Optimized culture medium for improving yield of mycophenolic acid and application of optimized culture medium

Abstract

The culture medium comprises methionine, a carbon source, a nitrogen source and a phosphorus source, and the contents of the methionine, the carbon source, the nitrogen source and the phosphorus source in the culture process are 0.3-0.5 g / L, 180-230 g / L, 19-27 g / L and 3-5 g / L respectively. The optimized culture medium for improving the yield of the intracellular antibiotics can be used for fermenting penicillium brevicompactum to produce mycophenolic acid, and the titer of the penicillium brevicompactum for producing the mycophenolic acid can be obviously improved in a short time by utilizing the optimized culture medium and the fermentation method which are prepared by the invention; by utilizing the culture medium prepared by the invention, the activity of the mycophenolic acid produced by the mould in the penicillium brevicompactum can be improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an optimized culture medium for improving mycophenolic acid production and an application thereof. Background Art

[0002] Intracellular antibiotics are a class of antibacterial substances synthesized and accumulated inside microbial cells, and are widely used in the fields of medicine and agriculture. However, due to the complex biosynthetic pathway, high production costs and insufficient yield, their application in industrial-scale production is limited. Traditional culture media often cannot fully meet the growth requirements of production strains and the high-yield expression of antibiotics. Therefore, optimizing the composition of the culture medium is of great significance for increasing the yield of intracellular antibiotics. Mycophenolic acid (MPA), as an intracellular antibiotic, has antifungal, antitumor and immunosuppressive effects. Secondly, it has a reversible inhibitory effect on xanthine mononucleotide dehydrogenase and selectively inhibits lymphocyte activity. Mycophenolic acid (MMF), an esterified derivative of mycophenolic acid, is widely used as a new generation of immunosuppressants in the clinical treatment of organ transplantation and autoimmune diseases. It is one of the most widely used immunosuppressants in the world, with good market prospects and huge demand. Therefore, as a precursor compound of MMF, improving the potency of MPA is of great significance to society and enterprises.

[0003] Chinese patent application CN102321697B discloses a method for efficiently accumulating and producing mycophenolic acid by adding Penicillium breviscompactum in the later stage, and discloses that the fermentation medium contains the following components in concentrations: glucose (C6H12O6·H2O) 80-120 g / L, glycine 12-15 g / L, potassium dihydrogen phosphate 2-4 g / L, magnesium sulfate (MgSO4·7H2O) 0.5-1 g / L, methionine 0.5-1 g / L, the solvent is distilled water, the pH value is 4.4-4.8, and the final titer reaches 5.0 g / L. Chinese patent application CN 112980902 A discloses a fermentation medium and a fermentation method for producing mycophenolic acid, which discloses that the fermentation medium contains the following components: 200-250 g / L glucose, 10-25 g / L aminoacetic acid, 6-10 g / L potassium dihydrogen sulfate, 1.0-2.0 g / L methionine, 1 g / L trace elements, 0.3 g / L folate, and the fermentation medium is fermented at a fermentation pH of 4.5 for 8 days, and the mycophenolic acid yield reaches 9.2 g / L.

[0004] Although the above method improves the potency of mycophenolic acid, the technology is complicated and not suitable for large-scale industrial production. In addition, the potency is low or the fermentation time is long. In order to meet the needs of society and factories, it is necessary to find a technology and fermentation method that can quickly increase the potency of mycophenolic acid. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a culture medium for optimizing the production of intracellular antibiotics, which can significantly increase the content of mycophenolic acid produced in the metabolism of Penicillium breviscompactum cells under low methionine content, thereby improving the mycophenolic acid yield and reducing production costs.

[0006] The invention provides an application of an optimized culture medium for increasing the yield of intracellular antibiotics in fermentation for increasing the titer of mycophenolic acid.

[0007] Technical solution: In order to achieve the above-mentioned purpose, the optimized culture medium for increasing the intracellular antibiotic production described in the present invention comprises methionine, a carbon source, a nitrogen source and a phosphorus source, wherein the content in the culture is 0.3-0.5 g / L methionine, 180-230 g / L carbon source, 19-27 g / L nitrogen source and 3-5 g / L phosphorus source.

[0008] Wherein, the optimized culture medium for increasing the intracellular antibiotic production is an optimized fermentation culture medium for increasing the production of mycophenolic acid.

[0009] The nitrogen source includes aminoacetic acid, yeast extract, corn syrup powder, soybean molasses and yeast cell wall; the carbon source is glucose, sucrose, fructose or soluble starch.

[0010] Wherein, the yeast extract is any one of YP600 Angel yeast powder, brewer's yeast powder, FM885 growth-promoting yeast powder and OXOID yeast extract.

[0011] Preferably, OXOID yeast extract is used.

[0012] Among them, glucose is preferably used as the carbon source.

[0013] Wherein, the culture medium also includes trace elements.

[0014] The culture medium comprises: 180-230 g / L glucose, 10-15 g / L aminoacetic acid, 3-5 g / L potassium dihydrogen phosphate, 0.3-0.5 g / L methionine, 7-8 g / L yeast extract, 1-2 g / L corn syrup powder, 0.5-1 g / L soybean molasses, 0.5-1 g / L yeast cell wall, 1-2 g / L trace elements, and the fermentation pH is 4.4-4.7.

[0015] Preferably, the optimized culture medium is a culture medium for improving the yield of mycophenolic acid, comprising 180-230 g / L of glucose, 10-15 g / L of aminoacetic acid, 0.3-0.5 g / L of methionine, 7-8 g / L of yeast extract, 1-2 g / L of corn syrup powder, 0.5-1 g / L of soy molasses, 0.5-1 g / L of yeast cell wall, 3-5 g / L of potassium dihydrogen phosphate, and 1-2 g / L of trace elements. Among the trace elements, the mass ratio of ferrous sulfate heptahydrate, anhydrous copper sulfate, manganese sulfate, and zinc sulfate heptahydrate is 0.020-0.022: 0.2-0.3: 0.1-0.12: 2.0.

[0016] Preferably, among the trace elements, the mass ratio of ferrous sulfate heptahydrate, anhydrous copper sulfate, manganese sulfate and zinc sulfate heptahydrate is 0.022:0.3:0.12:2.0.

[0017] Furthermore, the method for preparing the culture medium for improving the yield of mycophenolic acid is carried out according to the following steps:

[0018] (1) Weigh 19 g of glucose and dissolve it in water to make it 70 ml in bottle A. Add 100 μl of the trace elements prepared into solution.

[0019] (2) Weigh 1.2 g of aminoacetic acid, 0.04 g of methionine, 0.3 g of potassium dihydrogen phosphate, 0.75 g of OXOID yeast extract, 0.1 g of corn syrup powder, 0.1 g of soy molasses, and 0.05 g of yeast cell wall, dissolve in water to a volume of 30 ml in bottle B, adjust the pH to 4.5, and sterilize in an autoclave at 115°C for 20 min. Mix A and B to obtain 100 ml of culture medium, which is the optimized culture medium for improving mycophenolic acid yield.

[0020] The invention discloses an application of the optimized culture medium for improving the intracellular antibiotic yield in fermenting Penicillium breviscompactum to produce mycophenolic acid.

[0021] The fermentation of Penicillium breviscompactum to produce mycophenolic acid includes a seed culture stage and a fermentation culture stage; the Penicillium breviscompactum is CCTCC CF 2008508.

[0022] The seed culture stage comprises the following steps: taking a spore suspension of Penicillium breviscapus for slant culture for 8-10 days, then preparing a bacterial suspension with physiological saline, then inoculating it into a shake flask filled with seed culture medium at a spore inoculation rate of 2-3%, and shaking culture at 24-26°C and 220-260rpm for 26-28h to obtain a shake flask seed culture solution.

[0023] The fermentation culture stage comprises the following steps: inoculating the seed culture solution at a volume ratio of 5-10% into a fermentation shake flask containing an optimized culture medium for fermentation culture, the culture temperature is 24-26°C, 220-260rpm, and the fermentation cycle is 6-7 days.

[0024] Preferably, the method for producing intracellular antibiotics using the above-mentioned optimized culture medium comprises the following steps:

[0025] a) preparing the seed culture medium and the optimized fermentation medium;

[0026] b) inoculating the production strain into the seed culture medium, controlling the inoculation amount to 2% (volume ratio);

[0027] c) inoculating the seed culture medium into the optimized fermentation medium at an inoculation rate of 10% (volume ratio);

[0028] d) carrying out fermentation culture at a suitable temperature (24° C.) and stirring conditions (250 rpm);

[0029] e) After reaching the stable phase, the culture medium is harvested to extract the intracellular antibiotics.

[0030] The optimized culture medium and fermentation method for increasing the intracellular antibiotic yield of the present invention are to increase the content of mycophenolic acid in the metabolites of Penicillium breviscompactum, thereby increasing the yield of mycophenolic acid. The culture medium of the present invention is made of glucose, aminoacetic acid, methionine, OXOID yeast extract, corn slurry powder, soybean molasses, yeast cell wall, potassium dihydrogen phosphate, trace elements and water. The method of the present invention is: first, preparing a culture medium for increasing mycophenolic acid in Penicillium breviscompactum; second, culturing Penicillium breviscompactum according to the fermentation method. The optimized culture medium and fermentation method prepared by the present invention significantly increase the titer of mycophenolic acid produced by Penicillium breviscompactum; the culture medium prepared by the present invention can increase the activity of mycophenolic acid produced by molds in Penicillium breviscompactum.

[0031] The present invention is optimized on the basis of a medium of glucose, aminoacetic acid and low concentration of methionine, supplemented with a nitrogen source, and additionally added with a certain concentration of OXOID yeast extract, corn syrup powder, soybean molasses and yeast cell wall, so that the biomass in the early stage of fermentation is increased. Compared with a general culture medium, the types of nitrogen sources are richer, and the bacteria can be multiplied in large quantities in the early stage of fermentation, thereby quickly obtaining a large amount of mycophenolic acid.

[0032] In fact, the amount of aminoacetic acid as the main nitrogen source in fermentation has reached the optimal value, and adding aminoacetic acid again will not achieve the effect of increasing the titer. At the same time, increasing the methionine content will significantly reduce the fermentation effect. The present invention adds a new specific nitrogen source to increase the biomass so that the titer increases, optimizes the nitrogen source to be richer than the nitrogen source of the general culture medium, and promotes the short compact Penicillium to produce more biomass in the early stage. At the same time, the fermentation is faster and the use of methionine is effectively reduced. On the original basis, the present invention supplements a specific nitrogen source, optimizes the nitrogen source and the ratio, finds a nitrogen source that can increase the titer of intracellular antibiotics, and screens out an OXOID yeast extract that can increase the titer from various yeast powders.

[0033] The nitrogen source in the culture medium of the present invention is very important, and the combination of yeast extract with corn syrup powder, soy molasses, and yeast cell walls plays a very important role in the production of the antibiotic mycophenolic acid. Furthermore, the selection of yeast powder in the nitrogen source is also very important. The best effect in the present invention is OXOID yeast extract, which can significantly increase the yield of the antibiotic mycophenolic acid, and the addition of other yeast powders has almost no effect or even has an inhibitory effect. The intracellular antibiotics required by the present invention belong to the secondary metabolites of Penicillium breviscompactum, and more intracellular antibiotics are produced by accelerating cell metabolism by increasing the nitrogen source. The culture medium of the present invention is relatively simple in composition, and the titer is improved; the fermentation conditions are optimized, and the fermentation time is effectively shortened. Under the preferred formula, the present invention can achieve fermentation for 6.5 days to obtain 9.7g / L of mycophenolic acid.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] (1) The optimized formula improves the nutrient utilization rate of the culture medium and the production intensity of the antibiotic mycophenolic acid;

[0036] (2) There is sufficient nitrogen source in the early stage to accelerate cell metabolism, so there is no need for later feeding, and the titer can reach a relatively high level.

[0037] (2) The culture medium composition is relatively simple, and the fermentation time is significantly shortened, thereby reducing production costs.

[0038] (3) Suitable for the production of intracellular antibiotics, with good universality and application prospects.

[0039] In summary, the optimized culture medium of the present invention can significantly improve the yield and quality of microorganism or cell culture, and the target product mycophenolic acid can be synthesized to the maximum extent by accurately matching the components. The present invention can screen out the key components that have the greatest impact on cell growth yield, so as to perform targeted optimization. This optimization can improve the synthesis level of the target product for a specific metabolic pathway. The optimized culture medium can reduce production costs and improve economic benefits. By rationally configuring the components of the culture medium, not only can the use of expensive reagents be reduced, but also the cost per unit product can be reduced by increasing the yield. The optimization of the culture medium of the present invention can also enhance the scalability of the biomanufacturing process. Reasonable culture medium design helps to smoothly transition from laboratory scale to industrial production scale and maintain the stability and consistency of the product. For example, by gradually optimizing the culture medium, high density can be achieved to improve the efficiency and yield of the bioreactor. The optimized culture medium can also improve the predictability and repeatability of the production process. In summary, the optimized culture medium not only has obvious advantages in improving yield and quality, but also performs well in economic and technological innovation, providing a solid foundation for modern biopharmaceuticals and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The growth curves of antibiotic-producing strains using the culture medium before and after optimization are compared (A is the culture medium before optimization, and B is the culture medium after optimization);

[0041] Figure 2 To show the comparison of antibiotic production using the culture medium before and after optimization;

[0042] Figure 3 To optimize the effect of adding different yeast powders to the nitrogen source on the titer of mycophenolic acid;

[0043] Figure 4 The effect of adding OXOID yeast extract alone or a combination of corn syrup, soy molasses and yeast cell wall on the titer of mycophenolic acid was investigated. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0045] Unless otherwise specified, the raw materials and reagents in the present invention are commercially available.

[0046] Among them, Penicillium breviscompactum CCTCC CF 2008508.

[0047] YP600 Angel Yeast Powder and FM885 Growth-Promoting Yeast Powder were purchased from Angel Yeast Co., Ltd.;

[0048] Brewer's yeast powder (product number: MF-011010) and yeast cell wall (product number: MF-011009) were purchased from Xinyang Mufan Biotechnology Co., Ltd.;

[0049] Soy molasses can be purchased from Xuzhou MuTian Agricultural Technology Co., Ltd., or any commercially available soybean molasses fermentation liquid, which contains: sucrose content 15-20%, monosaccharide 5-10%, crude protein (soy whey protein) 5-8%, and solid foreign matter about 50%.

[0050] OXOID yeast extract (catalog number: LP0021) was purchased from OXOID, UK;

[0051] Corn syrup powder (cas: 66071-94-1, item number: C805620) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0052] The above raw materials can be the same type of raw materials available from other companies or manufacturers on the market.

[0053] Seed culture medium: sucrose 20g / L, raw soybean powder 5g / L, corn syrup powder 5g / L, yeast extract 5g / L, KH2PO41g / L, MgSO41g / L, initial pH value is 6.5, deionized water is the solvent.

[0054] Fermentation medium: glucose 190 g / L, aminoacetic acid 12 g / L, potassium dihydrogen phosphate 3 g / L, methionine 0.4 g / L, trace elements 1 g / L, initial pH value is 4.5 (trace elements: ferrous sulfate heptahydrate, anhydrated copper sulfate, manganese sulfate, zinc sulfate heptahydrate mass ratio is 0.022:0.3:0.12:2.0), deionized water is the solvent.

[0055] Optimized culture medium: glucose 190 g / L, aminoacetic acid 12 g / L, potassium dihydrogen phosphate 3 g / L, methionine 0.4 g / L, OXOID yeast extract 7.5 g / L, corn syrup powder 1 g / L, soy molasses 1 g / L, yeast cell wall 0.5 g / L, trace elements 1 g / L, initial pH value is 4.5 (trace elements: ferrous sulfate heptahydrate, anhydrated copper sulfate, manganese sulfate, zinc sulfate heptahydrate mass ratio is 0.022:0.3:0.12:2.0), deionized water is the solvent.

[0056] Example 1

[0057] 1. Prepare various culture media: seed culture medium, fermentation culture medium, and optimized culture medium.

[0058] 2. Microbial culture: Penicillium breviscapus CCTCC CF 2008508 was inoculated into PDA solid slant medium (potato dextrose agar medium) at 22°C and the humidity of the incubator was controlled at 60%. The spores of the cultured slant (eggplant bottle) were washed with 35 mL of physiological saline, and the spores in the spore solution were fully dispersed with sterilized glass beads. The spores were filtered with lens paper to remove the residual hyphae in the spore solution, and the number of spores in the spore solution was counted with a hemocytometer. The concentration of the spore solution was diluted to 10 7 CFU / mL.

[0059] 3. Seed culture: Inoculate the cell suspension of Penicillium breviscapus at a volume ratio of 2% (10 7 CFU / mL) in seed culture medium and cultured at 24°C and 250 r / min for 26 h to obtain seed solution.

[0060] 4. Microbial fermentation: The seed solution was inoculated into the fermentation medium and the optimized medium at a volume ratio of 10%, and cultured at 24°C and 250 r / min for 6.5 days.

[0061] 3. Wet weight test

[0062] Starting from the third day, the wet weight of cells was measured for five consecutive days to observe the growth status. Figure 1 As shown, it was found that the biomass using the optimized medium was about 10% higher than that of the fermentation medium.

[0063] Example 2

[0064] Under the same conditions, the experimental process of Example 1 was repeated. When the microbial fermentation time was 6.5 days, the fermentation liquid was taken for high performance liquid chromatography (HPLC) detection.

[0065] Detection method:

[0066] Since mycophenolic acid exists almost exclusively inside cells, intracellular substances need to be extracted outside cells. The method for determining the yield of mycophenolic acid in fermentation broth is as follows: Shake the fermentation broth before measuring, and use a 10mL pipette to draw 5mL of fermentation stock into a 250mL conical flask. Pour 95mL of anhydrous ethanol into the conical flask, seal it immediately with a sealing film, oscillate for 5 minutes, and after standing for 2 hours, take 1mL of fermentation broth and centrifuge (3500rpm / min) for 15 minutes. Take the supernatant and dilute it to the corresponding multiple with diluent, and shake it well. Filter it into an injection vial with a syringe-type microporous membrane filter, and use a high performance liquid chromatograph (HPLC) to detect the mycophenolic acid content. Chromatographic detection conditions: chromatographic column C18 (4.6mm×250mm, 5μm), flow rate 1.0mL / min, injection volume 20μL, wavelength 249nm, detection temperature 40°C. The results are as follows. Figure 2It shows that the optimized culture medium produces a high yield (titer) of the antibiotic mycophenolic acid, which can reach 9.7g / L, which is about 17% higher than the fermentation medium, and the effect is very significant.

[0067] Example 3

[0068] Compare the effects of adding different yeast powders in nitrogen sources on the titer of mycophenolic acid.

[0069] According to the method of Example 1, the OXOID yeast extract in the optimized culture medium was replaced with equal amounts of YP600 Angel yeast powder, brewer's yeast powder, and FM885 growth-promoting yeast powder, respectively, and fermentation was performed. At the same time, the test was performed according to the method of Example 2. The results are as follows Figure 3 shown.

[0070] Figure 3 It can be seen that compared with the fermentation medium without yeast, only the optimized medium with OXOID yeast extract added to the culture medium with different types of yeast can significantly increase the mycophenolic acid titer, while other types of yeast extracts will inhibit the mycophenolic acid titer.

[0071] Example 4

[0072] The effects of adding OXOID yeast extract alone or a combination of corn syrup, soy molasses and yeast cell walls on mycophenolic acid titer were compared.

[0073] The culture medium was prepared according to the method of Example 1, wherein the control group was the formula of Example 1; the OXOID group was the formula of Example 1 without adding corn slurry powder, soy molasses and yeast cell wall; the corn slurry powder, soy molasses, yeast cell wall group was the formula of Example 1 without adding OXOID, fermented according to the method of Example 1, and tested according to the method of Example 2. The results are as follows Figure 4 shown.

[0074] Figure 4 It can be seen that the control group (i.e., Example 1) has the best effect and the highest mycophenolic acid titer, while the mycophenolic acid titer of the OXOID group and the corn syrup powder, soy molasses, and yeast cell wall group is significantly reduced, indicating that in the formula of the present invention, the OXOID group and the corn syrup powder, soy molasses, and yeast cell wall group must be used at the same time to have a good synergistic effect and significantly improve the mycophenolic acid titer.

Claims

1. An optimized culture medium for increasing intracellular antibiotic production, It is characterized in that It includes methionine, a carbon source, a nitrogen source and a phosphorus source, and the contents thereof in the culture are 0.3-0.5 g / L of methionine, 180-230 g / L of carbon source, 19-27 g / L of nitrogen source and 3-5 g / L of phosphorus source.

2. The optimized culture medium for increasing intracellular antibiotic production according to claim 1, It is characterized in that The optimized culture medium for increasing the intracellular antibiotic production is preferably an optimized fermentation medium for increasing the production of mycophenolic acid.

3. The optimized culture medium for increasing intracellular antibiotic production according to claim 1, It is characterized in that The nitrogen source comprises aminoacetic acid, yeast extract, corn syrup, soybean molasses and yeast cell wall; the carbon source is glucose, sucrose, fructose or soluble starch.

4. The optimized culture medium for increasing intracellular antibiotic production according to claim 1, It is characterized in that The yeast extract is any one of YP600 Angel yeast powder, brewer's yeast powder, FM885 growth-promoting yeast powder, and OXOID yeast extract.

5. The optimized culture medium for increasing intracellular antibiotic production according to claim 1, It is characterized in that The yeast extract is OXOID yeast extract.

6. The optimized culture medium for increasing intracellular antibiotic production according to claim 1, It is characterized in that The culture medium comprises: 180-230 g / L of glucose, 10-15 g / L of aminoacetic acid, 3-5 g / L of potassium dihydrogen phosphate, 0.3-0.5 g / L of methionine, 7-8 g / L of yeast extract, 1-2 g / L of corn syrup powder, 0.5-1 g / L of soybean molasses, 0.5-1 g / L of yeast cell wall, 1 g / L of trace elements, and a fermentation pH of 4.4-4.7; wherein, among the trace elements, the mass ratio of ferrous sulfate heptahydrate, anhydrous copper sulfate, manganese sulfate, and zinc sulfate heptahydrate is 0.020-0.022:0.2-0.3:0.10-0.12:2.

0.

7. Use of the optimized culture medium for increasing intracellular antibiotic production according to claim 1 in fermenting Penicillium breviscompactum to produce mycophenolic acid.

8. The use according to claim 7, It is characterized in that The fermentation of Penicillium breviscompactum to produce mycophenolic acid includes a seed culture stage and a fermentation culture stage; the Penicillium breviscompactum is CCTCC CF 2008508 9. The use according to claim 8, It is characterized in that The seed culture stage comprises the following steps: taking a spore suspension of Penicillium breviscone for slant culture for 8-10 days, then preparing a bacterial suspension with physiological saline, then inoculating it into a shake flask filled with seed culture medium at a spore inoculation rate of 2-3%, and shaking culture at 24-26°C and 220-260rpm for 26-28h to obtain a shake flask seed culture solution.

10. The use according to claim 8, It is characterized in that The fermentation culture stage comprises the following steps: inoculating the seed culture solution at a volume ratio of 5-10% into a fermentation shake flask containing an optimized culture medium for fermentation culture at a culture temperature of 24-26° C., 220-260 rpm, and a fermentation cycle of 6-7 days.

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