度の高いホスファチジルコリ
ン、ホスファチジルエタノールアミンについても、同様
にして得ることができる。Further, phosphatidylcholine, phosphatidylethanolamine, etc. can be made to have a roughly high content of ω6 unsaturated fatty acids by a conventional method such as column chromatography (liquid chromatography is particularly preferred). For example, phosphatidylcholine is fractionated into a fraction collector for each peak by preparative liquid column chromatography, and each of the obtained fractions is saponified and decomposed using conventional methods and converted into methyl ester. The composition is measured, and the one with a high concentration of γ-linolenic acid is selected as the target product. Similarly, phosphatidylethanolamine with a high concentration of γ-linolenic acid can be obtained. Furthermore, phosphatidylcholine and phosphatidylethanolamine with high concentrations of arachidonic acid and bis-homo-γ-linolenic acid can also be obtained in the same manner.
[実施例] 次に、本発明を実施例により説明する。[Example] Next, the present invention will be explained by examples.
実施例1
第1表に示したムコール用培地(p)14)84!を1
0ρ容ジャーファーメンタ−に久れ、この培地にムコー
ル・シルシネロイデスHUT 1121(FEBMP−
9359)を接種し、30℃で3日間通気攪拌培養を行
なった。Example 1 Mucor culture medium shown in Table 1 (p) 14) 84! 1
I have been using a 0μ volume jar fermenter for a long time, and I have added Mucor circinelloides HUT 1121 (FEBMP-
9359) and cultured with aeration and agitation at 30°C for 3 days.
男
1表
ムコール
用培地
コニディオボラス
用培地
グルコース 150 g# 30 g
/l硫酸アンモニウム 19.8 g/ρ
酵母エキス 0.6 g711 5 g
#!MgSO41,Og# 1 ghFeSO
440mg71 0.01g/j!ペプトン
10g/オKH2PO49,Og71
3 g/l培養終了後、培養液をt濾過して菌
体を回収し、湿菌体を得た。この湿菌体7kgをメタノ
ール20Il。Male 1 table Mucor medium Conidiobolus medium Glucose 150 g # 30 g
/l Ammonium sulfate 19.8 g/ρ Yeast extract 0.6 g711 5 g
#! MgSO41, Og#1ghFeSO
440mg71 0.01g/j! peptone
10g/O KH2PO49, Og71
After the completion of the 3 g/l culture, the culture solution was T-filtered to collect the bacterial cells to obtain wet bacterial cells. 7 kg of this wet bacterial body was mixed with 20 Il of methanol.
と共に破砕抽出した。得られた抽出液をエバポレーター
で約50011111まで濃縮し、ジクロロメタン30
0IIIJを加えて二層分離した。次いで、ジクロロメ
タン層を回収し、濃縮して約501の濃縮液を得た。こ
れにアセトンを400mA加え、沈澱物をtp遇した。It was crushed and extracted together. The obtained extract was concentrated to about 50011111 in an evaporator, and dichloromethane 30
0IIIJ was added to separate the two layers. The dichloromethane layer was then collected and concentrated to obtain a concentrate of approximately 501. Acetone was added at 400 mA to this, and the precipitate was treated with tp.
沈澱物は2回アセトンで洗浄し、約20gの白色結晶を
得た。このものの脂肪酸組成を第3表に示す。このうち
2gをクロロホルムに溶解し、//
シリカゲルカラムクロマトグラフィー(カラム:フコ−
ゲル。和光純薬製)に注入した。展開溶媒はクロロホル
ム/メタノール=())4:1(I+) 3 : 2.
(IIT) 1 : 4と分けて行なった。The precipitate was washed twice with acetone to obtain about 20 g of white crystals. The fatty acid composition of this product is shown in Table 3. Dissolve 2g of this in chloroform and perform // silica gel column chromatography (column: Fuco-
gel. (manufactured by Wako Pure Chemical Industries, Ltd.). The developing solvent was chloroform/methanol = () 4:1 (I+) 3:2.
(IIT) 1:4.
この結果、(I)からはホスファチジルエタノールアミ
ンが(n )からはホスファチジルコリンが、(II+
)からはその他のリン脂質が分画された。As a result, phosphatidylethanolamine is obtained from (I), phosphatidylcholine is obtained from (n), and phosphatidylcholine is obtained from (II+
), other phospholipids were fractionated.
続いて、ホスファチジルコリン1gを分取用液体クロマ
トグラフィー(カラム、 oDs 、 m媒;メタノー
ル・水ニアセトニトリルー90.5ニア・2.5流速;
10m1’/m1n)にてUV205mmで検出でき
るビ一りごとのフラクションを分取した。薄層クロマト
グラフィーでホスファチジルコリンであることを確認し
た後、ガスクロマトグラフィーにて脂肪酸組成を調べた
ところ、分取したものの中からγ−リノレン酸を95%
含有したホスファチジルコリン400mgを得た。また
、ホスファチジルエタノールアミンを分取用液体クロマ
トグラフィーで分取したところ、約2oomgのγ−リ
ノレン酸を90%以上含んだホスファデジルエタノール
アミンが得られた。この結果を第2表に示す。Subsequently, 1 g of phosphatidylcholine was subjected to preparative liquid chromatography (column, oDs, m solvent; methanol/water niacetonitrile 90.5 nia/2.5 flow rate;
10 m1'/m1n), and a fraction of each Bi whole that could be detected with UV 205 mm was collected. After confirming that it was phosphatidylcholine using thin layer chromatography, we investigated the fatty acid composition using gas chromatography and found that 95% of the fractionated γ-linolenic acid was phosphatidylcholine.
400 mg of the contained phosphatidylcholine was obtained. Further, when phosphatidylethanolamine was separated by preparative liquid chromatography, about 2 oomg of phosphadecylethanolamine containing 90% or more of γ-linolenic acid was obtained. The results are shown in Table 2.
第 2 表
実施例2
第1表に示したコニディオボラス用培地にγリルン酸含
有油(モルティエレラ属菌体からの抽出油脂ニオレイン
酸43%、リノール酸11%1γ−リノレン酸8%)を
培地に対して3 voj!% となるように加えた培地
を作製した。この培地にコニディオボラス・ヘテロスポ
ラス八TCC12941を接種し、30℃で2日間振ど
う培養した。Table 2 Example 2 γ-linolenic acid-containing oil (43% nioleic acid, 11% linoleic acid, 8% γ-linolenic acid) was added to the Conidiobolus culture medium shown in Table 1. 3 voj for the medium! A medium was prepared in which the amount of 1% was added. This medium was inoculated with Conidiobolus heterosporus 8TCC12941, and cultured with shaking at 30°C for 2 days.
培養終了後、遠心分離により菌体を集菌し、湿菌体を得
た。この湿菌体から、実施例1と同様の方法でリン脂質
を抽出したところ、20gの粗リン脂質が得られた。こ
のものの脂肪酸組成を第3表に示す。After the culture was completed, the bacterial cells were collected by centrifugation to obtain wet bacterial cells. When phospholipids were extracted from the wet bacterial cells in the same manner as in Example 1, 20 g of crude phospholipids were obtained. The fatty acid composition of this product is shown in Table 3.
第3表
ルコリン203mgが得られた。また、ホスファチジル
エタノールアミン1gを分取用液体クロマトグラフィー
で分取したところ、ビス−ホモ−γ−リノレン酸を50
%以上含有したホスファチジルエタノールアミン12m
gとアラキドン酸を80%以上含有したホスファチジル
エタノールアミン178mgが得られた。この結果を第
4表に示す。Table 3 203 mg of lucorin was obtained. Furthermore, when 1 g of phosphatidylethanolamine was separated using preparative liquid chromatography, 50% of bis-homo-γ-linolenic acid was collected.
Phosphatidylethanolamine containing 12m or more
178 mg of phosphatidylethanolamine containing 80% or more of g and arachidonic acid was obtained. The results are shown in Table 4.
第4表
得られた粗リン脂質を、実施例1と同様の方法でシリカ
ゲルカラムクロマトグラフィーにて分画したところ、約
5gのホスファチジルコリンと約2gのホスファチジル
エタノールアミンが得られた。Table 4 When the obtained crude phospholipid was fractionated by silica gel column chromatography in the same manner as in Example 1, about 5 g of phosphatidylcholine and about 2 g of phosphatidylethanolamine were obtained.
得られたホスファチジルコリン1gを、実施例1と同様
の方法で分取用液体クロマトグラフィーにて分取したと
ころ、ビス−ホモ−γ−リノレン酸を50%以上含有し
たホスファチジルコリン24mgとアラキドン酸を80
%以上含有したホスファチジ[発明の効果]
本発明によれば、ω6系不飽和脂肪酸を高含量で含むリ
ン脂質を得ることができる上に、さらにカラムクロマト
グラフィー等で分画 精製することにより、ω6系不飽
和脂肪酸が高含量のボスファヂシルコリン、ホスファチ
ジルエタノールアミン等のリン脂質を容易に得ることが
できる。When 1 g of the obtained phosphatidylcholine was separated by preparative liquid chromatography in the same manner as in Example 1, 24 mg of phosphatidylcholine containing 50% or more of bis-homo-γ-linolenic acid and 80% of arachidonic acid were separated.
[Effects of the Invention] According to the present invention, it is possible to obtain phospholipids containing a high content of ω6-based unsaturated fatty acids, and by further fractionating and purifying them by column chromatography etc. Phospholipids such as bosphadicylcholine and phosphatidylethanolamine, which have a high content of unsaturated fatty acids, can be easily obtained.
得られたリン脂質は生理活性が高く、医薬品。The obtained phospholipids have high physiological activity and are used as pharmaceuticals.
リポソームの原料1食品、化粧品等の分野において利用
が期待される。Raw material for liposomes 1 It is expected to be used in the fields of food, cosmetics, etc.
Claims (2)
6系不飽和胞肪酸含有胞質生産能を有する微生物を、炭
素源を含む培地に培養し、培養物からω6系不飽和脂肪
酸含有リン脂質を採取することを特徴とするω6系不飽
和脂肪酸含有リン脂質の製造法。(1) Belongs to the genus Mucor or Conidiobolus, ω
An ω6 unsaturated fatty acid characterized by culturing a microorganism capable of producing spores containing a 6 unsaturated fatty acid in a medium containing a carbon source, and collecting phospholipids containing an ω6 unsaturated fatty acid from the culture. Method for producing phospholipid content.
モ−γ−リノレン酸およびアラキドン酸のいずれかであ
り、リン脂質がホスファチジルコリンまたはホスファチ
ジルエタノールアミンである請求項1記載の製造法。(2) The production method according to claim 1, wherein the ω6 unsaturated fatty acid is any one of γ-linolenic acid, bis-homo-γ-linolenic acid, and arachidonic acid, and the phospholipid is phosphatidylcholine or phosphatidylethanolamine.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP16666988A JPH062068B2 (en) | 1988-07-06 | 1988-07-06 | Method for producing ω6 unsaturated phospholipids containing unsaturated fatty acid |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP16666988A JPH062068B2 (en) | 1988-07-06 | 1988-07-06 | Method for producing ω6 unsaturated phospholipids containing unsaturated fatty acid |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH0216989A true JPH0216989A (en) | 1990-01-19 |
JPH062068B2 JPH062068B2 (en) | 1994-01-12 |
Family
ID=15835531
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP16666988A Expired - Lifetime JPH062068B2 (en) | 1988-07-06 | 1988-07-06 | Method for producing ω6 unsaturated phospholipids containing unsaturated fatty acid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH062068B2 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPWO2005083101A1 (en) * | 2004-03-01 | 2007-08-02 | サントリー株式会社 | Method for producing phospholipid containing long-chain polyunsaturated fatty acid as a constituent element and use thereof |
EP2251412A3 (en) * | 1996-03-28 | 2011-09-28 | DSM IP Assets B.V. | Preparation of microbial polyunsaturated fatty acid containing oil from pasteurised biomass |
US8217151B2 (en) | 2002-06-19 | 2012-07-10 | Dsm Ip Assets B.V. | Pasteurisation process for microbial cells and microbial oil |
CN116622594A (en) * | 2023-07-21 | 2023-08-22 | 深圳市东荣生物科技有限责任公司 | Compound microbial preparation for promoting digestion and preparation method thereof |
-
1988
- 1988-07-06 JP JP16666988A patent/JPH062068B2/en not_active Expired - Lifetime
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2251412A3 (en) * | 1996-03-28 | 2011-09-28 | DSM IP Assets B.V. | Preparation of microbial polyunsaturated fatty acid containing oil from pasteurised biomass |
EP2280062A3 (en) * | 1996-03-28 | 2011-09-28 | DSM IP Assets B.V. | Preparation of microbial polyunsaturated fatty acid containing oil from pasteurised biomass |
EP2251410A3 (en) * | 1996-03-28 | 2011-09-28 | DSM IP Assets B.V. | Preparation of microbial polyunsaturated fatty acid containing oil from pasteurised biomass |
EP2251411A3 (en) * | 1996-03-28 | 2011-10-12 | DSM IP Assets B.V. | Preparation of microbial polyunsaturated fatty acid containing oil from pasteurised biomass |
US8217151B2 (en) | 2002-06-19 | 2012-07-10 | Dsm Ip Assets B.V. | Pasteurisation process for microbial cells and microbial oil |
US9457108B2 (en) | 2002-06-19 | 2016-10-04 | Dsm Ip Assets B.V. | Pasteurisation process for microbial cells and microbial oil |
US10493174B2 (en) | 2002-06-19 | 2019-12-03 | Dsm Ip Assets B.V. | Pasteurisation process for microbial cells and microbial oil |
JPWO2005083101A1 (en) * | 2004-03-01 | 2007-08-02 | サントリー株式会社 | Method for producing phospholipid containing long-chain polyunsaturated fatty acid as a constituent element and use thereof |
JP4850060B2 (en) * | 2004-03-01 | 2012-01-11 | サントリーホールディングス株式会社 | Method for producing phospholipid containing long-chain polyunsaturated fatty acid as a constituent element and use thereof |
CN116622594A (en) * | 2023-07-21 | 2023-08-22 | 深圳市东荣生物科技有限责任公司 | Compound microbial preparation for promoting digestion and preparation method thereof |
CN116622594B (en) * | 2023-07-21 | 2023-10-17 | 深圳市东荣生物科技有限责任公司 | Compound microbial preparation for promoting digestion and preparation method thereof |
Also Published As
Publication number | Publication date |
---|---|
JPH062068B2 (en) | 1994-01-12 |
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