Fermentation process method for high yield of wuyiencin
By optimizing the fermentation medium and feeding strategy, using glucose and fish meal as the main ingredients, and controlling the glucose and ammonia nitrogen concentrations in the fermentation broth, the problems of low fermentation level and high cost of wuyimycin were solved, and efficient biosynthesis was achieved and production costs were reduced.
Patent Information
- Application Number
- CN202510784531.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-12
- Publication Date
- 2025-09-16
AI Technical Summary
The current fermentation level of wuyimycin is low and the production cost is too high, which limits its industrialization and marketization process.
The carbon, nitrogen and phosphorus sources of the fermentation medium were optimized, and combined with the feeding strategy, glucose and fish meal were used as the main ingredients. By controlling the glucose and ammonia nitrogen concentrations in the fermentation broth, a batch fermentation method was adopted to regulate the fermentation process.
The fermentation yield of wuyilabin was significantly improved, the production cost was reduced, the difficulty of fermentation regulation was reduced, and large-scale synthesis was achieved.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial fermentation, in particular to a fermentation process for high-yield wuyienin. Background Art
[0002] Wuyimycin is a new type of agricultural antibiotic with low toxicity, high efficiency, broad spectrum and environmental protection, with completely independent intellectual property rights in my country. It is produced by fermentation of the Wuyi strain of Streptomyces parviflorus. This biological preparation has a broad-spectrum antibacterial activity against fungal diseases of crops, among which the control efficiency of powdery mildew of cucumber reaches over 90% (equivalent to triadimefon), and the control effect against gray mold and black spot is significant. It achieves efficient prevention and control by inhibiting the expansion of pathogenic hyphae and the spread of spores. It has been applied on a large scale to control fungal diseases of fruit and vegetable crops, with the comprehensive benefits of reducing chemical pesticide residues, increasing crop yields and the sustainability of farmland ecology. However, the current production cost of wuyimycin is too high and the fermentation level is low, which greatly restricts its industrialization and marketization process. How to improve the fermentation level of wuyimycin by improving the fermentation process has become a goal pursued by the industry.
[0003] At present, the fermentation level is low and is limited by factors such as culture medium. Analysis of Wuyimycin shows that its main component, Wuyimycin A, is a nucleoside antibiotic with a molecular formula of C 13 H 21 N3O 14 , containing a cytidine backbone and peroxide bonds, with precursors primarily consisting of UDP-glucose, cytosine, and triphosphoglycerate. Glucose, as a fast-acting carbon source, provides a foundation for cells to rapidly replenish the carbon backbone, precursors for product synthesis, and ATP supply, thereby ensuring the ATP supply of wuyimycin and promoting product synthesis. Higher concentrations of glucose are beneficial for promoting cell growth and metabolism in the fermentation broth.
[0004] It is considered that the replacement of fish meal peptone has a positive impact on product synthesis. Fish meal peptone, which is rich in purine and pyrimidine substances, is used as a nitrogen source. Because purine and pyrimidine are important components of nucleic acids, fish meal peptone, as a natural nitrogen source, contains rich nucleic acid decomposition products, which are precursors for the synthesis of wuyi mycin and important components of cellular DNA synthesis. It is speculated that the choice of fish meal peptone as a nitrogen source may provide microorganisms with conditions closer to their natural growth environment, thereby facilitating their growth and product synthesis. At the same time, the market price of yeast powder is 30,000 yuan / ton, while the market price of fish meal is 12,000 yuan / ton, which is 40% of yeast powder. Therefore, it is expected to replace yeast powder to reduce raw material costs. However, the fermentation of fish meal instead of yeast powder may lead to problems such as excessive bacterial mass, insufficient oxygen supply, and abnormal metabolism, which in turn leads to low product synthesis levels.
[0005] In view of the current problems of low production efficiency and high production cost of wuyimycin synthesized by microbial fermentation, the development of an industrial fermentation method for the production of wuyimycin has important application value. Summary of the Invention
[0006] The purpose of the present invention is to provide a high-yield wuyimycin fermentation process method to solve the problems existing in the above-mentioned prior art. By optimizing the carbon source, nitrogen source and phosphorus source of the fermentation medium and combining the feeding strategy with batch fermentation, the fermentation yield of wuyimycin is significantly improved, the production cost is reduced, the feedback inhibition and control operation load of the fermentation process are avoided, and the difficulty of fermentation control is greatly reduced, which is of great value for realizing large-scale synthesis of wuyimycin.
[0007] To achieve the above object, the present invention provides the following solutions:
[0008] The present invention provides a high-yield wuyiin fermentation process, comprising the steps of inoculating a fermentation medium with Streptomyces noursei CK-15 and then controlling the concentrations of glucose and ammonia nitrogen in the fermentation broth by feeding;
[0009] When the glucose concentration of the fermentation broth is lower than 5 g / L, a glucose solution is added through an exogenous flow to maintain the glucose concentration in the fermentation broth at 5-15 g / L; when the ammonia nitrogen concentration of the fermentation broth is lower than 0.5 g / L, an ammonium sulfate solution is added through an exogenous flow to maintain the ammonia nitrogen concentration in the fermentation broth at 0.5-1.0 g / L.
[0010] Preferably, the fermentation medium comprises the following components at the following concentrations: 50 g / L glucose, 5 g / L fish meal, 10 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 0.34 g / L potassium dihydrogen phosphate, and 0.04 g / L zinc sulfate.
[0011] Preferably, the inoculation amount of the Streptomyces noursei CK-15 is 5%-10%, the fermentation temperature is 30° C., the rotation speed is 200-600 rpm, the dissolved oxygen is 10%-30%, and the initial pH value is regulated to 6.5.
[0012] Preferably, when the pH of the fermentation broth drops to 6.0, liquid ammonia is used for dynamic feeding to maintain the pH at 6.
[0013] Preferably, the fermentation time is 120-170 hours.
[0014] Preferably, the fermentation broth is separated and purified by HPLC to obtain wuyinomycin.
[0015] The present invention also provides the use of the fermentation broth obtained by the method in any of the following:
[0016] (1) Application in inhibiting red yeast;
[0017] (2) Application in the preparation of products that inhibit red yeast.
[0018] The present invention also provides the use of the fermentation broth obtained by the method in any of the following:
[0019] (1) Application in inhibiting plant pathogenic fungi;
[0020] (2) Application in the preparation of products for inhibiting plant pathogenic fungi;
[0021] The plant pathogenic fungi include tomato gray mold, apple rot, rice blast and wheat fusarium.
[0022] The present invention also provides a red yeast antibacterial agent, comprising the fermentation liquid obtained by the method or wuyimycin.
[0023] The present invention also provides a plant pathogenic fungus antibacterial agent, comprising the fermentation liquid or wuyimycin obtained by the method, wherein the plant pathogenic fungi include tomato gray mold, apple rot fungus, rice blast fungus and wheat fusarium.
[0024] The present invention discloses the following technical effects:
[0025] This invention combines culture medium optimization and feeding strategies to address the low production efficiency and high production costs of wuyimycin biosynthesis. A novel process has been developed using Streptomyces noursei CK-15 as the production strain, glucose and fish meal as the main components of the fermentation medium, along with a suitable feeding strategy and the regulation of the negative impact of phosphorus sources on the fermentation process. This method not only increases wuyimycin fermentation yield and reduces production costs, but also avoids feedback inhibition and operational load during the fermentation process, significantly reducing the difficulty of fermentation regulation, and possesses significant industrial application value in wuyimycin biosynthesis.
[0026] The new process provided by the present invention is characterized by selecting glucose as a fast-acting carbon source to promote cell growth and metabolism in the fermentation broth, screening fish meal as a suitable nitrogen source and the optimal phosphorus source content for the synthetic product, and combining it with a fed-batch fermentation method to control the carbon and nitrogen source levels during the fermentation process, thereby promoting the synthesis of wuyimycin. This method significantly improves the biosynthesis efficiency of wuyimycin by optimizing the metabolic regulatory network, while effectively reducing the complexity of the precise control of the feeding process in the fermentation system. It shows important application potential in industrial production, and its wuyimycin yield can reach 9.44g / L, which is much higher than the yield of wuyimycin biosynthesized by existing technologies. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0028] Figure 1 This is the statistical result of Wuyimycin production when yeast powder is used as nitrogen source;
[0029] Figure 2 This is the dry weight test result of mycelium when yeast powder is used as nitrogen source;
[0030] Figure 3 The results of the fermentation test of wuyinicin content and dry weight of the fungus using fish meal as nitrogen source are shown in the figure.
[0031] Figure 4 Results of wuyienin content detection when fish meal and yeast powder were used as nitrogen sources for fermentation; A: wuyienin content when fish meal was used as nitrogen source, B: wuyienin content when yeast powder was used as nitrogen source; C: comparison chart of wuyienin content detection when fish meal and yeast powder were used as nitrogen sources;
[0032] Figure 5 The chromatograms of the wuyimycin content at different time points were obtained using a 5L fermenter with fish meal as the nitrogen source for fermentation in Example 2; A: peak of the standard wuyimycin product; B: fermentation 12 h, C: fermentation 24 h, D: fermentation 36 h, E: fermentation 48 h, and F: fermentation 60 h.
[0033] Figure 6 The chromatograms of the wuyimycin content at different time points were obtained using a 5 L fermenter with fish meal as the nitrogen source for fermentation in Example 2; A: 72 h of fermentation, B: 84 h of fermentation, C: 96 h of fermentation, D: 108 h of fermentation, and E: 120 h of fermentation;
[0034] Figure 7 Effects of fermentation medium containing different phosphates on the dry weight of the bacteria and the content of wuyimycin; A: dry weight of the bacteria under three groups of phosphate concentrations, B: content of wuyimycin under three groups of phosphate concentrations;
[0035] Figure 8 To compare the content of wuyinicin in fermenters with and without organic nitrogen sources;
[0036] Figure 9 The results of the antibacterial activity test of fermentation broth against red yeast rice were obtained before and after fermentation condition optimization. Three culture dishes were used for three replicates. The left side of each culture dish shows the fermentation broth obtained under the optimized fermentation conditions of the present invention, and the right side shows the fermentation broth obtained using fermentation medium II before optimization.
[0037] Figure 10 The results of the antibacterial activity test on plant pathogenic fungi of the fermentation broth collected before and after the optimization of fermentation conditions;
[0038] Figure 11 To detect the content of wuyinomycin in the fermentation broth after 120 hours of fermentation after optimization of fermentation conditions. DETAILED DESCRIPTION
[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0040] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.
[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.
[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.
[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.
[0044] Example 1: Optimization of fast-acting carbon sources for wuyinicin biosynthesis
[0045] Streptomyces noursei CK-15, from the Agricultural Antibiotics Research Group at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, was previously known as Streptomyces albulus CK-15 (Genebank accession number NZ_CP026094.1). Following genome sequencing, the strain was renamed and classified by NCBI in 2024 as Streptomyces nourseistrain CK-15.
[0046] 1. Culture medium preparation:
[0047] Prepare SFM medium: Boil 20g of soybean meal in distilled water for 30 minutes. Filter through four layers of gauze and dilute to 1000mL. Aliquot 100mL into 250mL Erlenmeyer flasks containing 2.0g of mannitol and 1.7g of agar powder. Sterilize with moist heat at 121°C for 30 minutes. Eggplant spore culture conditions: 28-30°C for 6-8 days.
[0048] Solid Betana medium (g / L): 10 g / L glucose, 1 g / L yeast extract, 2 g / L peptone, and 20 g / L agar, adjusted to pH 7.5 with sodium hydroxide. The solute is water (the same applies to other culture media involved in the present invention, in which the solute is water).
[0049] Liquid seed culture medium (g / L): glucose 50 g / L, fish meal 5 g / L, ammonium sulfate 10 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.8 g / L, potassium dihydrogen phosphate 1.36 g / L, ferrous sulfate 0.03 g / L and zinc sulfate 0.04 g / L, adjusted to pH 7 with sodium hydroxide.
[0050] Wuyimycin fermentation medium (g / L):
[0051] (1) Fermentation medium I: glucose 20 g / L, soybean powder 20 g / L, ammonium sulfate 4 g / L, corn flour 30 g / L, and calcium carbonate 3 g / L.
[0052] (2) Fermentation medium II: glucose 50 g / L, yeast powder 5 g / L, ammonium sulfate 10 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 0.34 g / L, and zinc sulfate 0.04 g / L.
[0053] 2. Fermentation seed culture:
[0054] Streptomyces noursei CK-15 was inoculated onto SFM medium or solid Betana medium and cultured in a 30°C constant temperature incubator for 5-6 days until spores grew. Three rings of spores were picked with an inoculation loop and placed in a 500mL triangular flask containing 60mL of liquid seed culture medium. The mixture was cultured in a 30°C constant temperature shaking incubator at 200rpm for 1-2 days to obtain fermentation seeds. A 5L fermentor-fed batch fermentation experiment of Wuyimycin was conducted based on the excellent culture medium and the optimal feeding strategy. A 5L liquid fermentor was selected, and two different blade shapes were used: a disc-shaped six-blade turbine agitator was used for the lower blade, and a four-blade propeller-type agitator was used for the upper blade.
[0055] 3. Fermentation culture:
[0056] The wuyijinsu fermentation medium, minus glucose, was prepared and added to a 5L fermentor tank. The total volume was set to 3L and sterilized at 121°C for 21 minutes. Stirring was immediately started (200-600 rpm, speed coupled with dissolved oxygen) and the temperature control system was turned on to cool the fermentor tank to 30°C. After this work was completed, 500mL of a separately sterilized solution containing 150g of glucose (sterilized at 121°C for 21 minutes) was added using a peristaltic pump to form the fermentation medium. The fermentation seed obtained above was inoculated with an 8% inoculum size and fermented under the conditions of an initial pH of 6.5, a temperature of 30°C, and a dissolved oxygen content of at least 30%. The pH value of the fermentation was controlled throughout the fermentation process. When the glucose concentration was below 5g / L, a pre-sterilized glucose solution with a mass fraction of 60% was added from an external source to maintain the glucose concentration in the fermentation liquid at 5-15g / L. Fermentation ended when the wuyijinsu concentration no longer increased. During the fermentation process, samples were taken every 12 h to determine the concentration of wuyinicin and the dry weight of mycelium.
[0057] (1) Wuyimycin content detection:
[0058] Take the fermentation broth of fermentation 12-120h, centrifuge the fermentation broth (2000rpm, 15min) to take the supernatant, take 1mL of the supernatant, add 1mL of 10% trichloroacetic acid (TCA) solution and centrifuge again (8000rpm, 15min), take 200μL in a sampling bottle, and use high performance liquid chromatography (HPLC) to measure the content of the strain fermentation broth product. Use distilled water to prepare the standard product to 1000μg / mL, and use the wuyimycin standard product as the control to detect the wuyimycin content in the fermentation sample. The response peak area of the sample calculated by the liquid chromatograph analysis software can be compared with the standard product to obtain the content of wuyimycin in the fermentation broth of each strain (see the chromatogram of the wuyimycin content detected at different time points within 12-120h). Figure 5-Figure 6 ). HPLC conditions are as follows:
[0059] Instrument model: Thermofisher U3000 high performance liquid chromatograph;
[0060] Chromatographic column: Waters Symmetry C18 Column, 5 μm, 4.6 mm × 250 mm;
[0061] Mobile phase: 1.4 g / L TCA aqueous solution;
[0062] Flow rate: 1 mL / min;
[0063] Temperature: 40℃;
[0064] Detection wavelength: UV 254nm;
[0065] Injection volume: 20 μL;
[0066] Analysis time: 20 minutes.
[0067] In a 5L liquid fermentation tank, a total volume of 3L of liquid culture medium was prepared according to the formula for wuyijinsu fermentation medium. The carbon source was selected as a single carbon source, glucose at a concentration of 50g / L. The medium was sterilized in a high-temperature autoclave at 115°C for 15 minutes and then immediately removed to avoid the Maillard reaction. Mature fermentation seeds were inoculated with the above fermentation medium at an 8% inoculum and cultured in a 5L liquid fermentation tank for 5-7 days. Samples were taken every 12 hours to test the carbon and nitrogen source content to ensure that the fermentation process was not lacking nutrients. In particular, the glucose and ammonium sulfate in the feed both act as precursors in product synthesis. After completion, the wuyijinsu concentration in the fermentation broth was measured, with high wuyijinsu production as the primary indicator. The final wuyijinsu concentration in the fermentation broth was used as a control. Data showed that the original fermentation medium I, which contained the relevant corn flour slow-release carbon source, had a fermentation yield of 3-4g / L. The medium using glucose as the carbon source and yeast powder as the nitrogen source (i.e., fermentation medium II), which was fermented in a batch-fed manner, showed a peak elution time of about 7 minutes, as shown in Table 1. HPLC test results are as follows Figure 1 As shown in the figure, at 120 h of fermentation, the titer of wuyimycin reached a maximum of 6016.53 ppm, that is, the yield of wuyimycin was 6.017 g / L.
[0068] Table 1 Wuyisin content at each time point
[0069]
[0070] (2) Determination of mycelium dry weight:
[0071] Take the fermentation liquid after 12-120 hours of fermentation, filter the precipitate with filter paper, dry the filter paper and weigh it, filter the precipitate after suction, put it into an 80℃ oven to dry, wait until it is completely dried, and calculate the difference between the mycelium and the filter paper, which is the dry weight of the mycelium. Figure 2 As shown, when yeast powder was used as nitrogen source, mycelium grew fastest during 12-72 h, grew slowly during 72-96 h, and showed a downward trend during 96-120 h. It reached the highest point at 96 h of fermentation, with a dry weight of 43.72 g.
[0072] Example 2: Optimization of nitrogen sources for wuyinicin biosynthesis
[0073] In this example, fish meal was used instead of yeast powder as the nitrogen source.
[0074] 1. Fermentation medium
[0075] Improved M3G fermentation medium: glucose 50 g / L, fish meal 5 g / L, ammonium sulfate 10 g / L, magnesium sulfate 0.5 g / L, potassium dihydrogen phosphate 0.34 g / L, and zinc sulfate 0.04 g / L.
[0076] 2. Fermentation control parameters of 5L fermenter
[0077] 2.1 Pretreatment of 5 L fed-batch fermentation
[0078] Select 5L liquid fermentation tank, use two different blade forms, wherein the lower blade uses disc six straight blade turbine agitator, and the upper layer adopts four-blade propeller type agitator. The preparation removes the wuyi bacterium fermentation medium of glucose, adds in 5L fermentation tank, total loading liquid volume is set to 3L, sterilizes at 121 ℃ for 20min, starts stirring immediately after finishing (200-600rpm), and opens the temperature control system and makes fermentation tank cool to 30 ℃. After this work finishes, 500mL aqueous solution that is dissolved with 150g glucose, sterilized separately (115 ℃ sterilization for 15min, subsequent other feed raw material sterilization also all is carried out under this condition) is all filled in with peristaltic pump, final concentration 30%, is made into fermentation medium.
[0079] 2.2 Sterilization and feeding
[0080] (1) Sterilization of culture medium: 1.8m 3 A modified M3G culture medium was prepared according to the specifications, added to a 5-liter fermentor, and sterilized at 121°C for 20 minutes. Glucose was sterilized separately to prevent the Maillard reaction of glucose at high temperatures. The sterilized 5-liter fermentor was temperature-controlled at 30°C, with the dissolved oxygen level at 100% under factory-standard conditions, and the corresponding sterile air flux and stirring speed were maintained. A previously separately sterilized 30% glucose solution (the carbon source component of the modified M3G fermentation medium) was pumped into the fermentor using a liquid pump.
[0081] (2) Preparation and addition of feed substrate: Fermentation seeds (preparation method see Example 1) were inoculated into the fermentation medium at an inoculum size of 8%, and fermentation was carried out under the culture conditions of initial pH 6.5, temperature 30°C, and dissolved oxygen maintained at above 10-30%. The pH value of the whole fermentation process was controlled at 6. When the glucose concentration in the fermentation broth dropped to 5 g / L, 60% sterile glucose solution was added by exogenous flow to maintain the glucose concentration in the range of 5-15 g / L, and 40% ammonium sulfate solution was added to maintain the ammonia nitrogen concentration in the range of 0.5-
[0082] 1.0g / L until the synthesis of wuyinicin stops.
[0083] For carbon source supplementation, use 60% glucose solution; for ammonia nitrogen supplementation, use 40% ammonium sulfate solution; for alkali solution, use 50% ammonia solution. All supplements must be sterilized at 15°C for 15 minutes before use.
[0084] The control group used fermentation medium (see fermentation medium II in Example 1), and the experimental group used modified M3G fermentation medium (glucose 50g / L, fish meal 5g / L, ammonium sulfate 10g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 0.34g / L and zinc sulfate 0.04g / L) for fermentation. The fermentation tank is connected to a condensing water circulation machine to control the tank temperature to 30°C. The fermentation process is regulated by continuous feeding, in which ammonia water is used as an alkali liquid to dynamically control the pH of the fermentation liquid to 6.0. When adding the fermentation liquid, the pH needs to be controlled to 6 in advance. The addition of the fermentation liquid will cause the pH to decrease and needs to be adjusted in time. At the same time, the ventilation volume is maintained at 1vvm, and the rotation speed is coupled with the dissolved oxygen so that the dissolved oxygen is between 10% and 30%.
[0085] 2.3 Fermentation parameter control
[0086] (1) pH: Set the initial pH to 6.5. After the mature seed solution is transferred, adjust the pH of the fermentation liquid back to 6.5 with pre-sterilized liquid ammonia. When the pH spontaneously drops to 6.0, dynamically feed with ammonia solution to maintain the pH in the range of 5.8-6.4, preferably dynamically maintaining the pH at 6.0.
[0087] (2) Temperature: Start the circulating water device and control the temperature at 30℃.
[0088] (3) Dissolved oxygen: tank pressure 0.05MPa, stirring 25HZ, by adjusting the ventilation volume, tank pressure or slightly increasing the speed, the dissolved oxygen is kept between 10% and 30%.
[0089] (4) Feeding: The carbon source was provided by pre-sterilized 60% glucose, the nitrogen source was provided by 40% ammonium sulfate, and the pH was controlled by liquid ammonia (50%) mixed with 100% ammonia water and sterile water in a volume ratio of 1:1.
[0090] (5) Glucose was fed in a dynamic constant-rate flow manner, i.e., feeding was started from 24 h after fermentation, and the residual sugar level was measured every 6-12 h. When the sugar concentration was lower than 0.7%, the feeding rate was appropriately accelerated, and when it was higher than 1.3%, the feeding rate was reduced to control the residual sugar concentration in the range of 0.5-1.5%.
[0091] (6) Ammonium sulfate was fed in a dynamic constant-rate flow manner, i.e., feeding was started from 36 h of fermentation, and ammonia nitrogen (NH4 + -N) level, when the ammonia nitrogen concentration is lower than 0.5g / L, the feeding speed is appropriately accelerated, and when it is higher than 0.9, the feeding speed is appropriately slowed down, and the ammonia nitrogen concentration is dynamically controlled between 0.5-1.0g / L.
[0092] (7) Termination of fermentation: At the beginning of fermentation, sample and measure the concentration of wuyi mycin in the supernatant. When the rate of increase of product concentration slows down significantly, stop the fermentation.
[0093] 2.4 Detection of carbon sources
[0094] Take 1 mL of supernatant and dilute to 10 mL. Use a blood glucose meter to test the sugar concentration and adjust the sugar replenishment rate of the fermentation tank according to the measured sugar concentration.
[0095] Carbon source calculation formula: Residual sugar content (g / L) = blood glucose meter test value × 1.8
[0096] 2.5 Detection of ammonia nitrogen
[0097] Take 40 μL of supernatant and dilute to 10 mL. Take 1 mL of the diluted supernatant and add 5 mL of solution B (18 g / L NaOH, 0.04 M sodium hypochlorite) and 5 mL of solution A (35 g / L phenol, 0.2 g / L sodium nitrosoferricyanide) in sequence. Heat in a 37°C water bath for 35 min. Measure the ammonia nitrogen value at a wavelength of 625 nm using a spectrophotometer and change the feeding rate in time.
[0098] The calculation formula of ammonia nitrogen was derived by making a standard curve of ammonium sulfate.
[0099] Ammonia nitrogen calculation formula: Ammonia nitrogen value = (0.2004 × OD 625 -0.0018)×250×0.212
[0100] In a 5L liquid fermentation tank, a total volume of 3L of liquid culture medium is prepared according to the formula of Wuyimycin fermentation medium, wherein yeast powder is replaced with fish meal, and the carbon source is selected separately as 50g / L glucose, which is sterilized in a high-temperature sterilizer at 115°C for 15 minutes and then immediately removed; mature fermentation seeds are inoculated with the above fermentation medium at an 8% inoculation rate and cultured in a 5L liquid fermentation tank for 5-7 days. Samples are taken every 12 hours after the start of fermentation to detect the carbon source and nitrogen source content.
[0101] like Figure 3 As shown in the figure, the dry weight of the bacteria reached 36g / L at the 84th hour of fermentation when fish meal was used as a nitrogen source. In the later stages of fermentation, the dry weight of the bacteria fluctuated slightly between 35-40g / L. There were slight differences in the effects of yeast powder and fish meal as nitrogen sources on the dry weight of the bacteria. Fish meal showed a slight delaying effect as a nitrogen source, but this did not affect its participation in the normal fermentation process. Figure 5-Figure 6 The results showed that the yield of wuyinomycin using fish meal as nitrogen source was 7.5 g / L. Figure 4 It showed that the content of wuyimycin could reach 9.5 g / L in the late fermentation stage, which indicated that the replacement of fish meal peptone had a positive effect on the synthesis of the product.
[0102] The present invention utilizes a superior culture medium and an optimal feeding strategy for the fermentation of wuyimycin using glucose and a suitable nitrogen source. Through system optimization, the novel culture medium formulation and fermentation process enable the synthesis of 9.5g / L of wuyimycin in the late fermentation period (144 hours). This represents a 250% increase in wuyimycin production compared to current fermentation levels, demonstrating its potential application in the field of industrial microbial fermentation.
[0103] Compared with the traditional wuyimycin fermentation production method, the present invention adopts the optimized culture medium formula and related fed-batch fermentation method to produce wuyimycin, which has the following advantages: glucose as substrate ensures the continuous and rapid supply of carbon skeleton and ATP, thereby greatly improving the fermentation yield of wuyimycin; ammonium sulfate as inorganic nitrogen source ensures the product NH4 + -N long-term demand, promote cell growth and metabolism; appropriate phosphorus source concentration reduces the fermentation process's requirement for dissolved oxygen levels while reducing costs.
[0104] Example 3: Effect of phosphate content on the biosynthesis of wuyiencin
[0105] In a 5L liquid fermentation tank, the total volume of the liquid culture medium was prepared according to the formula of Wuyi mycin fermentation medium M3G. After passing through the 5L fermentation tank, the bacterial mass reached 40g / L. In the 5L tank of the fermentation plant, the fermentation with fish meal instead of yeast powder resulted in excessive bacterial mass, insufficient oxygen supply, abnormal metabolism and low product synthesis level. Therefore, based on fish meal as the nitrogen source, three different fermentation media were set up, including full phosphate (1.36g / L potassium dihydrogen phosphate), 1 / 4 phosphate (0.34g / L potassium dihydrogen phosphate) and no phosphate (no potassium dihydrogen phosphate). The test found that the bacterial growth in the fermentation tank with full phosphate was the fastest; the bacterial growth in the fermentation tank with 1 / 4 phosphate was second, with the highest bacterial dry weight (DCW) of 33.8g / L; the bacterial growth in the fermentation tank without phosphate was almost stagnant, with the highest DCW of 22.2g / L ( Figure 7A). This shows that 1 / 4 phosphate can significantly reduce the bacterial concentration. The 1 / 4 phosphate fermentor had the highest wuyimycin content, indicating that 1 / 4 phosphate did not reduce the level of product synthesis. In the 1 / 4 phosphate fermentor, the bacterial concentration was significantly reduced, but the wuyimycin content did not decrease. In the fermentor without phosphate, the bacterial dry weight was significantly reduced, and the wuyimycin content was significantly reduced ( Figure 7 (B) This indicates that 1 / 4 phosphate is the optimal concentration for fermentation, and that the fermentation system cannot lack phosphate.
[0106] Example 4: Organic nitrogen source replacement
[0107] On the basis of a fermentation medium of one-quarter phosphate, two groups of fermentation media containing different organic nitrogen sources, soybean powder and corn steep liquor, were set up as follows:
[0108] (1) Fermentation medium containing soybean meal: 50 g / L glucose, 2.5 g / L fish meal, 2.5 g / L soybean meal, 10 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 0.34 g / L potassium dihydrogen phosphate, and 0.04 g / L zinc sulfate. Adjust the pH to 7 with NaOH.
[0109] (2) Fermentation medium containing corn syrup: 50 g / L glucose, 2.5 g / L fish meal, 2.5 g / L corn syrup, 10 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 0.34 g / L potassium dihydrogen phosphate, 0.04 g / L zinc sulfate, and adjusted to pH 7 with NaOH.
[0110] Fish meal has a high phosphorus content. Corn syrup powder and soybean meal were mixed with fish meal in a 1:1 ratio to ferment wuyi mycin in order to further reduce the phosphorus content in the culture medium. The growth of the bacteria added with corn syrup was inhibited during the fermentation process, and the bacteria were almost dead after 60 hours of fermentation. The growth rate of the bacteria added with soybean meal was slow. After 60 hours of fermentation, the bacteria gradually recovered their activity after adding 1.5g / L fish meal. Figure 8 As shown in the figure, the wuyimycin content in fermentation without soybean meal was higher than that in fermentation with soybean meal, indicating that the phosphorus deficiency in the culture medium with the organic nitrogen source inhibited bacterial growth and reduced the wuyimycin content. Therefore, the organic nitrogen source was not added during the subsequent fermentation process.
[0111] According to the above experimental results, the optimal fermentation conditions for synthesizing wuyimycin can be obtained as follows:
[0112] The fermentation seeds are inoculated with a modified MG3 fermentation medium at an inoculum size of 8%, and fermentation is carried out under the culture conditions of an initial pH of 6.5, a temperature of 30°C, 200-600 rpm, and a rotation speed coupled with dissolved oxygen, with dissolved oxygen maintained at 10%-30%. The pH value of the entire fermentation process is dynamically controlled at 5.8-6.4 (preferably maintained at a pH of 6.0). When the glucose concentration is lower than 5g / L, a 60% pre-sterilized glucose solution is added through an exogenous flow to maintain the glucose concentration in the fermentation broth at 5-15g / L. When the ammonia nitrogen concentration is lower than 0.5g / L, a 40% ammonium sulfate solution is added through an exogenous flow to maintain the ammonia nitrogen concentration at 0.5-1.0g / L. Fermentation is stopped when the rate of increase of the wuyimycin concentration significantly slows down. Among them, the improved M3G fermentation medium: glucose 50g / L, fish meal 5g / L, ammonium sulfate 10g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 0.34g / L and zinc sulfate 0.04g / L.
[0113] Example 4: Fermentation evaluation after optimization of wuyinicin fermentation conditions
[0114] The seed liquid of Streptomyces noursei CK-15 was prepared according to the method of Example 1, and fermented in a 5 L fermentation tank according to the optimal fermentation conditions:
[0115] The fermentation seeds are inoculated with an optimized fermentation medium (i.e., improved MG3 fermentation medium) at an inoculum size of 8%, and fermented under culture conditions of an initial pH of 6.5, a temperature of 30°C, 200-600 rpm, a rotation speed coupled with dissolved oxygen, and dissolved oxygen maintained at 10%-30%. The pH value of the entire fermentation process is dynamically controlled at 5.8-6.4 (preferably maintained at a pH of 6.0). When the glucose concentration is lower than 5g / L, a 60% pre-sterilized glucose solution is added through an exogenous flow to maintain the glucose concentration in the fermentation broth at 5-15g / L. When the ammonia nitrogen concentration is lower than 0.5g / L, a 40% ammonium sulfate solution is added through an exogenous flow to maintain the ammonia nitrogen concentration at 0.5-1.0g / L. Fermentation is stopped when the rate of increase of the wuyimycin concentration is significantly reduced. Among them, the improved M3G fermentation medium: glucose 50g / L, fish meal 5g / L, ammonium sulfate 10g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 0.34g / L and zinc sulfate 0.04g / L.
[0116] The CK-15 fermentation broth was collected after 120 hours of fermentation and its antibacterial activity against red yeast and plant pathogenic fungi (both provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences). A fermentation broth obtained from simultaneous fermentation in optimized pre-fermentation medium II was used as a control (standard). First, the antibacterial activity of the fermentation broths obtained from CK-15 in two different fermentation media was determined using the tube-plate method with red yeast as the indicator bacteria. A seed solution of CK-15 was prepared and inoculated into the optimized fermentation medium at an inoculum size of 8%. The culture conditions were 28°C, 200-600 rpm, with the rotation speed coupled to dissolved oxygen, and incubation for 72 hours. After completion of the incubation, the wuyimycin fermentation broth was filtered through filter paper to obtain the wuyimycin fermentation broth. After the PDA medium was cooled to 37°C, 1 mL of red yeast solution with an OD value of 1.2 was added to every 100 mL of PDA medium. After mixing, 20 mL of PDA medium containing red yeast solution was added to each culture dish. Place the Oxford cup in a plate and add 200 μL of fermentation liquid. Set up 3 replicates for each treatment and observe and measure the diameter of the inhibition zone after culturing at 25°C for 48 hours. Figure 9 As shown in the results, the fermentation broth collected under the optimized wuyiimycin fermentation conditions of the present invention has higher antibacterial activity against red yeast.
[0117] After testing the antibacterial activity of fermentation broths using red yeast as a model strain, the antibacterial activity of fermentation broths against tomato gray mold (Botrytis cinerea), wheat head blight (Gibberella sanbinetti), apple rot (Valsamail), and rice blast (Magnaporthe oryzae) was also tested. The fermentation broths from the two fermentation media were added to PDA medium cooled to 37°C and mixed until the final fermentation broth content reached 1.2%. After the medium solidified, a sterile 0.6 cm diameter borer was used to shave off the aforementioned pathogen cakes of similar growth conditions. These cakes, mycelial side facing down, were placed in the center of a PDA plate and incubated at 25°C for 3-5 days. Colony growth was then observed. Each treatment was replicated five times. After 3-5 days of growth, colony diameters were measured using the cross-hatch method, and the inhibition rate for each treatment group was calculated. Inhibition rate = (control colony diameter - treatment colony diameter) / control colony diameter × 100%.
[0118] like Figure 10As shown in Table 2, the results show that the fermentation liquid collected under the optimized wuyinicin fermentation conditions of the present invention has a stronger inhibitory effect on tomato gray mold, apple rot pathogen, rice blast pathogen, and wheat fusarium sphaeroides; the inhibition rate of the optimized fermentation medium on tomato gray mold is 39.20%, the inhibition rate on apple rot pathogen is 80.45%, the inhibition rate on rice blast pathogen is 39.04%, and the inhibition rate on wheat fusarium sphaeroides is 47.76%.
[0119] Figure 11 After optimization, the fermentation broth was collected after 120 hours of fermentation, and the content of wuyimycin in the fermentation broth was determined to be 9.44 g / L. This shows that the present invention can significantly improve the synthesis efficiency and content of wuyimycin through the optimization of fermentation conditions, and has better antibacterial activity.
[0120] Table 2 Antibacterial effects of different fermentation media on four pathogenic fungi
[0121]
[0122] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.
Claims
1. A fermentation process for high-yield wuyienin, characterized in that: The method comprises the steps of inoculating a fermentation medium with Streptomyces noursei CK-15 and then controlling the concentrations of glucose and ammonia nitrogen in the fermentation liquid by feeding; When the glucose concentration of the fermentation broth is lower than 5 g / L, a glucose solution is added through an exogenous flow to maintain the glucose concentration in the fermentation broth at 5-15 g / L; when the ammonia nitrogen concentration of the fermentation broth is lower than 0.5 g / L, an ammonium sulfate solution is added through an exogenous flow to maintain the ammonia nitrogen concentration in the fermentation broth at 0.5-1.0 g / L.
2. The method according to claim 1, wherein The fermentation medium includes components with the following concentrations: 50 g / L glucose, 5 g / L fish meal, 10 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 0.34 g / L potassium dihydrogen phosphate, and 0.04 g / L zinc sulfate.
3. The method according to claim 1, wherein The inoculation amount of the Streptomyces noursei CK-15 is 5%-10%, the fermentation temperature is 30° C., the rotation speed is 200-600 rpm, the dissolved oxygen is 10%-30%, and the initial pH value is regulated to 6.
5.
4. The method according to claim 1, wherein When the pH of the fermentation liquid dropped to 6.0, liquid ammonia was used for dynamic feeding to maintain the pH at 6.
5. The method according to claim 1, wherein The fermentation time is 120-170h.
6. The method according to claim 1, wherein The fermentation broth is separated and purified by HPLC to obtain wuyinomycin.
7. Use of the fermentation broth obtained by the method according to any one of claims 1 to 6 in any one of the following: (1) Application in inhibiting red yeast; (2) Application in the preparation of products that inhibit red yeast.
8. Use of the fermentation broth obtained by the method according to any one of claims 1 to 6 in any one of the following: (1) Application in inhibiting plant pathogenic fungi; (2) Application in the preparation of products for inhibiting plant pathogenic fungi; in, The plant pathogenic fungi include tomato gray mold, apple rot fungus, rice blast fungus and wheat head blight fungus.
9. A red yeast antibacterial agent, characterized in that The invention comprises the fermentation liquid or wuyinicin obtained by the method according to claims 1-6.
10. A plant pathogenic fungus inhibitor, characterized in that: The invention comprises the fermentation liquid or wuyimycin obtained by the method according to claims 1-6, wherein the plant pathogenic fungi include tomato gray mold, apple rot fungus, rice blast fungus and wheat fusarium.