Maintenance solution for culturing influenza virus as well as preparation method and application of maintenance solution

Abstract

The invention relates to the technical field of biological medicines, in particular to a maintenance solution for culturing influenza viruses as well as a preparation method and application of the maintenance solution. The maintenance liquid comprises a basic culture medium and effective components, the effective components comprise amino acids, vitamins, salts and a virus nutrition enhancer, and the virus nutrition enhancer is a microorganism induced culture extract. The extract is prepared by inoculating fermentation microbial bacteria, performing induced culture through a plant induction factor extract containing American ginseng, polygonum cuspidatum and golden cypress, and then performing autolysis and enzymolysis in the presence of compound metabolic regulatory factors, namely trichosalix A and N-acetylcysteine. The maintenance fluid can significantly enhance host cell metabolism, inhibit cell antiviral response and delay cell apoptosis after infection, so that the culture yield and process stability of influenza viruses are significantly improved under the condition of no dependence on serum.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a maintenance solution for culturing influenza virus, its preparation method, and its application. Background Technology

[0002] The research and development, and production of influenza virus vaccines and antiviral drugs, are highly dependent on the efficient and stable amplification of viruses in in vitro cell culture systems. Among these factors, the cell maintenance medium used during viral infection is a key factor affecting virus yield, antigenic consistency, and process stability.

[0003] Current maintenance media for influenza virus amplification under cell culture conditions are mainly based on basal media such as DMEM, MEM, and DMEM / F-12, supplemented with certain amounts of amino acids, vitamins, salts, and complex nutrients such as peptones / extracts to maintain the host cell state during the infectious period and support viral replication. However, existing technologies generally have the following shortcomings: First, some maintenance media still rely on complex components such as serum or animal-derived proteins, which raises concerns about uncertain sources, large batch-to-batch variability, and high pressure for controlling exogenous risks; Second, cells during the infectious period are more sensitive to changes in pH, osmotic pressure, and ionic strength, while existing maintenance media lack adequate pH / osmotic pressure settings and drift control measures, which can easily lead to a decline in cell state and a shortened infection time, thereby affecting the consistency and stability of virus harvest.

[0004] Therefore, existing maintenance media for influenza virus culture still have shortcomings in terms of eliminating serum dependence, improving component clarity and quality control, and optimizing the cellular microenvironment during the infection period. There is an urgent need to provide a maintenance media for influenza virus culture and a method for preparing its key promoting components, enabling the maintenance media to stably maintain the host cell state during the infection period without serum dependence, and improving the consistency and reproducibility of virus harvesting; simultaneously, by limiting the process parameters and quality standards of the virus proliferation promoter, batch-to-batch variability can be reduced, meeting the requirements for large-scale preparation and quality control. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a maintenance solution for culturing influenza virus, its preparation method, and its application.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A maintenance medium for culturing influenza virus includes DMEM / F-12 basal medium and active ingredients; the active ingredients include amino acids, vitamins, salts, and a virus nutrient fortifier, wherein the virus nutrient fortifier includes a microbial induced culture extract; Currently, maintenance media for influenza virus culture generally rely on serum or complex animal and plant hydrolysates. The specific active components for promoting viral growth are unclear, and there are large batch-to-batch differences, resulting in unstable viral amplification efficiency. These traditional additives mainly provide basic nutrition and cannot actively intervene in and optimize the metabolic state and antiviral defense response of cells during the special stage of viral infection.

[0007] The microbial induced culture extract added to the maintenance solution in this invention is a complex active component obtained by multi-stage induction culture, autolysis and enzymatic hydrolysis of fermentation microorganisms. This extract is not a traditional nutritional supplement, but a virus nutritional enhancer with multiple biological functions: (1) This extract can regulate the metabolic state of host cells during infection, significantly enhance the levels of sugar metabolism and lipid metabolism in cells, and provide sufficient energy and metabolic precursors for viral replication; at the same time, it promotes nucleic acid translation and protein synthesis in host cells, and directly promotes the replication of the viral genome and the assembly of structural proteins; (2) This extract can inhibit the expression of interferon-stimulated genes by affecting the interferon effector pathway, thereby reducing the inherent antiviral response of host cells and creating a more favorable intracellular environment for viral replication; in addition, it can delay the cell apoptosis process caused by viral infection, effectively prolong the time window of viral replication, and help increase the viral yield.

[0008] The microbial induced culture extract was prepared by the following method: an induction regulator was added to a liquid culture medium, and fermentation microorganisms were inoculated and cultured with shaking; glycerol, plant inducing factor extract, and osmotic protectant were added under aseptic conditions for induction culture; after the culture was completed, the fermentation microbial cells were collected, the cell bodies were resuspended in phosphate buffer, and metabolic regulators were added for autolysis at 40-50℃; after adding glucoamylase for further autolysis, the enzyme was inactivated, the supernatant was collected by centrifugation, and then concentrated and dried to obtain the extract. The fermentation microorganism is any one of Yersinia lipolytica, Saccharomyces cerevisiae, or Bacillus subtilis.

[0009] The induction regulator is a mixture of L-tyrosine and tartaric acid.

[0010] The plant-inducing factor extract is obtained by enzymatic extraction from American ginseng, Polygonum cuspidatum, and Phellodendron amurense.

[0011] The metabolic regulator is at least one of compound A and N-acetylcysteine; preferably, the metabolic regulator is a mixture of compound A and N-acetylcysteine, wherein the final concentration of compound A in the system is 0.10-0.50 mg / L and the final concentration of N-acetylcysteine ​​in the system is 100-800 mg / L.

[0012] Compound A (trigulin A), as a histone deacetylase inhibitor, remodels Bacillus subtilis metabolites through epigenetic regulation, endowing the extract with the potential to activate host cell anabolic metabolism. N-acetylcysteine, as an antioxidant, protects the active ingredients and maintains cellular redox homeostasis. The combination of these two compounds forms a synergistic system of activation and protection, enabling the final extract to simultaneously enhance cellular metabolism, inhibit antiviral responses, and delay apoptosis, thereby optimizing the viral replication microenvironment and increasing viral yield.

[0013] During the induction culture stage of the fermentation microorganisms, the compound extracts of American ginseng, Polygonum cuspidatum, and Phellodendron amurense exhibit synergistic effects as exogenous metabolic regulatory signals. The active ingredients of these three plants collectively constitute a multi-target microbial metabolic pathway reprogramming induction system. They synergistically intervene in the metabolism of the fermentation microorganisms, not by directly providing nutrients, but by guiding a directional shift in their biosynthetic direction, thereby promoting the accumulation of a series of specific active substances in the microbial cells that can subsequently regulate host cell function: functional peptides, enzymes, and signaling molecules. When the microbial induced culture extract prepared by this method is applied to virus maintenance solutions, its active ingredients indirectly enhance host cell metabolism, inhibit innate immune responses, and delay apoptosis, thus creating a highly optimized intracellular environment for influenza virus replication and significantly improving virus yield and process stability.

[0014] Preferably, the preparation method of the plant inducing factor extract is as follows: 10-20 parts by weight of American ginseng, 7-14 parts by weight of Polygonum cuspidatum, and 7-10 parts by weight of Phellodendron chinense are washed, chopped to about 0.3-1 cm, and 0.05-0.2 wt% ascorbic acid is added. The mixture is then stirred with 80-120 parts by weight of PBS buffer (pH 7.0-7.8) at 2-8℃ to obtain a homogenate. Cellulase and pectinase are added to the homogenate, and the mixture is gently stirred at 35-55℃ for 40-90 minutes. min; squeeze and filter with sterile gauze, collect the filtrate, centrifuge at 400×g to obtain the supernatant; centrifuge the supernatant at 4℃ and 8000×g to obtain the centrifuged liquid, filter the centrifuged liquid through a 0.22-0.5μm polyethersulfone filter membrane for sterilization, transfer it to an ultrafiltration centrifuge tube, centrifuge at 4℃ and 4000×g to obtain the ultrafiltrate, concentrate it to 1 / 10 of the original volume to obtain the concentrate; centrifuge the concentrate at 4℃ and 100000×g to collect the precipitate, add PBS buffer to the precipitate, gently pipette and resuspend, wash, add 1-4wt% mannitol, freeze dry to obtain the plant inducing factor extract.

[0015] The ultracentrifugation precipitate is an enriched high molecular weight active component, such as polysaccharide-protein complexes / vesicle-like particles, which are more likely to induce the metabolism of fermenting bacteria than small molecule impurities in the supernatant; therefore, this invention selects to collect the precipitate and resuspend and freeze-dry it as a plant inducing factor extract.

[0016] Preferably, the final concentration of the active ingredient in the maintenance solution includes: L-glutamine 40-80 mg / L, L-arginine hydrochloride 20-30 mg / L, L-serine 3-8 mg / L, L-lysine hydrochloride 15-25 mg / L, L-proline 8-20 mg / L, glycine 8-15 mg / L, nicotinamide 0.2-0.6 mg / L, inositol 1.6-3.0 mg / L, D-calcium pantothenate 0.3-1.0 mg / L, sodium chloride 180-250 mg / L, anhydrous calcium chloride 5-10 mg / L, sodium selenite 0.03-0.1 mg / L, and microbial induced culture extract 220-320 mg / L.

[0017] Preferably, in the method for preparing the microbial induced culture extract, the inoculum amount of the fermenting microorganism is 2-6 wt%; in the induction culture step, the amount of glycerol added is 15-30 g / L, the final concentration of glycine betaine is 0.05-0.1 g / L, and the final concentration of the plant inducing factor extract is 0.8-1.5 g / L.

[0018] Preferably, in the method for preparing the microbial induced culture extract, in the autolysis step, the volume ratio of wet bacterial cells to resuspension is 1:(4-8), and the total concentration of metabolic regulatory factors in the system is 1.8-3.0% (w / v).

[0019] Preferably, in the method for preparing the microbial induced culture extract, the amount of glucoamylase added in the step of adding glucoamylase for continued autolysis is 300-600 U / mL.

[0020] Preferably, the permeation protectant is any one of glycine betaine, glycine betaine derivatives, choline, and carnitine.

[0021] Preferably, the pH of the maintenance solution is 7.0-7.4 and the osmotic pressure is 280-350 mOsm / kg.

[0022] A method for preparing a maintenance solution for culturing influenza virus includes the following steps: (1) Prepare each active ingredient separately and filter to remove bacteria; (2) Calculate and measure the amount of each substance to be added based on the final concentration; (3) Preheat the DMEM / F-12 basic culture medium, add each active ingredient in sequence under aseptic conditions, and mix well; (4) Adjust the pH of the maintenance solution to 7.0-7.4, and the osmotic pressure to 280-350 mOsm / kg; (5) Perform terminal sterilization filtration, and then repackage and store.

[0023] The filtration and sterilization described in steps (1) and (5) are performed using a 0.22 μm filter membrane.

[0024] The application of the maintenance solution in the culture or preparation of influenza virus.

[0025] The beneficial effects of this invention are: 1. This invention provides a maintenance medium for culturing influenza virus, its preparation method, and its application. Microbial-induced culture extract is added to the raw materials. This component is not a simple nutritional supplement, but a viral nutrient enhancer with biological functions. It can regulate the state of host cells during the infection period: by enhancing cellular glucose metabolism, lipid metabolism, and protein synthesis, it provides ample raw materials and energy for viral replication; by inhibiting the interferon effector pathway and interferon-stimulated gene expression, it reduces the cell's inherent antiviral defense; simultaneously, it effectively delays virus infection-induced apoptosis, thereby significantly prolonging the viral replication time and ultimately greatly increasing the yield of influenza virus, with effects superior to traditional serum-containing maintenance media.

[0026] 2. The maintenance solution of the present invention can be used for the efficient amplification of influenza virus in cell culture systems. The core active components in the formula, such as the production strains and plant inducing factors, can be equivalently replaced according to actual conditions, provided that the established quality standards are met. This provides flexibility for formula optimization, cost control and adaptation to different production conditions, and enhances the practical value and scope of application of the present invention. Detailed Implementation

[0027] The invention will now be described in further detail with reference to specific embodiments, but it should not be construed as limiting the scope of the invention to the following embodiments.

[0028] The raw materials described in this application are partially described; all other raw materials not described are commercially available. DMEM / F-12 basal culture medium was purchased from Shanghai Gesai Biotechnology Co., Ltd., model 384222.

[0029] YPD liquid culture medium was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., product number HB5193-1.

[0030] Compound A is qugulijunin A, CAS: 58880-19-6.

[0031] Bacillus subtilis was purchased from the China General Microbiological Culture Collection Center, strain number: CGMCC1.107.

[0032] Yersinia lipolytica was purchased from the China Medical Bacterial Culture Collection Center, strain number CMCC98025.

[0033] The brewer's yeast was purchased from the China Medical Bacterial Culture Collection Center, strain number CMCC98019.

[0034] The glucoamylase was purchased from Hebei Changxinghang Biotechnology Co., Ltd., product number CXH8123.

[0035] Cellulase was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., with an enzyme activity of 10,000 U / g and product number FFY-0673.

[0036] Pectinase was purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., product number FFY-3602.

[0037] Trypsin was purchased from Siterofan Biotechnology (Hangzhou) Co., Ltd., product number SH30042.02.

[0038] Example 1 A maintenance medium for culturing influenza virus, the virus maintenance medium comprising DMEM / F-12 basal medium and active ingredients; the active ingredients comprising amino acids, vitamins, salts, and virus nutrient fortifiers.

[0039] The amino acids include L-glutamine 60 mg / L, L-arginine hydrochloride 25 mg / L, L-serine 5 mg / L, L-lysine hydrochloride 18 mg / L, L-proline 12 mg / L, and glycine 10 mg / L; the vitamins include nicotinamide 0.3 mg / L, inositol 2.2 mg / L, and D-calcium pantothenate 0.5 mg / L; the salts include sodium chloride 220 mg / L, anhydrous calcium chloride 8 mg / L, and sodium selenite 0.05 mg / L; the virus nutrient fortifier includes a microbial induced culture extract, wherein the amount of the microbial induced culture extract added is 280 mg / L.

[0040] The microbial induced culture extract was prepared by the following method: Add 0.3 g / L L-tyrosine and 20 mM tartaric acid to YPD liquid medium, and inoculate 4 wt% fermentation microbial cells under aseptic conditions. Incubate at 30℃ and 200 rpm with shaking for 18 h. Then, under aseptic conditions, add 20 g / L glycerol, a plant inducing factor extract (final concentration 1 g / L), and glycine betaine (final concentration 0.08 g / L). Incubate at 30℃ and 200 rpm for 20 h. After incubation, centrifuge at 8000g for 10 min and collect the fermentation microbial cells. Resuspend the cells in sodium phosphate buffer at pH 7.4 at a wet cell to resuspended liquid volume ratio of 1:5. Add metabolic regulators to achieve a total concentration of 2.2% (w / v) in the system, and adjust the pH to 7.5. Incubate at 45℃ and 200 rpm for 20 h for autolysis. Then add 500... The enzyme was inactivated by U / mL glucoamylase and allowed to autolyze at 45°C for 18 hours. After enzyme inactivation, the supernatant was collected by centrifugation, vacuum concentration, and spray drying to obtain the microbial induced culture extract.

[0041] The fermentation bacteria are Yersinia lipophila.

[0042] The preparation method of the plant inducing factor extract is as follows: 15 parts by weight of American ginseng, 10 parts by weight of Polygonum cuspidatum, and 10 parts by weight of Phellodendron chinense are washed, chopped to about 0.5 cm, and 0.1 wt% ascorbic acid is added. The mixture is then stirred with 80 parts by weight of pH 7.2 PBS buffer at 4℃ to obtain a homogenate. 200 U / g cellulase and 150 U / g pectinase are added to the homogenate, and the mixture is gently stirred at 45℃ for 60 minutes. min; squeeze and filter with sterile gauze, collect the filtrate, centrifuge at 400×g to obtain the supernatant; centrifuge the supernatant at 4℃ and 8000×g to obtain the centrifuged liquid, filter the centrifuged liquid through a 0.45μm polyethersulfone filter membrane for sterilization, transfer it to an ultrafiltration centrifuge tube, ultrafilter and centrifuge at 4℃ and 4000×g to obtain the ultrafiltrate, concentrate it to 1 / 10 of the original volume to obtain the concentrate; ultracentrifuge the concentrate at 4℃ and 100000×g to collect the precipitate, add PBS buffer to the precipitate, gently pipette and resuspend, wash, add 2wt% mannitol, freeze dry to obtain the plant inducing factor extract.

[0043] The metabolic regulator is a mixture of compound A and N-acetylcysteine, wherein the final concentration of compound A in the system is 0.30 mg / L and the final concentration of N-acetylcysteine ​​in the system is 300 mg / L.

[0044] The method for preparing the maintenance solution for culturing influenza virus includes the following steps: (1) Prepare each active ingredient separately and filter it through a 0.22μm filter membrane for sterilization; (2) Calculate the amount of each substance to be added based on the final concentration mentioned above; (3) Preheat DMEM / F-12 basic culture medium to 37°C, add each active ingredient in sequence under aseptic conditions, and mix thoroughly; (4) Adjust the pH of the maintenance solution to 7.2 and the osmotic pressure to 325 mOsm / kg; (5) After terminal sterilization filtration through a 0.22μm filter membrane, the product is packaged and stored at 4℃ for later use.

[0045] Example 2 It is basically the same as Example 1, except that the fermentation microorganism is Saccharomyces cerevisiae.

[0046] Example 3 It is basically the same as Example 1, except that the fermentation bacteria are Bacillus subtilis.

[0047] Example 4 It is basically the same as Example 1, except that the metabolic regulator is compound A.

[0048] Example 5 It is basically the same as Example 1, except that the metabolic regulator is N-acetylcysteine.

[0049] Comparative Example 1 It is basically the same as Example 1, except that no virus nutrient fortifier is added to the formula of the maintenance solution used to culture the influenza virus.

[0050] Comparative Example 2 The method is basically the same as in Example 1, except that: no metabolic regulatory factors are added in the preparation method of the microbial induced culture extract, and it is prepared by the following method: 0.3 g / L L-tyrosine and 20 mM tartaric acid were added to YPD liquid medium. 4 wt% of fermentation microorganisms were inoculated under aseptic conditions and cultured at 30℃ and 200 rpm with shaking for 18 h. Then, under aseptic conditions, 20 g / L glycerol, a plant inducing factor extract (final concentration 1 g / L), and glycine betaine (final concentration 0.08 g / L) were added, and the mixture was cultured at 30℃ and 200 rpm for 20 h. After culture, the mixture was centrifuged at 8000g for 10 min to collect the fermentation microbial cells. The cells were resuspended in sodium phosphate buffer (pH 7.4) at a wet cell to resuspended liquid volume ratio of 1:5, and the pH was adjusted to 7.5. The mixture was then autolyzed at 45℃ and 200 rpm for 20 h. 500 U / mL glucoamylase was added, and the mixture was further autolyzed at 45℃ for 18 h. After enzyme inactivation, the supernatant was collected by centrifugation, vacuum concentration, and spray drying to obtain the microbial induced culture extract.

[0051] The fermentation bacteria are Yersinia lipophila.

[0052] Comparative Example 3 The method is basically the same as in Example 1, except that: no plant inducing factor extract is added in the preparation method of the microbial induced culture extract, and it is prepared by the following method: Add 0.3 g / L L-tyrosine and 20 mM tartaric acid to YPD liquid medium, and inoculate 4 wt% of fermentation microbial cells under aseptic conditions. Incubate at 30℃ and 200 rpm with shaking for 18 h. Then, add 20 g / L glycerol and glycine betaine under aseptic conditions, with a final glycine betaine concentration of 0.08 g / L, and induce culture at 30℃ and 200 rpm for 20 h. After incubation, centrifuge at 8000g for 10 min and collect the fermentation microbial cells. Resuspend the microbial cells in sodium phosphate buffer at pH 7.4 at a wet cell to resuspended liquid volume ratio of 1:5, and add metabolic regulators to achieve a total concentration of 2.2% (w / v). Adjust the pH to 7.5. Incubate at 45℃ and 200 rpm for 20 h for autolysis. Then add 500... The enzyme was inactivated by U / mL glucoamylase and allowed to autolyze at 45°C for 18 hours. After enzyme inactivation, the supernatant was collected by centrifugation, vacuum concentration, and spray drying to obtain the microbial induced culture extract.

[0053] The fermentation bacteria are Yersinia lipophila.

[0054] Comparative Example 4 It is basically the same as Example 1, except that the metabolic regulator is a commonly used trypsin.

[0055] Comparative Example 5 It is basically the same as Example 1, except that the metabolic regulatory factor is composed of trypsin and N-acetylcysteine ​​in a mass ratio of 2:1.

[0056] Test Example 1 Virus titer test: The experimental group used the maintenance medium for culturing influenza virus as described in this invention, while the control group used the control culture medium (containing 2% fetal bovine serum, 1% penicillin and streptomycin antibiotics, and 97% DMEM).

[0057] Cell lines and virus strains: canine kidney cells MDCK-v (Madin Darby Canine Kidney Cells, accession number: CCTCCNO:C2024173), influenza A virus strain H1N1 type IVR-215.

[0058] Experimental Methods: The MDCK cell culture conditions described are those commonly used in the field. During viral infection, the MOI is 0.001-0.1, and the medium is replaced with the appropriate maintenance medium after 30-60 min of adsorption. The maintenance medium may further contain 0.5-5 μg / mL TPCK-trypsin to promote HA lysis. Samples are taken every 24 h… TCID50 is calculated using the Reed-Muench method. Preferred culture conditions: seeding density of 5 × 10⁵ cells / ml, 37℃, dissolved oxygen (DO) of 50%, compressed air, oxygen, carbon dioxide, and nitrogen are introduced at a ratio of 70%, 20%, 5%, and 5% respectively, with an aeration rate of 1.0 and a rotation speed of 110 rpm. When MDCK cells grow to 10⁻¹² × 10⁵ cells / ml… 6 At a concentration of cells / mL, influenza A virus strain H1N1 was inoculated. The control group used control culture medium, while the experimental group used the maintenance medium for culturing influenza virus described in the examples and comparative examples of this invention to maintain virus proliferation. The virus titers of the maintenance medium for culturing influenza virus described in this invention and the control group culture medium were tested. Each group was tested 4 times, and the average value was taken. The results are shown in Table 1.

[0059] Table 1. Influenza virus titer test results (Log10(CID50) / 100μl) Test Example 2 CCK-8 assay to detect the effect of maintenance medium used for influenza virus culture on cell viability Experimental Procedure: MDCK cell suspension was washed with preheated α-MEM basal medium and collected by centrifugation. The supernatant was discarded, and the cells were gently resuspended in the maintenance solutions prepared in the examples and comparative studies, respectively. Viable cell counts were then performed (trypan blue staining). The cell concentration of all groups was uniformly adjusted to 4.0 × 10⁻⁶. 4 cells / mL. Add 100 μL of cell suspension (i.e., 4.0 × 10⁶ cells / mL) to each well of a 96-well plate. 3 (cells / well). Wells containing only maintenance medium and no cells were set up as blank controls.

[0060] The inoculated 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. This incubation period was designed to simulate the pre-infection and early infection stages of the virus, evaluating the sustained support of different maintenance media for basal cell viability and metabolic activity. After incubation, 10 μL of CCK-8 reagent was precisely added to each well, and incubation continued for 2 hours. The absorbance (OD value) of each well was measured using a microplate reader at 450 nm. Relative cell viability was calculated using the following formula: Relative cell viability (%) = (OD value / OD value) 样品组 -OD 空白组 ) / (OD 实施例1组 -OD 空白组() × 100%; where, the cell viability of the group in Example 1 was used as a 100% baseline for normalization, and the results were expressed as the difference rate of proliferation / survival. Each group was tested 4 times and the average value was taken, as shown in Table 2.

[0061] Table 2. Differential rate of cell proliferation The results above show that the maintenance solution for culturing influenza virus prepared in this invention has the effect of promoting influenza virus proliferation. The specific reasons are as follows: In Example 1, the virus titer reached 9.3 Log TCID after 96 hours. 50 At a concentration of 100 μl, the relative cell activity was 100%, significantly superior to all comparative examples. In contrast, Comparative Example 1 showed a sharp decrease in both virus titer and cell activity, indicating that the microbial induced culture extract prepared in this invention acts as a maintenance solution to enhance cell metabolism, inhibit antiviral responses, and delay apoptosis. The differences in effects between Example 1 (using *Yersinia lipolytica*), Example 2 (using *Saccharomyces cerevisiae*), and Example 3 (using *Bacillus subtilis*) suggest that *Yersinia lipolytica*, due to its superior lipid metabolism and synthesis capabilities, produces an extract that, after induction and autolysis, is more advantageous in optimizing the host cell lipid environment and supporting influenza virus envelope synthesis. Comparative Example 2 (without added metabolic regulators) and Comparative Example 3 (without added plant inducing factors) showed significantly lower virus titers and cell activity than Example 1, demonstrating that both are essential for achieving optimal results. Plant inducing factors act as multi-target metabolic programming signals during the fermentation induction stage, guiding the synthesis of specific active substances by the cells; while metabolic regulators, through the synergistic effect of epigenetic activation and redox homeostasis maintenance during the autolysis stage, deeply optimize the activity spectrum of the extract. Examples 4-5, using only a single metabolic regulator, were less effective than Example 1, which used a combination of both, further confirming the synergistic effect of the combined system. Comparative Examples 4 and 5, while slightly better than Comparative Example 2 (which completely lacked the regulator) when using the common protease trypsin instead of compound A, were far less effective than Example 1, which used compound A. This demonstrates that compound A, as a histone deacetylase inhibitor, has a unique mechanism for epigenetic remodeling of microbial metabolites.

Claims

1. A maintenance solution for culturing influenza virus, characterized in that, It includes a basic culture medium and active ingredients; the active ingredients include amino acids, vitamins, salts, and virus nutrient fortifiers, the virus nutrient fortifiers including microbial induced culture extracts; The microbial induced culture extract was prepared by the following method: an induction regulator was added to a liquid culture medium, and fermentation microorganisms were inoculated and cultured with shaking; glycerol, plant inducing factor extract, and osmotic protectant were added under aseptic conditions for induction culture; after the culture was completed, the fermentation microbial cells were collected, the cell bodies were resuspended in phosphate buffer, and metabolic regulators were added for autolysis at 40-50℃; after adding glucoamylase for further autolysis, the enzyme was inactivated, the supernatant was collected by centrifugation, and then concentrated and dried to obtain the extract. The induction regulator is a mixture of L-tyrosine and tartaric acid; The plant inducing factor extract is obtained by enzymatic hydrolysis of American ginseng, Polygonum cuspidatum, and Phellodendron amurense. The metabolic regulator is at least one of compound A and N-acetylcysteine; compound A is trichosuccinimide A. The fermentation microorganism is any one of Yersinia lipolytica, Saccharomyces cerevisiae, or Bacillus subtilis.

2. The maintenance solution for culturing influenza virus according to claim 1, characterized in that, The final concentrations of the active ingredients in the maintenance solution include: L-glutamine 40-80 mg / L, L-arginine hydrochloride 20-30 mg / L, L-serine 3-8 mg / L, L-lysine hydrochloride 15-25 mg / L, L-proline 8-20 mg / L, glycine 8-15 mg / L, nicotinamide 0.2-0.6 mg / L, inositol 1.6-3.0 mg / L, D-calcium pantothenate 0.3-1.0 mg / L, sodium chloride 180-250 mg / L, anhydrous calcium chloride 5-10 mg / L, sodium selenite 0.03-0.1 mg / L, and microbial induced culture extract 220-320 mg / L.

3. The maintenance solution for culturing influenza virus according to claim 1, characterized in that, In the method for preparing the microbial induced culture extract, the inoculum amount of the fermenting microorganism is 2-6 wt%; in the induction culture step, the amount of glycerol added is 15-30 g / L, the final concentration of the osmotic protectant is 0.05-0.1 g / L, and the final concentration of the plant inducing factor extract is 0.8-1.5 g / L.

4. The maintenance solution for culturing influenza virus according to claim 1, characterized in that, In the preparation method of the microbial induced culture extract, the volume ratio of wet bacterial cells to resuspension in the autolysis step is 1:(4-8), and the total concentration of metabolic regulatory factors in the system is 1.8-3.0% (w / v).

5. The maintenance solution for culturing influenza virus according to claim 1, characterized in that, In the preparation method of the microbial induced culture extract, in the step of adding glucoamylase for continued autolysis, the amount of glucoamylase added is 300-600 U / mL; the permeation protectant is any one of glycine betaine, glycine betaine derivatives, choline derivatives, and carnitine derivatives.

6. The maintenance solution for culturing influenza virus according to claim 1, characterized in that, The basal culture medium includes any one of DMEM, MEM, DMEM / F-12, M199, and 1640.

7. The maintenance solution for culturing influenza virus according to claim 1, characterized in that, The maintenance solution has a pH of 7.0-7.4 and an osmotic pressure of 280-350 mOsm / kg.

8. A method for preparing a maintenance solution for culturing influenza virus according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Prepare each active ingredient separately and filter to remove bacteria; (2) Calculate and measure the amount of each substance to be added based on the final concentration; (3) Preheat the DMEM / F-12 basic culture medium, add each active ingredient in sequence under aseptic conditions, and mix well; (4) Adjust the pH of the maintenance solution to 7.0-7.4, and the osmotic pressure to 280-350 mOsm / kg; (5) Perform terminal sterilization filtration, and then repackage and store.

9. The preparation method according to claim 8, characterized in that, The filtration and sterilization described in steps (1) and (5) are performed using a 0.22 μm filter membrane.

10. The use of the maintenance solution according to any one of claims 1-7 in the culture or preparation of influenza virus.

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