JPH0412956B2 - - Google Patents
Info
- Publication number
- JPH0412956B2 JPH0412956B2 JP63262575A JP26257588A JPH0412956B2 JP H0412956 B2 JPH0412956 B2 JP H0412956B2 JP 63262575 A JP63262575 A JP 63262575A JP 26257588 A JP26257588 A JP 26257588A JP H0412956 B2 JPH0412956 B2 JP H0412956B2
- Authority
- JP
- Japan
- Prior art keywords
- abscisic acid
- medium
- culture
- natural abscisic
- corn
- 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.)
- Expired - Lifetime
Links
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 claims description 80
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 claims description 40
- 239000002609 medium Substances 0.000 claims description 38
- 238000004519 manufacturing process Methods 0.000 claims description 28
- 240000008042 Zea mays Species 0.000 claims description 25
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 25
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 25
- 235000005822 corn Nutrition 0.000 claims description 25
- 241001465180 Botrytis Species 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 238000000034 method Methods 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 229910000019 calcium carbonate Inorganic materials 0.000 description 8
- 239000000284 extract Substances 0.000 description 8
- 239000012535 impurity Substances 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 241001672694 Citrus reticulata Species 0.000 description 5
- 239000000654 additive Substances 0.000 description 5
- 238000012258 culturing Methods 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000207199 Citrus Species 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 235000020971 citrus fruits Nutrition 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 239000003375 plant hormone Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 241000123650 Botrytis cinerea Species 0.000 description 2
- 241001157813 Cercospora Species 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000007102 metabolic function Effects 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229930192334 Auxin Natural products 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001064001 Rosisphaerella rosicola Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000002363 auxin Substances 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002595 cold damage Effects 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 1
- 239000004062 cytokinin Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000000469 ethanolic extract Substances 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000005089 fruit drop Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
（産業上の利用分野）
本発明は微生物の代謝機能を利用した天然型ア
ブシジン酸の製造法に関し、生産量を著しく増加
し、かつ夾雑物が少なく精製を容易にできる方法
に係るものである。
（従来の技術）
天然型アブシジン酸は1963年カリフオルニア大
学のアデイコツトらにより棉の幼果の落果促進物
質として初めて単離され、それ以後、種々の植物
細胞に対する重要な生理作用が見い出され、今日
では巾広い生理活性を有する植物ホルモンとして
認められている。
天然型アブシジン酸の植物生理作用の特徴は他
の植物ホルモンであるオーキシン、ジベレリン、
サイトカイニン等の促進作用と拮抗する抑制作用
をもつことにあるが、そのうちでも注目すべき作
用の一つは他の植物ホルモンと異なつて植物気孔
の閉鎖作用を持つことである。将来天然型アブシ
ジン酸はこのような生理作用を利用して、干魃、
冷害等の気候変化に対応できる物質として農業生
産への応用が期待され、さらにビール醸造におけ
る品質向上とか、価格低廉化（特開昭58−101677
号公報参照）等の醸造への利用、人工種子製造に
おける不定胚の発芽促進利用開発等が期待されて
いる。
従来アブシジン酸の製造法としては有機合成的
方法を利用していたものが殆どで、これらの製造
法では光学活性を有する天然型アブシジン酸を得
ることは極めて困難であり、これを得られる特殊
な製造法が考えられ無いでは無いが製造費が高価
となり、上記の農業、醸造とか食品製造に利用す
るにはコスト高になつて実質的に使用不可能であ
つた。
このため、有機合成法に代るものとして微生物
の代謝機能を利用した発酵生産が有力手段として
検討されるに至り、本発明者等の一人によりボト
リチス菌による製造法（特公昭61−35838）、及び
セルコスポラ・ロシコラ［Ｅ xperientia，33，
1556（1977）］とその改良法（特公昭59−45359、
特公昭60−6629）が発明されて公知になつてい
る。さらにボトリチス菌の培養において柑橘類果
皮の添加によりアブシジン酸の生産量が増加する
ことも確認されている。
しかし上記の微生物を利用した製造法の最大の
欠点は発酵生産による天然型アブシジン酸の蓄積
量が極めて低く、かつ発酵速度も遅く、天然型ア
ブシジン酸の工業的規模の生産手段としては極め
て効率の悪い方法である。また柑橘類果皮添加の
場合は夾雑物が多くて精製費が高価になる問題点
がある。
（発明が解決しようとする問題点・本発明の目
的）
本発明者等は、天然型アブシジン酸を効率良く
生産する方法に関して鋭意検討を加え、前記した
問題点を解決することを目的としたものである。
（問題点を解決するための手段）
本発明は、実験の繰り返しにより、不完全菌の
一種であるボトリチス（Botrytis）属に属し、天
然型アブシジン酸生産能を有する菌株を培養する
に当たり、培地中に窒素源および菌の生育あるい
は天然型アブシジン酸の生産に必要な微量成分の
補給源としてコーン、コーン胚芽、ホミニーフイ
ード（とうもろこしヌカ）を単独にまたは２種以
上混合して添加することにより天然型アブシジン
酸の生産量が増加し、また添加物によつては生産
量が多少劣つても夾雑物が少なく、精製費用を大
巾に低下させ得ること、および充分工業化し得る
ことを見い出して本発明を完成した。
すなわち本発明は、ボトリチス属に属し、天然
型アブシジン酸生産能を有する菌株を前記した剤
を添加した培地に接種し、適切な条件で培養し、
該培養物中より天然型アブシジン酸を採取するこ
とを特徴とする天然型アブシジン酸の製造方法で
ある。
以下本発明の詳細を述べる。
本発明に使用する菌株はボトリチス属に属する
天然型アブシジン酸生産菌であり、これには通常
の変異菌や変異処理によつて生じた菌株をも含む
ものとする。
ボトリチス菌に属する天然型アブシジン酸生産
菌種の具体例として、ボトリチス・シネレア
（Botrytis cinerea）微工研菌寄第6156号（特公
昭61−35838）があげられる。
本発明は、後に記載するように前記した培地に
前記した剤を添加し、適切な方法によつて滅菌
し、前記のボトリチス属に属する天然型アブシジ
ン酸生産菌株の胞子又は菌糸体部分の水懸濁液を
培地1g当たり１×102個細胞以上接種し培養する。
培養は静置培養、振とう培養、若しくは通気培養
で行う。培養条件は温度10〜35℃、好ましくは20
〜30℃、培地のPHは３〜12、好ましくは４〜８、
期間は１〜15日間、好ましくは３〜９日間であ
る。期間は培養状態により決める。
培養終了後、有機化合物の精製法を応用するこ
とも可能であり、一般的な精製法により単離精製
することが可能である。
（原料菌株の液体培養の検討）
現在のところ天然型アブシジン酸の微生物生産
はボトリチス菌とセルコスポラ菌のみで報告され
ている。このうちセルコスポラ菌は液体培養で実
施されているがその培養液からのアブシジン酸の
収量は低くて採算に合わない。
ボトリチス菌はこれまで天然型アブシジン酸を
固体培養でよく生産するが、液体培養では殆んど
生産しないとされていた。
本発明に用いる培地は固体培地、液体培地のい
ずれでも良い。
しかし液体培養が培養管理上極く有利であるか
ら、本発明の検討に当たりボトリチス菌が液体培
養で天然型アブシジン酸を生産するかを検討し
た。本検討の終始は次の通りである。
最近に本発明者の一人が他の者との共同研究に
より、フスマ熱水抽出物とみかん果皮熱水抽出物
を添加した培地により液体培養したところ高率の
収量を得た（未公知）。本発明者等はこれとは別
個に液体培養について検討し、前記のみかん果皮
の他に本発明の培地添加物であるコーン、ホミニ
ーフイード、コーン胚芽を添加し、従来の無添加
培地による培養とを比較検討した。
その比較検討に使用した培地と培養手順は、バ
レイシヨ抽出液の４倍希釈液（この100ml中には
グルコース0.5g、バレイシヨ5g相当分の抽出物を
含む）に、コーンスチープリカー0.1g、炭酸カル
シウム2gを加えた培地80mlを300ml容三角フラス
コに分注し、120℃20分間滅菌したものを対照培
地（基本培地）とした。この他に、みかん果皮添
加培地を第２対照培地とした。先の基本培地（対
照培地）に、コーン、コーン胚芽、ホミニーフイ
ードの一種又は二種類以上添加した本発明の培地
（添加物の添加量は表−１に示す）を調製し、こ
れらの二種の対照培地と本発明の培地とにボトリ
チス・シネレア菌（FERM、Ｐ−6156）の胞子
又は菌糸体の濃厚懸濁液を培地１ml当たり１×
102個細胞以上を接種し、25℃で７日間回転振と
う培養（200rpm）を行ない、得られた培養物を
前記と同様に処理した結果を表−１に示す。
(Industrial Application Field) The present invention relates to a method for producing natural abscisic acid using the metabolic functions of microorganisms, and relates to a method that can significantly increase the production amount, contain few impurities, and facilitate purification. (Prior art) Natural abscisic acid was first isolated in 1963 by Adikotsuto et al. of the University of California as a substance that promotes fruit drop in young cotton fruits.Since then, it has been discovered that it has important physiological effects on various plant cells, and today it is known as abscisic acid. It is recognized as a plant hormone with a wide range of physiological activities. The plant physiological action of natural abscisic acid is characterized by the presence of other plant hormones such as auxin, gibberellin,
It has an inhibitory effect that antagonizes the promoting effect of cytokinins, etc., but one of its most noteworthy effects is that, unlike other plant hormones, it has a closing effect on plant stomata. In the future, natural abscisic acid will utilize these physiological effects to help with drought,
It is expected to be applied to agricultural production as a substance that can cope with climate changes such as cold damage, and it can also be used to improve the quality of beer brewing and reduce the price (Japanese Patent Application Laid-Open No. 58-101677
It is expected to be used in brewing (see the above publication), and to promote the germination of somatic embryos in the production of artificial seeds. Conventionally, most methods for producing abscisic acid have used organic synthesis methods, and it is extremely difficult to obtain optically active natural abscisic acid using these production methods. Although the production method is not inconceivable, the production cost is high, and it is practically impossible to use it for the above-mentioned agriculture, brewing, or food production. For this reason, fermentation production using the metabolic functions of microorganisms has been considered as a promising alternative to organic synthesis methods, and one of the present inventors has developed a production method using Botrytis bacteria (Japanese Patent Publication No. 61-35838). and Cercospora rosicola [E xperientia, 33,
1556 (1977)] and its improved method (Special Publication No. 59-45359,
Japanese Patent Publication No. 60-6629) was invented and is now well known. Furthermore, it has been confirmed that the production of abscisic acid increases when citrus peel is added to the culture of Botrytis bacteria. However, the biggest drawback of the above production method using microorganisms is that the amount of natural abscisic acid accumulated through fermentation production is extremely low, and the fermentation rate is also slow, making it extremely inefficient as a means of producing natural abscisic acid on an industrial scale. That's a bad method. In addition, when citrus peel is added, there is a problem that there are many impurities and the refining costs are high. (Problems to be Solved by the Invention/Object of the Invention) The present inventors have conducted intensive studies on a method for efficiently producing natural abscisic acid, and have aimed to solve the above-mentioned problems. It is. (Means for Solving the Problems) The present invention, through repeated experiments, has revealed that, in culturing a strain belonging to the genus Botrytis, which is a type of Deuteromycetes, and having the ability to produce natural abscisic acid, Natural abscisin can be produced by adding corn, corn germ, and hominy feed (corn bran) singly or in a mixture of two or more as a nitrogen source and a supplementary source of trace components necessary for bacterial growth or production of natural abscisic acid. The present invention was developed based on the discovery that the production amount of acid can be increased, and even if the production amount is slightly inferior depending on the additive, there are fewer impurities, and that the refining cost can be greatly reduced, and that it can be fully industrialized. completed. That is, the present invention involves inoculating a strain belonging to the genus Botrytis and having the ability to produce natural abscisic acid into a medium supplemented with the above-mentioned agent, culturing it under appropriate conditions,
This is a method for producing natural abscisic acid, which is characterized by collecting natural abscisic acid from the culture. The details of the present invention will be described below. The strain used in the present invention is a naturally occurring abscisic acid-producing bacterium belonging to the genus Botrytis, and includes normal mutant bacteria and strains produced by mutation treatment. A specific example of a naturally occurring abscisic acid-producing bacterial species belonging to the Botrytis bacterium is Botrytis cinerea, Microtechnical Research Institute No. 6156 (Japanese Patent Publication No. 61-35838). The present invention involves adding the above-mentioned agent to the above-mentioned medium as described later, sterilizing it by an appropriate method, and suspending the spores or mycelium of the above-mentioned naturally occurring abscisic acid-producing strain belonging to the genus Botrytis in water. Inoculate the suspension at 1×10 2 or more cells per gram of medium and culture.
Culture is performed by static culture, shaking culture, or aeration culture. Culture conditions are temperature 10-35℃, preferably 20℃
~30°C, pH of the medium is 3-12, preferably 4-8,
The period is 1 to 15 days, preferably 3 to 9 days. The period is determined depending on the culture condition. After completion of the culture, it is also possible to apply a purification method for organic compounds, and it is possible to isolate and purify by a general purification method. (Study of liquid culture of raw material strains) Currently, microbial production of natural abscisic acid has been reported only in Botrytis and Cercospora bacteria. Among these, Cercospora bacteria are cultured in liquid, but the yield of abscisic acid from the culture solution is low and unprofitable. Until now, Botrytis bacteria was thought to produce natural abscisic acid well in solid culture, but hardly to produce it in liquid culture. The medium used in the present invention may be either a solid medium or a liquid medium. However, since liquid culture is extremely advantageous in terms of culture management, in studying the present invention, we investigated whether Botrytis bacteria can produce natural abscisic acid in liquid culture. The details of this study are as follows. Recently, one of the inventors of the present invention, in collaboration with others, obtained a high yield when liquid culturing was carried out using a medium to which a hot water extract of bran and a hot water extract of mandarin peel were added (unknown). Separately, the present inventors investigated liquid culture, and added corn, hominy feed, and corn germ, which are the medium additives of the present invention, in addition to the above-mentioned tangerine peel, thereby making it different from culture using conventional additive-free medium. A comparative study was conducted. The medium and culture procedure used for the comparative study were a 4-fold dilution of potato extract (100 ml contains extract equivalent to 0.5 g of glucose and 5 g of potato), 0.1 g of corn steep liquor, and calcium carbonate. 80 ml of the medium to which 2 g was added was dispensed into a 300 ml Erlenmeyer flask and sterilized at 120°C for 20 minutes, which was used as a control medium (basic medium). In addition, a medium supplemented with mandarin peel was used as a second control medium. A medium of the present invention was prepared by adding one or more types of corn, corn germ, and hominy feed to the above basic medium (control medium) (the amounts of additives are shown in Table 1). A concentrated suspension of spores or mycelium of Botrytis cinerea (FERM, P-6156) was added to the control medium and the medium of the present invention at 1× per ml of medium.
At least 10 2 cells were inoculated, cultured with rotational shaking (200 rpm) at 25°C for 7 days, and the resulting culture was treated in the same manner as described above. The results are shown in Table 1.
【表】
上表で分かるように第１対照の無添加培地によ
る天然型アブシジン酸生産量と第２対照を含むコ
ーン、ホミニーフイード、コーン胚芽を添加した
培地による生産量とは格段の違いがあつたが、培
養液の着色情況はみかん果皮を用いた第２対照の
場合は、顕著な着色と濁りが認められ、コーン、
ホミニーフイード、コーン胚芽を添加物とした培
養液は僅かしか着色せず濁りも少ないから精製上
で極めて有利で実用的と考えられた。
（本発明の固体培養による天然型アブシジン酸の
定量）
フスマ10g、炭酸カルシウム1g、水10mlを500
ml容三角フラスコ中で撹拌混合し、120℃20分間
加熱滅菌したものを対照培地とし、フスマ7g、
炭酸カルシウム1g、コーンフラワー3g、水10ml
を500ml容三角フラスコ中で撹拌混合し、120℃20
分間加熱滅菌したものを対照培地とする。次
に、フスマ、炭酸カルシウム、水からなる基本培
地にコーン、コーン胚芽、ホミニーフイード（と
うもろこしヌカ）の一種又は二種以上を添加した
培地を本発明の培地〜とし（培地組成は表−
２に示す）前記と同様に加熱滅菌して調製する。
これ等の対照培地，と本発明の培地〜と
にボトリチス、シネレア菌（FERM、Ｐ−6156）
の胞子又は菌糸体の濃厚懸濁液を培地1g当たり
１×102個細胞以上を接種し、25℃で７日間静置
培養した。
培養終了後、各試験区の培養物を夫々20倍量の
水、エタノールまたはメタノール中に約２時間浸
漬抽出し、次いで、水、エタノールまたはメタノ
ール抽出液を減圧濃縮した濃縮液をPH9.0に調節
し、酢酸エチルで抽出洗浄した。抽出から残つた
水溶液をPH3.0に調節した後、酢酸エチルで抽出
し、得られた酢酸エチル液を無水硫酸ナトリウム
で乾燥後濃縮し、酢酸エチル可溶性酸性区分を
得、その一定量をシリカゲル薄層にスポツトし、
酢酸エチル１：ｎ−ヘキサン１の溶媒で展開して
天然型アブシジン酸の蓄積量を表−２の通り定量
した。[Table] As can be seen from the table above, there was a marked difference between the production amount of natural abscisic acid in the first control medium without additives and the production amount in the second control medium containing corn, hominy feed, and corn germ. However, in the case of the second control using mandarin peel, the coloring of the culture solution was markedly colored and turbid, and corn,
A culture solution containing hominy feed or corn germ as an additive is only slightly colored and has little turbidity, so it is thought to be extremely advantageous and practical for purification. (Quantification of natural abscisic acid by solid culture of the present invention) 10 g of bran, 1 g of calcium carbonate, and 10 ml of water
A control medium was prepared by stirring and mixing in a ml Erlenmeyer flask and sterilizing the mixture by heating at 120°C for 20 minutes.
1g calcium carbonate, 3g cornflour, 10ml water
Stir and mix in a 500ml Erlenmeyer flask and heat at 120℃20
The control medium is one that has been heat sterilized for a minute. Next, a medium in which one or more types of corn, corn germ, and hominy feed (corn bran) were added to a basic medium consisting of bran, calcium carbonate, and water was used as the medium of the present invention (medium composition is shown in Table 1).
2) Prepare by heat sterilization in the same manner as above.
These control media, and the media of the present invention include Botrytis and Cinerea (FERM, P-6156).
A concentrated suspension of spores or mycelium was inoculated at 1×10 2 or more cells per gram of medium, and cultured stationary at 25° C. for 7 days. After culturing, the culture of each test plot was extracted by immersion in 20 times the volume of water, ethanol, or methanol for about 2 hours, and then the water, ethanol, or methanol extract was concentrated under reduced pressure to a pH of 9.0. The mixture was adjusted and extracted and washed with ethyl acetate. After adjusting the aqueous solution remaining from the extraction to PH3.0, it was extracted with ethyl acetate, and the obtained ethyl acetate solution was dried over anhydrous sodium sulfate and concentrated to obtain an ethyl acetate-soluble acidic fraction, and a certain amount of it was poured into a silica gel thin layer. Spotted on the layer,
It was developed with a solvent of ethyl acetate 1:n-hexane 1, and the accumulated amount of natural abscisic acid was quantified as shown in Table 2.
【表】【table】
【表】
上記の表−２によつて明らかであるように、本
発明の剤を加えないで炭酸カルシウムとフスマの
みを添加して調製した培地からなる対照区及び
炭素源であるコーンフラワーを添加した対照区
、本発明の実施例区〜の天然型アブシジン
酸の生産蓄積量は表−２に記載の通りで、対照区
の蓄積量８mgに対して実施例区〜は約17倍
〜14倍、対照区の蓄積量40mgに対して実施例区
〜は約3.5倍〜2.8倍であつて、窒素源および
菌の生育あるいは天然型アブシジン酸の生産に必
要な微量成分の補給源としてコーン、コーン胚
芽、ホミニーフイード（とうもろこしヌカ）の一
種又は二種以上添加した培地が天然型アブシジン
酸の製造に極めて適合することを前記の定量によ
り確認し得た。前記は標品（米国シグマケミカル
社製）と比較して確認したものである。
（実施例）
フスマ7g、みかん果皮（乾燥）3g、炭酸カル
シウム1g、水10mlを500ml容三角フラスコに分注
し120℃20分加圧滅菌した対照培地と表−３に示
す実施例区〜の通りの組成にした本発明の培
地とにより前記と同じ手順に従つて培養、抽出、
精製を行つて天然型アブシジン酸の生成を定量し
た。その結果を表−３に示した。[Table] As is clear from Table 2 above, a control group consisting of a medium prepared by adding only calcium carbonate and bran without the addition of the agent of the present invention, and a control group consisting of a medium prepared by adding only calcium carbonate and bran, and a control group consisting of a medium prepared by adding only calcium carbonate and bran, and a control group containing a medium prepared by adding corn flour as a carbon source The accumulated amount of natural abscisic acid produced in the control plot and the example plot of the present invention is as shown in Table 2, and the accumulation amount in the example plot is about 17 to 14 times that of 8 mg in the control plot. The accumulation amount in the example plots was approximately 3.5 to 2.8 times that of 40 mg in the control plot, and corn and corn were used as a nitrogen source and a supplementary source of trace components necessary for bacterial growth and production of natural abscisic acid. It was confirmed by the above quantitative determination that the medium containing one or more of germ and hominy feed (corn bran) is extremely suitable for producing natural abscisic acid. The above was confirmed by comparing with a standard product (manufactured by Sigma Chemical Co., USA). (Example) 7 g of bran, 3 g of tangerine peel (dried), 1 g of calcium carbonate, and 10 ml of water were dispensed into a 500 ml Erlenmeyer flask and sterilized under pressure at 120°C for 20 minutes.The control medium and the example groups shown in Table 3 were used. Cultivation, extraction and
Purification was performed and the production of natural abscisic acid was quantified. The results are shown in Table-3.
【表】
実施例区〜での培養物からの抽出液は、対
照区の抽出液と比較して色度（430nmに於ける吸
光度）において約1/9、乾物量において約1/4であ
つて、アブシジン酸以外の夾雑物の量が格段に低
く、精製費用の低廉さが収量の若干の低さを補つ
て余り有ることを確認し得た。
（作用及び効果）
本発明は前記によつて明らかにしたように、天
然型アブシジン酸生産能を持つ菌体を培養する培
地に窒素源および菌の生育あるいは天然型アブシ
ジン酸の生産に必要な微量成分の補給源としての
コーン、コーン胚芽、ホミニーフイード（とうも
ろこしヌカ）を単独にまたは２種以上混合して添
加することにより天然型アブシジン酸を、他の培
地により生産するに比して著増することが出来
た。柑橘類果皮を培地に添加するときは生産量の
み増加することが出来るが、この時は夾雑物が多
いため抽出液を濁らせかつ精製を困難にする。し
かるに本発明によつて生産した培養物には夾雑物
がほとんどなく澄んだ抽出液が得られ、若干の生
産量不足を充分に補うことが出来る効果を持つ。[Table] The extracts from the cultures in the example plots ~ have approximately 1/9 of the chromaticity (absorbance at 430 nm) and approximately 1/4 of the dry matter content compared to the extracts of the control plots. It was confirmed that the amount of impurities other than abscisic acid was extremely low, and that the low purification cost more than compensated for the slightly low yield. (Functions and Effects) As clarified above, the present invention provides a medium for culturing bacterial cells capable of producing natural abscisic acid, including a nitrogen source and a trace amount necessary for bacterial growth or production of natural abscisic acid. By adding corn, corn germ, and hominy feed (corn bran) as supplementary sources of ingredients, either singly or in combination of two or more, natural abscisic acid can be significantly increased compared to production using other media. was completed. When citrus peel is added to the culture medium, only the production can be increased, but in this case there are many impurities that make the extract cloudy and difficult to purify. However, the culture produced according to the present invention has almost no impurities and a clear extract can be obtained, which has the effect of being able to sufficiently compensate for the slight production shortage.
Claims (1)
産能を有する菌株を培地に培養し、培養物中に天
然型アブシジン酸を蓄積せしめ、該培養物から天
然型アブシジン酸を採取して天然型アブシジン酸
を製造するに際し、培地中に窒素源および菌の生
育あるいは天然型アブシジン酸の生産に必要な微
量成分の補給源としてコーン、コーン胚芽、ホミ
ニーフイード（とうもろこしヌカ）を単独にまた
は２種以上混合して添加することを特徴とする天
然型アブシジン酸の製造方法。1. A strain belonging to the genus Botrytis and having the ability to produce natural abscisic acid is cultured in a medium, natural abscisic acid is accumulated in the culture, and natural abscisic acid is collected from the culture to produce natural abscisic acid. During production, corn, corn germ, and hominy feed (corn bran) are added to the culture medium, either singly or in a mixture of two or more, as a nitrogen source and a supplementary source of trace components necessary for bacterial growth or production of natural abscisic acid. A method for producing natural abscisic acid, characterized by:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26257588A JPH02109988A (en) | 1988-10-18 | 1988-10-18 | Production of natural type abscisic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26257588A JPH02109988A (en) | 1988-10-18 | 1988-10-18 | Production of natural type abscisic acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02109988A JPH02109988A (en) | 1990-04-23 |
| JPH0412956B2 true JPH0412956B2 (en) | 1992-03-06 |
Family
ID=17377713
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26257588A Granted JPH02109988A (en) | 1988-10-18 | 1988-10-18 | Production of natural type abscisic acid |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02109988A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5299240A (en) * | 1976-02-16 | 1977-08-19 | Fuji Kouso Kk | Edible culture medium |
| JPS6135838A (en) * | 1984-07-28 | 1986-02-20 | Katsuhiko Ichikawa | Method and apparatus for preparing straw ash for tea ceremony |
| JPS63148980A (en) * | 1986-12-12 | 1988-06-21 | Okumoto Seifun Kk | Production of spongy grain wastage pellet |
-
1988
- 1988-10-18 JP JP26257588A patent/JPH02109988A/en active Granted
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5299240A (en) * | 1976-02-16 | 1977-08-19 | Fuji Kouso Kk | Edible culture medium |
| JPS6135838A (en) * | 1984-07-28 | 1986-02-20 | Katsuhiko Ichikawa | Method and apparatus for preparing straw ash for tea ceremony |
| JPS63148980A (en) * | 1986-12-12 | 1988-06-21 | Okumoto Seifun Kk | Production of spongy grain wastage pellet |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02109988A (en) | 1990-04-23 |
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