JPH03183498A - Production of beta-carotene - Google Patents
Production of beta-caroteneInfo
- Publication number
- JPH03183498A JPH03183498A JP32087689A JP32087689A JPH03183498A JP H03183498 A JPH03183498 A JP H03183498A JP 32087689 A JP32087689 A JP 32087689A JP 32087689 A JP32087689 A JP 32087689A JP H03183498 A JPH03183498 A JP H03183498A
- Authority
- JP
- Japan
- Prior art keywords
- carotene
- cells
- culture
- algae
- beta
- 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.)
- Pending
Links
- OENHQHLEOONYIE-UKMVMLAPSA-N all-trans beta-carotene Natural products CC=1CCCC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C OENHQHLEOONYIE-UKMVMLAPSA-N 0.000 title claims abstract description 55
- 235000013734 beta-carotene Nutrition 0.000 title claims abstract description 55
- TUPZEYHYWIEDIH-WAIFQNFQSA-N beta-carotene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)CCCC1(C)C)C=CC=C(/C)C=CC2=CCCCC2(C)C TUPZEYHYWIEDIH-WAIFQNFQSA-N 0.000 title claims abstract description 55
- 239000011648 beta-carotene Substances 0.000 title claims abstract description 55
- 229960002747 betacarotene Drugs 0.000 title claims abstract description 55
- OENHQHLEOONYIE-JLTXGRSLSA-N β-Carotene Chemical compound CC=1CCCC(C)(C)C=1\C=C\C(\C)=C\C=C\C(\C)=C\C=C\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C OENHQHLEOONYIE-JLTXGRSLSA-N 0.000 title claims abstract description 55
- 238000004519 manufacturing process Methods 0.000 title claims description 15
- 238000012258 culturing Methods 0.000 claims abstract description 16
- 229910001508 alkali metal halide Inorganic materials 0.000 claims abstract description 15
- 150000008045 alkali metal halides Chemical class 0.000 claims abstract description 15
- 241000195628 Chlorophyta Species 0.000 claims abstract description 13
- 239000013505 freshwater Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 10
- 241000195585 Chlamydomonas Species 0.000 abstract description 7
- 239000001963 growth medium Substances 0.000 abstract description 7
- 239000011780 sodium chloride Substances 0.000 abstract description 5
- 239000003513 alkali Substances 0.000 abstract 1
- 150000004820 halides Chemical class 0.000 abstract 1
- 241000195493 Cryptophyta Species 0.000 description 21
- 239000002609 medium Substances 0.000 description 21
- 239000000243 solution Substances 0.000 description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 230000035508 accumulation Effects 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000195649 Chlorella <Chlorellales> Species 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- 241000195620 Euglena Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- UPYKUZBSLRQECL-UKMVMLAPSA-N Lycopene Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1C(=C)CCCC1(C)C)C=CC=C(/C)C=CC2C(=C)CCCC2(C)C UPYKUZBSLRQECL-UKMVMLAPSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 241001148696 Stichococcus Species 0.000 description 2
- 230000005587 bubbling Effects 0.000 description 2
- 150000001746 carotenes Chemical class 0.000 description 2
- 235000005473 carotenes Nutrition 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000007154 intracellular accumulation Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- NCYCYZXNIZJOKI-UHFFFAOYSA-N vitamin A aldehyde Natural products O=CC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-UHFFFAOYSA-N 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000488537 Bracteacoccus Species 0.000 description 1
- 241000488538 Bracteacoccus minor Species 0.000 description 1
- 241000218637 Chlamydomonas nivalis Species 0.000 description 1
- 241000195634 Dunaliella Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000237502 Ostreidae Species 0.000 description 1
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910001514 alkali metal chloride Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- CBGUOGMQLZIXBE-XGQKBEPLSA-N clobetasol propionate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H](C)[C@@](C(=O)CCl)(OC(=O)CC)[C@@]1(C)C[C@@H]2O CBGUOGMQLZIXBE-XGQKBEPLSA-N 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006266 etherification reaction Methods 0.000 description 1
- 239000000576 food coloring agent Substances 0.000 description 1
- 230000026030 halogenation Effects 0.000 description 1
- 238000005658 halogenation reaction Methods 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229940112877 olux Drugs 0.000 description 1
- 235000020636 oyster Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 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）
本発明は、β−カロチンの製造法に関し、さらに詳しく
は淡水性緑藻類をハロゲン化アルカリ金属濃度が０．１
５Ｍ以上の培地で培養することにより、β−カロチンを
細胞内にＭ積することを特徴とするβ−カロチンの製造
方法に関する。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing β-carotene, and more specifically, the present invention relates to a method for producing β-carotene, and more specifically, the present invention relates to a method for producing β-carotene.
The present invention relates to a method for producing β-carotene, which is characterized in that β-carotene is accumulated in cells by culturing in a medium of 5M or more.
（従来の技術）
β−カロチンはカロチノイドに属する色素で、藻類にお
いては強光によって生成される電子を受容することによ
って細胞内での過度の活性酸素の生成を阻害している物
質である。食品用色素として用いられ、また最近では健
康食品としても利用されている。従来、β−カロチンは
合成法（特公昭５９−１．０６５９号）により生産され
ていたが、最近では合成品の安全性への不安から天然品
指向が強まり、天然β−カロチンとして海水性の藻類ド
ナリエラ（Ｄｕｎａｌｉｅｌｌａ）から抽出されたもの
が利用されているが、β−カロチン製造のためには４Ｍ
という高濃度のＮａＧを含む培地の調製が必要でありＣ
Ｊ、Ｐｈｙｃｏｌｏｇｙ、 １８．５２９−５３７（１
９８２）） 、 コストが高くなる欠点があった。(Prior Art) β-carotene is a pigment belonging to carotenoids, and in algae, it is a substance that inhibits the production of excessive active oxygen within cells by accepting electrons generated by strong light. It is used as a food coloring, and recently it has also been used as a health food. In the past, β-carotene was produced by a synthetic method (Japanese Patent Publication No. 1.0659/1982), but recently, due to concerns about the safety of synthetic products, there has been a growing preference for natural products, and natural β-carotene has been produced using seawater-based methods. Extracted from the algae Dunaliella is used, but 4M is used for β-carotene production.
It is necessary to prepare a medium containing a high concentration of NaG.
J, Physics, 18.529-537 (1
982)), had the disadvantage of high cost.
（発明が解決しようとする課題）
そこで本発明者らは、淡水性の緑藻類をハロゲン化アル
カリ金属濃度が０．１５Ｍ以上の培養液で培養すること
によりβ−カロチンが細胞内に多量に？！ｆ積されるこ
とを見い出し、収量の優れたβ−カロチンの製造方法、
また細胞重量当り０．１％以上のβ−カロチンを含む淡
水性緑藻類を完成するに到った。(Problem to be Solved by the Invention) Therefore, the present inventors have investigated the possibility of culturing freshwater green algae in a culture medium containing an alkali metal halide concentration of 0.15M or more, thereby increasing the amount of β-carotene within the cells. ! A method for producing β-carotene with an excellent yield by discovering that the f product is
Furthermore, freshwater green algae containing 0.1% or more of β-carotene per cell weight have been completed.
（課題を解決するための手段）
かくして本発明によれば、淡水性緑藻類をハロゲン化ア
ルカリ金属濃度が０．１５Ｎ以上の培養液で培養するこ
とにより、β−カロチンを細胞内に蓄積することを特徴
とするβ−カロチンの製造方法、また細胞重量当り ０
．１％以上のβ−カロチンを含む淡水性緑藻類が提供さ
れる。(Means for Solving the Problems) Thus, according to the present invention, accumulation of β-carotene in cells can be prevented by culturing freshwater green algae in a culture solution with an alkali metal halide concentration of 0.15N or more. Characterized method for producing β-carotene, and 0 per cell weight
．． Freshwater green algae containing 1% or more of β-carotene are provided.
藻類の培養は大別して２つの方法がある。一つは無菌的
に培養漕等で培養する方法であり、もう一つは無菌に保
つことをせず、プール、池、湖等により大規模に培養す
る方法である。品質の安定のためには無菌的な培養方法
が好ましいが、コストを下げる点からは無菌的でない培
養方法が好ましい。There are two main methods for culturing algae. One is to culture aseptically in a culture tank or the like, and the other is to culture on a large scale in a pool, pond, lake, etc. without maintaining sterility. Although aseptic culturing methods are preferable for stable quality, non-sterile culturing methods are preferable from the viewpoint of reducing costs.
本発明に用いることができる淡水性の単細胞の緑藻類で
、例えば、タラミドモナス
（Ｃｈｌａｍ ｄｏｍｏｎａｓ ）属、クロレラ（Ｃｈ
ｌｏｒｅｌｌａ ）属、ユーグレナ（いｔｌｕ）属、プ
ラクテアコツカス（ｒａｃｔｅａ ｏｃｃｕｓ ）属、
シリンドロシステス（’ ｄ ｏｃ ｓ ’ｓ）属、ス
ティココッカス（５ｅｉｃｈｏｃｏｃｃｕｓ）属などが
ある。これらの藻類は有菌状態で培養した場合には他の
菌類の増殖により死滅することが多く、無菌的に培養す
ることが好ましい。Freshwater unicellular green algae that can be used in the present invention include, for example, the genus Thalamydomonas, Chlorella
lorella) genus, Euglena (itlu) genus, lactea occus genus,
These include the genus Cylindrocystes ('docs') and the genus Stichococcus. When these algae are cultured in a sterile state, they often die due to the growth of other fungi, so it is preferable to culture them in an aseptic manner.
なお、これらの藻類は、例えば、米国テキサス大学オー
スチン校のコレクション（［ＩＴＥＸＣＯＬＬＥＣＴＩ
ＯＮ）などから容易に入手できる。In addition, these algae are available from the collection of the University of Texas at Austin ([ITEX COLLECTI]), for example.
It is easily available from sources such as ON.
培養にあたっては、培地は本発明に用いることができる
藻類が増殖しうる培地であれば特に限定されない。例え
ば、ＴＡＰ培地、ＭＩＮ培地などがある。In culturing, the medium is not particularly limited as long as the algae that can be used in the present invention can grow. Examples include TAP medium and MIN medium.
培養には、静置培養でも構わないが、培地や培養液中の
酸素濃度、二酸化炭素濃度を上げた方が増殖がよく、バ
ブリングを行ったり、振盪培養を行うことが好ましい。Although static culture may be used for culturing, increasing the oxygen concentration and carbon dioxide concentration in the medium or culture solution improves growth, and it is preferable to perform bubbling or shaking culture.
ＮａＣＱ濃度が０．１５Ｍ以上の培養液によるβ−カロ
チンの蓄積を促進する培養においても、増殖に適さない
ため増殖はしないが、十分にβ−カロチンを生合成する
まで、細胞が生存できるように、バブリングを行ったり
、振盪培養を行うことが好ましい。Even in a culture that promotes β-carotene accumulation using a culture solution with a NaCQ concentration of 0.15M or more, cells do not proliferate because they are not suitable for proliferation, but cells can survive until sufficient β-carotene is biosynthesized. It is preferable to perform bubbling or shaking culture.
本発明に用いることのできる藻類の培養温度は増殖およ
びβ−カロチンの生合成に適した温度であれば特に限定
されない。また、本発明に用いられる藻類は増殖、β−
カロチンの生合成のためには光照射が必要である。照射
する光はβ−カロチンの生合成に適した光であれば、特
に限定されない。例えば、白色光を用いることができる
。照射する光の強度も本発明に用いられる藻類の増殖、
β〜カロチンの生合成に適したものであれば特に限定さ
れない。The culture temperature of algae that can be used in the present invention is not particularly limited as long as it is suitable for growth and biosynthesis of β-carotene. In addition, the algae used in the present invention can be used for proliferation, β-
Light irradiation is necessary for carotene biosynthesis. The light to be irradiated is not particularly limited as long as it is suitable for biosynthesis of β-carotene. For example, white light can be used. The intensity of the irradiated light also affects the growth of algae used in the present invention,
There is no particular limitation as long as it is suitable for the biosynthesis of β-carotene.
本発明に用いる藻類の内、ブラシノコツカス属、プラク
テアコツカス属、シリンドロシスナス属。Among the algae used in the present invention, the genus Brassinococcus, the genus Prakteacoccus, and the genus Cylindrocysnus.
およびスティココッカス属などに属する属する藻類、ク
ラミドモナス属、クロレラ属、およびユーグレナ属など
に属する藻類の一部は、一般に、培養液が凍結したり、
２５℃を越えると死滅することが多いので、培養環境は
０℃以上（ただし、 ０℃の場合は培養液が凍結しない
場合に限る）２５℃以下に保つことが好ましい。増殖、
およびβ−カロチンの生産能のためには、 ４℃以上１
５℃以下に保つことが好ましく、特に８℃以上１２℃以
下が好ましい。また、これらの藻類は７０００１ ｕｘ
未満では増殖能・カンタザンチンの生産量が低下し、
４００００１ｕｘを越えるとカンタザンチンの有無に係
わらず細胞内で活性酸素が発生するため死滅しやすい。In general, the culture solution of some algae belonging to the genus Stichococcus, Chlamydomonas genus, Chlorella genus, and Euglena genus is frozen,
If the temperature exceeds 25°C, the cells often die, so it is preferable to keep the culture environment at 0°C or higher (however, if the temperature is 0°C, this is only if the culture solution does not freeze) and below 25°C. proliferation,
For β-carotene production capacity, the temperature should be 4°C or higher1.
It is preferable to maintain the temperature at 5°C or lower, particularly preferably 8°C or higher and 12°C or lower. Also, these algae are 70001 ux
If it is less than that, the proliferation ability and canthazanthin production decrease
If the concentration exceeds 400,001 ux, active oxygen is generated within the cells regardless of the presence or absence of canthazanthine, and cells tend to die.
そのため、好ましい光の強度は７０００１ｕｘ以上４０
０００１ｕｘ以下であり、特に好ましくは１００００１
ｕｘ以上２００００１ ｕｘ以下である。Therefore, the preferred light intensity is 70001ux or more 40
0001ux or less, particularly preferably 100001
ux or more and 200001 ux or less.
本発明に用いる藻類の内、クラミドモナス属、クロレラ
属、およびユーグレナ属などに属する藻類は、上記の一
部を除き、一般に２５℃を越えても４０″Ｃ以下であれ
ば、死滅することはほとんどないが、４０℃を越えると
死滅しやすい。そのため、 ０℃以上（ただし、 ０℃
の場合には凍結しない場合に限る）４０℃以下に保つこ
とが好ましく、増殖、β−カロチンの生産能のためには
、 ４℃以上３５℃以下に保つことが好ましく、特に８
℃以上１２℃以下が好ましい。また、これらの藻類では
、適する光の強度が異なり、照射する光の強度がｌｏｏ
ｏｌｕｘ未満では増殖能・生産量が低下する。２０００
０１ｕｘを越えると細胞内で活性酸素が発生するため死
滅する。そのため、好ましい光の強度は３０００１ｕｘ
以上１５０００１ｕｘ以下であり、特に好ましくは４０
００１ｕｘ以上１３０００１ｕｘ以下である。Among the algae used in the present invention, algae belonging to the genus Chlamydomonas, Chlorella, and Euglena, with the exception of some of the above, generally rarely die if the temperature exceeds 25°C but is below 40"C. However, if the temperature exceeds 40℃, they will easily die.Therefore, if the temperature exceeds 0℃ (but 0℃
It is preferable to keep the temperature at 40°C or lower (limited to cases where it is not frozen), and for growth and β-carotene production ability, it is preferable to keep it at a temperature of 4°C or higher and 35°C or lower, especially when 8°C
The temperature is preferably 12°C or higher. In addition, the intensity of light suitable for these algae differs, and the intensity of light irradiated may vary depending on the type of algae.
If the concentration is less than olux, the proliferation ability and production amount will decrease. 2000
If it exceeds 0.01 ux, active oxygen is generated within the cells and the cells die. Therefore, the preferred light intensity is 30001ux
more than 150,001 ux, particularly preferably 40
001ux or more and 130001ux or less.
β−カロチンの生合成の促進に用いる細胞は増殖期の細
胞でも飽和期の細胞でも構わない。効率の点から細胞数
は多い方が好ましい。The cells used to promote β-carotene biosynthesis may be cells in the proliferation phase or cells in the saturation phase. From the viewpoint of efficiency, it is preferable to have a large number of cells.
これらの細胞を培地からハロゲン化アルカリ金属濃度が
０．１５Ｍ以上の培養液に移す。ここでいう培養液は、
細胞が培養ｗＪ閏中に生存し続けられるものであれば、
特に限定されない。生存し続けられる限りにおいて、増
殖に必要な成分を含む必要はなく、単に純水にハロゲン
化アルカリ金属を溶解したものでも構わない。These cells are transferred from the culture medium to a culture solution with an alkali metal halide concentration of 0.15M or more. The culture solution here is
If the cells can continue to survive in culture wJ,
Not particularly limited. As long as it can continue to survive, it is not necessary to contain components necessary for proliferation, and a solution of an alkali metal halide simply dissolved in pure water may be used.
ハロゲン化アルカリ金属としては、塩化アルカリ金属が
好ましく、特に塩化ナトリウムまたは塩化カリウムが好
ましく、塩化ナトリウムが最も好ましい。As the alkali metal halide, alkali metal chlorides are preferred, particularly sodium chloride or potassium chloride, and sodium chloride is most preferred.
本発明に用いる藻類は淡水性の藻類であり、ハロゲン化
アルカリ金属濃度が高いと死滅することが多くなる。そ
のため、ハロゲン化アルカッ金属濃度は用いる藻類の生
存に影響しないものであることが好ましい。ハロゲン化
アルカリ金属の種類によって、また藻類の種類によって
、生存しうる濃度の上限は異なるが、塩化ナトリウムの
場合は、一般に０．５Ｍ以下であることが好ましく、特
に０．３Ｍ以下であることが好ましい、一方、藻類細胞
は生存している限りにおいて、ハロゲン化アルカリ金属
濃度の高い培養液はどβ−カロチンの細胞内の蓄積が促
進される。上記の観点から、最も好ましいハロゲン化ア
ルカリ金属濃度は、ハロゲン化アルカリ金属の種類によ
って、また、藻類の種類によって異なるが、塩化ナトリ
ウムの場合、−般に０．２５Ｍから０．３５Ｍの範囲に
最適濃度がある場合が多い。The algae used in the present invention are freshwater algae, and if the concentration of alkali metal halide is high, they often die. Therefore, it is preferable that the alkali metal halide concentration does not affect the survival of the algae used. The upper limit of the viable concentration differs depending on the type of alkali metal halide and the type of algae, but in the case of sodium chloride, it is generally preferably 0.5M or less, and particularly preferably 0.3M or less. Preferably, on the other hand, as long as the algal cells are alive, a culture solution with a high concentration of alkali metal halide promotes the accumulation of β-carotene in the cells. From the above point of view, the most preferable alkali metal halide concentration varies depending on the type of alkali metal halide and the type of algae, but in the case of sodium chloride, it is generally optimal in the range of 0.25M to 0.35M. There is often a concentration.
細胞を培養液に移すには、遠心等により培地と細胞を分
離して培地を除去し培養液を加える方法、細胞懸濁液を
高濃度のハロゲン化アルカリ金属を含む液で希釈する方
法等がある。To transfer cells to a culture medium, there are two methods: separating the medium and cells by centrifugation, removing the medium, and adding the culture medium, and diluting the cell suspension with a solution containing a high concentration of alkali metal halide. be.
ハロゲン化アルカリ金属濃度が０ 、１５ｍＭ以上の培
養液で２〜１２日程度培養することにより細胞内にβ−
カロチンの蓄積が促進される。それ以下の培養日数では
Ｍ積置は少なく、それ以上の培養日数ではβ−カロチン
の蓄積量の増加はほとんど認められない。好ましくは８
以上１２日以下がよいが。β-
Accumulation of carotene is promoted. If the number of culture days is less than that, M accumulation is small, and if the number of culture days is more than that, almost no increase in the amount of β-carotene accumulated is observed. Preferably 8
12 days or less is better.
１２日を過ぎても分解等による減少は認められないので
、特に問題はない。Since no decrease due to decomposition etc. is observed even after 12 days, there is no particular problem.
こうして得られた淡水性緑藻類は、β−カロチンを精製
する場合には不純物が少ない、効率がよいということに
より、水産用飼料として細胞のまま利用する場合には飼
料全体の量を増大させないため、好ましくは、細胞重量
当り０．１％以上のβカロチンを含んでいることが好ま
しい。本発明の培養方法によれば、細胞重量当り０．１
％以上のβ−カロチンを含む淡水性緑藻類が容易に得る
ことができる。The freshwater green algae obtained in this way have few impurities and are highly efficient when purifying β-carotene, and when used as aquaculture feed as cells, the total amount of feed does not increase. Preferably, it contains 0.1% or more of β-carotene per cell weight. According to the culture method of the present invention, 0.1 per cell weight
% or more of β-carotene can be easily obtained.
β−カロチンは必要に応じて精製されるが、精製法は特
に限定されない。例えば、β−カロチンをＭ積した細胞
を１０％ＫＯＨで６０℃、２０分間インキュベートし、
エーテルで溶媒抽出し、シリカゲルカラムでアセトンを
含む石油エーテルを溶媒として精製する。β-carotene is purified if necessary, but the purification method is not particularly limited. For example, cells loaded with β-carotene are incubated with 10% KOH at 60°C for 20 minutes,
Solvent extraction with ether and purification using a silica gel column using petroleum ether containing acetone as a solvent.
また、細胞のまま処理せず、あるいは乾燥させる、破砕
するなどの簡単な処理で、例えば、水産用飼料に混入し
て用いることもできる６（発明の効果）
かくして本発明によれば、ハロゲン化アルカリ金属濃度
が０．１５Ｍ以上の培養液で培養することにより、淡水
条件での培養時に比べ効率的に取得できるβ−カロチン
の製造方法、細胞重量当り０．１％以上のβ−カロチン
を含む淡水性緑藻類が提供される。In addition, the cells can be used as they are without being treated, or by simple treatment such as drying or crushing, for example, by mixing them into aquatic feed.6 (Effects of the Invention) Thus, according to the present invention, halogenation A method for producing β-carotene that can be obtained more efficiently by culturing in a culture medium with an alkali metal concentration of 0.15 M or more than when culturing in freshwater conditions, containing 0.1% or more β-carotene per cell weight Freshwater green algae are provided.
（実施例）
以下に実施例を挙げて本発明をさらに具体的に説明する
。(Example) The present invention will be described in more detail with reference to Examples below.
実施例１
クラミドモナス・ラインバーダイ
（Ｃｈｌａｍ ｄｏｍｏｎａｓ ｒｅｉｎｈａｒｄｔｉ
Ｌ ＵＴＥＸ Ｃ０ＬＬＥＣＴＩＯＮ２２４６株）を
、 ５００−のフラスコに入れた３００ｄのＴＡＰ培地
（Ｉｎ当りＮＨ４０Ｊ ０−４ｇ、Ｃａα２０．０３８
５ｇ、Ｍｇ５Ｏａ・７Ｈ２００，１ｇ、 Ｋ２ＨＰＯ
４０，１１９ｇ、 ＫＨ２ＰＯ４０，０８ｇ、
ＥＤＴＡ ５０ｍｇ、 ＺＪＩＳＯＪ ・７Ｈ２
０２２ｍｇ、 Ｈ３ＢＯ３１１，４ｍｇ、ＨｎＣＡ
２・７Ｈ２０５，０８ｅｇ、Ｆｅ５Ｏｒ７Ｈ２０４，９
９ｍｇ、Ｃｏα２・６Ｈ２０１，８１ｍｇ、 Ｃｕ５
Ｏａ・５Ｈ２０１，５７ｍｇ。Example 1 Chlamydomonas reinhardti
LUTEX COLLECTION 2246 strain) was placed in a 500-ml flask in 300d TAP medium (NH40J 0-4g per In, Caα20.038
5g, Mg5Oa・7H200,1g, K2HPO
40,119g, KH2PO40,08g,
EDTA 50mg, ZJISOJ・7H2
022mg, H3BO311.4mg, HnCA
2・7H205,08eg, Fe5Or7H204,9
9mg, Coα2・6H201, 81mg, Cu5
Oa・5H201, 57 mg.
（ＮＨｉ）ｅＭｏｒ０２ａ・４Ｈ２０１，１ｍｇ、
トリス２ 、４２ｇ、 酢酸７１を水に溶解し、
ＰＨ６，８にｔＪＲ整したもの）に細胞濃度が５Ｘ１０
５／ｍ１２になるように播種した。これを７０００１ｕ
ｘの白色光の下、２５℃で回転振盪培養（半径２７ｃＶ
１００ｒｐｍ） Ｌ／た。 ３日後、この細胞を１００
０ Ｘ Ｇで１０分間遠心し、培地を除去し、この細胞
をＴＡＰ培地、および０．３ＭのＮａαを含むＴＡＰ培
地に移す。さらに３日間、７０００１ｕｘの白色光の下
、２５℃で静置培養後、細胞を１００ＯＸＧで１０分間
遠心し、回収後細胞内のβ−カロチン量を高速液体クロ
マトグラフィー法で測定した。(NHi)eMor02a・4H201, 1mg,
Dissolve 42 g of Tris 2 and 71 g of acetic acid in water,
tJR adjusted to pH 6.8) with a cell concentration of 5X10.
The seeds were sown at a density of 5/m12. This is 70001u
Rotary shaking culture (radius 27 cV) at 25 °C under white light at x
100 rpm) L/ta. After 3 days, the cells were divided into 100
Centrifuge for 10 minutes at 0×G, remove the medium, and transfer the cells to TAP medium and TAP medium containing 0.3 M Naα. After static culture at 25° C. under 70,001 ux white light for an additional 3 days, the cells were centrifuged at 100 OXG for 10 minutes, and after collection, the amount of β-carotene in the cells was measured by high performance liquid chromatography.
５Ｘ１０６個の細胞のベレットに５００ ｄの１０％Ｋ
ＯＨ溶液を加え、６０℃で２０分間インキュベートした
。10% K for 500 d on pellets of 5 x 106 cells
OH solution was added and incubated at 60°C for 20 minutes.
２５０Ｉ６ｔのサンプルに１２５−のエチルエーテルを
加え撹拌後これに更に、 ３７５縛の３％のＮａＣＱ溶
液を加えよく撹拌した。これのエーテル層を回収濃縮し
容積を５−にした。これを逆相液体クロマトグラフィー
法で分析する。分析に際してカラムは００５８０−ＴＭ
（東ソー株式会社製）を用い、溶媒としてメタノール
：アセトン：塩化メチレン８： １： １を用いた。細
胞内のβ−カロチン量を第１表に示す。After adding ethyl ether of 125 to a sample of 250I6t and stirring, a 3% NaCQ solution of 375 was further added and stirred thoroughly. The ether layer was collected and concentrated to a volume of 5-. This is analyzed using reverse phase liquid chromatography. The column used for analysis was 00580-TM.
(manufactured by Tosoh Corporation), and methanol:acetone:methylene chloride 8:1:1 was used as the solvent. Table 1 shows the amount of β-carotene in the cells.
ＨＰＬＣ分析において保持時間からβ−カロチンと思わ
れる画分を分取し、減圧乾燥したところ赤外吸収スペク
トルがβ−カロチン標準品（和光紬薬、β−カロチン特
級）と一致した。A fraction believed to be β-carotene was collected from the retention time in HPLC analysis and dried under reduced pressure, and its infrared absorption spectrum matched that of a β-carotene standard product (Wako Tsumugi Pharmaceutical Co., Ltd., β-carotene special grade).
クラミドモナス・ラインバーダイは０．３ＭのＮａＣＱ
存在下で培養することにより、β−カロチンの細胞内蓄
積量が増大することがわかった。Chlamydomonas linebardaii is 0.3M NaCQ
It was found that culturing in the presence of β-carotene increased the intracellular accumulation of β-carotene.
実施例２
クラミドモナス・ニバリス（Ｃｈｌａｍ ｄｏｍｏｎａ
ｓｎｉｖａｌｉｓ、 ＵＴＥＸ Ｃ０ＬＬＥＣＴＩＯ
Ｎ １９６９株）を、細胞濃度が５Ｘ１０５／−になる
ように５００−フラスコに入れｆ： ３００ｄ（７）Ｍ
Ｉ Ｎ培地（１２当すＮＨ２Ｏ０−４ｇ、ＣａＣＪｈ
０．０３８５ｇ、Ｈｇ５Ｏｖ７Ｈ２００，１ｇ、ＫＨ
２Ｏ４０，７１７ｇ、 ＫＨ２ＰＯ４ｏ、３ｓ３．、、
ＥＤＴＡ ５０ｍｇ、 Ｚｎ５Ｏａ・７Ｈ２０２
２ｍｇ、Ｈ３ＢＯ３４ｒｎｇ、 Ｍｎα２・４Ｈ２０
５，０６＋ｆ！、、 Ｆｅ５Ｏａ４Ｈ２０９９ｍｇ、
Ｃｏα２・６Ｈ２０１，６１ｍｇ、 Ｃｕ５Ｏａ
・５１（２０１、５７ｍｇ、（ＮＨ４）ｅＭｏ７０２ａ
・４Ｈ２０１，１ｍｇの割合で水に溶解し、ｐＨを６．
８に調整したもの）に播種し、これを１００００１ｕｘ
の白色光の下、２０℃で回転振盪培養（半径２０ｃｆｆ
ｌ・１００「ＰＩ！＋〉シた。 ７日後、この細胞をＮ
ａＧを含まないＭＩＮ培地、ＮａＣＱを０．１Ｍ含むＭ
ＩＮ培地、Ｎａαを０．２Ｍ含むＭＩＮ培地に移した。Example 2 Chlamydomonas nivalis
snivalis, UTEX COLLECTIO
N 1969 strain) was placed in a 500-flask so that the cell concentration was 5X105/- f: 300d(7)M
IN medium (120-4 g of NH2O, CaCJh
0.0385g, Hg5Ov7H200,1g, KH
2O40,717g, KH2PO4o, 3s3. ,,
EDTA 50mg, Zn5Oa・7H202
2mg, H3BO34rng, Mnα2・4H20
5,06+f! ,, Fe5Oa4H2099mg,
Coα2・6H201,61mg, Cu5Oa
・51 (201, 57mg, (NH4)eMo702a
・Dissolve 1 mg of 4H201 in water and adjust the pH to 6.
8) and sow this at 100001ux.
Rotary shaking culture (radius 20 cff) at 20 °C under white light of
l・100 "PI!+>". After 7 days, this cell was
MIN medium without aG, M containing 0.1M NaCQ
The mixture was transferred to IN medium and MIN medium containing 0.2M Naα.
さらに７日間、上と同じ条件で培養後、細胞を１００Ｏ
ＸＧで１０分間遠心し、回収の後、細胞内のβ−カロチ
ン量を実施例１と同様に高速液体クロマトグラフィー法
で測定した。その結果を第１表に示す。After culturing for another 7 days under the same conditions as above, cells were incubated at 100
After centrifugation at XG for 10 minutes and collection, the amount of intracellular β-carotene was measured by high performance liquid chromatography in the same manner as in Example 1. The results are shown in Table 1.
タラミドマナス・ニバリスは０．１５Ｍ以上のＮａα存
在下で培養することにより、β−カロチンの細胞内蓄積
量が増大することがわかった。It was found that culturing Thalamidomanas nivalis in the presence of 0.15 M or more Naα increases the amount of β-carotene accumulated in the cells.
実施例３
クラミドモナス・イエローストネシス
（Ｃｈ ａｍ ｄｏｍｏｎａｓ ｅ Ｉｏｗ
ｓｔｏｎｅｓｉｓ、 ＵＴＥＸＣＯＬＬＥＣＴＩＯ
Ｎ １９７０株）、シリンドロシステス・レビッソニ（
江住餞■Ｕ牡り吐狙山糧吐量、ＵＴＥＸＣＯＬＬＥＣＴ
ＩＯＮ １２５９株）、スティココッヵス・バチラリス
（Ｓｔ’ｃｈｏｃｏｃｃｕｓ ｕ貝■虹す、 ＵＴＥ
ＸＣＯＬＬＥＣＴＩＯＮ １７８株）、プラクテアコ
ツカス・マイナー（Ｂｒａｃｔｅａｃｏｃｃｕｓ ｍ１
ｎｏｒ、ＵＴＥＸ−Ｃ０ＬＬＥＣＴＩＯＮ６６株） １
１８１１１度が５Ｘ１ｏ５／ｒｎ！２ニナルヨうニ５ｏ
。Example 3 Chlamydomonas yellowstonesis
stonesis, UTEX COLLECTIO
N 1970 strain), Cylindrocystes lebissoni (
Ezumi 餞■U oyster taiyama suction amount, UTEX COLLECT
ION 1259 strain), St'chococcus bacillus (St'chococcus spp.), UTE
XCOLLECTION 178 strains), Bracteacoccus minor (Bracteacoccus m1)
nor, UTEX-C0LLECTION66 stocks) 1
18111 degrees is 5X1o5/rn! 2 ninaruyo uni 5o
.
−のフラスコに入った３００−のＭＩＮ培地に播種し、
５０００１ｕｘの白色光のもとで回転振盪培養（半径
２０ｃｍ・１０１００ｒｐ Ｌ／た。 ７日後、この細
胞を１０００ＸＧで１０分間遠心し、培地を除去し、０
．３８のＮａαを含むＭＩＮ培地、およびＮａαを含ま
ないＭＩＮ培地を加えた。さらに７日後、上と同じ条件
で培養後、細胞を１００ＯＸＧで１０分間遠心し回収の
後、細胞内のβ−カロチン量を実施例１と同様に高速液
体クロマトグラフィー法で測定した。その結果を第１表
に示す。- Seed in 300- MIN medium in a flask,
Rotary shaking culture (radius 20 cm, 10,100 rp L/L) under 50,001 ux of white light. After 7 days, the cells were centrifuged at 1,000 x G for 10 minutes, the medium was removed, and
．． 38 MIN medium with Naα and MIN medium without Naα were added. After another 7 days, after culturing under the same conditions as above, the cells were centrifuged at 100 OXG for 10 minutes and recovered, and the amount of intracellular β-carotene was measured by high performance liquid chromatography in the same manner as in Example 1. The results are shown in Table 1.
淡水性の単細胞の緑藻類は０．１５Ｍ以上のＮａα存在
下でβ−カロチンの細胞内蓄積量が増大することがわか
った。It was found that the intracellular accumulation of β-carotene in freshwater unicellular green algae increases in the presence of 0.15 M or more Naα.
実施例４
０．３ＭのＮａαを含むＴＡＰ培地の代わりに０．３８
のにαを用いるほかは実施例１と同様にβ−カロチンを
製造し、細胞内のβ−カロチン量を測定した。Example 4 Instead of TAP medium containing 0.3M Naα, 0.38
β-carotene was produced in the same manner as in Example 1, except that α was used, and the amount of β-carotene in the cells was measured.
その結果を第１表に示す。The results are shown in Table 1.
クラミドモナス・ラインバーダイは０．３ＭのＫＣＱ存
在下で培養することにより、β−カロチンの細胞内蓄積
量が増大することがわかった。It was found that when Chlamydomonas linebardaii was cultured in the presence of 0.3M KCQ, the amount of β-carotene accumulated in the cells increased.
（以
下
余
白）
第
表
（注）ハロゲン化アルカリ金属は実施例１．２．３では
ＮａＣ５Ｌ、実施例４ではＫＣＱを用いた。(Margin below) Table (Note) As the alkali metal halide, NaC5L was used in Example 1.2.3, and KCQ was used in Example 4.
表中の単位のない数値は細胞重量あたりのβ−カロチン
のしめる割合（％）を示す。Numerical values without units in the table indicate the percentage (%) of β-carotene per cell weight.
Claims (1)
１５Ｍ以上の培養液で培養することにより、β−カロチ
ンを細胞内に蓄積することを特徴とするβ−カロチンの
製造方法。 ２、細胞重量当り０．１％以上のβ−カロチンを含む淡
水性緑藻類の細胞。[Claims] 1. Freshwater green algae with an alkali metal halide concentration of 0.
A method for producing β-carotene, which comprises accumulating β-carotene within cells by culturing in a culture solution of 15M or more. 2. Freshwater green algae cells containing 0.1% or more of β-carotene per cell weight.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32087689A JPH03183498A (en) | 1989-12-11 | 1989-12-11 | Production of beta-carotene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP32087689A JPH03183498A (en) | 1989-12-11 | 1989-12-11 | Production of beta-carotene |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH03183498A true JPH03183498A (en) | 1991-08-09 |
Family
ID=18126251
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP32087689A Pending JPH03183498A (en) | 1989-12-11 | 1989-12-11 | Production of beta-carotene |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH03183498A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010105959A (en) * | 2008-10-30 | 2010-05-13 | Nikken Sohonsha Corp | Composition containing highly pure 9-cis-beta carotene and method for preparing the same |
-
1989
- 1989-12-11 JP JP32087689A patent/JPH03183498A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010105959A (en) * | 2008-10-30 | 2010-05-13 | Nikken Sohonsha Corp | Composition containing highly pure 9-cis-beta carotene and method for preparing the same |
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