A method for improving the fermentation yield of ergot acid

Abstract

The present invention provides a method for increasing the fermentation yield of ergot acid. The seed liquid of Claviceps paspali is inoculated into a fermentation medium for liquid fermentation culture. When the content of succinic acid in the fermentation medium is lower than 13 g / L, an aqueous solution of succinic acid is supplemented to the fermentation broth so that the content of succinic acid in the fermentation broth is maintained at 10-15 g / L. The Claviceps paspali is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC NO. 41366. Using the method of the present invention, the fermentation yield of ergot acid can be stabilized above 5.0 g / L, and the highest reaches 5.172 g / L. Moreover, the raw materials of the fermentation medium used in this method are inexpensive, the feeding method is simple, and it is easy to be popularized and used in industrial production, which can significantly improve the production efficiency of enterprises.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation, and more specifically, to a method for increasing the fermentation yield of ergot acid. Background Art

[0002] Clavieps paspali belongs to the fungal kingdom, Ascomycota, Pyrenomycetes, Hypocreales, Clavicipitaceae, Claviceps. This fungus parasitizes on gramineous plants such as rye, wheat, barley, oats, and Setaria viridis. Ergot fungi are plant pathogens, but they have important industrial value and can produce many ergot alkaloids with medicinal value. Ergot alkaloids are important drugs commonly used in obstetrics and gynecology, and their application history is long.

[0003] Ergot acid is a part of the basic structure of natural ergot alkaloids. It is manufactured on a large scale as an intermediate for the synthesis of some semi-synthetic ergot alkaloids. The discovered semi-synthetic ergot alkaloids can be used as drugs, such as nicergoline, methylergonovine, nicergoline mesylate, and methysergide. They have not only achieved remarkable results in improving the mental health problems of the elderly and retina problems caused by blood vessels, but also played a prominent role in the treatment of acute cerebrovascular problems.

[0004] The existing ergot acid production process generally first ferments ergotamine through ergot fungi, and then destroys the amide bond by alkali hydrolysis to generate ergot acid. At present, this process has been relatively maturely studied, but the fermentation yield is low.

[0005] The prior art CN117126748A discloses a method for breeding ergot fungus resistant mutants with high ergot acid yield. Through strain mutagenesis breeding, a resistant mutant strain is obtained. After fermentation culture (the culture medium composition is: mannitol 100 g / L, succinic acid 35 g / L, yeast powder 0.5 g / L, ferrous sulfate 0.025 g / L, zinc sulfate 0.01 g / L, corn steep liquor dry powder 20 g / L, magnesium sulfate 0.7 g / L, antifoaming agent 0.8 g / L, adjust the pH to 7.0), the ergot acid yield reaches 3.82 g / L, and some batches reach 4.36 g / L.

[0006] The prior art CN116024107A discloses a method for constructing a Claviceps fungus producing ergot acid, including the step of blocking the expression of the non-ribosomal polypeptide synthetase g5167 gene in the Claviceps fungus, and achieving the accumulation of ergot acid by knocking out the g5167 gene to block the synthesis of ergotamide. This prior art also discloses that the engineering bacteria are cultured in a fermentation medium (80 - 100 g / L of sorbitol, 35 - 45 g / L of succinic acid, 0.25 - 0.50 g / L of yeast powder, 0.01 - 0.05 g / L of ferrous sulfate, 0.01 - 0.05 g / L of zinc sulfate, 10 - 20 g / L of corn steep liquor dry powder, 1.0 - 1.5 g / L of magnesium sulfate, adjusting the pH to 4.8 - 6.5 with ammonia water / sodium hydroxide) for 12 - 15 days, and the fermentation broth is treated with hydrochloric acid or acetic acid, and the final product is 3 - 4 g / L.

[0007] For industrial scale-up production, the yields of ergot acid obtained by the methods for fermentative production of ergot acid disclosed in the prior art are all unsatisfactory, and the use of relatively expensive mannitol or sorbitol as the main carbon source component results in high production costs. Improving the fermentation yield and reducing the medium cost are of great significance for industrial production. Summary of the Invention

[0008] Aiming at the defects existing in the production of Claviceps fungus in the prior art, the purpose of the present invention is to provide a method for improving the fermentation yield of ergot acid, which can stably maintain the ergot acid yield at 5.0 g / L or above.

[0009] The method for improving the fermentation yield of ergot acid provided by the present invention includes inoculating the seed liquid of Claviceps paspali into a fermentation medium for liquid fermentation culture. When the content of succinic acid in the fermentation medium is lower than 13 g / L, an aqueous solution of succinic acid is added to the fermentation broth to maintain the succinic acid content in the fermentation broth at 10 - 15 g / L. The Claviceps paspali is preserved in the China General Microbiological Culture Collection Center (Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), and the preservation number is CGMCC NO. 41366 (the preservation date is June 14, 2024).

[0010] In another preferred example, the aqueous solution of succinic acid is added to the fermentation broth in a fed-batch manner.

[0011] In another preferred example, the concentration of the added aqueous solution of succinic acid is 50 - 80 g / L.

[0012] In another preferred example, the feeding rate is 17 - 25 mL·h -1 .

[0013] In another preferred example, the temperature of the fermentation culture is 21 - 25 °C, and the rotation speed is 200 - 500 rpm.

[0014] In another preferred example, the fermentation medium contains the following components in the following amounts based on its total volume:

[0015] Sorbitol 5 - 25 g / L, corn starch hydrolyzed by α - amylase 50 - 100 g / L, succinic acid 30 - 40 g / L, yeast extract 0.4 - 0.6 g / L, cottonseed cake powder 15 - 25 g / L, dry corn steep liquor 10 - 20 g / L, ammonia water 30 - 40 g / L, ferrous sulfate heptahydrate 0.001 - 0.030 g / L, zinc sulfate heptahydrate 0.001 - 0.015 g / L, magnesium sulfate heptahydrate 0.6 - 0.8 g / L, wherein the mass concentration of the ammonia water is 15 - 25%.

[0016] In another preferred example, the fermentation medium contains the following components in the following amounts based on its total volume:

[0017] Sorbitol 5 - 25 g / L, corn starch hydrolyzed by α - amylase 60 - 80 g / L, succinic acid 30 - 40 g / L, yeast extract 0.4 - 0.6 g / L, cottonseed cake powder 15 - 25 g / L, dry corn steep liquor 10 - 20 g / L, ammonia water 30 - 40 g / L, ferrous sulfate heptahydrate 0.001 - 0.030 g / L, zinc sulfate heptahydrate 0.001 - 0.015 g / L, magnesium sulfate heptahydrate 0.6 - 0.8 g / L, wherein the mass concentration of the ammonia water is 15 - 25%.

[0018] In another more preferred example, the content of the corn starch hydrolyzed by α - amylase in the fermentation medium is 65 - 75 g / L based on its total volume.

[0019] In another preferred example, the yeast extract is Angel FM802.

[0020] In another preferred example, the corn starch hydrolyzed by α - amylase is obtained by hydrolyzing corn starch with α - amylase at 80 - 90 °C for 28 - 32 min.

[0021] In another preferred example, during the hydrolysis process, the mass ratio of α - amylase to corn starch is 0.0005:1 - 0.0009:1, more preferably 0.0006:1 - 0.0008:1, and most preferably 0.0007:1.

[0022] In another preferred example, the preparation method of the fermentation medium comprises the following steps:

[0023] After mixing α - amylase, corn starch and water, heat to 80 - 90 °C and keep warm for 28 - 32 min to hydrolyze the corn starch with α - amylase, then add the remaining components of the fermentation medium and heat to 110 - 130 °C for sufficient sterilization to obtain the fermentation medium.

[0024] In another preferred example, the method for increasing the yield of ergot acid fermentation includes the following steps:

[0025] (1) Strain activation: Spread the suspension of the working cell bank of Claviceps paspali on a PDA slant and culture at 26 - 30 °C for 7 - 8 days to obtain the GI slant strain;

[0026] Among them, the Claviceps paspali is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC NO.41366,

[0027] The PDA medium contains the following components in the following contents based on its total volume:

[0028] Potato dextrose agar 35 - 43 g / L, yeast extract 0.4 - 0.6 g / L, and adjust the pH to 7.10 - 7.30 with sodium hydroxide solution;

[0029] (2) Seed liquid preparation: Scrape the GI slant strain obtained in step (1), after homogenization, place it in the seed liquid medium for culture at a temperature of 21 - 25 °C and a rotation speed of 220 - 260 rpm for 96 - 144 h, and the wet weight of the thallus ≥ 45 g / L;

[0030] The seed liquid medium contains the following components in the following contents based on its total volume:

[0031] Mannitol 45 - 55 g / L, succinic acid 9 - 11 g / L, soybean powder 4 - 6 g / L, potassium dihydrogen phosphate 0.75 - 1.25 g / L, magnesium sulfate heptahydrate 0.25 - 0.35 g / L, and adjust the pH to 5.00 - 5.20 with ammonia water,

[0032] (3) Fermentation culture: Inoculate the seed liquid obtained in step (2) into the fermentation medium for liquid fermentation culture at an inoculation amount of 8 - 12% (v / v). When the content of succinic acid in the fermentation broth is lower than 13 g / L, supplement the succinic acid solution to the fermentation broth in a continuous feeding manner to maintain the succinic acid concentration in the fermentation broth at 10 - 15 g / L,

[0033] The fermentation medium, based on its total volume, contains the following components in the following amounts: sorbitol 5 - 25 g / L, α - amylase - hydrolyzed corn starch 50 - 100 g / L, succinic acid 30 - 40 g / L, yeast extract 0.4 - 0.6 g / L, ferrous sulfate heptahydrate 0.000 - 0.030 g / L, zinc sulfate heptahydrate 0.000 - 0.015 g / L, magnesium sulfate heptahydrate 0.6 - 0.8 g / L, corn steep liquor dry powder 10 - 20 g / L, ammonia water 30 - 40 g / L, cottonseed cake powder 15 - 25 g / L. During the process of hydrolyzing corn starch with α - amylase, the weight ratio of corn starch to α - amylase is 1:0.0005 - 1:0.0009.

[0034] The process for preparing the fermentation medium is as follows: Mix corn starch, α - amylase and water, then heat to 80 - 90 °C and keep warm for 28 - 32 min to hydrolyze corn starch with α - amylase. Then add the remaining components of the fermentation medium and heat to 110 - 130 °C for sufficient sterilization to obtain the fermentation medium. The weight ratio of corn starch to α - amylase is 1:0.0005 - 1:0.0009.

[0035] In another preferred example, in step (1), the concentration of the sodium hydroxide solution used to adjust the pH value of the PDA medium is 10 - 40%.

[0036] In another preferred example, in step (2), the mass concentration of the ammonia water used to adjust the pH value of the seed - liquid medium is 15 - 25%.

[0037] In another preferred example, in the method for increasing the fermentation yield of ergot acid described above, the temperature of fermentation culture is 21 - 25 °C, the aeration rate is 0.25 - 1.0 vvm, the dissolved oxygen is controlled above 30%, the tank pressure is 0.02 - 0.08 MPa, and the culture time is 13 - 14 days. Description of the Drawings

[0038] Figure 1 It is a graph showing the changing trends of the succinic acid concentration, pH and titer in the fermentation broth during the fermentation process in the fermenter in Example 7;

[0039] Figure 2 It is the HPLC chromatogram of the fermentation broth in Example 15 after alkali treatment. Detailed Description of the Invention

[0040] In view of the disadvantages of high production cost and low yield in the existing microbial fermentation process of ergot acid, the inventors of this application found that by using an improved fermentation medium and an improved cultivation process to cultivate Claviceps paspali (preserved in the China General Microbiological Culture Collection Center, preservation number CGMCC NO. 41366), ergot acid can be obtained with high yield and low cost. Specifically, on the one hand, the inventors conducted in-depth research on the medium and found that during the production of ergot acid by Claviceps paspali, using corn starch hydrolyzed by α-amylase as the main fermentation carbon source to replace part of sorbitol or mannitol in the existing fermentation medium, reducing the dosage of sorbitol or mannitol, not only reduced the production cost but also significantly increased the yield of ergot acid. And using cottonseed cake powder as one of the nitrogen source components can also significantly increase the total yield of ergot acid. On the other hand, the inventors conducted in-depth research on the feeding method of fermentation and found that when the seed liquid of the above-mentioned Claviceps paspali was transferred to a fermenter filled with the fermentation medium and fermented for 3 days (it was detected that the content of succinic acid in the fermentation broth was lower than 13 g / L at this time), a slow rebound sign of the pH of the fermentation broth began to appear. When fermented for 9 days, the concentration of succinic acid in the fermentation broth tended to 0, and from the 9th day, the rebound amplitude of the pH accelerated while the titer growth amplitude slowed down. Based on this, the inventors found that during the fermentation process, when the content of succinic acid in the fermentation broth is lower than 13 g / L, by continuously feeding a succinic acid solution into the fermentation broth to make the content of succinic acid in the fermentation broth 10 - 15 g / L, not only the titer of the fermentation broth is significantly increased, but also the rebound amplitude of the pH of the fermentation broth in the middle and late fermentation stages is slowed down. Finally, the inventors used the above-mentioned improved medium and by continuously feeding succinic acid and controlling the content of succinic acid in the fermentation broth at 10 - 15 g / L, after 14 days of fermentation culture, the total content of ergot acid can reach more than 5.0 g / L.

[0041] Term Definition

[0042] "Seed liquid" refers to a liquid containing the above-mentioned Claviceps paspali cells cultured in a seed culture medium. In a specific embodiment of the present invention, the preparation process of the "seed liquid" is as follows: (1) Coat the above-mentioned Claviceps paspali on a PDA slant medium (the PDA medium contains: 35-43 g / L of potato dextrose agar, 0.4-0.6 g / L of yeast extract, adjust the pH to 7.10-7.30 with sodium hydroxide solution, and the sterilization conditions are 121 °C, 20 min), and culture at 26-30 °C for 7-8 days to obtain a GI slant strain (mature mycelium); (2) Scrape the GI slant strain (mature mycelium) and inoculate it into a seed liquid medium (the seed liquid medium contains: 45-55 g / L of mannitol, 9-11 g / L of succinic acid, 4-6 g / L of soybean powder, 0.75-1.25 g / L of potassium dihydrogen phosphate, 0.25-0.35 g / L of magnesium sulfate heptahydrate, adjust the pH to 5.00-5.20 with ammonia water, and the sterilization conditions are 121 °C, 20 min), and culture at 21-25 °C at a rotation speed of 220-260 rpm for 4-6 days to obtain the seed liquid.

[0043] "Corn starch" can be prepared by methods well-known in the art or obtained through commercial channels, such as but not limited to being purchased from Pingliang Guowei Starch.

[0044] "Cottonseed cake powder" refers to the powder obtained by grinding the cake crushed from the cottonseed, that is, the seed of cotton, which contains high protein and is rich in various amino acids such as tryptophan, lysine, and methionine. It can be prepared by methods well-known in the art or obtained through commercial channels, such as but not limited to being purchased from Jinan Huilong Biology.

[0045] "Yeast extract" can be prepared by methods well-known in the art or obtained through commercial channels, such as but not limited to Angel FM802.

[0046] "Corn steep liquor dry powder" refers to a powdery substance obtained by low-temperature and instant spray drying of corn steep liquor, with a protein weight content of ≥42% and a moisture weight content of ≤10%, and is used as a supplement for water-soluble plant proteins and water-soluble vitamins and other nutrient elements during the biological fermentation process. It can be prepared by methods well-known in the art or obtained through commercial channels, such as but not limited to being purchased from Hongrun Baoshun.

[0047] "Ammonia water" is an aqueous solution of ammonia. The mass concentration of ammonia water used in the present invention is 15-25%, such as 20%.

[0048] "Titer" refers to the total content of ergine and isolysergic acid in the fermentation broth per unit volume, with the unit of g / L. The calculation method is as follows: Titer = (Sum of the peak areas of ergine and isolysergic acid in the product of the culture medium of the present invention determined by HPLC ÷ Sum of the peak areas of ergine and isolysergic acid in the standard product) × Concentration of the standard product × Content of the standard product × Dilution factor of the product of the culture medium of the present invention.

[0049] In the description of the present invention, the culture medium used for fermentation culture is liquid, and fermentation culture is also called liquid fermentation culture.

[0050] In the present invention, the carbon source and nitrogen source of the fermentation medium are understood according to the conventional understanding in the art. For example, the carbon source refers to the raw material providing the carbon source, and the nitrogen source refers to the raw material providing the nitrogen source.

[0051] The carbon source used in the present invention can be selected from sorbitol, corn starch hydrolyzed by α-amylase, succinic acid, glycerol, maltodextrin, etc., and more preferably from sorbitol, corn starch hydrolyzed by α-amylase, and succinic acid. Among them, the corn starch hydrolyzed by α-amylase is the main carbon source, and the hydrolyzed corn starch is rich in nutritional components, including slow-acting carbon sources and fast-acting carbon sources, such as short-chain dextrins with different lengths, a small amount of polysaccharides and glucose. In the early stage of fermentation culture, the fast-acting carbon source can promote the growth of mycelium of the bacteria and shorten the lag phase of the strain; in the middle and late stages of fermentation culture, the slow-acting carbon sources such as short-chain dextrins and a small amount of polysaccharides are hydrolyzed into fast-acting carbon sources by the enzymes produced by the bacteria themselves, continuously providing nutrients for the bacteria and prolonging the product generation time. The low price of corn starch replaces expensive sorbitol or mannitol, which not only significantly increases the yield of ergine, but also significantly reduces the cost of the culture medium.

[0052] The nitrogen source used in the present invention can be selected from yeast extract, cottonseed cake powder, dry corn steep liquor, ammonia water, soybean cake powder, etc., and more preferably from yeast extract, cottonseed cake powder, dry corn steep liquor, and ammonia water. Cottonseed cake powder is rich in various amino acids such as tryptophan, lysine, and methionine, and tryptophan is a precursor substance for the synthesis of ergine. In the middle and late stages of fermentation culture, the slow-acting nitrogen source cottonseed cake powder not only provides culture for the growth of the bacteria, but also provides precursor substances for the product synthesis. The inventors of the present application have proved through experiments that cottonseed cake powder as one of the fermentation nitrogen sources further increases the yield of ergine.

[0053] In the method for increasing the fermentation yield of ergot acid of the present invention, when the content of succinic acid in the fermentation culture medium is lower than 13 g / L (from the 3rd to the 13th day of fermentation culture), a succinic acid solution is added to the fermentation broth to make the content of succinic acid in the fermentation broth 10-15 g / L. Adding the succinic acid solution is a key step in the method for increasing the total ergot acid fermentation yield of the present invention. Succinic acid can participate in the synthesis of products as a carbon source to ensure that the ratio of carbon source to nitrogen source in the fermentation broth is appropriate. At the same time, in the later stage of fermentation, the continuously added succinic acid solution can also play a role in adjusting the pH of the fermentation medium, maintaining the pH at 5.05-5.15, which is beneficial to the synthesis of ergot acid products. In the present invention, during the fermentation process of Claviceps paspali, especially from the 3rd to the 13th day of fermentation, by continuously adding the succinic acid solution, the content of succinic acid in the medium is maintained at 10-15 g / L, significantly increasing the fermentation yield of total ergot acid.

[0054] Compared with the prior art, the advantages of the present invention are as follows:

[0055] (1) The present invention uses Claviceps paspali (preserved in the China General Microbiological Culture Collection Center, preservation number CGMCC NO. 41366) as the fermentation strain. By adding an aqueous succinic acid solution, the fermentation yield of ergot acid can be significantly increased to more than 5.0 g / L, and the highest reaches 5.172 g / L, which is significantly higher than the yield disclosed in the prior art.

[0056] (2) The fermentation medium of the present invention uses the hydrolyzate of corn starch as one of the fermentation carbon sources. The components obtained after hydrolyzing corn starch with α-amylase include slow-acting carbon sources and fast-acting carbon sources, such as short-chain dextrins with different lengths, a small amount of polysaccharides and glucose. In the early stage of fermentation culture, the fast-acting carbon source can promote the growth of bacteria and shorten the lag phase of the strain; in the middle and later stages of fermentation culture, the slow-acting carbon sources such as short-chain dextrins and a small amount of polysaccharides are hydrolyzed into fast-acting carbon sources by the enzymes produced by the bacteria themselves, continuously providing nutrients for the bacteria and prolonging the product formation time. In addition, compared with sorbitol or mannitol used in other prior arts, corn starch and α-amylase in the corn starch solution hydrolyzed by α-amylase are low in price.

[0057] (3) The fermentation medium of the present invention uses cottonseed cake powder as one of the fermentation slow-acting nitrogen sources. Cottonseed cake powder is rich in various amino acids such as tryptophan, lysine and methionine, and tryptophan is a precursor substance for the synthesis of ergot acid. In the middle and later stages of fermentation culture, the slow-acting nitrogen source cottonseed cake powder not only provides culture for the growth of bacteria, but also provides precursor substances for product synthesis.

[0058] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, all percentages, ratios, proportions, or parts are by volume.

[0059] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0060] The contents of succinic acid and ergot acid in the fermentation broth are both determined by commonly used methods in the art. For example, it is determined by the external standard method using HPLC.

[0061] For example, the method for determining succinic acid includes the following steps:

[0062] ① Detection of the standard: Weigh an appropriate amount of succinic acid reference substance accurately, dissolve it in ultrapure water to prepare a succinic acid solution containing 4 mg per 1 mL, and set aside.

[0063] ② Preparation of the solution to be measured: Take an appropriate amount of the fermentation filtrate, dilute and make up the volume with 0.02 mmol / L phosphate buffer solution, and filter it through a 0.22 μm filter membrane for HPLC analysis.

[0064] ③ Perform HPLC analysis on the standard dilution solution and the sample dilution solution to be measured respectively, and determine the content of succinic acid in the sample according to the relationship between the content and the area ratio.

[0065] The chromatograph used for HPLC is a UPLC type high performance liquid chromatograph, the chromatographic column is Kromasil C18 (150 mm × 4.5 mm, 5 μm), the detector is DAD; detection wavelength: 210 nm; column temperature: 40 °C; flow rate: 1 mL / min; mobile phase A: 0.02 mmol / L potassium dihydrogen phosphate solution (pH 2.9); mobile phase B: acetonitrile; running time: 18 min; gradient elution program: 5% B from 0.00 - 0.50 min, 12% B from 0.50 - 6.00 min, 40% B from 6.00 - 12.00 min, 40% B from 12.00 - 18.00 min.

[0066] In the following embodiments, the content of ergot acid in the ergot fermentation broth can also be determined by the external standard method using HPLC. Specifically as follows:

[0067] ① Detection of standard: Accurately weigh about 10 mg of the reference substance and place it in a 10 mL volumetric flask. Dissolve and dilute it to the scale with the diluent (acetonitrile: water = 50:50), and shake well. Accurately pipette 1.0 mL of the above solution into a 10 mL volumetric flask, dissolve and dilute it to the scale with the diluent, and shake well for standby.

[0068] ② Preparation of test solution: Accurately pipette 1.0 mL of the fermentation broth into a 1.5 mL microcentrifuge tube, add 0.39 mL of 5 M sodium hydroxide solution, mix well, and oscillate at 80 °C and 1500 rpm for 4 h to obtain the hydrolysis solution. Transfer all of the above hydrolysis solution to a 50 mL volumetric flask, add the diluent (acetonitrile: water = 50:50) to volume to the scale, shake well, and filter through a 0.22 μm filter membrane for HPLC analysis.

[0069] ③ Perform HPLC analysis on the standard dilution solution and the test sample dilution solution respectively, and determine the content of ergine in the sample according to the relationship between the content and the area ratio.

[0070] The chromatograph used in HPLC is an UPLC type high performance liquid chromatograph, the chromatographic column is a Waters Acquity UPLCBEH C18 column (100×2.1 mm 1.7 μm); the detector is DAD; the detection wavelength: 310 nm; the column temperature: 40 °C; the flow rate: 0.35 mL / min; mobile phase A: 8 mmol / L ammonium formate solution (pH 8.0); mobile phase B: acetonitrile: methanol (V / V = 80:20); running time: 17 min; gradient elution program: 97% A from 0.00 - 0.50 min, 70% A from 0.50 - 6.50 min, 38% A from 6.50 - 9.00 min, 20% A from 9.00 - 9.20 min, 20% A from 9.20 - 11.00 min, 97% A from 11.00 - 12.00 min, 97% A from 12.00 - 17.00 min.

[0071] For the materials, reagents, etc. used in the following examples, unless otherwise specified, they can all be obtained from commercial channels.

[0072] The paspali ergot strain used in the following examples is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC NO.41366.

[0073] The yeast extract used in the following examples is Angel FM802.

[0074] In the following examples, unless otherwise specified, the solution is an aqueous solution and the concentration is the mass concentration.

[0075] Example 1

[0076] (1) Preparation of PDA slant medium: Mix 39 g / L of potato dextrose agar, 0.5 g / L of yeast extract with water, and adjust the pH to 7.10 - 7.30 using sodium hydroxide solution (mass concentration about 20%); Pipette 18 mL of PDA liquid medium into a test tube (25 x 200 mm) and sterilize at 121 °C for 20 min;

[0077] (2) Activation of strains: Pipette 0.75 mL of the working cell bank suspension of Claviceps paspali into the PDA slant prepared in step (1), spread evenly using a spreader, and culture at 28 °C for 7 - 8 days to obtain the GI slant strains;

[0078] (3) Preparation of seed liquid medium: Mix 50 g / L of mannitol, 10 g / L of succinic acid, 5 g / L of soybean powder, 1 g / L of potassium dihydrogen phosphate, 0.3 g / L of magnesium sulfate heptahydrate with water, and adjust the pH to 5.00 - 5.20 using ammonia water (mass concentration 20%), transfer to an Erlenmeyer flask (for example, a 250 mL Erlenmeyer flask with a liquid loading of 60 mL), and sterilize at 121 °C for 20 min;

[0079] (4) Preparation of seed liquid: Scrape the GI slant strains obtained in step (2) into a centrifuge tube, add 10 mL of normal saline, and homogenize using a high - speed disperser. Pipette 1.1 mL of the homogenate into a 250 mL shake flask containing 60 mL of seed liquid medium, culture at a temperature of 23 °C, and culture at a rotation speed of 240 rpm for 96 - 144 h, with the wet weight of the thallus ≥ 45 g / L;

[0080] (5) Preparation of fermentation medium: Mix 100 g / L of sorbitol, 35 g / L of succinic acid, 0.5 g / L of yeast extract, 0.022 g / L of ferrous sulfate heptahydrate, 0.001 g / L of zinc sulfate heptahydrate, 15 g / L of corn steep liquor dry powder, 0.7 g / L of magnesium sulfate heptahydrate, 30 g / L of ammonia water (mass concentration 20%) with water, place in an Erlenmeyer flask (a 250 mL Erlenmeyer flask with a liquid loading of 60 mL), and sterilize at 121 °C for 20 min;

[0081] (6) Fermentation culture in shake flask: Inoculate the seed liquid obtained in step (4) into a 250 mL shake flask containing 60 mL of fermentation medium at an inoculation amount of 10% (v / v), culture at a temperature of 23 °C, and culture at a rotation speed of 240 rpm for 14 days to obtain the fermentation broth. The fermentation broth is processed by the conventional methods in the art to obtain ergot acid and isolysergic acid.

[0082] Product detection: Perform liquid chromatography analysis on the fermentation broth. The products include ergot acid and isolysergic acid.

[0083] Test result: After testing the fermentation broth, the total ergocristine (ergocristine and isolysergic acid) yield (titer) was approximately 2.732 g / L.

[0084] Examples 2-1, 2-2, and 2-3

[0085] Based on Example 1, these examples studied the effect of using corn starch, glycerol, or maltodextrin to replace part of the sorbitol in the fermentation medium on the yield of ergocristine in fermentation production.

[0086] Examples 2-1, 2-2, and 2-3 only differed from Example 1 in step (5) in the preparation of the fermentation medium, and the other steps were the same as those in Example 1.

[0087] (5) Preparation of the fermentation medium: Mix 50 g / L of organic carbon source (the types of organic carbon sources are corn starch hydrolyzed by α-amylase, glycerol, or maltodextrin), 15 g / L of sorbitol, 35 g / L of succinic acid, 0.5 g / L of yeast extract, 0.022 g / L of ferrous sulfate heptahydrate, 0.001 g / L of zinc sulfate heptahydrate, 15 g / L of corn steep liquor powder, 0.7 g / L of magnesium sulfate heptahydrate, 30 g / L of ammonia water (mass concentration 20%) with water, and place it in a 250 mL Erlenmeyer flask with a liquid loading of 60 mL, and sterilize it at 121 °C for 20 min.

[0088] The pretreatment method for hydrolyzing corn starch with α-amylase is as follows: Mix corn starch, α-amylase (enzyme activity unit is approximately 20,000 U / mL) with water (the weight ratio of corn starch to α-amylase is 1:0.0005 respectively), heat the mixture to 80-90 °C, and keep it warm for 30 min to hydrolyze the corn starch with α-amylase.

[0089] The preparation process of the fermentation medium containing corn starch hydrolyzed by α-amylase is as follows: Mix corn starch, α-amylase (enzyme activity unit is approximately 20,000 U / mL) with water (the weight ratio of corn starch to α-amylase is 1:0.0005 respectively), heat the mixture to 80-90 °C, and keep it warm for 30 min to hydrolyze the corn starch with α-amylase, and then add the remaining components of the fermentation medium and sterilize it at 121 °C for 20 min to obtain the fermentation medium containing corn starch hydrolyzed by α-amylase. The remaining components of the fermentation medium are: 15 g / L of sorbitol, 35 g / L of succinic acid, 0.5 g / L of yeast extract, 0.022 g / L of ferrous sulfate heptahydrate, 0.001 g / L of zinc sulfate heptahydrate, 15 g / L of corn steep liquor powder, 0.7 g / L of magnesium sulfate heptahydrate, 30 g / L of ammonia water (mass concentration 20%).

[0090] The products in the fermentation broth were detected, and the detection results are shown in Table 1. As can be seen from Table 1, when the organic carbon source was corn starch, the total ergocristine yield (titer) in the fermentation broth was the highest, but still slightly lower than the control (Example 1). Considering the raw material cost, corn starch was selected as the carbon source for the subsequent experiments.

[0091] Table 1 Effect of the type of organic carbon source on the product titer

[0092]

[0093] Examples 3-1, 3-2, and 3-3

[0094] Based on Example 2-2, these examples studied the effect of the hydrolysis of corn starch in the fermentation medium with different dosages of α-amylase on the yield of ergocristine in fermentation.

[0095] Examples 3-1, 3-2, and 3-3 were only different from Example 2-2 in step (5) for preparing the fermentation medium, and the other steps were the same as those in Example 2-2.

[0096] (5) Preparation of the shake flask fermentation medium: Mix corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water (where the content of corn starch is 50 g / L, and the weight ratios of corn starch to α-amylase are 1:0.0005, 1:0.0007, and 1:0.0009 respectively). Heat the mixture to 80-90 °C and keep it warm for 30 min to hydrolyze the corn starch with α-amylase, and then add the remaining components of the fermentation medium and sterilize at 121 °C for 20 min to obtain the fermentation medium.

[0097] The remaining components of the fermentation medium are: 15 g / L of sorbitol, 35 g / L of succinic acid, 0.5 g / L of yeast extract, 0.022 g / L of ferrous sulfate heptahydrate, 0.001 g / L of zinc sulfate heptahydrate, 15 g / L of corn steep liquor dry powder, 0.7 g / L of magnesium sulfate heptahydrate, and 30 g / L of ammonia water (mass concentration is 20%).

[0098] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid loading of 60 mL was used.

[0099] The products and initial reducing sugars in the fermentation broth were detected, and the detection results are shown in Table 2. As can be seen from Table 2, 50 g / L of corn starch was treated with three dosages of α-amylase at 80 - 90 °C for 30 min, and the obtained fermentation media were used for the fermentation of the seed liquid of Claviceps paspali. The initial reducing sugar concentrations in the fermentation broth were approximately 0.5%, 1.8%, and 2.9% respectively. Higher or lower reducing sugar concentrations would inhibit the growth of the bacteria in the early stage, prolong the culture cycle, and reduce the product yield. The total ergocristine yields (potencies) in the obtained fermentation broths were approximately 2.555 g / L, 2.903 g / L, and 2.343 g / L respectively. When the weight ratio of corn starch to α-amylase was 1:0.0007, the total ergocristine yield was the highest. Higher or lower reducing sugar concentrations would inhibit the growth of the bacteria in the early stage, prolong the culture cycle, and reduce the product yield.

[0100] Table 2 Effects of hydrolysis of corn starch with different dosages of α-amylase on product potency

[0101]

[0102] Examples 4-1, 4-2, and 4-3

[0103] Based on Example 3-2, these examples studied the effects of the content of hydrolyzed corn starch in the fermentation medium on the yield of ergocristine in fermentation production.

[0104] Examples 4-1, 4-2, and 4-3 were only different from Example 3-2 in step (5) of the preparation of the fermentation medium, and the other steps were the same as those in Example 3-2.

[0105] (5) Preparation of the fermentation medium: Mix corn starch, α-amylase (enzyme activity unit is approximately 20,000 U / mL) with water (where the weight ratio of corn starch to α-amylase is 1:0.0007, and the corn starch contents are 50 g / L, 70 g / L, and 100 g / L respectively), heat the mixture to 80 - 90 °C, keep it warm for 30 min to hydrolyze the corn starch with α-amylase, then add the remaining fermentation culture components, and sterilize at 121 °C for 20 min to obtain the fermentation medium.

[0106] The remaining fermentation culture components: 15 g / L of sorbitol, 35 g / L of succinic acid, 0.5 g / L of yeast extract, 0.022 g / L of ferrous sulfate heptahydrate, 0.001 g / L of zinc sulfate heptahydrate, 15 g / L of corn steep liquor powder, 0.7 g / L of magnesium sulfate heptahydrate, 30 g / L of ammonia water (mass concentration is 20%).

[0107] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid loading of 60 mL was used.

[0108] The products of the fermentation culture solution were detected, and the detection results are shown in Table 3. As can be seen from Table 3, for the fermentation of mature seeds of Claviceps paspali using corn starch hydrolyzed by α-amylase at three different concentrations (contents), the yields (potencies) of total ergot acid in the obtained fermentation broth were approximately 2.928 g / L, 3.692 g / L, and 3.114 g / L, respectively. When the concentration of corn starch was 70 g / L, the yield of total ergot acid was the highest.

[0109] Table 3 Influence of the content of corn starch hydrolyzed by α-amylase on the product potency

[0110] Example Corn starch (g / L) Titer (g / L) 4-1 50 2.928 4-2 70 3.692 4-3 100 3.114

[0111] Examples 5-1, 5-2, and 5-3

[0112] Based on Example 4-2, these examples studied the influence of different nitrogen sources in the fermentation medium on the yield of ergot acid in fermentation production.

[0113] Examples 5-1, 5-2, and 5-3 were only different from Example 4-2 in step (5) for preparing the fermentation medium, and the other steps were the same as those in Example 4-2.

[0114] (5) Preparation of the fermentation medium: Mix corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL), and water (where the content of corn starch is 70 g / L, and the weight ratio of corn starch to α-amylase is 1:0.0007). Heat the mixture to 80 - 90 °C and keep it warm for 30 min to hydrolyze the corn starch with α-amylase, and then add the remaining fermentation culture components and sterilize at 121 °C for 20 min to obtain the fermentation medium.

[0115] The remaining fermentation culture components: slow-acting nitrogen source 15 g / L (soybean cake powder, peanut cake powder, or cottonseed cake powder), sorbitol 15 g / L, succinic acid 35 g / L, yeast extract 0.5 g / L, ferrous sulfate heptahydrate 0.022 g / L, zinc sulfate heptahydrate 0.001 g / L, corn steep liquor dry powder 15 g / L, magnesium sulfate heptahydrate 0.7 g / L, ammonia water (mass concentration is 20%) 30 g / L.

[0116] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid loading of 60 mL was used.

[0117] The products of the fermentation culture solution were detected, and the detection results are shown in Table 4. As can be seen from Table 4, for the fermentation of mature seeds of Claviceps paspali using fermentation media supplemented with three different slow-acting nitrogen sources respectively, the yields (potencies) of total ergot acid in the obtained fermentation broth were approximately 1.523 g / L, 3.256 g / L, and 3.953 g / L, respectively. When cottonseed cake powder was added, the yield of total ergot acid was the highest.

[0118] Table 4 Influence of the type of slow-acting nitrogen source on the product titer

[0119] Example Type of slow-acting nitrogen source Titer (g / L) 5-1 Soybean cake powder 1.523 5-2 Peanut cake powder 3.256 5-3 Cottonseed cake powder 3.953

[0120] Examples 6-1, 6-2 and 6-3

[0121] On the basis of Example 5-3, these examples studied the influence of the concentration (content) of cottonseed cake powder in the fermentation medium on the yield of ergot acid in fermentation production.

[0122] Examples 6-1, 6-2 and 6-3 are only different from Example 5-3 in step (5) of preparing the fermentation medium, and the other steps are the same as those in Example 5-3.

[0123] (5) Preparation of the fermentation medium: Mix corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water (where the content of corn starch is 70 g / L, and the weight ratio of corn starch to α-amylase is 1:0.0007), heat the mixture to 80-90 °C, keep it warm for 30 min to hydrolyze the corn starch with α-amylase, then add the remaining fermentation culture components, sterilize at 121 °C for 20 min, and the obtained fermentation medium is obtained.

[0124] The remaining fermentation culture components: 15 g / L of sorbitol, 35 g / L of succinic acid, 0.5 g / L of yeast extract, 0.022 g / L of ferrous sulfate heptahydrate, 0.001 g / L of zinc sulfate heptahydrate, 15 g / L of corn steep liquor dry powder, 0.7 g / L of magnesium sulfate heptahydrate, 30 g / L of ammonia water (mass concentration is 20%), cottonseed cake powder (concentration gradients are 15 g / L, 20 g / L, 25 g / L).

[0125] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid loading of 60 mL is used.

[0126] Detect the products in the fermentation broth, and the detection results are shown in Table 5. It can be seen from Table 5 that when the fermentation media containing three concentrations (contents) of cottonseed cake powder are used for the fermentation of mature seeds of Claviceps paspali, the total ergot acid yields (titers) in the obtained fermentation broths are about 4.018 g / L, 4.513 g / L, and 4.214 g / L respectively. Among them, when the concentration of cottonseed cake powder is 20 g / L, the total ergot acid yield is the highest.

[0127] Table 5 Influence of the content of cottonseed cake powder on the product titer

[0128] Example Cottonseed cake powder (g / L) Titer (g / L) 6-1 15 4.018 6-2 20 4.513 6-3 25 4.214

[0129] Example 7

[0130] Example This example studied the changes in the succinic acid content (g / L) and pH of the fermentation broth when the fermentation and culture steps of Example 6-2 were carried out in a fermenter.

[0131] Example 7 The difference between Example 7 and Example 6-2 is only that the fermentation and culture in Example 7 were carried out in a fermenter. All steps were the same as those in Example 6-2. The total volume of the fermentation medium in the fermenter was about 10 L.

[0132] Fermenter culture: The seed liquid was inoculated into the fermenter at an inoculation amount of 10% (v / v). The fermentation temperature was 21-25 °C, the aeration rate was 0.25-1.0 vvm, the dissolved oxygen was controlled above 30%, the tank pressure was 0.02-0.08 MPa, and the culture time was 13-14 days to obtain the fermentation broth.

[0133] During the fermentation process, the succinic acid content (g / L), pH, and titer (g / L) in the fermentation broth were measured. The test results are shown in Figure 1 . The succinic acid content was as follows: 24.72 g / L on the 1st day, 18.08 g / L on the 2nd day, 12.37 g / L on the 3rd day, and 8.26 g / L on the 4th day. Starting from the 3rd day of fermentation, the pH rebounded, but the rebound amplitude was small. After the 9th day, the succinic acid content was very low, and the pH increase became steeper, while the titer increase slowed down. When the fermentation ended (on the 14th day), the titer of total ergot acid in the fermentation broth was measured to be 4.211 g / L.

[0134] Examples 8-10

[0135] These examples studied the effect of supplementing succinic acid on the fermentation yield based on Example 7.

[0136] The differences between Examples 8-10 and Example 7 were only that succinic acid was supplemented during the fermentation process, and other steps were the same as those in Example 7. The time and amount of succinic acid supplementation were determined according to the succinic acid content detected on the 2nd, 3rd, 4th, and 9th days in Example 7.

[0137] Referring to the succinic acid content on the 2nd day in Example 7, in Example 8, the succinic acid content was sampled and detected on the 2nd day of fermentation. When the succinic acid content was lower than 22 g / L, a succinic acid solution (content 60 g / L) was batch-supplemented on the same day to maintain the succinic acid content in the fermentation broth at 20-30 g / L. From the 2nd to the 13th day of fermentation, batch feeding was carried out according to this method (that is, the succinic acid solution was supplemented 2-3 times a day to maintain the succinic acid content in the fermentation broth at 20-30 g / L).

[0138] Refer to the succinic acid content on the third day of Reference Example 7. In Example 9, the succinic acid content was sampled and detected on the 3rd day of fermentation. When the succinic acid content was lower than 13 g / L, a succinic acid solution (content: 60 g / L) was added in batches on the same day to maintain the succinic acid content in the fermentation broth at 10 - 20 g / L. From the 3rd to the 13th day of fermentation, fed-batch operation was carried out according to this method (i.e., the succinic acid solution was added 2 - 3 times a day to maintain the succinic acid concentration in the fermentation broth at 10 - 20 g / L).

[0139] Refer to the succinic acid concentration on the fourth day of Reference Example 7. In Example 10, the succinic acid content was sampled and detected on the 4th day of fermentation. When the succinic acid concentration was lower than 9 g / L, a succinic acid solution (concentration: 60 g / L) was added in batches on the same day to maintain the succinic acid concentration in the fermentation broth at 5 - 10 g / L. From the 4th to the 13th day of fermentation, fed-batch operation was carried out according to the above method (i.e., the succinic acid solution was added 2 - 3 times a day to maintain the succinic acid concentration in the fermentation broth at 5 - 10 g / L).

[0140] At the end of fermentation (the 14th day of fermentation), the fermentation broth was detected by high performance liquid chromatography (HPLC). All the titers obtained below were compared with the titer result of Example 7 to obtain the titer increase. The results are shown in Table 6.

[0141] Table 6 Titers and their increases of total ergot acid in the hydrolyzed fermentation broth

[0142] Example Titer (g / L) Titer increase 8 4.483 6.34％ 9 4.927 16.79％ 10 4.620 9.63％

[0143] It can be seen from the detection results in Table 6 that in Example 9, when the succinic acid concentration was maintained at 10 - 20 g / L by feeding from the 3rd to the 13th day of fermentation culture, the highest yield of total ergot acid was obtained.

[0144] Examples 11 - 13

[0145] Based on Example 9, these examples studied the effect of differences in the control of succinic acid content on the fermentation yield from the 3rd to the 13th day of fermentation.

[0146] The differences between Examples 11 - 13 and Example 9 were only the different succinic acid contents maintained in the fermentation broth. All other steps were the same as those in Example 9. The feeding method was to add a succinic acid solution with a concentration of 60 g / L in batches from the 3rd to the 13th day.

[0147] In Example 11, the succinic acid solution was added in 3 batches to maintain the succinic acid content in the fermentation broth at 15 - 20 g / L.

[0148] In Example 12, the succinic acid solution was added in 3 batches to maintain the succinic acid content in the fermentation broth at 10 - 15 g / L.

[0149] In Example 13, the succinic acid solution was added in 3 batches to maintain the succinic acid content in the fermentation broth at 5 - 10 g / L.

[0150] At the end of fermentation (on the 14th day of fermentation), the fermentation broth was detected by high performance liquid chromatography (HPLC), and the results are shown in Table 7.

[0151] Table 7 Potency and increase of total ergot acid in the hydrolyzed fermentation broth

[0152] Example Titer (g / L) Titer increase 11 4.835 14.72％ 12 5.021 19.33％ 13 4.713 11.95％

[0153] It can be seen from the detection results in Table 7 that in Example 12, when the succinic acid content in the fermentation broth was maintained at 10-15 g / L by batch feeding of succinic acid solution during fermentation days 3-13, the highest yield of total ergot acid was obtained.

[0154] Example 14

[0155] In this example, based on Example 12, the effect of the feeding method of adding succinic acid solution on the potency was studied.

[0156] The difference between this example and Example 12 lies in the method of adding succinic acid solution, and other steps are the same as those in Example 12.

[0157] This example adopted continuous feeding, specifically: on the 3rd day of fermentation, the succinic acid content was sampled and detected. When the succinic acid concentration in the fermentation system was lower than 13 g / L, succinic acid solution (with a concentration of 60 g / L) was continuously fed at a feeding rate of 17-25 mL·h -1 to maintain the succinic acid concentration in the fermentation broth at 10-15 g / L. Continuous feeding was carried out until the 13th day of fermentation culture, and then the feeding was stopped.

[0158] At the end of fermentation (on the 14th day of fermentation), the fermentation broth was detected by high performance liquid chromatography (HPLC), and the results are shown in Table 8.

[0159] Table 8 Potency and increase of total ergot acid in the hydrolyzed fermentation broth

[0160] Example Titer (g / L) Titer increase 14 5.172 22.74％

[0161] It can be seen from the detection results in Table 8 that when continuous feeding was adopted during fermentation days 3-13, the potency of total ergot acid in the obtained fermentation broth was relatively high. And it was detected that during the middle and late stages of fermentation in this example, the pH rebound amplitude slowed down, and the pH of the fermentation broth was maintained at 5.05-5.15, which was more beneficial to the product synthesis of the bacteria. Therefore, the preferred feeding method is continuous feeding. The fermentation broth of this example was treated with alkali and detected by HPLC, and the obtained chromatogram is shown in Figure 2 .

[0162] In summary, the method for improving the fermentation yield of ergot acid provided by the present invention can significantly improve the fermentation level, stabilize the yield above 5.0 g / L, and reach a maximum of 5.172 g / L. Moreover, the raw materials for feeding are succinic acid and water, which are easily obtained. The feeding method is simple and can be used without complex production equipment, making it easy to be popularized in industrial production and significantly increasing the production efficiency of enterprises.

[0163] Although the present invention is disclosed above with preferred embodiments, it is not intended to limit the present invention. Without departing from the spirit and scope of the present invention, those skilled in the art can make various modifications and deformations based on this. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A method for increasing the fermentation yield of total ergot acid, wherein the total ergot acid is composed of ergine and isolysergic acid, and is characterized in that, Inoculate the seed liquid of Claviceps paspali into the fermentation medium for liquid fermentation culture. When the content of succinic acid in the fermentation medium is lower than 13 g / L, add an aqueous solution of succinic acid to the fermentation broth to maintain the succinic acid content in the fermentation broth at 10 - 15 g / L. The Claviceps paspali is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO. 41366. The aqueous solution of succinic acid is added to the fermentation broth in a continuous feeding manner. The concentration of the continuously fed aqueous solution of succinic acid is 50 - 80 g / L. The flow rate of continuous feeding is 17 - 25 mL·h -1 , The fermentation medium contains the following components in the following amounts: Sorbitol 5 - 25 g / L, corn starch hydrolyzed by α - amylase 50 - 100 g / L, succinic acid 30 - 40 g / L, yeast extract 0.4 - 0.6 g / L, cottonseed cake powder 15 - 25 g / L, dry corn steep liquor 10 - 20 g / L, ammonia water 30 - 40 g / L, ferrous sulfate heptahydrate 0.001 - 0.030 g / L, zinc sulfate heptahydrate 0.001 - 0.015 g / L, magnesium sulfate heptahydrate 0.6 - 0.8 g / L. The mass concentration of the ammonia water is 15 - 25%.

2. The method for increasing the fermentation yield of total lysergic acid according to claim 1, wherein The temperature of the fermentation culture is 21 - 25 °C, and the rotation speed is 200 - 500 rpm.

3. The method for increasing the total ergot acid fermentation yield according to claim 1, characterized in that The corn starch hydrolyzed by α - amylase is obtained by hydrolyzing corn starch with α - amylase at 80 - 90 °C for 28 - 32 min.

4. The method for increasing the total yield of lysergic acid fermentation according to claim 3, wherein During the hydrolysis process, the mass ratio of α - amylase to corn starch is 0.0005:1 - 0.0009:

1.

5. The method for increasing the total ergot acid fermentation yield according to claim 4, characterized in that, During the hydrolysis process, the mass ratio of α - amylase to corn starch is 0.0006:1 - 0.0008:

1.

6. The method for increasing the total ergot acid fermentation yield according to claim 5, characterized in that, During the hydrolysis process, the mass ratio of α - amylase to corn starch is 0.0007:

1.

7. The method for increasing the total ergot acid fermentation yield according to claim 1, characterized in that, The content of the corn starch hydrolyzed by α - amylase is 60 - 80 g / L.

8. The method for increasing the total ergot acid fermentation yield according to claim 7, characterized in that, The content of the corn starch hydrolyzed by α - amylase is 70 g / L.

9. The method for increasing the total ergot acid fermentation yield according to claim 1, wherein The preparation method of the fermentation medium includes the following steps: After mixing α - amylase, corn starch and water, heat to 80 - 90 °C and keep warm for 28 - 32 min to hydrolyze the corn starch with α - amylase, and then add the remaining components of the fermentation medium and heat to 110 - 130 °C for sufficient sterilization to obtain the fermentation medium.

10. The method for increasing the total ergot acid fermentation yield according to claim 1, characterized in that, The method includes the following steps: (1) Strain activation: Spread the suspension of the working cell bank of Claviceps paspali on a PDA slant and culture at 26 - 30 °C for 7 - 8 days to obtain the GI slant strain. Among them, the Claviceps paspali is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC NO. 41366. Among them, for the PDA medium, calculated by its total volume, it contains the following components in the following amounts: Potato dextrose agar 35 - 43 g / L, yeast extract 0.4 - 0.6 g / L, and adjust the pH to 7.10 - 7.30 with sodium hydroxide solution. (2) Seed culture preparation: Scrape the GI slant culture obtained in step (1), after homogenization, place it in a seed culture medium for cultivation at a temperature of 21 - 25 °C and a rotation speed of 220 - 260 rpm for 96 - 144 h, and the wet cell weight ≥ 45 g / L; The seed culture medium, based on its total volume, contains the following components in the following amounts: Mannitol 45 - 55 g / L, succinic acid 9 - 11 g / L, soybean powder 4 - 6 g / L, potassium dihydrogen phosphate 0.75 - 1.25 g / L, magnesium sulfate heptahydrate 0.25 - 0.35 g / L, and adjust the pH to 5.00 - 5.20 with ammonia water. (3) Fermentation culture: Inoculate the seed culture obtained in step (2) into the fermentation medium for liquid fermentation culture at an inoculation amount of 8 - 12% (v / v). When the content of succinic acid in the fermentation broth is lower than 13 g / L, supplement the succinic acid solution to the fermentation broth in a continuous feeding manner to maintain the succinic acid concentration in the fermentation broth at 10 - 15 g / L. The fermentation medium, based on its total volume, contains the following components in the following amounts: sorbitol 5 - 25 g / L, α - amylase hydrolyzed corn starch 50 - 100 g / L, succinic acid 30 - 40 g / L, yeast extract 0.4 - 0.6 g / L, ferrous sulfate heptahydrate 0.000 - 0.030 g / L, zinc sulfate heptahydrate 0.000 - 0.015 g / L, magnesium sulfate heptahydrate 0.6 - 0.8 g / L, corn steep liquor dry powder 10 - 20 g / L, ammonia water 30 - 40 g / L, cottonseed cake powder 15 - 25 g / L. During the process of α - amylase hydrolyzing corn starch, the weight ratio of corn starch to α - amylase is 1:0.0005 - 1:0.0009. The preparation method of the fermentation medium includes: Mix corn starch, α - amylase and water, heat to 80 - 90 °C, keep warm for 28 - 32 min to hydrolyze corn starch by α - amylase, and then add the remaining components of the fermentation medium, heat to 110 - 130 °C for sufficient sterilization to obtain the fermentation medium, where the weight ratio of corn starch to α - amylase is 1:0.0005 - 1:0.0009.

11. The method for increasing the total ergine fermentation yield according to claim 10, wherein The temperature of fermentation culture is 21 - 25 °C, the aeration rate is 0.25 - 1.0 vvm, the dissolved oxygen is controlled above 30%, the tank pressure is 0.02 - 0.08 MPa, and the culture time is 13 - 14 days.

Images