Method for preparing trans-aconitic acid by using aspergillus terreus fermentation and application thereof

Abstract

This invention provides a method for preparing trans-aconitic acid using Aspergillus terrestris fermentation and its application, relating to the field of fermentation engineering technology. By adjusting the pH and dissolved oxygen (DO) during the fermentation process, this invention can significantly increase the trans-aconitic acid content in the fermentation broth. Simultaneously, it solves the problem of excessively high production costs caused by low trans-aconitic acid content during subsequent separation and purification, greatly reducing the production cost of trans-aconitic acid, simplifying the production steps, and providing a simple and widely applicable method for trans-aconitic acid production.

Description

Technical Field

[0001] This application relates to the field of fermentation engineering technology, and in particular to a method for preparing trans-aconitic acid by fermentation of Aspergillus terrestris and its application. Background Technology

[0002] Aconitic acid is an unsaturated tricarboxylic acid, first discovered and named after the plant Aconitum carmichaelii, and widely found in sugarcane, corn, and wheat. Aconitic acid is further divided into cis-aconitic acid and trans-aconitic acid. Trans-aconitic acid can be used as a predator for brown planthoppers and also possesses nematicidal and other insecticidal activities. Furthermore, trans-aconitic acid can 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. Therefore, it has been listed by the U.S. Department of Energy as one of the top 30 potential building blocks.

[0003] Traditional trans-aconitic acid is produced by chemical methods, mainly through chlorination, saponification, dechlorination, and CO2 removal of 1,1,2,3-tetracarboxylic acid compounds, or by high-temperature hydrolysis with citric acid. This process is not only complex but also produces many byproducts and is costly.

[0004] Chinese patent CN110527637A discloses an engineered strain for preparing trans-aconitine. However, during fermentation, the pH of this strain decreases due to the production and accumulation of the target product, causing toxicity to the strain and affecting the yield of trans-aconitine, thus preventing its industrial application. Therefore, a practical and feasible method for producing high-yield trans-aconitine is urgently needed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing trans-aconic acid using Aspergillus terrestris fermentation and a method for increasing the yield of trans-aconic acid prepared by Aspergillus terrestris fermentation. Specifically, trans-aconic acid is produced using genetically engineered Aspergillus terrestris strains. Through fermentation process control and optimization, the yield of trans-aconic acid is significantly increased, which has high application value.

[0006] On the one hand, this application provides a method for preparing trans-aconic acid by fermentation of Aspergillus terrestris, the method comprising the following steps: inoculating Aspergillus terrestris into a fermentation medium, and using a pH adjuster to control the pH to 2-4.5 during the fermentation process.

[0007] Furthermore, at a pH range of 2.2-3.2, this application demonstrates that the yield of trans-aconitic acid can reach 75.21 g / L within this pH range.

[0008] During fermentation, a pH adjuster is used to control the pH to any one of the following values: 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 adjuster is urea.

[0010] This application is the first to use urea as a pH adjuster in the Aspergillus terreus fermentation process for preparing trans-aconitic acid, demonstrating its ability to effectively regulate the pH during fermentation and reduce damage to the engineered bacteria caused by excessively low pH. In other words, using urea as a pH adjuster can effectively improve the fermentation process, thereby increasing the yield of trans-aconitic acid. This is because the Aspergillus terreus engineered bacteria At-Δ used in the fermentation process of this application... cadA It can slowly utilize urea 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, thus avoiding the adverse effects of pH reduction on engineered bacteria. In contrast, adding other alkalis may cause localized excessive alkalinity in the fermentation process, affecting the growth and metabolism of the bacteria.

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

[0012] In a preferred embodiment, the pH during fermentation is adjusted by adding the pH adjuster. Specifically, in this application, the pH is adjusted according to the engineered Aspergillus terreus strain At-Δ. cadA The characteristics of pH changes during the fermentation preparation of trans-aconitic acid were investigated, and urea was selected as a pH regulator to control the fermentation process and increase the yield of trans-aconitic acid.

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

[0014] Preferably, 30%-70%.

[0015] As those skilled in the art will understand, the initial calibration value of dissolved oxygen (DO) refers to the DO value set at 100% after fixing the initial tank pressure, flow rate, and rotation speed of the fermenter. Subsequently, the DO value will show a trend of first decreasing and then increasing as microbial growth and metabolism change. In this application, by controlling the rotation speed to ensure that the DO value does not exceed 70%, i.e., not exceeding 70% of the initial calibration value, it is beneficial to increase the yield of trans-aconitic acid.

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

[0017] Those skilled in the art will understand that after determining the initial dissolved oxygen (DO) (initial calibration value), the DO value can be controlled in real time using conventional and common methods according to the actual situation, as long as the DO value can be controlled to not exceed 70% of the initial calibration value. This application will not make any specific limitations on this.

[0018] In a preferred embodiment, the aeration rate is kept constant 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;

[0020] 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, 0.15%-0.3% corn germ oil, and the balance being water;

[0021] 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, 0.2% corn germ oil, and the remainder being water.

[0022] 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 remainder being water.

[0023] Urea is added to the fermentation medium as a pH regulator and a slow-acting nitrogen source. The ammonia released after the strain slowly utilizes it can not only increase the pH during the fermentation process, but also replenish the nitrogen source consumed by the strain during fermentation, thus promoting product formation.

[0024] The fermentation medium can be prepared using a general method.

[0025] In a preferred embodiment, the fermentation medium is mixed according to the formula and then sterilized at 121°C for 20 min.

[0026] Preferably, the method further includes the addition of glucose.

[0027] As the fermentation process progresses, the glucose concentration in the culture medium gradually decreases, which can lead to a lack of carbon source in the later stages of fermentation and affect the yield of trans-aconitine. Therefore, it is necessary to replenish glucose in a timely manner when the glucose content decreases in order to maintain the normal progress of the fermentation reaction.

[0028] Optionally, glucose can be added when the residual sugar drops to 15-25 g / L to maintain the glucose concentration in the fermentation broth at 15-25 g / L.

[0029] Among them, the addition of glucose can replenish the carbon source consumed during fermentation in a timely manner, so that the fermentation efficiency can be maintained at a high level for a long time.

[0030] Further, the fermentation conditions include: an inoculum size of 5%-10%, an aeration rate of 0.2-1 vvm, an initial rotation speed of 100-400 r / min, and a tank pressure of 0.06-0.12 MPa; preferably, the fermentation conditions include: an inoculum size of 10%, an aeration rate of 0.4 vvm, an initial rotation speed of 220 r / min, and a tank pressure of 0.1 MPa, with the DO controlled to not exceed 70% by adjusting the rotation speed.

[0031] The inoculation rate can be any value among 5%, 6%, 7%, 8%, 9%, and 10%.

[0032] Here, the inoculum volume refers to the volume of seed liquid transferred in divided by (seed liquid volume + fermentation liquid volume).

[0033] The ventilation volume 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.

[0034] The starting speed can be any value among 100 r / min, 200 r / min, 220 r / min, 300 r / min, and 400 r / min.

[0035] 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.

[0036] In a preferred embodiment, the rotation speed refers to the initial fermentation speed, and the rotation speed is adjusted according to the DO value during fermentation.

[0037] Furthermore, the method also includes the step of inoculating Aspergillus terreus spores into a seed culture medium to obtain Aspergillus terreus seed culture.

[0038] In a preferred embodiment, the Aspergillus terreus seed liquid can be cultured using a primary fermentation method.

[0039] Preferably, the conditions for culturing the Aspergillus terrestris seed culture include: controlling the spore inoculation amount to 5.1 × 10⁻⁶ spores in a 30 L fermenter. 6 -3.8×10 7 1 spore / mL, aeration rate 0.2-1 vvm, rotation speed 100-400 r / min, tank pressure 0.06-0.12 MPa, culture time 18-30 h; preferably, the spore inoculum size is 1.5 × 10⁶ spores / mL. 7 1 spore / mL, aeration rate of 0.5 vvm, tank pressure of 0.12 MPa, initial rotation speed of 240 r / min, and incubation for 24 h; preferably, the seed culture medium comprises: 7%-10% glucose, 0.002%-0.01% potassium dihydrogen phosphate, 0.1%-1% corn steep liquor powder, 0.1%-1% yeast extract, 0.04%-0.08% magnesium sulfate, 0.2%-0.6% ammonium sulfate, 0.001%-0.01% ferrous sulfate, 0.001%-0.01% copper sulfate, 0.0002%-0.0007% manganese chloride, with the balance being water. More preferably, the composition is 10% glucose, 0.005% potassium dihydrogen phosphate, 0.1% corn steep liquor powder, 0.2% yeast extract, 0.06% magnesium sulfate, 0.3% ammonium sulfate, 0.001% ferrous sulfate, 0.002% copper sulfate, 0.0005% manganese chloride, with the balance being water.

[0040] Furthermore, the fermentation is carried out in a 30 or 100 L fermenter; preferably, 100 L.

[0041] Furthermore, the *Aspergillus terrestris* is modified, the modification including: silencing and / or weakening the encoding of aconitine decarboxylase (…). cadA ) genes.

[0042] Preferably, the modification includes: knocking out the encoding of Aspergillus terrestris cis-aconitate decarboxylase (… cadA ) genes, ku80 Genes and pyrG Gene.

[0043] More preferably, the Aspergillus terreus is the engineered Aspergillus terreus strain At-Δ disclosed in patent CN110527637A. cadA .

[0044] In a preferred embodiment, a method for preparing trans-aconitic acid using Aspergillus terrestris fermentation is provided. The method includes: inoculating Aspergillus terrestris seed liquid into the fermentation medium at an inoculation rate of 5%-10%; controlling the aeration rate at 0.2-1 vvm; the initial rotation speed at 100-400 r / min; the tank pressure at 0.06-0.12 MPa; and controlling the pH to 2-4.5 using a pH adjuster during fermentation, controlling the dissolved oxygen (DO) value to not exceed 70% of the initial calibration value. Fermentation is terminated when the trans-aconitic acid content no longer increases, thereby obtaining a fermentation broth containing trans-aconitic acid.

[0045] In a preferred embodiment, a method for preparing trans-aconitic acid using Aspergillus terrestris fermentation includes the following steps:

[0046] Step 1: Inoculate Aspergillus terreus spores into seed culture medium for Aspergillus terreus seed culture. The conditions for Aspergillus terreus seed culture include: spore inoculation amount of 5.1 × 10⁻⁶ spores. 6 -3.8×10 7 The seed culture of Aspergillus terreus was obtained by incubating at a concentration of 1 spore / mL, an aeration rate of 0.2-1 vvm, a rotation speed of 100-400 r / min, a tank pressure of 0.06-0.12 MPa, and a culture time of 18-30 h.

[0047] Step 2: Inoculate Aspergillus terrestris seed liquid into the fermentation medium at an inoculation rate of 5%-10%. Control the aeration rate at 0.2-1 vvm, the initial rotation speed at 100-400 r / min, and the tank pressure at 0.06-0.12 MPa. During the fermentation process, use a pH adjuster to control the pH at 2-4.5 and control the dissolved oxygen (DO) value to not exceed 70% of the initial calibration value. Stop the fermentation when the trans-aconitine content no longer increases, and you will get a fermentation broth containing trans-aconitine.

[0048] The seed culture medium includes:

[0049] The ingredients are: 7-10% glucose, 0.002%-0.01% potassium dihydrogen phosphate, 0.1-1% corn steep liquor powder, 0.1%-1% yeast extract, 0.04%-0.08% magnesium sulfate, 0.2%-0.6% ammonium sulfate, 0.001%-0.01% ferrous sulfate, 0.001%-0.01% copper sulfate, 0.0002%-0.0007% manganese chloride, with the balance being water.

[0050] The fermentation medium includes:

[0051] The ingredients are: 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 0.1%-1%, corn germ oil 0.15%-0.3%, and the balance being water.

[0052] 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.

[0053] On the other hand, this application also provides a fermentation broth containing trans-aconitine prepared by the method; preferably, the trans-aconitine content in the fermentation broth is greater than 32 g / L; more preferably, it is greater than 75 g / L.

[0054] Preferably, the yield of trans-aconitine in the fermentation broth is 75.21 g / L. In this application, the trans-aconitine content in the fermentation broth is detected by liquid chromatography, and the purity is 100%, which increases the yield by 717.5% compared with the prior art.

[0055] On the other hand, this application also provides the application of the method in the preparation of trans-aconitic acid and / or in increasing the yield of trans-aconitic acid.

[0056] The method described in this application can increase the yield of trans-aconitine, greatly improving production efficiency. Furthermore, the increased concentration of trans-aconitine in the fermentation broth obtained by this method saves subsequent purification steps, significantly reducing production costs, and is of great significance to actual production.

[0057] The present invention has the following beneficial effects:

[0058] This invention significantly increases the trans-aconitic acid content in the fermentation broth by adjusting the pH and dissolved oxygen (DO) during the trans-aconitic acid production process of Aspergillus terrestris. It also solves the problem of high production costs caused by excessively low trans-aconitic acid content during subsequent separation and purification, greatly reducing the production cost of trans-aconitic acid and simplifying the production steps. This provides a simple and widely applicable method for trans-aconitic acid production.

[0059] 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 yield reported in patent CN110527637A, and 147.56% higher than the control group in this group, greatly reducing production costs. Attached Figure Description

[0060] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0061] Figure 1 This is a graph showing the change in the fermentation yield of trans-aconitic acid.

[0062] Figure 2 This is a graph showing the pH change during the fermentation of trans-aconitic acid.

[0063] Figure 3 This is a graph showing the change in DO during the fermentation of trans-aconitic acid.

[0064] Figure 4 The graph shows the change in itaconic acid content in the experimental case. Detailed Implementation

[0065] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0066] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0067] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0068] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers.

[0069] In Examples 1-4 and Comparative Example 1, the Aspergillus terreus used was the engineered Aspergillus terreus strain At-Δ. cadA The Chinese patent document published in publication number CN110527637A.

[0070] The construction method includes: knocking out the enzyme encoding Aspergillus terrestris cis-aconitate decarboxylase ( cadA ) genes, ku80 Genes and pyrG Gene.

[0071] The Aspergillus terreus used in Example 5 was CICC40205, which was purchased from the China Industrial Microbial Culture Collection Center with the accession number CICC40205.

[0072] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in this invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, based on the prior art mastery of one of ordinary skill in the art and the description of this invention, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of this invention may be used to implement this invention. Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0073] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0074] The detection methods for itaconic acid and trans-aconitic acid are both liquid chromatography, and the operation methods are the same. The main difference is that the standards are different. For specific detection methods, please refer to reference Li. et al. Microb Cell Fact (2020) 19:174 pH was measured using a pH meter.

[0075] Engineered Aspergillus terreus At-Δ cadA Seed culture method: Aspergillus terreus spores were inoculated into a seed culture medium for Aspergillus terreus seed culture. The conditions for Aspergillus terreus seed culture included: preparing the seed culture medium at a volume ratio of 60% in a 30 L fermenter, and the inoculum size being 1.5 × 10⁻⁶. 7 Seed culture of Aspergillus terrestris was obtained by incubating at 1 spore / mL, aeration rate of 0.5 vvm, tank pressure of 0.12 MPa, rotation speed of 240 r / min for 24 h.

[0076] Engineered Aspergillus terreus At-Δ cadAThe seed culture medium consists of: 10% glucose, 0.005% potassium dihydrogen phosphate, 0.1% corn steep liquor powder, 0.2% yeast extract, 0.06% magnesium sulfate, 0.3% ammonium sulfate, 0.001% ferrous sulfate, 0.002% copper sulfate, 0.0005% manganese chloride, with the remainder being water.

[0077] Cultivation method of Aspergillus terreus CICC40205 seed culture: Prepare seed culture medium in a 30 L seed tank at a volume ratio of 60%, with an inoculum size of 1.5 × 10⁻⁶. 7 Spores / mL, aeration rate of 0.5 vvm, rotation speed of 240 r / min, tank pressure of 0.12 MPa, culture for 24 h.

[0078] The seed culture medium for Aspergillus terrestris CICC40205 consists of: glucose 120 g / L, magnesium sulfate 0.6 g / L, ammonium sulfate 4 g / L, dipotassium hydrogen phosphate 0.001 g / L, zinc sulfate 0.01 g / L, ferrous sulfate 0.4 g / L, copper sulfate 0.05 g / L, corn steep liquor 3 g / L, and the remainder being water.

[0079] Example 1

[0080] A method for preparing trans-aconic acid using Aspergillus terrestris fermentation includes:

[0081] Step 1: Prepare a fermentation medium in a 30 L fermenter at a volume ratio of 60%, which includes 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 remainder is water. Sterilize at 121℃ for 20 min to obtain the fermentation medium.

[0082] Step 2: Transfer 10% of the Aspergillus terrestris seed culture into the fermenter. The fermentation control conditions are as follows: ventilation rate 0.4 vvm, rotation speed 380 r / min, and tank pressure 0.1 MPa. During fermentation, the pH value is controlled by adding urea to maintain the fermentation process at 2.0-4.5 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 fermentation, the ventilation rate, rotation speed, and tank pressure are kept constant to allow the dissolved oxygen (DO) to fluctuate naturally. Fermentation is stopped when the trans-aconitine content no longer increases. After 120 h of fermentation, the trans-aconitine yield can reach 34 g / L.

[0083] Example 2

[0084] A method for preparing trans-aconic acid using Aspergillus terrestris fermentation includes:

[0085] Step 1: Prepare a fermentation medium in a 100 L fermenter at a volume ratio of 60%, which includes 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, 2 g / L corn germ oil, and the remainder is water. Sterilize at 121℃ for 20 min to obtain the fermentation medium.

[0086] Step 2: Transfer 10% of the Aspergillus terrestris seed culture into the fermenter. The fermentation control conditions are as follows: ventilation rate 0.4 vvm, rotation speed 220 r / min, and tank pressure 0.1 MPa. During fermentation, the pH value is controlled by adding urea to maintain the fermentation process at 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 fermentation, the ventilation rate, rotation speed, and tank pressure are kept constant to allow the dissolved oxygen (DO) to fluctuate naturally. Fermentation is stopped when the trans-aconitine content no longer increases. After 272 h of fermentation, the trans-aconitine yield is 60.22 g / L.

[0087] Example 3

[0088] A method for preparing trans-aconic acid using Aspergillus terrestris fermentation includes:

[0089] Step 1: Prepare a fermentation medium in a 100 L fermenter at a volume ratio of 60%, which includes 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, 2 g / L corn germ oil, and the remainder is water. Sterilize at 121℃ for 20 min to obtain the fermentation medium.

[0090] Step 2: Transfer 10% of the Aspergillus terrestris seed culture into the fermenter. The fermentation control conditions are as follows: control the aeration rate at 0.4 vvm, the initial rotation speed at 220 r / min, and the tank pressure at 0.1 MPa. During the fermentation process, control the pH value of the fermentation process at 2.2-3.2 by adding urea until the fermentation is completed. When the residual sugar drops to 15-25 g / L, add glucose to maintain its concentration at 15-25 g / L. While keeping the aeration rate constant, control the DO value by controlling the stirring speed so that it does not exceed 70% of the initial calibration value. Stop the fermentation when the trans-aconitine content no longer increases. After 334 h of fermentation, the trans-aconitine yield is 75.21 g / L.

[0091] Example 4

[0092] A method for preparing trans-aconic acid using Aspergillus terrestris fermentation includes:

[0093] Step 1: Prepare a fermentation medium in a 100 L fermenter at a volume ratio of 60%, which includes 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, 2 g / L corn germ oil, and the remainder is water. Sterilize at 121℃ for 20 min to obtain the fermentation medium.

[0094] Step 2: Transfer 10% of the Aspergillus terrestris seed culture into the fermenter. The fermentation control conditions are as follows: ventilation rate 0.4 vvm, initial rotation speed 220 r / min, tank pressure 0.1 MPa. When the residual sugar drops to 15-25 g / L, glucose is added to maintain its concentration at 15-25 g / L. During fermentation, ammonia is added to adjust the fermentation pH to 2.2-3.2. The DO value is controlled to not exceed 70% of the initial value by controlling the stirring speed. Fermentation is stopped when the trans-aconitine content no longer increases. After 179 h of fermentation, 41.18 g / L of acid is produced.

[0095] Comparative Example 1

[0096] A method for preparing trans-aconic acid using Aspergillus terrestris fermentation includes:

[0097] Step 1: Prepare a fermentation medium in a 100 L fermenter at a volume ratio of 60%, which includes 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, 2 g / L corn germ oil, and the remainder is water. Sterilize at 121℃ for 20 min to obtain the fermentation medium.

[0098] Step 2: Transfer 10% of the Aspergillus terrestris seed culture into the fermenter. The fermentation control conditions are as follows: control the ventilation volume at 0.4 vvm, the rotation speed at 220 r / min, and the tank pressure at 0.1 MPa. During the fermentation process, keep the ventilation volume, rotation speed, and tank pressure constant, allowing the pH and DO to fluctuate naturally. When the residual sugar drops to 15-25 g / L, add glucose to maintain its concentration at 15-25 g / L. Stop fermentation when the trans-aconitine content no longer increases. After 180 h of fermentation, the acid production is 30.38 g / L.

[0099] Conclusion Analysis:

[0100] The changes in trans-aconitic acid content, pH, and DO over time during the fermentation process of Examples 1-4 and Comparative Example 1 were monitored, and Tables 1, 2, and 3 were obtained. A graph was plotted from Table 1 to obtain... Figure 1 The graph obtained from Table 2 is shown below. Figure 2 Table 3 shows the results obtained from the graph. Figure 3 .

[0101] Table 1. Changes in trans-aconitic acid content

[0102]

[0103] Table 2 pH changes

[0104]

[0105] Table 3. Changes in DO

[0106]

[0107] From Table 1, Table 2, Table 3, Figure 1 , Figure 2 , Figure 3 The results show that

[0108] The results of Example 1 preliminarily demonstrate that the pH value of the fermentation process can be controlled by adding urea, thereby increasing the yield of trans-aconitic acid in the fermentation broth. Comparing the results of Example 1 with those of Example 2, it is also shown that the size of the fermenter and the specific pH control range have a certain impact on the yield of trans-aconitic acid. A 100 L fermenter and a pH value of 2.2-3.2 are preferred for the fermentation preparation of trans-aconitic acid.

[0109] The only difference between Example 2 and Example 3 is whether or not DO is controlled, demonstrating that DO control has a significant impact on the production yield of trans-aconitine. In Example 3, the aeration rate was kept constant, and the DO value was controlled to be no higher than 70% of the initial calibration value by controlling the stirring speed. This made the environment more favorable for Aspergillus terrestris fermentation to produce trans-aconitine, thereby increasing the yield of trans-aconitine.

[0110] The only difference between Example 4 and Example 3 is the use of different pH adjusters. Specifically, Example 3 used urea as the pH adjuster, while Example 4 used ammonia. The yield of trans-aconitic acid in Example 4 was lower than that in Example 3, demonstrating that not all pH adjusters can achieve high yields of trans-aconitic acid. On the other hand, the Aspergillus terreus engineered strain At-Δ used in this example for fermentation... cadA The ability to slowly utilize urea as a nitrogen source to sustain microbial growth aligns with the growth pattern of *Aspergillus terrestris*, which requires a low C / N ratio for growth and a high C / N ratio for fermentation and acid production. Furthermore, the ammonia produced during urea decomposition helps stabilize the pH during fermentation, thus preventing adverse effects of pH drops on the engineered bacteria. In this embodiment, controlling the pH between 2.2 and 3.2 maximized the yield of trans-aconitic acid.

[0111] The difference between Comparative Example 1 and Example 2 is that the fermentation pH was not adjusted, which proves that the yield of trans-aconitic acid is low when the pH is not adjusted during the fermentation process.

[0112] In summary, this embodiment is the first to use urea as a pH adjuster to control the pH of the trans-aconitine fermentation process, and the fermentation yield of trans-aconitine is greatly increased by controlling the pH and DO value, which provides support for the large-scale production and commercial production of trans-aconitine.

[0113] The optimal fermentation conditions given in this embodiment are:

[0114] In a 100 L fermenter, at a ratio of 1.5 × 10 7 Seed culture was prepared using 10 spores / mL and inoculated into a fermenter at a rate of 10%. The aeration rate was controlled at 0.4 vvm, the initial rotation speed was 220 r / min, and the tank pressure was 0.1 MPa. Urea was added during fermentation to control the fermentation pH at 2.2-3.2. When the residual sugar reached 15-25 g / L, glucose was added to maintain the concentration at 15-25 g / L. While keeping the aeration rate constant, the dissolved oxygen (DO) value was controlled by adjusting the stirring speed to ensure it did not exceed 70% of the initial calibration value. Fermentation was terminated when the trans-aconitine content no longer increased, thus obtaining a fermentation broth containing trans-aconitine.

[0115] 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, 2 g / L corn germ oil, with the remainder being water. The mixture was sterilized at 121℃ for 20 min to obtain the fermentation medium.

[0116] Example 5

[0117] To verify whether urea-controlled pH and DO control can increase the yield of other organic acids, this embodiment uses two methods to produce itaconic acid: natural fermentation and fed-batch urea with DO control.

[0118] Experimental Example 1

[0119] This embodiment provides a method for preparing itaconic acid using Aspergillus terrestris fermentation, including:

[0120] Step 1: Prepare a fermentation medium in a 100 L fermenter at a volume ratio of 60%, which includes 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 remainder is water. Sterilize at 121℃ for 20 min to obtain the fermentation medium.

[0121] Step 2: Transfer 10% of the Aspergillus terrestris seed culture into the fermenter. The fermentation control conditions are as follows: control the ventilation volume at 0.4 vvm, the rotation speed at 220 r / min, and the tank pressure at 0.1 MPa. Keep the ventilation volume, rotation speed, and tank pressure constant during the fermentation process, allowing the pH and DO to change naturally. Stop the fermentation when the glucose content is less than 0.5 g / L. After 71 h of fermentation, the itaconic acid content is 79.68 g / L.

[0122] Experimental Example 2

[0123] This embodiment provides a method for preparing itaconic acid using Aspergillus terrestris fermentation, including:

[0124] Step 1: Prepare a fermentation medium in a 100 L fermenter at a volume ratio of 60%, which includes 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 remainder is water. Sterilize at 121℃ for 20 min to obtain the fermentation medium.

[0125] Step 2: Transfer 10% of the Aspergillus terrestris seed culture into the fermenter. The fermentation control conditions are as follows: control the ventilation rate at 0.4 vvm, the rotation speed at 220 r / min, and the tank pressure at 0.1 MPa. Fermentation is carried out by adding urea and controlling DO (adding urea to maintain pH 2.2-3.2 while controlling DO to not exceed 70% of the initial DO by controlling the rotation speed). Fermentation is stopped when the glucose content is less than 0.5 g / L. Itaconic acid is 74.14 g / L after 71 h of fermentation.

[0126] The specific fermentation processes of Experiment 1 and Experiment 2 are shown in Table 4. A graph can be plotted from Table 4 to obtain... Figure 4 .

[0127] Table 4. Statistics on itaconic acid content

[0128]

[0129] The results are shown in Table 4 and Figure 4 As shown, the production of itaconic acid did not increase but decreased slightly after adding urea to control pH and DO value, proving that the method in this application is designed for trans-aconitic acid and is not applicable to itaconic acid.

[0130] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for preparing trans-aconic acid by fermentation of Aspergillus terrestris, characterized in that, The method includes the following steps: Step 1: Inoculate Aspergillus terreus spores into a seed culture medium for Aspergillus terreus seed culture. The conditions for Aspergillus terreus seed culture include: an inoculum size of 1.5 × 10⁻⁶ spores. 7 The seed culture of Aspergillus terreus was obtained by incubating at 1 spore / mL, aeration rate of 0.5 vvm, tank pressure of 0.12 MPa, rotation speed of 240 r / min for 24 h. Step 2: Inoculate the Aspergillus terrestris seed culture into the fermentation medium at an inoculation rate of 10%. Control the aeration rate at 0.4 vvm, the initial rotation speed at 220 r / min, and the tank pressure at 0.1 MPa. During fermentation, use a pH adjuster (urea) to control the pH at 2.2-3.

2. Control the dissolved oxygen value to be no higher than 30%-70% of the initial calibration value. Stop fermentation when the trans-aconitine content no longer increases, thus obtaining a fermentation broth containing trans-aconitine. The fermentation is carried out in a 100 L fermenter. The Aspergillus terreus is modified, the modification including: knocking out cadA Gene, ku80 Genes and pyrG Gene; The seed culture medium comprises: 10% glucose, 0.005% potassium dihydrogen phosphate, 0.1% corn steep liquor powder, 0.2% yeast extract, 0.06% magnesium sulfate, 0.3% ammonium sulfate, 0.001% ferrous sulfate, 0.002% copper sulfate, 0.0005% manganese chloride, with the remainder being water; 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, 0.2% corn germ oil, and the remainder being water; The fermentation broth contains more than 75 g / L of trans-aconitic acid.

2. The fermentation broth containing trans-aconitic acid prepared by the method described in claim 1, characterized in that, The fermentation broth contains more than 75 g / L of trans-aconitic acid.

3. The application of the method as described in claim 1 in the preparation of trans-aconitic acid and / or in increasing the yield of trans-aconitic acid.

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