Method for preparing trans-aconitic acid through aspergillus terreus fermentation and application of trans-aconitic acid

Abstract

The invention provides a method for preparing trans-aconitic acid through aspergillus terreus fermentation and application of the trans-aconitic acid, and relates to the technical field of fermentation engineering. By adjusting the pH and dissolved oxygen (DO) in the fermentation process, the content of trans-aconitic acid in the fermentation liquor can be greatly increased, meanwhile, the problem that the production cost is too high due to the fact that the content of trans-aconitic acid is too low in the subsequent separation and purification of trans-aconitic acid is solved, the production cost of trans-aconitic acid is greatly reduced, and the yield of trans-aconitic acid is increased. The production steps of the trans-aconitic acid are simplified, and a simple and convenient method which can be widely popularized is provided for the production of the trans-aconitic acid.

Description

Technical Field

[0001] The present application relates to the technical field of fermentation engineering, and in particular to a method for preparing trans-aconitic acid by fermentation with Aspergillus terreus and its application. Background Art

[0002] Aconitic acid is an unsaturated tricarboxylic acid, which was originally named after the plant Aconitum, and is widely found in sugarcane, corn, and wheat. Aconitic acid is divided into cis-aconitic acid and trans-aconitic acid. Among them, trans-aconitic acid can be used as a brown planthopper antifeedant, and also has anti-insect activity such as nematicide. In addition, trans-aconitic acid can also be used to modify chitosan to improve its water solubility, or combined with glycerol and cinnamic acid to form biocompatible polyesters for skin tissue engineering, or reacted with ethylene glycol to produce biodegradable biocompatible polyesters, etc. Therefore, it is also listed as one of the top 30 potential candidate building blocks by the U.S. Department of Energy.

[0003] The traditional method for producing trans-aconitic acid is chemical production, which is mainly produced by chlorination, saponification, dechlorination, deCO2 removal or high-temperature hydrolysis of 1,1,2,3-tetracarboxyl compounds with citric acid. Not only is the process complicated, but it also produces many by-products and is costly.

[0004] Chinese patent CN110527637A discloses an engineered bacterium for preparing trans-aconitic acid. During the fermentation process, the engineered strain will produce and accumulate the target product, which will cause the pH of the fermentation process to decrease, thereby causing toxicity to the strain, thereby affecting the yield of trans-aconitic acid, resulting in the inability of the engineered strain to be promoted and applied in industry. Therefore, a practical and high-yield trans-aconitic acid production method is urgently needed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing trans-aconitic acid by fermenting Aspergillus terreus and a method for improving the yield of trans-aconitic acid prepared by fermenting Aspergillus terreus. Specifically, trans-aconitic acid is produced by a genetically engineered Aspergillus terreus strain, and the yield of trans-aconitic acid is greatly improved through fermentation process control and optimization, which has high application value.

[0006] On the one hand, the present application provides a method for preparing trans-aconitic acid by fermenting Aspergillus terreus, the method comprising the following steps: inoculating Aspergillus terreus into a fermentation medium, and using a pH regulator to control the pH to 2-4.5 during the fermentation process.

[0007] Furthermore, the pH is 2.2-3.2, and it is demonstrated in the present application that the yield of trans-aconitic acid can reach 75.21 g / L in this pH range.

[0008] During the fermentation process, a pH regulator is used to control the pH to any value of 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, and 4.5.

[0009] Furthermore, the pH regulator is urea.

[0010] This application first uses urea as a pH regulator in the fermentation of Aspergillus terreus to prepare trans-aconitic acid, proving that it can effectively regulate the pH of the fermentation process and reduce the damage to the engineered bacteria caused by too low pH. That is, using urea as a pH regulator can effectively improve the fermentation process, thereby increasing the yield of trans-aconitic acid. This is because the Aspergillus terreus engineered bacteria At-Δ cadA Urea can be used slowly as a nitrogen source to maintain the growth of microorganisms. At the same time, the ammonia slowly produced when urea is decomposed can effectively maintain the pH of the fermentation process, thereby avoiding the adverse effects of pH reduction on the engineered bacteria. The addition of other alkalis may cause local over-alkalinity in the fermentation, affecting the growth and metabolism of the bacteria.

[0011] Those skilled in the art can select the method of adding the pH adjuster and the concentration of the pH adjuster according to actual conditions, as long as the pH can be controlled within the specified range.

[0012] In a preferred embodiment, the pH during the fermentation process is adjusted by adding the pH regulator. Specifically, in the present application, according to the Aspergillus terreus engineered bacteria At-Δ cadA Based on the characteristics of pH changes during the fermentation process of preparing trans-aconitic acid, urea was selected as a pH regulator to achieve the purpose of controlling the fermentation process and increasing the yield of trans-aconitic acid.

[0013] Furthermore, the method also includes controlling the dissolved oxygen (DO) value during the fermentation process to be no higher than 70% of the initial calibration value.

[0014] Preferably, 30%-70%.

[0015] Among them, those skilled in the art can understand that the initial calibration value of dissolved oxygen (DO) refers to the DO value at this time, that is, the initial calibration value, which is set to 100% after fixing the initial tank pressure, flow rate and rotation speed of the fermenter. Thereafter, as the growth and metabolism of the microorganisms change, the DO value will show a trend of first decreasing and then increasing. In this application, by controlling the rotation speed, the DO value is not higher than 70%, that is, not higher than 70% of the initial calibration value, which is conducive to the increase of trans-aconitic acid production.

[0016] Preferably, the method further comprises the step of determining an initial calibration value of dissolved oxygen (DO).

[0017] It will be understood by those skilled in the art that after determining the initial dissolved oxygen (DO) (initial calibration value), the DO value can be controlled in real time using conventional general methods according to actual conditions. As long as the DO value can be controlled to be no higher than 70% of the initial calibration value, no specific limitation is made in the present application.

[0018] In a preferred embodiment, the ventilation volume is controlled to remain unchanged during the fermentation process, and the dissolved oxygen (DO) value is controlled by controlling the stirring speed.

[0019] Furthermore, the fermentation medium includes glucose, ammonium sulfate, sodium nitrate, copper sulfate, manganese chloride, potassium dihydrogen phosphate, yeast extract, corn germ oil, and water; Preferably, the fermentation medium comprises: 10%-15% glucose, 0.01%-0.1% ammonium sulfate, 0.02-0.08% sodium nitrate, 0.001%-0.01% copper sulfate, 0.0002%-0.0007% manganese chloride, 0.0002%-0.001% potassium dihydrogen phosphate, 0.1%-1% yeast extract powder, 0.15-0.3% corn germ oil, and the balance is water; More preferably, the fermentation medium comprises: 12% glucose, 0.08% ammonium sulfate, 0.03% sodium nitrate, 0.001% copper sulfate, 0.0003% manganese chloride, 0.0005% potassium dihydrogen phosphate, 0.1% yeast extract powder, 0.2% corn germ oil, and the balance is water.

[0020] In a preferred embodiment, the fermentation medium comprises: 120 g / L glucose, 0.8 g / L ammonium sulfate, 0.3 g / L sodium nitrate, 0.01 g / L copper sulfate, 0.003 g / L manganese chloride, 0.005 g / L potassium dihydrogen phosphate, 1 g / L yeast extract, 2 g / L corn germ oil, and the balance is water.

[0021] Among them, urea is added to the fermentation medium as a pH regulator and slow-acting nitrogen source. The ammonia released after it is slowly utilized by the strain can not only increase the pH during the fermentation process but also replenish the nitrogen source consumed by the strain during the fermentation process, thereby promoting product formation.

[0022] The fermentation medium can be prepared by a general method.

[0023] In a preferred embodiment, the fermentation medium is mixed according to the ratio and then sterilized at 121° C. for 20 min.

[0024] Preferably, the method further comprises feeding glucose.

[0025] As the fermentation process progresses, the glucose concentration in the culture medium gradually decreases, which will lead to a lack of carbon source in the later fermentation process and affect the increase in trans-aconitic acid production. Therefore, it is necessary to supplement glucose in time when the glucose content decreases to maintain the normal progress of the fermentation reaction.

[0026] Optionally, when the residual sugar level drops to 15-25 g / L, glucose may be added to maintain the glucose concentration in the fermentation broth at 15-25 g / L.

[0027] Among them, the flow addition of glucose can timely replenish the carbon source consumed during the fermentation process, so that the fermentation efficiency can be maintained at a high level for a long time.

[0028] Furthermore, the fermentation conditions include: an inoculation amount of 5%-10%, a ventilation volume of 0.2-1vvm, a starting speed of 100-400 r / min, and a tank pressure of 0.06-0.12 MPa; preferably, the fermentation conditions include: an inoculation amount of 10%, a ventilation volume of 0.4 vvm, a starting speed of 220 r / min, a tank pressure of 0.1 MPa, and the DO is controlled to be no higher than 70% by adjusting the speed.

[0029] Among them, the inoculation amount can be any value among 5%, 6%, 7%, 8%, 9%, and 10%.

[0030] Here, the inoculum size refers to the volume of seed solution transferred in / (volume of seed solution + volume of fermentation solution).

[0031] The ventilation rate can be any value among 0.2 vvm, 0.3 vvm, 0.4 vvm, 0.5 vvm, 0.6 vvm, 0.7 vvm, 0.8 vvm, 0.9 vvm, and 1 vvm.

[0032] The starting speed can be any value among 100 r / min, 200 r / min, 220 r / min, 300 r / min, and 400 r / min.

[0033] The tank pressure can be any value among 0.06 MPa, 0.07 MPa, 0.08 MPa, 0.09 MPa, 0.1 MPa, 0.11 MPa, and 0.12 MPa.

[0034] In a preferred embodiment, the rotation speed refers to the rotation speed of the initial fermentation, and the rotation speed during the fermentation process is adjusted according to the DO value.

[0035] Furthermore, the method also includes the step of inoculating Aspergillus terreus spores into a seed liquid culture medium to carry out Aspergillus terreus seed liquid culture to obtain Aspergillus terreus seed liquid.

[0036] In a preferred embodiment, the Aspergillus terreus seed liquid can be cultured by primary fermentation.

[0037] Preferably, the conditions for culturing the Aspergillus terreus seed solution include: controlling the spore inoculation amount to 5.1*10 6 -3.8*10 7 spores / mL, ventilation volume 0.2-1vvm, rotation speed 100-400 r / min, tank pressure 0.06-0.12 Mpa, culture 18-30 h; preferably, the spore inoculation amount is 1.5*10 7 spores / mL, ventilation volume 0.5 vvm, tank pressure 0.12 Mpa, initial speed 240 r / min, culture 24 h; preferably, the seed liquid culture medium comprises: glucose 7%-10%, potassium dihydrogen phosphate 0.002%-0.01%, corn steep liquor dry powder 0.1%-1%, yeast extract powder 0.1%-1%, magnesium sulfate 0.04%-0.08%, ammonium sulfate 0.2%-0.6%, ferrous sulfate 0.001%-0.01%, copper sulfate 0.001%-0.01%, manganese chloride 0.0002%-0.0007%, and the balance is water. More preferably, glucose 10%, potassium dihydrogen phosphate 0.005%, corn steep liquor powder 0.1%, yeast extract powder 0.2%, magnesium sulfate 0.06%, ammonium sulfate 0.3%, ferrous sulfate 0.001%, copper sulfate 0.002%, manganese chloride 0.0005%, and the balance is water.

[0038] Furthermore, the fermentation is carried out in a 30 or 100 L fermenter; preferably, 100 L.

[0039] Furthermore, the Aspergillus terreus is transformed, and the transformation includes: silencing and / or weakening the gene encoding aconitate decarboxylase ( cadA ) genes.

[0040] Preferably, the transformation comprises: knocking out the gene encoding Aspergillus terreus aconitate decarboxylase ( cadA ) genes, ku80 Genes and pyr Gene.

[0041] More preferably, the Aspergillus terreus is the Aspergillus terreus engineered strain At-Δ disclosed in patent CN110527637A. cadA .

[0042] In a preferred embodiment, a method for preparing trans-aconitic acid by fermentation using Aspergillus terreus, the method comprising: inoculating Aspergillus terreus seed liquid into a fermentation medium at an inoculation rate of 5%-10%, controlling the ventilation volume to 0.2-1vvm, the initial rotation speed to 100-400 r / min, the tank pressure to 0.06-0.12 MPa, and using a pH regulator to control the pH to 2-4.5 during the fermentation process, controlling the dissolved oxygen (DO) value to no more than 70% of the initial calibration value, and ending the fermentation when the trans-aconitic acid content no longer increases, thereby obtaining a fermentation liquid containing trans-aconitic acid.

[0043] In a preferred embodiment, a method for preparing trans-aconitic acid by fermentation using Aspergillus terreus comprises the following steps: Step 1: Inoculate Aspergillus terreus spores into a seed liquid culture medium for Aspergillus terreus seed liquid culture. The conditions for Aspergillus terreus seed liquid culture include: a spore inoculation amount of 5.1*10 6 -3.8*10 7 spores / mL, ventilation volume 0.2-1vvm, rotation speed 100-400 r / min, tank pressure 0.06-0.12Mpa, culture 18-30 h, obtain Aspergillus terreus seed solution; Step 2, inoculating Aspergillus terreus seed liquid into the fermentation medium at an inoculation rate of 5%-10%, controlling the ventilation volume to 0.2-1vvm, the initial rotation speed to 100-400 r / min, the tank pressure to 0.06-0.12 MPa, and using a pH regulator to control the pH to 2-4.5 during the fermentation process, controlling the dissolved oxygen (DO) value to be no higher than 70% of the initial calibration value, and ending the fermentation when the trans-aconitic acid content no longer increases, thereby obtaining a fermentation liquid containing trans-aconitic acid.

[0044] The seed liquid culture medium comprises: Glucose 7-10%, potassium dihydrogen phosphate 0.002%-0.01%, corn steep liquor powder 0.1-1%, yeast extract powder 0.1%-1%, magnesium sulfate 0.04%-0.08%, ammonium sulfate 0.2%-0.6%, ferrous sulfate 0.001%-0.01%, copper sulfate 0.001%-0.01%, manganese chloride 0.0002%-0.0007%, and the balance is water.

[0045] The fermentation medium comprises: Glucose 10%-15%, ammonium sulfate 0.01%-0.1%, sodium nitrate 0.02-0.08%, copper sulfate 0.001%-0.01%, manganese chloride 0.0002-0.0007%, potassium dihydrogen phosphate 0.0002%-0.001%, yeast extract powder 0.1%-1%, corn germ oil 0.15-0.3%, and the balance is water.

[0046] In a preferred embodiment, the fermentation time is 334 h, and the yield of trans-aconitic acid in the fermentation broth is 75.21 g / L.

[0047] On the other hand, the present application also provides a fermentation broth containing trans-aconitic acid prepared by the method; preferably, the content of trans-aconitic acid in the fermentation broth is greater than 32 g / L; more preferably, greater than 75 g / L.

[0048] Preferably, the yield of trans-aconitic acid in the fermentation broth is 75.21 g / L. In the present application, the content of trans-aconitic acid in the fermentation broth is detected by liquid chromatography, and the purity is 100%, which is 717.5% higher than the yield in the prior art.

[0049] On the other hand, the present application also provides the use of the method in preparing trans-aconitic acid and / or increasing the yield of trans-aconitic acid.

[0050] The method described in the present application can achieve an increase in the yield of trans-aconitic acid, greatly improving the production efficiency. In addition, the concentration of trans-aconitic acid in the fermentation broth obtained by the method of the present application is increased, saving the subsequent purification steps, greatly reducing the production cost, and is of great significance to actual production.

[0051] The present invention has the following beneficial effects: The present invention can greatly increase the content of trans-aconitic acid in the fermentation broth by adjusting the pH and DO (dissolved oxygen) in the process of producing trans-aconitic acid by Aspergillus terreus, and solves the problem of excessively high production cost caused by too low content of trans-aconitic acid in subsequent separation and purification of trans-aconitic acid, greatly reduces the production cost of trans-aconitic acid, simplifies the production steps of trans-aconitic acid, and provides a simple method that can be widely promoted for the production of trans-aconitic acid.

[0052] By using this method for fermentation in a 100 L fermenter, the yield of trans-aconitic acid can be increased to 75.21 g / L, which is 717.5% higher than the 9.2 g / L trans-aconitic acid yield reported in patent CN110527637A and 147.56% higher than the control group, greatly reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings: Figure 1 It is the curve diagram of trans-aconitic acid fermentation yield change; Figure 2 This is the pH change curve of trans-aconitic acid fermentation; Figure 3This is the DO variation curve of trans-aconitic acid fermentation; Figure 4 It is a curve chart of the change of itaconic acid content in the test example. DETAILED DESCRIPTION

[0054] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0055] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0056] The Aspergillus terreus used in Examples 1-4 and Comparative Example 1 is an engineered strain of Aspergillus terreus At-Δ cadA Disclosed in the Chinese patent document with publication number CN110527637A.

[0057] The construction method includes: knocking out the gene encoding Aspergillus terreus aconitate decarboxylase ( cadA ) genes, ku80 Genes and pyr Gene.

[0058] The Aspergillus terreus used in Example 5 is CICC40205, which was purchased from the China Industrial Microbiological Culture Collection Center with a collection number of CICC40205.

[0059] When the embodiment gives numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention are identical with the meanings commonly understood by those skilled in the art. Except the specific method, equipment, material used in the embodiment, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment and material of the prior art similar or equivalent to the method, equipment, material in the embodiment of the present invention can also be used to realize the present invention. If not specifically stated, in the following embodiments, reagents used or instruments are not indicated by manufacturers, and are conventional products that can be obtained by commercial purchase.

[0060] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in microbiology, biochemistry, analytical chemistry, cell culture, and related fields conventional in the technical field.

[0061] The detection methods of itaconic acid and trans-aconitic acid are both liquid chromatography, and the operation methods are the same. The main difference is that the standard products are different. The specific detection methods can be referred to the literature Li et al. Microb Cell Fact (2020) 19:174 ; pH was measured using a pH meter.

[0062] Aspergillus terreus engineered strain At-Δ cadA The seed liquid culture method includes: inoculating Aspergillus terreus spores into a seed liquid culture medium for Aspergillus terreus seed liquid culture. The conditions for Aspergillus terreus seed liquid culture include: preparing a seed culture medium at a volume ratio of 60% in a 30 L fermentation tank, and the seed culture inoculation amount is 1.5*10 7 spores / mL, ventilation volume 0.5 vvm, tank pressure 0.12 MPa, rotation speed 240 r / min, and culture for 24 h to obtain Aspergillus terreus seed solution.

[0063] Aspergillus terreus engineered strain At-Δ cadA The seed liquid culture medium includes: 10% glucose, 0.005% potassium dihydrogen phosphate, 0.1% corn steep liquor powder, 0.2% yeast extract powder, 0.06% magnesium sulfate, 0.3% ammonium sulfate, 0.001% ferrous sulfate, 0.002% copper sulfate, 0.0005% manganese chloride, and the balance is water.

[0064] Cultivation method of Aspergillus terreus CICC40205 seed solution: prepare seed culture medium at a volume ratio of 60% in a 30 L seed tank, and the seed culture inoculation amount is 1.5*10 7 spores / mL, ventilation volume was controlled at 0.5 vvm, rotation speed was 240 r / min, tank pressure was 0.12 MPa, and culture was carried out for 24 h.

[0065] The seed liquid culture medium of Aspergillus terreus CICC40205 includes: 120 g / L glucose, 0.6 g / L magnesium sulfate, 4 g / L ammonium sulfate, 0.001 g / L dipotassium hydrogen phosphate, 0.01 g / L zinc sulfate, 0.4 g / L ferrous sulfate, 0.05 g / L copper sulfate, 3 g / L corn steep liquor, and the balance is water.

[0066] Example 1 A method for preparing trans-aconitic acid by fermentation with Aspergillus terreus, comprising: Step 1. Prepare fermentation medium in a 30 L fermenter at a volume ratio of 60%, including 120 g / L glucose, 0.8 g / L ammonium sulfate, 0.3 g / L sodium nitrate, 0.01 g / L copper sulfate, 0.003 g / L manganese chloride, 0.005 g / L potassium dihydrogen phosphate, 1 g / L yeast extract powder, 2 g / L corn germ oil, and the balance is water, and sterilize at 121°C for 20 min to obtain the fermentation medium.

[0067] Step 2: Transfer the Aspergillus terreus seed liquid into the fermentation tank at an inoculation rate of 10%. The fermentation control conditions are: control the ventilation volume to 0.4 vvm, the rotation speed to 380 r / min, and the tank pressure to 0.1 MPa. During the fermentation process, urea is added to control the pH value of the fermentation process to 2.0-4.5 until the end of fermentation, and glucose is added when the residual sugar is 15-25 g / L to maintain the glucose concentration at 15-25 g / L. During the fermentation process, the ventilation volume, rotation speed and tank pressure are kept unchanged to allow the DO to change naturally. Fermentation is stopped when the trans-aconitic acid content no longer increases. Finally, the trans-aconitic acid yield can reach 34 g / L after 120 h of fermentation.

[0068] Example 2 A method for preparing trans-aconitic acid by fermentation with Aspergillus terreus, comprising: Step 1. Prepare fermentation medium in a 100 L fermenter at a volume ratio of 60%, including 120 g / L glucose, 0.8 g / L ammonium sulfate, 0.3 g / L sodium nitrate, 0.01 g / L copper sulfate, 0.005 g / L potassium dihydrogen phosphate, 0.003 g / L manganese chloride, 1 g / L yeast extract powder, 2 g / L corn germ oil, and the balance is water, and sterilize at 121°C for 20 min to obtain the fermentation medium.

[0069] Step 2: Transfer the Aspergillus terreus seed liquid into the fermentation tank at an inoculation rate of 10%. The fermentation control conditions are: control the ventilation volume to 0.4 vvm, the rotation speed to 220 r / min, and the tank pressure to 0.1 MPa. During the fermentation process, urea is added to control the pH value of the fermentation process to 2.2-3.2 until the end of fermentation. When the residual sugar is 15-25 g / L, glucose is added to maintain the glucose concentration at 15-25 g / L. During the fermentation process, the ventilation volume, rotation speed and tank pressure are kept unchanged to allow the DO to change naturally. Fermentation is stopped when the trans-aconitic acid content no longer increases. After 272 h of fermentation, the trans-aconitic acid yield is 60.22 g / L.

[0070] Example 3 A method for preparing trans-aconitic acid by fermentation with Aspergillus terreus, comprising: Step 1. Prepare fermentation medium in a 100 L fermenter at a volume ratio of 60%, including 120 g / L glucose, 0.8 g / L ammonium sulfate, 0.3 g / L sodium nitrate, 0.01 g / L copper sulfate, 0.005 g / L potassium dihydrogen phosphate, 0.003 g / L manganese chloride, 1 g / L yeast extract powder, 2 g / L corn germ oil, and the balance is water, and sterilize at 121°C for 20 min to obtain the fermentation medium.

[0071] Step 2, transfer the Aspergillus terreus seed liquid into the fermentation tank at an inoculation rate of 10%, and the fermentation control conditions are: control the ventilation volume to 0.4 vvm, the initial speed to 220 r / min, the tank pressure to 0.1 MPa, and control the pH value of the fermentation process to 2.2-3.2 by adding urea until the end of fermentation, and when the residual sugar drops to 15-25 g / L, add glucose to maintain its concentration at 15-25 g / L, and control the DO value by controlling the stirring speed while keeping the ventilation volume unchanged so that it is not higher than 70% of the initial calibration value. Fermentation is stopped when the trans-aconitic acid content no longer increases. After 334 h of fermentation, the trans-aconitic acid yield is 75.21 g / L.

[0072] Example 4 A method for preparing trans-aconitic acid by fermentation with Aspergillus terreus, comprising: Step 1. Prepare fermentation medium in a 100 L fermenter at a volume ratio of 60%, including 120 g / L glucose, 0.8 g / L ammonium sulfate, 0.3 g / L sodium nitrate, 0.01 g / L copper sulfate, 0.005 g / L potassium dihydrogen phosphate, 0.003 g / L manganese chloride, 1 g / L yeast extract powder, 2 g / L corn germ oil, and the balance is water, and sterilize at 121°C for 20 min to obtain the fermentation medium.

[0073] Step 2: Transfer the Aspergillus terreus seed liquid into the fermentation tank at an inoculation rate of 10%. The fermentation control conditions are: ventilation volume 0.4vvm, initial speed 220 r / min, tank pressure 0.1 MPa, and when the residual sugar drops to 15-25 g / L, glucose is added to maintain its concentration at 15-25 g / L. During the fermentation process, ammonia water is added to adjust the fermentation pH to 2.2-3.2. The DO value is controlled to be no higher than 70% of the initial value by controlling the stirring speed. Fermentation is stopped when the trans-aconitic acid content no longer increases. After 179 h of fermentation, 41.18 g / L of acid is produced.

[0074] Comparative Example 1 A method for preparing trans-aconitic acid by fermentation with Aspergillus terreus, comprising: Step 1. Prepare fermentation medium in a 100 L fermenter at a volume ratio of 60%, including 120 g / L glucose, 0.8 g / L ammonium sulfate, 0.3 g / L sodium nitrate, 0.01 g / L copper sulfate, 0.005 g / L potassium dihydrogen phosphate, 0.003 g / L manganese chloride, 1 g / L yeast extract powder, 2 g / L corn germ oil, and the balance is water, and sterilize at 121°C for 20 min to obtain the fermentation medium.

[0075] Step 2: Transfer the Aspergillus terreus seed liquid into the fermentation tank at an inoculation rate of 10%. The fermentation control conditions are: control the ventilation volume to 0.4 vvm, the rotation speed to 220 r / min, and the tank pressure to 0.1 MPa. Keep the ventilation volume, rotation speed and tank pressure unchanged during the fermentation process to allow the pH and DO to change naturally. When the residual sugar drops to 15-25 g / L, add glucose to maintain its concentration at 15-25 g / L. Stop the fermentation when the trans-aconitic acid content no longer increases. After 180 h of fermentation, the acid production is 30.38 g / L.

[0076] Conclusion analysis: Monitor the changes of trans-aconitic acid content, pH and DO over time during the fermentation process of the above Examples 1-4 and Comparative Example 1 to obtain Tables 1, 2 and 3. Figure 1 , we can get from Table 2 Figure 2 , Table 3 is plotted to obtain Figure 3 .

[0077] Table 1 Changes in trans-aconitic acid content

[0078] Table 2 pH changes

[0079] Table 3 DO changes

[0080] From Table 1, Table 2, Table 3, Figure 1 , Figure 2 , Figure 3 The results show that The results of Example 1 preliminarily prove that the pH value of the fermentation process can be regulated by adding urea, thereby increasing the yield of trans-aconitic acid in the fermentation broth. The results of Example 1 compared with those of Example 2 also prove that the scale of the fermentation tank and the specific control range of the pH value have a certain influence on the yield of trans-aconitic acid, and preferably a 100 L fermentation tank and a pH value of 2.2-3.2 are used for fermentation to prepare trans-aconitic acid.

[0081] The difference between Example 2 and Example 3 is only whether DO is controlled, which proves that DO control has a great influence on the production yield of trans-aconitic acid. In Example 3, the ventilation volume is controlled unchanged, and the stirring speed is controlled to control the DO value to be no higher than 70% of the initial calibration value, so that the environment is more conducive to the fermentation of trans-aconitic acid by Aspergillus terreus, thereby increasing the yield of trans-aconitic acid.

[0082] The difference between Example 4 and Example 3 is that different pH regulators are used for pH adjustment. Specifically, Example 3 uses urea as the pH regulator, while Example 4 uses ammonia water as the pH regulator. The trans-aconitic acid yield in Example 4 is lower than that in Example 3, which proves that not all pH regulators can increase the trans-aconitic acid yield. On the one hand, the Aspergillus terreus engineered bacteria At-Δ cadA Urea can be used slowly as a nitrogen source to maintain the growth of microorganisms, which is consistent with the growth law that the growth process of Aspergillus terreus requires a low carbon-nitrogen ratio, while the fermentation and acid production process requires a high carbon-nitrogen ratio. On the other hand, the ammonia produced when urea is decomposed can make the pH of the fermentation process more stable, thereby avoiding the adverse effects of the decrease in pH on the engineered bacteria. In addition, it was found in this embodiment that controlling the pH at 2.2-3.2 can maximize the production of trans-aconitic acid.

[0083] The difference between Comparative Example 1 and Example 2 is that the fermentation pH is not regulated, which proves that the trans-aconitic acid yield is low when the pH is not regulated during the fermentation process.

[0084] In summary, in this example, urea was used as a pH regulator for the first time to control the pH of the fermentation process of trans-aconitic acid, and the fermentation yield of trans-aconitic acid was greatly improved by controlling the pH and DO values, providing support for the expanded production and commercial production of trans-aconitic acid.

[0085] The optimal fermentation conditions provided in this embodiment are: In a 100 L fermenter, 1.5*10 7The seed solution was prepared with spores / mL and inoculated into the fermentation tank at a 10% inoculation rate. The ventilation volume was controlled at 0.4 vvm, the initial rotation speed was 220 r / min, the tank pressure was 0.1 MPa, and urea was added during the fermentation process to control the fermentation pH to 2.2-3.2. When the residual sugar was 15-25 g / L, glucose was added to keep its concentration at 15-25 g / L. While keeping the ventilation volume unchanged, the stirring speed was controlled to make the dissolved oxygen (DO) value not higher than 70% of the initial calibration value. The fermentation was terminated when the trans-aconitic acid content no longer increased, and the fermentation liquid containing trans-aconitic acid was obtained.

[0086] Fermentation medium: 120 g / L glucose, 0.8 g / L ammonium sulfate, 0.3 g / L sodium nitrate, 0.01 g / L copper sulfate, 0.005 g / L potassium dihydrogen phosphate, 0.003 g / L manganese chloride, 1 g / L yeast extract powder, 2 g / L corn germ oil, and the balance is water. The fermentation medium is sterilized at 121°C for 20 min.

[0087] Example 5 In order to verify whether urea can regulate pH and control DO to increase the yield of other organic acids, in this example, two methods, natural fermentation and urea addition with DO control, were used to ferment and produce itaconic acid.

[0088] Test Example 1 This example provides a method for preparing itaconic acid by fermentation with Aspergillus terreus, comprising: Step 1. Prepare fermentation medium in a 100 L fermenter at a volume ratio of 60%, including 150 g / L glucose, 0.6 g / L magnesium sulfate, 3 g / L ammonium sulfate, 0.01 g / L diammonium hydrogen phosphate, 0.01 g / L zinc sulfate, 0.4 g / L ferrous sulfate, 2 g / L corn steep liquor, 0.05 g / L cysteine, and the balance being water. Sterilize at 121°C for 20 min to obtain fermentation medium.

[0089] Step 2: Transfer the Aspergillus terreus seed liquid into the fermentation tank at an inoculation rate of 10%. The fermentation control conditions are as follows: control the ventilation volume to 0.4 vvm, the rotation speed to 220 r / min, and the tank pressure to 0.1 MPa. Keep the ventilation volume, rotation speed and tank pressure unchanged during the fermentation process to allow the pH and DO to change naturally. The fermentation is terminated when the glucose content is less than 0.5 g / L. The itaconic acid content is 79.68 g / L at 71 h of fermentation.

[0090] Test Example 2 This example provides a method for preparing itaconic acid by fermentation with Aspergillus terreus, comprising: Step 1. Prepare fermentation medium in a 100 L fermenter at a volume ratio of 60%, including 150 g / L glucose, 0.6 g / L magnesium sulfate, 3 g / L ammonium sulfate, 0.01 g / L diammonium hydrogen phosphate, 0.01 g / L zinc sulfate, 0.4 g / L ferrous sulfate, 2 g / L corn steep liquor, 0.05 g / L cysteine, and the balance being water. Sterilize at 121°C for 20 min to obtain fermentation medium.

[0091] Step 2: Transfer the Aspergillus terreus seed liquid into the fermentation tank at an inoculation rate of 10%. The fermentation control conditions are as follows: control the ventilation volume to 0.4 vvm, the rotation speed to 220 r / min, the tank pressure to 0.1 MPa, and fermentation by adding urea superimposed on DO control (adding urea to maintain pH 2.2-3.2 while controlling DO not higher than 70% of the initial DO by the rotation speed). The fermentation is terminated when the glucose content is less than 0.5 g / L. The itaconic acid content is 74.14 g / L at 71 h of fermentation.

[0092] The specific fermentation process of Experimental Example 1 and Experimental Example 2 is shown in Table 4. Figure 4 .

[0093] Table 4 Statistics of itaconic acid content

[0094] The results are shown in Table 4 and Figure 4 As shown, after adding urea to control pH and DO values, the yield of itaconic acid did not increase but slightly decreased, proving that the method of the present application is designed for trans-aconitic acid and is not applicable to itaconic acid.

[0095] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for preparing trans-aconitic acid by fermentation with Aspergillus terreus, characterized in that: The method comprises the following steps: inoculating Aspergillus terreus into a fermentation medium, and using a pH regulator to control the pH value to be 2-4.5 during the fermentation process, wherein the pH regulator is urea.

2. The method according to claim 1, characterized in that The method also includes controlling the dissolved oxygen value in the fermentation process to be no higher than 70% of the initial calibration value.

3. The method according to claim 1, characterized in that The fermentation medium comprises glucose, ammonium sulfate, sodium nitrate, copper sulfate, manganese chloride, potassium dihydrogen phosphate, yeast extract powder and corn germ oil.

4. The method according to claim 1, characterized in that: The fermentation conditions include: inoculation amount of 5%-10%, ventilation volume of 0.2-1vvm, initial rotation speed of 100-400 r / min, and tank pressure of 0.06-0.12 MPa.

5. The method according to claim 1, characterized in that The method further comprises inoculating the Aspergillus terreus spores into a seed liquid culture medium to carry out Aspergillus terreus seed liquid culture.

6. The method according to claim 1, characterized in that The fermentation was carried out in a 30 or 100 L fermentor.

7. The method according to claim 1, characterized in that The Aspergillus terreus is transformed, and the transformation includes: silencing and / or weakening the gene encoding aconitate decarboxylase.

8. A fermentation broth containing trans-aconitic acid prepared by the method according to any one of claims 1 to 7.

9. The fermentation broth according to claim 8, characterized in that The content of trans-aconitic acid in the fermentation broth is greater than 32 g / L.

10. Use of the method according to any one of claims 1 to 7 in preparing trans-aconitic acid and / or increasing the yield of trans-aconitic acid.

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