CN1182798A - Method for producing natural abscisic acid by fungus fermentation - Google Patents
Method for producing natural abscisic acid by fungus fermentation Download PDFInfo
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- CN1182798A CN1182798A CN 96117784 CN96117784A CN1182798A CN 1182798 A CN1182798 A CN 1182798A CN 96117784 CN96117784 CN 96117784 CN 96117784 A CN96117784 A CN 96117784A CN 1182798 A CN1182798 A CN 1182798A
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Abstract
The present invention belongs to a new method for producing natural active abscisic acid by means of gungus fermentation. Said invention is characterized by improving existent abscisic acid bactreia, changing formula of culture medium, changing liquid batch fermentation process to continuous-flow feeding and discharging process, adopting inoculation fixation and adding key substrate to stabilize acid yield so as to greatly raise yield, reduce nergy consumption and dosage of raw material, and greatly reduce production cost and provide possibility for industrial scale production.
Description
The invention belongs to the fermentation engineering of producing natural active dormin with the fungi fermentation method.
Dormin (Abscisic Acid is called for short ABA) is one of five big plant hormone of having found in the world at present.The ABA of natural type is owing to have very strong regulation activity to growing of farm crop, can promote the maturation of fruit class, cereal, beans to grow, can increase substantially its output and quality, can strengthen greatly again that it is cold-resistant, drought resisting and salt resistance ability, thereby have broad application prospects.At present, ABA has been deep into vegetable cell and genetically engineered level in the research of basic field.Yet, owing to be present in the intravital natural radioactivity ABA optical configuration of plant only is (S)-(+)-ABA, the production cost of simple (S)-(+)-ABA is high, fetch long price, and the ABA of synthetic, what obtain is the Racemid type, active ABA much smaller than natural type, therefore, ABA be applied to agriculture production almost be the empty talk.In order to solve and to satisfy the problem that ABA is applied to agriculture production, recent two decades comes, and abroad begins to utilize microbe fermentation method to produce natural active dormin.1977, Italy G.Assante at first adopts Cercospora Rosicola fermentative production natural active dormin, be mainly solid fermentation, but because its output is very low, its commodity price very high (193.4 dollars/milligram), nineteen eighty-two, but I finds the luxuriant Shanxi of ball of Japan to utilize fungi Botrytis Cinerea production natural radioactivity ABA, and has applied for patent, thereafter, in 1987,1988, nineteen ninety, the Song Benjun first-class of toray company and new Japanese chemical industrial company, produce natural active dormin with the Botrytis bacterial strain in succession, six patents have been applied for, production technique is respectively solid fermentation, also educates the liquid shaking bottle fermentation, has adopted the Mierocrystalline cellulose foam to make the liquid fermenting of carrier simultaneously, fermentation yield rises substratum by 15-20mg/ and brings up to 23.8-85mg/ and rise fermented liquid, and the highest output has reached 300mg/L.But above patent also exists: 1. fermentation yield is low; 2. zymotechnique still is confined to solid fermentation and liquid batch fermentation, does not make full use of the characteristic that thalline produces dormin continuously.Thalline is grown in a fixed culture environment, produces acid, when product (dormin) when reaching finite concentration, produce restraining effect, thalline is secretory product no longer, causes the waste of nutritive substance, thalline and the energy, output can not be improved, and production efficiency is low, the cost height.Still can not solve the problem that is applied in the agriculture production.
Task of the present invention is to seek the novel process that can increase substantially dormin output, reduces the production cost of dormin significantly, makes commercial scale production become possibility.
Technical solution of the present invention:
1. transforming existing dormin generation bacterial strain is superior strain, increases substantially the fermentation activity of bacterial classification.
2. changing liquid batch zymotechnique is (initial PH control, PH control in vegetative period, PH control production phase, fermentation end of a period phase PH control) zymotechnique of the continuous flow feeding of immobilized thallus, discharging, PH control.
3. select to be more suitable in thalli growth, produce the culture medium prescription of acid.
4. by adding the pathways metabolism of crucial substrate, the fermentation of week control thalline, increase substantially dormin output.
The bacterial classification that the present invention uses is Botrytis Cinerea-Cercospoxa RosicolaFD338 (being called for short B.C.FD338).This bacterium is to carry out carrying out the high yield dormin bacterial strain that protoplastis merges and the correlated inheritance mutagenic treatment is obtained with another dormin generation bacterium Cercospora Rosicola216 again after the protoplastis mutagenesis with Botrytis bacterial strain Botrytis CinereaT-1.
The present invention adopts three cascade supervention ferment, according to three grades of different requirements, has selected three kinds of different culture medium A, B, C.It consists of: culture medium A:
Glucose 0.2-2% extractum carnis 0.1-1% yeast extract paste 0.1-1%
Seminose 0.1-0.5% wort 5-20% corn steep liquor 0.001-0.05%
(or sucrose 0.1-0.5%, rhamnosyl 0.1-0.5%, peptone 0.1-1%
Glycerine 0.1-1%, vitamin H 0.001-0.05%)
NH
4NO
3?0.1-0.5％?MgSO
4?0.05-0.3％，KCl?0.1-0.3％
K
2HPO
40.05-0.5% substratum B:
Rice bran juice 20-70% (or starch 0.5-5%, dextrin 0.1-2%)
Waste molasses 0.5-7% (or cellobiose 0.5-5%, or lactose 0.5-5%
Or semi-lactosi 0.5-5%, sucrose 0.1-3%)
Soybean cake powder 0.1-2% (or groundnut meal 0.1-2%, or cottonseed meal 0.1-3%)
L-glutamic acid 0.01-0.5%, (NH
4)
2SO
40.01-0.8% (or urea 0.1-1%)
MgSO
40.05-0.3% KCl 0.1-0.3% VitB1 0.0001-0.05% culture medium C:
Dextrin 0.1-3% wheat bran juice 20-70% (or Semen Maydis powder 0.1-3%)
Glucose 0.5-5% (or sucrose 0.5-1.0% or waste molasses 0.5-7% or lactose
0.1--7%, or semi-lactosi 0.1-7%
Cottonseed meal 0.1-2% (or soybean cake powder 0.1-8%, whey 0.1-9%,
Or groundnut meal 0.1-8%)
Citrus juice 0.5-5% (or cellobiose 0.5-15%, or glycerine 0.1-5%,
Or soya-bean oil 0.5-5%, or Trisodium Citrate 0.1-5%)
L-glutamic acid 0.01-5% (or halfcystine 0.01-5%, or L-glutamic acid+halfcystine
0.01-5％)
(NH
4)
2SO
40.01-5% (or NH
4NO
30.1-7%, or NaNO
30.1-7%,
Or ammonia 0.1-7%)
VitB1 0.001-0.5% (or zeatin 0.001-0.5%)
MgSO
4?0.05-0.5％ NaCl?0.05-0.5％
FeSO
4?0.05-0.5％ CuSO
4?0.05-0.5％
Trace elements such as boron, molybdenum, cobalt, nickel (0.1-100 μ mol/L)
Fermenting process of the present invention is:
With the activation after bacterial classification inoculation in culture medium A, in triangular flask after shake-flask culture 24-72 hour, be inoculated in the seeding tank that is placed with substratum B fermentation culture 24-60 hour with the inoculum size of 5-10%, still be inoculated in the three grade fermemtation jar then and ferment by the inoculum size of 5-10%.The three grade fermemtation jar as fermented nutritive liquid, and adds the micropore ceramics material with culture medium C in tank body, (or micropore nethike embrane, cinder, polyurethane foam, brick slag or sodium alginate, polyvinyl alcohol, gelatin etc.) the thalline immobilization is handled.In the three grade fermemtation process, when treating that thalli growth enters stationary phase, reduce biomass growth rate by reduction dissolved oxygen concentration, reduction leavening temperature means, and continuous lower concentration fed-batch medium C and interpolation precursor substrate, discharging at regular time and quantity.With the fermented liquid organic solvent extractionprocess after the fermentation, the ion exchange column method after silica gel column layer folding method and active carbon adsorption etc. extract, promptly gets white pure product dormin.
The precursor substrate that adds at fermentation system mainly contains: imidazoles, morpholine, pyridine and derivative thereof, ethanamide, amine acetate, β. β-two formylamino acid, Sour quinoline, any in the mevalonic acid.
Reagent used in the leaching process is: methyl alcohol, ethanol, ethyl acetate, acetone, chloroform, ether, cyclohexane, lower alcohols such as sherwood oil.The used resin of ion exchange column is a strong base anion resins, also can be macroporous resin.Fermentation condition: temperature: 10 ℃ of-40 ℃ of PH:3-12
The PH regulation and control are with the stream ammonification or add CaCO
3Or the mode that adds NaOH, KOH is carried out:
Fermentation time: 1-30 days, precursor substrate addition: 0.1-16%
Whole process flow of the present invention is seen accompanying drawing.
Advantage of the present invention:
1. produce the domestic and international at present high 3-4 of production peak that reports of output times (Japanese Patent is up to the 300mg/L fermented liquid, and this bacterial classification B.C.FD338 output is the 900-1200mg/L fermented liquid) of bacterial classification;
2. fermentation system adopts immobilization technology, has avoided the loss of thalline in continuously feeding discharging process, makes product acid amount stable;
3. fermentation system can be stablized 10-30 days in the product acid phase, thereby has improved production efficiency greatly, has reduced energy consumption and raw material usage quantity, and production cost is reduced significantly.
4. the interpolation of key precursor substrate has promoted the fermentating metabolism forward to carry out smoothly, has increased substantially output.
Embodiment:
With 10 of 1000ml triangular flasks, every bottled 300ml culture medium A is in 120 ℃ of sterilizations, and after the cooling, the B.C.FD338 bacterium spore suspension after the inoculation activation places on 25 ℃ of shaking tables under-28 ℃ of temperature shake-flask culture 24-72 hour.
Adorn in the 100 liter fermentor tanks of 50 liters of substratum B in cultured primary seed solution is inoculated in by 5% inoculum size, aeration-agitation was cultivated 24-60 hour under 24-28 ℃ of temperature.
Do three grades of jar fermentations with 1 ton of fermentor tank.Dress culture medium C 750 liters and micropore ceramics pearl (particle diameter 0.9-160mm in jar
31.0-5g/L), behind conventional hot high pressure vapor sterilization, by 5% inoculum size inoculation secondary seed solution, aeration-agitation 48 hours, feed ammonia then PH is controlled at 4-8, reduce dissolved oxygen concentration (reduction air flow), reduce leavening temperature to 10-25 ℃ of velocity flow and add culture medium C, with the ratio adding mevalonic acid of 0.1%-16% with 0.01-5L/ hour, make fermentation system in stable condition, once every discharging in 10 hours.The liquid material that gone out is reclaimed product in the fermented liquid with ion-exchange-resin process, and use washing with alcohol, must white crystals shape dormin product.Specific rotation [α]
D 28=+419 °.
After testing: fermentation system is through 25 days stable product acid, and output can reach 1.2 gram dormins/rise fermented liquid.Reclaim through ion exchange method, product recovery rate reaches 80%.
Claims (7)
1 usefulness fungi fermentation is produced the novel method of natural active dormin, its feature: three cascade supervention ferment, fungi immobilization technology are adopted in fermentation.In third stage fermenting process, wait stable product acid amount by control nutritive ingredient (fed-batch medium C), control dissolved oxygen concentration, temperature, PH, and discharging at regular time and quantity.Extraction process adopts organic solvent extraction, ion-exchange, column chromatography and absorption method.Whole process flow is seen accompanying drawing.
2 methods according to the described fungi fermentation production of claim 1 natural active dormin, it is characterized in that: it is A that the substratum of three cascade supervention ferment is respectively the one-level substratum, and secondary medium is B, and three-stage culture medium is C, and it consists of: culture medium A:
Glucose 0.2-2% extractum carnis 0.1-1% yeast extract paste 0.1-1%
Seminose 0.1-0.5% wort 5-20% corn steep liquor 0.001-0.05%
(or sucrose 0.1-0.5%, rhamnosyl 0.1-0.5%, peptone 0.1-1%
Glycerine 0.1-1%, vitamin H 0.001-0.05%)
NH
4NO
3?0.1-0.5％MgSO
4?0.05-0.3％，KCl?0.1-0.3％
K
2HPO
40.05-0.5% substratum B:
Rice bran juice 20-70% (or starch 0.5-5%, dextrin 0.1-2%)
Waste molasses 0.5-7% (or cellobiose 0.5-5%, or lactose 0.5-5%
Or semi-lactosi 0.5-5%, sucrose 0.1-3%)
Soybean cake powder 0.1-2% (or groundnut meal 0.1-2%, or cottonseed meal 0.1-3%)
L-glutamic acid 0.01-0.5% (NH
4)
2SO
40.01-0.8% (or urea 0.1-1%)
MgSO
40.05-0.3% KCl 0.1-0.3% VitB1 0.0001-0.05% culture medium C:
Dextrin 0.1-3% wheat bran juice 20-70% (or Semen Maydis powder 0.1-3%)
Glucose 0.5-5% (or sucrose 0.5-1.0% or waste molasses 0.5-7% or lactose
0.1--7%, or semi-lactosi 0.1-7%
Cottonseed meal 0.1-2% (or soybean cake powder 0.1-8%, whey 0.1-9%,
Or groundnut meal 0.1-8%)
Citrus juice 0.5-5% (or cellobiose 0.5-15%, or glycerine 0.1-5%,
Or soya-bean oil 0.5-5%, or Trisodium Citrate 0.1-5%)
L-glutamic acid 0.01-5% (or halfcystine 0.01-5%, or L-glutamic acid+halfcystine
0.01-5％)
(NH
4)
2SO
40.01-5% (or NH
4NO
30.1-7%, or NaNO
30.1-7%,
Or ammonia 0.1-7%)
VitB1 0.001-0.5% (or zeatin 0.001-0.5%)
MgSO
4?0.05-0.5％ NaCl?0.05-0.5％
FeSO
4?0.05-0.5％ CuSO
40.05-0.5％
Micro-0.1-100 μ mol/L such as boron, molybdenum, cobalt, nickel
3 novel methods according to the described fungi fermentation production of claim 2 natural active dormin, it is characterized in that: first order seed is inoculated in secondary seed, and secondary seed is inoculated in the three grade fermemtation jar, and its inoculum size is 5-10%.
4 novel methods by claim 1 or 2 described fungi fermentation production PBI 58 is characterized in that: the material that is used for fixing fungi is micropore ceramics (or sodium alginate, polyvinyl alcohol, gelatin, micropore nethike embrane, clinker brick slag, polyurethane foam etc.).
5 novel methods according to the described fungi fermentation production of claim 3 PBI 58, it is characterized in that: the precursor substrate that adds in the three grade fermemtation system is mainly: imidazoles, morpholine, pyridine and derivative thereof, ethanamide, amine acetate, β, β-two formylamino acids; Sour quinoline, any in the mevalonic acid etc., addition are 0.1-16%.
6 novel methods by claim 1 or 5 described fungi fermentations production dormins, it is characterized in that: in the extraction process, used eluent is a lower alcohols.
7 novel methods by claim 2 or 4 described fungi fermentation production PBI 58, it is characterized in that: fermentation condition is: temperature: 10 ℃-40 ℃, PH3-12, the PH regulation and control are with the stream ammonification or add CaCO
3Or the mode that adds NaOH, KOH is carried out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 96117784 CN1067724C (en) | 1996-11-18 | 1996-11-18 | Method for producing natural abscisic acid by fungus fermentation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 96117784 CN1067724C (en) | 1996-11-18 | 1996-11-18 | Method for producing natural abscisic acid by fungus fermentation |
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| Publication Number | Publication Date |
|---|---|
| CN1182798A true CN1182798A (en) | 1998-05-27 |
| CN1067724C CN1067724C (en) | 2001-06-27 |
Family
ID=5124604
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 96117784 Expired - Lifetime CN1067724C (en) | 1996-11-18 | 1996-11-18 | Method for producing natural abscisic acid by fungus fermentation |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1076587C (en) * | 1998-11-21 | 2001-12-26 | 云南农业大学和昆明恒溢隆经贸有限责任公司 | Process for preparing natural bud inhibitor and its application in inhibiting axillary bud of tobacco |
| WO2008092297A1 (en) * | 2007-01-24 | 2008-08-07 | Chengdu Institute Of Biology, The Chinese Academy Of Sciences | A new process for preparing natural abscisic acid |
| CN100427606C (en) * | 2005-10-08 | 2008-10-22 | 中国科学院成都生物研究所 | Method for preparing 14C or 3H marked natural active abscisic acid |
| CN100513377C (en) * | 2005-10-08 | 2009-07-15 | 中国科学院成都生物研究所 | Method for separating and extracting abscisic acid from fermented liquid by ionic exchanging and reversed phase chromatography |
| CN101662934B (en) * | 2007-01-31 | 2013-09-18 | 瓦伦特生物科学公司 | Stable s-(+)-abscisic acid liquid and soluble granule formulations |
| CN104988188A (en) * | 2015-07-20 | 2015-10-21 | 中国药科大学 | Method for increasing fermentation output of abscisic acid |
| CN113981016A (en) * | 2021-12-17 | 2022-01-28 | 四川龙蟒福生科技有限责任公司 | Fermentation formula for reducing S-ABA impurities in fermentation production |
-
1996
- 1996-11-18 CN CN 96117784 patent/CN1067724C/en not_active Expired - Lifetime
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1076587C (en) * | 1998-11-21 | 2001-12-26 | 云南农业大学和昆明恒溢隆经贸有限责任公司 | Process for preparing natural bud inhibitor and its application in inhibiting axillary bud of tobacco |
| CN100427606C (en) * | 2005-10-08 | 2008-10-22 | 中国科学院成都生物研究所 | Method for preparing 14C or 3H marked natural active abscisic acid |
| CN100513377C (en) * | 2005-10-08 | 2009-07-15 | 中国科学院成都生物研究所 | Method for separating and extracting abscisic acid from fermented liquid by ionic exchanging and reversed phase chromatography |
| WO2008092297A1 (en) * | 2007-01-24 | 2008-08-07 | Chengdu Institute Of Biology, The Chinese Academy Of Sciences | A new process for preparing natural abscisic acid |
| CN101662934B (en) * | 2007-01-31 | 2013-09-18 | 瓦伦特生物科学公司 | Stable s-(+)-abscisic acid liquid and soluble granule formulations |
| CN104988188A (en) * | 2015-07-20 | 2015-10-21 | 中国药科大学 | Method for increasing fermentation output of abscisic acid |
| CN104988188B (en) * | 2015-07-20 | 2019-01-22 | 中国药科大学 | A method of improving abscisic acid fermentation yield |
| CN113981016A (en) * | 2021-12-17 | 2022-01-28 | 四川龙蟒福生科技有限责任公司 | Fermentation formula for reducing S-ABA impurities in fermentation production |
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| Publication number | Publication date |
|---|---|
| CN1067724C (en) | 2001-06-27 |
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