【０００１】[0001]
【産業上の利用分野】本発明は、微生物を利用したパル
ミトオレイン酸の製造方法に関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing palmitooleic acid using a microorganism.
【０００２】[0002]
【従来の技術】パルミトオレイン酸は不飽和脂肪酸の一
つであり、これまでに抗腫瘍効果、血管障害防護作用等
の優れた薬理作用、生理作用を示すことが明らかにされ
ており、今後ますます医薬品、化粧品、食品分野での利
用が期待されている。このパルミトオレイン酸の工業的
な製造方法としては、マカデミアナッツ等のナッツ類か
らの抽出による方法や、酵母からの抽出による方法（特
開昭62-257391 号等) が知られている。2. Description of the Related Art Palmitooleic acid is one of unsaturated fatty acids and has been shown to exhibit excellent pharmacological and physiological actions such as an antitumor effect and a protective effect against vascular disorders. It is increasingly expected to be used in the pharmaceutical, cosmetic and food fields. As an industrial production method of palmitooleic acid, a method by extraction from nuts such as macadamia nuts and a method by extraction from yeast (Japanese Patent Application Laid-Open No. 62-257391) are known.
【０００３】ところが、マカデミアナッツから抽出され
る全脂肪酸中のパルミトオレイン酸の含有率は約20％に
過ぎない。また従来用いられている酵母の場合にも、全
脂肪酸中のパルミトオレイン酸の含有率は30〜45％程度
に止まっている。このため、高純度のパルミトオレイン
酸を得るためには脂肪酸をマカデミアナッツや酵母から
抽出した後、パルミトオレイン酸のみを分画分取する複
雑な操作が必要であった。However, the content of palmito oleic acid in the total fatty acids extracted from macadamia nuts is only about 20%. In addition, in the case of a conventionally used yeast, the content of palmitooleic acid in all fatty acids is limited to about 30 to 45%. For this reason, in order to obtain high purity palmitooleic acid, a complicated operation of extracting and separating only palmitooleic acid after extracting fatty acids from macadamia nuts or yeast was required.
【０００４】[0004]
【発明が解決しようとする課題】本発明は上記した従来
の問題点を解決して、従来よりも簡単かつ安定に高純度
のパルミトオレイン酸を製造することができるパルミト
オレイン酸の製造方法を提供するためになされたもので
ある。SUMMARY OF THE INVENTION The present invention solves the above-mentioned conventional problems and provides a method for producing palmitooleic acid which can more easily and stably produce high-purity palmitooleic acid. It was made to provide.
【０００５】[0005]
【課題を解決するための手段】上記の課題を解決するた
めになされた本発明は、酵母の一種である、含有全脂肪
酸中の６０％以上の割合でパルミトオレイン酸を含有す
るクリベロマイセス・ポリスポラス（Kluyveromyces po
lysporus) を培地で培養することにより、菌体内にパル
ミトオレイン酸を蓄積させ、これを採取することを特徴
とするものである。Means for Solving the Problems] The present invention has been made in order to solve the aforementioned problem, is a type ofyeast, containing total fat
Contains palmito oleic acid in more than 60% of the acid
That Kluyveromyces Porisuporasu (Kluyveromyces po
lysporus) in a culture medium, thereby accumulating palmitooleic acid in the cells and collecting the palmitooleic acid.
【０００６】従来、パルミトオレイン酸含有酵母として
知られていたものは、サッカロマイセス属（Saccharomy
ces)、キャンディダ属(Candida) 、ロドトルラ属(Rhodo
torula) 等であるが、本発明者はスクリーニングを行っ
た結果、これらとは全く異なるクリベロマイセス・ポリ
スポラス（Kluyveromyces polysporus) が、全脂肪酸中
の60％という高い割合でパルミトオレイン酸を含有する
ことを発見した。そしてこのクリベロマイセス・ポリス
ポラスを炭素源、窒素源、無機塩類、成長因子等を含む
栄養培地または合成培地により培養し、菌体内にパルミ
トオレイン酸含有油を蓄積させ、これを採取することに
よって高純度のパルミトオレイン酸が得られることを確
認した。[0006] Conventionally, yeast known as palmitooleic acid-containing yeast is of the genus Saccharomyces.
ces), Candida, Rhodotorula (Rhodo
As a result of the screening, the present inventors have found that Kluyveromyces polysporus, which is completely different from these, contains palmitooleic acid in a high proportion of 60% of the total fatty acids. discovered. Then, the Kluyveromyces polysporus is cultured in a nutrient medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, growth factors, and the like. It was confirmed that palmitooleic acid was obtained.
【０００７】本発明では、菌株としてクリベロマイセス
・ポリスポラス（Kluyveromyces polysporus) IFO 0996
またはこれを親株として変異処理を施したものを用いる
ことができる。なお、この菌株は市販されているので容
易に入手可能である。変異誘起手法としては、紫外線ま
たはγ線などの照射や、ニトロソグアニジン、EMF など
の薬剤処理が挙げられる。In the present invention, Kluyveromyces polysporus IFO 0996 is used as a strain.
Alternatively, a mutant obtained by subjecting the parent strain to a mutation treatment can be used. In addition, since this strain is commercially available, it can be easily obtained. Examples of the mutagenesis method include irradiation with ultraviolet rays or γ rays, and treatment with drugs such as nitrosoguanidine and EMF.
【０００８】前記した培養培地中の炭素源としては、ブ
ドウ糖、ショ糖、糖蜜等の糖質、乳酸、酢酸等の有機
酸、エタノール等のアルコール類を用いる。窒素源とし
ては、硫酸アンモニウム、リン酸アンモニウム、尿素、
アンモニア等あるいはカザミノ酸、ペプトン、肉エキス
等の有機窒素源を用いる。さらにリン酸塩、硫酸塩、マ
グネシウム塩、カリウム塩、鉄塩、マンガン塩、亜鉛
塩、その他無機塩類を必要に応じて添加する。As the carbon source in the culture medium, sugars such as glucose, sucrose and molasses, organic acids such as lactic acid and acetic acid, and alcohols such as ethanol are used. Nitrogen sources include ammonium sulfate, ammonium phosphate, urea,
Ammonia or an organic nitrogen source such as casamino acid, peptone or meat extract is used. Further, phosphates, sulfates, magnesium salts, potassium salts, iron salts, manganese salts, zinc salts, and other inorganic salts are added as necessary.
【０００９】このような培地で培養することにより菌体
内にパルミトオレイン酸を蓄積させた酵母菌体中から、
公知の方法で脂質を抽出することができる。例えば、湿
菌体を凍結乾燥後、クロロホルム：メタノール混合溶液
や、ｎ−ヘキサン：イソプロパノル混合溶液等で抽出す
る方法を採ることができる。また、湿菌体をエタノール
等の極性溶媒とともに機械的破砕した後、ｎ−ヘキサ
ン：イソプロパノル混合溶液等で抽出する方法を採って
もよい。このようにしてパルミトオレイン酸高含有油を
生産することができる。[0009] By culturing in such a medium, the yeast cells having palmitooleic acid accumulated in the cells,
The lipid can be extracted by a known method. For example, a method may be employed in which the wet cells are lyophilized and then extracted with a mixed solution of chloroform: methanol or a mixed solution of n-hexane: isopropanol. Alternatively, a method may be employed in which the wet cells are mechanically crushed together with a polar solvent such as ethanol and then extracted with a mixed solution of n-hexane and isopropanol. Thus, palmito oleic acid-rich oil can be produced.
【００１０】[0010]
【実施例】次に本発明を実施例によって更に詳細に説明
する。 〔実施例１〕ポリペプトン５g/L 、酵母エキス３g/L 、
麦芽エキス３g/L 、ブドウ糖10g/Lの組成の培地100mL
の入った容量500mL のフラスコに、７種類のパルミトオ
レイン酸生産酵母をそれぞれ植菌し、振盪数150/m 、温
度30℃で48時間培養した。培養液から菌体を水洗分離し
て乾燥菌体を得た。この乾燥菌体にクロロホルム：メタ
ノール混合溶液を加え、常温で３時間、抽出を３回繰り
返し行った。得られた抽出油の脂肪酸組成比をガスクロ
マトグラフィーにより分析した結果、表１に示すとおり
の結果となった。Next, the present invention will be described in more detail by way of examples. [Example 1] Polypeptone 5 g / L, yeast extract 3 g / L,
Malt extract 3g / L, glucose 10g / L composition medium 100mL
Each of seven kinds of palmitooleic acid-producing yeasts was inoculated into a flask having a capacity of 500 mL and cultured at a shaking frequency of 150 / m at a temperature of 30 ° C. for 48 hours. The cells were washed with water and separated from the culture solution to obtain dried cells. A mixed solution of chloroform and methanol was added to the dried cells, and extraction was repeated three times at room temperature for 3 hours. As a result of analyzing the fatty acid composition ratio of the obtained extracted oil by gas chromatography, the results shown in Table 1 were obtained.
【００１１】[0011]
【表１】[Table 1]
【００１２】上記の表１から、本発明のクリベロマイセ
ス・ポリスポラス（K.polysporus）においては全脂肪酸
中にパルミトオレイン酸（C16:1）が61.9％を占め、他
の菌株に比較してパルミトオレイン酸の含有率がはるか
に大きく、かつ乾燥菌体当たりの脂肪酸含有率も高いこ
とがわかる。From Table 1 above, palmitooleic acid (C16: 1 ) accounts for 61.9% of the total fatty acids in K. polysporus of the present invention, and palmitooleic acid (C16: 1 ) accounts for more than other strains. It can be seen that the content of toleic acid is much higher and the content of fatty acid per dried cell is high.
【００１３】〔実施例２〕実施例１に示したと同一の培
地３Ｌの入ったジャーファーメンタ（容量５Ｌ）にクリ
ベロマイセス・ポリスポラス（Kluyveromyces polyspor
us) IFO 0996を植菌し、攪拌数400rpm、温度30℃にて48
時間培養した。pHの変動に対しては、塩酸または水酸化
ナトリウムを添加してpH6.0 に保った。培養液から遠心
分離した菌体を乾燥し、この乾燥菌体12.68gに対してク
ロロホルムとメタノールとの混合液(2:1) を20倍量添加
し、常温にて４時間、抽出を３回繰り返しパルミトオレ
イン酸を含む油1.06g を得た。この油中のパルミトオレ
イン酸の含有率をガスクロマトグラフィー分析した結
果、61.3％であった。Example 2 Kluyveromyces polysporus (Kluyveromyces polysporus) was placed in a jar fermenter (5 L capacity) containing 3 L of the same medium as shown in Example 1.
us) Inoculate IFO 0996, stirring at 400 rpm, temperature 30 ° C, 48
Cultured for hours. For pH fluctuation, the pH was maintained at 6.0 by adding hydrochloric acid or sodium hydroxide. The cells centrifuged from the culture solution are dried, and a mixed solution (2: 1) of chloroform and methanol (20: 1) is added to 12.68 g of the dried cells, and extraction is performed three times at room temperature for 4 hours. 1.06 g of oil containing palmito oleic acid was obtained repeatedly. Gas chromatographic analysis of the palmito oleic acid content in the oil showed 61.3%.
【００１４】[0014]
【発明の効果】以上に詳細に説明したように、本発明は
含有全脂肪酸中パルミトオレイン酸含有率が６０％以上
である脂肪酸を菌体内に蓄積できるクリベロマイセス・
ポリスポラスを用いたので、従来法のような酵母抽出液
からパルミトオレイン酸を分画分取する複雑な操作を必
要とせず、はるかに簡単な工程でかつ安定してパルミト
オレイン酸を製造することができる。また乾燥菌体当た
りの脂肪酸含有率も高く、工業的な生産に適した方法で
ある。As explained in detail above, the present invention
Palmito oleic acid content intotal fatty acidsis 60% or more
Which can accumulate fatty acids thatare
The use of polysporus eliminates the need for complicated procedures for fractionating and separating palmitooleic acid from yeast extracts as in the conventional method, and enables palmitooleic acid to be produced stably and in a much simpler process. be able to. In addition, the fatty acid content per dry cell is high, and this method is suitable for industrial production.
───────────────────────────────────────────────────── フロントページの続き (58)調査した分野(Int.Cl.7，ＤＢ名) C12P 7/64 ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ) ＢＩＯＳＩＳ（ＤＩＡＬＯＧ) ＷＰＩ（ＤＩＡＬＯＧ)──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl.7 , DB name) C12P 7/64 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP05048112AJP3090810B2 (en) | 1993-03-09 | 1993-03-09 | Method for producing palmito oleic acid |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP05048112AJP3090810B2 (en) | 1993-03-09 | 1993-03-09 | Method for producing palmito oleic acid |
| Publication Number | Publication Date |
|---|---|
| JPH06253872A JPH06253872A (en) | 1994-09-13 |
| JP3090810B2true JP3090810B2 (en) | 2000-09-25 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP05048112AExpired - Fee RelatedJP3090810B2 (en) | 1993-03-09 | 1993-03-09 | Method for producing palmito oleic acid |
| Country | Link |
|---|---|
| JP (1) | JP3090810B2 (en) |
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| JPH06253872A (en) | 1994-09-13 |
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