CN104988188B - A method of improving abscisic acid fermentation yield - Google Patents
A method of improving abscisic acid fermentation yield Download PDFInfo
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- CN104988188B CN104988188B CN201510429182.9A CN201510429182A CN104988188B CN 104988188 B CN104988188 B CN 104988188B CN 201510429182 A CN201510429182 A CN 201510429182A CN 104988188 B CN104988188 B CN 104988188B
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Abstract
The present invention relates to a kind of production method of abscisic acid, especially a kind of method for improving abscisic acid fermentation yield belongs to technical field of bioengineering.The method of the present invention includes inclined-plane culture, seed culture and zymotechniques, and one or more kinds of combinations of molybdic acid and its esters are added in zymotechnique, to improve the yield that Botrytis cinerea fermentation produces natural abscisic acid.Compared with prior art, the present invention can overcome the problems such as mycelium clustering and high fermentation broth viscosity in Botrytis cinerea fermentation process, bacterial strain is improved to the utilization rate of oxygen, promote the biosynthesis of abscisic acid, fermentation yield improves 50.1%-162.5% compared with original process, and abscisic acid fermentation level is up to 2.1g/L or more.Fermentation accelerant used in this technique is at low cost, simple process, is convenient for industrial application.
Description
Technical field
The present invention relates to a kind of production method of abscisic acid, especially a kind of method for improving abscisic acid fermentation yield belongs to
In technical field of bioengineering.
Background technique
Abscisic acid is one of big hormone of plant five, is served in plant growth and development many-sided: as adjusted stomatal movement,
Inhibit germination, control embryo and seed development is mature, is related to degeneration-resistant effect and is such as dehydrated, arid, salt stress, low temperature, disease pest
The environmental factors responses such as harmful and mechanical trauma.It in agricultural, can be used for improving drought resisting, the cold-resistant, saline-alkaline tolerance of crop, answer at it
Had a large amount of patent reports with field, as the patent application of Publication No. CN104671979A disclose it is a kind of containing abscisic acid
No. 1 cold tolerance of eastern agriculture winter wheat can be improved in seed dressing；The patent application of Publication No. CN101199284 discloses a kind of containing de-
Fall the Kernel apricot tree flowers and fruits antifreezing agent of acid；The patent application of Publication No. CN1229015C discloses a kind of florescence containing abscisic acid
Extension agent etc..The production method of abscisic acid can be mainly divided into three classes: (1) being extracted from plants；(2) chemical synthesis；(3) micro- life
Object fermentation method.The method wherein extracted in plant because in plant content it is extremely low, it is difficult to it is a large amount of to obtain, cost it is excessively high and without the valence of applying
Value.Artificial synthesized abscisic acid is the mixture of a variety of optical isomers, splits difficult, the active abscisic acid for being much smaller than natural type,
Price is expensive, limits its application.And microbe fermentation method is the natural abscisic acid production method of rising in recent years, in work
It is promoted and applied in industry.Production by Microorganism Fermentation abscisic acid mainly uses fungi, such as Botrytis cinerea
(B.cinerea), because the bacterium fermentation yield is higher than other microorganisms, this has had more detailed report (Lu Li in the literature
Treasure etc., amino acid and living resources 2011,33 (2): 23-26).To improve its fermentation yield, people mainly to its fermentation liquid at
Point, nutriment, addition precursor etc. studied.Such as: Japan Patent JP 5836393 is reported in C.rosicola liquid
Guanine is added in body fermentation medium, fermentation yield can be made to improve；But costly due to guanine price, in process of production
Higher cost；Japan Patent JP 0260590 reports the addition citrus dry fruit skin in B.cinerea liquid fermentation medium and mentions
Object is taken, fermentation yield can be improved, but due to the substance containing inhibition fungi growth in orange peel, and its content is by citrus kind
The influence of the factors such as class, the place of production, its additive amount more difficult to control in fermentation.The report such as Zheng Hang (amino acid and living resources 2002,
24 (4): 25-27) addition nutriment such as corn flour, sodium citrate etc., the production of Botrytis cinerea liquid fermentation can be improved
The yield of abscisic acid, the acid yield that falls off after optimization is up to 0.212 grams per liter.The patent application publication of Publication No. CN1182798A
It is a kind of to use three grade fermemtation method, using the discharging of continuous flow feeding and strain immobilization in third level fermentation, and add
Add any one of key substrate such as imidazoles, morpholine, pyridine and its derivatives, acetamide, ammonium acetate, mevalonic acid etc., makes to take off
The yield for falling acid can reach 1.2 grams per liter fermentation liquids；The patent application of Publication No. CN 101041837A discloses one kind and passes through
Stream plus inositol release the technique of glucose repression to improve the yield of abscisic acid during the fermentation.Publication No. CN
The patent application of 102399827 A then passes through the precursor of stream during the fermentation plus acetyl coenzyme A as bacterial strain synthesis abscisic acid
Substance, meanwhile, the method that addition biosurfactant Tween-20 improves fermentation liquid dissolved oxygen situation to prepare natural abscisic acid,
The fermentation yield of abscisic acid can be improved.Nevertheless, there are still some problems for the above-mentioned prior art, if technique is more complex, add
Substrate, precursor or the promotor added is at high cost, is unfavorable for cost reduction of production etc., in addition, also holding during liquid fermentation
Easily there is the disadvantages of mycelium conglomeration, fermentation broth viscosity is big, dissolved oxygen reduces, so that fermentation yield is difficult to further increase.
Summary of the invention
The purpose of the present invention is in view of the defects existing in the prior art, propose a kind of side for improving abscisic acid fermentation yield
Method solves the problems, such as that mycelium conglomeration, fermentation broth viscosity are big.
The present invention solves technical problem by the following technical programs: a method of abscisic acid fermentation yield is improved, including
Following steps:
The first step carries out Botrytis cinerea inclined-plane culture 3-5 days, 24-30 DEG C of cultivation temperature；
Second step, the Botrytis cinerea through inclined-plane culture carry out seed culture 48-72 hours, 24-28 DEG C of cultivation temperature；
Third step, the Botrytis cinerea through seed culture carry out fermented and cultured 8-11 days, 24-28 DEG C of cultivation temperature, train
The fermentation accelerant that addition concentration is 0.01-200mM when supporting, the fermentation accelerant are at least one in molybdic acid or molybdate
Kind；
4th step, fermented culture Botrytis cinerea extraction purification, obtain the de- of high yield
Fall acid.
The above method adds one or more kinds of combinations of molybdic acid and its esters in zymotechnique, to improve
Botrytis cinerea fermentation produces the yield of natural abscisic acid.Botrytis cinerea As 3.4584 in the above method, purchased from China
General Microbiological Culture preservation administrative center.
The present invention further solves technical problem by the following technical programs, and in the first step, inclined-plane culture uses PDA
Plating medium.
In the second step, the ingredient of seed culture medium is as follows by weight percentage, glucose 1-3%, MgSO4 0.01-
0.1%, KCl 0.01-0.1%, KH2PO40.01-0.1%, monosodium glutamate 0.1-0.5%, FeSO4·7H2O
0.5mg/L, ZnSO4·7H2O 2.5mg/L, CuSO4·5H2O 4.0mg/L, the water of surplus.
In the third step, the ingredient of fermentation medium is as follows, sucrose or glucose
10-40/L, monohydrate potassium 1-10g/L, Tween-80 1-30g/L, corn flour or corn pulp 1-15g/L, soya-bean oil
1-25g/L, sweet potato powder 1-15g/L, molybdate 1-100mM.The molybdate be sodium molybdate, potassium molybdate, ammonium molybdate, calcium molybdate,
At least one of molybdic acid ferrous iron, magnesium molybdate, copper molybdate, zinc molybdate, molybdate individually in 121 DEG C of sterilizing 25min, were fermenting
Primary or gradation is added in fermentation medium in journey.The additive amount of the molybdate is 0.01-200mM.
Seed culture medium containing Botrytis cinerea is seeded to fermentation medium culture with the inoculum concentration of 1%-10%.
In 4th step, extraction purification is that product will be centrifuged after fermenting, and collects fermentation liquid, with salt acid for adjusting pH to 2-4,
Isometric ethyl acetate extraction is added and crystallizes to obtain product after extract liquor concentration.
The present invention can inhibit mycelia to grow using molybdate to a certain extent, induction mycelia from filiform become corynebacterium or
Pellet shapes, significantly dispersion mycelia, change hypha form, reduce fermentation broth viscosity, improve bacterial strain to the utilization rate of oxygen, greatly improve
The yield of abscisic acid.The present invention improves bacterial strain to the utilization rate of oxygen, promotes the biosynthesis of abscisic acid, sends out compared with original process
Ferment output increased 50.1%-162.5%, abscisic acid fermentation level is up to 2.1g/L or more.Fermentation accelerant used in this technique at
This low, simple process is convenient for industrial application.
Detailed description of the invention
Fig. 1 is the effect contrast figure in one embodiment of the invention, and A figure is not plus the hypha growth condition of molybdate, B scheme
For the hypha growth condition after addition molybdate.It can be seen that mycelia is more dispersed after molybdate is added, not easy to knot groups, so as to improve
Oxygen and nutrient between mycelia and culture medium exchange, and promote the raising of fermentation yield.
Specific embodiment
Botrytis cinerea As 3.4584 used in embodiment is purchased from China General Microbiological culture presevation administrative center,
It repeats no more below.
Embodiment 1
Actication of culture is potato glucose agar medium (PDA) with culture medium: weighing fresh potato 200g, goes after cleaning
Skin is cut into small pieces and boils 30min, and four layers of-eight layers of filtered through gauze are rear that glucose 20g and agar 20g is added, and mend after completely dissolution
Water is to 1L, 121 DEG C of sterilizing 20min.On the Botrytis cinerea picking that freeze-drying is saved to activation plate, 26 DEG C of culture activation 5
It.Thallus after activation is smashed and stirred evenly, is placed at 26 DEG C into liquid seed culture medium with sterile spatula picking, shaking flask training
It supports 72 hours (200rpm).Liquid seed culture medium group becomes (containing in every liter): glucose 20g, MgSO40.2g, KCl0.5g,
KH2PO40.8g, monosodium glutamate 3.0g, FeSO4·7H2O0.5mg, ZnSO4·7H2O2.5mg, CuSO4·5H2O4.0mg。
Fermentation medium composition are as follows: sucrose 20/L, monohydrate potassium 6g/L, Tween-80 10g/L, corn flour 3g/L, beans
Oily 10g/L, sweet potato powder 10g/L.Configured fermentation medium is divided into 2 groups, wherein one group is not added fermentation accelerant, as sky
White control；Another group is added fermentation accelerant sodium molybdate 5mmol/L in the medium.Every group be inoculated with respectively by volume 5% liquid
Body seed, at 27.5 DEG C, 200 revs/min shaken cultivation 8 days, measuring the sodium molybdate group acid yield that falls off is 1.26g/L, and hair is not added
The control group yield of ferment promotor is 0.77g/L, than control output increased 63.6%.As a result as shown in Figure 1, A is control group,
B is embodiment, and it is obvious compared with the mycelium dispersion in A culture dish to be added to the mycelium in the B culture dish of molybdate.
Embodiment 2
Actication of culture and seed culture method are the same as embodiment 1.Fermentation medium composition are as follows: sucrose 30/L, a citrate hydrate
Sour 4g/L, Tween-80 5g/L, corn flour 4g/L, soya-bean oil 6g/L, sweet potato powder 15g/L.Configured fermentation medium is divided into 2
Group, wherein one group is not added fermentation accelerant, as blank control；Another group of preparation 100mmol/L molybdic acid magnesium solution, after sterilizing
Ferment 0h, 12h, for 24 hours, the 48h time-division 4 times, be separately added into the molybdic acid magnesium solution of fermentating liquid volume 2.5%.This two groups fermentation item
Part are as follows: liquid seeds inoculum concentration 10%, cultivation temperature be 24 DEG C, 200 revs/min shaken cultivation 10 days, measure magnesium molybdate group and fall off
Acid yield is 2.1g/L, and the control group yield that fermentation accelerant is not added is 0.80g/L, than control output increased 162.5%.
Embodiment 3
Actication of culture and seed culture method are the same as embodiment 1.Fermentation medium composition are as follows: sucrose 10/L, a citrate hydrate
Sour 10g/L, Tween-80 15g/L, corn flour 5g/L, soya-bean oil 15g/L, sweet potato powder 5g/L.Configured fermentation medium is divided into
2 groups, wherein one group is not added fermentation accelerant, as blank control；Another group of addition fermentation accelerant 0.1mmol/L molybdic acid and
5mmol/L molybdic acid is ferrous, is inoculated with liquid seeds 5%, and cultivation temperature is 26 DEG C, 200 revs/min shaken cultivation 10 days, measure molybdic acid
Magnesium group falls off acid yield for 1.7g/L, and the control group yield that fermentation accelerant is not added is 0.84g/L, than compareing output increased
103.3%.
Embodiment 4
Actication of culture and seed culture method are the same as embodiment 1.Fermentation medium composition are as follows: sucrose 40/L, a citrate hydrate
Sour 3g/L, Tween-80 5g/L, corn pulp 3g/L, soya-bean oil 5g/L, sweet potato powder 5g/L.Configured fermentation medium is divided into 2
Group, wherein one group is not added fermentation accelerant, as blank control；Another group of fermentation accelerant 0.5mmol/L molybdenum in the medium
Then sour ammonium is separately added into 0.5mmol/L molybdic acid potassium solution as fermentation accelerant when fermenting 20h and 30h.This two groups connect
Kind of liquid seeds amount is 5%, and cultivation temperature is 28 DEG C, 200 revs/min shaken cultivation 11 days, measure addition fermentation abscisic acid and produce
Amount is 1.88g/L, and the control group yield that fermentation accelerant is not added is 0.74g/L, than control output increased 154.1%.
In addition to above-mentioned implementation, the present invention can also have other embodiments.It is all to be formed using equivalent substitution or equivalent transformation
Technical solution, fall within the scope of protection required by the present invention.
Claims (7)
1. a kind of method for improving abscisic acid fermentation yield, comprising the following steps:
The first step carries out Botrytis cinerea inclined-plane culture 3-5 days, 24-30 DEG C of cultivation temperature；
Second step, the Botrytis cinerea through inclined-plane culture carry out seed culture 48-72 hours, 24-28 DEG C of cultivation temperature；
Third step, Botrytis cinerea through seed culture carry out fermented and cultured 8-11 days, and 24-28 DEG C of cultivation temperature, when culture
The fermentation accelerant that concentration is 1.5-200mM is added, the fermentation accelerant is at least one of molybdic acid or molybdate；
4th step, fermented culture Botrytis cinerea extraction purification, obtain the abscisic acid of high yield.
2. improving the method for abscisic acid fermentation yield according to claim 1, it is characterised in that: in the first step, inclined-plane
Culture uses PDA plate culture medium.
3. improving the method for abscisic acid fermentation yield according to claim 1, it is characterised in that: in the second step, seed
The ingredient of culture medium is as follows by weight percentage, glucose 1-3%, MgSO4 0.01-0.1%, KCl 0.01-0.1%, KH2PO4
0.01-0.1%, monosodium glutamate 0.1-0.5%, FeSO4·7H2O 0.5mg/L, ZnSO4·7H2O 2.5mg/L, CuSO4·
5H2O 4.0mg/L, the water of surplus.
4. improving the method for abscisic acid fermentation yield according to claim 1, it is characterised in that: in the third step, fermentation
The ingredient of culture medium is as follows, sucrose or glucose 10-40g/L, monohydrate potassium 1-10g/L, Tween-80 1-30g/L,
Corn flour or corn pulp 1-15g/L, soya-bean oil 1-25g/L, sweet potato powder 1-15g/L, molybdate 1.5-200mM.
5. improving the method for abscisic acid fermentation yield according to claim 4, it is characterised in that: the molybdate is molybdic acid
At least one of sodium, potassium molybdate, ammonium molybdate, calcium molybdate, molybdic acid ferrous iron, magnesium molybdate, copper molybdate, zinc molybdate, molybdate is independent
In 121 DEG C of sterilizing 25min, primary during the fermentation or gradation is added in fermentation medium.
6. improving the method for abscisic acid fermentation yield according to claim 4, it is characterised in that: Botrytis cinerea will be contained
Seed culture medium fermentation medium culture is seeded to the inoculum concentration of 1%-10%.
7. improving the method for abscisic acid fermentation yield according to claim 1, it is characterised in that: in the 4th step, extract
Purifying is that product will be centrifuged after fermenting, and collects fermentation liquid and is extracted with ethyl acetate with salt acid for adjusting pH to 2-4, extract liquor concentration
Afterwards, product is crystallized to obtain.
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| CN105400833A (en) * | 2015-12-11 | 2016-03-16 | 山东营养源食品科技有限公司 | A method of increasing a fermentation yield of natural abscisic acid |
| CN106995826A (en) * | 2017-04-18 | 2017-08-01 | 四川龙蟒福生科技有限责任公司 | A kind of fermentation medium for being used to produce S abscisic acids |
| CN107058410A (en) * | 2017-04-18 | 2017-08-18 | 四川龙蟒福生科技有限责任公司 | A kind of preparation method of S abscisic acids |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1182798A (en) * | 1996-11-18 | 1998-05-27 | 中国科学院成都生物研究所 | Method for producing natural abscisic acid by fungus fermentation |
| CN102399827A (en) * | 2011-11-18 | 2012-04-04 | 中国科学院成都生物研究所 | Method for efficiently preparing natural abscisic acid |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1182798A (en) * | 1996-11-18 | 1998-05-27 | 中国科学院成都生物研究所 | Method for producing natural abscisic acid by fungus fermentation |
| CN102399827A (en) * | 2011-11-18 | 2012-04-04 | 中国科学院成都生物研究所 | Method for efficiently preparing natural abscisic acid |
Non-Patent Citations (1)
| Title |
|---|
| 灰绿葡萄孢发酵液中天然型脱落酸的分离纯化;郑珩，等;《中国抗生素杂志》;20021031;第27卷(第10期);第589页第1-2.2节 |
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