A high-temperature-resistant eurotium cristatum fermented composition and domestication fermentation method

Abstract

This invention discloses a composition and domestication fermentation method for highly thermotolerant *Aurogonium cristatum*, aiming to solve the problems of low survival rate and metabolic abnormalities of this strain during high-temperature hypoxic fermentation. The composition comprises a pre-excitation composition, a quenching and cofactor composition, and a high-temperature electron compensation composition. The process includes: inoculating the strain into a system containing calcium pyroglutamate and sodium selenite, and pre-exciting it with hydrogen peroxide at room temperature; subsequently, adding L-ascorbic acid to quench residual hydrogen peroxide, followed by adding riboflavin and ferrous citrate for sequential targeted loading of cofactors; and during the high-temperature hypoxic stage, adding disodium fumarate, manganese sulfate, and L-malic acid for electron compensation. This invention activates thermotolerant pathways through room-temperature controlled stress, avoids Fenton reaction toxicity, and completes respiratory chain enzyme cofactor assembly, effectively reconstructing metabolic flux under high-temperature microaerobic conditions, significantly improving viable bacterial survival rate, reducing organic acid accumulation, and increasing the yield of secondary metabolites.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a composition for fermenting highly thermotolerant *Aurotriarcha* and a method for domestication and fermentation. Background Technology

[0002] *Aspergillus cristatus* is a core strain in food fermentation and the extraction of bioactive metabolites, and it has high application value in industrial production. The optimal growth and fermentation temperature for this strain is typically between 28°C and 32°C. During large-scale industrial submerged fermentation or solid-state fermentation at room temperature in summer, the accumulated bioheat in the fermentation system can easily raise the ambient temperature to above 40°C, severely affecting the growth and metabolism of the bacteria.

[0003] Currently, the existing technology for culturing and acclimatizing highly thermotolerant strains typically employs a physical gradient heating method, which involves continuously subculturing the strains at gradually increasing temperatures. This method, which relies purely on physical heat stimulation for acclimatization, results in a high strain mortality rate, and the acquired thermotolerance is often phenotypic adaptation or physiological acclimatization, with unstable gene expression. Once removed from the high-temperature environment, the thermotolerance trait is highly susceptible to degradation.

[0004] To mitigate the damage to microorganisms caused by high temperatures, some conventional techniques involve adding traditional heat protectants such as sodium alginate, glycerol, or trehalose to the culture medium. These substances utilize their polyhydroxyl properties to replace water molecules and maintain protein conformation. However, adding large amounts of these substances, especially large molecules or high-viscosity substances like sodium alginate, can significantly increase the viscosity of the fermentation broth, increase gas-liquid mass transfer resistance, and lead to a sharp drop in dissolved oxygen levels.

[0005] During the high-temperature fermentation stage, the solubility of oxygen in the liquid is inevitably reduced due to thermodynamic laws. As an aerobic fungus, *Aspergillus cristatus* experiences a disruption of its conventional aerobic respiratory chain in a high-temperature, low-dissolved-oxygen environment. To survive, the fungal cells undergo metabolic pathway shifts, initiating an anaerobic fermentation pathway, leading to the production of metabolic byproducts such as lactic acid, acetic acid, and ethanol, with a significant accumulation of organic acids. This accumulation not only causes an abnormal decrease in the pH of the fermentation broth, which in turn inhibits the growth of the fungus itself, but also prevents the efficient flow of cellular substances and energy to the synthesis pathways of target secondary metabolites, ultimately resulting in a substantial reduction in the yield of the target product. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the issues of low survival rate of *Aurotriarcha cristatus* in high-temperature fermentation systems, abnormal metabolism due to hypoxia, and decreased product yield in the prior art, and to provide a composition and domestication fermentation process for fermenting highly thermotolerant *Aurotriarcha cristatus*.

[0007] To address the problems mentioned in the background art, the first aspect of the present invention provides a composition for fermentation of highly thermostable *Aurogonium cristatum*, comprising a pre-excitation composition, a quenching and cofactor composition, and a high-temperature electron compensation composition; Based on the final concentration in the fermentation system, the pre-excitation composition comprises: 0.5-1.0 g / L calcium pyroglutamate and 0.01-0.03 mg / L sodium selenite; The quenching and cofactor composition comprises component A, which is composed of a free oxygen quencher, and component B, which is composed of a cofactor donor complex; wherein component A is 0.05-0.1 g / L L-ascorbic acid; and component B comprises 0.5-1.0 mg / L riboflavin and 1.5-2.0 mg / L ferrous citrate. The high-temperature electronic compensation composition comprises: 2.0-3.5 g / L disodium fumarate, 0.05-0.08 g / L manganese sulfate monohydrate, and 0.1-0.2 g / L L-malic acid.

[0008] Further, in the high-temperature electronic compensation composition, the molar ratio of L-malic acid to disodium fumarate is limited to 1:15 to 1:20; preferably, the concentration of L-malic acid is 0.15-0.20 g / L, and the concentration of disodium fumarate is 2.5-3.5 g / L.

[0009] Furthermore, components A and B in the pre-excitation composition, quenching and auxiliary composition, and high-temperature electronic compensation composition are all prepared separately as aqueous solutions, and each component aqueous solution is sterilized by filtration membrane, wherein the aqueous solutions of components A and B are stored in the dark.

[0010] Furthermore, the composition further includes an aqueous solution of hydrogen peroxide as a dynamic cross-stressing agent, wherein the aqueous solution of hydrogen peroxide has a mass fraction of 0.1%.

[0011] A second aspect of the present invention provides a method for the domestication and fermentation of highly thermotolerant *Aurogonium cristatum* based on the aforementioned composition for fermentation, comprising the following steps: Step S1, Strain Activation and Seed Culture Preparation: *Aspergillus cristatus* was inoculated into a basal fermentation medium and cultured to obtain a seed culture containing homogeneous mycelia in the logarithmic growth phase. The culture parameters were: temperature 28.0-30.0℃, shaking speed 150-200 rpm, and culture time 48-72 h.

[0012] Step S2, Room Temperature Oxidative Stress Pre-excitation: The pre-excitation composition is added to a fermenter containing the basic fermentation medium, and the seed culture is inoculated at an inoculum rate of 5.0%-10.0% (v / v) for fermentation. Fermentation parameters are: constant temperature at 30.0-31.0℃, stirring speed at 150-250 rpm, and aeration rate of 0.5-1.0 vvm. Hydrogen peroxide aqueous solution is added, and with online redox potential electrode feedback control, the free hydrogen peroxide concentration in the fermentation system is dynamically maintained at 0.1-0.2 mmol / L for a duration of 20-24 h.

[0013] Step S3, Targeted Loading of the Prosthetic Group: Stop the flow of hydrogen peroxide aqueous solution, and pump component A of the quencher and prosthetic group composition into the fermenter in one go. Maintain stable stirring for 10-15 minutes to reduce the remaining free hydrogen peroxide in the system. After confirming that there is no hydrogen peroxide residue, add component B of the quencher and prosthetic group composition into the system, maintaining the system temperature at 30.0-32.0℃ and the stirring speed at 150-200 rpm. The transition period should last for 4-6 hours.

[0014] Step S4, Electron Acceptor Compensated High-Temperature Fermentation: After the transition period, the temperature is slowly increased to 40.0-45.0℃ at a constant rate of 1.0-1.5℃ / h via the fermenter's temperature control jacket. During the high-temperature fermentation process, the dissolved oxygen level of the fermentation system is monitored in real time. When the dissolved oxygen is less than or equal to 15.0%, the high-temperature electron compensation composition is added at a constant rate. During the addition of the high-temperature electron compensation composition and during continuous fermentation, ammonia or hydrochloric acid is used to dynamically maintain the pH of the fermentation broth between 4.5 and 5.5. The continuous fermentation time at 40.0-45.0℃ is 72-120 hours.

[0015] Step S5, Fermentation product harvesting and separation: After fermentation, the system temperature is lowered to 20.0-25.0℃, and the fermentation broth is centrifuged at 4000-6000 rpm for 10-15 min, or solid-liquid separation is performed by plate and frame filtration at 0.2-0.4 MPa pressure, and the solid phase mycelium and liquid phase supernatant are collected separately.

[0016] The present invention, by adopting the above technical solution, can bring the following beneficial effects: 1. This invention pre-excites the bacterial strain under oxidative stress at room temperature by adding a pre-excitation composition consisting of calcium pyroglutamate, sodium selenite, and hydrogen peroxide to the fermentation system. This technique utilizes controlled hydrogen peroxide concentration to induce a stress response in the cells, prompting them to express relevant stress-resistant enzymes before physical heating, thus avoiding the mass cell death caused by traditional physical high-temperature acclimatization and improving the viable survival rate of *Aspergillus cristatus* in the subsequent high-temperature fermentation environment.

[0017] 2. This invention employs a prosthetic group sequential targeted loading process. After pre-excitation, L-ascorbic acid is first added to quench residual hydrogen peroxide, followed by the addition of riboflavin and ferrous citrate. This technical feature eliminates the conditions that trigger the Fenton reaction, avoids lipid peroxidation damage to cells caused by the combination of free iron ions and hydrogen peroxide, and simultaneously provides the necessary iron-sulfur clusters and flavin adenine dinucleotide prosthetic groups for the synthesis of key enzymes in the bacterial respiratory chain, laying the material foundation for subsequent high-temperature microaerobic metabolism.

[0018] 3. This invention reconstructs the metabolic flux of cells under anaerobic conditions by adding a high-temperature electron compensation composition consisting of disodium fumarate, manganese sulfate, and L-malic acid during the high-temperature, low-dissolved-oxygen fermentation stage. This technique utilizes disodium fumarate as an exogenous electron acceptor analog (fumarate respiratory co-substrate) to maintain the redox balance of cells under anaerobic conditions. Furthermore, L-malic acid activates bypass metabolism to relieve substrate feedback inhibition, preventing the metabolic flux from shifting to the anaerobic acid-producing pathway, reducing the accumulation of organic acid byproducts, and increasing the yield of target secondary metabolites. Attached Figure Description

[0019] Figure 1 This is a flowchart of the domestication and fermentation process of *Aurogonium cristatum*, a highly thermotolerant strain, according to the present invention. Figure 2 This is a comparison diagram of intracellular catalase activity in various experimental groups of this invention; Figure 3 This is a comparison chart of fumarate reductase activity in different experimental groups of this invention; Figure 4 This is a comparison chart of the concentration of viable bacterial spores at the fermentation endpoint of each experimental group in this invention; Figure 5 This is a comparison chart of lactic acid accumulation in the fermentation broth of each experimental group in this invention; Figure 6 This is a comparison chart of lovastatin titers in different experimental groups of this invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see the appendix Figure 1-6 This invention provides a composition for fermentation of highly thermotolerant *Aurotriarcha* and a method for domestication and fermentation.

[0022] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0023] Eurotium cristatum strains are available for purchase from the China General Microbiological Culture Collection Center, which provides standard slant spores or freeze-dried powder strains. Calcium pyroglutamate, chemically known as bis(5-oxo-L-proline) calcium salt, has the molecular formula: The CAS number is 31821-43-3. It is a commercially available conventional fine chemical raw material with a definite chemical structure and molecular stereochemistry. It is a white crystalline powder at room temperature. Sodium selenite, molecular formula is CAS number 10102-18-8, commercially available analytical grade product; Hydrogen peroxide, CAS number 7722-84-1, is a commercially available 30% (w / w) aqueous solution of hydrogen peroxide, which is a transparent and colorless liquid. L-Ascorbic acid, molecular formula is CAS number 50-81-7, commercially available analytical grade product; Riboflavin, molecular formula is CAS number 83-88-5, commercially available analytical grade product; Ferrous citrate, with the molecular formula: CAS number 23383-11-1, commercially available biochemical reagent grade product; Disodium fumarate, chemically known as disodium fumarate, has the molecular formula: CAS number 17013-01-3, commercially available biochemical reagent grade product; Manganese sulfate monohydrate, molecular formula is CAS number 10034-96-5, commercially available analytical grade product; L-malic acid, chemically known as L-hydroxysuccinic acid, has the molecular formula [missing information]. CAS number 97-67-6, commercially available analytical grade product; Malt extract, CAS number 8002-48-0, is a commercially available product for use with microbial culture media. Peptone, CAS No. 91079-40-2, is a commercially available product specifically for microbial culture media. Potassium dihydrogen phosphate, molecular formula is CAS number 7778-77-0, commercially available analytical grade product; Magnesium sulfate heptahydrate, molecular formula is CAS number 10034-99-8, commercially available analytical grade product.

[0024] Preparation Example 1: This preparation example provides a combined broth for the fermentation of highly thermostable *Aurogonium cristatum*, including the following steps: Weigh 0.5g of calcium pyroglutamate and 0.01mg of sodium selenite, dissolve them in purified water, and bring the volume to 1L. Filter the solution through a 0.22μm microporous membrane to obtain the pre-excitation solution for later use. Weigh 0.05g of L-ascorbic acid, dissolve it in purified water, and bring the volume to 0.5L to obtain the quenching solution. Weigh 0.5mg of riboflavin and 1.5mg of ferrous citrate, dissolve them in purified water, and bring the volume to 0.5L to obtain the auxiliary base solution. Filter both the quenching solution and the auxiliary base solution through a 0.22μm microporous membrane for sterilization and store them in the dark for later use. Weigh 2.0g of disodium fumarate, 0.05g of manganese sulfate monohydrate, and 0.1g of L-malic acid, dissolve them in purified water, and bring the volume to 1L. Filter the solution through a 0.22μm microporous membrane for sterilization to obtain the high-temperature electron compensation solution for later use.

[0025] Preparation Example 2: This preparation example provides a combined broth for the fermentation of highly thermostable *Aurogonium cristatum*, including the following steps: Weigh 0.75g of calcium pyroglutamate and 0.02mg of sodium selenite, dissolve them in purified water, and bring the volume to 1L. Filter the solution through a 0.22μm microporous membrane to obtain the pre-excitation solution for later use. Weigh 0.075g of L-ascorbic acid, dissolve it in purified water, and bring the volume to 0.5L to obtain the quenching solution. Weigh 0.75mg of riboflavin and 1.75mg of ferrous citrate, dissolve them in purified water, and bring the volume to 0.5L to obtain the auxiliary base solution. Filter both the quenching solution and the auxiliary base solution through a 0.22μm microporous membrane for sterilization and store them in the dark for later use. Weigh 2.75g of disodium fumarate, 0.065g of manganese sulfate monohydrate, and 0.15g of L-malic acid, dissolve them in purified water, and bring the volume to 1L. Filter the solution through a 0.22μm microporous membrane for sterilization to obtain the high-temperature electron compensation solution for later use.

[0026] Preparation Example 3: This preparation example provides a combined broth for the fermentation of highly thermostable *Aurogonium cristatum*, including the following steps: Weigh 1.0 g of calcium pyroglutamate and 0.03 mg of sodium selenite, dissolve in purified water and bring the volume to 1 L. Filter through a 0.22 μm microporous membrane to obtain the pre-excitation solution for later use. Weigh 0.1 g of L-ascorbic acid, dissolve in purified water and bring the volume to 0.5 L to obtain the quenching solution. Weigh 1.0 mg of riboflavin and 2.0 mg of ferrous citrate, dissolve in purified water and bring the volume to 0.5 L to obtain the auxiliary base solution. Filter both the quenching solution and the auxiliary base solution through a 0.22 μm microporous membrane for sterilization and store in the dark for later use. Weigh 3.5 g of disodium fumarate, 0.08 g of manganese sulfate monohydrate and 0.18 g of L-malic acid, dissolve in purified water and bring the volume to 1 L. Filter through a 0.22 μm microporous membrane for sterilization to obtain the high-temperature electron compensation solution for later use. Example Example 1

[0027] This embodiment provides a method for the domestication and fermentation of highly thermostable *Aurogonium cristatum*, including the following steps: The first step, strain activation and seed culture preparation: The preserved slant spores of *Aspergillus cristatus* were inoculated into a shake flask containing basic fermentation medium and cultured at 28.0℃ and 150 rpm for 48 h to obtain a homogeneous mycelial suspension as the seed culture.

[0028] The second step was pre-excitation under ambient temperature oxidative stress: the pre-excitation combination solution obtained in Preparation Example 1 was added to the basic fermentation medium in the fermenter, and the seed liquid was inoculated at an inoculation rate of 5.0% (v / v); the temperature of the fermenter was kept constant at 30.0℃, the stirring speed was 150 rpm, and the aeration rate was 0.5 vvm; starting from the inoculation, a 0.1% hydrogen peroxide aqueous solution was continuously pumped into the fermenter, and with the help of online redox potential electrode feedback control, the concentration of free hydrogen peroxide in the fermentation broth was dynamically maintained at 0.1 mmol / L. This dynamic fed-batch stress process lasted for 20 h.

[0029] The third step is the sequential targeted loading of the auxiliary group: After the stress process ends, the hydrogen peroxide feed path is immediately cut off, and the quenching solution obtained in Preparation Example 1 is quickly pumped into the fermenter in one go. The stirring is kept stable for 10 minutes to reduce the remaining free hydrogen peroxide in the system. After confirming that there is no hydrogen peroxide residue, the auxiliary group solution obtained in Preparation Example 1 is added to the system. The system temperature is maintained at 30.0℃, the stirring speed is 150 rpm, and the transition period is maintained for 4 hours.

[0030] Step 4, electron acceptor compensated high-temperature fermentation: After the transition period, the temperature was slowly increased to 40.0℃ at a constant rate of 1.0℃ / h through the temperature control jacket of the fermenter; during the high-temperature fermentation process, the dissolved oxygen level of the fermentation system was monitored in real time. When the online sensor showed dissolved oxygen ≤15.0%, the feeding program was started to add the high-temperature electron-compensated combined solution obtained in Example 1 to the fermentation system at a constant rate; during the fermentation, 2.0mol / L ammonia or 2.0mol / L hydrochloric acid was used to dynamically maintain the pH of the fermentation broth at 4.5; fermentation was continued at 40.0℃ for 72h.

[0031] Step 5, Fermentation product harvesting and separation: After fermentation, the system temperature is lowered to 20.0℃, and the fermentation broth is centrifuged at 4000rpm for 10min to separate the solid and liquid phases. The solid phase mycelium and the liquid phase supernatant are collected separately for later use. Example 2

[0032] This embodiment provides a method for the domestication and fermentation of highly thermostable *Aurogonium cristatum*, including the following steps: The first step, strain activation and seed culture preparation: The preserved slant spores of *Aspergillus cristatus* were inoculated into a shake flask containing basic fermentation medium and cultured at 29.0℃ and 175 rpm for 60 h to obtain a homogeneous mycelial suspension as the seed culture.

[0033] The second step was pre-excitation under ambient temperature oxidative stress: the pre-excitation combination solution obtained in Preparation Example 2 was added to the basic fermentation medium in the fermenter, and the seed liquid was inoculated at an inoculation rate of 8.0% (v / v); the temperature of the fermenter was kept constant at 30.5℃, the stirring speed was 200 rpm, and the aeration rate was 0.8 vvm; starting from the inoculation, a 0.1% hydrogen peroxide aqueous solution was continuously pumped into the fermenter, and with the help of online redox potential electrode feedback control, the concentration of free hydrogen peroxide in the fermentation broth was dynamically maintained at 0.15 mmol / L. This dynamic fed-batch stress process lasted for 22 h.

[0034] The third step is the sequential targeted loading of the auxiliary group: After the stress process ends, the hydrogen peroxide feed path is immediately cut off, and the quenching solution obtained in Preparation Example 2 is quickly pumped into the fermenter in one go. The stirring is kept stable for 12 minutes to reduce the remaining free hydrogen peroxide in the system. After confirming that there is no hydrogen peroxide residue, the auxiliary group solution obtained in Preparation Example 2 is added to the system. The system temperature is maintained at 31.0℃, the stirring speed is 175 rpm, and the transition period is maintained for 5 hours.

[0035] Step 4, electron acceptor compensated high-temperature fermentation: After the transition period, the temperature was slowly increased to 42.0℃ at a constant rate of 1.2℃ / h through the temperature control jacket of the fermenter; during the high-temperature fermentation process, the dissolved oxygen level of the fermentation system was monitored in real time. When the online sensor showed dissolved oxygen ≤15.0%, the feeding program was started to add the high-temperature electron-compensated combined solution obtained in Example 2 to the fermentation system at a constant rate; during the fermentation, 2.0 mol / L ammonia or 2.0 mol / L hydrochloric acid was used to dynamically maintain the pH of the fermentation broth at 5.0; fermentation was continued at 42.0℃ for 96 hours.

[0036] Step 5, Fermentation product harvesting and separation: After fermentation, the system temperature is lowered to 22.0℃, and the fermentation broth is centrifuged at 5000rpm for 12min to separate the solid and liquid phases. The solid phase mycelium and the liquid phase supernatant are collected separately for later use. Example 3

[0037] This embodiment provides a method for the domestication and fermentation of highly thermostable *Aurogonium cristatum*, including the following steps: The first step, strain activation and seed culture preparation: The preserved slant spores of *Aspergillus cristatus* were inoculated into a shake flask containing basic fermentation medium and cultured at 30.0℃ and 200rpm for 72h to obtain a homogeneous mycelial suspension as the seed culture.

[0038] The second step was pre-excitation under ambient temperature oxidative stress: the pre-excitation combination solution obtained in Preparation Example 3 was added to the basic fermentation medium in the fermenter, and the seed liquid was inoculated at an inoculation rate of 10.0% (v / v); the temperature of the fermenter was kept constant at 31.0℃, the stirring speed was 250 rpm, and the aeration rate was 1.0 vvm; starting from the inoculation, a 0.1% hydrogen peroxide aqueous solution was continuously pumped into the fermenter, and with the help of online redox potential electrode feedback control, the concentration of free hydrogen peroxide in the fermentation broth was dynamically maintained at 0.2 mmol / L. This dynamic fed-batch stress process lasted for 24 h.

[0039] The third step is the sequential targeted loading of the auxiliary group: After the stress process ends, the hydrogen peroxide feed path is immediately cut off, and the quenching solution obtained in Preparation Example 3 is quickly pumped into the fermenter in one go. The stirring is kept stable for 15 minutes to reduce the remaining free hydrogen peroxide in the system. After confirming that there is no hydrogen peroxide residue, the auxiliary group solution obtained in Preparation Example 3 is added to the system. The system temperature is maintained at 32.0℃, the stirring speed is 200 rpm, and the transition period is maintained for 6 hours.

[0040] Step 4, electron acceptor compensated high-temperature fermentation: After the transition period, the temperature was slowly increased to 45.0℃ at a constant rate of 1.5℃ / h through the temperature control jacket of the fermenter; during the high-temperature fermentation process, the dissolved oxygen level of the fermentation system was monitored in real time. When the online sensor showed dissolved oxygen ≤15.0%, the feeding program was started to add the high-temperature electron-compensated combined solution obtained in Example 3 to the fermentation system at a constant rate; during the fermentation, 2.0 mol / L ammonia or 2.0 mol / L hydrochloric acid was used to dynamically maintain the pH of the fermentation broth at 5.5; fermentation was continued at 45.0℃ for 120 h.

[0041] Step 5, Fermentation product harvesting and separation: After fermentation, the system temperature is lowered to 25.0℃, and the fermentation broth is centrifuged at 6000rpm for 15min to separate the solid and liquid phases. The solid phase mycelium and the liquid phase supernatant are collected separately for later use. Comparative Example

[0042] Comparative Example 1:

[0043] Compared with Example 2, the difference is that the second step of room temperature oxidative stress pre-excitation and the third step of cofactor sequential targeted loading are not performed, and the high temperature electron compensation combination liquid is not added in the fourth step. Instead, the seed liquid is directly introduced into the fermenter and then heated to 42.0°C at the same rate for physical high temperature fermentation. All other aspects are the same.

[0044] Comparative Example 2: Compared with Example 2, the difference is that in the third step of the auxiliary base timing targeted loading stage, after the hydrogen peroxide flow path is cut off, the quenching liquid obtained in Preparation Example 2 is not pumped in, but the auxiliary base liquid obtained in Preparation Example 2 is directly flow-added. All other aspects are the same.

[0045] Comparative Example 3: Compared with Example 2, the difference is that in the third step of the auxiliary base timing targeted loading stage, after the quenching liquid obtained in Preparation Example 2 is pumped in and stirred, the auxiliary base liquid obtained in Preparation Example 2 is not added; otherwise, they are the same.

[0046] Comparative Example 4: Compared with Example 2, the difference is that in the fourth step of the electron acceptor compensated high-temperature fermentation stage, L-malic acid was removed from the high-temperature electron compensation combination liquid formulation fed into the feed program, while the rest are the same.

[0047] Comparative Example 5: Compared with Example 2, the difference is that in the fourth step of the electron acceptor compensated high-temperature fermentation stage, the high-temperature electron compensation combination liquid formulation added by starting the feeding program has removed disodium fumarate, while the rest are the same.

[0048] Test Case Description and Experimental Methods Test Example 1: Comprehensive Evaluation of Physicochemical and Biochemical Indicators and Target Products of Fermentation System This test case aims to quantitatively analyze the fermentation process and end products of Examples 2 and Comparative Examples 1 to 5. Test parameters include intracellular catalase activity, fumarate reductase activity, viable spore concentration at the fermentation endpoint, accumulation of organic acid byproducts, and the titer of the target secondary metabolite, lovastatin.

[0049] (I) Experimental Procedure 1. Sampling and pretreatment for enzyme activity determination: At the end of the transition period of the third step of cofactor sequential targeted loading, 50 mL of sample was aseptically taken from each fermenter. The fermentation broth was centrifuged at 4000 rpm for 10 min to collect the cell pellet. The pellet was washed twice with pre-cooled phosphate buffer. The cells were suspended in the buffer and the cells were disrupted by an ultrasonic disruptor under ice bath conditions. The parameters were: power 300 W, ultrasonic 3 s, interval 5 s, total time 15 min. The disrupted liquid was centrifuged at 10000 rpm for 15 min at low temperature. The supernatant was used as crude enzyme solution for later use.

[0050] 2. Intracellular catalase activity was determined using ultraviolet spectrophotometry. The reaction system contained 2.9 mL of phosphate buffer and 0.1 mL of crude enzyme solution. A quantitative amount of hydrogen peroxide was added to start the reaction. The rate of decrease of absorbance at 240 nm wavelength over time was measured. The unit of catalase activity was defined as the amount of enzyme that breaks down 1 micromole of hydrogen peroxide per milligram of protein per minute.

[0051] 3. Fumarate reductase activity was determined by a colorimetric method using sodium 2,6-dichlorophenolindophenol as the electron acceptor. The reaction system included crude enzyme solution, sodium fumarate substrate, and indicator. The absorbance change was measured at a wavelength of 600 nm. The activity unit was defined as the amount of enzyme that reduces 1 nanomolar of indicator per milligram of protein per minute. Protein concentration was determined using the BCA method.

[0052] 4. Determination of viable spore concentration at the fermentation endpoint: Samples were taken at the 96-hour fermentation endpoint. After serial dilution of the fermentation broth, 0.1 mL of the sample was spread onto potato dextrose agar plates. The plates were incubated upside down in a 28°C incubator for 72 hours. Colony counting was performed to calculate the viable spore concentration.

[0053] 5. High-performance liquid chromatography (HPLC) analysis of metabolites: Samples were centrifuged at the fermentation endpoint, and the supernatant was filtered through a 0.22 μm organic phase filter membrane. The contents of lovastatin and lactic acid were determined by HPLC using a C18 reversed-phase column. The mobile phase for lactic acid detection was 0.05% phosphoric acid aqueous solution, and the detection wavelength was 210 nm. The mobile phase for lovastatin detection was a mixture of methanol and 0.1% phosphoric acid aqueous solution in a volume ratio of 75:25, and the detection wavelength was 238 nm. Quantitative calculation was performed using the external standard method.

[0054] (II) Experimental Data The following is a summary of the test data for each experimental group under the same parallel operating conditions.

[0055]

[0056] (III) Results Analysis The difference between the results of Example 2 and Comparative Example 1 confirms the limitations of conventional physical heating fermentation. Comparative Example 1 lacks oxidative stress pre-excitation and compensation mechanisms. Under high temperature and accompanying low dissolved oxygen conditions, the expression level of intracellular stress-resistant enzyme systems is low, resulting in a significant decrease in viable cell count. At the same time, hypoxia leads to the blockage of the conventional aerobic respiratory chain, and the metabolic flow shifts to the anaerobic fermentation branch, causing a large accumulation of lactic acid and inhibiting the synthesis of the target secondary metabolite lovastatin. Example 2 significantly improved the synthesis efficiency of the target product and reduced the production of harmful organic acids through pre-process control.

[0057] The viable bacterial concentration in Comparative Example 2 was extremely low. This group retained hydrogen peroxide stress but removed the ascorbic acid quenching step during the transition period. The residual hydrogen peroxide reacted with the subsequently added ferrous citrate in a Fenton reaction, generating highly reactive free radicals that caused lipid peroxidation damage, resulting in the death of a large number of mycelial cells. The experimental data proved the necessity of the transition quenching step in the process connection.

[0058] Comparative Example 3 verified the mechanism of action of targeted loading of the transitional prosthetic donor. The catalase activity in this group was induced normally, but due to the lack of loading of riboflavin and ferrous citrate, sufficient flavin adenine dinucleotide and iron-sulfur clusters could not be formed intracellularly, resulting in fumarate reductase only expressing the protein skeleton and lacking the catalytic activity core. The enzyme activity data was significantly lower than that in Example 2. This led to the inability of the bacteria to effectively initiate alternative respiration in the high temperature and low oxygen stage, resulting in high lactic acid accumulation and a decrease in viable bacteria count.

[0059] Comparative Example 4 reflects the role of the metabolic regulator L-malic acid in eliminating product feedback inhibition. In this group, the fumarate reductase activity remained at a moderate to high level. However, due to the lack of L-malic acid to drive metabolic flux through the replenishment pathway, succinic acid generated from fumarate reduction accumulated, causing the reaction to tend towards thermodynamic equilibrium. The inhibition of electron acceptor reduction slowed down the overall efficiency of the anaerobic respiratory chain, resulting in lovastatin titers and viable bacterial concentrations only reaching part of the levels of Example 2.

[0060] Comparative Example 5 illustrates the fermentation state in the absence of an exogenous electron acceptor analog (fumarate respiratory aid substrate) disodium fumarate. Although the enzyme system was assembled in the early stage and the fumarate reductase activity was high, the lack of an actual electron acceptor substrate prevented effective electron transfer in the entire respiratory chain. The cells still entered a state of hypoxia and death due to the stagnation of the electron transport chain in the high-temperature environment, resulting in abnormal lactic acid accumulation and a significant decrease in product titer. This confirms that pre-acclimatization must be combined with post-electron acceptor compensation (fumarate respiratory aid) to solve the problem of limited high-temperature fermentation.

Claims

1. A composition for fermentation of highly thermostable *Aurogonium cristatum*, characterized in that, Including pre-excitation compositions, quenching and auxiliary group compositions, and high-temperature electronic compensation compositions; Based on the final concentration in the fermentation system, the pre-excitation composition comprises: 0.5-1.0 g / L calcium pyroglutamate and 0.01-0.03 mg / L sodium selenite; The quenching and cofactor composition comprises component A, which is composed of a free oxygen quencher, and component B, which is composed of a cofactor donor complex; wherein component A is 0.05-0.1 g / L L-ascorbic acid; and component B comprises 0.5-1.0 mg / L riboflavin and 1.5-2.0 mg / L ferrous citrate. The high-temperature electronic compensation composition comprises: 2.0-3.5 g / L disodium fumarate, 0.05-0.08 g / L manganese sulfate monohydrate, and 0.1-0.2 g / L L-malic acid.

2. The composition for fermentation of highly thermostable *Aurogonium cristatum* according to claim 1, characterized in that, In the high-temperature electronic compensation composition, the molar ratio of L-malic acid to disodium fumarate is limited to 1:15-1:20, and the concentration of L-malic acid is 0.15-0.20 g / L, and the concentration of disodium fumarate is 2.5-3.5 g / L.

3. The composition for fermentation of highly thermostable *Aurogonium cristatum* according to claim 1, characterized in that, The pre-excitation composition, components A and B in the quenching and auxiliary composition, and the high-temperature electronic compensation composition are all prepared separately as aqueous solutions, and the aqueous solutions of each component are sterilized by filtration membrane. The aqueous solutions of components A and B are stored in the dark.

4. The composition for fermentation of highly thermostable *Aurogonium cristatum* according to claim 1, characterized in that, The composition further includes an aqueous solution of hydrogen peroxide as a dynamic cross-stressing agent, wherein the aqueous solution of hydrogen peroxide has a mass fraction of 0.1%.

5. A method for the domestication and fermentation of *Aurogonium cristatum* based on a composition for fermentation according to any one of claims 1-4, characterized in that, Includes the following steps: Step S1, strain activation and seed culture preparation: Inoculate *Aspergillus cristatus* into a basic fermentation medium and culture to obtain a seed culture of homogeneous mycelium in the logarithmic growth phase; Step S2, room temperature oxidative stress pre-excitation: The pre-excitation composition is added to a fermenter containing a basic fermentation medium, the seed liquid is inoculated for fermentation, hydrogen peroxide aqueous solution is added, the concentration of free hydrogen peroxide in the fermentation system is dynamically maintained, and the stress process is completed. Step S3, Targeted loading of cofactor: Stop the flow of hydrogen peroxide aqueous solution, pump component A of the quencher and cofactor composition into the fermenter in one go, and stir to reduce the remaining free hydrogen peroxide in the system; after confirming that there is no hydrogen peroxide residue, add component B of the quencher and cofactor composition into the system, and maintain the system temperature to pass through the transition period; Step S4, Electron acceptor compensated high-temperature fermentation: After the transition period, the temperature is increased. During the high-temperature fermentation process, the dissolved oxygen level of the fermentation system is monitored in real time. When the dissolved oxygen drops to or below the preset threshold, the feeding program is started to add the high-temperature electron compensation composition to the fermentation system and fermentation continues. Step S5, Fermentation product harvesting and separation: After fermentation, the temperature is lowered and solid-liquid separation is performed to collect the solid phase mycelium and the liquid phase supernatant separately.

6. The method for domesticating and fermenting highly thermotolerant *Aurogonium cristatum* using the composition for fermentation according to claim 5, characterized in that, In step S1, the cultivation parameters are: temperature 28.0-30.0℃, shaking speed 150-200rpm, and cultivation time 48-72h; In step S2, the inoculation amount of the seed liquid is 5.0%-10.0% (v / v); the fermentation parameters are: constant temperature at 30.0-31.0℃, stirring speed at 150-250 rpm, and aeration rate at 0.5-1.0 vvm; the hydrogen peroxide aqueous solution is fed in conjunction with online redox potential electrode feedback control, the free hydrogen peroxide concentration is dynamically maintained at 0.1-0.2 mmol / L, and the feeding duration is 20-24 h.

7. The method for domesticating and fermenting highly thermotolerant *Aurogonium cristatum* using the composition for fermentation according to claim 5, characterized in that, In step S3, after pumping in component A, the stirring is kept running stably for 10-15 minutes; after adding component B, the system temperature is maintained at 30.0-32.0℃ and the stirring speed is 150-200 rpm. The duration of the transition period is 4-6 hours.

8. The method for domesticating and fermenting highly thermotolerant *Aurogonium cristatum* using the composition for fermentation according to claim 5, characterized in that, In step S4, during the heating stage, the temperature is slowly increased to 40.0-45.0℃ at a constant rate of 1.0-1.5℃ / h through the temperature control jacket of the fermenter; the preset threshold for dissolved oxygen is 15.0%, and the high-temperature electronic compensation composition is added at a constant rate when the dissolved oxygen is less than or equal to 15.0%.

9. The method for domesticating and fermenting highly thermotolerant *Aurogonium cristatum* using the composition for fermentation according to claim 5, characterized in that, In step S4, during the addition of the high-temperature electron compensation composition and the continuous fermentation, ammonia or hydrochloric acid is used to dynamically maintain the pH of the fermentation broth between 4.5 and 5.5; the continuous fermentation time is 72-120 h at 40.0-45.0℃.

10. The method for domesticating and fermenting highly thermotolerant *Aurogonium cristatum* using the composition for fermentation according to claim 5, characterized in that, In step S5, after the system temperature is lowered to 20.0-25.0℃, the fermentation broth is centrifuged at 4000-6000 rpm for 10-15 min, or solid-liquid separation is performed by plate and frame filtration at a pressure of 0.2-0.4 MPa.

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