CN1067724C - Method for producing natural abscisic acid by fungus fermentation - Google Patents

Abstract

The invention belongs to a new method for producing natural active abscisic acid by fungal fermentation. In this method, by transforming the existing abscisic acid bacteria, changing the formula of the culture medium, changing the liquid batch fermentation process to continuous flow feeding and discharging, immobilizing the strains, and adding key substrates to stabilize the acid production, thereby greatly improving The output is reduced, the energy consumption and raw material usage are reduced, the production cost is greatly reduced, and the industrial scale production becomes possible.

Description

A new method for the production of natural abscisic acid by fungal fermentation

The invention belongs to the fermentation engineering of producing natural active abscisic acid by fungal fermentation method.

Abscisic acid (ABA) is one of the five major plant hormones found in the world. Natural ABA has a strong regulatory activity on the growth and development of crops, can promote the maturity and development of fruits, cereals, and beans, can greatly improve their yield and quality, and can greatly enhance their cold resistance, drought resistance and salt tolerance. ability, so it has broad application prospects. At present, the research of ABA in the basic field has reached the level of plant cells and genetic engineering. However, since the optical configuration of natural active ABA in plants is only (S)-(+)-ABA, the production cost of pure (S)-(+)-ABA is extremely high, and the price is expensive, while synthetic The obtained ABA is the Racemid type, which is much less active than the natural type of ABA. Therefore, the application of ABA in agricultural production is almost empty talk. In order to solve and meet the problem of ABA applied in agricultural production, in the past two decades, foreign countries have begun to use microbial fermentation to produce natural active abscisic acid. In 1977, Italian G.Assante first fermented Cercospora Rosicola to produce natural active abscisic acid, mainly solid fermentation, but because of its low output, its commodity price was very high ($193.4/mg). In 1982, Marumo Shingo of Japan discovered The fungus Botrytis Cinerea can be used to produce natural active ABA, and a patent was applied for, and then, in 1987, 1988, and 1990, Matsumoto Shunichi of Toray Corporation and New Japan Chemical Industry Corporation. Successively use Botrytis strains to produce natural active abscisic acid, and applied for six patents. The production process is solid fermentation and liquid shake flask fermentation. At the same time, cellulose foam is used as the carrier of liquid fermentation. The fermentation output has increased from 15-20mg/liter The culture medium is increased to 23.8-85mg/liter of fermentation broth, and the highest yield has reached 300mg/L. However, the above patents still exist: 1. the fermentation yield is low; 2. the fermentation process is still limited to solid fermentation and liquid batch fermentation, and the characteristics of continuous production of abscisic acid by the bacteria are not fully utilized. The bacteria grow in a fixed culture environment and produce acid. When the product (abscisic acid) reaches a certain concentration, it will have an inhibitory effect, and the bacteria will no longer secrete products, resulting in waste of nutrients, bacteria and energy, so that the output cannot be achieved. Improvement, low productivity, high cost. Still can not solve the problem that is applied to agricultural production.

The task of the present invention is to find a new process that can greatly increase the output of abscisic acid, greatly reduce the production cost of abscisic acid, and make industrial scale production possible.

Technical solution of the present invention:

1. The existing abscisic acid-producing strains are transformed into high-yielding strains, and the fermentation activity of the strains is greatly improved.

2. The liquid batch fermentation process is changed to a fermentation process of immobilized bacteria continuous feeding, feeding, discharging, and pH control (initial pH control, growth phase pH control, production phase pH control, and fermentation end phase pH control) fermentation process.

3. Choose a medium formula that is more suitable for bacterial growth and acid production.

4. By adding key substrates, regulating the metabolic pathway of bacterial fermentation, the production of abscisic acid is greatly increased.

The strain used in the present invention is Botrytis Cinerea-Cercospoxa Rosicola FD338 (abbreviated B.C.FD338). The bacterium is a high-yield abscisic acid strain obtained by protoplast mutagenesis with Botrytis Cinerea T-1 and then protoplast fusion with another abscisic acid producing bacterium Cercospora Rosicola216 and related genetic mutagenesis.

The present invention adopts three-stage continuous fermentation, and three different culture media A, B, and C are selected according to different requirements of the three stages. It consists of:

Medium A:

Glucose 0.2-2% Beef Extract 0.1-1% Yeast Extract 0.1-1%

Mannose 0.1-0.5% Wort 5-20% Corn steep liquor 0.001-0.05%

(or 0.1-0.5% sucrose, 0.1-0.5% rhamnose, 0.1-1% peptone

Glycerol 0.1-1%, Biotin 0.001-0.05%)

NH ₄ NO ₃ 0.1-0.5%, MgSO ₄ 0.05-0.3%, KCl 0.1-0.3%

K ₂ HPO ₄ 0.05-0.5%

Medium 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 galactose 0.5-5%, sucrose 0.1-3%)

Bean cake powder 0.1-2% (or peanut cake powder 0.1-2%, or cottonseed cake powder 0.1-3%)

Glutamic acid 0.01-0.5%, (NH ₄ ) ₂ SO ₄ 0.01-0.8% (or urea 0.1-1%)

MgSO ₄ 0.05-0.3%, KCl0.1-0.3% Thiamine 0.0001-0.05%

Medium C: dextrin 0.1-3%, wheat bran juice 20-70% (or corn flour 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 galactose 0.1-7%

Cottonseed cake powder 0.1-2% (or bean cake powder 0.1-8%, whey 0.1-9%,

(or peanut powder 0.1-8%)

Citrus juice 0.5-5% (or cellobiose 0.5-15%, or glycerin 0.1-5%,

(or soybean oil 0.5-5%, or sodium citrate 0.1-5%)

Glutamic acid 0.01-5% (or cysteine 0.01-5%, or glutamic acid + cysteine

O.01-5%)

(NH ₄ ) ₂ SO ₄ 0.01-5% (or NH ₄ NO ₃ 0.1-7%, or NaNO ₃ 0.1-7%,

or ammonia 0.1-7%)

Thiamine 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%

Boron, molybdenum, cobalt, nickel and other trace elements (0.1-100μmol/L)

Fermentation process of the present invention is:

Inoculate the activated strains in the culture medium A, after 24-72 hours of shaking flask culture in the Erlenmeyer flask, inoculate 5-10% of the inoculum in the seed tank that has been placed in the medium B for fermentation and culture for 24- 60 hours, then still inoculate in a three-stage fermenter with an inoculation amount of 5-10% for fermentation. The three-stage fermentation tank uses medium C as the fermentation medium, and adds microporous ceramic materials (or microporous omentum, cinder, polyurethane foam, brick slag or sodium alginate, polyvinyl alcohol, gelatin, etc.) Immobilize the bacteria. In the three-stage fermentation process, when the growth of the bacteria enters the stable period, the growth rate of the bacteria is reduced by reducing the dissolved oxygen concentration and the fermentation temperature, and the medium C is continuously fed at a low concentration and the precursor substrate is added, and the quantity is determined regularly Discharge. After the fermented fermentation broth is extracted by organic solvent extraction, ion exchange column method, silica gel column chromatography and activated carbon adsorption method, the pure white abscisic acid is obtained.

The precursor substrates added in the fermentation system mainly include any of imidazole, morpholine, pyridine and its derivatives, acetamide, acetamide, β,β-diformyl acid, morphine, and mevalonic acid.

The reagents used in the extraction process are: methanol, ethanol, ethyl acetate, acetone, chloroform, ether, cycloethane, petroleum ether and other lower alcohols. The resin used in the ion exchange column is a strong base anion resin or a macroporous resin. Fermentation conditions: Temperature: 10°C-40°C pH: 3-12

pH control is carried out by adding ammonia or adding CaCO ₃ or adding NaOH, KOH:

Fermentation time: 1-30 days, precursor substrate addition amount: 0.1-16%

Whole process flow of the present invention is shown in accompanying drawing.

Advantages of the present invention:

①The output of the production strain is 3-4 times higher than the highest yield reported at home and abroad (the highest yield in Japanese patents is 300mgL fermentation broth, and the yield of this strain B.C.FD33S is 900-1200mg/L fermentation broth);

② The fermentation system adopts immobilization technology, which avoids the loss of bacteria in the process of continuous feeding and discharging, and stabilizes the acid production;

③ The fermentation system can be stable for 10-30 days during the acid production period, thereby greatly improving production efficiency, reducing energy consumption and raw material usage, and greatly reducing production costs.

④ The addition of key precursor substrates promotes the smooth progress of fermentation metabolism and greatly increases the yield.

Example:

Use 10 1000ml Erlenmeyer flasks, sterilize each bottle with 300ml medium A at 120°C, inoculate the activated spore suspension of B.C.FD33S bacteria after cooling, place on a shaking table at a temperature of 25°C-28°C, and shake the flask for 24 -72 hours.

Inoculate the cultivated primary seed liquid into a 100-liter fermenter with 50 liters of medium B inside according to the inoculum amount of 5%, and cultivate it with aeration and stirring at a temperature of 24-28° C. for 24-60 hours.

A 1-ton fermenter is used as a three-stage tank for fermentation. The tank contains 50 liters of medium C7 and microporous ceramic beads (particle size 0.9-160mm ³ , 1.0-5g/L). After sterilizing with conventional high-pressure hot steam, inoculate the secondary seed liquid with 5% inoculation amount, ventilate and stir for 48 hour, then feed ammonia to control the pH at 4-8, reduce the dissolved oxygen concentration (reduce aeration), reduce the fermentation temperature to 10-25°C, add medium C at a rate of 0.01-5L/hour, and add medium C at a rate of 0.1%- Add mevalonic acid at a ratio of 16% to stabilize the state of the fermentation system, and discharge once every 10 hours. Recover the product in the fermented liquid from the liquid material by ion exchange resin method, and wash it with ethanol to obtain the white crystalline abscisic acid product. Optical rotation [α] _D ²⁸ =+419°.

After testing: after 25 days of stable acid production in the fermentation system, the output can reach 1.2 grams of abscisic acid per liter of fermentation broth. Recovered by ion exchange method, the product recovery rate reaches 80%.

Claims (4)

1. The method for producing natural active abscisic acid by fungal fermentation is characterized in that the fermentation adopts three-stage continuous fermentation and fungal immobilization process, and in the third-stage fermentation process, precursor substrates: imidazole, morpholine, pyridine and derivatives thereof are added Any one of substance, acetamide, ammonium acetate, β, β-diformyl acid, coline or mevalonic acid, the addition amount is 0.1-16%, and the medium C with the following composition is added by feeding: Dextrin 0.1-3% Wheat Bran Juice 20-70% Glucose 0.5-5% Cottonseed meal 0.1-2% Citrus juice 0.5-5% Glutamic acid 0.01-5% Ammonium sulfate 0.01-5% Thiamine 0.001-0.5% Magnesium sulfate 0.05-0.5% Sodium Chloride 0.05-0.5% Ferrous Sulfate 0.05-0.5% Copper Sulfate 0.05-0.5% Trace elements boron, molybdenum, cobalt, nickel 0.1-100μmol/L Control the dissolved oxygen concentration, temperature, and pH to stabilize the acid production, and regularly and quantitatively discharge the material; the extraction process uses organic solvent extraction, ion exchange, column chromatography and adsorption. 2. According to the method for producing natural abscisic acid by fungal fermentation according to claim 1, it is characterized in that the material used to fix the fungi is microporous ceramics, sodium alginate, polyvinyl alcohol, gelatin, microporous omentum, cinder, brick slag or polyurethane foam. 3. According to the method for producing natural active abscisic acid by fungal fermentation according to claim 1, it is characterized in that, in the extraction process, the eluent used is lower alcohols. 4. According to the method for producing natural active abscisic acid by fungal fermentation according to claim 1, it is characterized in that the fermentation conditions are: temperature 10-40 ° C, pH 3-12, pH regulation and control by adding ammonia or adding CaCO ₃ or adding NaOH, KOH way.

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