CN107988305B - Preparation method of gibberellic acid - Google Patents

Abstract

The invention provides a preparation method of gibberellic acid, which comprises the following steps: s1) preparing a seed solution of gibberellic acid; s2) transferring the seed liquid of gibberellic acid into a fermentation tank for fermentation to obtain gibberellic acid fermentation liquid; s3) filtering and concentrating the gibberellic acid fermentation liquor by a membrane to obtain a concentrated solution; s4) refining the concentrated solution to obtain the gibberellic acid. Compared with the prior art, the method adopts sugar and oil mixed supplementation in the fermentation process, improves the acid production, simultaneously takes the corn protein powder as an organic nitrogen source, has higher protein content of more than 50 percent, can realize high-density fermentation of gibberellin, can avoid the hidden trouble of producing aspergillus flavus like peanut cake powder, ensures the synthesis and metabolism of thalli, and can realize rapid and stable fermentation; moreover, the added vegetable oil can not only provide a carbon source for fermentation, but also effectively reduce the occurrence of conditions of fermentation bubble liquid escape and the like, and avoid potential risks of material loss, bacterial contamination and the like.

Description

Preparation method of gibberellic acid

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a preparation method of gibberellic acid.

Background

Gibberellic acid is a secondary metabolite obtained by fermentation and metabolism of a kind of gibberella, and 116 kinds of gibberellic acid are isolated, identified and named, among which GA is the most widely used in agriculture₃Plays a great role in agricultural production in China.

GA₃Has very high plant regulating activity and obvious regulation on the growth and development of various cropsHas the effect of saving energy. In the north, GA₃The gibberellic acid solution with a certain concentration is sprayed in the flowering phase, so that the fruit setting rate of fruit trees can be effectively improved, the fruit growth and development are promoted, and the fruit yield is improved by about 20-30%. In the south, gibberellic acid is mainly applied to hybrid rice seed production, and male and female parents of rice are regulated to bloom at the same time, so that the pollination rate of the female parents is greatly improved, the yield of hybrid rice seeds is further improved, the production cost of the seeds is reduced, and the burden of farmers is reduced.

The existing gibberellic acid is usually prepared by a liquid submerged fermentation method, and batch/fed-batch soybean oil or glucose and other modes are adopted in the fermentation process, so that the obtained fermentation acid yield is 2800-3000 ppm at a high level, but the fermentation acid yield is still to be improved.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing gibberellic acid, which has high and stable yield.

The invention provides a preparation method of gibberellic acid, which comprises the following steps:

s1) preparing a seed solution of gibberellic acid;

s2) transferring the seed liquid of gibberellic acid into a fermentation tank for fermentation to obtain gibberellic acid fermentation liquid; a fermentation medium in the fermentor, comprising: 10-40 g/L of corn protein powder; 1-10 g/L potassium dihydrogen phosphate; 5-30 g/L of a small-molecular organic carbon source; 0.5-5 g/L of vegetable oil; 0.5-2 g/L magnesium sulfate; 0.5-2 g/L of ammonium sulfate; 0.1-1 g/L of trace elements; in the fermentation process, when the dissolved oxygen is higher than 20-30%, glucose and vegetable oil are supplemented;

s3) filtering and concentrating the gibberellic acid fermentation liquor by a membrane to obtain a concentrated solution;

s4) refining the concentrated solution to obtain the gibberellic acid.

Preferably, the step S1) is specifically:

A1) the frozen GA₃Inoculating the strain into a seed culture medium for shake flask culture to obtain a suspension; the seed culture medium comprises 5-15 g/L of first organic nitrogen source and 5-15 g/L of first micromolecule organic nitrogen sourceA carbon source, 1-5 g/L potassium dihydrogen phosphate, 0.1-1 g/L magnesium sulfate and 0.1-1 g/L polypropylene glycol;

A2) transferring the suspension into a seeding tank for fermentation culture to obtain a seed solution; the culture medium of the seeding tank comprises 5-15 g/L of second organic nitrogen source, 5-15 g/L of second micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol.

Preferably, the step a2) further includes:

after fermentation culture, transferring the mixture into a secondary seed tank for continuous fermentation culture to obtain a seed solution; the culture medium of the secondary seed tank comprises 5-15 g/L of a third organic nitrogen source, 5-15 g/L of a third micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol.

Preferably, the first organic nitrogen source, the second organic nitrogen source and the third organic nitrogen source are respectively and independently selected from one or more of soybean protein, peanut cake powder, cottonseed cake powder, corn steep liquor, corn protein powder, peptone, yeast powder and fish meal;

the small molecule organic carbon source, the first small molecule organic carbon source, the second small molecule organic carbon source and the third small molecule organic carbon source are respectively and independently selected from one or more of sucrose, glucose, molasses, amylodextrin, mannose and galactose.

Preferably, the step S3) is specifically:

B1) filtering the gibberellic acid fermentation liquor by using a tubular membrane to obtain a tubular membrane filtration clear liquid and a tubular membrane filtration residual liquid;

B2) performing ultrafiltration on the tubular membrane filtered clear liquid to obtain an ultrafiltration clear liquid and an ultrafiltration residual liquid;

B3) and (4) carrying out nanofiltration on the ultrafiltration clear liquid to obtain a concentrated solution.

Preferably, the step B1) is specifically:

filtering the gibberellic acid fermentation liquor by a tubular membrane until the volume of clear liquid is 0.2-0.6 times of the volume of the fermentation liquor, and then continuously adding an aqueous solvent until the volume of the clear liquid is 1-2.5 times of the volume of the fermentation liquor to obtain tubular membrane filtered clear liquid and tubular membrane filtered residual liquid.

Preferably, the step S4) is specifically:

C1) extracting the concentrated solution by using an extracting agent to obtain an extract liquid;

C2) and concentrating and crystallizing the extract liquor to obtain the gibberellic acid.

Preferably, the step C1) is specifically:

mixing the concentrated solution with an extracting agent, adjusting the pH value to 2-3, extracting, standing and layering to obtain an extract liquid and a water phase; the volume ratio of the concentrated solution to the extracting agent is 1: (0.8 to 1.5);

and continuously extracting the water phase and the organic solvent to obtain an organic phase as an extractant of the concentrated solution.

Preferably, the step C2) is specifically:

and (3) carrying out reduced pressure evaporation concentration on the extract liquor, concentrating to one third to two thirds of the original volume, cooling, carrying out suction filtration, then carrying out secondary concentration, stopping concentration after crystallization, cooling, crystallizing, and filtering to obtain the gibberellic acid.

Preferably, the fermentation temperature in the step S2) is 28-30 ℃; the fermentation time is 8-10 days; the pH value of the fermentation is 5.0-5.2; the fermentation pressure is 0.01-0.05 MPa; the air flow rate of the fermentation is 2000-3000 Nm³H; the mass ratio of the glucose to the vegetable oil is (1-3): 1.

the invention provides a preparation method of gibberellic acid, which comprises the following steps: s1) preparing a seed solution of gibberellic acid; s2) transferring the seed liquid of gibberellic acid into a fermentation tank for fermentation to obtain gibberellic acid fermentation liquid; a fermentation medium in the fermentor, comprising: 10-40 g/L of corn protein powder; 1-10 g/L potassium dihydrogen phosphate; 5-30 g/L of a small-molecular organic carbon source; 0.5-5 g/L of vegetable oil; 0.5-2 g/L magnesium sulfate; 0.5-2 g/L of ammonium sulfate; 0.1-1 g/L of trace elements; in the fermentation process, when the dissolved oxygen is higher than 20-30%, glucose and vegetable oil are supplemented; s3) filtering and concentrating the gibberellic acid fermentation liquor by a membrane to obtain a concentrated solution; s4) refining the concentrated solution to obtain the gibberellic acid. Compared with the prior art, the method adopts sugar and oil mixed supplementation in the fermentation process, improves the acid production, simultaneously takes the corn protein powder as an organic nitrogen source, has higher protein content of more than 50 percent, can realize high-density fermentation of gibberellin, can avoid the hidden trouble of producing aspergillus flavus like peanut cake powder, ensures the synthesis and metabolism of thalli, and can realize rapid and stable fermentation; moreover, the addition of the vegetable oil can not only provide a carbon source for fermentation, but also effectively reduce the occurrence of conditions of fermentation bubble liquid escape and the like, and avoid potential risks of material loss, bacterial contamination and the like; in addition, the activity of various enzymes and metabolites in the microbial growth and metabolism process can be comprehensively ensured by adding trace elements into the fermentation medium.

Drawings

Fig. 1 is a schematic flow chart of embodiment 1 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a preparation method of gibberellic acid, which comprises the following steps:

s1) preparing a seed solution of gibberellic acid;

s2) transferring the seed liquid of gibberellic acid into a fermentation tank for fermentation to obtain gibberellic acid fermentation liquid; a fermentation medium in the fermentor, comprising: 10-40 g/L of corn protein powder; 1-10 g/L potassium dihydrogen phosphate; 5-30 g/L of a small-molecular organic carbon source; 0.5-5 g/L of vegetable oil; 0.5-2 g/L magnesium sulfate; 0.5-2 g/L of ammonium sulfate; 0.1-1 g/L of trace elements; in the fermentation process, when the dissolved oxygen is higher than 20-30%, glucose and vegetable oil are supplemented;

s3) filtering and concentrating the gibberellic acid fermentation liquor by a membrane to obtain a concentrated solution;

s4) refining the concentrated solution to obtain the gibberellic acid.

The present invention is not particularly limited in terms of the source of all raw materials, and may be commercially available.

Firstly, preparing seed liquid of gibberellic acid; the preparation method of the seed solution is a preparation method well known to those skilled in the art, and is not particularly limited, and the wet weight of the seed solution is preferably 30-40%; the preparation method of the invention preferably comprises the following steps: A1) the frozen GA₃Inoculating the strain into a seed culture medium for shake flask culture to obtain a suspension; the seed culture medium comprises 5-15 g/L of a first organic nitrogen source, 5-15 g/L of a first micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol; A2) transferring the suspension into a seeding tank for fermentation culture to obtain a seed solution; the culture medium of the seeding tank comprises 5-15 g/L of second organic nitrogen source, 5-15 g/L of second micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol.

The frozen GA₃Inoculating the strain into a seed culture medium for shake flask culture to obtain a suspension; the GA₃The inoculation amount of the strain is preferably 0.4-1.2%, more preferably 0.5-1%, still more preferably 0.6-1%, still more preferably 0.8-1% of the volume of the seed culture solution; the seed culture medium comprises 5-15 g/L of first organic nitrogen source, 5-15 g/L of first micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol, preferably comprises 8-12 g/L of first organic nitrogen source, 8-12 g/L of first micromolecular organic carbon source, 2-4 g/L of monopotassium phosphate, 0.3-0.8 g/L of magnesium sulfate and 0.3-0.8 g/L of polypropylene glycol, more preferably comprises 9-11 g/L of first organic nitrogen source, 9-11 g/L of first micromolecular organic carbon source, 2-4 g/L of monopotassium phosphate, 0.5-0.7 g/L of magnesium sulfate and 0.4-0.5 g/L of polypropylene glycol, still more preferably comprises 10g/L of first organic nitrogen source, 10g/L of first micromolecular organic carbon source, 3g/L potassium dihydrogen phosphate, 0.6g/L magnesium sulfate and 0.5g/L polypropylene glycol; the first organic nitrogen source is not particularly limited as long as it is known to those skilled in the art, and in the present invention, it is preferably one or more of soybean protein, peanut cake powder, cottonseed cake powder, corn steep liquor, corn gluten meal, peptone, yeast powder and fish meal; under the action of protease secreted by microbe,decomposing into amino acids, absorbing by thallus to obtain thallus protein, and using organic nitrogen source as precursor of thallus growth and propagation; the first small molecule organic carbon source is not particularly limited as long as it is a small molecule organic carbon source well known to those skilled in the art, and in the present invention, one or more of sucrose, glucose, molasses, amylodextrin, mannose and galactose are preferred; the small molecular organic carbon source can provide energy for the growth and the propagation of microbial cells and carbon components necessary for synthesizing thalli, and can also provide carbon components required for synthesizing target products; potassium dihydrogen phosphate in the culture medium is used for providing phosphorus element, phosphorus is an essential component of nucleic acid and protein and is also an important component for transferring Adenosine Triphosphate (ATP), and in the aspect of regulation of metabolic pathways, phosphorus also plays an important role in facilitating the progress of sugar metabolism, so that the growth of microorganisms can be promoted; the magnesium sulfate used in the present invention is not particularly limited as long as it is well known to those skilled in the art, and magnesium sulfate heptahydrate is preferred in the present invention; magnesium sulfate is used for providing magnesium element which is an activator of a plurality of enzymes and can promote metabolism of carbohydrate, synthesis of nucleic acid, conversion of phosphate and the like; polypropylene Glycol (PPG) is used as a defoaming agent in a culture medium, so that the phenomenon that the escaped liquid loses nutrition and thalli due to bubbles can be avoided, and the escaped liquid can also increase the risk of bacterial contamination; the method of shake flask culture is a method well known to those skilled in the art, and is not particularly limited, and the temperature of shake flask culture in the present invention is preferably 28 ℃ to 30 ℃, more preferably 28.5 ℃ to 29.5 ℃, and still more preferably 28.8 ℃ to 29.2 ℃; the shake flask culture time is preferably 60-80 h, more preferably 62-75 h, still more preferably 64-70 h, still more preferably 66-70 h, and most preferably 68 h.

Transferring the suspension into a seeding tank for fermentation culture; the volume ratio of the transferred suspension to the culture solution in the seeding tank is preferably 0.01 to 0.02%, more preferably 0.013 to 0.018%, still more preferably 0.014 to 0.018%, most preferably 0.016%; the culture medium of the seeding tank comprises 5-15 g/L of second organic nitrogen source, 5-15 g/L of second micromolecular organic carbon source and 1-5 g/L of phosphoric acidMonopotassium, 0.1-1 g/L magnesium sulfate and 0.1-1 g/L polypropylene glycol, preferably comprises 8-12 g/L second organic nitrogen source, 8-12 g/L second small molecular organic carbon source, 2-4 g/L monopotassium phosphate, 0.3-0.8 g/L magnesium sulfate and 0.3-0.8 g/L polypropylene glycol, more preferably comprises 9-11 g/L second organic nitrogen source, 9-11 g/L second small molecular organic carbon source, 2-4 g/L monopotassium phosphate, 0.5-0.7 g/L magnesium sulfate and 0.4-0.5 g/L polypropylene glycol, and further preferably comprises 10g/L second organic nitrogen source, 10g/L second small molecular organic carbon source, 3g/L monopotassium phosphate, 0.6g/L magnesium sulfate and 0.5g/L polypropylene glycol; the second organic nitrogen source is not particularly limited, but is preferably one or more of soybean protein, peanut cake powder, cottonseed cake powder, corn steep liquor, corn protein powder, peptone, yeast powder and fish powder; the second small molecule organic carbon source is not particularly limited, but is preferably one or more of sucrose, glucose, molasses, amylodextrin, mannose and galactose; in the invention, the pH value of the culture medium is preferably 4.5-5.5, more preferably 4.8-5.4, still more preferably 5-5.2, and most preferably 5.1, namely the pH value of the culture medium is adjusted to the above value by using sulfuric acid before sterilization; the fermentation culture method is a method well known to those skilled in the art, and is not particularly limited, and the temperature in the present invention is preferably 28 to 30 ℃, more preferably 28.5 to 29.5 ℃, and still more preferably 28.8 to 29.2 ℃; the fermentation culture time is preferably 48-72 h, more preferably 48-60 h, still more preferably 48-55 h, and most preferably CO₂Rising to the highest point, and finishing fermentation culture after 0.2 or 2 hours of decline; the pressure of the fermentation culture is preferably 0.01-0.05 MPa, more preferably 0.02-0.04 MPa, still more preferably 0.03-0.04 MPa, and most preferably 0.035 MPa; the air flow rate of the fermentation culture is preferably 60-70 Nm³More preferably 62 to 68Nm³Perh, and preferably 64 to 66Nm³H, most preferably 65Nm³/h。

After the fermentation culture is finished, preferably performing seed transfer and amplification culture, namely transferring the seeds into a secondary seed tank for continuous fermentation culture to obtain a seed solution; this step is carried outThe inoculation amount in the step is preferably 10-15% of the volume of the culture solution in the secondary seeding tank; the culture medium of the secondary seed tank comprises 5-15 g/L of third organic nitrogen source, 5-15 g/L of third micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol, preferably comprises 8-12 g/L of third organic nitrogen source, 8-12 g/L of third micromolecular organic carbon source, 2-4 g/L of monopotassium phosphate, 0.3-0.8 g/L of magnesium sulfate and 0.3-0.8 g/L of polypropylene glycol, more preferably comprises 9-11 g/L of third organic nitrogen source, 9-11 g/L of third micromolecular organic carbon source, 2-4 g/L of monopotassium phosphate, 0.5-0.7 g/L of magnesium sulfate and 0.4-0.5 g/L of polypropylene glycol, more preferably comprises 10g/L of third organic nitrogen source, 10g/L of third micromolecular organic carbon source, 3g/L potassium dihydrogen phosphate, 0.6g/L magnesium sulfate and 0.5g/L polypropylene glycol; the third organic nitrogen source is not particularly limited, but is preferably one or more of soybean protein, peanut cake powder, cottonseed cake powder, corn steep liquor, corn protein powder, peptone, yeast powder and fish powder; the third small molecule organic carbon source is not particularly limited as long as it is a small molecule organic carbon source well known to those skilled in the art, and in the present invention, one or more of sucrose, glucose, molasses, amylodextrin, mannose and galactose are preferred; in the invention, the pH value of the culture medium is preferably 4.5-5.5, more preferably 4.8-5.4, still more preferably 5-5.2, and most preferably 5.1, namely the pH value of the culture medium is adjusted to the above value by using sulfuric acid before sterilization; the method for continuing the fermentation culture is a method well known to those skilled in the art, and is not particularly limited, and the temperature in the present invention is preferably 28 ℃ to 30 ℃, more preferably 28.5 ℃ to 29.5 ℃, and still more preferably 28.8 ℃ to 29.2 ℃; the time for continuous fermentation culture is preferably 15-25 h, more preferably 18-25 h, still more preferably 20-24 h, and most preferably CO₂Rising to the highest point, and finishing fermentation culture after 0.2 or 2 hours of decline; the pressure of the fermentation culture is preferably 0.01-0.05 MPa, more preferably 0.02-0.04 MPa, still more preferably 0.03-0.04 MPa, and most preferably 0.035 MPa; the air flow rate of the fermentation culture is preferably 600-800 Nm³More preferably 650 to 800Nm³Perh, preferably 700 to 800Nm³Per hour, more preferably 740 to 780Nm³H, most preferably 760Nm³/h。

The invention directly inserts the frozen strains into the shake flask, avoids the strain variation risk of the slant activator in the slant growth process, and the freezing tube can well keep the parallelism of the strains, thereby ensuring the stability of the subsequent enlarged growth.

Transferring the seed liquid of gibberellic acid into a fermentation tank for fermentation to obtain gibberellic acid fermentation liquid; a fermentation medium in the fermentor, comprising: 10-40 g/L of corn protein powder; 1-10 g/L potassium dihydrogen phosphate; 5-30 g/L of a small-molecular organic carbon source; 0.5-5 g/L of vegetable oil; 0.5-2 g/L magnesium sulfate; 0.5-2 g/L of ammonium sulfate; 0.1-1 g/L of trace elements; and supplementing glucose and vegetable oil when the dissolved oxygen is higher than 20% in the fermentation process.

The content of the corn protein powder in the fermentation medium provided by the invention is preferably 15-35 g/L, more preferably 15-30 g/L, further preferably 20-25 g/L, and most preferably 22 g/L. The corn protein powder is rich in protein, reaches more than 50 percent, can provide sufficient nitrogen source for high-density fermentation of gibberellin, and ensures the synthesis and metabolism of thalli.

The content of the monopotassium phosphate is preferably 3-10 g/L, more preferably 4-8 g/L, further preferably 5-7 g/L, and most preferably 6 g/L. Phosphorus is an essential component of nucleic acids and proteins and also an important energy transmitter, Adenosine Triphosphate (ATP), and plays an important role in the regulation of metabolic pathways, and is advantageous for the progress of sugar metabolism, and thus it promotes the growth of microorganisms. Phosphorus is an essential element in the gibberellin synthesis process, and the lack of phosphorus can cause the synthesis of gibberellin precursors to be hindered, so that the concentration of potassium dihydrogen phosphate is increased, and the increase of labor and operation cost caused by adding potassium dihydrogen phosphate in the fermentation process is avoided.

According to the invention, the content of the small molecular organic carbon source is preferably 5-25 g/L, more preferably 8-15 g/L, even more preferably 8-12 g/L, and most preferably 10 g/L; the small-molecule organic carbon source is an organic carbon source well known to those skilled in the art, and is not particularly limited, and in the present invention, one or more of sucrose, glucose, molasses, amylodextrin, mannose and galactose are preferred, and sucrose and glucose are more preferred; the mass ratio of the sucrose to the glucose is preferably (1-2): (2-1), more preferably (1-1.5): (1.5-1), and more preferably (1-1.2): (1.2-1), most preferably 1: 1. in the invention, the small molecular organic carbon source is used as a fermentation carbon source, so that a fast and slowly-utilized carbon source can be provided for fermentation metabolism, energy sources are provided for the growth and the propagation of microbial cells, carbon components necessary for synthesizing thalli are provided, and carbon components required for synthesizing a target product are provided. In the invention, the addition of sucrose avoids feedback inhibition of gibberellin fermentation caused by excessive glucose to influence the synthesis of gibberellin, and sucrose as a substitute carbon source of glucose can decompose and supply thallus metabolism in time, thereby reducing the occurrence of feedback inhibition.

The content of the vegetable oil is preferably 1-4 g/L, more preferably 2-3 g/L, and further preferably 2 g/L; the vegetable oil is a vegetable oil known to those skilled in the art, and is not particularly limited, but in the present invention, one or more of salad oil, sunflower oil, linseed oil, ethyl palm oil and olive oil are preferable, but stearic acid inhibits fermentation of gibberellin, and thus salad oil is more preferable in the present invention. The vegetable oil can reduce the generation of foam in the fermentation process, avoid the phenomena of liquid escape and the like, and further avoid potential risks of material loss, bacterial contamination and the like; meanwhile, the vegetable oil can also provide a carbon source for fermentation, provide a carbon skeleton for the synthesis of gibberellin, and accelerate the synthesis of gibberellin.

The content of magnesium sulfate in the fermentation medium for producing gibberellic acid provided by the invention is preferably 0.5-1.5 g/L, more preferably 0.8-1.2 g/L, and further preferably 1 g/L; the magnesium sulfate is not particularly limited as long as it is known to those skilled in the art, and magnesium sulfate heptahydrate is preferred in the present invention. Magnesium is an activator of many enzymes, and promotes metabolism of carbohydrates, synthesis of nucleic acids, conversion of phosphates, and the like.

The content of the ammonium sulfate is preferably 0.5-1.5 g/L, more preferably 0.8-1.2 g/L, and still more preferably 1 g/L.

According to the invention, the content of the trace elements is preferably 0.2-0.8 g/L, more preferably 0.2-0.6 g/L, still more preferably 0.3-0.5 g/L, and most preferably 0.39 g/L; the trace elements are known to those skilled in the art, and are not particularly limited, but the present invention preferably includes ferrous sulfate, zinc sulfate, manganese sulfate, sodium molybdate, copper sulfate and cobalt chloride; the mass ratio of the ferrous sulfate, the zinc sulfate, the manganese sulfate, the sodium molybdate, the copper sulfate and the cobalt chloride is preferably (0.5-1.5): (0.5-1.5): (0.5-1.5): (0.1-0.5): (0.1-0.5): (0.1 to 0.5), more preferably (0.8 to 1.2): (0.8-1.2): (0.8-1.2): (0.2-0.4): (0.2-0.4): (0.2 to 0.4), and preferably 1: 1: 1: 0.3: 0.3: 0.3. the composition added with the trace elements can meet the requirement of the trace elements required by the growth of microorganisms, comprehensively ensure the activity of various enzymes and metabolites in the growth and metabolism process of the microorganisms, increase the activity of certain enzymes participating in product synthesis, and obviously improve the fermentation yield.

In the present invention, the fermentation medium most preferably comprises:

the fermentation medium provided by the invention takes corn protein powder as an organic nitrogen source, the protein content is higher and reaches more than 50%, gibberellin high-density fermentation can be realized, the hidden danger of aspergillus flavus generated by peanut cake powder can be avoided, the synthesis and metabolism of thalli are ensured, and the fermentation can be rapidly and stably performed; meanwhile, the addition of the vegetable oil can not only provide a carbon source for fermentation, but also effectively reduce the occurrence of conditions of fermentation bubble liquid escape and the like, and avoid potential risks of material loss, bacterial contamination and the like; in addition, the activity of various enzymes and metabolites in the microbial growth and metabolism process can be comprehensively ensured by adding trace elements into the fermentation medium.

Transferring the seed liquid of gibberellic acid into a fermentation tank for fermentation to obtain gibberellic acid fermentation liquid. The transfer amount is preferably 10 to 15 percent of the mass of the culture solution of the fermentation tank; the fermentation temperature is preferably 28-30 ℃, more preferably 28.5-29.5 ℃, and further preferably 28.8-29.2 ℃; what is needed isThe fermentation time is preferably 8 to 10 days, more preferably 8.5 to 9.5 days, and further preferably 9 days; the pH value of the fermentation is preferably 5.0-5.2; after fermentation is started, the pH value is gradually increased by 0.1/half hour each time, and preferably ammonia water is fed to control the pH value to be 5.0-5.2; the fermentation pressure is preferably 0.01-0.05 MPa, more preferably 0.02-0.04 MPa, still more preferably 0.03-0.04 MPa, and most preferably 0.035 MPa; the air flow rate of the fermentation is preferably 2000-3000 Nm³More preferably 2000 to 2800Nm³Perh, more preferably 2000 to 2500Nm³(iii)/h, most preferably 2200 to 2300Nm³H is used as the reference value. In the fermentation process, the dissolved oxygen rebounds gradually, and when the dissolved oxygen is higher than 20% -30%, glucose and vegetable oil are supplemented; the vegetable oil is the same as the above, and is not described again; when the supplemented glucose is solid, the mass ratio of the glucose to the vegetable oil is preferably (1-3): 1, more preferably (1.5 to 2.5): 1, more preferably 2: 1; when glucose is added in the form of a glucose solution, the mass concentration of glucose in the glucose solution is preferably 40% to 50%, more preferably 42% to 48%, and still more preferably 45% to 48%; the volume ratio of the glucose solution to the vegetable oil is preferably (2-4): 1, more preferably (2.5 to 3.5): 1, and preferably (3-3.5): 1, most preferably 3.2: 1.

the fermentation method provided by the invention adopts the processes of sugar and oil mixed supplementation and fed-batch, is superior to the fermentation yield of the single fed-batch of sugar or oil, increases the oil supplementation amount or the single oil supplementation, and generates the byproduct GA₁However, the amount of impurities in the post-extraction is small, and the amount of sugar is increased or increased, which results in increased fermentation impurities, impure proteins, and the like.

According to the invention, sugar and oil are mixed and supplemented in the fermentation process, the acid production is improved, meanwhile, the corn protein powder is used as an organic nitrogen source, the protein content is higher and reaches more than 50%, the high-density fermentation of gibberellin can be realized, the hidden danger of aspergillus flavus generated by peanut cake powder can be avoided, the synthesis and metabolism of thalli are ensured, and the fermentation can be rapidly and stably carried out; moreover, the addition of the vegetable oil can not only provide a carbon source for fermentation, but also effectively reduce the occurrence of conditions of fermentation bubble liquid escape and the like, and avoid potential risks of material loss, bacterial contamination and the like; in addition, the activity of various enzymes and metabolites in the microbial growth and metabolism process can be comprehensively ensured by adding trace elements into the fermentation medium.

Filtering and concentrating the gibberellic acid fermentation liquor by using a membrane to obtain a concentrated solution; the method of concentration is known to those skilled in the art, and is not particularly limited, and the present invention is preferably embodied as follows: B1) filtering the gibberellic acid fermentation liquor by using a tubular membrane to obtain a tubular membrane filtration clear liquid and a tubular membrane filtration residual liquid; B2) performing ultrafiltration on the tubular membrane filtered clear liquid to obtain an ultrafiltration clear liquid and an ultrafiltration residual liquid; B3) and (4) carrying out nanofiltration on the ultrafiltration clear liquid to obtain a concentrated solution.

In the present invention, the sources of all raw materials are not particularly limited, and they may be commercially available.

Filtering gibberellic acid fermentation liquor by a tubular membrane to obtain ultrafiltration clear liquid and ultrafiltration residual liquid; the gibberellic acid fermentation broth is a gibberellic acid fermentation broth well known to those skilled in the art, and is not limited at all; the specification of the tubular membrane is preferably 30-50 KD, and more preferably 40 KD; the material of the tubular membrane is preferably a metal membrane, a ceramic membrane, a natural polymer membrane or a synthetic polymer membrane. According to the invention, the steps are preferably embodied as follows: filtering gibberellic acid fermentation liquor by a tubular membrane until the volume of clear liquid is 0.2-0.6 times of the volume of the fermentation liquor, more preferably 0.3-0.5 times, still more preferably 0.35-0.5 times, most preferably 0.375-0.5 times, then continuously adding a water solvent until the volume of clear liquid is 1-2.5 times, more preferably 1.5-2.2 times, still more preferably 1.5-1.8 times, most preferably 1.5-1.75 times of the volume of the fermentation liquor, and obtaining tubular membrane filtration clear liquid and tubular membrane filtration residual liquid; the water solvent can be clear water or recycled water.

Preferably, the residual liquid after tubular membrane filtration is subjected to plate-and-frame filtration to obtain plate-and-frame filtration clear liquid and filter residue; the specification of the membrane filtered by the plate frame is preferably 600-900B, more preferably 700-800B, and still more preferably 750B; the material is preferably polypropylene. Clear liquid obtained by plate-frame filtration can be used as an aqueous solvent for tubular membrane filtration; and drying the filter residue to be used for producing bacterial manure.

Performing ultrafiltration on the tubular membrane filtered clear liquid to obtain an ultrafiltration clear liquid and an ultrafiltration residual liquid; the cut-off molecular weight of the ultrafiltration membrane is preferably 4000-6000, more preferably 4500-5500, and further preferably 5000; the material of the ultrafiltration membrane is preferably an organic membrane, and more preferably one of cellulose and derivatives thereof, polycarbonate, polyvinyl chloride, polyvinylidene fluoride, polysulfone, polyacrylonitrile, polyamide, polysulfonamide, sulfonated polysulfone, cross-linked polyvinyl alcohol and modified acrylic acid polymer; the ultrafiltration method is a method well known to those skilled in the art, and is not particularly limited, and continuous ultrafiltration is preferred in the present invention; and preferably, stirring the ultrafiltration residual liquid, and then performing plate-frame filtration to obtain plate-frame filtration clear liquid and filter residues. Clear liquid obtained by plate-frame filtration can be used as an aqueous solvent for tubular membrane filtration; and drying the filter residue to be used for producing bacterial manure.

Carrying out nanofiltration on the ultrafiltration clear liquid to obtain a concentrated solution; the nanofiltration method is a method well known to those skilled in the art, and is not particularly limited, and in the present invention, the first nanofiltration and the second nanofiltration are preferably performed sequentially; the first nanofiltration is preferably four-stage continuous nanofiltration concentration to obtain a concentrated solution and four-stage drainage water; the first stage effluent and the second stage effluent are preferably recycled, and the third stage effluent and the fourth stage effluent are preferably subjected to secondary nanofiltration concentration to obtain a concentrated solution and effluent; the second nanofiltration concentration is preferably high-power nanofiltration concentration; combining the concentrated solution obtained by the two times of nanofiltration, and recycling the discharged water obtained by the second time of nanofiltration; the specification of the membrane for the first nanofiltration is preferably 80-200D, more preferably 100-150D, and further preferably 100D; the material of the membrane for the first nanofiltration is preferably an aromatic and polyacid hydrogen composite nanofiltration membrane; the specification of the membrane for the second nanofiltration is preferably 80-200D, more preferably 100-150D, and further preferably 100D; the material of the membrane for the second nanofiltration is preferably an aromatic and polyacid hydrogen composite nanofiltration membrane.

According to the invention, the combined concentrate is preferably extracted with an extractant to give an extract; the step preferably comprises the following specific steps: mixing the concentrated solution with an extracting agent, adjusting the pH value to 2-3, extracting, standing and layering to obtain an extract liquid and a water phase; and continuously extracting the water phase and the organic solvent to obtain an organic phase as an extractant of the concentrated solution. The volume ratio of the concentrated solution to the extracting agent is preferably 1: (0.8 to 1.5), more preferably 1: (0.9-1.2), and preferably 1: 1; the pH value is preferably adjusted by adopting sulfuric acid; the pH value is preferably 2.5-3, more preferably 2.8-3, and further preferably 2.8; the organic solvent is not particularly limited as long as it is well known to those skilled in the art, and in the present invention, ethyl acetate is preferable; the continuous extraction method is a method well known to those skilled in the art, and is not particularly limited, and in the present invention, cross-flow extraction is preferred, and six-stage cross-flow extraction is more preferred; the content of the target product in the water phase is reduced to below 100ppm through continuous extraction. The water phase after continuous extraction is preferably discharged into a sewage treatment station for treatment after solvent is recovered by evaporation.

Concentrating and crystallizing the extract liquor to obtain gibberellic acid; the method for concentrating and crystallizing is a method well known by those skilled in the art, and is not particularly limited, in the invention, crystallization is preferably performed through two evaporation and concentration processes, namely, evaporation and concentration are performed on an extract under reduced pressure, the concentrate is concentrated to one third to two thirds of the original volume, after cooling and suction filtration, secondary concentration is performed, the concentrate is stopped after crystallization, and the concentrate is cooled and crystallized, and after filtration, gibberellic acid is obtained; and (3) preferably, evaporating and concentrating the mother liquor of the primary crystallization under reduced pressure until crystallization exists, cooling and crystallizing, and filtering to obtain the gibberellic acid.

According to the present invention, it is preferable that the gibberellic acid obtained by crystallization is dried and pulverized. The drying is preferably boiling drying; the pulverization is preferably carried out by a micronizer.

The invention sequentially adopts microfiltration, ultrafiltration and nanofiltration for concentration, extracts with extract liquor, and concentrates and crystallizes to obtain the gibberellic acid, has short production period, reduces energy consumption, improves concentration yield, simultaneously avoids the problem of inactivation of the effective components of the heat-sensitive substances of the gibberellic acid product, and improves the product quality.

In order to further illustrate the present invention, the following examples are provided to describe the preparation method of gibberellic acid.

The reagents used in the following examples are all commercially available.

Example 1

1.1 mixing GA₃The strain freezing tube is directly inoculated into a shake flask, the inoculation amount is 0.8 percent of the volume of the shake flask culture medium, the formula of the culture medium in the shake flask is shown in table 1, the fermentation temperature is 29 +/-0.2 ℃, and the shake flask culture is carried out for about 68 hours to obtain suspension.

TABLE 1 Shake flask culture Medium formulation

1.2 the suspension was inoculated into a first seed tank at an inoculum size of 0.016% of the volume of the broth, the first seed tank medium formulation is shown in Table 2.

TABLE 2 first-class seed tank culture medium formulation

Fermentation volume: the volume is 1.3t, and the volume after digestion is 1.5t (sulfuric acid is added to adjust the pH to 5.1 before sterilization).

Fermentation temperature: 29. + -. 0.2 ℃.

Operating the tank pressure: 0.035 MPa.

Air flow rate: 65Nm³/h。

Seed transferring time: CO 2₂Raising to the highest point, and transferring seeds after 0.2 or 2 hours of descent (48-72 hours).

1.3 inoculating the culture solution in the first-stage seeding tank into the second-stage seeding tank, wherein the inoculation amount is 8 percent of the volume of the culture solution, and the formula of the culture medium in the second-stage seeding tank is shown in Table 3.

TABLE 3 Secondary seeding tank Medium formulation

Fermentation volume: the volume is 15t, the volume after digestion is 16.5t, and the volume after conversion is 18t (sulfuric acid is added before sterilization to adjust the pH to 5.1).

Fermentation temperature: 29. + -. 0.2 ℃.

Operating the tank pressure: 0.035 MPa.

Air flow rate: 760Nm³/h。

Seed transferring time: CO 2₂Rising to the highest point, and obtaining the seed liquid after 0.2 or 2 hours of decline (estimated about 20 hours).

The shake flask, primary and secondary seed tank broth assay data are shown in Table 4.

TABLE 4 Shake flask, Primary and Secondary seed tank broth test data

Seed tank

Wet weight

pH

Length of hypha

Thickness of hypha

Whether the cytoplasm is uniform or not

With or without cavitation

Viscosity of fermentation broth

Shake flask seed liquid

31％

4.52

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

First-class seed liquid

16.5％

4.99

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

Second-stage seed liquid

22.5％

5.53

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

1.4 the seed solution obtained in 1.3 is transferred into a fermentation tank for fermentation according to 10-15% of the mass of the culture solution, and the formula of the fermentation medium is shown in Table 5.

TABLE 5 fermentation Medium recipe

Fermentation volume: the volume is fixed to 82t, the volume after the extinction is 90t, and the volume after the rotation is 100 t.

Fermentation temperature: 29. + -. 0.2 ℃.

Operating the tank pressure: 0.035 MPa.

Air flow rate: 2200Nm³/h。

Controlling the pH: after the operation is started, the PH is gradually increased, the PH is increased by 0.1/half hour each time, and the PH is controlled to be 5.0-5.2 by adding ammonia water in a flowing mode.

And (3) supplementary material control: the dissolved oxygen rebounds by 20 percent, and the automatic feeding of glucose, salad oil and glucose (45 percent) solution is started: oil (3.2L:1L), dissolved oxygen control point 20% -30%.

And (3) fermentation period: and 9 days.

The fermentation liquids obtained in example 1 were examined, and the results are shown in tables 6 and 7.

TABLE 6 fermentation broth assay data

TABLE 7 fermentation broth wet weight

1.5 tubular membrane filtration:

and (3) sterilizing the 120t fermentation broth after fermentation is finished, transferring the fermentation broth into a storage tank, and directly starting tubular membrane filtration (without adjusting the pH). And continuously adding tap water after filtering out about 45T of clear liquid of the fermentation liquid, and stopping tubular membrane filtration when filtering out about 260T of clear liquid. (specification of tubular membrane: 40KD, material: ceramic membrane)

The clear liquid after membrane filtration enters the next main process (ultrafiltration), and the residual liquid enters the next auxiliary process (plate-and-frame filtration).

1.6, plate and frame filtration:

and (3) directly carrying out plate-and-frame filtration (the specification of a plate-and-frame filtration membrane is 750B, and the material is polypropylene) on the residual liquid about 55T after the tubular membrane filtration, using the filtered clear liquid for the next pot of tubular membrane filtration, and drying the filter residue to produce bacterial manure.

1.7 ultrafiltration:

and (3) carrying out ultrafiltration on the clear liquid after the tubular membrane, wherein the ultrafiltration is an organic material membrane with the molecular weight of 5 kilo, and the membrane material is polyvinylidene fluoride. The ultrafiltration is continuous ultrafiltration, the ultrafiltration clear liquid enters the next procedure, and the residual liquid enters a filter residue stirring tank and then enters a plate frame for filtration.

1.8 nanofiltration

And (3) performing nanofiltration concentration on the clear liquid after ultrafiltration, wherein 2 times of concentration are adopted in the nanofiltration concentration, the first concentration is 4-stage continuous concentration, the concentrated solution enters the next procedure, the 1-2-stage discharged water is recycled, and the 3-4-stage discharged water is highly concentrated (the concentrated solution enters the next procedure, and the discharged water is recycled). During production, a primary high-pressure nanofiltration membrane is adopted. (nanofiltration membrane specification 100D, material aromatic and polyacid hydrogen composite nanofiltration membrane)

1.9 extraction

Transferring the nanofiltration concentrated solution to a refining plant, adding an equal volume of continuous extraction solvent (the solvent is obtained by 6-stage continuous extraction and contains a certain amount of target product), adding sulfuric acid to adjust the pH value to 2.8, and performing single-batch extraction. And standing for layering after extraction is finished. And (4) after layering, enabling the upper solvent phase to enter the next procedure.

And carrying out continuous 6-stage cross-flow extraction on the lower-layer water phase and a fresh solvent (ethyl acetate) after layering to reduce the content of a target product in the water phase to be less than 100 ppm.

After continuous extraction, the water phase is treated by a membrane scraper evaporator to recover part of the solvent and then discharged into a sewage treatment station.

And transferring the solvent phase after continuous extraction into a storage tank for temporary storage, and using the solvent phase for single-batch extraction of the next batch.

Concentration by 1.102000L evaporator does not crystallize

And (3) carrying out reduced pressure evaporation concentration on the solvent phase after single batch extraction at the temperature of 45 ℃, evaporating out about 2T of recovered solvent when concentration is carried out, putting the recovered solvent into a crystallizing tank for cooling, carrying out suction filtration, and entering the next procedure.

1.111000L evaporator concentration crystallization

Evaporating and concentrating 2000L of the concentrated liquid under reduced pressure at 50 deg.C, concentrating until there is crystal, and cooling again in a crystallizing tank. Then, carrying out suction filtration, and taking the solid as a product to enter the next working procedure. And (4) carrying out reduced pressure evaporation concentration on the primary mother liquor again, and putting the concentrated mother liquor into a crystallizing tank for cooling and crystallizing again after the concentrated mother liquor is crystallized. Then, carrying out suction filtration, and taking the solid as a product to enter the next working procedure. The liquid is used as the final mother liquor to enter a mother liquor storage tank and then is prepared into the cream preparation.

1.12 drying and pulverizing

And (3) drying and crushing the crystallized product (a boiling dryer is adopted at present for drying, and a universal micronizer is adopted at present for crushing) and subpackaging to obtain a final product, wherein the performance detection results are shown in table 8.

Table 8 results of measuring properties of the crystalline product obtained in example 1

Serial number	Inspection item	Quality standard
			1	Content (wt.)	≥90％
2	Moisture content	≤0.5％
			3	Specific rotation	＞+80°
4	Yield of	≥70％

The schematic flow diagram of example 1 is shown in FIG. 1.

Example 2

2.1 tubular membrane filtration:

120t of the fermentation broth obtained in example 1 was sterilized after the end of fermentation, transferred to a storage tank, and then directly subjected to tubular membrane filtration (without pH adjustment). And continuously adding tap water after filtering out the clear liquid about 45T of the fermentation liquid, and stopping tubular membrane filtration when filtering out the clear liquid about 260T of the fermentation liquid. (specification of tubular film: 4KD and Material: Metal film)

The clear liquid after membrane filtration enters the next main process (ultrafiltration), and the residual liquid enters the next auxiliary process (plate-and-frame filtration).

2.2, plate and frame filtration:

and (3) directly carrying out plate-frame filtration (the specification of a plate-frame filtration membrane is 750B, the material is polypropylene) on the residual liquid about 35T after the tubular membrane filtration, using the filtered clear liquid for the next pot of tubular membrane filtration, and drying the filter residue to produce bacterial manure.

2.3, ultrafiltration:

and (3) carrying out ultrafiltration on the clear liquid after the tubular membrane, wherein the ultrafiltration is carried out by using an organic material membrane with 4 thousand molecular weight, and the membrane material is polycarbonate. The ultrafiltration is continuous ultrafiltration, the ultrafiltration clear liquid enters the next procedure, and the residual liquid enters a filter residue stirring tank and then enters a plate frame for filtration.

2.4 nanofiltration

And (3) performing nanofiltration concentration on the clear liquid after ultrafiltration, wherein 2 times of concentration are adopted in the nanofiltration concentration, the first concentration is 4-stage continuous concentration, the concentrated solution enters the next procedure, the 1-2-stage discharged water is recycled, and the 3-4-stage discharged water is highly concentrated (the concentrated solution enters the next procedure, and the discharged water is recycled). During production, a primary high-pressure nanofiltration membrane is adopted. (nanofiltration membrane 200D, aromatic material and polyacid hydrogen composite nanofiltration membrane)

2.5 extraction

Transferring the nanofiltration concentrated solution to a refining plant, adding an equal volume of continuous extraction solvent (the solvent is obtained by 6-stage continuous extraction and contains a certain amount of target product), adding sulfuric acid to adjust the pH value to 2.8, and performing single-batch extraction. And standing for layering after extraction is finished. And (4) after layering, enabling the upper solvent phase to enter the next procedure.

And carrying out continuous 6-stage cross-flow extraction on the lower-layer water phase and a fresh solvent (ethyl acetate) after layering to reduce the content of a target product in the water phase to below 200 ppm.

After continuous extraction, the water phase is treated by a membrane scraper evaporator to recover part of the solvent and then discharged into a sewage treatment station.

And transferring the solvent phase after continuous extraction into a storage tank for temporary storage, and using the solvent phase for single-batch extraction of the next batch.

Concentration by 2.62000L evaporator does not crystallize

And (3) carrying out reduced pressure evaporation concentration on the solvent phase after single batch extraction at the temperature of 45 ℃, evaporating out about 2T of recovered solvent when concentration is carried out, putting the recovered solvent into a crystallizing tank for cooling, carrying out suction filtration, and entering the next procedure.

2.71000L evaporator concentration crystallization

Evaporating and concentrating 2000L of the concentrated liquid under reduced pressure at 50 deg.C, concentrating until there is crystal, and cooling again in a crystallizing tank. Then, carrying out suction filtration, and taking the solid as a product to enter the next working procedure. And (4) carrying out reduced pressure evaporation concentration on the primary mother liquor again, and putting the concentrated mother liquor into a crystallizing tank for cooling and crystallizing again after the concentrated mother liquor is crystallized. Then, carrying out suction filtration, and taking the solid as a product to enter the next working procedure. The liquid is used as the final mother liquor to enter a mother liquor storage tank and then is prepared into the cream preparation.

2.8 drying and pulverizing

And (3) drying and crushing the crystallized product (a boiling dryer is adopted at present for drying, and a universal micronizer is adopted at present for crushing) and then subpackaging to obtain a final product, wherein the performance detection results are shown in table 9.

Table 9 results of measuring properties of the crystalline product obtained in example 2

Serial number	Inspection item	Quality standard
			1	Content (wt.)	≥90％
2	Moisture content	≤0.5％
			3	Specific rotation	＞+80°
4	Yield of	≥70％

Example 3

The medium formulation and culture conditions were the same as in example 1.

And (3) supplementary material control: the dissolved oxygen rebounds by 20 percent, the automatic fed-batch glucose (45 percent) solution is started, and the dissolved oxygen control point is 20 to 30 percent.

The fermentation liquids obtained in example 3 were examined, and the results are shown in tables 10 and 11.

TABLE 10 fermentation broth assay data

TABLE 11 wet weight of fermentation broth

Example 4

The medium formulation and culture conditions were the same as in example 1.

And (3) supplementary material control: the dissolved oxygen rebounds by 20 percent, the automatic flowing salad oil is started, and the dissolved oxygen control point is 20 to 30 percent.

The fermentation liquids obtained in example 4 were examined, and the results are shown in tables 12 and 13.

TABLE 12 fermentation broth assay data

TABLE 13 fermentation broth wet weight

Example 5

The medium formulation and culture conditions were the same as in example 1.

And (3) supplementary material control: the dissolved oxygen rebounds by 20 percent, and the automatic feeding of glucose, salad oil and glucose (45 percent) solution is started: salad oil (4L:1L) and dissolved oxygen control point of 20-30 percent.

The fermentation liquids obtained in example 5 were examined, and the results are shown in tables 14 and 15.

TABLE 14 fermentation broth assay data

TABLE 15 wet weight of fermentation broth

It can be seen from examples 3 and 4 that glucose supplementation or salad oil alone directly affects the final GA target product₃The amount of (c); further, it can be seen from example 5 that the final target product GA is obtained at different ratios of glucose (45%) to salad oil₃Much lower than in example 1. Therefore, the feed ratio of example 1 was the optimum feed combination, and the results obtained by batch fermentation using the medium of example 1 are shown in Table 16. It can be seen from Table 16 that it gives a stable and high yield of GA₃。

TABLE 16 GA₃Batch production test data

Example 6

6.1 mixing GA₃The strain freezing tube is directly inoculated into a shake flask, the inoculation amount is 1.2 percent of the volume of the shake flask culture medium, the formula of the culture medium in the shake flask is shown in table 1, the fermentation temperature is 29 +/-0.2 ℃, and the shake flask culture is carried out for about 68 hours to obtain suspension.

6.2 Primary and Secondary seed tanks the same as in example 1.

6.3 Shake flask, primary and secondary seed tank broth test data are shown in Table 17.

TABLE 17 Shake flask, Primary and Secondary seed tank broth test data

Seed tank

Wet weight

pH

Length of hypha

Thickness of hypha

Whether the cytoplasm is uniform or not

With or without cavitation

Viscosity of fermentation broth

Shake flask seed liquid

22％

4.0

Long and long

Thin and thin

Uniformity

A large number of

++

First-class seed liquid

19％

3.99

Long and long

Is thinner

Uniformity

Small amount of

++

Second-stage seed liquid

20％

4.58

Long and long

Thin and thin

Is relatively uniform

Small amount of

+++

Example 7

7.1 mixing GA₃The strain freezing tube is directly inoculated into a shake flask, the inoculation amount is 0.4 percent of the volume of the shake flask culture medium, the formula of the culture medium in the shake flask is shown in table 1, the fermentation temperature is 29 +/-0.2 ℃, and the shake flask culture is carried out for about 68 hours to obtain suspension.

7.2 Primary and Secondary seed tanks the same as in example 1.

7.3 Shake flask, primary and secondary seed tank broth assay data are shown in Table 18.

TABLE 18 Shake flask, Primary and Secondary seed tank broth test data

Seed tank

Wet weight

pH

Length of hypha

Thickness of hypha

Whether the cytoplasm is uniform or not

With or without cavitation

Viscosity of fermentation broth

Shake flask seed liquid

18.5％

4.2

Long and long

Is thinner

Is relatively uniform

A large number of

+++

First-class seed liquid

17.7％

4.33

Is longer

Is thinner

Uniformity

A large number of

+++

Second-stage seed liquid

18.6％

4.12

Long and long

Thin and thin

Is relatively uniform

A large number of

+++

Comparative example 1

1.1 multiple activation of GA₃Inoculating 1-2 ring strains from the preservation strain inclined plane into a seed shaking bottle, and performing shake culture; then GA is added₃Inoculating the strain into a seeding tank for culturing to obtain a suspension; and continuously culturing the obtained suspension in a seed tank, and finally inoculating the suspension into a fermentation tank for fermentation to obtain fermentation liquor.

1.2 Shake flask, Primary and Secondary seed tank media and fermentation conditions were the same as in example 1.

1.3 Shake flask, primary and secondary seed tank broth test data are shown in Table 19.

TABLE 19 Shake flask, Primary and Secondary seed tank broth test data

Seed tank

Wet weight

pH

Length of hypha

Thickness of hypha

Whether the cytoplasm is uniform or not

With or without cavitation

Viscosity of fermentation broth

Shake flask seed liquid

25.3％

4.55

Is longer

Thin and thin

Uniformity

A large number of

+++

First-class seed liquid

16.5％

4.99

Long and long

Is thinner

Is relatively uniform

A large number of

+++

Second-stage seed liquid

22.5％

5.53

Is longer

Thin and thin

Uniformity

Small amount of

++++

Comparative example 2

2.1 multiple activation of GA₃Inoculating 1-2 ring strains from the preservation strain inclined plane into a seed shaking bottle, and performing shake culture; then GA is added₃Inoculating the strain into a seeding tank for culturing to obtain a suspension; and continuously culturing the obtained suspension in a seed tank, and finally inoculating the suspension into a fermentation tank for fermentation to obtain fermentation liquor.

2.2 Shake flask, Primary and Secondary seed tank media and fermentation conditions were the same as in example 1.

2.3 Shake flask, primary and secondary seed tank broth test data are shown in Table 20.

TABLE 20 Shake flask, Primary and Secondary seed tank broth test data

Seed tank

Wet weight

pH

Length of hypha

Thickness of hypha

Whether the cytoplasm is uniform or not

With or without cavitation

Viscosity of fermentation broth

Shake flask seed liquid

22.6％

4.71

Long and long

Is thinner

Uniformity

Small amount of

++++

First-class seed liquid

16.5％

4.99

Is longer

Thin and thin

Uniformity

A large number of

+++

Second-stage seed liquid

22.5％

5.53

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

GA comparison of examples 6-7 with example 1₃The amount of the strain freezing pipe directly connected into the shake flask has certain influence on the quality of the seeds, but the strain can be normally supplied to the fermentation tank, and finally the shake flask inoculation amount in the embodiment 1 is determined to be optimal; by comparing comparative examples 1-2 with example 1, the shake flask, primary and secondary seed tank culture broths were all in the normal range, but multiple activation of GA was reduced₃The step of preserving the slant of the strain shortens the time of preparing the seed solution and simplifies the process flow of preparing the seeds, and the shake flask data obtained by the batch production of example 1 are shown in table 21.

TABLE 21 Shake flask batch production data

Batches of

Wet weight

pH

Length of hypha

Thickness of hypha

Whether the cytoplasm is uniform or not

With or without cavitation

Viscosity of fermentation broth

1751

31％

4.54

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

1752

27％

4.26

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

1753

26％

4.46

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

1754

23％

4.73

Is longer

Is thinner

Is relatively uniform

Small amount of

++++

Example 8

8.1 transferring the seed liquid obtained in 1.3 into a fermentation tank according to 10-15% of the mass of the culture liquid for fermentation, wherein the formula of the fermentation culture medium is shown in Table 22.

TABLE 22 fermentation Medium formulation

The fermentation conditions were the same as in example 1.

The fermentation liquids obtained in example 8 were examined, and the results are shown in tables 23 and 24.

TABLE 23 fermentation broth assay data

TABLE 24 fermentation broth wet weight

Example 9

9.1 the seed solution obtained in 1.3 is transferred into a fermentation tank for fermentation according to 10-15% of the mass of the culture solution, and the formula of the fermentation medium is shown in Table 25.

TABLE 25 fermentation Medium recipe

The fermentation conditions were the same as in example 1.

The fermentation liquids obtained in example 9 were examined, and the results are shown in tables 26 and 27.

TABLE 26 fermentation broth assay data

TABLE 27 wet weight of fermentation broth

Example 10

10.1 the seed solution obtained in 1.3 is transferred into a fermentation tank for fermentation according to 10-15% of the mass of the culture solution, and the formula of the fermentation medium is shown in Table 28.

TABLE 28 fermentation Medium formulation

The culture conditions were the same as in example 1.

The fermentation liquids obtained in example 10 were examined, and the results are shown in tables 29 and 30.

TABLE 29 fermentation broth assay data

TABLE 30 wet weight of fermentation broth

It can be seen from examples 8 and 9 that adjusting the amounts of corn gluten meal and monopotassium phosphate directly affects the final target product GA₃The amount of (a) in example 1 medium is better than in examples 8, 9; further, example 10 shows that the final target product of peanut cake meal instead of corn gluten meal is much lower than that of example 1. The example 1 formulation is therefore the optimal medium combination.

Claims (10)

1. A method for preparing gibberellic acid, comprising:

s1) preparing a seed solution of gibberellic acid;

s2) transferring the seed liquid of gibberellic acid into a fermentation tank for fermentation to obtain gibberellic acid fermentation liquid; the fermentation medium in the fermentation tank consists of the following components: 10-40 g/L of corn protein powder; 1-10 g/L potassium dihydrogen phosphate; 5-30 g/L of a small-molecular organic carbon source; 0.5-5 g/L of vegetable oil; 0.5-2 g/L magnesium sulfate; 0.5-2 g/L of ammonium sulfate; 0.1-1 g/L of trace elements; in the fermentation process, when the dissolved oxygen is higher than 20-30%, supplementing glucose and vegetable oil; the micromolecular organic carbon source is sucrose and glucose; the mass ratio of the sucrose to the glucose is (1-2): (2-1); the vegetable oil is salad oil; the microelements are ferrous sulfate, zinc sulfate, manganese sulfate, sodium molybdate, copper sulfate and cobalt chloride; the mass ratio of the ferrous sulfate to the zinc sulfate to the cobalt chloride is (0.5-1.5): (0.5-1.5): (0.5-1.5): (0.1-0.5): (0.1-0.5): (0.1 to 0.5);

s3) filtering and concentrating the gibberellic acid fermentation liquor by a membrane to obtain a concentrated solution;

s4) refining the concentrated solution to obtain the gibberellic acid.

2. The preparation method according to claim 1, wherein the step S1) is specifically:

A1) the frozen GA₃Inoculating the strain into a seed culture medium for shake flask culture to obtain a suspension; the seed culture medium comprises 5-15 g/L of a first organic nitrogen source, 5-15 g/L of a first micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol;

A2) transferring the suspension into a seeding tank for fermentation culture to obtain a seed solution; the culture medium of the seeding tank comprises 5-15 g/L of second organic nitrogen source, 5-15 g/L of second micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol.

3. The method for preparing as claimed in claim 2, wherein the step a2) further comprises:

after fermentation culture, transferring the mixture into a secondary seed tank for continuous fermentation culture to obtain a seed solution; the culture medium of the secondary seed tank comprises 5-15 g/L of a third organic nitrogen source, 5-15 g/L of a third micromolecular organic carbon source, 1-5 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate and 0.1-1 g/L of polypropylene glycol.

4. The method according to claim 2 or 3, wherein the first organic nitrogen source, the second organic nitrogen source and the third organic nitrogen source are each independently selected from one or more of soybean protein, peanut cake powder, cottonseed cake powder, corn steep liquor, corn gluten meal, peptone, yeast powder and fish meal;

the first small molecule organic carbon source, the second small molecule organic carbon source and the third small molecule organic carbon source are respectively and independently selected from one or more of sucrose, glucose, molasses, amylodextrin, mannose and galactose.

5. The preparation method according to claim 1, wherein the step S3) is specifically:

B1) filtering the gibberellic acid fermentation liquor by using a tubular membrane to obtain a tubular membrane filtration clear liquid and a tubular membrane filtration residual liquid;

B2) performing ultrafiltration on the tubular membrane filtered clear liquid to obtain an ultrafiltration clear liquid and an ultrafiltration residual liquid;

B3) and (4) carrying out nanofiltration on the ultrafiltration clear liquid to obtain a concentrated solution.

6. The preparation method according to claim 5, wherein the step B1) is specifically:

filtering the gibberellic acid fermentation liquor by a tubular membrane until the volume of clear liquid is 0.2-0.6 times of the volume of the fermentation liquor, and then continuously adding an aqueous solvent until the volume of the clear liquid is 1-2.5 times of the volume of the fermentation liquor to obtain tubular membrane filtered clear liquid and tubular membrane filtered residual liquid.

7. The preparation method according to claim 1, wherein the step S4) is specifically:

C1) extracting the concentrated solution by using an extracting agent to obtain an extract liquid;

C2) and concentrating and crystallizing the extract liquor to obtain the gibberellic acid.

8. The preparation method according to claim 7, wherein the step C1) is specifically:

mixing the concentrated solution with an extracting agent, adjusting the pH value to 2-3, extracting, standing and layering to obtain an extract liquid and a water phase; the volume ratio of the concentrated solution to the extracting agent is 1: (0.8 to 1.5);

and continuously extracting the water phase and the organic solvent to obtain an organic phase as an extractant of the concentrated solution.

9. The preparation method according to claim 7, wherein the step C2) is specifically:

and (3) carrying out reduced pressure evaporation concentration on the extract liquor, concentrating to one third to two thirds of the original volume, cooling, carrying out suction filtration, then carrying out secondary concentration, stopping concentration after crystallization, cooling, crystallizing, and filtering to obtain the gibberellic acid.

10. The preparation method of claim 1, wherein the temperature of the fermentation in the step S2) is 28-30 ℃; the fermentation time is 8-10 days; the pH value of the fermentation is 5.0-5.2; the fermentation pressure is 0.01-0.05 MPa; the air flow rate of the fermentation is 2000-3000 Nm³H; when the supplemented glucose is solid, the mass ratio of the glucose to the vegetable oil is (1-3): 1.

Images