WO2008092297A1

WO2008092297A1 - A new process for preparing natural abscisic acid

Info

Publication number: WO2008092297A1
Application number: PCT/CN2007/000268
Authority: WO
Inventors: Hong Tan; Baoliang Lei; Zhidong Li; Jinyan Zhou; Jie Yang
Original assignee: Chengdu Institute Of Biology, The Chinese Academy Of Sciences
Priority date: 2007-01-24
Filing date: 2007-01-24
Publication date: 2008-08-07

Abstract

A process for preparing natural abscisic acid by feeding inositol to derepress glucose repression during fermentation, and the novel bacterial strain for the process are provided. The process includes the follow steps: culturing fungi producing natural abscisic acid in the primary liquid culture medium, inoculating the cultured primary seed liquid to the secondary liquid culture medium and culturing, feeding culture medium during the tertiary fermentation and feeding inositol simultaneously, and recovering abscisic acid from fermented broth.

Description

A new method for preparing natural abscisic acid

Technical field

The present invention relates to a method for preparing natural abscisic acid by adding a solution of inositol to glucose repression during fermentation, and a novel strain used in the method, wherein the strain is subjected to gene-directed mutagenesis Obtained by improving the natural abscisic acid producing strains. BACKGROUND OF THE INVENTION Abscisic acid (ABA) is one of the five major plant hormones that have been discovered in the world. The natural type of abscisic acid has strong regulatory activity on the growth and development of crops, can promote the ripening and development of fruits, cereals and beans, can greatly improve its yield and quality, and greatly enhance its cold tolerance and drought resistance. With the ability to resist salt and alkali, it has broad application prospects. At present, the research of abscisic acid in the basic field has penetrated into the level of plant cells and genetic engineering. However, since the optical configuration of natural abscisic acid present in plants is only (S) - (+) - abscisic acid, the production cost of pure (S) - (+) - abscisic acid is extremely high and expensive. The artificially synthesized abscisic acid is a racemic body, and its activity is much smaller than that of the natural type of abscisic acid. Therefore, the application of abscisic acid to agricultural production is almost empty talk. In order to solve and meet the needs of abscisic acid for agricultural production, in the past two decades, foreign countries (especially Japan) have been studying the use of microbial fermentation to produce natural abscisic acid. For example, it is known that the fungus Botrytis cinerea can be used to produce natural abscisic acid, and liquid fermentation using, for example, cellulose foam as a carrier is disclosed, and the fermentation yield is increased, and the highest yield is 300 mg/liter. (See, for example, JP-A-6-247927, JP-A-6-197775, JP-A-6-133786, JP-A-6-7180, JP-A-2-10988, JP-A- 2- In addition, CN1182798A discloses a method for producing natural abscisic acid by fungal fermentation, which adopts tertiary fermentation and is employed in tertiary fermentation. 2克每升发酵液。 The continuous flow of feed and discharge and the immobilization of the strain, and the addition of key substrates, so that the acid production is stable, the highest yield of abscisic acid reached 1. 2 g / liter of fermentation broth. Patent ZL001.32024. 6 discloses a process for preparing natural abscisic acid by a method of preparing a natural abscisic acid by using a fungal batch liquid medium fed-feed process. However, the microbial strains in the prior art are products of mutagenesis and improvement at the cell level, and the production of abscisic acid has certain limitations; fermentation process (solid fermentation, liquid batch fermentation, continuous flow plus feed discharge process, batch) Liquid medium flow plus feeding process, etc.) is still limited by glucose repression. When the substrate sugar concentration is high, cell metabolism inhibition is produced, the utilization rate of other sugars by the bacteria is reduced, and even no product is secreted, resulting in yield. Low, high cost consequences. SUMMARY OF THE INVENTION The present inventors conducted intensive research on the deficiencies of the glucose repression phenomenon, low fermentation yield, and high production cost in the process of producing natural abscisic acid by microbial fermentation, which has been known in the prior art. The present invention has been completed by an improved process which is simpler and easier, has a higher yield of abscisic acid, and has a lower production cost. It is an object of the present invention to provide a method for fermentative production of natural abscisic acid by a fermentation process in which inositol is added to relieve glucose repression. Another object of the present invention is to provide a novel strain for producing natural abscisic acid.

More specifically, the present invention provides a novel method for preparing a natural abscisic acid comprising the steps of: cultivating a fungus capable of producing a natural abscisic acid in a primary liquid medium (for example, medium A described below) The fungus is selected from the group consisting of: Botrytis (for example, Botrytis cinerea) ■> Cercospora (for example, Cercospora rosicola), Penicillium (Penici lliura), Aspergillus (Aspergillus) And a genetically modified strain of the above strain; and the first-stage seed liquid cultured in the above-mentioned first-stage liquid medium is inoculated into a second-stage liquid medium (for example, the medium B described below); The second-stage seed liquid cultured in the above second liquid medium is inoculated into a third-stage liquid medium (for example, the medium C described below) for a suitable period of time, and then the third-stage liquid is started. The medium is fed with fed fermentation and a certain concentration of inositol is added to relieve glucose repression and increase the utilization rate of other sugars in the third-stage liquid medium; ABA said fermentation broth was collected. A specific embodiment of the present invention is the use of Botrytis cinerea (for example, Botrytis cinerea), Cercospora (for example, Cercospora rosicola), Aspergi llus or other fungi producing natural abscisic acid which produce natural abscisic acid and The genetically modified bacteria are subjected to tertiary fermentation culture on a liquid medium, and different culture media are used in each fermentation stage. In the third-stage fermentation, a liquid medium suitable for the third-stage fermentation, such as the medium C described below, is used as the fermentation medium, and the culture is carried out at a suitable temperature for a period of time, for example, at about 26 ° C. After 12-72 hours of incubation, the liquid medium is fed and fed, and a certain concentration of inositol is added at the same time. The third-stage liquid medium (for example, medium C) can be fed continuously (continuous or non-uniform) and/or intermittent, and intermittent addition is preferred. The method is known. Patent It is disclosed in ZL00132024. 6 that the inositol addition method can adopt continuous (uniform or non-uniform) flow addition and/or intermittent flow addition mode, and continuous (constant speed or non-hook speed) flow addition mode is preferable. The continuous (constant or non-uniform) flow addition method is to add a certain concentration of inositol and a continuous flow to the third-stage fermenter at a certain flow rate (hook speed or non-uniform speed) until the fermentation is stopped (the lower tank) ) For example, about 10 hours before. The intermittent flow adding method is to intermittently add a certain concentration of inositol to the third-stage fermenting tank in a manner of adding a batch at intervals. The intermittent time is preferably 1 to 5 times every 8 to 30 hours, more preferably about 1 hour at intervals of 10 hours. Those skilled in the art can also use other suitable time interval feed streams as needed. Fermentation conditions: temperature 23 ° C - 29 ° C, pH: 4-7 Fermentation time: 8-9 days. With the process of the present invention, in the third stage of fermentation, the liquid medium is only fed and fed without discharging. By adding a certain concentration of inositol, the glucose repression is relieved, and the utilization rate of the cells to other sugars in the medium such as sucrose, molasses, lactose, dextrin and other starch hydrolyzates is increased, the acid production rate is increased, and the production is lowered. cost. The abscisic acid in the fermentation broth is recovered in a conventional manner, for example, by extraction with an organic solvent extraction method, an ion exchange resin method, a macroporous adsorption resin method, a silica gel column chromatography method, and an activated carbon adsorption method, thereby obtaining a white crystalline abscisic acid. . In a preferred embodiment, the invention also employs a technical scheme such as a new strain to further increase the yield of natural fall acid. The method of the present invention may employ known strains which produce natural abscisic acid, for example, Botrytis cinerea which is known, or obtained by genetic engineering or genetic engineering methods, which produces natural abscisic acid, for example, Botrytis cinerea, Cercospora (for example, Cercospora rosicola), Aspergillus (Aspergi llus), or other fungi that produce natural abscisic acid and genetically modified bacteria thereof. In a preferred method of the invention, known high-yielding strains which produce abscisic acid are used, such as Botrytis cinerea TB-3-H8, TBI-9 (CGMCC No. 0500, which is disclosed in the patent ZL-00132024. 6), TBC-10, Ferm P-6156, BC FD338, etc. The new strain is used in the most preferred method of the invention: the abscisic acid high-yield strain Botrytis cinerea TBC 10, which was deposited on December 15, 2006 at the General Microbiology Center of the China Collection of Microorganisms Collections (CGMCC) ), the accession number is CGMCC No. 1889, which uses the gene-directed mutagenesis technique of Botrytis cinerea TB-3-A3 to obtain a change of 24 bases and leads to the corresponding coded five. A change in amino acids The new strain TBC-10, (the sequence of the gene sequence and amino acid sequence is from the strain Botrytis cinerea 3. 3790, purchased by the Institute of Microbiology, Chinese Academy of Sciences, or other homology on Genebank) High species of FPPS).

The gene sequence of the TBC-10 strain has the following characteristics: The strain is aligned with the gene sequence of the strain Farethyl diphosphate/pyphosphate synthase (Bosstis cinerea) 3. 3790, There are 2 base changes.

The amino acid sequence of the TBC-10 strain has the following characteristics: The amino acid sequence alignment of the strain with the strain Farithyl diphosphate/pyphosphate synthase of Botrytis cinerea 3. 3790, There are five amino acid changes (1 to 5 sites); amino acid sequence alignments with farnesyl diphosphate/pyphosphate synthase (FPPS) from other highly homologous species on Genebank, There are four amino acid changes (1, 3, 4, 5 sites). The process of the present invention employs a tertiary fermentation technique, and the use of tertiary fermentation to prepare abscisic acid is known (see, for example, CN1182798A). According to the different requirements of the tertiary fermentation, three different culture media are separately selected for the fermentation, and the culture medium may be a medium suitable for the purpose known to those skilled in the art or the culture disclosed in the invention patent ZL00132024. base. The use of the medium disclosed in the invention patent ZL00132024. 6 is preferred. The invention discloses a medium A, B, and C, and the specific composition thereof is as follows: glucose·0. 2%-3% beef extract 0. 1%-1% wheat bran 1%-5 sucrose 1%~4 % ammonium nitrate 0. 1%-2% magnesium sulfate 0. 01%-0. 1% maltose 0. 1%-3% medium B starch 1%-5% wheat bran 1%-5% glucose 1%-4 %% maltose 0. 1%-3% Bean cake powder 0. 1%-2% · 0. 1%-5% Medium c

A preferred embodiment of the invention is based on the known fermentation process (disclosed in the patent ZL00132024. 6) in which a certain concentration of inositol is added. That is: the activated strain is inoculated into the medium A, and cultured in a solid culture or a flask at 23 ° C - 29 ° C for 24 to 76 hours, then inoculated with 10% - 50% of the inoculum. Fermentation in a seed tank with sterile medium B at 23 C-29 °C The culture is carried out for 24 to 60 hours, and then inoculated into a third-stage fermentor at a seeding rate of preferably 10% to 40% for fermentation production. The third-stage fermentation uses medium C as the fermentation medium, and after fermentation for 12-72 hours at about 26 ° C, the feed of medium C and a certain concentration of inositol is started to be fed. There are two ways of adding inositol: one is continuous (uniform or non-uniform) flow, and the other is intermittent flow, the former is preferred. In the former method, inositol was continuously fed at a rate of 0.01 to 10 g/L * h (either at a constant rate or at a non-uniform rate) until about 10 hours before the fermentation was stopped (down can). In the latter case, inositol is added intermittently, and the intermittent time is 1 to 5 times every 8-30 hours, preferably about 1 hour at intervals of 10 hours, and the amount of supplement is 0. 05 - 50 mg / L fermentation broth, preferably 0. 1 - 10 _m g / L fermentation broth. When the process of the present invention is used, the bottom stream is fed without feeding. On the basis of adjusting (increasing or lowering) the dissolved oxygen concentration in the fermentation broth, controlling the temperature, foam and other means to reduce the metabolism of the bacteria, by adding a certain concentration of inositol, the glucose repression can be relieved, and the cells are further enhanced in the medium. The utilization rate of sugars such as sucrose, molasses, lactose, dextrin and other starch hydrolysates increases the acid production rate and reduces the production cost. After the fermentation, the fermentation broth is extracted by an organic solvent extraction method, an ion exchange resin method, a macroporous adsorption resin method, a silica gel column chromatography method, and an activated carbon adsorption method to obtain a white crystal abscisic acid.

Fermentation conditions: Temperature: 23 ° C - 29. C, pH: 4-7 Fermentation time: 8-9 days. The full process flow of the present invention is shown in Figure 1. In the most preferred method of the present invention, the production of the product is promoted by using a strain which produces a natural abscisic acid high-yield, and the production of abscisic acid is greatly improved. Fermentation of natural abscisic acid by the method of the invention can artificially regulate the ratio of carbon and nitrogen in the fermentation system, relieve glucose repression, increase the utilization rate of other sugars in the culture medium, and make the strain have a higher substrate conversion rate. And the product synthesis rate produces abscisic acid, thereby increasing the yield, greatly saving energy consumption and raw materials, improving production efficiency, reducing production cost, and simplifying the fermentation production process of microbial production of abscisic acid. The comparison results of the production of abscisic acid by the existing process and the process of the present invention are shown in the following Table 1. Process species: Abscisic acid production Fermentation cycle Miscellaneous contamination (except glucose) (g/L). Dyeing rate (%)

(days) other sugar utilization

(%) Liquid batch fermentation ΤΒ-3-Η8 1. 0 30 30% continuous flow plus feed ΤΒ-3-Η8 1. 2 10-30 50 40%

Discharge batch bottom flow plus ΤΒ-3-Η8 1. 6 8-9 10 50%

Feeding batch bottom flow plus ΤΒ-3-Η8 2. 0 8-9 8 80% feeding and flowing muscle

Alcohol (invention)

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*TB- 3- H8 (Botrytis cinerea CO ) (Tan Hong et al., using protoplast mutagenesis techniques o

Screening for high-yield strains of abscisic acid, Journal of Applied and Environmental Biology, 1998, 4 (3): 281-285), (attached to the open certificate). BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a process flow diagram of the present invention. DETAILED DESCRIPTION OF THE INVENTION The method of the present invention is further illustrated by the following non-limiting examples. Example 1 25 pieces of 1000 ml flasks were sterilized by dissolving 300 ml of medium A in each bottle at 120 Torr. After cooling, the activated oxalic acid high-yield strain Botrytis cinerea TBC-10 CGMCC No. 1889 spore suspension was inoculated. Shake the flask for 24 to 46 hours at 25 °C. The cultured first-class seed solution is inoculated into a 100-liter fermenter (secondary seed tank) containing 50 liters of medium B at a dose of 13%-15%, and aeration is stirred at a temperature of 26 ° C to 28 ° C. Cultivate for 24-40 hours. Use a 1 ton fermenter to make a three-stage tank fermentation. The tank contains about 500 L of medium C. After sterilization by conventional high-pressure steam sterilization, the seed tank of the second tank is inoculated with 10%-15% inoculum. 1 % - 0. 5 %。 The amount of the total volume of the fermentation broth is 0. 1% - 0. 5 % . 5-3. 0 mg/L fermentation broth. The infusion of the inositol was intermittently added at intervals of 2 hours. The combination of media used in the implementation process is as follows:

The fermentation temperature was 27 ° C, the fermentation pH was 4-7, and the fermentation time reached 9 days. The can fermentation broth is recovered by ion exchange resin method, eluted with ethanol, and recrystallized to obtain a white crystalline abscisic acid product. After the fermentation, the fermentation system was subjected to stable fermentation for 9 days, the contamination rate of the bacteria was 5.0%, and the product yield was 2.1 g of abscisic acid/liter of fermentation broth. After ion exchange and recrystallization recovery, the product recovery rate is 85%. Example 2 50 pieces of 1000 ml triangular flasks were used, and each bottle of 300 ral medium A was sterilized at 12 CTC, and after cooling, the activated abscisic acid high-yield strain Botrytis cinerea TBC-10 CGMCC No. 1889 spore suspension was placed and placed. Shake the flask for 24 to 33 hours at 25 °〇. The cultured first-class seed liquid is inoculated into a 200-liter fermenter (secondary seed tank) containing 100 liters of medium B according to the inoculation amount of 15%-18%, and agitated and cultured at a temperature of about 26 ° C. 30 hours. Use a 1 ton fermenter to make a three-stage tank fermentation. The tank contains a medium C of about 500 L. After sterilization by conventional high-pressure steam sterilization, the second-stage tank seed solution is inoculated with 20% - 25% inoculum. 05%-0. The total volume of the fermentation broth is 0. 05%-0. 3%. Inositol was added continuously (either at a constant rate or at a constant rate) at a rate of 0.1 to 1.0 g/L · h until about 10 hours before the fermentation was stopped (down can). 5% 麦糖糖 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% Maltose 0. 0% 糖糖 5. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 % medium ^C

2% Lactose 1. 2% 0% 0. 01% 糖糖0. 0% 豆粉粉1. 0% glycerin 1. 0% thiamine 0. 1% fatty acid 0. 5% potassium hydrogen phosphate 0. 05% glucose 20. 0% potassium dihydrogen phosphate 0. 01% 01% 硫酸糖0. 01% 硫酸硫酸0. 01% 硫酸硫酸0. 01% 硫酸硫酸0. 01%

The fermentation temperature was about 27 ° C, the fermentation pH was 4-7, and the fermentation time was 9 days. The can fermentation broth is recovered by ion exchange resin method, eluted with ethanol, and recrystallized to obtain a white crystalline abscisic acid product. After the fermentation, the fermentation system was subjected to stable fermentation for 9 days, the contamination rate of the bacteria was 5.0%, and the product yield was 2.4 g of abscisic acid/liter of fermentation broth. After ion exchange and recrystallization recovery, the product recovery rate is 85%. Example 3 30 bottles of 1000 ml flasks were used, and 300 ml of medium A per bottle was sterilized at 120 ° C. After cooling, the activated abscisic acid high-yield strain B. cinerea TBI-9 CGMCC No. 0500 spore suspension was inoculated. Shake the flask for 24 to 33 hours at 27 °C. The cultured first-class seed liquid is inoculated into a 100-liter fermenter (second-stage seed tank) containing 50 liters of medium B according to the inoculation amount of 15%-20%, and the mixture is ventilated at a temperature of about 26 ° C. 30 hours. Using a 1 ton fermenter to make a three-stage tank fermentation, the tank contains about 500 L of medium C. After sterilization by conventional high-pressure steam sterilization, the seed tank of the second tank is inoculated with 10%-15% inoculum. — After 24 hours, the medium C was continuously fed at a rate of 0.1 to 5 L/h until 10 hours before the fermentation (lower tank) was stopped. Inositol was added continuously (either at a constant rate or at a constant rate) at a rate of 0.1 to 5. 0 yg/L, h until about 10 hours before the fermentation was stopped (down can). The combination of media used in the implementation process is as follows: Glucose 3.0% Beef Cream 0.5% Wheat Bran 5.0% Sucrose 4.0%

2.0% magnesium sulfate 0.1% maltose 1.0%

Starch 5.0% Wheat bran 5.0% Glucose 4.0% Maltose 2.0% Bean cake powder 2.0% Sodium nitrate 5.0%

The fermentation temperature was about 26 ° C, the fermentation pH was 4-7, and the fermentation time was 9 days. The can fermentation broth is recovered by ion exchange resin method, eluted with ethanol, and recrystallized to obtain a white crystalline abscisic acid product. After the fermentation, the fermentation system was subjected to stable fermentation for 9 days, the contamination rate of the bacteria was 8. 0%, and the product yield was 2.1 g of abscisic acid/liter of fermentation broth. After ion exchange and recrystallization recovery, the product recovery rate is 84%. Example 4 25 pieces of 1000 ml flasks were used, and 300 ml of medium A per bottle was sterilized at 120 ° C. After cooling, the activated abscisic acid high-yield strain TB-3 - H8 spore suspension was inoculated and placed at about 26 ° C. Shake the flask for 40 - 56 hours at temperature. The cultured first-class seed solution is 15%-20°/. The inoculation amount was inoculated into a 100-liter fermenter (secondary seed tank) containing 50 liters of medium B, and agitated and cultured at a temperature of 26 ° C - 27 ° C for 24-26 hours. Use a 1 ton fermenter to make a three-stage tank fermentation. The tank contains about 500 L of medium C. After sterilization by conventional high-pressure steam sterilization, the seed tank of the second tank is inoculated with 10%-15% inoculum. 1%— 0. 0. 1%— 0. The total volume of the fermentation broth is 0.1% - 0. 5%. Inositol was added continuously (either at a constant rate or at a constant rate) at a rate of 0.1 to 3. 0 μg/L · h until about 10 hours before the fermentation was stopped (down can). The composition of the medium used in the process is as follows: glucose 0. 2%-3% beef extract 0. wheat bran 1 - 5 % sucrose 1% - 4% ammonium nitrate 0. 1% - 2% magnesium sulfate 0. 01% - 0 1%-% 麦糖糖0. 1%-3% Medium B Starch 1%-5% Wheat bran 1%-5% Glucose 1%-4% Maltose 0. 1%~3% Bean cake powder 0. 1%-2% 0% 0% 0% 糖糖0. 0% 糖 glycol 0. 03% 豆饼粉 14. 0% 糖糖0. 03% 糖糖。 0% glycerol 0. 03% sucrose 0. 03% sucrose 0. 03% sucrose 0. 02% sucrose 0. 02% sucrose 0. 03% sucrose 1. 0% manganese sulfate 0. 01% nickel sulfate 0. 03% Fermentation temperature is about 27 ° C, fermentation pH 4-7. The fermentation tank can reach the tank when it reaches 9 days. The can fermentation broth is recovered by ion exchange resin method, eluted with ethanol, and recrystallized to obtain a white crystalline abscisic acid product. After the fermentation, the fermentation system was subjected to stable fermentation for 9 days, the contamination rate of the bacteria was 8. 0%, and the product yield was 2.0 g of abscisic acid/liter of fermentation broth. After ion exchange and recrystallization recovery, the product recovery rate is 82%. Comparative Example 5 A continuous flow fed feed discharge process was employed. 30 bottles of 1000 ml flasks were used, and 300 ml of medium A per bottle was sterilized at 12 CTC. After cooling, the activated abscisic acid high-yield strain TB-3-H8 was sprayed, and the spore suspension was placed at a temperature of about 26 ° C. The flask was incubated for 40-56 hours. The cultured first-class seed solution is inoculated into a 100-liter fermenter (second-stage seed tank) containing 50 liters of medium B at a 15%-20% inoculum, and aeration is stirred at a temperature of 26 ° C - 27 ° C. Cultivate for 24-26 hours. Three-stage tank fermentation was carried out with a 1 ton fermenter. The tank was filled with medium C of about 500 L and microporous ceramic beads (particle size of 0.9-160 mm3, 1. 0-5 g/L, purchased from Jiangxi Jingdezhen Special Ceramics Research Institute). After sterilization by conventional high-pressure steam sterilization, the seed tank of the second tank is inoculated with 10%-15% inoculation amount, and the fermentation is stirred for 20-48 hours, then the chlorine is introduced to control the pH to 3-8, and the fermentation is lowered. The temperature is 10 ° C - 25 ° C, and the medium C is continuously fed at a rate of 0.01 to 5 L / h, continuously at a rate of 0.5 - 1. 0 yg / L * h (uniform or non-uniform) Add inositol. Discharge once every 10 hours. The fermentation temperature was about 27 ° C and the fermentation pH was 4-7. The medium combination used in the course of the implementation was the same as in Comparative Example 4. The fermentation time is up to 25 days when the cans are placed. The next tank fermentation broth recovers the product by ion exchange resin method, elutes with ethanol, and recrystallizes to obtain white Crystalline abscisic acid product. After the fermentation is carried out for 25 days, the contamination rate of the bacteria is 30.0%, and the product yield is 1.6 g of abscisic acid/liter of fermentation broth. After ion exchange and recrystallization recovery, the product recovery rate reached 82%. Example 6 50 pieces of 1000 ml flasks were used, and 300 ml of medium A per bottle was sterilized at 12 CTC. After cooling, the activated abscisic acid high-yield strain Botrytis cinerea TBC-10 CGMCC No. 1889 spore suspension was inoculated. Incubate at 25 ° (temperature, shake flask for 24-33 hours. Inoculate the cultured first-class seed solution at a 15% '-18% inoculum in a 200 liter fermentor containing 100 liters of medium B (secondary Seed tank), agitated and cultured for 24 to 30 hours at a temperature of about 26 ° C. ' Three-stage tank fermentation with 1 ton fermenter, medium C of about 500 L in tank, sterilized by conventional high-pressure steam sterilization Inoculate the secondary tank seed solution according to the inoculation amount of 20% - 25%, and agitate and ferment the fermentation. After 15-24 hours, the feed medium C is added intermittently, and the intermittent time is fed every 20-28 hours. 3 times, each feeding amount is 0. 05% - 0. 3 ° /.. until about 10 hours before stopping the fermentation (under can). The medium used in the implementation is as follows: Glucose 3. 0% 牛糖0. 0% 糖糖0. 0% Ammonium nitrate 1. 5% Magnesium sulphate 0. 05% Maltose 0. 3% Medium B 0% 小麦糖1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0% 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 01% 硫酸糖0. 01% 硫酸糖 0. 01% 硫酸糖 0. 01% 硫酸硫酸0. 01% 硫酸硫酸0. 01%

The fermentation temperature was about 27 ° C and the fermentation pH was 4-7. The can is fermented for 9 days. The can fermentation broth was recovered by ion exchange resin method, eluted with ethanol, and recrystallized to obtain a white crystalline abscisic acid product. After testing: After 9 days of stable fermentation, the fermentation system has a contamination rate of 5. 0% and a product yield of 2. 0 g of abscisic acid per liter of fermentation broth. After ion exchange and recrystallization recovery, the product recovery rate is 85%.

Claims

Rights request

A method for preparing a natural abscisic acid, which comprises the steps of:

The fungus capable of producing natural abscisic acid is cultured in a first stage liquid medium selected from the group consisting of: Bo rytis, Cercospora, Aspergillus, Penicillium a genus (Penicillium), or a genetically modified bacterium of the above strain; inoculating the first-stage seed liquid cultured in the above first-stage liquid medium onto the second-stage liquid medium for cultivation;

The second-stage seed liquid cultured in the above second-stage liquid medium is inoculated into the third-stage liquid medium, and after the fermentation is carried out at 26 ° C for 12-72 hours, the third-stage liquid medium flow is started. Feeding, and simultaneously adding inositol fermentation culture;

The resulting abscisic acid was collected from the above fermentation broth.

2. A method according to claim 1 wherein the inositol flow plus feed is a continuous flow addition and/or a batch flow addition.

3. A method according to claim 2 wherein the continuous flow is added as a uniform or non-uniform flow.

4. The method according to claim 2, wherein said continuous flow is carried out by adding a continuous flow of inositol to the third-stage fermenter at a flow rate of 0.01 - 10 g/L * h until 10 hours before the fermentation is stopped. .

5. The method according to claim 2, wherein said intermittent infusion of inositol is fed one to five times every 8 to 30 hours.

6. A method according to claim 5, wherein the feeding is carried out once every 10 hours, and each time the amount is 0.05 - 50 mg / L of fermentation broth.

7. A method according to claim 6 wherein the amount of each additional dose is from 0.1 to 10 mg/L of fermentation broth.

The method according to any one of claims 1 to 7, wherein the fungus used is Grifola

(Botrytis cinerea).

9. A method according to claim 8 wherein said fungus is Botrytis cinerea TBC-10 CGMCC No. 1889.

10. Botrytis cinerea used in the method of any one of claims 1 to 9 (Botrytis

Cinerea) TBC-10 CGMCC No.1889.