Fermentation medium for producing Ppd A by using marine fungi and production method thereof

Abstract

The invention discloses a culture medium for improving Ppd A production of marine fungi, which comprises the following components in volume of 1000mL: 2.25 to 3.75 g of sodium nitrate, 0.75 to 1.25 g of dipotassium phosphate, 0.375 to 0.625 g of sodium chloride, 0.375 to 0.625 g of magnesium sulfate, 0.0075 to 0.0125 g of ferrous sulfate, 22.5 to 37.5 g of maltose, 7.5 to 12.5 g of yeast extract and the balance of deionized water, and the pH value of the culture medium is 7.0 + / -0.1. The invention also provides a method for producing Ppd A by using the culture medium to ferment and culture deep-sea fungi. When the culture medium and the method provided by the invention are adopted to ferment and culture the marine fungi Didmela sp.FATR0054, the strain can grow quickly in the culture medium and improve the flux of a secondary metabolic pathway, the yield of the target product Ppd A can be improved by using the culture medium to ferment and culture the marine fungi Didmela sp.FATR0054 to produce the Ppd A, the difficulty is reduced for later purification work, and the highest yield of the Ppd A can reach 255.83 mg / L and is 3.98 times of the original level.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation culture medium technology, specifically relating to the technical field of culture medium for culturing marine fungi, and more specifically, to a fermentation culture medium for producing Ppd A using marine fungi and its production method. Background Technology

[0002] Marine fungi are important members of the marine ecosystem, capable of surviving and reproducing under extreme environmental conditions such as high salinity, low temperature, and low light. Due to their long-term adaptation to complex ecological environments, they often carry more diverse and inactive biosynthetic gene clusters, possessing the potential to synthesize novel and uniquely functional metabolites. The natural products of marine fungi exhibit extremely high bioactivity and chemical structural diversity, thus holding enormous potential in the pharmaceutical, agricultural, and industrial fields. With the development of marine resources, it is expected that more bioactive compounds with medicinal potential will be discovered and developed.

[0003] The marine fungus Didymella sp. FATR0054 (GenBank accession number: OQ875733) has the ability to produce a variety of secondary metabolites. It can produce a class of PKS-NRPS natural products, including the macrocyclic alkaloid compound phomapyrrolidone A (Ppd A) with significant anticancer activity, which shows great promise for development.

[0004] However, the low yield of Ppd A under the initial fermentation process has become a bottleneck restricting its application in preclinical studies. Furthermore, this strain is wild-type and has not undergone any genetic engineering modification, so its metabolic potential has not been fully explored. Therefore, there is a need in this field to improve the Ppd A synthesis capacity of Didymella sp. FATR0054 by designing high-yield culture media and optimizing fermentation process parameters.

[0005] Culture media are the essential material basis for microbial growth and metabolism. Their composition directly affects the growth rate, biosynthetic products, and regulation of metabolic pathways. Optimizing culture media composition can not only improve microbial growth efficiency but also regulate the synthesis of secondary metabolites, with significant applications in natural product screening, biosynthesis optimization, and industrial fermentation. Summary of the Invention

[0006] This invention addresses the shortcomings of existing technologies by first optimizing the culture medium for fermentation production of Ppd A to obtain a high-yield culture medium formulation. This is achieved by first optimizing the types of carbon and nitrogen sources, then using PB experimental design to screen components in the culture medium that significantly affect Ppd A yield, and finally optimizing the concentration of these significantly influencing factors using response surface methodology. Ultimately, a suitable high-yield culture medium formulation for Ppd A is obtained.

[0007] Therefore, in a first aspect, the present invention provides a culture medium for improving the production of Ppd A by marine fungi, comprising the following components per 1000 mL volume:

[0008] Sodium nitrate 2.25–3.75 g, dipotassium hydrogen phosphate 0.75–1.25 g, sodium chloride 0.375–0.625 g, magnesium sulfate 0.375–0.625 g, ferrous sulfate 0.0075–0.0125 g, maltose 22.5–37.5 g, yeast extract 7.5–12.5 g, balance deionized water, pH 7.0 ± 0.1.

[0009] According to a preferred embodiment of the present invention, the culture medium comprises the following components per 1000 mL volume:

[0010] Sodium nitrate 2.25g, dipotassium hydrogen phosphate 1.25g, sodium chloride 0.625g, magnesium sulfate 0.42g, ferrous sulfate 0.0084g, maltose 35g, yeast extract 12.5g, balance deionized water, pH 7.0±0.1.

[0011] According to the present invention, the marine fungus is Didymella sp. FATR0054.

[0012] In a second aspect, the present invention provides a method for preparing the culture medium, comprising accurately weighing each component, dissolving it in deionized water, bringing the volume to 1000 mL, and adjusting the pH to 7.0 ± 0.1.

[0013] A third aspect of the present invention provides a method for producing Ppd A by fermenting and culturing deep-sea fungi using the aforementioned culture medium.

[0014] Preferably, the marine fungus is Didymella sp. FATR0054.

[0015] Furthermore, the method includes the following steps:

[0016] Step 1: After thawing the marine fungal spore solution frozen at -80℃, streak it in a solid culture medium and grow it for 7-14 days to obtain a fresh solid culture medium rich in spores.

[0017] Step 2: Scoop out a piece approximately 0.5 cm in size from the fresh marine fungus solid culture medium. 2 Agar blocks were inoculated into seed culture medium and fermented for 72 hours to obtain fresh seed liquid.

[0018] Step 3: Take the fresh seed liquid and inoculate it into the culture medium described in claim 1 or 2 at a volume ratio of 5%. After culturing in a shaker at 28°C and 175 rpm for 168 h, the fermentation broth is obtained.

[0019] Step 4: Take the fermentation broth and perform filtration, extraction and purification to obtain Ppd A.

[0020] According to the present invention, in step one, the formulation of the solid culture medium, based on a volume of 1000 mL, is as follows:

[0021] 30g glucose, 10g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 20g agar powder, and the remainder is deionized water;

[0022] According to the present invention, in step two, the formulation of the seed culture medium, based on a volume of 1000 mL, is as follows:

[0023] 30g glucose, 10g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, with the remainder being deionized water.

[0024] According to the present invention, in step four, the filtration is to perform vacuum filtration on the fermentation broth to separate the bacterial cells; the extraction is to dissolve the bacterial cells obtained by filtration in methanol; and the purification is to centrifuge the extracted bacterial cells and then filter them through a microfiltration membrane.

[0025] The present invention has the following beneficial effects:

[0026] 1. The culture medium of the present invention comprises sodium nitrate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, maltose, and yeast extract. The raw materials are readily available and inexpensive, which is conducive to its widespread application.

[0027] 2. The preparation of the culture medium of the present invention only requires dissolving the above raw materials in deionized water. The method is simple, easy, and requires no special technical training or special instruments and equipment.

[0028] 3. The marine fungus Didymella sp. FATR0054 was fermented and cultured using the culture medium and method of the present invention. This strain grows rapidly in this culture medium, increasing the flux of secondary metabolic pathways. Fermenting and culturing marine fungus Didymella sp. FATR0054 using this culture medium to produce Ppd A can increase the yield of the target product Ppd A, reducing the difficulty of subsequent purification work. The highest yield of Ppd A can reach 255.83 mg / L, which is 3.98 times the original level. Attached Figure Description

[0029] Figure 1 The result is from carbon source optimization.

[0030] Figure 2 The results are from nitrogen source optimization.

[0031] Figure 3 Examples 1, 2, 3, 4, 5 and 6 are comparisons of Ppd A yield and strain growth with the control group.

[0032] Figure 4 , Figure 5 , Figure 6 The results of single-factor gradient experiments on the effects of ferrous sulfate, maltose, and magnesium sulfate on Ppd A yield are shown.

[0033] Figure 7 , Figure 8 , Figure 9 The response surfaces are the effects of ferrous sulfate, maltose, and magnesium sulfate on Ppd A yield, respectively.

[0034] Figure 10 The fermentation process parameter curves of Didymella sp. FATR0054 in a 5L bioreactor are shown. Detailed Implementation

[0035] The present invention will be further explained and illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions provided by the manufacturer.

[0036] All culture medium components used in the following examples are commercially available products. Specifically, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, and maltose were purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium nitrate and sodium chloride were purchased from Shanghai Titan Technology Co., Ltd.; and yeast extract was purchased from Shanghai Chaorui Biotechnology Co., Ltd.

[0037] 1. The bacterial strain used in this invention:

[0038] Marine fungus Didymella sp. FATR0054: GenBank accession number OQ875733. The marine fungus Didymella sp. FATR0054 used in the following examples has accession number CCTCC NO:M 20241087.

[0039] 2. Solid culture medium:

[0040] Accurately weigh 30g of glucose, 10g of peptone, 2g of yeast extract, 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, and 20g of agar powder. Dissolve them in deionized water and bring the volume to 1000mL. Adjust the pH to 7.0±0.1 with sodium hydroxide solution.

[0041] 3. Seed culture medium:

[0042] Accurately weigh 30g of glucose, 10g of peptone, 2g of yeast extract, 1g of potassium dihydrogen phosphate, and 0.5g of magnesium sulfate. Dissolve them in deionized water and bring the volume to 1000mL. Adjust the pH to 7.0±0.1 with sodium hydroxide solution.

[0043] Example 1

[0044] 1.1 Optimization of carbon and nitrogen sources

[0045] By screening more than 10 different culture media, the culture medium formula was determined to be: 30g glucose, 10g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 10g sodium chloride and 1L deionized water.

[0046] The glucose in the basal fermentation medium was replaced with fructose, maltodextrin, sucrose, vegetable oil, mannitol, soluble starch, glycerol, maltose, glucose, and cane molasses, respectively. The mass fraction of each carbon source was the same, and other components remained unchanged. Each group was divided into 3 replicates.

[0047] The organic nitrogen source yeast extract was replaced with soybean flour, peptone, urea, beef extract, corn steep liquor, and malt extract, respectively, with the same mass fraction of each nitrogen source and other components remaining unchanged. Each group had 3 replicates.

[0048] Fermentation was performed using the marine fungus Didymella sp. FATR0054, and the yield of Ppd A and the dry weight of the cell were measured. The experimental results are as follows: Figure 1 and Figure 2 As shown. Figure 1 Based on the results of carbon source optimization, maltose is the optimal carbon source. Figure 2 Based on the results of nitrogen source optimization, it can be seen that the optimal nitrogen source is yeast extract.

[0049] 1.2 Preparation of Culture Medium

[0050] 1.2.1 Preparation of the original culture medium (control group)

[0051] Accurately weigh 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of sodium chloride, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate, 30g of glucose, and 10g of yeast extract. Dissolve them in deionized water and bring the volume to 1000mL. Adjust the pH to 7.0±0.1 with sodium hydroxide solution.

[0052] 1.2.2 Preparation of Fermentation Culture Medium (Experimental Group)

[0053] Accurately weigh 3g of sodium nitrate, 1g of dipotassium hydrogen phosphate, 0.5g of sodium chloride, 0.5g of magnesium sulfate, 0.01g of ferrous sulfate, 30g of maltose, and 10g of yeast extract. Dissolve them in deionized water and bring the volume to 1000mL. Adjust the pH to 7.0±0.1 with sodium hydroxide solution.

[0054] 1.3 Fermentation process

[0055] After thawing the spore suspension of the marine fungus Didymella sp. FATR0054, which had been frozen at -80℃, it was streaked and cultured on a solid medium for 7–14 days, and then cultured on fresh solid medium until conidia were produced. A small agar piece, approximately 0.5 cm in size, was then taken from a fresh solid medium containing abundant spores of the marine fungus Didymella sp. FATR0054. 2 Agar blocks were inoculated into 250 mL shake flasks containing 100 mL of seed culture medium and fermented at 28 °C and 175 rpm for 72 h to obtain fresh seed liquid. The seed liquid was then inoculated into the high-yield culture medium at an inoculation rate of 5% (v / v) and cultured at 28 °C and 175 rpm for 168 h.

[0056] 1.4. Ppd A Yield Detection

[0057] Take 100 mL of fermentation broth and separate the cells from the fermentation broth by vacuum filtration. Extract the cells with excess methanol using ultrasound, evaporate to dryness under reduced pressure using a rotary evaporator (55℃), completely dissolve in 10 mL of methanol, transfer 1.5 mL of the methanol solution to a 2.0 mL EP tube, centrifuge at 12000 rpm, collect the supernatant to a 2.0 mL EP tube, filter through a 0.22 μm filter membrane to a liquid chromatography vial, and store at 4℃ for HPLC analysis.

[0058] The content of Ppd A was determined by external standard method using high performance liquid chromatography. The content of Ppd A in the supernatant and bacterial cells was also determined. The experimental results are as follows: Figure 3 As shown.

[0059] Depend on Figure 3 The results showed that the yield of the target Ppd A measured using the basal culture medium of the experimental group in Example 1 was 141.95 mg / L, which was significantly better than that of the control group; the dry weight of the cells was 15.32 g / L, which was comparable to that of the control group.

[0060] Example 2: Experimental Design, Data Analysis, and Detection Results

[0061] The Plackett-Burman experimental design can analyze the significance of responses to multiple different factors, thereby identifying the most important culture medium components for fermentation yield or cell growth.

[0062] For each factor, two levels, high (+1) and low (-1), were selected. The significance of each factor for the corresponding variable was determined by statistical analysis of the difference between the two levels of each factor and the overall difference.

[0063] Designed using Design Expert 12 data processing software, this experiment selected seven influencing factors and adopted a PB experimental design table with N=12. The factors and levels represented by each parameter are shown in the tables (see Table 1 and Table 2). In Table 1, X1, X2, X3, X4, X5, X6, and X7 represent sodium nitrate, dipotassium hydrogen phosphate, sodium chloride, magnesium sulfate, ferrous sulfate, maltose, and yeast extract, respectively.

[0064] The marine fungus used was Didymella sp. FATR0054, and the experimental results are shown in Table 3.

[0065] Table 1: Plackett-Burman Design and Results

[0066]

[0067] Table 2: Concentration of culture medium in Plackett-Burman design experiments

[0068]

[0069] Table 3: Plackett-Burman Design Analysis of Variance

[0070]

[0071]

[0072] The influence of seven factors on Ppd A yield was analyzed, and the results are shown in Tables 1 and 3. A p-value less than 0.05 indicates that the factor has a significant impact on Ppd A yield. Therefore, magnesium sulfate, ferrous sulfate, and maltose, the three most significant factors affecting Ppd A yield, were selected as the targets for response surface methodology optimization. A total of 12 experimental groups were set up in the PB design. Analysis of variance of the Ppd A yield data showed that groups 1, 2, 3, 5, and 8 were high-yield experimental groups, with Pd A yields all greater than 200 mg / L (see Table 1). This indicates that a maltose concentration of 37.5 g / L, a ferrous sulfate concentration range of 0.0075 g / L to 0.0125 g / L, and a magnesium sulfate concentration range of 0.375 g / L to 0.625 g / L in the culture medium have good yield-enhancing effects and practicality. Example 3 further investigated the center points of magnesium sulfate, ferrous sulfate, and maltose concentrations.

[0073] Group 2 had the highest yield among the 12 groups. The culture medium formula for Group 2 was: sodium nitrate 2.25g, dipotassium hydrogen phosphate 1.25g, sodium chloride 0.625g, magnesium sulfate 0.375g, ferrous sulfate 0.0125g, maltose 37.5g, yeast extract 12.5g, with the remainder being deionized water, and a pH of 7.0±0.1. Figure 3 The results showed that the target Ppd A yield measured using the culture medium of the experimental group in Example 2 reached 237.15 mg / L, which was significantly better than that of the control group.

[0074] The remaining four factors in the second group had a relatively small impact on Ppd A yield. Therefore, the concentration levels of these four factors in the second group were selected for the CCD experiment: Sodium nitrate (-1): 2.25 g / L, dipotassium hydrogen phosphate (+1): 1.25 g / L, sodium chloride (+1): 0.625 g / L, and yeast extract (+1): 12.5 g / L. Further CCD optimization was performed in Example 3, taking into account the concentration ranges of magnesium sulfate, ferrous sulfate, and maltose.

[0075] Example 3: Single-factor gradient experiment and response surface methodology

[0076] 3.1 Single-factor gradient experiment

[0077] Based on the Plackett-Burman experimental results, magnesium sulfate (X4), ferrous sulfate (X5), and maltose (X6) were selected for response surface methodology to investigate their interactions and optimize their composition.

[0078] To obtain more accurate response surface methodology results, the center point near the optimal region was first determined. A single-factor gradient variation was used to determine the center point, and the experimental results are as follows: Figure 4 , Figure 5 and Figure 6 As shown, ferrous sulfate 0.01 g / L, maltose 35 g / L, and magnesium sulfate 0.4 g / L are most favorable for the production of Ppd A. Therefore, this combination was ultimately selected as the center point for the next response surface methodology experiment.

[0079] 3.2 Response Surface Design

[0080] The response surface methodology employed a three-factor central composite design (CCD), with three replicates per group. The experimental design was constructed using the statistical analysis software Design Expert 12, and the specific factor levels and corresponding Ppd A yields are listed in Table 4.

[0081] The three factors are ferrous sulfate (X1), magnesium sulfate (X2), and maltose (X3). Each factor has five levels, with corresponding code values ​​of -1.68, -1, 0, +1, and +1.68. The specific concentrations are as follows:

[0082] X1: Ferrous sulfate—0.0058 g / L, 0.0075 g / L, 0.01 g / L, 0.0125 g / L, 0.0142 g / L;

[0083] X2: Magnesium sulfate—0.064 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.736 g / L;

[0084] X3: Maltose—26.6g / L, 30g / L, 35g / L, 40g / L, 43.4g / L.

[0085] This design incorporates combinations of center points, pivot points, and factor levels, effectively evaluating single-factor effects, interactions, and secondary effects, providing a reliable foundation for model building and optimal condition prediction. The CCD design possesses high statistical power and is suitable for establishing response surface models and finding the optimal ratio of factors.

[0086] Table 4: Central Composite Design Table and Ppd A Yield

[0087]

[0088] This study used Design Expert 12 software to perform regression analysis on the results of the central composite experiment. The results of the analysis of variance are shown in Table 5.

[0089] Table 5: Analysis of Variance of Experimental Results for Central Composite Design

[0090]

[0091] The results showed that when the p-value was less than 0.05, the regression model was statistically significant overall, and the lack-of-fit term was not significant, indicating that the model fit was good and there was no serious bias. The model's coefficient of determination R0... 2 The coefficient of variation (COP) is 0.8965, indicating a high degree of fit and the ability to accurately explain 89.65% of the variation in the experimental data. Furthermore, the COP is 4.45%, which is within a reasonable range, indicating good experimental repeatability and reliable results. By performing a quadratic polynomial regression on the experimental data, the following regression equation was obtained:

[0092] R=242.88-14.93A+11.63B+11.13C+2.40AB+6.46AC-7.78BC-5.40A 2 -10.18B 2 +1.10C 2

[0093] Figure 7 , Figure 8 and Figure 9 The response surface plots for the three factors (ferrous sulfate X1, magnesium sulfate X2, and maltose X3) in the central composite design experiment are presented. The figures show that the interaction surfaces of each pair of factors exhibit significant peaks, indicating that under certain specific combinations, they contribute to increased Ppd A yield. This also demonstrates a synergistic effect among the three factors, collectively influencing the synthesis efficiency of Ppd A.

[0094] Based on the above analysis, partial derivatives of the model equations were solved and response surface optimization predictions were performed using Design Expert 12 software. The results showed that the yields of groups 1, 3, 9, 12, and 14 to 20 were all above 230 mg / L. This indicates that when the concentrations of ferrous sulfate (X1) in the culture medium are in the range of 0.0075–0.0125 g / L, magnesium sulfate (X2) in the range of 0.2–0.6 g / L, and maltose (X3) in the range of 30–35 g / L, a good product enhancement effect is achieved.

[0095] Preferably, group 3 yielded the highest Ppd A. The culture medium formulation was: sodium nitrate 2.25 g / L, dipotassium hydrogen phosphate 1.25 g / L, magnesium sulfate 0.6 g / L, ferrous sulfate 0.0075 g / L, maltose 30 g / L, yeast extract 12.5 g / L, and pH 7.0 ± 0.1. Under these conditions, combined with... Figure 3 The target Ppd A yield measured using the culture medium of the experimental group in Example 3 reached 262.70 mg / L, which was significantly better than that of the control group.

[0096] Design Expert 12 software predicted that when the culture medium formulation was: sodium nitrate 2.25 g / L, dipotassium hydrogen phosphate 1.25 g / L, sodium chloride 0.625 g / L, magnesium sulfate 0.42 g / L, ferrous sulfate 0.0084 g / L, maltose 35 g / L, yeast extract 12.5 g / L, and pH 7.0 ± 0.1, the Ppd A yield could reach 258.58 mg / L. Example 4 conducted a shake-flask verification experiment.

[0097] Example 4: Shaking test

[0098] To verify the effectiveness and reliability of this statistical method, the predicted formula from Example 3 was tested three times, with three replicates each time. The predicted formula was as follows: 2.25g sodium nitrate, 1.25g dipotassium hydrogen phosphate, 0.625g sodium chloride, 0.42g magnesium sulfate, 0.0084g ferrous sulfate, 35g maltose, and 12.5g yeast extract were weighed, dissolved in deionized water, and brought to a final volume of 1000mL. The pH was adjusted to 7.0±0.1 with 3M sodium hydroxide solution.

[0099] Ultimately, the experimental result was 255.63 mg / L. Optimization and prediction were performed using Design Expert 12 software. The partial derivative of the fitted equation was calculated, and the predicted result was 258.58 mg / L. The validation value was close to the predicted value, demonstrating the reliability of the regression equation and the effectiveness of the statistical method.

[0100] Table 6: Validation of Optimized Culture Medium Yield

[0101]

[0102] After confirming the culture medium components, the final yield of Ppd A reached 255.63 mg / L.

[0103] Example 5

[0104] 5.1 Preparation of Fermentation Culture Medium

[0105] Weigh out 3.75g sodium nitrate, 1.25g dipotassium hydrogen phosphate, 0.625g sodium chloride, 0.625g magnesium sulfate, 0.0125g ferrous sulfate, 37.5g maltose, and 7.5g yeast extract. Dissolve them in deionized water and bring the volume to 1000mL. Adjust the pH to 7.0±0.1 with 3M sodium hydroxide solution.

[0106] 5.2 Fermentation Operation Process

[0107] The fermentation process is the same as in Example 1.

[0108] 5.3 Ppd A Yield Detection

[0109] The extraction and detection methods are the same as in Example 1.

[0110] The final yield of the target compound Ppd A was measured to be 227.82 ± 15.93 mg / L.

[0111] Example 6

[0112] 6.1 Preparation of Fermentation Culture Medium

[0113] Weigh out 2.25g sodium nitrate, 0.75g dipotassium hydrogen phosphate, 0.375g sodium chloride, 0.375g magnesium sulfate, 0.0075g ferrous sulfate, 22.5g maltose, and 7.5g yeast extract. Dissolve them in deionized water and bring the volume to 1000mL. Adjust the pH to 7.0±0.1 with 3M sodium hydroxide solution.

[0114] 6.2 Fermentation Operation Process

[0115] The fermentation process is the same as in Example 1.

[0116] 6.3 Ppd A Yield Detection

[0117] The extraction and detection methods are the same as in Example 1.

[0118] The final yield of the target compound Ppd A was 181.20 ± 2.53 mg / L.

[0119] The results showed that optimizing the carbon and nitrogen sources of the selected preferred culture media (Example 2) yielded the optimal culture medium formulation described in this invention (Example 4), resulting in a Ppd A yield of 255.63 mg / L. Simultaneously, the Ppd A yields in Examples 5 and 6 reached 227.82 mg / L and 181.20 mg / L, respectively. Figure 3 The results showed that, compared with the control group, the production of PpdA by culturing marine fungus Didymella sp. FATR0054 using the fermentation medium used in this invention was significantly improved.

[0120] Example 7

[0121] 7.1 Preparation of Fermentation Culture Medium

[0122] Using the formulation of Example 4, 6.75g of sodium nitrate, 3.75g of dipotassium hydrogen phosphate, 1.875g of sodium chloride, 1.26g of magnesium sulfate, 0.0252g of ferrous sulfate, 105g of maltose, and 37.5g of yeast extract were weighed, dissolved in deionized water, and brought to a final volume of 3000mL. The pH was adjusted to 7.0±0.1 with 3M sodium hydroxide solution.

[0123] 7.2 Fermentation Operation Process

[0124] After thawing the spore suspension of the marine fungus Didymella sp. FATR 0054, which had been frozen at -80℃, it was streaked and cultured on a solid medium for 7–14 days, and then cultured on fresh solid medium until conidia were produced. A small agar sample, approximately 0.5 cm in size, was then taken from a fresh solid agar plate containing abundant spores of the marine fungus Didymella sp. FATR 0054. 2 Agar blocks were inoculated into 250mL shake flasks containing 100mL of seed culture medium and fermented at 28℃ and 175rpm for 72h to obtain fresh seed liquid. The seed liquid was then inoculated into a 5L fermenter at an inoculation rate of 5% (v / v) and cultured at 28℃ and 175rpm for 168h before collecting the fermentation broth.

[0125] 7.3 Ppd A Yield Detection

[0126] The extraction and detection methods are the same as in Example 1.

[0127] The fermentation process for this batch was as follows: aeration rate of 1.0 vvm, tank pressure of 0.03 MPa, and temperature of 28℃. The stirring rate was automatically adjusted based on dissolved oxygen levels. During fermentation, changes in cell dry weight (DCW), PpdA yield, pH, and dissolved oxygen (DO) were monitored. Relevant data are as follows: Figure 10 As shown, the bacterial cells grew rapidly in the first 48 hours, with DCW reaching 4.56 g / L and DO decreasing to 3%, indicating active cell metabolism. Subsequently, product accumulation began, with Ppd A concentration reaching 70.51 mg / L at 96 hours and peaking at 257.00 mg / L at 168 hours, with a cell dry weight of 13.57 g / L. The pH stabilized at around 9, and DO fluctuated between 2.5% and 18%. These results indicate that the formulation can support high-density growth and high-yield accumulation, making it suitable for pilot-scale production.

[0128] In summary, the culture medium of the present invention has simple components, convenient preparation method, low cost, and does not require special equipment, and can significantly improve the yield of Ppd A produced by marine fungus Didymella sp. FATR0054.

[0129] The above description is merely an example of the embodiments of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A culture medium for improving the production of Ppd A by marine fungi, characterized in that, Based on a volume of 1000 mL, it includes the following components: Sodium nitrate 2.25–3.75 g, dipotassium hydrogen phosphate 0.75–1.25 g, sodium chloride 0.375–0.625 g, magnesium sulfate 0.375–0.625 g, ferrous sulfate 0.0075–0.0125 g, maltose 22.5–37.5 g, yeast extract 7.5–12.5 g, balance deionized water, pH 7.0 ± 0.

1.

2. The culture medium according to claim 1, characterized in that, Based on a volume of 1000 mL, it includes the following components: Sodium nitrate 2.25g, dipotassium hydrogen phosphate 1.25g, sodium chloride 0.625g, magnesium sulfate 0.42g, ferrous sulfate 0.0084g, maltose 35g, yeast extract 12.5g, balance deionized water, pH 7.0±0.

1.

3. The culture medium according to claim 1 or 2, characterized in that, The marine fungus is Didymella sp. FATR0054.

4. The method for preparing the culture medium according to any one of claims 1 to 3, characterized in that, This includes accurately weighing each component, dissolving it in deionized water, bringing the volume to 1000 mL, and adjusting the pH to 7.0 ± 0.

1.

5. A method for producing Ppd A by fermenting and culturing deep-sea fungi, characterized in that, Use the culture medium according to any one of claims 1 to 3.

6. The method according to claim 5, characterized in that, The marine fungus is Didymella sp. FATR0054.

7. The method according to claim 5 or 6, characterized in that, Includes the following steps: Step 1: After thawing the marine fungal spore solution frozen at -80℃, streak it in a solid culture medium and grow it for 7-14 days to obtain a fresh solid culture medium rich in spores. Step 2: Scoop out a piece approximately 0.5 cm in size from the fresh marine fungus solid culture medium. 2 Agar blocks were inoculated into seed culture medium and fermented for 72 hours to obtain fresh seed liquid. Step 3: Take the fresh seed liquid and inoculate it into the culture medium described in claim 1 or 2 at a volume ratio of 5%. After culturing in a shaker at 28°C and 175 rpm for 168 h, the fermentation broth is obtained. Step 4: Take the fermentation broth and perform filtration, extraction and purification to obtain Ppd A.

8. The method according to claim 5 or 6, characterized in that, In step one, the formula for the solid culture medium, based on a volume of 1000 mL, is as follows: 30g glucose, 10g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 20g agar powder, and the remainder is deionized water.

9. The method according to claim 5 or 6, characterized in that, In step two, the seed culture medium formula is as follows, based on a volume of 1000 mL: 30g glucose, 10g peptone, 2g yeast extract, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, with the remainder being deionized water.

10. The method according to claim 5 or 6, characterized in that, In step four, the filtration is to perform vacuum filtration on the fermentation broth to separate the bacterial cells; the extraction is to dissolve the bacterial cells obtained by filtration in methanol; and the purification is to centrifuge the extracted bacterial cells and then filter them through a microfiltration membrane.

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