A method for producing lysergic acid by fermentation

Abstract

The invention provides a method for producing lysergic acid by fermentation, wherein the seed liquid of Claviceps paspali is inoculated in a fermentation medium for liquid fermentation culture, wherein the Claviceps paspali is deposited in the China General Microbiological Culture Collection Center, and the deposit number is CGMCC NO.41366, and the fermentation medium, based on its total volume, comprises 80-150 g / L of carbon source and 40-90 g / L of nitrogen source, wherein the carbon source comprises corn starch hydrolyzed by alpha-amylase, and the content thereof is 50-100 g / L. The content of lysergic acid in the fermentation liquid obtained by the inventive method can reach 4.5 g / L, and the fermentation medium uses corn starch as the main carbon source, and the cost is low.

Description

Technical Field

[0001] The present invention relates to the field of biological fermentation technology, and more specifically to a method for producing lysergic acid by fermentation. Background Art

[0002] Lysergic acid is a type of indole compound extracted from ergot. It is a white crystalline powder that is easily soluble in solutions such as pyridine, sodium hydroxide and hydrochloric acid, and slightly soluble in dilute sulfuric acid. As the core structure of ergot alkaloids, lysergic acid plays an important role in the pharmaceutical field, such as participating in the synthesis of drugs such as nicergoline, methylergonovine and dimethylergonovine.

[0003] At present, the production of lysergic acid mainly adopts microbial fermentation. Prior art CN111334439A discloses a ergot mutant and its application in preparing lysergic acid fermentation liquid, and the prior art adopts mutation breeding and utilizes metabolite feedback inhibition mechanism to increase the tolerance of ergot to its own metabolite lysergic acid to improve lysergic acid output. The prior art also discloses that the ergot mutant is cultivated for 15 days through fermentation medium (sorbitol 80g / L, succinic acid 35g / L, yeast powder 0.5g / L, ferrous sulfate 0.02g / L, zinc sulfate 0.01g / L, corn steep liquor dry powder 18g / L, defoamer 0.5g / L), and the content of lysergic acid in the obtained fermentation liquid is about 3g / L.

[0004] Prior art CN116024107A discloses an engineering bacterium of lysergic acid and its construction method and application, which blocks the synthesis of lysergic amide by knocking out the g5167 gene, thereby achieving the accumulation of lysergic acid, and based on this, performs fermentation process optimization and fermentation liquid treatment optimization. The prior art also discloses that the engineering bacterium is cultured for 12-15 days through a fermentation medium (sorbitol 80-100g / L, succinic acid 35-45g / L, yeast powder 0.25-0.50g / L, ferrous sulfate 0.01-0.05g / L, zinc sulfate 0.01-0.05g / L, corn steep liquor dry powder 10-20g / L, magnesium sulfate 1.0-1.5g / L, ammonia / sodium hydroxide is used to adjust pH to 4.8-6.5), and the content of lysergic acid in the obtained fermentation liquid is 3-4g / L.

[0005] Prior art CN117126748A discloses a method for breeding a resistant mutant of ergot with high lysergic acid production. The obtained resistant mutant is fermented and cultured in a fermentation medium (100 g / L mannitol, 35 g / L succinic acid, 0.5 g / L yeast powder, 0.025 g / L ferrous sulfate, 0.01 g / L zinc sulfate, 20 g / L corn steep liquor powder, 0.7 g / L magnesium sulfate, 0.8 g / L defoamer, and the pH is adjusted to 7.0). The content of lysergic acid in the obtained fermentation liquid is about 4 g / L.

[0006] In summary, the prior art of producing lysergic acid by fermentation of ergot fungi not only has unsatisfactory yield but also high cost (generally using mannitol or sorbitol as the main carbon source component, resulting in high culture medium cost), which is difficult to meet industrial demand. Therefore, a production process of lysergic acid with low cost and high yield is needed. Summary of the invention

[0007] In view of the problems existing in the prior art of producing lysergic acid by fermentation, the present invention aims to provide a method for producing lysergic acid by fermentation. The method utilizes a new fermentation strain, has high yield, and utilizes corn starch as the main carbon source, so the cost is low.

[0008] The method for producing lysergic acid by fermentation of the present invention comprises inoculating seed liquid of Claviceps paspali into a fermentation medium for liquid fermentation culture, wherein the Claviceps paspali is deposited in the China General Microbiological Culture Collection Center (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), with a deposit number of CGMCCNO.41366 (deposit date: June 14, 2024), and the fermentation medium, based on its total volume, comprises 80-150 g / L of a carbon source and 40-90 g / L of a nitrogen source, wherein the carbon source comprises corn starch hydrolyzed by alpha-amylase, and the content thereof is 50-100 g / L, preferably 60-80 g / L.

[0009] In another preferred embodiment, the carbon source comprises 65-75 g / L corn starch hydrolyzed by α-amylase.

[0010] In another preferred embodiment, the fermentation medium, based on its total volume, contains 50-80 g / L of nitrogen source, more preferably 60-75 g / L.

[0011] In another preferred embodiment, the nitrogen source comprises cottonseed cake powder, and the content thereof is 12-30 g / L, more preferably 15-25 g / L.

[0012] In another preferred embodiment, the fermentation medium further comprises a metal salt, and its content is 0.2-1.0 g / L based on the total volume of the fermentation medium. In another more preferred embodiment, the content of the metal salt is 0.5-0.9 g / L based on the total volume of the fermentation medium. In another preferred embodiment, the metal salt is selected from ferrous sulfate, zinc sulfate, magnesium sulfate or a combination thereof.

[0013] In another preferred example, the carbon source further comprises sorbitol, succinic acid or a combination thereof.

[0014] In another preferred embodiment, the nitrogen source further comprises yeast extract, corn steep liquor powder, cottonseed cake powder, ammonia water or a combination thereof.

[0015] In another preferred embodiment, the fermentation medium, based on its total volume, comprises the following components:

[0016] 5-25 g / L sorbitol, 50-100 g / L corn starch hydrolyzed by alpha-amylase, 30-40 g / L succinic acid, 0.4-0.6 g / L yeast extract, 15-25 g / L cottonseed cake powder, 10-20 g / L corn steep liquor powder, 30-40 g / L ammonia water, 0.001-0.030 g / L ferrous sulfate heptahydrate, 0.001-0.015 g / L zinc sulfate heptahydrate, 0.6-0.8 g / L magnesium sulfate heptahydrate, wherein the mass concentration of the ammonia water is 15-25%.

[0017] In another preferred embodiment, the fermentation medium, based on its total volume, comprises the following components:

[0018] 5-25 g / L sorbitol, 60-80 g / L corn starch hydrolyzed by alpha-amylase, 30-40 g / L succinic acid, 0.4-0.6 g / L yeast extract, 15-25 g / L cottonseed cake powder, 10-20 g / L corn steep liquor powder, 30-40 g / L ammonia water, 0.001-0.030 g / L ferrous sulfate heptahydrate, 0.001-0.015 g / L zinc sulfate heptahydrate, 0.6-0.8 g / L magnesium sulfate heptahydrate, wherein the mass concentration of the ammonia water is 15-25%.

[0019] In another preferred example, the fermentation medium, based on its total volume, contains 65-75 g / L of corn starch hydrolyzed by the α-amylase.

[0020] In another preferred embodiment, the mass concentration of the ammonia water is 20%.

[0021] In another preferred embodiment, the yeast extract is Angel Yeast FM802.

[0022] In another preferred embodiment, the method for preparing 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 minutes so that the corn starch is hydrolyzed by the α-amylase, and then add the remaining fermentation medium components (for example, sorbitol, succinic acid, yeast extract, cottonseed cake powder, corn steep liquor powder, ammonia water, ferrous sulfate heptahydrate, zinc sulfate heptahydrate and magnesium sulfate heptahydrate) and heat to 110-130° C. for sufficient sterilization to obtain the fermentation medium.

[0024] In another preferred embodiment, during the hydrolysis of corn starch by α-amylase, the weight ratio of corn starch to α-amylase is 1:0.0005-1:0.0009, more preferably, 1:0.0006-1:0.0008.

[0025] In another preferred embodiment, in the method for producing lysergic acid by fermentation, the fermentation culture temperature is 21-25°C.

[0026] In another preferred embodiment, in the method for producing lysergic acid by fermentation, the fermentation culture is carried out at a rotation speed of 220-260 rpm for 12-14 days.

[0027] In another preferred embodiment, the method for producing lysergic acid provided by the present invention comprises the steps of:

[0028] (1) Activation of bacterial strains: Spread the suspension of the working cell bank of Claviceps paspali onto a PDA slant and culture at 26-30°C for 7-8 days to obtain a GI slant bacterial strain;

[0029] The Claviceps paspali is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.41366.

[0030] The PDA medium, based on its total volume, contains the following components:

[0031] Potato dextrose agar 35-43 g / L, yeast extract 0.4-0.6 g / L, pH adjusted to 7.10-7.30 with sodium hydroxide solution;

[0032] (2) Seed liquid preparation: scrape the GI slant strain obtained in step (1), homogenize it, and place it in a seed liquid 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 weight of the bacteria is ≥45 g / L;

[0033] The seed liquid culture medium, based on its total volume, comprises the following components:

[0034] Mannitol 45-55g / L, succinic acid 9-11g / L, soybean powder 4-6g / L, potassium dihydrogen phosphate 0.75-1.25g / L, magnesium sulfate heptahydrate 0.25-0.35g / L, ammonia water is used to adjust the pH to 5.00-5.20;

[0035] (3) Fermentation culture: The seed liquid obtained in step (2) is inoculated into a fermentation medium at an inoculum amount of 8-12% (v / v) for liquid fermentation culture at a temperature of 21-25° C., a pH of 4.95-5.80, and a rotation speed of 220-260 rpm for 12-14 days to obtain a fermentation liquid.

[0036] The fermentation medium, based on its total volume, comprises the following components:

[0037] Sorbitol 5-25g / L, corn starch hydrolyzed by α-amylase 50-100g / L, succinic acid 30-40g / L, yeast extract 0.4-0.6g / L, ferrous sulfate heptahydrate 0.000-0.030g / L, zinc sulfate heptahydrate 0.000-0.015g / L, magnesium sulfate heptahydrate 0.6-0.8g / L, corn steep liquor powder 10-20g / L, ammonia water 30-40g / L, cottonseed cake powder 15-25g / L, wherein in the process of α-amylase hydrolyzing corn starch, the weight ratio of corn starch to α-amylase is 1:0.0005-1:0.0009,

[0038] The fermentation medium preparation process is as follows: corn starch, α-amylase and water are mixed, heated to 80-90°C, and kept warm for 28-32 minutes so that the corn starch is hydrolyzed by the α-amylase, and then the remaining fermentation medium components are added, and heated to 110-130°C for sufficient sterilization to obtain the fermentation medium, wherein the weight ratio of corn starch to α-amylase is 1:0.0005-1:0.0009.

[0039] In another preferred embodiment, in step (1), the concentration of the sodium hydroxide solution used to adjust the pH value of the PDA culture medium is 4.0-6.0 M.

[0040] In another preferred embodiment, in step (2), the mass concentration of ammonia water used to adjust the pH value of the seed liquid culture medium is 15-25%. DETAILED DESCRIPTION

[0041] In view of the disadvantages of low yield and high production cost in the method of producing lysergic acid by microbial fermentation in the prior art, the inventors of the present application have found through in-depth research that lysergic acid can be obtained in high yield by culturing Clavicepspaspali (deposited in the China General Microbiological Culture Collection Center, with a deposit number of CGMCC NO.41366) through an improved fermentation medium. The improved fermentation medium uses corn starch hydrolyzed by α-amylase as the main fermentation carbon source, which significantly increases the yield of lysergic acid while reducing the amount of sorbitol or mannitol, thereby reducing the cost of the culture medium. In addition, the improved fermentation medium uses cottonseed meal as one of the nitrogen source components, further increasing the yield of lysergic acid, which can reach 4.5 g / L, and the titer is increased by about 12.5% ​​compared with the culture medium in the prior art.

[0042] Definition of terms

[0043] "Seed liquid" refers to the culture liquid containing the above-mentioned Claviceps spaspali fungus obtained by culturing in a seed culture medium. In a specific embodiment of the present invention, the preparation process of the "seed liquid" is as follows: (1) the above-mentioned Claviceps spaspali fungus is spread on a PDA slant culture medium (the PDA culture medium contains: 35-43 g / L potato dextrose agar, 0.4-0.6 g / L yeast extract, the pH is adjusted to 7.10-7.30 with sodium hydroxide solution, and the sterilization condition is 121°C, 20 min), and cultured at 26-30°C for 7-8 days to obtain GI slant strains (mature mycelium); (2) scrape the G I slant strain (mature mycelium) was inoculated with seed liquid culture medium (seed liquid culture medium contained: 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, ammonia water was used to adjust the pH to 5.00-5.20, and the sterilization conditions were 121°C, 20 min), and cultured at 21-25°C and a rotation speed of 220-260 rpm for 4-6 days to obtain seed liquid.

[0044] As described herein, "corn starch" can be prepared using methods well known in the art, or can be obtained through commercial channels, such as, but not limited to, corn starch purchased from Pingliang Guowei Starch Co., Ltd.

[0045] As described herein, "cottonseed cake powder" refers to a powder obtained by crushing cottonseed (i.e., cotton seeds) cakes, which is high in protein and rich in various amino acids such as tryptophan, lysine, and methionine. It can be prepared using methods well known in the art, or obtained through commercial channels, such as, but not limited to, cottonseed cake powder purchased from Jinan Huilong Biotechnology Co., Ltd.

[0046] As described herein, "yeast extract" can be prepared using methods well known in the art, or can be obtained through commercial channels, such as but not limited to Angel FM802.

[0047] As described herein, "corn syrup powder" refers to a powdered substance obtained by low-temperature, instantaneous spray drying of corn syrup, with a protein content of ≥42% and a moisture content of ≤10%, which is used as a nutritional supplement for water-soluble plant protein, water-soluble vitamins, and other nutrients during the biological fermentation process. It can be prepared using methods well known in the art, or obtained through commercial channels, such as, but not limited to, corn syrup powder purchased from Beijing Hongrun Baoshun Technology Co., Ltd.

[0048] As used herein, "aqueous ammonia" is an aqueous solution of ammonia with a mass concentration of 15-25%, such as 20%.

[0049] As described herein, "titer" refers to the total content of lysergic acid and isolysergic acid in a unit volume of fermentation broth, in g / L, and is calculated as follows: Titer = (the sum of the peak areas of lysergic acid and isolysergic acid of the products in the culture medium of the present invention determined by HPLC / the sum of the peak areas of lysergic acid and isolysergic acid of the standard) × concentration of the standard × content of the standard × dilution multiple of the product of the culture medium of the present invention.

[0050] The culture medium used in the fermentation culture described in the present invention is liquid, and the fermentation culture is also called liquid fermentation culture.

[0051] The contents of carbon source and nitrogen source in the fermentation medium of the present invention are generally understood in the art. For example, the content of carbon source refers to the weight of the raw material providing the carbon source in the total volume of the fermentation medium, and the content of nitrogen source refers to the weight of the raw material providing the nitrogen source in the total volume of the fermentation medium.

[0052] 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 is more preferably selected from sorbitol, corn starch hydrolyzed by α-amylase and succinic acid. Based on the total volume of the culture medium, the content of the carbon source is 80-150 g / L, more preferably 100-140 g / L, and further more preferably 120-130 g / L.

[0053] The nitrogen source used in the present invention can be selected from yeast extract, cottonseed cake powder, corn steep powder, ammonia water, soybean cake powder, etc., and is more preferably selected from yeast extract, cottonseed cake powder, corn steep powder and ammonia water. Based on the total volume of the culture medium, the content of the nitrogen source is 40-90 g / L, more preferably 50-80 g / L, for example 60.5-70.5 g / L.

[0054] Metal salts may also be added to the culture medium of the present invention, for example, ferrous sulfate heptahydrate, zinc sulfate heptahydrate, magnesium sulfate heptahydrate, etc.

[0055] The pH of the fermentation medium of the present invention is about 4.95-5.80.

[0056] The fermentation broth obtained by the present invention can be treated with a sodium hydroxide solution and then analyzed by liquid chromatography. For example, the fermentation broth is treated with a sodium hydroxide solution having a final concentration of 1.0-2.0 M at 75-85° C. for 4-5 hours, filtered, and the filtrate is analyzed by liquid chromatography.

[0057] Compared with the prior art, the advantages of the present invention are:

[0058] (1) The present invention uses Claviceps paspali (deposited in China General Microbiological Culture Collection Center, with a deposit number of CGMCC NO.41366) as a fermentation strain and a low-cost culture medium to obtain lysergic acid in high yield and at low cost. The fermentation yield can reach 4.5 g / L, which is at least 12.5% ​​higher than the existing fermentation yield.

[0059] (2) The fermentation medium of the present invention uses corn starch hydrolyzate as one of the fermentation carbon sources. The components obtained after the corn starch is hydrolyzed by α-amylase include slow-acting carbon sources and fast-acting carbon sources, such as short-chain dextrins of varying 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 period of the strain; in the middle and late stages of fermentation culture, the slow-acting carbon source such as short-chain dextrin and a small amount of polysaccharides are hydrolyzed into a fast-acting carbon source by enzymes produced by the bacteria themselves, continuously providing nutrition to the bacteria and prolonging the product production time. In addition, in the corn starch solution hydrolyzed by α-amylase, corn starch and a-amylase are cheaper than sorbitol or mannitol used in other prior arts.

[0060] (3) The fermentation medium of the present invention uses cottonseed cake powder as one of the slow-release nitrogen sources for fermentation. Cottonseed cake powder is rich in various amino acids such as tryptophan, lysine and methionine, and tryptophan is a precursor for the synthesis of lysergic acid. In the middle and late stages of fermentation culture, the slow-release nitrogen source cottonseed cake powder not only provides nutrients for bacterial growth, but also provides precursors for product synthesis.

[0061] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually performed under normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, all percentages, ratios, proportions, or parts are by weight.

[0062] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0063] In the following examples, the lysergic acid content in the ergot fermentation broth was determined by external standard method using HPLC.

[0064] The details are as follows:

[0065] ① Detection of standard substance: Accurately weigh about 10 mg of the reference substance and place it in a 10 mL volumetric flask, dissolve it with diluent (acetonitrile: water = 50:50) and dilute it to the scale, shake well. Accurately transfer 1.0 mL of the above solution to a 10 mL volumetric flask, dilute it to the scale with diluent, shake well, and set aside.

[0066] ② Preparation of samples to be tested: Accurately transfer 1.0 mL of fermentation broth to a 1.5 mL microcentrifuge tube, add 0.39 mL of 5 M sodium hydroxide solution, mix well, and shake at 80 ° C and 1500 rpm for 4 hours to obtain a hydrolyzate. Transfer all of the above hydrolyzate to a 50 mL volumetric flask, add diluent (acetonitrile: water = 50:50) to the scale, shake well, and filter through a 0.22 μm filter membrane for HPLC analysis.

[0067] ③ The standard dilution and the sample dilution were subjected to HPLC analysis respectively, and the content of lysergic acid in the sample was determined according to the relationship between content and area ratio.

[0068] The chromatograph used in HPLC is a UPLC high performance liquid chromatograph, and the chromatographic column is a Waters Acquity UPLCBEH C18 column (100×2.1mm 1.7μm); the detector is DAD; the detection wavelength is 310nm; the column temperature is 40°C; the flow rate is 0.35mL / min; the mobile phase A is 8mmol / L ammonium formate solution (pH 8.0); the mobile phase B is acetonitrile:methanol (V / V=80:20); the running time is 17min; the gradient elution program is: 0.00-0.50min 97%A, 0.50-6.50min70%A, 6.50-9.00min 38%A, 9.00-9.20min 20%A, 9.20-11.00min 20%A, 11.00-12.00min97%A, 12.00-17.00min97%A.

[0069] In the following examples, Claviceps paspali was used, which was deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.41366.

[0070] The yeast extract used in the following examples is Angel FM802.

[0071] In the following examples, unless otherwise specified, the solution is an aqueous solution.

[0072] Example 1

[0073] In this embodiment, lysergic acid is produced by fermentation using existing fermentation medium.

[0074] (1) Preparation of PDA medium: 39 g / L potato dextrose agar, 0.5 g / L yeast extract and water were mixed, and the pH was adjusted to 7.10-7.30 with 5 M sodium hydroxide solution; 18 mL of PDA liquid medium was pipetted into a test tube (25 x 200 mm) and sterilized at 121°C for 20 min;

[0075] (2) Activation of bacterial strains: Pipette 0.75 mL of the bacterial suspension of the working cell bank of Claviceps paspali onto the PDA slant obtained in step (1), apply it evenly using a spreader, and culture at 28° C. for 7-8 days to obtain the GI slant bacterial strains;

[0076] (3) Preparation of seed liquid culture medium: 50 g / L mannitol, 10 g / L succinic acid, 5 g / L soybean powder, 1 g / L potassium dihydrogen phosphate, and 0.3 g / L magnesium sulfate heptahydrate were mixed with water, and the pH was adjusted to 5.00-5.20 with ammonia water (mass concentration of 20%), and the mixture was placed in a conical flask (for example, a 250 mL conical flask with a liquid volume of 60 mL), and sterilized at 121° C. for 20 min;

[0077] (4) Preparation of seed solution: scrape the GI slant strain obtained in step (2) into a centrifuge tube, add 10 mL of physiological 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 solution culture medium. Cultivate at 23° C. and 240 rpm for 96-144 h, and the wet weight of the bacteria is ≥45 g / L.

[0078] (5) Preparation of fermentation medium: 100 g / L sorbitol, 35 g / L succinic acid, 0.5 g / L yeast extract, 0.022 g / L ferrous sulfate heptahydrate, 0.001 g / L zinc sulfate heptahydrate, 15 g / L corn steep liquor powder, 0.7 g / L magnesium sulfate heptahydrate, and 30 g / L ammonia water (mass concentration of 20%) were mixed with water, placed in a 250 mL flask (250 mL flask with a liquid volume of 60 mL), and sterilized at 121° C. for 20 min;

[0079] (6) Fermentation culture: The seed solution obtained in step (4) was inoculated into a 250 mL shake flask containing 60 mL of fermentation medium at a 10% (v / v) inoculation rate, the culture temperature was 23° C., and the rotation speed was 240 rpm for 14 days to obtain a fermentation liquid;

[0080] Product detection: The fermentation broth obtained in step (6) was detected by liquid chromatography, and the products included lysergic acid and isolysergic acid. Detection result: The total lysergic acid yield (titer) was 2.732 g / L.

[0081] Examples 2-1, 2-2 and 2-3

[0082] These examples, based on Example 1, studied the effect of using corn starch hydrolyzed by α-amylase, glycerol or maltodextrin in the fermentation medium to replace part of sorbitol on the yield of lysergic acid produced by fermentation.

[0083] In Examples 2-1, 2-2 and 2-3, only step (5) of the preparation of the fermentation medium is different from that in Example 1, and the other steps are the same as those in Example 1.

[0084] (5) Preparation of fermentation medium: 50 g / L of organic carbon source (the types of organic carbon sources are corn starch hydrolyzed by α-amylase, glycerol and 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, and 30 g / L of ammonia water (mass concentration of 20%) were mixed with water to prepare a fermentation medium, which was filled into a 250-mL conical flask with a liquid volume of 60 mL and sterilized at 121° C. for 20 min.

[0085] The pretreatment method for hydrolyzing corn starch with α-amylase is as follows: corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water are mixed (the weight ratio of corn starch to α-amylase is 1:0.0005, respectively), the mixed solution is heated to 80-90°C, and kept warm for 30 minutes, so that the corn starch is hydrolyzed by α-amylase.

[0086] The preparation process of the fermentation medium containing corn starch hydrolyzed by α-amylase is as follows: corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water are mixed (wherein the weight ratio of corn starch to α-amylase is 1:0.0005), the mixed solution is heated to 80-90°C, kept warm for 30 minutes, so that the corn starch is hydrolyzed by α-amylase, and then the remaining fermentation medium components are added, sterilized at 121°C for 20 minutes, and the fermentation medium containing corn starch hydrolyzed by α-amylase is obtained. The remaining fermentation culture components are: sorbitol 15g / L, succinic acid 35g / L, yeast extract 0.5g / L, ferrous sulfate heptahydrate 0.022g / L, zinc sulfate heptahydrate 0.001g / L, corn steep liquor dry powder 15g / L, magnesium sulfate heptahydrate 0.7g / L, ammonia water (mass concentration is 20%) 30g / L.

[0087] The product of the fermentation broth was tested, and the test results are shown in Table 1. As can be seen from Table 1, when the organic carbon source is corn starch hydrolyzed by α-amylase, the total lysergic acid yield (titer) in the fermentation broth is the highest, but still slightly lower than the control (Example 1). Based on the cost of raw materials, corn starch hydrolyzed by α-amylase was selected as the carbon source for later experiments.

[0088] Table 1 Effect of organic carbon source type on product titer

[0089]

[0090] Examples 3-1, 3-2 and 3-3

[0091] These examples, based on Example 2-2, studied the effects of different ratios of corn starch to α-amylase in the fermentation medium on the yield of lysergic acid.

[0092] In Examples 3-1, 3-2 and 3-3, only step (5) of the preparation of the fermentation medium is different from that in Example 2-2, and the other steps are the same as those in Example 2-2.

[0093] (5) Preparation of fermentation medium: corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water are mixed (the corn starch content 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). The mixed solution is heated to 80-90° C. and kept warm for 30 min to allow the corn starch to be hydrolyzed by the α-amylase. The remaining fermentation medium components are then added and sterilized at 121° C. for 20 min to obtain the fermentation medium.

[0094] The remaining fermentation culture components are: 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 powder 15 g / L, magnesium sulfate heptahydrate 0.7 g / L, and ammonia water (mass concentration is 20%) 30 g / L.

[0095] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid volume of 60 mL was used.

[0096] The product and initial reducing sugar of the fermentation broth were tested, and the test 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 medium was used for the fermentation of ergot seed liquid of Paspalum cuneiformis. The initial reducing sugar concentrations in the fermentation broth were 0.5%, 1.8%, and 2.9%, respectively, and the total lysergic acid production (titer) in the fermentation broth was 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 lysergic acid production was the highest. Higher or lower reducing sugar concentrations will inhibit the early growth of the bacteria, prolong the culture cycle, and reduce the amount of products.

[0097] Table 2 Effect of different dosages of α-amylase on the titer of corn starch hydrolyzed

[0098]

[0099] Examples 4-1, 4-2 and 4-3

[0100] These examples, based on Example 3-2, studied the effect of the hydrolyzed corn starch content in the fermentation medium on the yield of lysergic acid produced by fermentation.

[0101] In Examples 4-1, 4-2 and 4-3, only step (5) of the preparation of the fermentation medium is different from that in Example 3-2, and the other steps are the same as those in Example 3-2.

[0102] (5) Preparation of fermentation medium: corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water are mixed (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). The mixed solution is heated to 80-90° C. and kept warm for 30 min to allow the corn starch to be hydrolyzed by α-amylase. Then, the remaining fermentation culture components are added and sterilized at 121° C. for 20 min to obtain the fermentation medium.

[0103] The remaining fermentation culture components: sorbitol 15g / L, succinic acid 35g / L, yeast extract 0.5g / L, ferrous sulfate heptahydrate 0.022g / L, zinc sulfate heptahydrate 0.001g / L, corn steep liquor powder 15g / L, magnesium sulfate heptahydrate 0.7g / L, ammonia water (mass concentration is 20%) 30g / L.

[0104] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid volume of 60 mL was used.

[0105] The products of the fermentation culture liquid were tested, and the test results are shown in Table 3. As can be seen from Table 3, three different concentrations (contents) of corn starch hydrolyzed by α-amylase were used for fermentation culture of Claviceps paspalum seed liquid, and the total lysergic acid yield (titer) in the obtained fermentation liquid was 2.928g / L, 3.692g / L and 3.114g / L respectively. When the concentration of corn starch was 70g / L, the total lysergic acid yield was the highest.

[0106] Table 3 Effect of the concentration (content) of corn starch hydrolyzed by α-amylase on the titer of the product

[0107] Serial number Corn starch hydrolyzed by α-amylase (g / L) Potency (g / L) 4-1 50 2.928 4-2 70 3.692 4-3 100 3.114

[0108] Examples 5-1, 5-2 and 5-3

[0109] These examples, based on Example 4-2, studied the effects of different nitrogen sources in the fermentation medium on the yield of lysergic acid produced by fermentation.

[0110] In Examples 5-1, 5-2 and 5-3, only step (5) of the preparation of the fermentation medium is different from that in Example 4-2, and the other steps are the same as those in Example 4-2.

[0111] (5) Preparation of fermentation medium: corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water (wherein the corn starch content is 70 g / L and the weight ratio of corn starch to α-amylase is 1:0.0007), the mixed solution is heated to 80-90° C. and kept warm for 30 min so that the corn starch is hydrolyzed by α-amylase, and then the remaining fermentation culture components are added and sterilized at 121° C. for 20 min to obtain the fermentation medium.

[0112] The remaining fermentation culture components: slow-release nitrogen source 15g / L (soybean cake powder, peanut cake powder or cottonseed cake powder), sorbitol 15g / L, succinic acid 35g / L, yeast extract 0.5g / L, ferrous sulfate heptahydrate 0.022g / L, zinc sulfate heptahydrate 0.001g / L, corn steep liquor powder 15g / L, magnesium sulfate heptahydrate 0.7g / L, ammonia water (mass concentration is 20%) 30g / L.

[0113] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid volume of 60 mL was used.

[0114] The products of the fermentation culture medium were tested, and the test results are shown in Table 4. As can be seen from Table 4, the fermentation mediums to which three slow-acting nitrogen sources were added were used for the fermentation culture of the ergot fungus seed liquid, and the yields (titer) of total lysergic acid in the obtained fermentation liquid were 1.523 g / L, 3.256 g / L and 3.953 g / L, respectively, among which the yield of total lysergic acid was the highest when cottonseed cake powder was added.

[0115] Table 4 Effect of slow-release nitrogen source types on product titer

[0116]

[0117]

[0118] Examples 6-1, 6-2 and 6-3

[0119] These examples, based on Example 5-3, studied the effect of cottonseed meal concentration (content) in the fermentation medium on the yield of lysergic acid produced by fermentation.

[0120] The only difference between Examples 6-1, 6-2 and 6-3 in terms of the preparation of the fermentation medium in step (5) is that in Example 5-3, and the other steps are the same as those in Example 5-3.

[0121] (5) Preparation of fermentation medium: corn starch, α-amylase (enzyme activity unit is about 20,000 U / mL) and water (wherein the corn starch content is 70 g / L and the weight ratio of corn starch to α-amylase is 1:0.0007), the mixed solution is heated to 80-90° C. and kept warm for 30 min so that the corn starch is hydrolyzed by α-amylase, and then the remaining fermentation culture components are added and sterilized at 121° C. for 20 min to obtain the fermentation medium.

[0122] The remaining fermentation culture components: sorbitol 15g / L, succinic acid 35g / L, yeast extract 0.5g / L, ferrous sulfate heptahydrate 0.022g / L, zinc sulfate heptahydrate 0.001g / L, corn steep liquor powder 15g / L, magnesium sulfate heptahydrate 0.7g / L, ammonia water (mass concentration is 20%) 30g / L, cottonseed cake powder (concentration gradients are 15g / L, 20g / L, 25g / L respectively).

[0123] During the preparation of the fermentation medium, a 250 mL Erlenmeyer flask with a liquid volume of 60 mL was used.

[0124] The product of the fermentation culture liquid was tested, and the test results are shown in Table 5. As can be seen from Table 5, the fermentation medium with three concentrations (contents) of cottonseed meal powder added was used for the fermentation culture of Claviceps paspalum seed liquid, and the total lysergic acid yield (titer) in the obtained fermentation liquid was 4.018 g / L, 4.513 g / L, and 4.214 g / L, respectively, among which the total lysergic acid yield was the highest when the concentration of cottonseed meal powder was 20 g / L.

[0125] Table 5 Effect of cottonseed cake powder concentration (content) on product titer

[0126] Serial number Cottonseed cake powder(g / L) Potency (g / L) 6-1 15 4.018 6-2 20 4.513 6-3 25 4.214

[0127] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in the specification of this case can be used in any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

Claims

1. A method for producing total lysergic acid by fermentation, wherein the total lysergic acid consists of lysergic acid and isolysergic acid, characterized in that: The seed liquid of Claviceps paspali is inoculated into the fermentation medium for liquid fermentation culture. The Claviceps paspali is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.41366. The fermentation medium, based on its total volume, contains 80-150 g / L of carbon source and 40-90 g / L of nitrogen source, wherein the carbon source contains 50-100 g / L of corn starch hydrolyzed by α-amylase, During the hydrolysis of corn starch by α-amylase, the weight ratio of corn starch to α-amylase is 1:0.0005-1:0.0009.

2. The method according to claim 1, characterized in that: The carbon source contains 60-80 g / L corn starch hydrolyzed by α-amylase.

3. The method according to claim 1, characterized in that During the hydrolysis of corn starch by α-amylase, the weight ratio of corn starch to α-amylase is 1:0.0006-1:0.0008.

4. The method according to claim 1, characterized in that: The nitrogen source comprises cottonseed meal, the content of which is 12-30 g / L, and / or The fermentation medium further comprises a metal salt, and the content of the metal salt is 0.2-1.0 g / L based on the total volume of the fermentation medium.

5. The method according to claim 4, characterized in that The cottonseed cake powder contained in the nitrogen source has a content of 15-25 g / L.

6. The method according to claim 4, characterized in that The metal salt is selected from ferrous sulfate or its hydrate, zinc sulfate or its hydrate, magnesium sulfate or its hydrate, or a combination thereof.

7. The method according to claim 1, characterized in that The carbon source further comprises sorbitol, succinic acid or a combination thereof, and The nitrogen source also includes yeast extract, corn steep liquor powder, ammonia water or a combination thereof.

8. The method according to claim 7, characterized in that The fermentation medium, based on its total volume, comprises the following components: Sorbitol 5-25g / L, corn starch hydrolyzed by α-amylase 50-100g / L, succinic acid 30-40g / L, yeast extract 0.4-0.6g / L, cottonseed cake powder 15-25g / L, corn steep liquor powder 10-20g / L, ammonia water 30-40g / L, ferrous sulfate heptahydrate 0.001-0.030g / L, zinc sulfate heptahydrate 0.001-0.015g / L, magnesium sulfate heptahydrate 0.6-0.8g / L, Wherein, the concentration of the ammonia water is 15-25%.

9. The method according to any one of claims 1 to 8, characterized in that: The method for preparing the fermentation medium comprises the following steps: After mixing α-amylase, corn starch and water, heat to 80-90° C. and keep warm for 28-32 minutes so that the corn starch is hydrolyzed by the α-amylase. Then, add the remaining fermentation medium components and heat to 110-130° C. for sufficient sterilization to obtain the fermentation medium.

10. The method according to any one of claims 1 to 8, characterized in that: The fermentation temperature is 21-25°C.

11. A method for producing total lysergic acid by fermentation, wherein the total lysergic acid consists of lysergic acid and isolysergic acid, characterized in that: The method comprises the steps of: (1) Activation of bacterial strains: Spread the suspension of the working cell bank of Claviceps paspali onto a PDA slant and culture at 26-30°C for 7-8 days to obtain a GI slant bacterial strain; The Claviceps paspali is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.41366. The PDA medium, based on its total volume, contains the following components: Potato dextrose agar 35-43 g / L, yeast extract 0.4-0.6 g / L, pH adjusted to 7.10-7.30 with sodium hydroxide solution; (2) Seed liquid preparation: scrape the GI slant strain obtained in step (1), homogenize it, and place it in a seed liquid 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 weight of the bacteria is ≥45 g / L; The seed liquid culture medium, based on its total volume, comprises the following components: Mannitol 45-55g / L, succinic acid 9-11g / L, soybean powder 4-6g / L, potassium dihydrogen phosphate 0.75-1.25g / L, magnesium sulfate heptahydrate 0.25-0.35g / L, ammonia water is used to adjust the pH to 5.00-5.20; (3) Fermentation culture: The seed liquid obtained in step (2) is inoculated into a fermentation medium at an inoculum amount of 8-12% (v / v) for liquid fermentation culture at a temperature of 21-25° C., a pH of 4.95-5.80, and a rotation speed of 220-260 rpm for 12-14 days to obtain a fermentation liquid; The fermentation medium, based on its total volume, comprises the following components: Sorbitol 5-25g / L, corn starch hydrolyzed by α-amylase 50-100g / L, succinic acid 30-40g / L, yeast extract 0.4-0.6g / L, ferrous sulfate heptahydrate 0.000-0.030g / L, zinc sulfate heptahydrate 0.000-0.015g / L, magnesium sulfate heptahydrate 0.6-0.8g / L, corn steep liquor powder 10-20g / L, ammonia water 30-40g / L, cottonseed cake powder 15-25g / L, wherein in 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 fermentation medium preparation process is as follows: corn starch, α-amylase and water are mixed, heated to 80-90°C, and kept warm for 28-32 minutes so that the corn starch is hydrolyzed by the α-amylase, and then the remaining fermentation medium components are added, and heated to 110-130°C for sufficient sterilization to obtain the fermentation medium, wherein the weight ratio of corn starch to α-amylase is 1:0.0005-1:0.0009.

12. A Claviceps paspali, which is deposited in China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.41366.

13. Use of Claviceps paspali according to claim 12 in the fermentation production of total lysergic acid, wherein the total lysergic acid consists of lysergic acid and isolysergic acid.

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