JP4344827B2 - Method for producing phospholipids containing long-chain highly unsaturated fatty acids using microorganisms of the genus Schizochytrium - Google Patents
Method for producing phospholipids containing long-chain highly unsaturated fatty acids using microorganisms of the genus Schizochytrium Download PDFInfo
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- JP4344827B2 JP4344827B2 JP2005342182A JP2005342182A JP4344827B2 JP 4344827 B2 JP4344827 B2 JP 4344827B2 JP 2005342182 A JP2005342182 A JP 2005342182A JP 2005342182 A JP2005342182 A JP 2005342182A JP 4344827 B2 JP4344827 B2 JP 4344827B2
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/74—Recovery of fats, fatty oils, fatty acids or other fatty substances, e.g. lanolin or waxes
Description
本発明は、PC-DHAである新規化合物に関する。本発明はまた、スキゾキトリウム属の微生物を用いた該化合物及び長鎖高度不飽和脂肪酸含有リン脂質、特にPC-DHAの製造方法に関する。本発明は、食品、医薬の分野において特に有用である。 The present invention relates to a novel compound which is PC-DHA. The present invention also relates to a method for producing the compound and a long-chain polyunsaturated fatty acid-containing phospholipid, particularly PC-DHA, using a microorganism belonging to the genus Schizochytrium. The present invention is particularly useful in the fields of food and medicine.
ドコサヘキサエン酸（DHA）は、22の炭素鎖、6個の二重結合から成るn-3系高度不飽和脂肪酸である。DHAは特にイワシやサバなどの青魚に多く含まれる脂肪酸で、海産魚においてはその生育に重要な必須脂肪酸であることが知られている。DHAが多様な生理活性を有することも報告され、多くの健康食品や乳児用ミルクに添加されている。また近年では、DHAのみならずDHA含有リン脂質の機能にも注目が集まっている。 Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid consisting of 22 carbon chains and 6 double bonds. DHA is a fatty acid that is abundant in blue fish such as sardines and mackerel, and is known to be an essential fatty acid that is important for the growth of marine fish. DHA has also been reported to have a variety of physiological activities and has been added to many health foods and infant milk. In recent years, attention has been focused not only on DHA but also on the function of DHA-containing phospholipids.
このようなDHA及びDHA含有リン脂質の機能に注目が集まり需要が高まる一方で、現在の主な供給源であるイワシやサバ、マグロは資源の減少の問題を抱えている。また、海洋汚染により、例えばダイオキシン、PCB（ポリ塩化ビフェニール）、DDT、BHC等の化学物質や、有機スズ化合物等の重金属化合物等の海洋汚染物質は食物連鎖の過程で生物濃縮を起こし、魚体内に高濃度に蓄積される。そこで、魚に代わる新たなDHA及びDHA含有リン脂質の供給源が求められている。 While attention has been focused on the functions of DHA and DHA-containing phospholipids and demand has increased, sardines, mackerel, and tuna, which are the current main sources, have a problem of resource reduction. Also, due to marine pollution, for example, chemical substances such as dioxin, PCB (polychlorinated biphenyl), DDT, and BHC, and marine pollutants such as heavy metal compounds such as organotin compounds cause bioconcentration in the process of the food chain. Accumulated in high concentration. Thus, there is a need for a new source of DHA and DHA-containing phospholipids to replace fish.
特許文献1は、PC、LPC、PF、LPE、PE等をリン脂質とし、DHA、EPAを主要な構成脂肪酸とする、イカ皮から得た脂肪酸含有グリセロリン脂質が、脳卒中易発症自然発症高血圧ラットの死亡を予防することについて記載する。 Patent Document 1 discloses that fatty acid-containing glycerophospholipids obtained from squid skin, which use PC, LPC, PF, LPE, PE, etc. as phospholipids and DHA, EPA as the main constituent fatty acids, are a stroke-prone spontaneously hypertensive rat. Describes prevention of death.
近年、海洋性単細胞真核生物のラビリンチュラ (Labyrinthula) が高度にDHAを産生することが報告され（非特許文献1）、新たなDHA供給源として注目されている。豊富にDHAを蓄積するラビリンチュラ類をワムシやアルテミアの餌料の栄養強化に用いる試みについても報告されている（非特許文献2）。しかし、DHAがどのようなリン脂質にどのように組み込まれているかについては明らかにされていなかった。リン脂質の形のDHA(例えばPC-DHA)を用いることで、DHA単独時と比較して付加価値があることが期待できる。 In recent years, the marine unicellular eukaryote Labyrinthula has been reported to produce DHA highly (Non-Patent Document 1) and has attracted attention as a new source of DHA. There has also been reported an attempt to use labyrinthula, which accumulates DHA abundantly, for nutritional enhancement of rotifer and artemia feed (Non-patent Document 2). However, it has not been clarified how DHA is incorporated into which phospholipid. By using DHA in the form of phospholipid (for example, PC-DHA), it can be expected to have added value compared to DHA alone.
特許文献2は、ラビリンチュラ目（Labyrinthulales）に分類されるシゾキトリウム属SR21株の微生物をポリペプトン及び／又はコーンスチープリカーを含む培地で培養し、(n-6)系DPA及び(n-3)系DHAの含有量が多い油脂（全脂肪酸中のC15:0は最大で10.1%）を得る方法について記載する。該油脂中の脂肪酸含有PCの脂質について、主に16:0-22:6、16:0-22:5、22:5-22:6、22:6-22:6であることが記載されている。 Patent Document 2 cultivates a microorganism of Schizochytrium SR21 strain classified as Labyrinthulales in a medium containing polypeptone and / or corn steep liquor, and (n-6) system DPA and (n-3) system A method for obtaining fats and oils with high DHA content (C15: 0 in all fatty acids is 10.1% at maximum) will be described. The lipid of fatty acid-containing PC in the fat is mainly described as 16: 0-22: 6, 16: 0-22: 5, 22: 5-22: 6, 22: 6-22: 6. ing.
特許文献3は、培養方法等について特に記載のないスキゾキトリウム種（ラビリンチュラ目）の微生物から抽出したリン脂質の各脂肪酸含量をGCにより決定した結果、C16:0、C22:5n6、C22:6n3が主に含まれていたことを記載する。 Patent Document 3 is a result of determining each fatty acid content of phospholipids extracted from microorganisms of Schizochytrium species (Labyrinthula) that are not particularly described with respect to the culture method, etc., as a result of C16: 0, C22: 5n6, C22: Indicate that 6n3 was mainly included.
特許文献4は、シゾキトリウム属KH105株(FERM P-18431)の微生物、及び該微生物を培地中で培養し、培養物からカロテノイド系物質及び／又はDHAを多量に含む高度不飽和脂肪酸を得るのための培養条件の検討について記載する。 Patent Document 4 discloses a microorganism of Schizochytrium genus KH105 (FERM P-18431) and culturing the microorganism in a medium to obtain a highly unsaturated fatty acid containing a large amount of carotenoid substances and / or DHA from the culture. The examination of the culture conditions is described.
本発明者らは、高度不飽和脂肪酸含有リン脂質生産能、特にDHA含有PC生産能を有し、かつ培養の容易な海洋性微生物を見出し、更には、該海洋性微生物が産生するDHA含有PCについてDHAがどのように組み込まれているか明らかにし、新規化合物を見出して該化合物を特定し、本発明を完成させた。 The present inventors have found a marine microorganism having a highly unsaturated fatty acid-containing phospholipid-producing ability, particularly a DHA-containing PC-producing ability, and can be easily cultured, and further, a DHA-containing PC produced by the marine microorganism. It was elucidated how DHA was incorporated for, and a new compound was found and identified to complete the present invention.
化合物
すなわち、本発明は、式Ｉ:
The compound, i.e.
の構造を有する化合物を提供する。
式Iに示すように、本発明の化合物（1-Pentadecanoyl-2-docosahexanoyl-sn-glycero-3-phosphocholine: 通称 Pentadecanoyl-docosahexanoyl-lecithine）は、PCのグリセロールの1位にC15:0の脂肪酸が、2位にC22:6の脂肪酸がそれぞれエステル結合した化合物であり、以下に詳しく説明するPC-DHAの一種である。
A compound having the structure:
As shown in Formula I, the compound of the present invention (1-Pentadecanoyl-2-docosahexanoyl-sn-glycero-3-phosphocholine: commonly known as Pentadecanoyl-docosahexanoyl-lecithine) has a C15: 0 fatty acid at the 1-position of PC glycerol. A compound in which a C22: 6 fatty acid is linked to the 2-position by an ester bond, and is a kind of PC-DHA described in detail below.
本発明の化合物は様々な光学異性体として存在することができ、そのすべてが本発明の範囲に含まれる。 The compounds of the present invention can exist as various optical isomers, all of which are within the scope of the present invention.
微生物
本発明はまた、スキゾキトリウム属F26-b株の微生物（FERM AP-20727）又は、18S rRNAの配列が配列番号:1と99.3%以上相同であって、請求項1に記載の化合物の生産能を有する、その変異体を提供する。
Microorganism The present invention also provides a microorganism of the genus Schizochytrium F26-b (FERM AP-20727) or a sequence of 18S rRNA that is 99.3% or more homologous to SEQ ID NO: 1, A mutant thereof having production ability is provided.
本明細書中において、スキゾキトリウム属の微生物とは、クロミスタ界(Chromista)、ラビリンチュラ亜門(Labyrinthista)、ラビリンチュラ綱(Labyrinthulae)、ラビリンチュラ目(Labyrinthulales)、ヤブレツボカビ科(Thraustochytriaceae)、スキゾキトリウム属(SChizochytrium、シゾキトリウム属ともいう)に属する微生物であり（地球環境調査計測辞典第3巻沿岸域編第3編第3章第5節参照）、より具体的には、後述の実施例5に記載の、スキゾキトリウム属 F26-b株（FERM AP-20727）の微生物である。本明細書中において、微生物を菌（体）ということもある。なお、ラビリンチュラ目の微生物についてはその分類体系が確立していないのが現状ではあるが、以下に述べる手法により判断される、F26-b株と18S rRNAの配列が99.3%以上相同であって、本発明の化合物の生産能を有する、その変異体又はF26-b株と実質的に同一の菌学的性質を有する変異体も、本発明の範囲に含まれる。 In the present specification, microorganisms belonging to the genus Schizochytrium include Chromista, Labyrinthista, Labyrinthulae, Labyrinthulales, Thraustochytriaceae, A microorganism belonging to the genus SChizochytrium (also referred to as the genus SChizochytrium) (see Global Environment Survey and Measurement Dictionary, Volume 3, Coastal Volume, Volume 3, Chapter 3, Section 5). It is a microorganism of the genus Schizochytrium F26-b (FERM AP-20727) described in Example 5. In the present specification, the microorganism may be referred to as a fungus (body). Although the classification system for Labyrinthula microorganisms has not yet been established, the sequence of F26-b strain and 18S rRNA is 99.3% or more homologous as judged by the method described below. Also included within the scope of the present invention are mutants having the ability to produce the compound of the present invention or mutants having substantially the same mycological properties as the F26-b strain.
微生物採取
本発明の微生物は、例えば次のような、非特許文献2に記載のフィルター法により、天然海水中から容易に単離することができる。すなわち、沿岸から堆積物や落葉、海藻などと共に採取した沿岸海水を濾紙（例えば平均孔径25μm）で濾過することにより大きな浮遊物等を除去し、次いでメンブランフィルター（例えば孔径0.8μm）で濾過し、目的の微生物を含む粒径0.8〜25μmの粒子をメンブランフィルター上に捕捉する。このフィルターを、培地（例えばペニシリンG及びストレプトマイシンをそれぞれ800及び365u/mL含有するPDA培地）上にのせて培養する。フィルター上に多数出現したコロニーの中から、例えば色、艶、形状、そして顕微鏡観察により所望の微生物のコロニーを選別し、培地（例えばGY平板培地）上に接種する。培地上で増殖の認められる株をスラント等に保存すると共に、当業者に公知の手法を用いて液体培養して脂質含量と脂肪酸組成を分析し、例えば培養液の光学密度（OD610）、乾燥細胞中のDHA含有率等を指標に、増殖性及びDHA生産性の良好な株を選抜することが出来る。菌株の分離及び／又は保存には、例えばポテト−デキストロース寒天培地を50%人工海水で調製し、必要に応じスラントあるいは滅菌シャーレ中に平板培地として使用することができる。
Microorganism Collection The microorganism of the present invention can be easily isolated from natural seawater by the filter method described in Non-Patent Document 2, for example. That is, remove the large suspended matter by filtering the coastal seawater collected along with sediments, fallen leaves, seaweed, etc. from the coast with filter paper (for example, average pore diameter 25 μm), then filter with a membrane filter (for example, pore diameter 0.8 μm), Particles having a particle size of 0.8 to 25 μm containing the target microorganism are captured on the membrane filter. This filter is cultured on a medium (for example, PDA medium containing 800 and 365 u / mL of penicillin G and streptomycin, respectively). A colony of a desired microorganism is selected from colonies appearing on the filter by, for example, color, gloss, shape, and microscopic observation, and inoculated on a medium (for example, a GY plate medium). In addition to storing strains that can be grown on the medium in slants, etc., liquid culture is performed using techniques known to those skilled in the art, and the lipid content and fatty acid composition are analyzed. For example, the optical density of the culture (OD 610 ), drying A strain having good growth and DHA productivity can be selected using the DHA content in the cell as an index. For isolation and / or storage of the strain, for example, a potato-dextrose agar medium can be prepared with 50% artificial seawater and used as a plate medium in a slant or sterilized petri dish as necessary.
F26-b株は、前記非特許文献2に記載の方法により、石垣島マングローブ落葉から単離された株である。F26-b株の微生物は図1のような外観を有し、18S rRNAをコードする遺伝子（18S rDNA）の全配列をNCBI blast検索した結果、標準菌株のSchizochytrium sp. FJU-512と99.2601%の同一性を有し（実施例5参照）、顕微鏡下の観察で二分裂することが確認できたので、その配列の同一性及び分裂様式から、スキゾキトリウム属であると同定された。18S rRNAを用いたラビリンチュラ目の同定方法については、J. Eukaryot. Microbiol., 46(6), 1999, 637-647を参照した。なお、F26-b株は、種、属の分類体系の確立していない分野の微生物であるが、18S rRNAの配列が配列番号:1（F26-b株の18S rRNAの配列）と99.3%以上相同であって、本発明の化合物の生産能を有する微生物であれば、本発明の微生物の変異体として、本発明の範囲に含まれる。 The F26-b strain is a strain isolated from Ishigakijima mangrove fallen leaves by the method described in Non-Patent Document 2. The microorganism of the F26-b strain has the appearance shown in FIG. 1. As a result of NCBI blast search of the entire sequence of the gene encoding 18S rRNA (18S rDNA), the standard strain Schizophytrium sp. FJU-512 and 99.2601% Since it was confirmed that it had identity (see Example 5) and was split into two by observation under a microscope, it was identified as a genus Schizochytrium from the identity of the sequence and the mode of division. For the method for identifying Labyrinthula using 18S rRNA, see J. Eukaryot. Microbiol., 46 (6), 1999, 637-647. The F26-b strain is a microorganism in a field where the species and genus classification system has not been established. However, the 18S rRNA sequence is 99.3% or more with SEQ ID NO: 1 (the 18S rRNA sequence of the F26-b strain). Any microorganism that is homologous and capable of producing the compound of the present invention is included in the scope of the present invention as a mutant of the microorganism of the present invention.
上記F26-b株と同一の種に属する菌株、又は該株と実質的に同一の菌学的性質を有する菌株か否かは、菌学的性質を観察することにより、及び／又は18S rDNA解析により、当業者であれば容易に判断することが出来る。 Whether it is a strain belonging to the same species as the F26-b strain or a strain having substantially the same mycological properties as the strain is determined by observing the mycological properties and / or 18S rDNA analysis Thus, those skilled in the art can easily determine.
微生物の培養
本発明の微生物の培養は、海水及び／又は人工海水を用いて調製した適当な培地に微生物を接種し、当業者に公知の手法を用いて行うことが出来る。
Cultivation of microorganisms The microorganisms of the present invention can be cultured by inoculating microorganisms in a suitable medium prepared using seawater and / or artificial seawater and using techniques known to those skilled in the art.
培地
培地には、例えば液体培地及び／又は寒天培地を用いることができる。より具体的には、例えば、3%グルコースと1%酵母エキスとを含むGY培地を50%人工海水で調製し、液体培地又は1.5%寒天で平板培地として使用することができる。
As the medium , for example, a liquid medium and / or an agar medium can be used. More specifically, for example, a GY medium containing 3% glucose and 1% yeast extract can be prepared with 50% artificial seawater and used as a plate medium with a liquid medium or 1.5% agar.
培地に添加することができる炭素源としては、例えばグルコース、フルクトース、サッカロース等の糖類、酢酸、リンゴ酸、コハク酸、プロピオン酸、パルミチン酸等の低級脂肪酸、オレイン酸、大豆油等の油脂類、デンプン、グリセリン等が用いられるが、これらに限定されるものではない。これらの炭素源を、例えば、培地1Lあたり20〜100g（好ましくは25〜40g、より好ましくは30g程度）の濃度で使用することができる。なお、本発明の特徴として、培地に脂肪酸等の油脂類を添加せずに培養物から高度不飽和脂肪酸含有リン脂質等の油脂類を採取することができる。 Examples of carbon sources that can be added to the medium include sugars such as glucose, fructose, and saccharose, lower fatty acids such as acetic acid, malic acid, succinic acid, propionic acid, and palmitic acid, and fats and oils such as oleic acid and soybean oil, Although starch, glycerol, etc. are used, it is not limited to these. These carbon sources can be used, for example, at a concentration of 20 to 100 g (preferably about 25 to 40 g, more preferably about 30 g) per liter of the medium. As a feature of the present invention, fats and oils such as highly unsaturated fatty acid-containing phospholipids can be collected from the culture without adding fats and oils such as fatty acids to the culture medium.
培地に添加することができる窒素源としては、酵母エキス、ペプトン、尿素、グルタミン酸ソーダ、酢酸アンモニウム、硝酸アンモニウム等が用いられるが、これらに限定されるものではない。これらの窒素源を、例えば、培地1Lあたり5g〜20g（好ましくは8g〜14g、より好ましくは10g程度）の濃度で使用することができる。 As a nitrogen source that can be added to the medium, yeast extract, peptone, urea, sodium glutamate, ammonium acetate, ammonium nitrate, and the like are used, but are not limited thereto. These nitrogen sources can be used at a concentration of, for example, 5 g to 20 g (preferably 8 g to 14 g, more preferably about 10 g) per liter of the medium.
さらに、必要に応じて、リン酸イオン、カリウムイオン、マグネシウムイオン、カルシウムイオン、鉄イオン、銅イオン、マンガンイオン等の無機イオン、及びビタミン類も微量栄養源として使用できる。所望の微生物以外の生育を阻害する目的で、抗生物質を添加することも出来る。例えば、ストレプトマイシンを0.05%添加することが出来る。これらの培地成分は所望の微生物の成長を害しない濃度であれば特に制限はない。 Furthermore, if necessary, inorganic ions such as phosphate ions, potassium ions, magnesium ions, calcium ions, iron ions, copper ions, manganese ions, and vitamins can also be used as trace nutrient sources. Antibiotics can be added for the purpose of inhibiting the growth of microorganisms other than the desired microorganism. For example, 0.05% of streptomycin can be added. These medium components are not particularly limited as long as they do not impair the growth of desired microorganisms.
培養に伴い消費される上記炭素源、窒素源等を、培養中に補ってもよい。
培地を調製した後、適当な酸又は塩基を用いて、pHを4.0〜9.5、好ましくは6.0〜7.0の範囲に調製し、オートクレーブ等を用いて滅菌することができる。
You may supplement the said carbon source, nitrogen source, etc. which are consumed with culture | cultivation during culture | cultivation.
After preparing the medium, the pH can be adjusted to 4.0 to 9.5, preferably 6.0 to 7.0 using an appropriate acid or base, and sterilized using an autoclave or the like.
培養条件
微生物の培養は、培養温度10℃〜45℃、好ましくは25〜28℃にて、通常3〜14日間（特に培養量が多い場合には14日程度）、振とう培養、静地培養、タンク中での工業的培養、寒天培地等の固形培地での培養等により、培地中で微生物を増殖させることができる。例えば、pHを6.0に調整後、28℃のインキュベータ内で、寒天平板培地で4〜5日、液体培地では110strokes/分で振とうしながら3日間行うことができる。大量培養には、例えば10L容ジャーファーメンター中に6LのGY培地を0.1%のシリコン系消泡剤と共に入れ、121℃で20分間オートクレーブ滅菌した後、GY培地中で定常期に達した100mLの培養液を接種し、28℃、0.5vvmの空気通気、攪拌速度400rpmで5日間培養することができる。
Culture conditions Microorganisms are cultured at 10 to 45 ° C., preferably 25 to 28 ° C., usually for 3 to 14 days (especially about 14 days when the amount of culture is large), shake culture, static culture The microorganisms can be grown in the medium by industrial culture in a tank, culture in a solid medium such as an agar medium, or the like. For example, after adjusting the pH to 6.0, it can be performed in a 28 ° C. incubator for 3 to 5 days with shaking on an agar plate medium at 110 strokes / minute in a liquid medium. For large-scale culture, for example, 6 L of GY medium is placed in a 10 L jar fermenter together with 0.1% silicon-based antifoaming agent, sterilized by autoclaving at 121 ° C. for 20 minutes, and then 100 mL of the stationary phase in GY medium is reached. The culture solution can be inoculated and cultured for 5 days at 28 ° C, 0.5 vvm air aeration, and a stirring speed of 400 rpm.
本発明の微生物の採取、培養、分離方法については、上述の方法に特に限定されず、当業者に公知の適切な手法を用いることが出来る。 The method for collecting, culturing, and separating the microorganism of the present invention is not particularly limited to the above-described method, and an appropriate method known to those skilled in the art can be used.
変異体
上記の微生物は天然から採取したものでもよく、また、本発明の化合物、長鎖高度不飽和脂肪酸含有リン脂質、PC-DHA生産能を有するよう設計された変異体及び／又は組換え体も、本発明の範囲内に含まれる。このような変異体及び／又は組換え体は、同組成の培地を用いて培養したときに野性株と比べてこれらの生産能が高くなるよう意図して設計されたものが含まれる。上記化合物の「生産能を有する」とは、有効な条件で培養したときに上記の化合物を生産することをいう。有効な条件は、当業者であれば適宜決定できる。
Mutant The above-mentioned microorganism may be collected from nature, and the compound of the present invention, a long-chain highly unsaturated fatty acid-containing phospholipid, a mutant and / or recombinant designed to have the ability to produce PC-DHA Are also included within the scope of the present invention. Such mutants and / or recombinants include those designed with the intention of increasing their productivity when compared to wild strains when cultured using a medium of the same composition. “Having the ability to produce” the above compound means producing the above compound when cultured under effective conditions. Effective conditions can be appropriately determined by those skilled in the art.
本明細書中において、微生物の所望の化合物等の生産能は、微生物（微生物自体、微生物の乾燥物、微生物の培養液、微生物の抽出物等も含む）を、例えば、以下の実施例中で記載するように、GC等を用いて定法に従って分析した場合に、所望の化合物等が含まれることで確認できる。このような生産能を有する微生物には、培養条件（培地の組成、培養時の温度条件、培養時のpH条件、培養密度、等）を適宜調整した場合に長鎖高度不飽和脂肪酸含有リン脂質の生産能を有するようになる微生物も含む。 In the present specification, the ability to produce desired compounds and the like of microorganisms refers to microorganisms (including microorganisms themselves, dried microorganisms, microorganism cultures, microorganism extracts, etc.), for example, in the following examples. As described, it can be confirmed that a desired compound or the like is contained when analyzed according to a conventional method using GC or the like. For microorganisms having such productivity, phospholipids containing long-chain highly unsaturated fatty acids when the culture conditions (medium composition, temperature conditions during culture, pH conditions during culture, culture density, etc.) are appropriately adjusted. It also includes microorganisms that become capable of producing
化合物の製造方法
本発明はまた、上記の微生物又は変異体を用いることを特徴とする、上記の化合物の製造方法を提供する。製造方法は、下記に詳しく述べる。
Method for Producing Compound The present invention also provides a method for producing the above-described compound, characterized by using the above-described microorganism or mutant. The manufacturing method will be described in detail below.
組成物の製造方法
本発明はまた、上記の微生物又は変異体を培地で培養し；得られた培養物から、長鎖高度不飽和脂肪酸（好ましくは炭素数16以上で不飽和結合2以上の脂肪酸、より好ましくは炭素数20以上で不飽和結合2以上の脂肪酸、さらに好ましくはDHA）含有リン脂質（好ましくはホスファチジルコリン（PC））を採取することを含む、PC-DHAを含有する食品又は医薬用組成物の製造方法を提供する。このような製造方法で得られた組成物は、海水等に由来する不純物をほとんど含まないものになるため、食品や医薬品用に特に適する。
Method for Producing Composition The present invention also comprises culturing the above microorganism or mutant in a medium; from the obtained culture, a long-chain highly unsaturated fatty acid (preferably a fatty acid having 16 or more carbon atoms and 2 or more unsaturated bonds). More preferably a fatty acid having 20 or more carbon atoms and two or more unsaturated bonds, more preferably DHA, and a phospholipid (preferably phosphatidylcholine (PC))-containing food or pharmaceutical containing PC-DHA A method for producing the composition is provided. Since the composition obtained by such a manufacturing method contains almost no impurities derived from seawater or the like, it is particularly suitable for foods and pharmaceuticals.
本発明はまた、前記培地がDHAを含まない培地である、上記の製造方法を提供する。
本明細書中において、リン脂質とは、グリセロリン脂質及びスフィンゴリン脂質を指し、特にグリセロリン脂質を指す。更に特定すれば、ホスファチジルグリセロール(PG)、ホスファチジルコリン(PC)、リゾホスファチジルコリン(LPC)、ホスファチジルエタノールアミン(PE)、ホスファチジルセリン(PS)及びホスファチジルイノシトール(PI)を指す。グリセロールの1位及び／又は2位にエステル結合した脂肪酸の少なくとも1つが長鎖高度不飽和脂肪酸である場合、該化合物を特に長鎖高度不飽和脂肪酸含有リン脂質という。エステル結合した脂肪酸の少なくとも1つが長鎖高度不飽和脂肪酸であれば、他の脂肪酸の炭素数及び不飽和度に関わらず、該化合物を長鎖高度不飽和脂肪酸含有リン脂質という。
The present invention also provides the above production method, wherein the medium is a medium not containing DHA.
In the present specification, phospholipid refers to glycerophospholipid and sphingophospholipid, and particularly refers to glycerophospholipid. More specifically, it refers to phosphatidylglycerol (PG), phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). When at least one fatty acid ester-bonded to the 1-position and / or 2-position of glycerol is a long-chain highly unsaturated fatty acid, the compound is particularly referred to as a long-chain highly unsaturated fatty acid-containing phospholipid. If at least one of the ester-bonded fatty acids is a long-chain highly unsaturated fatty acid, the compound is referred to as a long-chain highly unsaturated fatty acid-containing phospholipid regardless of the carbon number and degree of unsaturation of the other fatty acids.
本明細書中において長鎖高度不飽和脂肪酸とは、炭素数16以上、好ましくは炭素数20以上の脂肪酸であって、不飽和結合を2以上、好ましくは4以上有する脂肪酸を指し、例えば、リノール酸(C18:2n6)、α-リノレン酸(C18:3n3)、アラキドン酸(C20:4n6)、エイコサペンタエン酸(C20:5n4、EPA)、ドコサテトラエン酸(C22:4)、ドコサペンタエン酸(C22:5、DPA)、ドコサヘキサエン酸(C22:6、DHA)、C16:2n4、C18:4n3、C20:4n3等を挙げることができるが、これらに限定されない。 In the present specification, the long-chain highly unsaturated fatty acid refers to a fatty acid having 16 or more carbon atoms, preferably 20 or more carbon atoms, and having 2 or more, preferably 4 or more unsaturated bonds, such as linole. Acid (C18: 2n6), α-linolenic acid (C18: 3n3), arachidonic acid (C20: 4n6), eicosapentaenoic acid (C20: 5n4, EPA), docosatetraenoic acid (C22: 4), docosapentaenoic acid (C22: 5, DPA), docosahexaenoic acid (C22: 6, DHA), C16: 2n4, C18: 4n3, C20: 4n3, and the like, but are not limited thereto.
本発明の方法で得られる組成物が含有する長鎖高度不飽和脂肪酸含有リン脂質に含まれ得る長鎖高度不飽和脂肪酸以外の脂肪酸としては、例えばC15:0、C13:0、C14:0、C16:0、C16:1n7、C18:0、C18:1n7等が挙げられるが、これらに限定されない。 Examples of fatty acids other than long-chain highly unsaturated fatty acids that can be contained in the long-chain highly unsaturated fatty acid-containing phospholipid contained in the composition obtained by the method of the present invention include C15: 0, C13: 0, C14: 0, Examples thereof include, but are not limited to, C16: 0, C16: 1n7, C18: 0, C18: 1n7, and the like.
本明細書中において、PC-DHAとは、前記長鎖高度不飽和脂肪酸含有リン脂質において長鎖高度不飽和脂肪酸がDHAであり、リン脂質がPCであって、PCのグリセロールの1位及び／又は2位の炭素にDHAがエステル結合した化合物の総称である。該化合物において、例えば1位（又は2位）にDHAが、2位（又は1位）に他の脂肪酸がエステル結合している場合、他の脂肪酸の炭素数及び不飽和度は特に制限されず、飽和脂肪酸であっても、不飽和脂肪酸であってもよい。 In the present specification, PC-DHA refers to the long-chain highly unsaturated fatty acid-containing phospholipid, wherein the long-chain highly unsaturated fatty acid is DHA, the phospholipid is PC, Or, it is a general term for compounds in which DHA is ester-bonded to carbon at the 2-position. In the compound, for example, when DHA is linked to the 1-position (or 2-position) and other fatty acid is linked to the 2-position (or 1-position), the number of carbon atoms and the degree of unsaturation of the other fatty acids are not particularly limited. It may be a saturated fatty acid or an unsaturated fatty acid.
本発明の製造方法で得られるPC-DHAを含有する組成物は、PC-DHAを含む限り、2以上の脂肪酸含有リン脂質からなる組成物であっても、他の構成成分を含むものであっても良い。含まれ得る他の構成成分としては、糖脂質、中性脂質、ステロール類等が挙げられる。好ましくは、上記の組成物は、リン脂質、糖脂質、中性脂質等の脂肪酸を含有する構成成分からなる。 The composition containing PC-DHA obtained by the production method of the present invention, as long as it contains PC-DHA, is a composition comprising two or more fatty acid-containing phospholipids and contains other components. May be. Other constituents that can be included include glycolipids, neutral lipids, sterols and the like. Preferably, the above composition is composed of constituents containing fatty acids such as phospholipids, glycolipids, and neutral lipids.
高度不飽和脂肪酸含有リン脂質、特にPC-DHA生産能を有する微生物を含む培養物から、当業者に公知の手法を用いて、上記微生物に含まれる高度不飽和脂肪酸含有リン脂質を含む組成物を回収することができる。例えば：培養物中の微生物を破砕しその破砕した微生物から直接ヘキサン等の有機溶媒によって抽出する；微生物を濾取し得られた微生物を直接圧搾して油分を取り出す；微生物を乾燥後、粉砕機等で粉砕しヘキサン等で抽出する；等の手法を用いることができるが、これらに限定されない。 From a culture containing a highly unsaturated fatty acid-containing phospholipid, particularly a microorganism capable of producing PC-DHA, using a technique known to those skilled in the art, a composition containing the highly unsaturated fatty acid-containing phospholipid contained in the microorganism is prepared. It can be recovered. For example: crushing microorganisms in the culture and extracting directly from the crushed microorganisms with an organic solvent such as hexane; removing microorganisms by directly pressing the microorganisms obtained by filtering the microorganisms; Or the like, and extraction with hexane or the like can be used, but it is not limited thereto.
本発明の方法ははまた、DHAを添加しない培地で培養した上記の微生物の培養物からPC-DHA含有組成物を得ることができるため、DHAを効率よく生産することができる。
本発明はまた、前記組成物中において、前記培養物由来である全脂質についてGCを用いて各脂肪酸を定量した場合、全脂質の15%以上（好ましくは30%以上、より好ましくは35%以上）がC15:0であり、全脂質の15%以上（好ましくは30%以上、より好ましくは33%以上）がDHAである、前記の製造方法を提供する。本発明はまた、前記組成物中において、前記培養物由来である全リン脂質についてGCを用いて各脂肪酸を定量した場合、脂肪酸のの10%以上（好ましくは15%以上、より好ましくは20%以上）がC15:0であり、脂肪酸の30%以上（好ましくは40%以上、より好ましくは50%以上）がDHAである、前記の製造方法を提供する。本発明はまた、前記組成物中において、前記培養物由来である脂肪酸含有PCについてGCを用いて1位の脂肪酸を定量した場合に脂肪酸の35%以上（好ましくは45%以上、より好ましくは50%以上）がC15:0である、前記の製造方法を提供する。本発明はまた、前記培養物由来である脂肪酸含有PCについてGCを用いて2位の脂肪酸を定量した場合に脂肪酸の40%以上（好ましくは50%以上、より好ましくは55%以上）がDHAである、前記の製造方法を提供する。
The method of the present invention can also produce DHA efficiently because a PC-DHA-containing composition can be obtained from a culture of the above microorganisms cultured in a medium not containing DHA.
The present invention also provides that in the composition, when each fatty acid is quantified using GC for the total lipid derived from the culture, 15% or more (preferably 30% or more, more preferably 35% or more) of the total lipid. ) Is C15: 0, and 15% or more (preferably 30% or more, more preferably 33% or more) of the total lipid is DHA. The present invention also provides that in the composition, when each fatty acid is quantified using GC for the total phospholipids derived from the culture, 10% or more (preferably 15% or more, more preferably 20%) of the fatty acid. The above production method is provided wherein C15: 0 is above, and 30% or more (preferably 40% or more, more preferably 50% or more) of the fatty acid is DHA. In the composition, the fatty acid-containing PC derived from the culture in the composition is 35% or more (preferably 45% or more, more preferably 50% or more, preferably 50% or more of the fatty acid when the first fatty acid is quantified using GC. % Or more) is C15: 0. In the present invention, when the fatty acid at position 2 is quantified using GC for the fatty acid-containing PC derived from the culture, 40% or more (preferably 50% or more, more preferably 55% or more) of the fatty acid is DHA. The manufacturing method is provided.
分析方法
前記のように微生物から抽出した高度不飽和脂肪酸含有リン脂質を含む組成物について、例えば以下の実施例に記載するような当業者に公知の分析手法を用いて、全脂質、中性脂質、糖脂質、リン脂質等を分画し、各画分を定量することができる。また、分画した各脂質についても、例えば以下の実施例に記載するような当業者に公知の手法を用いて、それぞれ脂肪酸組成を調べることができる。
Analytical method About the composition containing the highly unsaturated fatty acid-containing phospholipid extracted from the microorganism as described above, for example, using an analytical method known to those skilled in the art as described in the following examples, total lipid, neutral lipid , Glycolipids, phospholipids, etc. can be fractionated and each fraction can be quantified. In addition, for each fractionated lipid, the fatty acid composition can be examined using a method known to those skilled in the art as described in the following examples, for example.
全リン脂質について、HPLCを用いた分析により、全ピーク面積に対する各脂肪酸のピーク面積の割合を算出することで各リン脂質クラス（PC、LPC、PE、PS、PI）の定量的な分析を行うことができる。また、各リン脂質クラスの１位又は2位の脂肪酸について、以下の実施例3のような手法によりGCを用いて脂肪酸組成を分析し、全ピーク面積に対する各脂肪酸のピーク面積の割合を算出することにより、定量的に分析を行うことができる。 Quantitative analysis of each phospholipid class (PC, LPC, PE, PS, PI) by calculating the ratio of the peak area of each fatty acid to the total peak area by analysis using HPLC for all phospholipids be able to. In addition, the fatty acid composition of the first or second fatty acid in each phospholipid class is analyzed by GC using the technique as in Example 3 below, and the ratio of the peak area of each fatty acid to the total peak area is calculated. Thus, the analysis can be performed quantitatively.
用途
本発明はまた、上記の化合物を含む、食品又は医薬用組成物を提供する。
本発明はまた、癌又はレム睡眠に関連した疾患又は状態を処置するための、上記の組成物を提供する。
Use The present invention also provides a food or pharmaceutical composition comprising the above compound.
The present invention also provides a composition as described above for treating a disease or condition associated with cancer or REM sleep.
本発明の製造方法を用いて製造された組成物並びに本発明の化合物、組成物、及び微生物は、例えば、食品、飼料、医薬、化粧用、特に食品又は医薬品用に用いることができ、例えば食品、飼料、医薬品、化粧品に添加して用いることができる。本発明の製造方法において、例えばタンク培養等の閉鎖培養系を用いれば、得られる組成物への海洋汚染の影響は限りなくゼロに近くなる。海洋汚染物質による汚染の度合いが低いという観点からは、本発明の製造方法で得た組成物は特に、粉ミルク等の乳児用食品、アルテミアやワムシなど食物連鎖の初期の段階に位置する生物用の飼料、乳幼児や妊婦用の食品、医薬品及び敏感肌用化粧品等にも利用することができる。種菌の汚染は、例えば、18S rDNA解析等の手法を用いて調べることが可能である。 The composition produced by using the production method of the present invention and the compound, composition and microorganism of the present invention can be used for, for example, food, feed, medicine, cosmetics, particularly food or medicine, for example, food It can be used by adding to feed, pharmaceuticals and cosmetics. In the production method of the present invention, for example, if a closed culture system such as tank culture is used, the influence of marine contamination on the resulting composition is almost zero. From the viewpoint of low pollution by marine pollutants, the composition obtained by the production method of the present invention is particularly suitable for infant foods such as powdered milk, and for organisms located in the early stages of the food chain such as artemia and rotifers. It can also be used for feed, food for infants and pregnant women, pharmaceuticals, and cosmetics for sensitive skin. The contamination of the inoculum can be examined using a technique such as 18S rDNA analysis.
該利用に際しては、上記の化合物、組成物、組成物を精製して得られる脂肪酸含有リン脂質、上記の微生物若しくはその乾燥物、微生物を培養して得られる培養物若しくはその殺菌した培養物、又は微生物を培養して培養物から長鎖高度不飽和脂肪酸含有リン脂質を採取した後の残渣も使用することができる。 In the use, the above-mentioned compound, composition, fatty acid-containing phospholipid obtained by purifying the composition, the above microorganism or a dried product thereof, a culture obtained by culturing the microorganism or a sterilized culture thereof, or The residue after culturing the microorganism and collecting the long-chain highly unsaturated fatty acid-containing phospholipid from the culture can also be used.
上記の食品の例としては、栄養補助食品、乳幼児用調製乳、未熟児用調整乳、幼児用食品、妊婦用食品、老人用食品、健康食品、機能性食品、特定保健用食品、油脂を含む食品（肉、魚、卵又はナッツ等の天然食品、調理時、加工時、加工仕上げ時に油脂を用いる食品等）、油脂を含まない食品（農産食品、発酵食品、畜産食品、水産食品等）、飲料等が挙げられ、当業者に公知の手法を用いてこれらに本発明の組成物等を配合することができる。配合量は、当業者であれば適宜決定できる。 Examples of the above foods include nutritional supplements, infant formulas, formulas for premature babies, infant foods, pregnant foods, foods for the elderly, health foods, functional foods, foods for specified health use, fats and oils Foods (natural foods such as meat, fish, eggs or nuts, foods that use fats and oils during cooking, processing, and finishing), foods that do not contain fats and oils (agricultural foods, fermented foods, livestock foods, marine foods, etc.), A drink etc. are mentioned, The composition of this invention etc. can be mix | blended with these using a method well-known to those skilled in the art. Those skilled in the art can appropriately determine the blending amount.
また、本明細書中において、本発明の組成物等を飼料として育てた動物に由来する、本発明の組成物等を含む食品（肉、魚、卵等）も、本発明の特徴を有する限り、本発明の組成物を添加した食品に含まれる。 In the present specification, a food (meat, fish, egg, etc.) containing the composition of the present invention derived from an animal grown using the composition of the present invention as a feed also has the characteristics of the present invention. And contained in foods to which the composition of the present invention is added.
上記の飼料の例としては、動物用飼料（例えば、犬、猫等のペット用飼料、鶏、豚、牛等の家畜用飼料、魚介類や甲殻類用の飼料、魚介類や甲殻類の養殖において種苗（稚仔魚）に用いる動物プランクトン（例えばワムシ、アルテミア）等の微小飼料生物用飼料が挙げられ、当業者に公知の手法を用いてこれらに本発明の組成物等を配合することができる。 Examples of the above feed include animal feeds (eg, pet feeds for dogs, cats, etc., livestock feeds for chickens, pigs, cattle, etc., feeds for seafood and crustaceans, seafood and shellfish farms, etc. And feeds for micro feed organisms such as zooplankton (for example, rotifer, artemia) used for seedlings (larvae), and the composition of the present invention and the like can be added to these using methods known to those skilled in the art. .
上記の化粧品の例としては、例えば、乳液、クリーム、化粧水、パック、洗浄剤等が挙げられ、当業者に公知の手法を用いてこれらに本発明の組成物等を配合することができる。 Examples of the above cosmetics include, for example, emulsions, creams, lotions, packs, detergents, and the like, and the composition of the present invention can be added to these using methods known to those skilled in the art.
DHAは、アレルギーやガンに対する抑制効果（Duchen K, Casas R, Fageras-Bottcher M, Yu G, Bjorksten B. Pediatr Allergy Immunol. (2000) 11, 29）等多様な生理活性を有することが知られる。また、PC-DHAはレム睡眠の増加（Cheruku SR, Montgomery-Downs HE, Farkas SL, Thoman EB, Lammi-Keefe CJ. Am J Clin Nutr. (2002) 76, 608）、脳卒中の抑制効果（井上 良計, 金田 輝之; オレオサイエンス (2002) 2, 76 ）を有することが知られている。さらに、ホスホリパーゼA2(PLA2)によって遊離したDHAは、哺乳類の脳において、核内レセプターのリガンドとして転写を調節するシグナル分子の機能をもつことが報告され（de Urquiza AM, Liu S, Sjoberg M, Zetterstrom RH, Griffiths W, Sjovall J, Perlmann T. Science. (2000) 290, 2140）、遊離したDHAがエイコサノイド様の機能を持つことも注目されている。2位にDHAが導入されている本発明のPC-DHAをヒトに与えた時には、体内でホスホリパーゼ A2で切れることによりDHAが遊離し、エイコサノイド様の作用をすることが期待できる。 DHA is known to have various physiological activities such as an inhibitory effect on allergy and cancer (Duchen K, Casas R, Fageras-Bottcher M, Yu G, Bjorksten B. Pediatr Allergy Immunol. (2000) 11, 29). PC-DHA also increased REM sleep (Cheruku SR, Montgomery-Downs HE, Farkas SL, Thoman EB, Lammi-Keefe CJ. Am J Clin Nutr. (2002) 76, 608), and stroke suppression effect (Yoshi Inoue) Total, Teruyuki Kaneda; Oreo Science (2002) 2, 76). Furthermore, DHA released by phospholipase A 2 (PLA 2 ) has been reported to function as a signal molecule that regulates transcription as a ligand for nuclear receptors in the mammalian brain (de Urquiza AM, Liu S, Sjoberg M Zetterstrom RH, Griffiths W, Sjovall J, Perlmann T. Science. (2000) 290, 2140), and it is also noted that the released DHA has an eicosanoid-like function. When the PC-DHA of the present invention in which DHA is introduced at position 2 is given to humans, it can be expected that DHA is released by cleaving with phospholipase A2 in the body and acts like an eicosanoid.
中性脂質形態の高度不飽和脂肪酸を含む魚油に比べ、リン脂質形態の高度不飽和脂肪酸の方が、脳卒中自然発症高血圧ラットを長生きさせる効果が高いという報告もある（「食品と開発」Vol.34, No.8, P41-43, 1999, 健康産業新聞社発行）。これらのことから、上記の医薬品の例としては、上記のようなDHA又はPC−DHAの機能に関連した疾患又は状態を処置するための医薬品が挙げられ、例えば、アレルギー、癌、脳卒中、レム睡眠に関連した疾患又は状態を処置するための医薬品が挙げられる。特に、抗癌作用を有する食品やレム睡眠を誘導するサプリメント等への利用が期待できる（日経バイオビジネス、2004、10巻、p29）。該医薬品は、当業者に公知の手法を用いて本発明の組成物等を配合して散剤、錠剤、カプセル剤、内用液剤、乳剤等、適切な形態に調製することができる。配合量は、当業者であれば適宜決定できる。 There is a report that phospholipid form of highly unsaturated fatty acid is more effective in prolonging spontaneously hypertensive rats with stroke compared to fish oil containing highly unsaturated fatty acid in neutral lipid form ("Food and Development" Vol. 34, No.8, P41-43, 1999, published by Health Industry Newspaper). From these facts, examples of the above-mentioned pharmaceuticals include pharmaceuticals for treating diseases or conditions related to the function of DHA or PC-DHA as described above. For example, allergy, cancer, stroke, REM sleep Medicaments for treating diseases or conditions associated with. In particular, it can be expected to be used for foods with anticancer effects, supplements that induce REM sleep, etc. (Nikkei Biobusiness, 2004, 10, p29). The pharmaceutical preparation can be prepared in an appropriate form such as a powder, tablet, capsule, liquid for internal use, emulsion and the like by blending the composition of the present invention using a method known to those skilled in the art. Those skilled in the art can appropriately determine the blending amount.
本発明の組成物には、その具体的な用途（例えば、栄養補助のため、成長促進のため、体質改善のため、必須脂肪酸（例えばDHA）供給のため、脳卒中予防／抑制のため、レム睡眠増加のため、アレルギー抑制のため、ガン予防のため等）及び／又はその具体的な用い方（例えば、量、回数、期間等）を表示することが出来る。 The composition of the present invention has specific uses (for example, for nutritional support, for promoting growth, for improving constitution, for supplying essential fatty acids (for example, DHA), for preventing / suppressing stroke, for REM sleep) It is possible to display an increase, an allergy suppression, a cancer prevention, etc.) and / or a specific usage (eg, amount, number of times, period, etc.).
以下に本発明を実施例により更に詳しく説明するが、本発明はこれら実施例に限定されるものではない。 EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
実施例1
F26-b株における全脂肪酸の組成分析 （Qiu X, Hong H, MacKenzie SL. J Biol Chem. (2001) 276, 31561の手法に従って行った）
（1．菌体の培養と回収）
ラビリンチュラ目に属するF26-b株の菌体（林雅弘先生（宮崎大学）より御分与頂いた。FERM AP-20727）をGY培地（pH 6.0、グルコース 3.18g、酵母エキス 1.06g、ストレプトマイシン 0.05g、50%人工海水 100ml） 300ml中で4日間、25℃、150rpmで培養した。3,000rpmで10分遠心し、上清を除去した。0.9% NaClを適当量加え、よく懸濁し、次いで3,000rpmで10分遠心し、上清を除去する作業を繰り返した。超純水を適当量加え、よく懸濁し、3,000rpmで10分遠心し、上清を除去した後、回収した菌体を凍結乾燥した（1.0g）。
Example 1
Composition analysis of total fatty acids in F26-b strain (performed according to the method of Qiu X, Hong H, MacKenzie SL. J Biol Chem. (2001) 276, 31561)
(1. Culture and recovery of bacterial cells)
The F26-b strain belonging to the Labyrinthula (Mr. Masahiro Hayashi (Miyazaki University). Ferm AP-20727) was added to GY medium (pH 6.0, glucose 3.18 g, yeast extract 1.06 g, streptomycin 0.05 g, 50% artificial seawater 100 ml) The cells were cultured in 300 ml for 4 days at 25 ° C. and 150 rpm. The supernatant was removed by centrifugation at 3,000 rpm for 10 minutes. An appropriate amount of 0.9% NaCl was added, suspended well, and then centrifuged at 3,000 rpm for 10 minutes to remove the supernatant. An appropriate amount of ultrapure water was added, well suspended, centrifuged at 3,000 rpm for 10 minutes, the supernatant was removed, and the recovered cells were lyophilized (1.0 g).
（2．Folch法による全脂質抽出）
1の工程で回収した菌体に20mlのクロロホルム／メタノール混液(2/1)を加え、超音波処理で菌体を破壊した。3,000rpmで15分間遠心し、上清を回収した。沈殿に20mlのクロロホルム／メタノール混液(2/1)を加え、再度脂質を抽出した。上清を遠心で集め、先の上清合わせ、分液ロートに移した。8mlの2% KCl溶液を加え、二層分配した。下層を回収し、エバポレーターで濃縮乾固した。乾固物を10mlのクロロホルム／メタノール混液(2/1)に溶解し、適当量を試験管に移しメチル化後、脂肪酸の分析を行った。
(2. Total lipid extraction by Folch method)
20 ml of a chloroform / methanol mixture (2/1) was added to the cells recovered in the step 1, and the cells were destroyed by ultrasonic treatment. The supernatant was collected by centrifugation at 3,000 rpm for 15 minutes. To the precipitate, 20 ml of a chloroform / methanol mixture (2/1) was added, and the lipid was extracted again. The supernatant was collected by centrifugation, combined with the previous supernatant, and transferred to a separatory funnel. 8 ml of 2% KCl solution was added and the two layers were partitioned. The lower layer was collected and concentrated to dryness with an evaporator. The dried product was dissolved in 10 ml of a chloroform / methanol mixture (2/1), an appropriate amount was transferred to a test tube, methylated, and then analyzed for fatty acids.
（3．FAのメチル化）
2で得た濃縮乾固したサンプルに2mlの3N methanolic HClを添加し、80℃で、一晩インキュベートした。室温まで冷まし、1mlの0.9% NaClを添加した。2mlのn-ヘキサンを加えてボルテックスし、次いで3,000rpmで10分遠心し、上層（ヘキサン層）を回収する作業を繰り返した。無水硫酸ナトリウムを少量添加した後、ろ過によって無水硫酸ナトリウムを除去し、ろ液を窒素ガスで濃縮乾固した。濃縮乾固したサンプルを200μlのn-ヘキサンで溶解した。
(3. Methylation of FA)
2 ml of 3N methanolic HCl was added to the concentrated and dried sample obtained in 2 and incubated at 80 ° C. overnight. Cool to room temperature and add 1 ml of 0.9% NaCl. 2 ml of n-hexane was added, vortexed, and then centrifuged at 3,000 rpm for 10 minutes to repeat the operation of recovering the upper layer (hexane layer). After a small amount of anhydrous sodium sulfate was added, anhydrous sodium sulfate was removed by filtration, and the filtrate was concentrated to dryness with nitrogen gas. The concentrated and dried sample was dissolved in 200 μl of n-hexane.
（4．GCによるFA分析）
3で得た試料を以下の条件で分析した。使用機材:Shimazu GC-14A (Shimazu Co., Japan)、カラム:HR-SS-10, 30 m x 0.25 mm i.d. (Shinwa Chemical Ind. Ltd., Japan) 、カラム温度: 150℃から220℃に昇温（2℃／分）。各ピークの同定はPUFA 3 standard mixture（Matreya）を使用した。
(4. FA analysis by GC)
The sample obtained in 3 was analyzed under the following conditions. Equipment: Shimazu GC-14A (Shimazu Co., Japan), Column: HR-SS-10, 30 mx 0.25 mm id (Shinwa Chemical Ind. Ltd., Japan), Column temperature: 150 ° C to 220 ° C (2 ° C / min). Each peak was identified using a PUFA 3 standard mixture (Matreya).
結果:
図2は、F26-b株の菌体からn-ヘキサンで抽出した全脂肪酸の組成をGCを用いて分析した結果である。保持時間23分台のピークがマーカーのDHAメチルエステル（SIGMA）の保持時間と一致することが確認されたので、さらにこのピークについてGC/MS解析を行った（図3）。その結果、標品のDHAとフラグメントが一致したので23分台のメジャーなピークはDHAであることが同定された。GC AREAをもとに算出したF26-b株の菌体の全脂質の脂肪酸組成を、表2中に示す。
この結果、F26-b株の菌体において全脂肪酸の約33%がDHAであることがわかった。また興味深いことに、飽和脂肪酸、特に哺乳動物には存在しない奇数飽和脂肪酸(C13:0、C15:0)、特にC15:0が非常に多く含まれていることが分かった。
result:
FIG. 2 shows the result of GC analysis of the composition of all fatty acids extracted with n-hexane from the cells of F26-b strain. Since it was confirmed that the peak at the retention time of 23 minutes coincided with the retention time of the marker DHA methyl ester (SIGMA), GC / MS analysis was further performed on this peak (FIG. 3). As a result, since the DHA and the fragment of the sample matched, the major peak at 23 minutes was identified as DHA. Table 2 shows the fatty acid composition of total lipids of the F26-b strain cells calculated based on GC AREA.
As a result, it was found that about 33% of all fatty acids were DHA in the F26-b strain. Interestingly, it was found that saturated fatty acids, particularly odd saturated fatty acids (C13: 0, C15: 0) that are not present in mammals, especially C15: 0, are contained in a large amount.
実施例2
F26-b株における中性脂質、糖脂質、リン脂質の脂肪酸分析
（全脂質の中性脂質、糖脂質、リン脂質への分離）
Sep-Pak Silica (Waters)を30mlのクロロホルムで平衡化した。濃縮乾固した全脂質を、2mlのクロロホルムに溶解し、サンプルを全量、カラムに負荷した。溶出は次の溶媒を用いて行った:クロロホルム30ml（中性脂質）、アセトン50ml（糖脂質）、メタノール20ml（リン脂質）。この順で溶出し、5mlずつのフラクションを回収した。以下の条件で、TLCによって各脂質が回収できていることを確認した。展開溶媒：n-ヘキサン／ジエチルエーテル／酢酸＝80:20:1(v/v/v)(中性脂質)、クロロホルム／メタノール／水＝65:25:4 (v/v/v) (糖脂質、リン脂質)。発色試薬：50%硫酸 (中性脂質)、オルシノール硫酸 (糖脂質)、Dittmer 試薬（リン脂質）。各フラクションをエバポレーターで濃縮した。FAの抽出、FAのメチル化、GCによるFA分析については、実施例1と同様の手法を用いて行った。
Example 2
Fatty acid analysis of neutral lipids, glycolipids and phospholipids in F26-b strain (separation of total lipids into neutral lipids, glycolipids and phospholipids)
Sep-Pak Silica (Waters) was equilibrated with 30 ml of chloroform. The concentrated and solidified total lipid was dissolved in 2 ml of chloroform, and the entire sample was loaded onto the column. Elution was performed using the following solvents: 30 ml chloroform (neutral lipid), 50 ml acetone (glycolipid), 20 ml methanol (phospholipid). Elution was performed in this order, and 5 ml fractions were collected. It was confirmed that each lipid was recovered by TLC under the following conditions. Developing solvent: n-hexane / diethyl ether / acetic acid = 80: 20: 1 (v / v / v) (neutral lipid), chloroform / methanol / water = 65: 25: 4 (v / v / v) (sugar Lipids, phospholipids). Coloring reagent: 50% sulfuric acid (neutral lipid), orcinol sulfate (glycolipid), Dittmer reagent (phospholipid). Each fraction was concentrated with an evaporator. FA extraction, FA methylation, and FA analysis by GC were performed using the same methods as in Example 1.
結果:
表1は、抽出に用いた菌体の乾燥重量と抽出した脂質重量を示す。
result:
Table 1 shows the dry weight of the cells used for extraction and the weight of the extracted lipid.
表2は、GC AREAからF26-b株の全脂質、中性脂質、糖脂質、リン脂質に画分おける脂肪酸組成を算出した結果を示す。 Table 2 shows the results of calculating the fatty acid composition in the fractions of total lipid, neutral lipid, glycolipid, and phospholipid of F26-b strain from GC AREA.
乾燥菌体から抽出した全脂質量及び、該全脂質中のDHA含有量から算出したところ、F26-b株の乾燥菌体1gから約87mg(8.67%)のDHAが抽出できたことが分かった。以上のことから、F26-b株の菌体は従来の報告どおり、高度にDHAを産生することが確認された。一般に、DHAを高度に含む魚とされるイワシの場合、1匹(100g)あたり1g程度のDHAを含むとされる。このデータと比較しても本株はDHAを豊富に含有しており、DHAの単離源として優れているといえる。また、リン脂質画分のDHAは約51%であることが分かった。 When calculated from the total lipid amount extracted from the dry cells and the DHA content in the total lipid, it was found that about 87 mg (8.67%) of DHA could be extracted from 1 g of the dry cells of the F26-b strain. . From the above, it was confirmed that the cells of F26-b strain produced DHA highly as reported previously. In general, in the case of a sardine that is considered to contain a high amount of DHA, it is assumed that about 1 g of DHA is contained per animal (100 g). Compared with this data, this strain is rich in DHA and can be said to be an excellent source of DHA isolation. The DHA of the phospholipid fraction was found to be about 51%.
実施例3
F26-b株におけるDHA含有リン脂質の分子種の同定（Mawatari S, Murakami K. Analytical Biochemistry. (1998) 264, 118の手法を用いて行った）
（1．HPLCを用いたリン脂質画分のクラス分離）
実施例2で回収したリン脂質画分をエバポレーターで濃縮し、200μlのn-ヘキサン／イソプロパノール＝3:1(v/v)に溶解した。次の条件で分離を行った。カラム:wakosil 5 NH2 Column, 250 x 4.6 mm (和光純薬)、移動相:アセトニトリル／メタノール／0.2%トリエチルアミン＝67:22:11(v/v/v)（0.2%トリエチルアミンはリン酸でpH4.0に調整）、流速:1ml／分、カラム温度:40℃、検出波長:210nm。
PC (Avanyi Polar Lipids)、PE(SIGMA)、PS (DOOSAN Serdary Research Laboratories)、PI (DOOSAN Serdary Research Laboratories)、LPC (1-Palmitoyl-sn-glycero-3-phosphocholine; Matreya)（各1μg/μl）をマーカーとして20μlを負荷し、F26-b株由来のリン脂質画分を20μl負荷した。マーカーの保持時間に従って、クラスごとに試験管に回収した。エバポレーターで回収したサンプルを濃縮し、2mlのクロロホルム／メタノール＝2:1(v/v)を加え、400μlの2%KCl溶液を添加した。よくボルテックスし、3,000rpmで5分遠心をかけ、上層を除き、残った下層を窒素ガスで濃縮乾固した。
Example 3
Identification of molecular species of DHA-containing phospholipids in the F26-b strain (using the method of Mawatari S, Murakami K. Analytical Biochemistry. (1998) 264, 118)
(1. Class separation of phospholipid fraction using HPLC)
The phospholipid fraction collected in Example 2 was concentrated with an evaporator and dissolved in 200 μl of n-hexane / isopropanol = 3: 1 (v / v). Separation was performed under the following conditions. Column: wakosil 5 NH 2 Column, 250 x 4.6 mm (Wako Pure Chemical Industries), mobile phase: acetonitrile / methanol / 0.2% triethylamine = 67: 22: 11 (v / v / v) (0.2% triethylamine is phosphoric acid, pH 4 0.0), flow rate: 1 ml / min, column temperature: 40 ° C., detection wavelength: 210 nm.
PC (Avanyi Polar Lipids), PE (SIGMA), PS (DOOSAN Serdary Research Laboratories), PI (DOOSAN Serdary Research Laboratories), LPC (1-Palmitoyl-sn-glycero-3-phosphocholine; Matreya) (each 1μg / μl) As a marker, 20 μl was loaded, and 20 μl of the phospholipid fraction derived from the F26-b strain was loaded. Each class was collected in a test tube according to the marker retention time. The sample collected by the evaporator was concentrated, 2 ml of chloroform / methanol = 2: 1 (v / v) was added, and 400 μl of 2% KCl solution was added. Vortexed well, centrifuged at 3,000 rpm for 5 minutes, the upper layer was removed, and the remaining lower layer was concentrated to dryness with nitrogen gas.
（2．PLA2によるFAの遊離）
濃縮乾固したサンプルに2mlのジエチルエーテルを加え、よく懸濁した。酵素液（200 mM Tris-HCl buffer (pH9.0)、6 mM CaCl2、PLA2(ホスホリパーゼA2、Naja mossambica mossambica由来; SIGMA)）を調整し、5Unitを100μl加え、2分ボルテックスした。37℃で2時間インキュベートした。窒素ガスでジエチルエーテルを除き、2mlのクロロホルム／メタノール＝2:1(v/v)を加え、400μlの2%KCl溶液を添加した。よくボルテックスし、3,000rpmで5分遠心した。下層を回収し、残った上層に1mlのクロロホルムを添加した。よくボルテックスし、3,000rpmで5分遠心した。下層を回収し、先に回収した分と合わせ、300μlを分取し、以下の条件でTLCによって反応産物を確認した。展開溶媒：クロロホルム／メタノール／水＝65:25:4(v/v/v)、発色試薬：50%硫酸、Dittmer試薬。残りは窒素ガスで濃縮乾固した。
(2. Release of FA by PLA 2 )
2 ml of diethyl ether was added to the concentrated and dried sample and well suspended. An enzyme solution (200 mM Tris-HCl buffer (pH 9.0), 6 mM CaCl 2 , PLA 2 (phospholipase A2, derived from Naja mossambica mossambica; SIGMA)) was prepared, 100 μl of 5 Unit was added, and vortexed for 2 minutes. Incubated for 2 hours at 37 ° C. Diethyl ether was removed with nitrogen gas, 2 ml of chloroform / methanol = 2: 1 (v / v) was added, and 400 μl of 2% KCl solution was added. Vortex well and centrifuge at 3,000 rpm for 5 minutes. The lower layer was collected, and 1 ml of chloroform was added to the remaining upper layer. Vortex well and centrifuge at 3,000 rpm for 5 minutes. The lower layer was recovered, combined with the previously recovered amount, 300 μl was collected, and the reaction product was confirmed by TLC under the following conditions. Developing solvent: chloroform / methanol / water = 65: 25: 4 (v / v / v), coloring reagent: 50% sulfuric acid, Dittmer reagent. The rest was concentrated to dryness with nitrogen gas.
（3．FAの精製）
Sep-Pak Silica (Waters)を30mlのクロロホルムで平衡化し、濃縮乾固したサンプルを、2mlのクロロホルムに溶解した。サンプルを全量、カラムに負荷し、次の溶媒で溶出した:クロロホルム30ml（遊離脂肪酸）、アセトン10ml、メタノール20ml（リゾリン脂質）。この順で溶出し、溶媒ごとにフラクションを回収した。以下の条件で、TLCによって脂肪酸が精製できていることを確認した。展開溶媒：クロロホルム／メタノール／水＝65:25:4(v/v/v)、発色試薬：50%硫酸（遊離脂肪酸）、Dittmer試薬（リゾリン脂質）。クロロホルム画分とアセトン画分を合わせて、濃縮乾固し、sn-2位の脂肪酸組成を分析した。さらに、メタノール画分も濃縮乾固し、sn-1位の脂肪酸組成を分析した（PLA2によって遊離する脂肪酸を2位、残りの脂肪酸（リゾリン脂質の脂肪酸）を1位の脂肪酸とする）。
FAのメチル化、GCによるFA分析については、実施例1と同様の手法を用いて行った。
(3. Purification of FA)
Sep-Pak Silica (Waters) was equilibrated with 30 ml of chloroform, and the concentrated and dried sample was dissolved in 2 ml of chloroform. The entire sample was loaded onto the column and eluted with the following solvent: 30 ml chloroform (free fatty acid), 10 ml acetone, 20 ml methanol (lysophospholipid). Elution was performed in this order, and fractions were collected for each solvent. It was confirmed that the fatty acid could be purified by TLC under the following conditions. Developing solvent: chloroform / methanol / water = 65: 25: 4 (v / v / v), coloring reagent: 50% sulfuric acid (free fatty acid), Dittmer reagent (lysophospholipid). The chloroform fraction and the acetone fraction were combined, concentrated and dried, and the fatty acid composition at the sn-2 position was analyzed. Further, the methanol fraction was also concentrated and dried, and the fatty acid composition at the sn-1 position was analyzed (the fatty acid liberated by PLA 2 was the second position, and the remaining fatty acid (the fatty acid of lysophospholipid) was the first position fatty acid).
FA methylation and FA analysis by GC were performed in the same manner as in Example 1.
結果:
F26-b株から抽出したリン脂質画分を、HPLCを用いてPC、LPC、PE、PI、PSの各種リン脂質クラスに分離した（図4）。HPLCのクロマトグラムから5分台にPC、10分台にLPC、13分台にPE、18分台にPI、25分台にPSが溶出することがわかった。
表3は、HPLCのAREA比から算出した各リン脂質クラスの割合を示す。
result:
The phospholipid fraction extracted from the F26-b strain was separated into various phospholipid classes of PC, LPC, PE, PI, and PS using HPLC (FIG. 4). From the chromatogram of HPLC, it was found that PC was eluted at 5 minutes, LPC at 10 minutes, PE at 13 minutes, PI at 18 minutes, and PS at 25 minutes.
Table 3 shows the ratio of each phospholipid class calculated from the AREA ratio of HPLC.
また各種リン脂質クラスごとに2位に含まれるFAの組成をGCで分析し、各画分のFAの割合をGC AREAから算出した結果を表4に示す。 Table 4 shows the results of GC analysis of the FA composition contained in the second position for each phospholipid class, and the proportion of FA in each fraction calculated from GC AREA.
さらに、各種リン脂質クラスごとに1位に含まれるFAの組成をGCで分析し、各画分のFAの割合をGC AREAから算出した結果を表5に示す。 Furthermore, the composition of FA contained in the first position for each phospholipid class was analyzed by GC, and the results of calculating the FA ratio of each fraction from GC AREA are shown in Table 5.
HPLC AREAより、F26-b株に含まれるリン脂質は約70%以上がPC画分から構成されており、次いでPE画分が約16%、PS画分が約9%、LPC及びPI画分が共に約3%の順に多く含まれていることが分かった。つまりF26-b株においてPC-DHAが豊富に存在することが確認された。これまでの研究で、DHA含有リン脂質の主な単離源の1つであるムラサキイカの場合、リン脂質中の50%がコリン含有リン脂質であり、そのコリン含有リン脂質に含まれる脂肪酸の50%がDHAであることが報告されている。今回の結果と比較すると、F26-b株はムラサキイカに匹敵するPC-DHAの供給源となりうることが示唆された。 From the HPLC AREA, the phospholipids contained in the F26-b strain consist of about 70% or more of the PC fraction, followed by the PE fraction of about 16%, the PS fraction of about 9%, and the LPC and PI fractions. Both were found to be included in the order of about 3%. That is, it was confirmed that PC-DHA is abundant in the F26-b strain. In previous studies, Murasaki squid, one of the main sources of DHA-containing phospholipids, is choline-containing phospholipids, and 50% of the fatty acids contained in the choline-containing phospholipids. 50% are reported to be DHA. Compared with this result, it was suggested that F26-b strain could be a source of PC-DHA comparable to purple squid.
実施例4
新規PC-DHAの構造決定
（1．HPLCを用いたPC画分の脂肪酸種による分離）
F26-b株に最も多く含まれる、DHA含有PCの構造を決定するため、さらに含有する脂肪酸種によるPCの分離を行った。分離にはMalissa Smith and Firoze B. Jungalwala. 1981. J. Lipid Res. 22: 697-704 中に記載の手法を参照した。
分離条件は以下の通りである。カラム:Inertsil ODS-3 (4.0×250 mm)、移動相:メタノール／1 mM リン酸カリウム緩衝液 (pH 7.4)＝9.5:0.5、検出波長:205 nm、流速:1 ml/min、温度:30℃。
実施例3で回収したPC画分を窒素で乾固し、クロロホルム（適当量、約100〜200μｌ）に溶解した。10μlを上記の条件でHPLCに負荷した。各ピークを回収し、エバポレーターで濃縮した。HPLCのチャートを図5に示す。
Example 4
Structure determination of new PC-DHA (1. Separation of PC fraction by fatty acid species using HPLC)
In order to determine the structure of the DHA-containing PC that is most abundant in the F26-b strain, the PC was further separated by the fatty acid species contained therein. For the separation, the method described in Malissa Smith and Firoze B. Jungalwala. 1981. J. Lipid Res. 22: 697-704 was referred to.
The separation conditions are as follows. Column: Inertsil ODS-3 (4.0 × 250 mm), mobile phase: methanol / 1 mM potassium phosphate buffer (pH 7.4) = 9.5: 0.5, detection wavelength: 205 nm, flow rate: 1 ml / min, temperature: 30 ° C.
The PC fraction collected in Example 3 was dried with nitrogen and dissolved in chloroform (appropriate amount, about 100 to 200 μl). 10 μl was loaded on the HPLC under the above conditions. Each peak was collected and concentrated with an evaporator. The HPLC chart is shown in FIG.
（2．各PC画分の脂肪酸組成分析）
HPLCによって分離した各PC画分において最も占有率の高かった、図5中3番のピークについて、GCを用いて、脂肪酸組成を分析した。GCのチャートを図6に示す。
(2. Fatty acid composition analysis of each PC fraction)
Fatty acid composition was analyzed by GC for the peak No. 3 in FIG. 5, which had the highest occupancy in each PC fraction separated by HPLC. A GC chart is shown in FIG.
（3．FAB-MS測定及びNMR測定）
GC分析により図5中3番ピークにおける脂肪酸組成が、C15:0及びDHAを含むPCであることが確認されたので（図6）、FAB-MS及びNMR測定を行い、構造決定を行った。図7はFAB-MS(negative)、図8はFAB-MS(positive)の結果を示す。また、図9は1H NMR、図10は2次元NMRの結果である。その結果、図11に示すような構造を持つ化合物であることがわかった。測定に際し、Nancy J. Jensen, Kenneth B. Tomer and Michael L. Gross. 1986. Lipids. 21: 580-581及び Holly C. Gaede, Ruth E. Stark. 2001. J. Chem. EduC. 78: 1248-1250を参照した。
今回決定された、2位にDHA、1位にC15:0の奇数の脂肪酸が結合しているPCは世界で最初の発見である。また哺乳類の脳の場合、PLA2の働きでリン脂質から遊離したDHAは核内レセプターのリガンドとして、転写を調節するシグナル分子の働きをもつことが報告されている（de Urquiza AM, Liu S, Sjoberg M, Zetterstrom RH, Griffiths W, Sjovall J, Perlmann T. Science. (2000) 290, 2140）。ラビリンチュラ目の微生物においてホスホリパーゼによってDHAが遊離する場合、哺乳類と同様に原生生物であるラビリンチュラ目の微生物においても、DHAがシグナル分子として働く可能性が示唆される。
(3. FAB-MS measurement and NMR measurement)
GC analysis confirmed that the fatty acid composition at the third peak in FIG. 5 was PC containing C15: 0 and DHA (FIG. 6), and the structure was determined by performing FAB-MS and NMR measurements. FIG. 7 shows the results of FAB-MS (negative), and FIG. 8 shows the results of FAB-MS (positive). FIG. 9 shows the results of 1 H NMR, and FIG. 10 shows the results of two-dimensional NMR. As a result, it was found that the compound had a structure as shown in FIG. In the measurement, Nancy J. Jensen, Kenneth B. Tomer and Michael L. Gross. 1986. Lipids. 21: 580-581 and Holly C. Gaede, Ruth E. Stark. 2001. J. Chem. EduC. 78: 1248- See 1250.
This is the first discovery in the world of a PC with an odd number of fatty acids of DHA in the 2nd position and C15: 0 in the 1st position. In the mammalian brain, DHA released from phospholipids by PLA 2 has been reported to act as a signal molecule that regulates transcription as a ligand for nuclear receptors (de Urquiza AM, Liu S, Sjoberg M, Zetterstrom RH, Griffiths W, Sjovall J, Perlmann T. Science. (2000) 290, 2140). When DHA is liberated by phospholipase in Labyrinthula microorganisms, it is suggested that DHA may act as a signal molecule in probiotic Labyrinthia microorganisms as well as mammals.
実施例5
F26-b株の18S rDNA解析
F26-b株の分子系統解析を行った。菌体から分離、精製した全ゲノムDNAをもとに、PCRで18S rRNA遺伝子(18S rDNA)を増幅した。このPCR産物をシークエンサーで全塩基配列を解析した。得られたF26-b株の18S rDNAの全塩基配列（1755bp、配列番号:1）をもとに、データベース(NCBI blast)で検索したところ、Schizochytrium sp. FJU-512と高い同一性(1744/1757 x100= 99.2601%)を持つことが分かり、また、その分裂様式についても、顕微鏡下の観察で二分裂することが確認されたので、F26-b株はスキゾキトリウム属の菌体であると結論した。図12は、18S rRNA遺伝子について、F26-b株（上段）と、FJU-512（下段）とを比較した図である。また、18S rRNA遺伝子の解析に基づく系統樹を、図13に示す。
Example 5
18S rDNA analysis of F26-b strain
Molecular phylogenetic analysis of F26-b strain was performed. The 18S rRNA gene (18S rDNA) was amplified by PCR based on the whole genomic DNA isolated and purified from the cells. The entire nucleotide sequence of this PCR product was analyzed with a sequencer. Based on the entire base sequence (1755 bp, SEQ ID NO: 1) of the 18S rDNA of the obtained F26-b strain, a database (NCBI blast) was searched, and it was found to be highly identical to Schizochchytrium sp. FJU-512 (1744 / 1757 x100 = 99.2601%), and the division pattern was confirmed to divide into two by observation under a microscope, so that the F26-b strain is a bacterium belonging to the genus Schizochytrium. I concluded. FIG. 12 is a diagram comparing the F26-b strain (upper) and FJU-512 (lower) for the 18S rRNA gene. A phylogenetic tree based on the analysis of 18S rRNA gene is shown in FIG.
Claims (8)
得られた培養物から、長鎖高度不飽和脂肪酸含有リン脂質を採取すること；
を含む、ＰＣ−ＤＨＡを含有する食品又は医薬用組成物の製造方法。 Culturing the microorganism of claim 2 in a medium;
Collecting long-chain polyunsaturated fatty acid-containing phospholipids from the resulting culture;
A method for producing a food or pharmaceutical composition containing PC-DHA.
得られた培養物から、炭素数１６以上で２２以下の不飽和結合２以上の長鎖高度不飽和脂肪酸含有リン脂質を採取すること；
を含む、ＰＣ−ＤＨＡを含有する食品又は医薬用組成物の製造方法。 Culturing the microorganism of claim 2 in a medium;
Collecting a long-chain highly unsaturated fatty acid-containing phospholipid having 2 or more unsaturated bonds having 16 or more and 22 or less carbon atoms from the obtained culture;
A method for producing a food or pharmaceutical composition containing PC-DHA.
得られた培養物から、炭素数２０以上で２２以下の不飽和結合２以上の長鎖高度不飽和脂肪酸含有リン脂質を採取すること；
を含む、ＰＣ−ＤＨＡを含有する食品又は医薬用組成物の製造方法。 Culturing the microorganism of claim 2 in a medium;
Collecting a long-chain highly unsaturated fatty acid-containing phospholipid having 2 or more unsaturated bonds having 20 or more and 22 or less carbon atoms from the obtained culture;
A method for producing a food or pharmaceutical composition containing PC-DHA.
得られた培養物から、ＤＨＡ含有リン脂質を採取すること；
を含む、ＰＣ−ＤＨＡを含有する食品又は医薬用組成物の製造方法。 Culturing the microorganism of claim 2 in a medium;
Collecting DHA-containing phospholipids from the resulting culture;
A method for producing a food or pharmaceutical composition containing PC-DHA.
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